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Design, synthesis and evaluation of novel cardiotonic agents based on 5-(2'-aminoethyl)carbostyril and (2'-aminoethyl)-1-hydroxy-2-pyridone systems

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Design, synthesis and evaluation of novel cardiotonic agents based on 5-(2'-aminoethyl)carbostyril and (2'-aminoethyl)-1-hydroxy-2-pyridone systems
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Yoon, Sung-Hwa, 1955-
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English
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xv, 149 leaves : ill. ; 29 cm.

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Acetates ( jstor )
Amines ( jstor )
Chlorides ( jstor )
Chromatography ( jstor )
Nitrogen ( jstor )
Plasmas ( jstor )
Protons ( jstor )
Room temperature ( jstor )
Sodium ( jstor )
Sulfates ( jstor )
Cardiotonic Agents -- chemical synthesis ( mesh )
Cardiotonic Agents -- pharmacology ( mesh )
Cardiovascular Agents -- chemical synthesis ( mesh )
Cardiovascular Agents -- pharmacology ( mesh )
Department of Medicinal Chemistry thesis Ph. D ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Medicinal Chemistry -- UF ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF
Dobutamine -- analogs & derivaties ( mesh )
Dobutamine -- pharmacology ( mesh )
Drug Design ( mesh )
Drug Evaluation ( mesh )
Medicinal Chemistry thesis, Ph. D
Research ( mesh )
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bibliography ( marcgt )
non-fiction ( marcgt )

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Thesis:
Thesis (Ph. D.)--University of Florida, 1989.
Bibliography:
Bibliography: leaves 143-148.
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Typescript.
General Note:
Vita.
Statement of Responsibility:
by Sung-Hwa Yoon.

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Full Text
DESIGN, SYNTHESIS AND EVALUATION OF NOVEL CARDIOTONIC
AGENTS BASED ON 5-(2'-AMINOETHYL)CARBOSTYRIL AND
(2'-AMINOETHYL)-I-HYDROXY-2-PYRIDONE SYSTEMS
BY
SUNG-HWA YOON
A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA 1989




Copyright 1989
by
Sung-Hwa Yoon




To my wife, Saun-Joo
for
her love, patience and encouragement




ACKNOWLEDGEMENTS
I would first like to thank Dr. Nicholas Bodor for giving me the privilege to learn the medicinal chemistry field in his research group. This work would never have been finished without his continuous advice and generosity. I would also like to thank the members of my supervisory committee, especially Dr. James Simpkins for his help in in vitro studies.
Sincere thanks are extended to several members of the group: Dr. Shinji Nishitani for sharing the invaluable chemistry knowledge and the more pleasant time during all my research, Dr. Whei Mei Wu for helping me in the analytical experiments, Dr. Peter Polgar for his invaluable assistance in the animal experiments, Dr. Marcus Brewster for reviewing my writing and all our office teams, Laurie Johnston, Joan Martignago, Julie Drigger for providing me the friendly atmosphere.
I would also like to thank all my Korean friends in Gainesville, especially, Mr. Seung Hoon Park for allowing me to use his computer system.
I especially thank my parents, Maeong-Ho and Yon-Yo, my brothers, Sung-Jae and Sung-Woo, my sisters, Yang-Soon and Jung-Mi, my parents-in-law, Young-Kook and Sook-In for their encouragement throughout this work.
iv




Finally, I would like to express my deepest appreciation to my wife, Saun-Joo, and my precious little girl, Alyssa. They are really everything in my life.
v




TABLE OF CONTENTS
page
ACKNOWLEDGEMENTS .......... ................... iv
LIST OF TABLES ....... ................... viii
LIST OF FIGURES ........ .................... ix
KEY TO ABBREVIATIONS ...... ................. xi
ABSTRACT ......... ...................... xiii
CHAPTERS
I. INTRODUCTION ........ ................. 1
Pathophysiology of Congestive Heart Failure 1 Development of Inotropic Agents .... ........ 3
Mechanism of Action ...... ............ 3
Glycosides ....... ................ 5
Catecholamines and Sympathomimetic Amines 7 Other Inotropic Agents .... .......... 8
Problems with Current P-Agonists ....... 13 Objectives of Research .... ............ 14
II. DESIGN ........ .................... 15
Pharmacology of Dobutamine and Its Analogues. 15
Mechanism of Action ... ........... 15
Metabolism of Dobutamine .. ......... .18
Adverse Effects ..... .............. 19
Design of Dobutamine Analogues As Selective 0,Agonist ....... .............. .19
Design of 6'-Substituted Dobutamine
Analogues ..... ............... ..19
Design of 5-(2'-Aminoethyl)-carbostyril
system ..... ............... 21
Design of (2'-Aminoethyl)-l-hydroxy-2-pyridone
system ...... ................ 24
III. EXPERIMENTAL ....... ................. 28
Materials and Methods ..... ...... 28
Synthesis ....... .................. 29
High Pressure Liquid Chromatography System. 75 Chemical Stability .... .............. 76
vi




In Vitro Studies ..... ............... 76
In Vivo Studies ..... ............... 79
IV. RESULTS AND DISCUSSION .... ............ 83
Synthesis ....... .................. 83
High Pressure Liquid Chromatography System. 116 Chemical Stability ..... .............. 120
In Vitro Studies ..... ............... ..120
In Vivo Studies ........................... 128
AM-i Calculation of (2-Aminoethyl)-l-hydroxy2-pyridone systems .... ........... 134
V. SUMMARY AND CONCLUSIONS .... ........... 140
REFERENCES ......... ...................... 143
BIOGRAPHICAL SKETCH ....... .................. 149
vii




LIST OF TABLES
Table page
1-1 Some receptor actions of catecholamines. . 8 1-2 Inotropic drugs that activate #,-receptors. 9 1-3 Inotropic drugs that inhibit c-AMP PDE. . 10 2-1 Adrenergic-receptor activity of sympathomimetic amines .... ............ 17
4-1 Substitution reaction of compound(18). .. 84 4-2 Replacement reaction of compound(35) ..... 93 4-3 The results of Fridel-Craft acylation reaction of compound(53,54) ... .......... ..98
4-4 Cyclization reaction of compound(67) ..... .101 4-5 Synthetic attempts for compound(84)... .. 112 4-6 Rearrangement reaction of N-oxide(88) ... 114 4-7 Half-life(hr) of disappearance and correlation coefficent for carbostyril compounds in
pH=7.40, in 100% human blood, in 80% human
plasma and in 20% rat liver homogenate. . 121 4-8 In vitro activity of the compound(15) ... 126 4-9 In vitro activity of the compound(16) ... 127 4-10 Electrophysiological data of compound(13). 131 4-11 Electrophysiological data of compound(14). 132 4-12 Physio-chemical data of compound(15), compound(16) and dopamine ... .......... 136
4-13 AM-l calculation results of compound(15). 137 4-14 AM-i calculation results of compound(16). . 138 4-15 AM-I calculation results of dopamine ....... ..139
viii




LIST OF FIGURES
Figure page
1-1 The c-AMP cascade in cardiac muscle cell. 4 1-2 The mechanism of the digitalis glycosides. 6 2-1 COMT reaction of catechol ... ........... ..18
2-2 Tautomers of 8-hydroxycarbostyril ......... ..24
2-3 Tautomers of l-hydroxy-2-pyridone ......... 26
4-1 Synthetic attempts of compound(8) ......... 83
4-2 Synthetic reaction sequence for compound(8). 85 4-3 The proton NMR spectrum of compound(8). . 88 4-4 Synthetic reaction sequence for compound(9). 89 4-5 The proton NMR spectrum of compound(9). .. 91 4-6 Attempts of synthetic reaction sequence for compound(41) ...... ................. 92
4-7 Attempts of synthetic reaction sequence for compound(41) ..... ............... .. 94
4-8 Attempts of synthetic reaction sequence for compound(41) ...... ................. 97
4-9 Retrosynthesis of carbostyril compound. .. 99 4-10 Synthetic reaction sequence for compound(12), compound(13) and compound(14) ........... .100
4-11 The proton NMR spectrum of compound(12). . 104 4-12 The proton NMR spectrum of compound(13). . 105 4-13 The proton NMR spectrum of compound(14). . 106 4-14 Synthetic reaction sequence for compound(15). 107 4-15 The proton NMR spectrum of compound(15). . 110 4-16 Attempts of synthetic reaction sequence for compound(16) ...... ................. ii1
ix




4-17 The proton NMR spectrum of compound(83). . 113 4-18 Synthetic reaction sequence for compound(16). 115 4-19 The proton NMR spectrum of compound(16). . 117 4-20 The HPLC chromatogram of compound(12) and its
metabolite in 80% human plasma at 23 hrs and
72 hrs ........ .................... 119
4-21 In vitro results of compound(12,13,14) in
80% human plasma ..... ............... 123
4-22 Cardiovascular effects of compound(8),
dobutamine and KM-13 .... ............. 129
4-23 In vivo pharmacokinetic result of compound(12)
in rat ........ .................... 133
x




KEY TO ABBREVIATIONS
AlCl3 aluminum chloride
BH3 borane
C degree centigrade
CDCl3 deuterated chloroform
CD3OD deuterated methanol
CHC13 chloroform
CH2C12 methylene chloride CS2 carbon disulfide
dec decomposition
DMF dimethylformamide
DMSO dimethylsulfoxide
DMSO-d6 deuterated dimethylsulfoxide
g gram
hr hour
'H NMR proton nuclear magnetic resonance HPLC high pressure liquid chromatography
IR infra-red
KBr potassium bromide
M molar
mg milligram
MgSO4 magnesium sulfate min minute
xi




ml milliliter mmol millimole m.p. melting point N normal
KH2PO4 potassium phosphate monobasic
nm nanometer
r correlation coefficent
rpm revolutions per minute
sec second
THF tetrahydrofuran
t1r half-life
UV ultraviolet ul microliter
> greater than
< less than
xii




Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy
DESIGN, SYNTHESIS AND EVALUATION OF NOVEL CARDIOTONIC
AGENTS BASED ON 5-(2'-AMINOETHYL)CARBOSTYRIL AND
(2'-AMINOETHYL)-l-HYDROXY-2-PYRIDONE SYSTEMS By
Sung-Hwa Yoon
May 1989
Chairman: Nicholas S. Bodor Major Department: Medicinal Chemistry
In order to develop novel, improved cardiotonic drugs, three different types of chemical manipulations of dobutamine were investigated.
Two 6'-substituted analogues of dobutamine were synthesized by coupling dopamine and the corresponding ketones, and the cardiovascular effects of these novel compounds were evaluated in dogs. The 6'-methoxy analogue of dobutamine showed five times higher inotropic activity than dobutamine without significant changes in mean arterial blood pressure and pheripheral vascular resistance.
Three new analogues based on the carbostyril system were designed, in which the m-hydroxy group of dobutamine was isosterically modified to an amide type carbostyril system. The compounds were synthesized from p-methoxyphenethylamine and their stabilities in chemical and biological media in xiii




vitro conditions were studied. All carbostyril analogues were extremely stable in pH = 7.40 phosphate buffer, whole human blood, 80% human plasma and 20% rat liver homogenate. In order to evaluate the effects on the function of sinus node and the cardiac conduction system, in vivo cardiac electrophysiological studies in dogs were performed. The 8hydroxycarbostyril analogue and the 3,4-dihydroxy-8-methoxy carbostyril analogue did not change the cardiac electrophysiological parameters.
Two analogues of (2'-aminoethyl)-l-hydroxy-2-pyridone system which has isosteric structural similarity with dopamine without having the COMT vulnerable m-hydroxy group were synthesized via 12 synthetic steps. Their dopaminergic activities were evaluated by measuring the inhibitory effects of prolactin secretion from the anterior pituitary in rats. 4-(2'-Aminoethyl)-l-hydroxy-2-pyridone caused a continuous reduction of prolactin secretion at 10 9 10- M concentration ranges while 5-(2 '-aminoethyl) -1-hydroxy-2-pyridone showed its activity at 106 M concentration. These results indicate that this system may be used as a starting material for new cardiotonic drugs.
Before the syntheses of the two analogues of (2'aminoethyl)-l-hydroxy-2-pyridone, semiempirical MO calculations(at the AM-1 level) were performed in order to understand the structural and electronic features as compared to dopamine. The results of AM-i calculation indicate that both compounds were less lipophilic than dopamine and have a xiv




weak interaction between the hydrogen in the N-hydroxy group and the adjacent oxygen atom.
xv




CHAPTER I
INTRODUCTION
Pathophysiology of Congestive Heart Failure
Congestive Heart Failure(CHF), which is defined as the circumstance which occurs when the ventricle is unable to provide a cardiac output sufficient to meet the metabolic demands of the body, is a common clinical manifestation associated with many different forms of heart disease. It is a leading cause of death throughout the United States and in other industralized countries. 23This disease is caused by a variety of abnormalities, including pressure and volume load changes, muscle atrophy, primary muscle disease or excessive peripheral demands such as high cardiac output failure. In the typical forms of heart failure, the contractility of the heart muscle reduced due to the inability of the left ventricle to adequately pump blood. This produces a reduction in cardiac output and, as a result, the heart is unable to meet the peripheral demands of the body.
Pathophysiologically, there are four main factors affecting left ventricular(LV) performance.4 The first is preload, which relates to the filling pressure of the left ventricle. The second factor, afterload, represents the load against which the heart must work, i.e., systemic arterial pressure and systemic vascular resistance. Third, the
1




2
contractility of the heart represents the intrinsic ability of the muscle to develop force and constrict. Contractility can be altered by the administration of positive or negative inotropic agents. The fourth factor, heart rate, is adjusted and controlled by the balance of sympathetic and parasympathetic tone.
As a number of pathophysiologic alterations occur during the development of CHF5, the four factors of LV performance are generally altered as follows:
(1) There is an intrinsic decrease in muscle contractility due to prolonged pressure or volume overload.
(2) Preload or left atrial filling pressure is increased due to retention of salt and water. An increased circulating blood volume results in pulmonary congestion and dyspnea.
(3) Although systemic blood pressure is often reduced in CHF, there is an increase in systemic vascular resistance (afterload), which can further reduce cardiac output.
(4) Heart rate is generally increased as a result of compensatory mechanisms.
The alterations in contractile state, preload, afterload and heart rate provide the basis for most of the therapeutic interventions. For example, in order to counteract the marked decrease in intrinsic contractility which occurs in heart failure, inotropic agents such as digitalis or catecholamines are administered and can improve contractile state and cardiovascular performance. The increased preload that exists in CHF can be lowered with diuretics and vasodilator drugs to




3
reduce the filling pressure to a more optimal level. The increase in systemic vascular resistance can be reduced by arteriolar vasodilators. As a result, these drugs cause an increase in forward cardiac output. Although the heart rate in patients in CHF is not generally manipulated, in patients with tachycardias, great benefits can be achieved by reducing the ventricular response. It is evident, therefore, that most of current therapeutic intervensions in heart diseases are designed to counter the pathophysiologic changes which occur as a result of CHF.
Development of Positive Inotropes for CHF Mechanism of Action
Since digitalis and its related cardiac glycosides have been employed in the treatment of CHF for nearly two centuries6, the mechanism of action of these compounds as positive inotropes, i.e., agents which increase the force of contraction of the heart muscle, has been extensively studied7-1 and then the cascade of events between extracellular stimulation and contraction of cardiac muscle cell are 12
recently established, as shown in Fig. 1-11.
Stimulation of either f-adrenergic receptors(a) or H2histaminergic receptors(b) activates the catalytic component(f) of the adenylate cyclase complex via close association of these cell surface receptors with the cell membrane bound adenylate cyclase regulatory component(d). Since this enzyme system is responsible for the conversion of




4
ATP to c-AMP, its activation results in increased cellular concentration of c-AMP. Higher intracellular c-AMP concentration leads to greater interaction of c-AMP with the regulatory subunit of protein kinase(h), which, in turn, increase its catalytic activity and cause enhanced phosphorylation(i) of various cellular proteins. These proteins are of particular importance for cardiac muscle contraction. Phosphorylation of specific proteins associated with the Ca2+ channel in the sarcolemma enhances the
ATP
a d
b d f
9
C e
c-AMP 5-AMP
h + C a
--+
cca
Figure 1-1 The c-AMP cascade in cardiac muscle cell




5
responsiveness of the channels to voltage activation (prolongs the gate(j) open time), which results in a greater influx of Ca 2+.13 The entering Ca +is thought to act as a trigger14 to release intracellular Ca2 stored in the sarcoplasmic reticulum(k). This release of Ca subsquently causes contraction through direct interaction with the contractile proteins(l). Phosphorylation(i) of specific proteins associated with sarcoplasmic reticulum(k) allows for a more rapid and increased re-uptake of Ca after a contractile event has occured. Thus, when intracellular c-AMP levels are raised, both the rate of contraction(df/dt) and the rate of relaxation(-df/dt) are increased and the shape of the resulting contractile force/time curve retains its symmetry. The latter is useful for characterizing the mechanism of compounds suspected to act on the c-AMP cascade. Two important ATP regulatory systems are depicted in Figure 1-1. At least one consequence of stimulating muscarinic cholinergic receptors(c) is to decrease the conversion of ATP to c-AMP5 through association of these receptors with an inhibitory component(e) of the adenylate cyclase complex. Similarly, the degradative enzyme phosphodiesterase(g;PDE III) decreases intracellular c-AMP by converting it to 5'-AMP. Glycosides
Digitalis glycosides have occupied a prominent place in the management of CHF and certain arrhythmias since Withering recognized that this steroid was useful in the treatment of dropsy in 17856. The manner in which digitalis exerts its




6
direct positive inotropic effect is most probably explained by the ability of digitalis to inhibit membrane bound Na+,K activated adenosine triphosphatase (Na+, K -ATPase). As shown in Figure 1-2, it is thought that glycosides(a) bind to cell membrane(b) Na K -ATPase(c) and inhibit the pumping of Na+ out of the cell. An increased intracellular Na+ concentration then increases Na+/Ca2+ exchange(d) or alternatively, increases the displacement of Ca2+ from membrane-associated
2+ 16 2+ Ca pools(e) Increased intracellular Ca concentration is directly associated with increased contraction as show in Fig.l-l.
Although digitalis is still prescribed to increase the force of myocardial contractions for patients with CHF, the toxicity of glycosides limits their utility.7 Most investigators18 engaged in Na-K-ATPase research agree that
b
I / Na +
d
2eg
Caa
Figure 1-2 :The mechanism of the digitalis glycosides




7
the toxicity of the glycosides is intimately related to their binding to the Na pump. This situation has prompted researchers to find non-glycoside cardiotonics. Catecholamines and Sympatomimetic Amines
For many years, catecholamines and other sympathomimetic amines have been used in an attempt to replace the cardiac glycosides in that stimulation of A-receptors by catecholamines increases c-AMP availability in the cell and, as a result, increases calcium availability for contractile proteins. These drugs showed initial promise in the treatment of heart failure when given intravenously (i.v.) infusion over short periods of time and are still useful as an inotropes when such therapy is required. 19 However, their use in CHF was limited due to their lack of oral bioavailability.
Their limitations also include the lack of P1-receptor vs 02- and a-receptors selectivity As shown in Table 1-15, activation of various sites on different adrenergic receptors results in different effects. In addition, as CHF progresses, the P-adrenergic receptors appear to down-regulate with the cascade becoming less sensitive to catecholamine stimulation.2123 Due to these problems, this approach has been avoided. The work which has continued in this area has dealt mostly with attempts to improve the poor oral bioavailabiliy associated with better receptor selectivity Adrenergic receptor activity of sympathomimetic amines under development for use in the treatment of CHF is listed12 in Table 1-2.




Table 1-1 : Some receptor actions of catecholamines
Aderenergic receptor site action
Beta, Myocardium Increase Contractility S-A node Increase Heart Rate Atrioventricular Enhance conduction Conduction System
Beta2 Arterioles Vasodilation
Lungs Bronchodilation Alpha Pheripheral Vasoconstriction Arterioles
Other Inotropic Aqents
Recent interest has been focused on an altogether new class of inotropic drugs which defy simple classification and are distinctive in that their mechanism does not involve the p1-receptor or sodium-potassium stimulated ATPase. The first breakthrough occurred nearly 10 years ago with the discovery that amrinone had positive inotropic activity. 24-2 Although its mechanism of action was at first unknown, it is thought to inhibit specific phosphodiesterase(PDE III). 27, As a result, significant attention had turned toward PDE inhibition. Several related heterocyclic compounds that inhibit c-AMP PDE have been prepared and these drugs are now entering clinical studies for the treatment of CHF. Representative compounds are listed in table 1-3.
These compounds also generally exhibit pronounced vasodilator properties and cause only modest inotropy in CHF




Table 1-2 Inotropic drugs that activate 01-receptors.
OH ON
R -0NHRIIt" NHR t1" 0j NHRII R tR IRHR
2-5 6,7 1,8.9
drug R R' R" I (prenalterol) OH H CH(CH3)2
2 (dopamine) OH OH H
3 (dobutamine) OH OH CHCH/CH, OH CH3
4 (dopexamine) OH OH (CH2)eNHCH2CH5 (ibopamine) OCOCH(CH3)2 OCOCH(CH3)2 CH3
6 (butopamine) Oil H CHCH2D OH CH3
7 (denopamine) OH H OCH3 CH2CH2, 0 OCH3
8 (xamoterol) OH H
CH2CH2NHCON 0
\-j
9 (doxaminol)b H H 0
CH2CH2 0 and OH3




10
Table 1-3 : Inotropic drugs that inhibit c-AMP PDE.
NHCOCH,
OCHH
xx
N N R
H oH
0 0 0 0
11-13 14 15 16
P OCH,
N N X--y NH R
o 0 0 0
HN NH r
2 n n N CH
00
0 0
17, 18 19.20 21 22
OCH
0 (OCH,
ft (prxmn)N ftCH
CH, e
CH, 0C1-9 OC H,
N
O OH,
H N H H H
00 0 0
23. 24 25 26, 27 28
drug X R R
I1 (amrinone) N H NH2 12 (milrinone) N CH" CN 13 (APP-201533) C'H C'H, NH,
14 (SKF-94120)
15 (perinone) N CH, CN
16 (BDF-8634) CHCH 17 (piroximone) N CHCH3
18 (enoximone) CHSCH, C H,
19 (imazodan) Hf
20 (CI-930) ('H3
21 (pimobendan)'
22 (LY.1955(62)d CH2 H H
23 (buquineran) (CH N (CH,)3('H,
24 (carbazeran)' N CH CH2CH,
25 (OPC-8212)
26 (quazinone) cl H C'H,
27 (RS-82856) H R/ H
28 (ORF-16600)




11
patient. In fact, their predominant afterload reduction effect is due to vasodilation property although it is not established that the vascular profile obtained from PDE inhibition is ideally suited for treating CHF. Therefore, these compounds should not be viewed as derivatives with pure cardiotonic action.
In addition to PDE inhibition, a small effort29 has been directed toward preparing c-AMP analogues with the thought that they might penetrate the sarcolemma of cardiac cells and stimulate c-AMP. The dibutyryl derivative of c-AMP known as bucladesine(l) has shown a promise for this purpose although c-AMP is chemically fragile and susceptible to PDE inactivation.
NIICO(C H2)1c 14
N
0
0
O=P
I ~ C0(CN)ICH'
ONa
(1)
Alternatively, histaminergic receptor agonists(H,agonists) were tested in the management of cardiac




12
decompensation because H2-agonists don't cause down regulation as CHF progresses."'3' Impromidine(2) is a potent and selective H,-agonist which has been studied for its cardiovascular effects in man and was shown to significantly improve myocardial function after treatment.32 However, at least three challanging points must be considered when H,agonists are used as potential new inotropic drugs: (1) Agents should have selective cardiovascular action in difference to H2-receptor mediated gastric acid secretion, (2) agents should have selective cardiotonic activity related to vascular and respiratory action, and (3) histaminic drugs should produce positive inotropic activity rather than heart rate increase.
Direct activation of adenylate cyclase was also considered as inotropic way. The natural product forskolin(3) is thought to interact with regulatory subunit or catalytic subunit of the adenylate cyclase system.33 This compound exhibits both inotropic and vasodilatory properties.
CH,
N H OH O ,, HI
0 H CH,
(C H), NHCNIH(C H,),SCH, CH, 0 OH
OCOCH,
CH, CH, O
(2) (3)




13
Problems with Current 01-Aconists
As discussed previously, clinical use of most catecholamines and sympathomimetic amines is largely limited by poor oral bioavailabily even though intravenous sympathomimetic agents are used for the acute treatment of heart failure. The use of parenteral drugs requires hospitalization, usually in intensive care units, and hemodynamic monitoring is necessary to optimize therapy. Moreover, these catecholamines can not be used chronically because tolerance develops rapidly(48 to 72 hours in some cases). 34 Therefore, the development of oral agents for chronic maintenance therapy of CHF is a high priority goal. A number of the sympathomimetic amines are available in oral forms and some data have accumulated regarding their use in patients with heart failure.
Another limitation of these agents results from their positive chronotropic(increase heart rate) effect which causes tachycardia and from their action to either increase or decrease peripheral resistance and thus, to change arterial pressure.35 Furthermore, the stimulation of P2-adrenergic receptors in the peripheral arterial system can decrease peripheral resistance and arterial pressure, which may not be desirable when myocardial ischemia is a potential problem.
In addition to problems of selectivity, a "downregulation,20 in P1-adenergic receptor density may occur following chronic use of sympathomimetic amines.3 This desensitization of physiologic responsiveness of ventricular




14
tissue to a P1-adrenergic agonist may be primarily due to a marked diminution in adenylate cyclase responsiveness rather than to change in the adrenergic receptor properties per se.37
From the described liabilities associated with 01agonists, it is easily concluded that the ideal 01-agonist should have (1) inotropic effect, but not chronotropic effects; (2) potent with long half life; (3) oral bioavailabilty; (4) no change in blood pressure and (5) no down-regulation.
Objectives of Research
This research is designed to develop novel cardiotonics which have long duration of action as well as oral bioavailability. These new agents were designed by chemical manipulation of dobutamine. Three different chemical manipulations were considered.
After the synthesis, the stability in biological media and the in vitro activity of these compounds were studied. Finally, in vivo activity was assessed by observing the changes in cardiac electrophysiological parameters caused by these compounds in dogs.




CHAPTER II
DESIGN
PharmacoloQy of Dobutamine and Its Analogues Mechanism of Action
Sympathomimetic amines generally augment myocardial contractility by stimulating 01-adrenergic receptor sites in the myocardium directly or by releasing endogenous norepinephrine as in the case of Aramine. Norepinephrine is an endogeneous catecholamine that is synthesized and stored in granules in adrenergic nerve endings in the myocardium. When sympathetic nerves to the heart are activated, norepinephrine is released from its stores and stimulates specific sites on the myocardial cell surface, termed 31adrenergic receptors. Stimulation of these receptors increases the rate of discharge of the sinoatrial node, thereby augmenting heart rate and enhancing atrioventricular conduction and ventricular myocardium contraction.
Dobutamine(4), which resulted from systematic modification of isoproterenol(5) by Tuttle and Mills acts directly on )1-adrenergic receptors in the myocardium to selectively increase the myocardial contractility with less side effects than is seen with other sympathomimetic agents. Dobutamine acts selectively on adrenergic 01-receptors sparing 92 and a receptors from stimulation (see Table 215




16
1). In comparison to dopamine(6) which is a precursor in the biosynthesis of norepinephrine and which stimulates the myocardium directly and indirectly through release of norepinephrine from its stores, dobutamine does not stimulate the heart indirectly and lacks the direct vasodilator effect of dopamine on the renal vasculature. In addition, dobutamine does not also show the predominant vasoconstrictor(aadrenergic) effect seen with dopamine. Furthermore, dobutamine has been shown not to increase heart rate substantially even when given in a dose that produces a prominent increase in myocardial contractility.42 High-doses of dobutamine(>7 ug/kg/min) raise heart rate appreciably. However, this raise in heart rate is usually less than that seen with high doses of dopamine(>10 ug/kg/min).43
HON
(4)
NO 1OH
OH
HO NH-CH(CH3)2 (5)
HO (6) HO




17
Table 2-1 : Adrenergic-receptor activity of sympathomimetic amines.
a1 02 Peripheral Cardiac Peripheral Norepinephrine .... .... 0 Epinephrine .... .... ++ Dopamine .... .... ++
Isoproterenol 0 .... .... Dobutamine + ++
Similarly, in the animal experiments, dobutamine exerts a more prominent inotropic than chronotropic action as compared to isoprotereno39. The reason for this difference in response has not been explained, but dobutamine appears to exert a relatively less pronounced effect on the sinoatrial node than on the ventricular contractile tissue.42
Since dobutamine was approved for clinical use in this country in 1978, it has been widely used to treat severe cardiac failure,4 heart failure following acute myocardial infarction4548 and for hemodynamic support in patients 49
following open heart surgery. Intravenous infusions are necessary at rates ranging from approximately 2.5 to 15.0 ug/kg/min. and produce a progressive increase in cardiac output. Pulmonary wedge pressure is decreased, reflecting a fall in diastolic filling pressure in the left ventricle. Generally, the improvement is more substantial in more severe states of cardiac failure.50




18
Metabolism of Dobutamine
The major route of metabolism of catecholamines includes 0-methylation of the catechol catalyzed by catechol 0methyltransferase(COMT). As shown in Figure 2-1, this enzyme catalyzes the transfer of a methyl group from S-adenosyl-Lmethionine to a catechol substrate, resulting in the formation of the meta and para 0-methylated products.52 COMT is widely distributed in mammalian tissues and plays a primary role in the extraneuronal inactivation of endogeneous catecholamines (dopamine, norepinephrine, epinephrine) as well as the detoxification of catechol drugs(isoproterenol, L-dopa).
OH OH OCHS
p I OH COMT. MgI OH
AdoMet AdoHcy
R R R
Figure 2-1 : COMT reaction of catechol.
Recently, Patrick et al.53 reported their metabolic studies in dogs, focusing on the fate of dobutamine after intraveneous dosing. When they examined dobutamine as a substrate of COMT, they found that dobutamine has a K, and Vm x similar to that of dopamine, the natural substrate. They also reported that metabolites are excreted mostly in the urine and feces and the major urinary metabolites are the glucuronide conjugates of dobutamine and meta-0-methyldobutamine. The




19
relative amount of dobutamine glucuronide in the urine was estimated to be 7%, whereas the amount of meta-Omethyldobutamine was estimated as 82% at 24 hours post i.v. administration. Like most of the intraveneous sympathomimetic amines, the slow steady-state plasma levels of dobutamine are increased in proportion to the infusion rate. The elimination half-life of dobutamine is approximately two minutes.54 Adverse Effects
The most serious adverse effect of all the sympathomimetic amines is the precipitation of arrhythmias. The electrophysiologic properties of dobutamine are similar to those of isoproterenol, and ventricular arrhythmias have been associated with the use of both drugs. However, it is claimed that dobutamine causes a lower incidence of
55 56
arrhythmias as compared with isopreterenol and dopamine
Dobutamine may cause a marked increase in heart rate or systolic blood pressure at high dosage, but reduction of dosage usually reverses these effects promptly. Other minor side effects reported include nausea, headache, anginal pain, palpitation and shortness of breath.
Design of Dobutamine Analogues As Selective 01-Agonists Design of 6'-Substituted Dobutamine Analogues
Since the pharmacologic profile of the action of dobutamine is particularly desirable, especially due to better selectivity of fl over P2 and a receptors, a number of studies have been done to modify the chemical structure of dobutamine




20
to make it orally effective conserving in the same time its pharmacological profile.
Recently, Tuttle et al.57 reported that replacement of the para hydroxyl group in dobutamine with carboxyamide at the end of the molecule increased inotropic potency threefold, but it introduced pressor activity that detracted from the inotropic selective profile of dobutamine. However, shifting the carboxyamide to the meta position avoided pressor activity and further enhanced inotropic potency to nine times that of dobutamine as measured in the anesthetized dog. In contrast to dobutamine, this compound(7), KM-13, produces a sustained
com 2
(7)
increase in left ventricular dP/dt with only immediate change in heart rate when administered orally to conscious dogs. It was also reported that the isobutyl bridge between the amine and phenyl ring bearing the carboxyamide is required to achieve the cardiac potency observed and blood pressure criteria.
Based on the above report, it became of interest to study the substituent effects on the phenyl ring and to investigate




21
the further structural requirements needed to meet the potency and blood pressure criteria as a part of structure-activity studies. Therefore, we have synthesized two dobutamine analogues which are substituted with R(=OCH3, CH3) at 6position and have examined their pharmacological activity.
Com2 R= OCH3 (8)
= CH3 (9)
Design of 5-(2'-Aminoethvl)carbostyril System
The intraveneous route of administration greatly limits the number of patients who can get benefit from inotropes because this mode of administration requires the close supervision of medical personnel and usually requires hospitalization. Clearly, the development of oral agents for chronic maintenance therapy of CHF is a high priority goal. Although a number of the symphatomimetic amines are available in an oral form, a new class of dobutamine analogue which would have longer durations of action and oral bioavailability are needed because of their potentially beneficial pharmacological profile.
According to metabolism of dobutamine,53 the major reason of short duration of action of dobutamine is its fast




22
elimination from the body by transformation in the liver to inactive glucuronide conjugates of the meta-Omethyldobutamine. A number of different approaches have been suggested to prolong the duration of similar catecholamines.
The first approach was based on esterification of the vulnerable phenolic OH groups, which might reduce the inactivation rate and result in prolonged activity. Bretschneider59 synthesized the 3,4-diacetate and 3,4dipropionate esters of isoproterenol and other catecholamines but did not report their pharmacological studies. The synthesis and biological testing of various esters were initiated in 1966.6061 In 1975, Tullar et al.62 reported that aromatic esters of N-tert-butylarterenol resulted in a long acting derivative and significant separation of bronchodilator-cardiovascular activity. Similarily, the replacement of the m-OH group of catecholamines by various substituents such as a methanesulfonamide, 63-65 a CH2OH,6 a ureido group67,8 or the aromatic N atom of 8-hydroxyquinoline was also investigeted.69
In 1976, Yoshizaki et al.70 reported that sympathomimetic amines containing 8-hydroxycarbostyril moeity probably exist as resonance hybrids having two acidic hydrogen atoms in a configuration approximating the hydroxyl groups of catecholcontaining adrenergic agents. When this carbostyril system was applied to salbutamol(10), it was found that the resulting procaterol(ll) possesses more potent bronchorelaxing activities than isoproterenol and better selectivity for 02-




23
receptors than salbutamol(10). Recently, Kaiser et al." reported that similar modification of the catechol ring on dopamine produced measurable activation of dopamine-sensitive adenylate cyclase and the potency of carbostyrils was enhanced by 8-hydroxylation and appropriate substitution of amino group of ethylamine side chain.
OH
MCLIM2NHHRO H
CHOH-CHHIICH (CH3)2
OH 1
C2K5
Y = CHIOH; R = t-Bu
(10) (11)
Since sympathomimetic amines with an 8-hydroxycarbostyril moeity exist as resonance hybrids which possess two weekly acidic hydrogen atoms in about the same general vicinity as those in catecholamine but are not good substrates for COMT(see Figure 2-2), it is evident that one of the most effective hydroxyl replacement group in catecholamines is the NH of a carbostyril derivative to produce longer duration of action. The recent results of Kaiser et al.71 have reinforced our strategies to improve dobutamine and to develop longer acting, orally effective cardiotonic agents.




24
N N OH OH H OH
Figure 2-2 Tautomers of 8-hydroxycarbostyril.
In order to determine if the better potency, high Pjselectivity and prolonged effectiveness accompanied isosteric modification of m-hydroxyl of dobutamine with an amide type carbostyril system, we have synthesized some modified 5-(2'aminoethyl)carbostyril derivatives(12,13,14) and examined their pharmacological effects.
0
HN HN CHCO
RO 2 3CONI2
R=CH3(12), R=H(13) (14)
Design of (2'-Aminoethyl)-l-hydroxy-2-pyridone System as
Dopamine Receptor Agonists
Structure-activity relationship studies among dopamine receptor agonists are ambiguous regarding the significance of the catechol system for binding to, and activation of dopamine receptors. Both hydroxyl groups apparently are important for




25
stimulation of the D-172 or DA173 subpopulations of dopamine receptors that are involved in activation of dopaminesensitive adenylate cyclase and initiation of smooth muscle relaxation. However, there are notable exceptions to this generalization. Thus, some 2-aminotetralins that bear only a single hydroxy group in a position meta to the embedded ethylamine side chain, retain a marked degree of D-1 agonist activity. Although selective noncatechol D-1 receptor agonists have not been identified, stimulation of this receptor subtype is also dependent upon the pattern of substitution of the amine nitrogen.74 Clearly, the catecholic system is not required for activation of D-2 72(not associated, or negatively linked with cyclic-AMP) and DA273(located on sympathetic nerve endings and subserving inhibition of norepinephrine release) receptor. Thus, the monohydroxylated tyramine derivative RU 24213(N-n-propyl-N-(2phenylethyl)-tyramine) is a selective D2 receptor agonist.75 However, some of the D-2 agonists are also capable of stimulating dopamine-sensitive adenylate cyclase. Miller's group76 also found that the nitrogen atom of dopamine is not essential for dopaminergic agonist activity and sulfonium analogues of dopamine possess significant activity in both dopamine binding and behavior studies. As a generalization, the presence of a hydroxyl group or a moiety that can imitate this functionality in a position meta to ethylamine is considered to constitute the dopaminergic pharmacophore. 76




26
From the previous SAR studies among dopamine receptor agonists and metabolic pathway of dopamine by COMT, it can be speculated that drugs should have the ethylamine unit if they are to show dopaminergic activity, but a hydroxyl group meta to the ethylamine chain catechol is not necessary to achieve long duration and/or oral effectiveness. On the basis of the above hypothesis, the l-hydroxy-2-pyridone system attached to the ethylamine side chain was considered as a new class of potential dopaminergic agonists as the l-hydroxy-2-pyridone moiety can tautomerize to the 2-hydroxypyridine-l-oxide form, as shown in Figure 2-3. This system has isosteric structural similarity with dopamine without having the vulnerable m-OH
N IN
N C N 0 I :I I O-H -o- H
Figure 2-3 : Tautomers of l-hydroxy-2-pyridone.
group which is a substrate to COMT. This may give long duration of activity and/or oral effectiveness to the dopamine analogues.
Accordingly, the synthesis and pharmacological evaluation of 5-(2'-aminoethyl)-l-hydroxy-2-pyridone(15) and 4-(2'aminoethyl)-l-hydroxy-2-pyridone(16) were undertaken.




27
H2.HU ri oyvw 2 *.Mc
(15) (16)




CHAPTER III
EXPERIMENTAL
Materials and Methods
Salts and nondeutrated solvents were obtained from Aldrich Chemical Co. and Fischer Scientific Co., unless otherwise noted. All salts were reagent grade. Solvents used for high pressure liquid chromatography were of spectral grade. All other nondeutrated solvents were of either reagent or spectral grade. Deutrated solvents were obtained from Aldrich Chemical Co.
Melting points, given in degree Celsius, were determined on a Fischer-Johns melting point apparatus and were uncorrected. Ultraviolet spectroscopy was performed on a Varian Cary 210 spectrophotometer. Proton nuclear magnetic resonance spectra were recorded on a Varian EM 390 spectrophotometer. Chemical shifts are reported in parts per million units(ppm) on the 6 scale downfield from tetramethylsilane which was used as an internal standard. The solvent used are given in parentheses for each spectrum reported. Multiplicities of proton are designated as singlet(s), doublet(d), triplet(t), quartet(q) or multiplet(m). Infrared(IR) spectra were recorded on PerkinElmer 281 spectrophotometer. Solid samples were run as either 28




29
a KBr pellet or a nujol mull; liquid samples were analyzed neat as a thin film between NaCl plates. Mass spectra were performed by Department of Medicinal Chemistry, University of Florida, Gainesville, Florida. Elemental analyses were performed by Atlantic Microlab. Inc., Atlanta, Georgia.
High pressure liquid chromatography was carried out on a modular system composed of a Autochrom M 500 pump a Rheodyne 7125 injector with a 20 ul loop and a Spectroflow 757 variable-wavelength detector. Chromatograms were recorded on a Fischer Scientific Series 5000 chart recorder. A Bio-Sil ODS-5, C18, 10 um particle size, reversed phase silica gel column 30 cm x 4.1 mm internal diameter was used. The Bio-Sil column was protected with a guard column packed with Whatman pellicular ODS-C,8 media. A Dynamic centrifuge having a maximum spin rate of 3,000 rpm was used to spin down tissue homogenates. Chromatographic samples in vitro were centrifuged using a Beckman Microfuge 11 capable of a 13,000 rpm spin rate.
Synthesis
Synthesis of 6'-Substituted Dobutamine Analoques(8,9) Preparation of 3-iodo-4-methoxybenzamide(18)
To a solution of 3.00 g(0.0108 mol) of 3-iodo-4methoxybenzoic acid in 150 ml of benzene was added 1.54 g(l.2 eq) of thionyl chloride and one drop of pyridine at room temperature. The reaction mixture was heated for 2 hrs at 65C and then cooled in ice-water bath before the reaction was




30
quenched with an excess of 28% saturated ammonium hydroxide solution. The white precipitate which formed immediately was collected and recrystallized from 50% aqueous ethanol to give
2.12 g of the desired product(70.6% yield).
m.p. : 151-152.5C.
IR(nujol mull) : 3370, 3170, 2930, 2860, 1650, 1620, 1590, 1470, 1440, 1275, 1050, 1020 cm'.
1H NMR(DMSO-d6) : 3.96(3H,s,-OCH3),7.18(IH,d,JAB=9Hz),
8. 10 (1H,m,JAB=9HzJAC=2Hz), 8.45(IH,d,JAc=2Hz), 7.1-7.5(2H, br.).
Preparation of 4-methoxy-3-(3'-oxybut-2'-envl)benzamide(19)
A solution of 0.276 g(10 mmol) of compound(18), a catalytic amount of 5% Pd-C, 0.081 ml of methyl vinyl ketone (1.1 eq) and 0.15 ml of triethyl amine in 10 ml of acetonitrile was refluxed for 2.5 hrs. Next, the solution was cooled to room temperature, and another 0.073 ml of methyl vinyl ketone(l.0 eq) and 0.115 ml of triethyl amine(l.0 eq) was added. After being refluxed again for 2 hrs, the solution was filtered to remove the catalyst and was concentrated to give a crude product which was identified as starting material with a small amount of desired product. The product was collected by column chromatography on silica-gel with ethyl acetate as an eluent to give a trace amount of product which was identified only by NMR.
IH NMR(CDCI3) : 2.32(3H,s,-COCH3), 3.95(3H,s,-OCH3),
6.83(iH,d,J=16Hz,vinylic H), 7.16(iH,d,J=9Hz), 7.25(2H,br),
7.80(1H,d,J=16Hz,Vinylic H), 8.10(iH,m,JAB3=9Hz,JAC=2Hz),




31
8.45 (lH,d,JAc=2HZ).
Preparation of 5-nitrosalicylaldehyde(21)
Nitric acid (8.0 g) was added dropwise to the solution of 10 g(0.081 mol) of commercially available salicylaldehyde
(20) in 40 ml of acetic acid while stirring in an ice-water bath. The temperature was kept below 15*C during addition of first portion(i/3) of nitric acid. The resulting mixture was stirred at 15C for 25 min., followed by for 2 hrs at 45C, and then poured immediately into 100 g of ice-water. The yellow precipitates which were identified as a mixture of 3and 5-nitro isomers were collected by filtration and dried to give a 12.03 g of product(89.0% yield).
Separation of 5-nitro isomer from mixture was performed as follow : The mixture of isomers was dissolved in 60 ml 0.83 N NaOH while warming. On standing overnight, the 5-nitro isomer precipitated and was filtered, washed with cold 0.83 N NaOH and redissolved in water. Finally, the resulting sodium salt solution of 5-nitro isomer was neutralized with dilute HCl solution to give a yellow precipitate which was dried to give 4.72 g of 5-nitrosalicylaldehyde (34.89% yield from salicylaldehyde).
m.p : 127-128.5C ( ref77 : 126C ).
IR(KBr) : 3450, 3030, 2940, 1665, 1630, 1590, 1530, 1480, 1340, 1290, 1100, 930, 780 cmI.
IH NMR(CDC13 + DMSO-d6) : 7.14(1H,d,JAB = 9Hz),
8.10-8.62 (2H,m), 10.33(1H,s,-CHO).




32
Preparation of 2-methoxy-5-nitrobenzaldehyde(22)
A solution of 2.47 g(0.0148 mol) of 5nitrosalicylalderhyde(21) and 2.65 g(l.3 eq) of K2CO3 in 30 ml of DMF was stirred at 60C for 1 hr and was treated with 1.10 ml(l.2 eq) of methyl iodide dropwise at room temperature. The resulting mixture was stirred for 2 hrs at 60C again. When reaction was completed, the reaction mixture was poured into 50 ml of ice-water. The yellow precipitate was collected, washed with water and dried to give 2.52 g of crude product. Recrystallization from ethanol gave 2.31 g of pure product(86.3% yield).
m.p : 85.5-87.5C.
IR(KBr) : 3045, 1680, 1590, 1520, 1485, 1340, 1275, 1170, 1030, 1010, 970, 840 cm1.
IH NMR(CDCI3) : 4.13(lH,s,-OCH3), 7.20(1H,dJAB=9Hz),
8. 40-8.75(2H,m), 10.57(IH,s,-CHO). Preparation of 2-methoxy-5-nitro-3- (3 -oxobut-l-enyl) benzene
(23)
A mixture of 9.47 g(0.0523 mol) of compound(22) and 20.0 g(l.2 eq) of l-triphenylphosphoanylidene-2-propanone in 250 ml of benzene was heated between 50 and 60C degree overnight. After all solvent was removed in vacuo, the remained crude product was purified by column chromatography on silica-gel with ethyl acetate-hexane(1:2) as an eluent to give a 9.13 g of pure product(79.0% yield).
m.p. : 139.5-141.5-C.




33
IR(KBr) : 3050, 1690, 1610, 1580, 1470, 1340, 1270, 1090, 1020, 830, 750 cm1.
1H NMR(CDCI3) : 2.43(3H,s,-COCH3), 4.05(3H,s,-OCH3),
6.85(1H,d,J=16.6Hz,vinylic H), 7.04(1H,d,JAB=9.2Hz), 7.81(1H,
d,J=16.6Hz,vinylic H) 8.26(1H,dd,JAB=9.2HzJAc=3.OHz) 8.42 (iH,d,JAc=3.0Hz).
Elemental Analysis for C11H1N02 Cald. : C:59.72, H:5.01, N:6.33 Found : C:59.80, H:5.06, N:6.30. Preparation of 5-amino-2-methoxy-3-(3'-oxobutyl)benzene(24)
A mixture of 2.00 g(9.04 mmol) of compound(23) and 65 mg of 10% Pd-C in 90 ml of ethyl acetate was hydrogenated at 25C and initial pressure of 15 psi for 80 min. After the reaction was completed as indicated by TLC, the reaction mixture was filtered and concentrated to give the desired product, which was used in the next reaction without further purification.
IR(KBr) : 3330(br.), 2960, 1705, 1510, 1330 cm'.
IH NMR(CDC13) ; 2.12(3H,s,-COCH3), 2,75(4H,s), 3.40(2H,s,NH2), 3.73(3H,s,-OCH3), 6.40-6.85(3H,m,aromatic H). Preparation of 5-cyano-2-methoxy-3-(3'-oxobutyl)benzene(25)
A mixture of 2.40 g of the crude amine(24) in 5 ml of cHCl and 5 ml of water was chilled to a slurry in ice-water bath. To this mixture, 1.02 g(l.2 eq) of sodium nitrite in 4 ml of water was added dropwise and stirred for 20 min. at 05C. After addition of a few crystal of urea to remove excess of sodium nitrite, the resulting mixture was carefully neutralized with an excess amount of calcium carbonate and




34
filtered into a warm solution of mixture of 1.73 g of CuCN and 2.31 g of NaCN which was prepared in 25 ml of water and 25 ml of benzene. The resulting mixture was stirred for 1 hr at room temperature, and then extracted with 100 ml of ether. Combined organic layers were washed with 5% NaOH solution, 5% HCl solution, water and brine continuously. Finally, evaporation of solvent in vacuo gave a brown colored crude product, which was purified on silica-gel column chromatography with ethyl acetate-hexane(l:3) to give 840 mg of pure product(33.3% yield).
m.p. : 64.5-66C.
IR(nujol mull) : 2920, 2860, 2230, 1710, 1605, 1500, 1430, 1380, 1260, 1030, 820 cm'.
IHNMR(CDCI3) : 2.12(3H,s,-COCH3), 2.80(4H,m), 3.88(3H,s,OCH3), 6.90(IH,d,JAI3=9Hz), 7.40-7.65(2H,m).
Elemantal Analysis for C12H13NO
Cald. : C:70.91, H:6.44, N:6.89 Found : C:70.84, H:6.47, N:6.86. Preparation of 4-methoxy-3-(3'-oxobutvl)benzamide(26)
A mixture of 300 mg(l.40 mmol) of compound(25), 170 mg of potassium bicarbonate and 1.00 ml of 30% hydrogen peroxide in 3.0 ml of methanol was stirred overnight at room temperature. After the reaction was completed, 25 ml of water was added to the reaction mixture and the resulting solution was extracted with 100 ml of ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give




35
287 mg of crude product which was recrystallized from EtOHEtOAc(l:l) to give 265 mg of pure product(85.6% yield).
m.p. : 128.5-130C.
IR(KBr) : 3420, 3365, 3180, 1720, 1660, 1605, 1505, 1440, 1390, 1260, 1170, 1030 cmI.
IH NMR(CDC13 + DMSO-d6) : 2.15(3H,s,-COCH3), 2.83(4H,m), 3.90 (3H, s, -OCH3) 6.85 (1H, d,JAB=9Hz), 6.00-7.50 (2H,br. ,-CONH2),
7.65-7.85(2H,m).
Elemental Analysis for C12H15NO2 Cald. : C:65.14, H:6.83, N:6.33 Found : C:65.02, H:6.89, N:6.28. Preparation of 3,4-dihydroxy-N-(3-(2'-methoxy-5'carbamoylphenyl)-l-methyl-n-propyl)-B-phenethylamine hydrochloride(8)
A mixture of 900 mg(4.07 mmol) of compound(26), 580 mg of dopamine(3.79 mmol), 10 mg of 10% PtO and 200 mg of 10% Pd-C in 25 ml of methanol and 8 ml of acetic acid was hydrogenated for 72 hrs at an initial pressure of 30 psi. After the reaction was completed as indicated by TLC, the reaction mixture was filtered and added 1 ml of c-HCl was added. The solution was concentrated to give a white colored foam product, which was subjected to column chromatography on silica-gel with CHCI3-MeOH-Acetic acid(6:2:0.5) and treated with 20% hydrogen chloride-methanol to afford 1.21 g of the desired hydrogen chloride salt of compound(8) as a powder(80.9% yield).
IH NMR(CD3OD) : 1.75(3H,d,-CH3), 1.70-2.40(3H,m), 2.673.37(8H,m), 3.40(lH,m), 3.95(3H,s,-OCH3), 6.55-6.87(3H,m),




36
7.05-7.10(iH,d), 7.90(2H,m).
Preparation of 2-methyl-5-nitrobenzyl alcohol(28)
To a solution of 24.50 g(0.135 mol) of acid(27) in 140 ml of anhydrous THF cooled in an ice bath was added 145 ml of 1.0 M BH3-THF solution dropwise via a syringe. After the resulting solution was stirred for 14 hrs at room temperature, it was carefully quenched with 10 ml of water and the solvent evaporated under reduced pressure. The residue was dissolved in 50 ml of ethyl acetate and extracted with 5% NaOH solution and water, dried over anhydrous magnesium sulfate and concentrated in vacuo to give 20.70 g of benzyl alcohol(91.8% yield).
m.p. : 73.5-75C.
IR(KBr) : 3250(-OH), 1600, 1530, 1340, 1040, 740 cm-.
IH NMR(CDCI3) : 2.37(3H,s,-CH3), 3.20(1H,s,-OH), 4.73(2H, s, -CH2-OH), 7.27(1H,d,JAB=5Hz), 7.97(1H,d,JAB=5Hz), 8.21(1H,s). Preparation of 2-methyl-5-nitrobenzvlaldehyde(29)
Chromium trioxide, 18.00 g(0.180 mol) was added to a magnetically stirred solution of 28.5 g(0.360 mol) pyridine in 450 ml of methylene chloride for 15 min. at room temperature to give a deep burgundy solution. To this mixture, a solution of 7.00 g(0.042 mol) of alcohol(28) in 10 ml of methylene chloride was added in one portion. A tarry, black deposit separated immediately. After stirring an additional 20 min., the solution was passed through a 5-cm thick silica gel colummn to remove inorganic salt and residue and then was washed with 5% NaOH solution, 5% HCI solution, saturated




37
NaHCO3 solution and brine and dried over anhydrous magnesium sulfate Finally, evaporation of solvent afforded 6.35 g of the aldehyde. Recrystallization from ethyl acetate-hexane(l:2) gave a 6.30 g of pure product(91.3% yield).
m.p. : 54.5-55.5C (ref.78 : 55C).
IR(KBr) : 3035, 2860, 2750, 1700, 1605, 1580, 1505, 1340, 1180, 1100, 830, cm1.
IH NMR(CDCI3) : 2.83(3H,s,-CH3), 7.50(1H,d,JAB=5Hz), 8.35 (1H,dd,JA=5HzJAc=l.5Hz), 8.65(1H,d,JAc=l.5Hz), 10.40(1H,s,CHO).
Preparation of 2-methyl-5-nitro-3-(3-oxobut-l-enyl)benzene(30)
A mixture of 10.43 g(0.0632 mol) of aldehyde(29) and 24.14 g(l.2 eq.) of l-triphenylphosponylidene-2-propanone in 300 ml of benzene was heated between 50 and 60C for 7 hrs. After all solvent was removed in vacuo, the remaining crude product was dissolved in minimum amount of chloroformand was transferred into column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent to give 12.30 g of crude product, which was recrystallized from ethyl acetatehexane mixture to give 11.65 g of pure product(90.0% yield).
m.p. : 95.5-96.5C.
IR(KBr) : 1705, 1630, 1510, 1355, 1280, 980 cm'.
IH NMR(CDCI3) : 2.43(3H,s,-COCH3), 2.57(3H,s,-CH3),
6.80(lH,d,vinyl H,J=16.5Hz), 7.42(lH,d,JAB=5Hz), 7.80(lH, d, vinyl H,J=16.5Hz), 8.15(1H,d,JAB=5Hz), 8.40(iH,s).
Elemental Analysis for CIIHIIN03 Cald. : C:64.38, H:5.40, N:6.82




38
Found : C:64.42, H:5.41, N:6.79. Preparation of 5-amino-2-methyl-3-(3'-oxobutyl)benzene(31)
A mixture of 5.00 g(0.0239 mol) of compound(30), 1.00 g of 10% Pd-C in 110 ml of ethyl acetate was hydrogenated at room temperature and an initial pressure of 15 psi for 80 min. After reaction was completed, the reaction mixture was filtered and concentrated to give the desired product quantatively. This material was directly used in the next reaction without further purification.
IH NMR(CDC3) : 2.10(3H,s,-COCH3), 2.18(3H,s,-CH3), 2.70 (4H,m,-CH2-CH2-), 3.53(2H,s,-NH2), 6.45(2H,m), 7.92(lH,d, JAB=5Hz).
Preparation of 5-cyano-2-methyl-3-(3'-oxobutvl)benzene(32)
A solution of 4.85 g of the crude amino compound(31) in 10 ml of c-HCI and 10 ml of distilled water was chilled to a slurry in an ice bath. To this solution, 1.97 g(l.2 eq.) of sodium nitrite in 10 ml of water was added dropwise and stirred for 1 hr at 0-5C. After addition of few crystals of urea to remove excess sodium nitrite, the solution was carefully neutralized by adding sodium carbonate while vigrously stirring and was filtered into a solution of 3.46 g(2.0 eq.) of cuprous cyanide and 4.62 g(2.5 eq.) of sodium cyanide in 50 ml of water and 50 ml of benzene at room temperature. The resulting dark solution was stirred for 2 more hrs at room temperature and was extracted with 200 ml of ether. The combined organic layer was washed with 5% NaOH solution, 5% HCl solution, saturated NaHCO3 solution and




39
brine, dried over anhydrous magnesium sulfate and concentrated to give a dark brown colored crude oil, which was purified by Krugnonal distillation, followed by column chromatography on silica-gel with ethyl acetate-hexane(l:2) to give 920 mg of white colored product(20.1% yield from compound(30)).
m.p. : 52.5-53.5C.
IR(KBr) : 2900, 2050(-CN), 1700, 1340, 1270 cm-.
1H NMR(CDCI3) : 2.20(3H,s,-COCH3), 2.40(3H,s,-CH3), 2.652.95(4H,m), 7.20-7.45(3H,m).
Elemental Analysis for C12H13NO Cald. : C:76.97, H:7.00, N:7.48 Found : C:76.84, H:7.01, N:7.42. Preparation of 4-methyl-3-(3'-oxobutvl)benzamide(33)
A mixture of 900 mg(4.71 mmol) of the cyano compound(32), 1.50 g of potassium bicarbonate and 3.0 ml of 30% hydrogen peroxide in 6 ml of methanol was stirred for 2 days at room temperature. After the reaction was completed, the solution was added to 20 ml of water and then extracted with 50 ml of ethyl acetate. The combined organic layer was washed with 20 ml of water, dried over anhydrous magnesium sulfate, filtered and concentrated to give 960 mg of the desired product, which was recrystallized from ethyl acetate-hexane(l:l) mixture to give 945 mg of an analytically pure compound(97.8% yield).
m.p. : 127-128C.
IR(KBr) : 3370, 3180, 1705, 1640, 1615, 1420, 1340, cm-I
IH NMR(DMSO-d6) : 2.13(3H,s,-COCH3), 2.30(3H,s,-CH3),
2.80(4H,s,-CH,-CH-), 7.22(2H,d,J=5Hz), 7.65(lH,d), 7.60-7.95




40
(2H,br.-CONH).
Elemental Analysis for C12H15NO2 Cald. : C:70.22, H:7.37, N:6.82 Found : C:69.66, H:7.41, N:6.76. Preparation of 3,4-dihydroxy-N-(3-(2'-methyl-5'carbamoylphenyl)-l-methyl-n-propyl)-B-phenethylamine. hydrochloride(9)
A mixture of 1.14 g(0.0056 mol) of the keto compound(34), 0.772 g(0.9 eq.) of dopamine and 20 mg of PtO2 and 250 mg of Pd-C in 31 ml of methanol and 10 ml of acetic acid was hydrogenated for 48 hrs with an initial pressure of 32 psi. After reaction was completed the mixture was filtered. 5 ml of 20% HCl-methanol was added to the filterate and the resultant solution was concentrated under reduced pressure to give white foam, which was subjected into column chromatography on silica-gel with chloroform-methanol-acetic acid(30:10:0.1) to give 1.45 g of the product.(75.9% yield)
m.p. : 165-169C.
IH NMR(CD30D) : 1.75(3H,d,-CH-CH3), 1.90-2.40(3H,m), 2.70 (3H,s,-CH3), 2.90-3.30(4H,m), 3.40-3.60(2H,m), 6.95(3H,m),
7.55(lH,d) 7.95(2H,m).
Synthesis of 5-(2'-Aminoethyl)carbostyril Derivatives(12,13, 14)
Preparation of 5-chloromethyl-8-hydroxyQuinoline(35)
To a 14.6 g(0.10 mol) of 8-hydroxyquinoline(34) in 100 ml two necked round bottom flask were added 16 ml of c-HCI slowly. The reaction pot was cooled in an ice-water bath and




41
16 ml of 37% formaldehyde was added. Hydrogen chloride gas was passed into the reaction mixture over a period of 3 hrs. The mixture was stirred overnight at room temperature. The yellow crystals which formed were filtered, washed with ether and dried in vacuo to give 20.7 g of product(90.0% yield).
m.p. : 281-283C (ref. 79 : 283oC dec., ref.80 : 250C). Preparation of 5-cyanomethyl-8-hydroxypuinoline(36)
To a solution of sodium cyanide(10.0 g, 0.20 mol) in 150 ml of DMSO at 80-90C was slowly added a hot solution of 9.2 g(0.04 mol) of compound(35) in 100 ml of DMSO. The mixture was stirred at 80-90'C for 45 min. It was allowed to cool and was carefully acidified with 15 ml of c-HCl in order to decompose excess sodium cyanide. The resulting brownish solution was then poured into 500-700 g of crushed ice and neutralized with 10% aqueous NaOH. The precipitate was filtered, washed well with water, and dried. Crystallization from benzene gave 1.20 g of product(16.3% yield).
m.p. : 179-182C (ref. 80 : 178-180oC, ref. 81 : 300'C).
IR(nujol mull) : 3350, 2970, 2230(CN), 1510, 1470, 1425, 1380, 1280, 1195, 835, 795 cm-.
'H NMR(CDCl3 + DMSO-d6) : 4.20(2H,s,-CH2-CN), 6.95-7.60 (3H,m) 8.35(1H,dd,JAB=9HzJAC=2Hz) 8.85(1H,ddJAB=9Hz, JAc=2Hz).
Preparation of 5-cyanomethyl-8-methoxyQuinoline(37)
To 0.866 g(l.4 eq.) of a 60% dispersion of sodium hydride in mineral oil was added 2.70 g(15 mmol) of compound(36) in 25 ml of anhydrous DMF dropwise over 5 min. at room




42
at room temperature. After the addition was completed, the solution was stirred at 50'C for 2 hrs. Next, the solution was cooled to room temperature and 1.16 ml (1.2 eq.) of methyl iodide in 3.0 ml of DMF was added dropwise. The resulting mixture was stirred at 50C for 30 min. again, and then, it was poured into 30 ml of ice water. The mixture was extracted with 160 ml of ethyl acetate. After being washed with water, the extracts were dried over anhydrous magnesium sulfate, decolorized with charcoal and concentrated to give a deep orange colored semisolid. This material was washed with nhexane to give 0.79 g of solid product(27.2% yield).
m.p. : 152.5-155C (ref.71 : 153-155C).
IR(nujol mull) : 2940, 2860, 2260(CN), 1620, 1580, 1510, 1470, 1380, 1270, 1110, 800 cm.
*H NMR(CDCI3) : 4.05(3H,s,-OCH3), 4.20(2H,s,-CH2-), 7.10 (lH,d,J=9Hz), 7.40-7.65(2H,m), 8.40(iH,dd,JJ=9HzJAc=2Hz),
8.85-9.05(lH,m).
Preparation of 8-hydroxy-5-methylauinoline(42)
To a suspension of 10.0 g(0.0434 mol) of compound(35) in 200 ml of ethyl alcohol was added 1.0 g of 10% Pd-C. The resulting mixture was hydrogenated at room temperature and an initial pressure of 50 psi for 3 hrs. After the reaction was completed, the mixture was filtered and washed with water. The combined aqueous alcoholic solution was concentrated in vacuo to give a yellow residue, which was dissolved in 100 ml of water then neutralized with dilute ammonium hydroxide to give a yellowish precipitate. The resulting precipitate was




43
collected, washed with water and dried to give the desired product(6.42 g, 93.0% yield). Recrystallization from ethyl acetate gave the pure compound.
m.p. : 123-124.5C (ref. 122-123C).
IR(KBr) : 3220(br.), 1580, 1470, 1410, 1270, 1180, 960, 780 cm1.
1H NMR(CDCI3) : 2.55(3H,s,-CH3), 7.05-7.50(3H,m), 8.27 (lH,dd,JAB=6HzJAc='.5Hz), 8.80(iH,dd,J=3Hz,J=iHz). Preparation of 8-hydroxy-5-methvlauinoline-N-oxide(43)
A solution of 12.5 g(0.0786 mol) of compound(42) and 29.5 g(0.170 mol) of 80% m-chloroperbenzoic acid in 270 ml of chloroform was stirred at room temperature for 8 hrs. The resulting solution was filtered through 5-cm layer of neutral alumina(Activity grade I). Concentration of the filtrate gave a crude product, which was purified by column chromatography on silica-gel with chloroform as an eluent to give 9.78 g of pure product(70.1% yield).
m.p. : 131-132.5C.
IR(KBr) : 3430(br.), 1605, 1530, 1480, 1430, 1395, 1310, 1270, 1135, 1050, 880, 820, 735 cm-.
IH NMR(CDC3) : 2.53(3H,s,-CH3), 7.02(iH,d,J=6Hz), 7.257.50(2H,m), 7.95(IH,d,J=6Hz), 8.40(lH,d,J=4Hz). Preparation of 8-acethoxy-5-methylcarbostyril(44)
A mixture of 9.78 g(0.0558 mol) of compound(43) and 60 ml of acetic anhydride was heated between 80-90C for 5 hrs. The white solid which had formed was filtered and filtrate was poured into 100 ml of water, neutralized with dilute ammonium




44
hydroxide to give another white solid, which was subsquently collected. The combined solid was washed with water again and dried to give 8.50 g of product, which was used in the next reaction without further purification(70.2% yield).
IR(KBr) : 2950(br.), 1750, 1645, 1490, 1435, 1360, 1180, 1145, 1040, 885, 835 cm-.
IH NMR(DMSO-d6) : 2.37(3H,s,-COCH3), 2.52(3H,s,-CH3),
6.60(1H,d,J=6Hz), 7.17(1H,d,J=6Hz), 7.65(1H,d,J=6Hz), 8.08(1H, d,J=6Hz).
Preparation of 8-hydroxy-5-methvlcarbostyril(45)
A suspension of 8.50 g(0.0392 mol) of compound(44) in 100 ml of c-HCl was heated at steam bath temperature for 7 hrs. Following dilution with 100 ml of water and cooling in ice-bath, a pink colored precipitate was collected, washed with water and dried to give 4.05 g of product(59.2% yield). Recrystallization from ethanol gave an analytically purre product.
IR(KBr) : 3100(br.), 1630, 1610, 1545, 1390, 1285 1160, 1060, 840cm-1.
IH NMR(DMSO-d6) : 2.40(3H,s,-CH), 6.57(iH,d,J=6Hz),
6.90(2H,s), 8.00(iH,d,J=6Hz). Preparation of 8-methoxy-5-methvlcarbostyril(46)
To a stirred suspension of 4.00 g(0.0228 mol) of compound(45) in a solution of 3.76 g(l.0 eq.) of K2CO3.1/2HO in 6.5 ml of water and 32 ml of acetone were added in one portion 2.16 ml(l.0 eq.) of dimethylsulfate. The resulting mixture was refluxed for 1 hr and then concentrated. The




45
residue was extracted with chloroform. The chloroform layer was washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated to give 3.89 g of the desired product(89.7% yield). Recrystallization from chloroform-hexane mixture gave an analytically pure product.
m.p. : 188-191C.
1H NMR(CDCI3) : 2.50(3H,s,-CH3), 3.93(3H,s,-OCH3), 6.70 (lH,d,J=6Hz), 6.90(2H,m), 7.92(lH,d,J=6Hz)
Elemental Analysis for CIIHIIN02
Cald. : C:69.82, H:5.86, N:7.40 Found : C:69.58, H:5.91, N:7.34. Preparation of 5-formyl-8-methoxvcarbostyril(49)
To a cold solution of 2.55 g(0.0134 mol) of 8-methoxy-5methylcarbostyril(46) in 15 ml of acetic anhydride were slowly added 3.0 ml of concentrated sulfuric acid. After the mixture was cooled in an ice-salt bath(about -5 0C), a solution of 3.60 g(0.036 mol) of chromium trioxide in 25 ml of acetic anhydride was added, with stirring at such a rate that did not cause the temperature to exceed 10'C and stirring was continued for 3 hrs at 5-10C in an ice-water bath. The contents of the flask were poured into 200 ml of ice-water, which was allowed to stand overnight. The resulting dark solution was extracted with 200 ml of chloroform and organic layer was washed with water, saturated sodium bicarbonate and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product, which was purified by




46
column chromatography on silica gel to give 320 mg of product(12.0% yield).
m.p. : 227.5-229C.
'H NMR(DMSO-d) : 4.10(3H,s,-OCH3), 6,85(lH,d,J=6Hz),
7.10(1H,d,J=5Hz), 7.60(lH,d,J=5Hz), 9.l5(iH,d,J=6Hz), 10.50 (IH,s,CHO).
IR(nujol mull) : 3170(br.), 1690, 1670, 1605, 1555, 1290, 1270, 1215, 1090, 1070, 840 cm1.
Elemental analysis for CjIH9N03 Cald. : C:65.03, H:4.46, N:6.98 Found : C:64.14, H:4.50, N:6.72. Preparation of 8-hydroxvuinoline-N-oxide(51)
To a solution of 11.14 g of 80% m-chloroperbenzoic acid in 80 ml of chloroformwas added 5.00 g(0.0344 mol) of 8hydroxyquinoline(34) in 40 ml of chloroform at room temperature. The resulting solution was stirred for 16 hrs at room temperature and filtered through 5-cm layer of neutral alumina(activity grade I). The filtrate was evaporated to give a yellow colored crude product which was recrystallized from ethyl actate-hexane mixture to give 2.72 g of the pure product(49.1% yield).
m.p. : 141-143C.
IR(KBr) : 3410(br.), 1600, 1530, 1460, 1405, 1280, 1155, 1050, 820 cm".
IH NMR(CDC13) : 7.05-7.50(4H,m), 7.78(1H,d,J=5.5Hz),
8.24(lH,d,J=4Hz).
Preparation of 8-acetoxvcarbostyril(52)




47
A mixture of 1.15 g(7.14 mmol) of compound(51) and 15 ml of acetic anhydride was heated in a steam bath for 5 hrs. The precipitate which had formed was filtered, washed with water and dried. The brown filtrate was neutralized with dilute ammonium hydroxide to yield another precipitate, which was also collected by filtration, washed with water and dried. The combined white precipitates were used in the next reaction without further purification.
m.p. : 252-255C (ref.82 : 251-154C, ref." : 250C).
IR(KBr) : 1760, 1660, 1640, 1600, 1270, 1185 cm-.
IH NMR(DMSO-d6) : 2.40(3H,s,-COCH3), 6.60(lH,d,J=6Hz), 7.10-7.40(2H,m), 7.65(1H,dd,JAB=5HzJAc='.5Hz), 8.00(1H,d, J=6Hz).
Preparation of 8-hydroxycarbostyril(53)
A suspension of 22.15 g(0.108 mol) of compound(52) in 200 ml of c-HCl was heated in a steam bath for 5 hrs. Following dilution with 200 ml of water and cooling in an ice bath, the tan solid which formed was collected(14.8 g, 84.7% yield). Recrystallization from EtOH gave an analytically pure sample as light tan needles.
m.p. : 289-295C (ref.82 : 297-299C, ref.84 : 287-288C).
IR(KBr) : 3100(br.), 1635, 1600, 1545, 1285, 830 cm'.
1H NMR(DMSO-d6) : 6.58(1H,d,J=6Hz) 7.02-7.30(3H,m), 7.93 (lH,d,J=6Hz).
Preparation of 8-methoxycarbostyril(54)
To a solution of 8.0 g(0.050 mol) of compound(53) in a solution of 4.3 g(0.027 mol) of potassium carbonate, 12.5 ml




48
of water and 62.5 ml of acetone were added dropwise 6.3 g(0.05 mol) of dimethyl sulfate during stirring and refluxing. Refluxing was continued for another 1 hr. The reaction mixture was concentrated and extracted with chloroform. The chloroform layer was evaporated to dryness and the residue was recrystallized from ethyl acetate-hexane mixture to give 7.45 g of pure product(85.1% yield).
m.p. : 108-109.5C (ref.70 : 108-109C).
IR(KBr) : 1645, 1600, 1465, 1270, 1090, 830 cm-.
IH NMR(CDC3) : 3.97(3H,s,-OCH3), 6.65(iH,d,J=6Hz), 6.957.20(3H,m), 7.73(IH,d,J=6Hz).
Preparation of 5-chloroacethyl-8-methoxvcarbostyril(55)
To a suspension of 1.61 g(0.01 mol) of compound(54) and 2.82 ml(2.5 eq.) of chloroacethyl chloride in 25 ml of carbon disulfide were added 4.40 g(3.3 eq.) of aluminum chloride in small portions while stirring and cooling in an ice-water bath. The reaction mixture was refluxed for 2 hrs. After the reaction was completed, the reaction mixture was cooled and the carbon disulfidelayer was decanted. The residue was mixed with chipped ice to give a crude solid product which was collected and washed with water and methanol to give the desired product(l.36 g, 54.0% yield). The product was used in the next reaction without further purification.
IH NMR(CDCI3) : 4.01(3H,s,-OjH3), 5.20(2H,s,-CH2-Cl),
6.70(1H,d,J=6Hz), 7.08(iH,d,J=5Hz), 7.90(iH,d,J=5Hz), 8.55(lH, d,J=6Hz).
Preparation of 5-(2'-chloroethyl)-8-hydroxycarbostyril(57)




49
To a suspension of 100 mg(0.42 mmol) of compound(55) in 4 ml of trifluoroacetic acid were added 2.0 ml of triethylsilane dropwise via a syringe under nitrogen atmosphere. The resulting suspension was stirred overnight to give a yellow solution, which was diluted with water to produce a white precipitate. The precipitate was collected and dried to give 62 mg of product(65.8% yield).
1H NMR(DMSO-d6) : 3.00(2H,m,-CH-), 3.55(2H,t,-CH2-Cl), 6.42(iH,d,J=6Hz), 6.80(2H,s,aromatic H), 7.90(IH,d,J=6Hz). Preparation of N-(2-(4'-methoxyphenyl)ethyl)-2,2,2trifluoroacetamide(60)
To a cold solution of 36.0 g(0.238 mol) of pmethoxyphenethylamine(59) in 360 ml of methylene chloride under nitrogen atmosphere was added dropwise with stirring a solution of 100 g(0.476 mol) of trifluoroacetic anhydride in 50 ml of methylene chloride. After the mixture was stirred for 1.5 hr at room temperature, the volatile material was removed in vacuo, toluene was added and removed, and residue was crystallized from 600 ml of mixture of ethyl ether-petroleum ether(l:l) to give a 48.70 g of white crystal(82.8% yield).
m.p. : 84-85C(ref.85 : 84C).
1H NMR(CDCI3) : 2.80(2H,m,-CH2-), 3.60(2H,m,-CH2-NH-) ,
3.80(3H,s,-OCH3), 6.95(2H,d,JAB=6Hz), 7.20(2H,d,JAB=6Hz). Preparation of N-(2-(4'-methoxy-3'-nitrophenyl)ethyl)-2,2,2trifluoroacetamide(61)
To a cold solution of 15.0 g(0.0605 mol) of compound(60) in 127 ml of trifluoroacetic acid under nitrogen atmosphere was added dropwise while stirring 3.9 ml (1.2 eq.) of




50
concentrated nitric acid. After the mixture was stirred for 3 hrs at room temperature, the solvents were removed and the residue was dissolved in 150 ml of ethyl acetate, which was successively washed with 5% HCl solution, dilute sodium bicarbonate solution and brine and dried over anhydrous magnesium sulfate-activated carbon. The mixture was filtered and the filtrate was concentrated. The resulting crude reddish solid was crystallized from ethyl acetate-hexane(l:l) to give 15.0 g of product(85.2% yield).
m.p. : 92-93*C(ref.85 : 92.5-93C).
IH NMR(CDC3) : 2.93(2H,t,-CH2-), 3.62(2H,m,-CH2-NH-),
7.15(1H,d,JAB=5Hz), 7.47(1H,dd,JAB=5HzJAc=I.5Hz), 7.77(1H,d, JAC=l.5Hz).
Preparation of N-(2-(3'-amino-4'-methoxvphenyl)ethyl)-2,2,2trifluoroacetamide(62)
A solution of 12.10 g(0.0414 mol) of compound(61), 1.20 g of 10% Pd-C in 160 ml of ethanol-ethyl acetate(l:l) was hydrogenated at room temperature and an initial pressure of 50 psi for 1 hr. After reaction completion, the reaction mixture was filtered and concentrated to give a crude product, which was crystallized from ethyl ether-hexane mixture to give 10.27 g of product(94.5% yield).
m.p. : 87-88C(ref.85 : 87-88C).
IH NMR(CDCI3) : 2.70(2H,t,-CH2-), 3.47(2H,m,-CH2-NH-),
3.83(5H,s,-OCH3 & -NH2), 6.50-6.85(3H,m,aromatic H), 7.10(1H, br.,-NH-).
Preparation of N-(2-(3'-(N-acetoacetyl)amino-4'methoxyphenyl)ethyl)-2.2,2-trifluoroacetamide(63)




51
The following reaction was modified from the reported method(ref 13). To a solution fo 5.82 g(0.0222 mol) of amine(62) in 20 ml of anhydrous THF were added 2.05 ml(l.2 eq.) of diketene dropwise via a syringe under nitrogen atmosphere. The reaction mixture was refluxed for 3.5 hrs and solvent was removed in vacuo. The resulting reddish thick oil was subjected to column chromatography on silica-gel with ethyl acetate-hexane(l:l) to give 6.12 g of product(79.6% yield).
m.p. : 104-105C.
*H NMR(CDCI3) : 2.32(3H,s,-COCH3), 2.75(2H,t,-CH2-), 3.60 (2H,-COCH2-CO-), 3.50-3.70(2H,m,-CH2-NH-), 3.90(3H,s,-OCH3),
6.75(lH,br.,-NH-CO-), 6.90(2H,s,aromatic H), 8.23(lH,s, aromatic H), 9.27(lH,br.,-NH-CO-).
IR(nujol mull) : 3290(amide), 1720(C=O), 1675(C=O), 1600, 1465, 1380, 1185, 1145, 1035 cm-.
Elemental analysis for C15HI7N204F3 Cald. : C:52.20, H:4.95, N:8.09 Found : C:51.98, H:4.96, N:8.05. Preparation of 5-(2'-trifluoroacetamido)ethyl-8-methoxy-4methvlcarbostyril(64)
A solution of 720 mg(2.08 mmol) of compound(63) in 25 ml of concentrated sulfuric acid was heated between 80 to 90C overnight. After the reaction mixture was cooled to room temperature, it was carefully poured into crushed ice. The resulting precipitate was filtered, washed with cold water and dried to give 292 mg of a grey colored compound which was




52
insoluble in most solvents except organic acids such as trifluoroacetic acid(45% yield).
m.p.: 224-227C.
1H NMR(TFA) : 2.90(3H,s,-CH3), 3.25-3.80(4H,m,-CH2-CH2-), 3. 9 5(3H, s, -OCH3) 7.17(IH,s,=CH-), 7.15-7.45(2H,m,aromatic
),7.70(IH,br.,-NH-CO-).
IR(nujol mull) : 3245(br., amide), 1715(C=O), 1645(C=O), 1605, 1545, 1210, 865 cm1.
Mass spec.(70eV) : 328(M+).
Preparation of 5-(2'-aminoethyl)-8-methoxy-4-methylcarbostyril hydroQen chloride(65)
A solution of 10.50 g(0.0320 mol) of compound(64) in 31.6 ml of ethanol and 72 ml of water containing 36 ml of concentrated HCI was refluxed for 10 hrs under nitrogen atmosphere. After the solution was cooled, the resultant white precipitate was collected and the filtrate was concentrated to give another brownish solid. This solid was dissolved in a small amount of methanol and diluted with ether to give a white precipitate. The combined white material was reprecipitated from MeOH-ether solution to give 8.20 g of HCI salt(95.43% yield).
The free base of compound(65) was obtained as follows: 3.00 g(0.0112 mol) of compound(65) were dissolved in a small amount of water. The resultant solution was made basic with dilute NH4OH, and then extracted with 200 ml of methylene chloride. The methylene chloride layer was dried over anhydrous magnesium sulfate, filtered and concentrated to give 2.34 g of




53
a white product(90.2% yield).
m.p.(HCl salt) : 235-245C(dec.).
m.p.(Free base) 159-160C.
1H NMR(DMSO-d6;HCl salt) : 2.70(3H,s,-CH3), 2.90-3.15(2H, br. -CH2-) 3. 20-3. 55 (2H, br. -CH2-) 3. 92 (3H, s, -OCH3) 6. 53 (1H,
s), 7.03-7.27(2H,m,aromatic H), 8.40(3H,br.-NLHH3).
Elementary Analysis for C13H16N202 (free base) Cald. : C:67.22, H:6.94, N:12.06 Found : C:67.07, H:7.00, N:12.00.
Mass spec.(70eV,free base) : 232(M+). Preparation of 3-(3'-oxobutvl)benzamide(66)
38.2 g of ethyl 2-(3'-cyanophenylmethyl)-3-oxobutyrate, which was prepared by heating m-cyanobenzyl bromide with acetoacetate for 2 hrs under reflux in 500 ml of concentrated hydrochloric acid. To the reaction mixture were added 500 ml of water, followed by extraction with ethyl acetate(3 x 500 ml). The organic phase was washed with water and dried over anhydrous sodium sulfate after which the solvent was removed. The resultant 3-(3'-oxobutyl)benzoic acid was mixed with 500 ml of benzene and 17 ml of thionyl chloride and heated for 2 hrs under reflux. The reaction solution was poured into an ice-cooled concentrated aqueous ammonia solution and the resulting amide extracted three times with 500 ml portions of ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was recrystallized from ethyl acetate-hexane mixture to give 23.1 g of the product(59.8% yield).




54
m.p. : 122-125C.
'H NMR(CDCI3) : 2.10(3H,s,-COCH3), 2.80(4H,s,-CH2-CH2-),
7.32(2H,d,aromatic H), 7.75(2H,m,aromatic H), 7.90(2H,br.,CONH2).
Preparation of N-(2-(3'-(N-cinnamoyl)amino-4'methoxvphenvl)ethyl)-2.2,2-trifluoroacetamide(67)
To an ice-cooled solution of 2.62 g(0.010 mol) of compound(62) in 20 ml of benzene and 20 ml of anhydrous THF containing 1.5 ml of pyridine were added 1.83 g(l.l eq.) of cinnamoyl chloride in 20 ml of anhydrous THF dropwise while stirring. After stirring in an ice-cooled water bath for 1 hr and subsquently at room temperature overnight, the reaction mixture was filtered to remove the formed white precipitate which was pyridine salt, and then it was concentrated. The resulting mixture was dissolved in 100 ml of ethyl acetate and organic layer was washed with 5% HCl solution, saturated sodium bicarbonate solution and brine. The dried organic layer was concentrated to give 3.47 g of product(88.5% yield).
m.p. : 139-140C.
IH NMR(CDC3) : 2.83(2H,t,-OCH2-), 3.65(2H,m,-CH2-NH),
3.90(3H,s,-OCH3), 6.68(1H,d,J=IOHz,-OCH=), 6.95(1H,s,aromatic
11), 7.00(2H,br.,-NH-), 7.38-7.70(5H,m,phenyl H), 7.78(iH,d, J=lOHz,=CH-NH-), 8.12(1H,s,aromaticH), 8.45(1H,s,aromaticH).
IR(nujol mull) : 3380, 3200-3060(br.), 1720, 1665, 1630, 1595, 1545, 1490, 1355, 1265, 1225, 1195, 1180, 1145, 1030, 805 cm-.




55
Elemental analysis for C20HI9N203F3 Cald. : C:61.22, H:4.88, N:7.14 Found : C:61.17, H:4.91, N:7.11. Synthetic attempts of ring-closure of compound(67)
To a solution of 1.40 g(3.57 mmol) of compound(67) in 30 ml of chlorobenzene were added portionwise under stirring 2.38 g(5.0 eq.) of aluminum chloride. The reaction mixture was heated 80 to 90C for 3 hrs. The resulting dark solution was carefully poured into ice and was extracted with ethyl acetate(2 x 100ml). The combined organic layers were washed with 5% HCI solution, saturated sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a reddish oil, which was identified as a mixture of products.
Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-methoxy-4-methvlcarbostyril(12)
Reductive Amination Method : To a suspension of 1.34 g(0.0050 mol) of HCl salt of compound(65) and 0.95 g(0.0050 mol) of keto compound(66) in 35 ml of methanol and 25 ml of ethanol were added 250 mg of sodium cyanoborohydride, portionwise at room temperature. The resulting solution was stirred for 24 hrs at which time another 150 mg of sodium cyanoborohydride was added and stirred for additional 24 hrs. After the reaction was completed, the solution was concentrated in vacuo to give a white solid which was subjected to chromatography on silica-gel with methanolchloroform(3:l) as an eluent to give 450 mg of product and 430




56
mg of starting material(22.0% yield). The acetate salt as a white powder was obtained from an acetic acid-methanol solution, followed by dilution with ether.
Hydrogenation Method : A mixture of 800 mg(0.00343 mol) of the free base of compound(65), 0.72 g(l.l eq.) of the keto compound(66), 300 mg of 10% Pd-C and 20 mg of PtO2 in 30 ml of MeOH and 10 ml of acetic acid was hydrogenated for 24 hrs at an initial pressure of 32 psi. After the reaction was completed as indicated by TLC, the mixture was filtered and concentrated to give a residue which was subjected to column chromatography on silica-gel with chloroform-methanolto yield a white solid following evaporation of the solvents.. The acetate salt of the product was obtained from an acetic acidmethanol solution, followed by dilution with ether.
m.p.(AcOH salt) : 141-145C.
IH NMR(AcOH salt in DMSO-d6) : 1.35(3H,d,-CH3), 1.60-2.20 (3H,br.-CH-CH2-CH2-) 2.00(3H,s,-CH3COOH) 2.80(5H,s + br.-CH3 & -CH2-CH2-) 3.10-3.35(2H,br.), 3.50-3.75(2H,br.) 3.98(3H,s,OCH3), 6.63(1H,s,), 7.20(2H,m), 7.57(2H,m), 7.95(2H,m).
U.V.(MeOH) : 258 nm(max.).
Elementary Analysis for C26H33N305(AcOH salt) Cald. : C:66.79, H:7.11, N:8.98 Found : C:66.53, H:7.18, N:8.90. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-hydroxy-4-methvlcarbostyril(13)
A solution of 200 mg of compound(12) in 7 ml of 48% HBr was refluxed for 20 hrs. After the solution was cooled to room




57
temperature, a white precipitate was formed which was filtered while the brownish filtrate was stored in a refrigerator. The filtrate gave a pale brownish solid, which was filtered, washed with acetone and dried.
m.p. : 153-155C.
1HNMR(CDCI3+DMSO-d6) : 1.50(3H,d,-CH3) 1.80-2.20(3H,br.), 2.80(3H, s,-CqH3) 2.75-2.85(2H,br.) 3.10-3.70(4H,br.) 6.67
(lH,s,), 7.20(2H,m,carbostyril ring H), 7.55(2H,m,o-Phenyl H to amide), 7.97(2H,m,phenyl H).
Elemental Analysis for C2H27N303.3HBr.3H20 Cald. : C:40.02, H:5.25, N:6.08, Br:34.72 Found : C:40.60, H:5.26, N:6.16, Br:34.98. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-hydroxy-4-methvlcarbostyril(13)
I) Generation of the free amine of compound(65) : 1.00 g of acetate salt of compound(65) was dissolved in minimum amount of water and made basic with dilute ammonium hydroxide The resulting basic aqueous solution was extracted with chloroform(2 X 50ml). The chloroform layers were washed with loml of water, dried over anhydrous magnesium sulfate filtered and concentrated to give 570 mg of a white solid. II) Reaction with BBr3 : A solution of 570 mg(0.00140 mol) of free amine(65) in 50 ml of dry dichloromethane was added to 30 ml of 1.0 M BBr3 solution dropwise via a syringe in an icewater bath under nitrogen atmosphere. After stirring the resulting suspension overnight at room temperature, it was carefully quenched with methanol and concentrated in vacuo to




58
give a yellowish foam, which was directly subjected into column chromatography on silica-gel with chloroformmethanol(l:3) as an eluent to yield a pale yellowish product. This product was purified by reprecipitation from a methanolether mixture to obtain 263 mg of pure product. (48.1% yield).
IH NMR(CD3OD) : 1.50(3H,d,-CH-CH3), 1.80-2.20(3H,br.-CHCH2-), 2.80(3H,s,-CH3), 2.75-2.85(2H,br,-CH2-NH-), 3.10-3.70
(4H,br.,2 of -CH2-ring), 6.67(lH,s), 7.20(2H,m), 7.55(2H,m),
7.97(2H,m).
Elemental Analysis for C23H27N303.l.6HBr.i.3H20 Cald. : C:50.48, H:5.72, N:7.69, Br:23.40 Found : C:50.43, H:5.55, N:7.52, Br:23.25. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl) ) aminoethyl) -8-methoxy-4-methyl-3,4dihydrocarbostyril (14)
A solution of 400 mg of the free base form of compound
(12) in 100 ml of methanol and 400 mg of 10% Pd-C was hydrogenated at an initial pressure of 58 psi for 3 days. After the solution was filtered, the filtrate was concentrated to give a white solid which was dissolved in chloroform and filtered. The filtrate was diluted with ether to give a pale yellow precipitate.
IR(nujol mull) : 3170-3450(br.-CONH2), 1660(C=O), 1460, 1375 cm'.
1H NMR(CD3OD) : 1.15(3H,d,-CH3), l.45(3H,d,-CH3), 1.752.40(4H,br.), 2.60-3.45(8H,br.), 3.85(3H,s,-OCH3), 6.90(2H,m),
7.43(2H,m), 7.72(2H,m).




59
Elemental analysis for C2H31N303.0.45CHCl3 Cald. : C:63.39, H:6.84, N:9.07, C1:10.32 Found : C:63.39, H:7.17, N:9.12, C1:10.14. Synthesis of (2'-Aminoethyl)-l-hydroxy-2-pyridone Analogues (15.16)
Preparation of 6-hydroxvpyridine-3-ethvlcarboxylate(71)
A suspension of 5.00 g(0.0359 mol) of 6-Hydroxynicotinic acid(69) and 7 ml of c-H2SO4 in 60 ml of absolute ethanol was refluxed for 12 hrs. After the reaction mixture became homogeneous, it was concentrated and then diluted with 50% of ammonia solution. The resultant white precipitate was filtered and dried to give 2.70 g of product. The filtrate was extracted with 200 ml of chloroform, the chloroform layer was dried over anhydrous magnesium sulfate filtered and concentrated to give 1.97 g of product. Recrystallization from ethyl acetate gave 4.67g of a white product(77.9% yield).
m.p. : 151-152C(ref. ; 150"C).
IH NMR(CDC3) : 1.40(3H,t,-CH3), 4.37(2H,m,-OCH2-), 6.65 (1H,d,JAB=7Hz,5-py-H), 8.10(1H,dd,JAB=7Hz,JAc=l.5Hz,4-py-H),
8.30(iH,dJBc=l.5Hz,2-py-H).
Preparation of 2-methoxy-5-carbethoxy pyridine(72)
Silver carbonate(5.95 g, 0.02 mol) and 2-hydroxy-5carbethoxypyridine(3.35 g, 0.02 mol) were reacted with methyl iodide(20.5 g, 0.14 mol) for 24 hrs in 30 ml of benzene at room temperature in the dark. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give a sticky oil which was directly subjected to column




60
chromatogrphy on silica gel with ethyl acetate-hexane(l:4) as an eluent to yield 2.72 g of the product(75.1% yield).
IR(neat) : 3050, 1710(C=O), 1600, 1490, 1370, 1260, 1110, 1020, 840, 780 cm'.
1H NMR(CDCI3) : l.45(3H,t,-CH2-CH3), 4.00(3H,s,-OCH3), 4.45(2H,m,-OCH2-), 6.80(1H,d,JAB=6Hz,3-py-H), 8.25(1H,dd, JAB=6Hz,JAC='.5Hz,4-py-H), 8.87(1H,d,JAC=l.5Hz,6-py-H). Preparation of 2-methoxy-5-hydroxymethylpyridine(73)
To a suspension of 0.85 g of LiAlH4 in 3 ml of anhydrous THF were added 1.38 g (0.00762 mol) of compound(72) in 7 ml of anhydrous THF dropwise in an ice-water bath under nitrogen atmosphere. The reaction mixture was stirred for 1 hr at room temperature and quenched with 0.85 ml of water, 0.85 ml of 15% NaOH solution and 3 x 0.85 ml of water. After the resulting solid was filtered and washed with ether(2 x 15 ml), the combined ether fractions were concentrated, the residue was redissolved in ethyl acetate, washed with brine and dried over anhydrous magnesium sulfate. This suspension was filtered and concentrated to give a pale yellowish liquid which was purified by silica-gel column chromatography with an ethyl acetate-hexane mixture to give 870 mg of the pure product (82.1% yield).
IR(neat) : 3360(-OH), 2960, 1610, 1570, 1490, 1390, 1285, 1210, 1150, 1015 cm'.
IH NMR(CDC13) : 3.90(3H,s,-OCH3), 4.35(lH,br.,-OH), 4.55 (2H,s,-CH2-), 6.72(lH,d,3-py-H), 7.67(lH,dd,4-py-H), 8.07(lH, d,6-py-H).




61
Preparation of 2-methoxy-5-chloromethylpyridine(74)
To a solution of 780 mg(0.00561 mol) of compound(73) in 5 ml of chloroform were added 0.80 ml(2.0 eq.) of thionyl chloride dropwise at room temperature under nitrogen atmosphere. After the resulting mixture was stirred for 4 days at room temperature, it was concentrated to give a sticky yellowish oil, which was dissolved in 20 ml of water, made basic with dilute ammonia solution and then extracted with chloroform(2 x 30 ml). The combined chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give a yellow liquid which was used in the next reaction without further purification.
IR(neat) : 2960, 1605, 1570, 1490, 1385, 1285, 1255, 1120, 1020, 825, 750 cmt.
IH NMR(CDC13) : 3.93(3H,s,-OCH3), 4.55(2H,s,-CH-Cl), 6.80 (IH,d,3-py-H1), 7.68(IH,dd,4-py-H), 8.20(iH,d,6-py-H). Preparation of 2-methoxy-5-cyanomethylpyridine(75)
Method 1 : The mixture of 760 mg(0.00501 mol) of compound
(74), a catalytic amount of sodium iodide and 0.50 g of sodium cyanide in 8 ml of methanol and 1.5 ml of water were refluxed for 1.5 hr. The resulting brownish solution was cooled and concentrated. The residue was dissolved in 30 ml of water and extracted with chloroform(2 x 20 ml). The combined chloroform layers were washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a yellowish oil, which was subjected to column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent to




62
give a white solid after evaporation of the solvents. An analytically pure product was obtained from recrystallization from ether-petroleum ether mixture(400 mg, 50.5% yield).
Method 2 : A mixture of 440 mg of compound(74), an excess of sodium cyanide and a catalytic amount of sodium iodide in 15 ml of dry acetone were stirred for 2 days under nitrogen atmosphere. After the reaction was completed as indicated by TLC, the reaction mixture was concentrated and redissolved in chloroform. The chloroform layer was washed with water and brine, dried and filtered. The filtrate was concentrated to give a reddish residue, which was purified by silica-gel column chromatography with ethyl acetate-hexane(2:3) to give 220 mg of product(78.5% yield).
m.p. : 53-54C.
IR(nujol mull) : 2250(-CN) cm1.
IH NMR(CDCI3) : 3.67(2H,s,-CH-CN), 3.95(3H,s,-OQH3), 6.80 (iH,d,3-py-jj), 7.60(iH,dd,4-py-jj), 8.17(iH,d,6-py-Hi).
Elemental Analysis for C8H8N20
Cald. : C:64.85, H:5.44, N:18.90 Found : C:64.92, H:5.48, N:18.85. Preparation of 2-methoxy-5-(2'-aminoethyl)pvridine(76)
To a solution of 220 mg(l.39 mmol) of compound(75) in 8 ml of anhydrous THF in an ice bath were added 7.0 ml(5.0 eq.) of 1.0 M BH3-THF complex dropwise by syringe under nitrogen atmosphere. After reaction mixture stirred for 24 hrs, it was quenched with methanol and concentrated in vacuo to give a residue, which was subsquently dissolved in 30 ml of 5% HCl




63
solution. The acidic aqueous layer was washed with ethyl acetate, and then made basic with 10% NaOH solution and finally extracted with chloroform(2 x 30 ml). The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give the yellow oil, which was used directly in the next reaction.
1H NMR(CDCI3) : 1.23(2H,s,-NH 2), 2.60-3.00(4H,m.-C2-CH2)6.70(iH,d,3-py-Hf), 7.40(iH,dd,4-py-H), 8.03(iH,d,6-py-H).
Preparation of 2-methoxy-5-(2'-acetaminoethvl)pyridine(77)
A mixture of crude compound(76), 0.5 ml of acetic anhydride and a catalytic amount of pyridine in 5 ml of dichloromethane was stirred for 40 min. at room temperature under nitrogen atmosphere. The reaction mixture was concentrated in vacuo and stripped with toluene once. The resulting oil was dissolved in 10 ml of water and made basic with 10% NaOH solution, and then extracted with chloroform(2 x 30 ml). The combined chloroform layers were washed with brine, dried, filtered and concentrated to give a crude product, which was purified by column chromatography on silica-gel with ethyl acetate to give a white solid.(151 mg, 52.7% yield from compound(74)).
m.p. : 54.5-55.5C.
IH NMR(CDCI3) : 1.93(3H,s,-COCH3), 2.70(2H,t,-CH2-), 3.45 (2H,m,-CH2-NH-), 3.90(3H,s,-OCH3), 6.20(IH,br.,-NH-), 6.78(IH, d,3-py-H), 7.45(IH,dd,4-py-H), 7.97(iH,d,6-py-H).
Elemental Analysis for CloH14N202:




64
Cald. : C:61.83, H:7.26, N:14.42 Found : C:61.87, H:7.31, N:14.37. Preparartion of 2-methoxy-5-(2'-acetaminoethyl)pvridine-loxide(78)
A solution of 300 mg(0.00154 mol) of compound(77) and 0.47 g(l.5 eq.) of 85% m-chloroperbenzoic acid in 5 ml of dichloromethane was stirred for 24 hrs at room temperature under nitrogen atmosphere. After the reaction was completed as indicated by TLC, the reaction mixture was concentrated in vacuo to give a residue, which was directly subjected to column chromatogrphy on silica-gel with ethyl acetatemethanol(2:l) as an eluent to give 180 mg of the desired product(55.6% yield).
m.p. : 128-129C.
IH NMR(CDCI3) : 1.93(3H,s,-COCH3), 2.78(2H,t,-CH2-), 3.45 (2H,m,-CH2-NH-), 4.05(3H,s,-OCH3), 6.95(iH,d,3-py-H) 7.30(IH, dd,4-py-H), 7.62(IH,br.,-NH-), 8.15(IH,d,6-py-H).
Mass spec. : 211(M+).
Preparation of 5-(2'-acetaminoethyl)-l-hydroxy-2-pyridone(79)
To 410 mg(0.00195 mol) of a white solid compound(78) were added 6.0 ml of acetyl chloride dropwise under nitrogen atmosphere. The resulting mixture was refluxed for 1 hr and the excess acetyl chloride was evaported under reduced pressure. The resulting sticky yellowish residue was identified as 1-acetoxypyridone from NMR, which was converted to 1-hydroxypyridone without further purification.




65
Hydrolysis of l-acetoxypyridone : The yellowish residue was dissolved in 10 ml of water and was stirred overnight and concentrated in vacuo to give a sticky residue. This was stripped with methanol-toluene twice and extracted with hot chloroform. The combined chloroform layers were concentrated to give a white solid, which was crystallized from chloroformether mixture to give the product(195 mg, 51.2% yield).
m.p. : 124.5-125.5C.
NMR(CD3OD) : 1.98(3H,s,-COCH3), 2.70(2H,t,-CH2-), 3.37(2H, m, -CH2-NH-) 6. 82 (1H, d, 3-py-1) 7. 15 (1H,dd, 4-py-H) 7. 95 (1H, d, 6-py-H).
IR(nujol mull) : 3320(N-OH), 3100(NH-C=O), 1695(NH-C=0), 1580, 1365, 910 cm'.
Elemental Analysis for C9H12N203.0.5H20 Cald. : C:52.67, H:6.36, N:13.65 Found : C:52.93, H:6.06, N:13.62. Preparation of 5-(2'-aminoethyl)-l-hydroxy-2-pyridone hydrogen chloride(15)
A solution of 1.10 g of compound(79) in 8 ml of MeOH-HOc-HCI(I:l:2) mixture was refluxed for 10 hrs under nitrogen atmosphere. After the reaction was completed, the solution was concentrated to give a crude solid product, which was recrystallized from ethanol-water mixture to give the desired compound(740 mg, 76.0% yield).
m.p. : 242-244C.
IR(nujol mull) : 3150-2700(br,OH & NH3+), 1650(C=O) cm'




66
H NMR(DMSO-d6) : 2.65-2.72(2H,m,-CH2-), 2,85(2H,br., -CH2NH3), 6.50(iH,d,3-py-H) 7.37(iH,dd,4-py-H) 7.80(iH,d,-6-py),8.25(3H,br.,-NH3 + ).
Mass spec. : 155(M+).
Elemental Analysis for C7Hn1N202CI
Cald. : C:44.10, H:5.81, N:14.69, C1:18.59 Found : C:44.19, H:5.82, N:14.64, C1:18.52. Preparation of 4-(2'-(N-benzoyl)aminoethvl)pyridine(81)
To a solution of 12.20 g(0.10 mol) of 4-(2'
aminoethyl)pyridine, 15.03 ml(l.l eq.) of triethylamine in 70 ml of chloroform was added dropwise benzoyl chloride(12.78 ml, 1.1 eq. in 20 ml of chloroform) in an ice-water bath under nitrogen atmosphere. The resulting dark solution was stirred for 1.5 hrs at room temperature and then poured into 100ml of ice-water. The solution was made basic with aqueous ammonia solution and extracted with 200 ml of chloroform. The combined chloroform layers were washed with water, dried over anhydrous magnesium sulfate and concentrated to give a yellow solid, which was recrystallized from acetone to give 15.5 g of white product(70.6% yield).
m.p. : 119-120'C.
IHNMR(CDCI3) : 2.90(2H,m,-CH2-) 3.65(2H,m,-CH,-NH-), 7.10 (2H,m,pyridine H), 7.42(3H,m,phenyl H), 7.35-7.70 (1H,br.,NH),
7.78(2H,m,phenyl H), 8.37(2H,m,pyridine H). Preparation of 4-(2'-(N-benzoyl)aminoethyl)pyridine-l-oxide
(82)




67
Method A : A mixture of 1.10 g(0.0050 mol) of compound
(81) and 1.21 g(l.2 eq.) of 80 85% m-chloroperbenzoic acid in 25 ml chloroform was stirred for 2.5 hrs at room temperature under nitrogen atmosphere. After the reaction was complete, the mixture was concentrated and then chromatographed on silica-gel with ethyl acetate-methanol(3:l) mixture as an eluent. The combined fractions were concentrated to give a white product(76.3% yield).
Method B : A solution of 1.10 g(O.0050 mol) of compound(81) and 3 ml of 30% hydrogen peroxide in 15 ml of acetic acid was heated to between 70 and 80C for 24 hrs under nitrogen atmosphere. After the mixture had cooled down, it was concentrated in vacuo and made basic with dilute ammonia. The aqueous solution was extracted with 100 ml of chloroform The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a viscous oil. The oil was subjected to column chromatography on silicagel with ethyl acetate-methanol mixture to give 285 mg of the white product(24.1% yield).
m.p. : 139-140C.
'HNMR(CDCI3) : 2.91(2H,m,-CH-), 3.63(2H,m,-CH2-NH-), 7.10 (2H,d,J=6.5Hz,pyridine H), 7.40(3H,m,phenyl H), 7.80-7.95(4H, m,phenyl H & pyridine H), 8.23(IH, br.-NH-).
Mass spec.(70eV) : 243(M+1).
Synthetic attempts for the rearrangement of N-oxide(82)
A solution of 500 mg(2.12 mmol) of N-oxide(82) in 10 ml of acetic anhydride was heated to between 90 and 100C for 40




68
min. under nitrogen atmosphere. After excess acetic anhydride was removed, the resulting black tarry residue was subjected to column chromatography on silica-gel with ethyl acetatemethanol(3:l) to give yellowish oil, which was identified as 4-(2'-(N-benzoyl)aminoetheneyl)pyridine by NMR data.
IH NMR(CDCI3) : 3.93(1H,dd,JAB=9Hz,JAx=5Hz),
4.50(IH,dd,JAB=9Hz,JAx=5Hz), 5.70(iH,ddJBx=7Hz,JAx=5Hz), 7.30 (2H,m,pyridine H), 7.50(3H,m,phenyl H), 8.15(3H,m,phenyl H),
8.65(2H,m,pyridine H).
General synthetic attempts for 4-(2'-(N-benzoyl)aminoethyl)2-chloropyridine with POC13
A mixture of 540 mg(2.28 mmol) of N-oxide and 3.0 ml of neat phosphorous trichloride(or alternately, dissolved in 15 ml of methylene chloride) was refluxed for 4 hrs under nitrogen atmosphere. The resulting yellow solution was poured onto 20 ml of ice-water and made basic with 10% NaOH. The pink colored suspension was extracted with chloroform(2 x 50 ml) and the combined chloroform layers were washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated to give a yellow oil which was composed of many products.
Preparation of 4-carbomethoxvpvridine-l-oxide(88)
Method 1 : A solution of 10.0 g(0.0729 mol) of methyl isonicotinate(87) and 17.3 g(l.l eq.) of 85% mchloroperbenzoic acid in 150 ml of dichloromethane was stirred overnight at room temperature under nitrogen atmosphere. After the resulting white precipitate was filtered, the filterate




69
was diluted with 100 ml of chloroform. The organic layer was washed with saturated sodium bicarbonate solution(200 ml) and aqueous layer was re-extracted with chloroform(2 x 50 ml). The combined organic layers were dried over anhydrous magnesium sulfate filtered and concentrated to give 8.94 g of white product(80.1% yield).
Method 2 : A solution of 10.0 g(0.0729 mol) of methyl isonicotinate, 20 ml of 30% hydrogen peroxide and 50 ml of acetic acid were refluxed for 6 hrs under nitrogen atmosphere. The solution was poured into 50 g of ice and made basic with sodium carbonate. The basic aqueous solution was exhaustively extracted with chloroform(3 x 50 ml). The combined chloroform layers were dried, filtered and concentrated to give the product(72.3% yield).
m.p. : 122-123C.
IH NMR(CDCI3) : 3.97(3H,s,-OCH3), 7.97(2H,d,3 & 5-py-H),
8.36(2H,d,2 & 6-py-H).
Preparation of 4-carbomethoxy-2-pyridone(89)
A solution of 5.4 g(0.0352 mol) of carbomethoxypyridineN-oxide(88) in 50 ml of acetic anhydride was refluxed for 18 hrs under nitrogen atmosphere. After removal of excess acetic anhydride in vacuo, the resulting black tarry residue was taken up in hot methanol, treated with charcoal and filtered, cooled to give a brown solid. This was recrystallized from methanol to give a pale yellowish product(767 mg, 14.3% yield).
m.p. : 213-215'C (ref.92 ; 211-213C).




70
1H NMR(CDC3+CD3OD) : 3.98(3H,s,-OCH3), 6.98(iH,dd,5-pyH,7.23(iH,s,6-py-H), 7.56(iH,d,3-py-Hj).
Preparation of 4-carbomethoxy-2-ethoxvpvridine(90)
Silver carbonate(5.76 g, 1.0 eq.), compound(89, 3.20 g, 0.0209 mol) and 3.3 ml(2.0 eq.) of ethyl iodide in 40 ml of benzene were stirred for 48 hrs at ambient temperature under nitrogen atmosphere in the dark. The reaction mixture was filtered and washed with 50 ml of benzene. The filtrate was washed with 10 % sodium bicarbonate solution and brine successively, dried and concentrated to give a crude liquid product, which was purified by column chromatography on silica-gel with ethyl acetate-hexane mixture as an eluent to give 3.14 g of the pure product(82.9% yield).
IR(neat) : 3000, 1735(C=O), 1600, 1560, 1100 cm-.
1H NMR(CDC13) : 1.33(3H,t,-CH2-CH3), 3.90(3H,s,-OCH3), 4. 3 5(2H, m, -OCH2-) 7. 27 (1H,s, 3-py-H) 7. 35 (1H, dd, 5-py-H) ,
8.22(IH,d,6-py-H).
Preparation of 2-ethoxy-4-hydroxymethylpyridine(91)
To a suspension of 0.66 g(2.0 eq.) of LiAlH4 in 10 ml of anhydrous THF cooled in an ice bath were added 3.20 g(0.0176 mol) of compound(90) in 15 ml of anhydrous THF dropwise under nitrogen atmosphere. The mixture was stirred for 20 min. and then quenched with 0.66 ml of water, 0.66 ml of 15% NaOH solution and 3 x 0.66 ml of water successively. The resulting precipitate was filtered and washed with 25 ml of ethyl acetate. The combined filtrate was washed with brine, dried,




71
filtered and concentrated to give 2.61 g of the product(96.1% yield).
IR(neat) : 3470(-OH), 1610, 1555, 1145, 805 cm-.
1H NMR(CDCI3) : 1.37(3H,t,-CH2-_H3), 3.65(1H,br.,--OH),
4.30(2H,m,-OCH2-CH3), 4.65(2H,s,-CH2-OH), 6.70(1H,s,3-py-H),
6.78(iH,dd,5-py-H), 8.00(iH,d,6-py-H). Preparation of 4-chloromethyl-2-ethoxvpvridine(92)
The mixture of 2.60 g(0.169 mol) of compound(91) and 1.86 ml(l.5 eq.) of thionyl chloride in 30 ml of chloroform was stirred overnight at room temperature under nitrogen atmosphere. After the reaction mixture was concentrated in vacuo, the residue was dissolved in 30 ml of water and made basic with dilute ammonium hydroxide and extracted with chloroform(2 x 50 ml). The chloroform layer was washed with water and brine, dried over anhydrous magnesium sulfate filtered and concentrated to give the desired product(2.83 g, 97.1% yield).
IR(neat) : 3020, 1615, 1570, 1170, 1050, 725 cm".
IH NMR(CDCI3) ; 1.42(3H,t,-CH2-CH3), 4.35(2H,m,-OCH2-), 4.48(2H,s,-CH2-Cl), 6.72(1H,s,3-py-H), 6.85(1H,dd,5-py-H),
8.14(lH,d,6-py-H).
Preparation of 4-cyanomethyl-2-ethoxypyridine(93)
A mixture of 0.53 g(0.00309 mol) of compound(92) and excess amount of potassium cyanide in 4 ml of ethanol and 0.8 ml of water was refluxed for 2 hrs under nitrogen atmosphere. The reaction mixture was cooled, diluted with chloroform and filtered. The filtrate was washed with water and brine, dried,




72
filtered and concentrated to give reddish oil, which was subjected into column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent and 313 mg of product was obtained(64.0% yield).
m.p. : 55.5-56C.
IR(nujol mull) : 2270(CN) cm'.
IH NMR(CDCI3) ; l.40(3H,t,-CH2-CH3), 3.72(2H,s,-CH2-CN),
4.35(2H,m,-OCH2-), 6.70(iH,s,3-py-H), 6.87(iH,s,5-py-H), 8.17 (lH,d,6-py-H).
Elemental Analysis for C9HION20
Cald. : C:66.64, H:6.21, N:17.27 Found : C:66.53, H:6.22, N:17.24.
Mass spec. : 163(M+).
Preparation of 4-(2'-aminoethyl)-2-ethoxypyridine(94)
To a solution of 440 mg(O.00271 mol) of compound(93) in 3.0 ml of anhydrous THF were added 6.0 ml(2.2 eq.) of 1.0 M diborane solution via a syringe at ice-water temperature under nitrogen atmosphere. The suspension was stirred overnight and resulting solution was quenched with methanol, and then concentrated to give a reddish oil. The oil was dissolved in 30 ml of 5% HCl solution and washed with 20 ml of ethyl acetate. The acidic aqueous layer was basified with 10% NaOH solution and extracted with chloroform The combined chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give an orange colored oil, which was directly used to next reaction.




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1H NMR(CDC3) : 1.40(3H,t,-OCH2-CH3), 2.73(2H,m,-CH2-CH2NH2), 2.93(2H,m,-CH2-CH2-NH2), 4.32(3H,qt,-OCH2-CH3), 6.70(lH, s,3-py-H), 6.87(IH,dd,5-py-H), 8.17(iH,d,6-py-H).
Preparation of 4-(2'-acetaminoethyl)-2-ethoxvpvridine(95)
A mixture of the crude amine(94), 0.5 ml of acetic anhydride and two drops of pyridine in 5 ml of methylene chloride were stirred for 1 hr at room temperature under nitrogen atmosphere. The mixture was concentrated to give a reddish oil, which was dissolved in water, made basic with 10% NaOH and extracted twice with 20 ml of chloroform. The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give a crude product, which was purified by column chromatogrphy on silicagel with ethyl acetate-methanol mixture.
m.p. : 84.5-85.5C.
IH NMR(CDCI3) : 1.37(3H,t,-OCH2-CH3), 1,93(3H,s,-COCH3),
2. 7 0(2H, t, -CH2-CH2-NH-) 3. 4 0(2H, m, -CH2-NH-) 4. 3 0(3H, qt, -OCH,CH3), 6.45(IH,br.,-NH-), 6.52(iH,s,3-py-H), 6.70(lH,dd,5-pyH), 8.00(IH,d,6-py-H).
Mass spec. (70 eV) : 208(M ).
Elemental analysis for C11HI6N202 Cald. : C:63.44, H:7.74, N:13.45 Found : C:63.51, H:7.75, N:13.44. Preparation of 4-(2'-acetaminoethyl)-2-ethoxypyridine-l-oxide
(96)
A solution of 500 mg(0.00240 mol) of compound(95) and 0.62 g of m-chloroperbenzoic acid(l.2 eq.) in 7.0 ml of




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dichlorometane was stirred overnight at room temperature under nitrogen atmosphere. The reaction mixture was concentrated in vacuo to give a yellowish residue, which was directly subjected to column chromatography on silica-gel with ethyl acetate-methanol mixture to give 380 mg of product(70.6% yield).
m.p. : 133.5-135C.
1H NMR(CDCI3+CD3OD(3:I)) : 1.40(3H,t,-OCH2-CH3), 1.97(3H, s,-COCH3), 2.60(2H,t,-CH2-CH2-NH-), 3.25(2H,m,-CH2-NH-),
4.17(2H,m,-OCH2-) 6.77(IH,dd,5-py-H) 6.82(iH,s,3-py-H) ,
7.97(iH,d,6-py-H).
Mass spec. : 225(M+1).
Preparation of 4-(2'-acetaminoethyl)-l-hydroxy-2-pvridone(86)
A suspension of 300 mg of compound(85) in 5.0 ml of acetyl chloride was stirred for 20 min. at room temperature and refluxed for 2 hrs under nitrogen atmosphere. Excess acetyl chloride was removed in vacuo and resulting residue was dissolved in 10 ml of water. The aqueous solution was stirred overnight and concentrated to give a yellow sticky oil. This oil was stripped with methanol twice to give a very hygroscopic yellow colored amorphous type solid.
IH NMR(CD3OD) : 1.93(3H,s,-COCH3)' 2.72(2H,m,-CH2-CH2-NH), 3.40(2H,m,-CH2-NH-), 6.60(iH,dd,5-py-H), 6.70(lH,s,3-py H), 7.97(iH,d,6-py-H).
Mass spec. : 225(M+1).
Preparation of 4-(2'-aminoethyl)-l-hydroxy-2-pyridone hydrogen chloride(16)




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A solution of crude compound(86) in 8.0 ml of methanolwater-c-HCl(l:1:2) mixture was refluxed overnight under nitrogen atmosphere. After the reaction mixture was concentrated in vacuo, the resulting reddish sticky residue was dried under reduced pressure, and then washed with ethanol to give the crude solid product, which was recrystallized from ethanol-water mixture to give a yellow product(630 mg : 41.1% from N-oxide).
m.p. : 224-226C.
IR(nujol mull) : 2600-3300(br.,-OH & -NH3 ), 1650(C=O) cm
H NMR(DMSO-d6) 2.62-3.25(4H,m,-CH2-CH2-), 6.17(lH,dd,5PY-H), 6.40(IH,s,3-py-H), 7.83(iH,d,6-py-H), 8.25(3H,br.,NH3+ ).
U.V.(MeOH) : 228, 303 nm.
Mass spec. : 155(M+).
Elemental analysis for C7HIIN202CI
Cald. : C:44.10, H:5.81, N:14.69, C1:18.59 Found : C:44.18, H:5.86, N:14.62, C1:18.51.
High Pressure Liguid Chromatography System
A mobile phase system was developed in order to analyze compounds produced in the in vitro hydrolysis experiments. The composition of the mobile phase for the carbostyril compounds consisted of acetonitrile:water:acetic acid:hexanesulfonic acid sodium salt(40:59:1:0.1). The carbostyril compounds(12, 13 and 14) had retention times between 2.6 and 4.2 min when




76
the flow rate was 1.5 ml/min. using a Bio-Sil-ODS 5 column. The metabolites of the carbostyril compounds in 80% human plasma had retention time between 3.2 min. and 5.6 min. The chart recorder was set at 0.25 cm/min. All carbostyril compounds were analyzed using ultraviolet detection at 258 nm. A BiO-Sil-ODS 5 reversed phase silica column was used in all analyses.
Chemical Stability
Stability of the Carbostyril Compounds(12, 13 and 14) in PH
7.40 Phosphate Buffer
Solutions of monobasic potassium phosphate(0.2 N) and dibasic potassium phosphate(0.2 N) were made and used to prepare a pH 7.40 phosphate buffer by mixing together.
Buffer solutions(4.9 ml) were equilibriated at 37C. At time zero, 50 ul of a 5.3 x 102 M stock solution of test compound in methanol were added to a buffer solution. At designated time points, 100 ul samples were removed and added to 900 ul of ice-cold 40 % acetonitril-water. The final concentration of test compound would be 5.3 x 10-5 M at time zero. Samples were stored at 0C until analyzed by HPLC.
In Vitro Studies
Stability of the Carbostyril Compounds(12, 13 and 14) in 100% Whole Human Blood
Freshly collected heparinized blood was obtained from the Civitan Regional Blood Center, Inc. (Gainesville, FL). The blood was stored in a refrigerator and used the next day.




77
Thirty microliters of a freshly prepared 0.061 M solution of test compound in methanol was added to 3.0 ml of blood, previouly equilibrated to 37C in a water bath and mixed throughly to result an initial concentration of 6.1 x 10-4 M. At designated time intervals, 100 ul aliquots were withdrawn from the test medium, added immediatly to 900 ul of ice-cold acetonitrile, shaken vigorously and placed in a freezer. The final test compound concentration was 6.1 x 10-5 M at time zero. When all samples had been collected, they were centrifuged at 13000 rpm for 5 min. The samples were kept at 0C until analyzed by HPLC.
Stability of the Carbostyril Compounds(12, 13 and 14) in 80% Human Plasma
Freshly collected plasma used was obtained from the Civitan Regional Blood Center, Inc.(Gainesville,FL) and contained about 80% plasma diluted with anticoagulant citrate phosphate-dextrose solution U.S.P. The plasma was stored in a refrigerator and used the next day. Thirty microliters of a freshly prepared 0.061 M solution of test compound in methanol was added to 3.0 ml of plasma, previously equilibrated to 37C in a water bath and mixed throughly to result an initial concentration of 6.1 x 10-4 M. At designated time intervals, 100 ul aliquots were withdrawn from test medium, added immediately to 900 ul of ice-cold acetonitrile, shaken vigorously and placed in a freezer. The final test compound concentration was 6.1 x 10'5 M at time zero. When all samples had been collected, they were centrifuged at 13,000




78
rpm for 5 min. The samples were kept at 0C until they were analyzed by HPLC.
Stability of the Carbostyril Compounds(12, 13 and 14) in 20% Rat Liver Homogenate
The liver homogenate was prepared by the following method. One Sprague-Dawley rat was killed by decapitation, and liver was removed, weighed and homogenated in a tissue homogenizer in 0.05 M aqueous phosphate buffer(pH 7.4) to make 20% liver homogenate. Thirty microliters of 5.3 x 10'3 M solution of test compound in methanol were added to 3.0 ml of the homogenate, previously equilibrated to 37C in a water bath, to result in an initial concentration of 5.3 x 10'5 M. At various time points, 100 ul of samples were withdrawn from the test medium, added immediately to 400 ml of ice-cold acetonitrile, shaken vigrously and placed in a freezer. The final test sample concentration was 1.0 x 105 M. When all samples had been collected, they were centrifuged at 13,000 rpm for 5 min. and were stored at 0C until analyzed by HPLC. In Vitro Evaluation of the Prolactin Inhibitory Effects of the Pyridones(15,16)
Adult female rats(Charles Rivers Labs), weighing 220 250 mg, were maintained on food and water ad libitum. Animals were sacrificed by decapitation; their pituitary glands were quickly removed from the cranium. The anterior pituitary(AP) of each animal was dissected into two equal halves and placed into incubation media.(media 199 supplied by Grand Island Biological Co.) The incubation was conducted at 37C, under continuous aeration(95% 025% C02); the pH was 7.61. After 30




79
min., the media was discarded and replaced with fresh media. After one-half hour additional preincubation, the media were discarded and replaced with fresh media containing either compound(15, 1 x 10-9 M) or compound(16, 1 x 10-9 M). In all cases, one-half of AP received the test drug; the other, the media 199 control. After 30 minutes, samples were taken from the media, and the remaining media were discarded. Fresh media containing compound(15, 1 x 10-8 M), compound(16, 1 x 10-8 M), respectively, were then added. Thirty minutes later, the second samples were taken. Same procedure was continued through the 1 x 10 M doses of these compounds. At end of experiment, each half of the AP was weighed. The samples were diluted 1:50 with phosphate-buffered saline and then assayed in triplicate by the radioimmunoassay method described. The data are given as nanograms of prolactin released per milligram of wet tissue weight. Paired Student's test was used to evaluate the significance of the inhibitory effects of the test drugs on prolactin secretion. The control AP half and the drug-treated half were employed in each paired comparision.
In Vivo Studies
Cardiovascular Effects of the Compound(8) in Dogs
This test was followed by the reported method.57 Adult mongrel dogs were anethestized with sodium pentobarbital, 30 mg/kg i.v., and given supplemental doses(i.m.) as needed. The femoral artery was cannulated to the level of the abdominal aorta for measurement of arterial blood pressure. Both femoral




80
veins were cannulated for the infusion of drug. A T-shaped tube was inserted into the trachea and the dog was placed on the respirator at 25 ml/kg tidal volume at 10-12 breaths/min. In some dogs, the chest was opened via a mid-sternum incision. The pericardium was removed and any fat on the ascending aorta was dissected away. A snug-fitting, calibrated electromagnetic flow probe was placed on the aorta and connected to a Carolina flowmeter. A Walton-Brodie strain gauge was used to measure cardiac contractile force. It was sewn on the surface of the right ventricle and stretched until a maxmimum contraction occured(the top of Starling's curve). Lead II ECG was recorded and was used to trigger a cardiotachometer. Total peripheral vascular resistance was estimated as mean arterial blood pressure/aortic blood flow. In other dogs, anesthetized as above but with closed chest and unsupported respiration, cardiac contractility was assessed as the maximum change in left ventricular(LV) blood pressure per unit time(LV/dP/dt,,,). This parameter was computed with a calibrated Grass differentiator(model 7P20) from a LV pressure signal obtained from a Millar transducer-tip catheter after introduction through the left carotid artery. Drug was introduced as an intravenous infusion for five minutes. For intravenous infusion, compound(8) was dissolved in saline. Infusions of drug were given in random order and doses were maintained for 5 minutes or until a steady state of cardiovascular responses occured at volume rates of 0.5 2.0 ml/min. The volume of vehicle did not affect cardiovascular variables.




81
Electrophysiological Studies of the Compounds(13,14)
Adult mongrel dogs were anethestized with sodium pentobarbital(30 mg/kg, i.v.), intubated and ventilated with room air. Bipolare and hexapolare catheters(French 5 and 6) were introduced percutaneously and positioned under X-ray and ECG control in four locations; atriocaval junction, right artrial appendage, right ventricular apex and aortic root. Three standard surface ECG leads were recorded simultaneously with intracardiac electrograms. After insertion of catheters, the following basic parameters of conduction intervals were recorded and measured; intraatrial conduction(PA) which is the interval from oneset of the P wave on surface leads to oneset of the low atrial activity in the His bundle recording, atrial and atrioventricular/AV-node conduction(AH) which is the interval from oneset of low atrial activity to oneset of His potential, His potential which is the interval from oneset to conclusion of the His potential in the His bundle recording, and His-Purkinje conduction(HV) which is the interval from oneset of His potential in the His bundle recording to the earliest oneset of ventricular activation in any intracardiac or surface leads. In order to measure the programmed electrical stimulation, atrial pacing was accomplished using a battery powered programmable stimulator, delivering pulses of 2 ms duration at twice diastolic threshold. The following two parameters of sinus node function were determined by atrial pacing and programmed atrial stimulation; sinus node recovery time(SNRT) which is the interval from the last paced




82
complex to the oneset of the first sinus beat as measured from the right atrial electrogram, and sinuatrial conduction time(SACT) which is the interval from the last paced beat to the first return sinus beat as recorded in the high right atrium. At same time, by using programmed electrical stimulation of the right atrium, the following parameters of AV node His-Purkinje function were determined; atrial effective refractory period(AERP), atrial fuctional refractory period(AFRP), effective refractory period of the atrioventricular node(AVNERP), functional refractory period of the atrioventricular node(AVNFRP) and effective refractory period of His bundle(HisERP). Drugs were dissolved in saline and introduced as an intrveneous infusion through the femoral vein.
Pharmacokinetic and Metabolism Studies of Compound(12) in Rat
An adult female Sprague-Dawley rat(255 g) was anethestized with sodium pentobarbital. 200 ul of solution of compound(12, 10mg/ml) in 20% DMSO-water were administered intraveneously through the femoral vein. At selected time points after drug administration, 100 ul of blood were withdrawn from the juglar vein, added immediately to 200 ul of 5% DMSO-acetonitrile and vortexed. When all samples had been collected, they were centrifuged at 12,000 rpm for 5 min. The samples were kept at 0C until they were analyzed by HPLC.




CHAPTER IV
RESULTS AND DISCUSSION
Synthesis
Synthesis of 3.4-Dihvdroxy-N-(6'-methoxy-3'-carbamylphenyl)l-methyl-n-phenyl-B-phenethylamine Hydrochloride(8)
The synthesis of compound(8) was initially attempted according to Figure 4-1.
OCR OCH 3 CH 3 0
I i)SOCl2 1 I MVK Pd-C/H2
- ()(8)
Sii)NH3 10% Pd-C
COOH iNNH2 CONH2
(17) (18) (19)
Figure 4-1 : Synthetic attempts of compound(8).
The required starting material(17) in Figure 4-1 was prepared from commercially available 4-methoxy-3-nitrobenzoic acid via reduction of nitro group under hydrogenation condition followed by diazotization and the Sandmeyer reaction. Conversion of the carboxylic acid to amide was successful after generation of the corresponding benzoyl chloride using thionyl chloride in the presence of catalytic amounts of pyridine, followed by treatment with saturated ammonia. However, substitution of the iodine atom by methyl vinyl
83




84
ketone(MVK) in the presence of catalytic amounts of 10% Pd-C and triethylamine was not successful although similar reaction with compounds which do not have the methoxy group in the ortho position to iodine gave moderate yields. In order to find the right conditions, the above reaction was further studied. The results of attempts were summerized in Table 41.
Table 4-1 : Substitution reaction of compound(18).
Rxn # Ratio(IV:MVK:NEt) Rxn Time Yield
1 1 : 1.1 : 1.1 2 hrs trace 2 1 : 1.1 : 1.1 overnight trace 3 1 : 1.5 : 1.5 6 hrs trace
4 1 : 1.5 : 1.5 24 hrs intractable 5 1 : 2.0 : 2.0 48 hrs intractable
From the results of Table 4-1, it might be concluded that electronic and steric effects of the methoxy group is very important in the above reaction as formation of a palladium complex with iodine is the rate controlling step in the above reaction and the methoxy group may inhibit formation of palladium complex.
After the unsuccessful results shown in Figure 4-1, another sequence outlined in Figure 4-2 was employed to synthesize the compound(6). Salicylaldehyde(20) was utilized as starting material. This was nitrated with c-HNO3/acetic acid to give a mixture of 3- and 5-nitrosalicylaldehyde(21),




85
OH OH OH PCH3
CH -O340 2 N- OH OCiO
o r AcOH CH 3I
02 NO2
(20) (21) (22)
I3 P=CHCCH 3
NC i)NaNO Pd-C/H2 0
o2Q
ii)CuCN
NH2 NO2
(25) (24) (23)
R202 /KHCO3
OCH3 OCH3 HO N
Dopamine HO
Pd-C ,PtO2/H2 HO CONH2
CONH 2
(26) (8)
Figure 4-2 Synthetic reaction sequence for compound(8).




Full Text

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DESIGN, SYNTHESIS AND EVALUATION OF NOVEL CARDIOTONIC AGENTS BASED ON 5-(2'-AMINOETHYL)CARBOSTYRIL AND (2'-AMINOETHYL)-1-HYDROXY-2-PYRIDONE SYSTEMS BY SUNG-HWA YOON A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY UNIVERSITY OF FLORIDA 1989

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Copyright 1989 by Sung-Hwa Yoon

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To my wife, Saun-Joo for her love, patience and encouragement

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ACKNOWLEDGEMENTS I would first like to thank Dr. Nicholas Bodor for giving me the privilege to learn the medicinal chemistry field in his research group. This work would never have been finished without his continuous advice and generosity. I would also like to thank the members of my supervisory committee, especially Dr. James Simpkins for his help in in vitro studies. Sincere thanks are extended to several members of the group: Dr. Shinji Nishitani for sharing the invaluable chemistry knowledge and the more pleasant time during all my research, Dr. Whei Mei Wu for helping me in the analytical experiments, Dr. Peter Polgar for his invaluable assistance in the animal experiments, Dr. Marcus Brewster for reviewing my writing and all our office teams, Laurie Johnston, Joan Martignago, Julie Drigger for providing me the friendly atmosphere. I would also like to thank all my Korean friends in Gainesville, especially, Mr. Seung Hoon Park for allowing me to use his computer system. I especially thank my parents, Maeong-Ho and Yon-Yo, my brothers, Sung-Jae and Sung-Woo, my sisters, Yang-Soon and Jung-Mi, my parents-in-law, Young-Kook and Sook-In for their encouragement throughout this work. iv

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Finally, I would like to express my deepest appreciation to my wife, Saun-Joo, and my precious little girl, Alyssa. They are really everything in my life. V

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TABLE OF CONTENTS ACKNOWLEDGEMENTS LIST OF TABLES LIST OF FIGURES. iv viii ix KEY TO ABBREVIATIONS ABSTRACT xi xiii CHAPTERS I. INTRODUCTION ... 1 Pathophysiology of Congestive Heart Failure 1 Development of Inotropic Agents 3 Mechanism of Action. . . 3 Glycosides . . . 5 Catecholamines and Sympathomimetic Amines 7 Other Inotropic Agents. . 8 Problems with Current ~1-Agonists. 13 Objectives of Research. . 14 II. DESIGN 15 Pharmacology of Dobutamine and Its Analogues. 15 Mechanism of Action ........... 15 Metabolism of Dobutamine ......... 18 Adverse Effects .............. 19 Design of Dobutamine Analogues As Selective ~1 -Agonist . . . 19 Design of 6'-Substituted Dobutamine Analogues ............... 19 Design of 5-(2'-Aminoethyl)-carbostyril system . . . 21 Design of (2'-Aminoethyl)-1-hydroxy-2-pyridone system . . . . 2 4 III. EXPERIMENTAL ................. 28 Materials and Methods ..... Synthesis ............ High Pressure Liquid Chromatography Chemical Stability ........ vi System. 28 29 75 76

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IV. v. In Vitro Studies .. In Vivo Studies .. RESULTS AND DISCUSSION ... 7 6 79 83 Synthesis . . 83 High Pressure Liquid Chromatography System. 116 Chemical Stability. 120 In Vitro Studies. . . . 120 In Vivo Studies . . . 128 AM-1 Calculation of (2-Aminoethyl)-1-hydroxy-2-pyridone systems 134 SUMMARY AND CONCLUSIONS REFERENCES BIOGRAPHICAL SKETCH. 140 143 149 vii

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LIST OF TABLES Table 1-1 Some receptor actions of catecholamines. 8 1-2 Inotropic drugs that activate ~1-receptors. 9 1-3 Inotropic drugs that inhibit c-AMP PDE .... 1 0 2-1 Adrenergic-receptor activity of sympathomimetic amines. . 17 4-1 Subs ti tut ion reaction of compound ( 18) 84 4-2 Replacement reaction of compound(35). 9 3 4-3 The results of Fridel-Craft acylation reaction of compound(53,54) .......... 98 4-4 Cyclization reaction of compound(67) ..... 1 0 1 4-5 4-6 Synthetic attempts for compound(84) Rearrangement reaction of N-oxide(88) .. 4-7 Half-life(hr) of disappearance and correlation coefficent for carbostyril compounds in pH=7.40, in 100% human blood, in 80% human 112 114 plasma and in 20% rat liver homogenate .... 121 4-8 4-9 4-10 4-11 4-12 4-13 4-14 4-15 In vitro activity of the compound(15). In vitro activity of the compound(16). Electrophysiological data of compound(13). Electrophysiological data of compound(14). Physic-chemical data of compound(15), compound(16) and dopamine ...... AM-1 calculation results of compound(15). AM-1 calculation results of compound(l6). AM-1 calculation results of dopamine ... viii 126 127 1 3 1 1 32 136 137 138 139

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Figure 1-1 1-2 2-1 2-2 2-3 4-1 4-2 4-3 4-4 4-5 4-6 4-7 4-8 4-9 4-10 4-11 4-12 4-13 4-14 4-15 4-16 LIST OF FIGURES The c-AMP cascade in cardiac muscle cell. 4 The mechanism of the digitalis glycosides. 6 COMT reaction of catechol. 18 Tautomers of 8-hydroxycarbostyril. 24 Tautomers of l-hydroxy-2-pyridone. 26 Synthetic attempts of compound(8). 83 Synthetic reaction sequence for compound(8) .. 85 The proton NMR spectrum of compound(8). 8 8 Synthetic reaction sequence for compound(9) .. 89 The proton NMR spectrum of compound(9). 91 Attempts of synthetic reaction sequence for compound ( 41) . . . 9 2 Attempts of synthetic reaction sequence for compound ( 41) . . . 9 4 Attempts of synthetic reaction sequence for compound ( 41) . . . 9 7 Retrosynthesis of carbostyril compound. 99 Synthetic reaction sequence for compound(l2}, compound(l3} and compound(l4). . 100 The proton NMR spectrum of compound(l2}. 104 The proton NMR spectrum of compound(l3}. 105 The proton NMR spectrum of compound(l4}. 106 Synthetic reaction sequence for compound(l5}. 107 The proton NMR spectrum of compound(l5} .... 110 Attempts of synthetic reaction sequence for compound(l6}. . ...... 111 ix

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4-17 4-18 4-19 4-20 4-21 4-22 4-23 The proton NMR spectrum of compound(83}. 113 Synthetic reaction sequence for compound(l6}. 115 The proton NMR spectrum of compound(l6}. 117 The HPLC chromatogram of compound(l2) and its metabolite in 80% human plasma at 23 hrs and 72 hrs. . . . . 119 In vitro results of compound(l2,13,14) in 80% human plasma. . . 123 Cardiovascular effects of compound(8), dobutamine and KM-13. . 129 In vivo pharmacokinetic result of compound(l2} in rat. . . . . 133 X

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c CDC13 CD30D CHC13 CH2Cl2 CS2 dee DMF DMSO DMS0-d6 g hr 1H NMR HPLC IR KBr M mg MgS04 min KEY TO ABBREVIATIONS aluminum chloride borane degree centigrade deuterated chloroform deuterated methanol chloroform methylene chloride carbon disulfide decomposition dimethylformamide dimethylsulfoxide deuterated dimethylsulfoxide gram hour proton nuclear magnetic resonance high pressure liquid chromatography infra-red potassium bromide molar milligram magnesium sulfate minute xi

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ml mmol m. P. N KH2P04 nm r rpm sec THF t112 UV ul > < milliliter millimole melting point normal potassium phosphate monobasic nanometer correlation coefficent revolutions per minute second tetrahydrofuran half-life ultraviolet microliter greater than less than xii

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy DESIGN, SYNTHESIS AND EVALUATION OF NOVEL CARDIOTONIC AGENTS BASED ON 5-(2'-AMINOETHYL)CARBOSTYRIL AND (2'-AMINOETHYL)-1-HYDROXY-2-PYRIDONE SYSTEMS By Sung-Hwa Yoon May 1989 Chairman: Nicholas s. Bodor Major Department: Medicinal Chemistry In order to develop novel, improved cardiotonic drugs, three different types of chemical manipulations of dobutamine were investigated. Two 61-substituted analogues of dobutamine were synthesized by coupling dopamine and the corresponding ketones, and the cardiovascular effects of these novel compounds were evaluated in dogs. The 61-methoxy analogue of dobutamine showed five times higher inotropic activity than dobutamine without significant changes in mean arterial blood pressure and pheripheral vascular resistance. Three new analogues based on the carbostyril system.were designed, in which the m-hydroxy group of dobutamine was isosterically modified to an amide type carbostyril system. The compounds were synthesized from p-methoxyphenethylamine and their stabilities in chemical and biological media in xiii

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vitro conditions were studied. All carbostyril analogues were extremely stable in pH= 7.40 phosphate buffer, whole human blood, 80% human plasma and 20% rat liver homogenate. In order to evaluate the effects on the function of sinus node and the cardiac conduction system, in vivo cardiac electrophysiological studies in dogs were performed. The 8-hydroxycarbostyril analogue and the 3,4-dihydroxy-8-methoxy carbostyril analogue did not change the cardiac electrophysiological parameters. Two analogues of (2'-aminoethyl)-1-hydroxy-2-pyridone system which has isosteric structural similarity with dopamine without having the COMT vulnerable m-hydroxy group were synthesized via 12 synthetic steps. Their dopaminergic activities were evaluated by measuring the inhibitory effects of prolactin secretion from the anterior pituitary in rats. 4-(2'-Aminoethyl)-1-hydroxy-2-pyridone caused a continuous reduction of prolactin secretion at 10~ 10~ M concentration ranges while 5-(2 1-aminoethyl) -1-hydroxy-2-pyridone showed its activity at 10~ M concentration. These results indicate that this system may be used as a starting material for new cardiotonic drugs. Before the syntheses of the two analogues of (2'aminoethyl)-1-hydroxy-2-pyridone, semiempirical MO calculations(at the AM-1 level) were performed in order to understand the structural and electronic features as compared to dopamine. The results of AM-1 calculation indicate that both compounds were less lipophilic than dopamine and have a xiv

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weak interaction between the hydrogen in the N-hydroxy group and the adjacent oxygen atom. xv

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CHAPTER I INTRODUCTION Pathophysiology of Congestive Heart Failure Congestive Heart Failure(CHF), which is defined as the circumstance which occurs when the ventricle is unable to provide a cardiac output sufficient to meet the metabolic demands of the body1 is a common clinical manifestation associated with many different forms of heart disease. It is a leading cause of death throughout the United States and in other industralized countries. 2 3 This disease is caused by a variety of abnormalities, including pressure and volume load changes, muscle atrophy, primary muscle disease or excessive peripheral demands such as high cardiac output failure. In the typical forms of heart failure, the contractility of the heart muscle reduced due to the inability of the left ventricle to adequately pump blood. This produces a reduction in cardiac output and, as a result, the heart is unable to meet the peripheral demands of the body. Pathophysiologically, there are four main factors affecting left ventricular (LV) performance. 4 The first is preload, which relates to the filling pressure of the left ventricle. The second factor, afterload, represents the load against which the heart must work, i.e.; systemic arterial pressure and systemic vascular resistance. Third, the 1

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2 contractility of the heart represents the intrinsic ability of the muscle to develop force and constrict. Contractility can be altered by the administration of positive or negative inotropic agents. The fourth factor, heart r~te, is adjusted and controlled by the balance of sympathetic and parasympathetic tone. As a number of pathophysiologic alterations occur during the development of CHF5 the four factors of LV performance are generally altered as follows: (1) There is an intrinsic decrease in muscle contractility due to prolonged pressure or volume overload. (2) Preload or left atrial filling pressure is increased due to retention of salt and water. An increased circuiating blood volume results in pulmonary congestion and dyspnea. (3) Although systemic blood pressure is often reduced in CHF, there is an increase in systemic vascular resistance (afterload), which can further reduce cardiac output. (4) Heart rate is generally increased as a result of compensatory mechanisms. The alterations in contractile state, preload, afterload and heart rate provide the basis for most of the therapeutic interventions. For example, in order to counteract the marked decrease in intrinsic contractility which occurs in heart failure, inotropic agents such as digitalis or catecholamines are administered and can improve contractile state and cardiovascular performance. The increased preload that exists in CHF can be lowered with diuretics and vasodilator drugs to

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reduce the filling pressure to a more optimal level. The increase in systemic vascular resistance can be reduced by arteriolar vasodilators. As a result, these drugs cause an increase in forward cardiac output. Although the heart rate in patients in CHF is not generally manipulated, in patients with tachycardias, great benefits can be achieved by reducing the ventricular response. It is evident, therefore, that most of current therapeutic intervensions in heart diseases are designed to counter the pathophysiologic changes which occur as a result of CHF. Development of Positive Inotropes for CHF Mechanism of Action Since digitalis and its related cardiac glycosides have been employed in the treatment of CHF for nearly t w o t <, cen ur1es the mechanism of action of these compounds as positive inotropes, i.e., agents which increase the force of contraction of the heart muscle, has been extensively studied7 -11 and then the cascade of events between extracellular stimulation and contraction of cardiac muscle cell are 1 2 recently established, as shown in Fig. 1-1 Stimulation of either ~-adrenergic receptors(a) or H ~ histaminergic receptors(b) activates the catalytic component(f) of the adenylate cyclase complex via close association of these cell surface receptors with the cell membrane bound adenylate cyclase regulatory component(d). Since this enzyme system is responsible for the conversion of 3

PAGE 19

4 ATP to c-AMP, its activation results in increased cellular concentration of c-AMP. Higher intracellular c-AMP concentration leads to greater interaction of c-AMP with the regulatory subunit of protein kinase (h) which, in turn, increase its catalytic activity and cause enhanced phosphorylation(i) of various cellular proteins. These proteins are of particular importance for cardiac muscle contraction. Phosphorylation of specific proteins associated with the Ca2+ channel in the sarcolemma enhances the 5'-AMP Ca2+-----x 2~ a k c.r Figure 1-1 The c-AMP cascade in cardiac muscle cell

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5 responsiveness of the channels to voltage activation(prolongs the gate(j) open time), which results in a greater influx of C 2 + 13 a 2 + t 1 4 The entering ca is thought to act as a rigger to release intracellular ca2+ stored in the sarcoplasmic reticulum (k) This release of Ca2+ subsquently causes contraction through direct interaction with the contractile proteins(l). Phosphorylation(i) of specific proteins associated with sarcoplasmic reticulum(k) allows for a more rapid and increased re-uptake of Ca2+ after a contractile event has occured. Thus, when intracellular c-AMP levels are raised, both the rate of contraction(df/dt) and the rate of relaxation(-df/dt) are increased and the shape of the resulting contractile force/time curve retains its symmetry. The latter is useful for characterizing the mechanism of compounds suspected to act on the c-AMP cascade. Two important ATP regulatory systems are depicted in Figure 1-1. At least one consequence of stimulating muscarinic cholinergic receptors(c) is to decrease the conversion of ATP to c-AMP1 5 through association of these receptors with an inhibitory component(e) of the adenylate cyclase complex. Similarly, the degradative enzyme phosphodiesterase(g;PDE III) decreases intracellular c-AMP by converting it to 5'-AMP. Glycosides Digitalis glycosides have occupied a prominent place in the management of CHF and certain arrhythmias since Withering recognized that this steroid was useful in the treatment of dropsy in 17856 The manner in which digitalis exerts its

PAGE 21

6 direct positive inotropic effect is most probably explained by the ability of digitalis to inhibit membrane bound Na+,K+ activated adenosine triphosphatase (Na+, K+-ATPase). As shown in Figure 1-2, it is thought that glycosides(a) bind to cell membrane(b) Na+, K+ -ATPase(c) and inhibit the pumping of Na + out of the cell. An increased intracellular Na + concentration then increases exchange(d) or alternatively, increases the displacement of Ca2+ from membrane-associated ca2+ pools ( e) 1 6 Increased intracellular Ca2+ concentration is directly associated with increased contraction as show in Fig. 1-1. Although digitalis is still prescribed to increase the force of myocardial contractions for patients with CHF, the toxicity of glycosides limits their t 1 t 17 u l l y. Most investigators1 8 engaged in Na-K-ATPase research agree that b Figure 1-2 The mechanism of the digitalis glycosides

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7 the toxicity of the glycosides is intimately related to their binding to the Na pump. This situation has prompted researchers to find non-glycoside cardiotonics. Catecholamines and Sympatomimetic Amines For many years, catecholamines and other sympathomimetic amines have been used in an attempt to replace the cardiac glycosides in that stimulation of fi-receptors by catecholamines increases c-AMP availability in the cell and, as a result, increases calcium availability for contractile proteins. These drugs showed initial promise in the treatment of heart failure when given intravenously (i.v.) infusion over short periods of time and are still useful as an inotropes when such therapy is required. 1 9 However, their use in CHF was limited due to their lack of oral bioavailability. Their limitations also include the lack of fi1-receptor vs fi, -and a-receptors selectivity As shown in Table 1-15 activation of various sites on different adrenergic receptors results in different effects. In addition, as CHF progresses, the fi-adrenergic receptors appear to down-regulate20 with the cascade becoming less sensitive to catecholamine stimulation. 2 1 -23 Due to these problems, this approach has been avoided. The work which has continued in this area has dealt mostly with attempts to improve the poor oral bioavailabiliy associated with better receptor selectivity Adrenergic receptor activity of sympathomimetic amines under development for use in the treatment of CHF is listed1 2 in Table 1-2.

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Table 1-1 : Some receptor actions of catecholamines Aderenergic receptor site Beta1 Myocardium S-A node action Increase Contractility Increase Heart Rate 8 Atrioventricular Enhance conduction Conduction System Beta2 Alpha Other Inotropic Agents Arterioles Lungs Pheripheral Arterioles Vasodilation Bronchodilation Vasoconstriction Recent interest has been focused on an altogether new class of inotropic drugs which defy simple classification and are distinctive in that their mechanism does not involve the ~ 1-receptor or sodium-potassium stimulated ATPase. The first breakthrough occurred nearly 10 years ago with the discovery that amrinone had positive inotropic activity.~~6 Although its mechanism of action was at first unknown, it is thought to inhibit specific phosphodiesterase(PDE III) .2~~ As a result, significant attention had turned toward PDE inhibition. Several related heterocyclic compounds that inhibit c-AMP PDE have been prepared and these drugs are now entering clinical studies for the treatment of CHF. Representative compounds are listed in table 1-3. These compounds also generally exhibit pronounced vasodilator properties and cause only modest inotropy in CHF

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Table 1-2 drug I (prennllerol) 2 (dopamine) 3 (dobulamine) 4 (dopexamine) 5 (ibopamine) 6 (bulopnmine) 7 (denopamine) 8 (xamolerol) 9 (doxaminol)b Inotropic drugs that activate B 1-receptors. OH I .~""'" R Rt 2-5 6, 7 R R' OH H OH OH OH OH OH OH OCOCH(CH3 ) 2 OCOCH(CH3)2 OH H OH H OH H H H NHRII ,Jo/o~""'" 1, 8 9 R" CH(CH3h H IHCH2CH2-@--0H CH3 ICH 2leNHCH2CH2-@ CH3 IH2CH2-@--0H CH3 OCH3 CH2CH 2-@--0CH3 I\ CH2CH 2 NHCON 0 \_/ '"'"'"' c,,

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Table 1-3 Inotrot;:>ic drugs that inhibit c-Af~P PDE. Co) ~'" Cg\ -~ "YJ(NH HN~ HN I 0 0 0 11-13 14 15 16 %"" NH ~o .:,,-CH, t-4N NH y 1 HN 0 17, 18 drug l l (amrinone) 12 (milrinone) 13 (APP-2015:13) 14 (Sl
PAGE 26

11 patient. In fact, their predominant afterload reduction effect is due to vasodilation property~ although it is not established that the vascular profile obtained from PDE inhibition is ideally suited for treating CHF. Therefore, these compounds should not be viewed as derivatives with pure cardiotonic action. In addition to PDE inhibition, a small effort29 has been directed toward preparing c-AMP analogues with the thought that they might penetrate the sarcolemma of cardiac cells and stimulate c-AMP. The dibutyryl derivative of c-AMP known as bucladesine(l) has shown a promise for this purpose although c-AMP is chemically inactivation. 0 I O=P f "-.o ONa fragile and susceptible to PDE <~o(CH,),
PAGE 27

1 2 decompensation because H2-agonists don't cause down regulation as CHF progresses. z1.30 3 1 Impromidine ( 2) is a potent and selective H2-agonist which has been studied for its cardiovascular effects in man and was shown to significantly improve myocardial function after treatment. 32 However, at least three challanging points must be considered when H., agonists are used as potential new inotropic drugs: (1) Agents should have selective cardiovascular action in difference to H2-receptor mediated gastric acid secretion, (2) agents should have selective cardiotonic activity related to vascular and respiratory action, and (3) histaminic drugs should produce positive increase. inotropic activity rather Direct activation of adenylate than heart rate cyclase was also considered as inotropic way. The natural product forskolin(J) is thought to interact with regulatory subunit or catalytic subunit of the adenylate cyclase ~ system.--' This compound exhibits both inotropic and vasodilatory properties. NH II r=<.(CH,) NHCNH(CH,),SCH, A CH, H~N NVNH ( 2) CH, ,'~ ( 3)

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13 Problems with Current 81-Agonists As discussed previously, clinical use of most catecholamines and sympathomimetic amines is largely limited by poor oral bioavailabily even though intravenous sympathomimetic agents are used for the acute treatment of heart failure. The use of parenteral drugs requires hospitalization, usually in intensive care units, and hemodynamic monitoring is necessary to optimize therapy. Moreover, these catecholamines can not be used chronically because tolerance develops rapidly ( 4 8 to 7 2 hours in some cases).~ Therefore, the development of oral agents for chronic maintenance therapy of CHF is a high priority goal. A number of the sympathomimetic amines are available in oral forms and some data have accumulated regarding their use in patients with heart failure. Another limitation of these agents results from their positive chronotropic ( increase heart rate) effect which causes tachycardia and from their action to either increase or decrease peripheral resistance and thus, to change arterial pressure.35 Furthermore, the stimulation of ~ 2-adrenergic receptors in the peripheral arterial system can decrease peripheral resistance and arterial pressure, which may not be desirable when myocardial ischemia is a potential problem. In addition to problems of selectivity, a "downregulation1120 in ~1-adenergic receptor density may occur following chronic use of sympathomimetic amines.36 This desensitization of physiologic responsiveness of ventricular

PAGE 29

14 tissue to a fi1-adrenergic agonist may be primarily due to a marked diminution in adenylate cyclase responsiveness rather t t. 37 than to change in the adrenergic recep or proper ies per se. From the described 1 iabil i ties associated with fi1 -agonists, it is easily concluded that the ideal fi1-agonist should have (1) inotropic effect, but not chronotropic effects; ( 2) potent with long half life; ( 3) oral bioavailabilty; (4) no change in blood pressure and (5) no down-regulation. Objectives of Research This research is designed to develop novel cardiotonics which have long duration of action as well as oral bioavailability. These new agents were designed by chemical manipulation of dobutamine. Three different chemical manipulations were considered. After the synthesis, the stability in biological media and the in vitro activity of these compounds were studied. Finally, in vivo activity was assessed by observing the changes in cardiac electrophysiological parameters caused by these compounds in dogs.

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CHAPTER II DESIGN Pharmacology of Dobutamine and Its Analogues Mechanism of Action sympathomimetic amines generally augment myocardial contractility by stimulating #1-adrenergic receptor sites in the myocardium directly or by releasing endogenous norepinephrine as in the case of Aramine.~ Norepinephrine is an endogeneous catecholamine that is synthesized and stored in granules in adrenergic nerve endings in the myocardium. When sympathetic nerves to the heart are activated, norepinephrine is released from its stores and stimulates specific sites on the myocardial cell surface, termed #1adrenergic receptors. Stimulation of these receptors increases the rate of discharge of the sinoatrial node, thereby augmenting heart rate and enhancing atrioventricular conduction and ventricular myocardium contraction. Dobutamine(4), which resulted from systematic , 1 9 modification of isoproterenol(5) by Tuttle and Mills, acts directly on #1-adrenergic receptors in the myocardium to selectively increase the myocardial contractility with less side effects than is seen with other sympathomimetic agents. Dobutamine acts selectively on adrenergic #1-receptors sparing #2 and a receptors from stimulatio~0 (see Table 2-15

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1 6 1). In comparison to dopamine(6) which is a precursor in the biosynthesis of norepinephrine and which stimulates the myocardium directly and indirectly through release o f norepinephrine from its stores, dobutamine does not stimulate the heart indirectly and lacks the direct vasodilator effect of dopamine on the renal vasculature. In addition, dobutamine does not also show the predominant vasoconstrictor(a-adrenergic) effect seen with d 41 opamine. Furthermore, dobutamine has been shown not to increase heart rate substantially even when given in a dose that produces a prominent increase in myocardial contractility.42 High-doses of dobutamine ( >7 ug/kg/min) raise heart rate appreciably. However, this raise in heart rate is usually less than that seen with high doses of dopamine(>lO ug/kg/min) .43 00~ I ~OH ( 4) ( 5) (6)

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Table 2-1 Adrenergic-receptor activity of sympathomimetic amines. Q 13, Peripheral Cardiac Norepinephrine ++++ ++++ Epinephrine ++++ ++++ Dopamine ++++ ++++ Isoproterenol 0 ++++ Dobutamine + ++++ 17 132 Peripheral 0 ++ ++ ++++ ++ Similarly, in the animal experiments, dobutamine exerts a more prominent inotropic than chronotropic action as compared to isoproterenol~ The reason for this difference i n response has not been explained, but dobutamine appears to exert a relatively less pronounced effect on the sinoatrial node than on the ventricular contractile tissue.42 Since dobutamine was approved for clinical use in this country in 1978, it has been widely used to treat severe cardiac failure, 4 4 heart failure following acute myocardial infarction45-48 and for hemodynamic support in patients following open heart surgery. 49 Intravenous infusions are necessary at rates ranging from approximately 2. 5 to 15. o ug/kg/min. and produce a progressive increase in cardiac output. Pulmonary wedge pressure is decreased, reflecting a fall in diastolic filling pressure in the left ventricle. Generally, the improvement is more substantial in more severe states of cardiac failure. 50

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1 8 Metabolism of Dobutamine The major route of metabolism of catecholamines includes 0-methylation of the catechol catalyzed by catechol 0-methyl transferase (COMT). 51 As shown in Figure 2-1, this enzyme catalyzes the transfer of a methyl group from S-adenosyl-Lmethionine to a catechol substrate, resulting in the formation of the meta and para a-methylated products.5 2 COMT is widely distributed in mammalian tissues and plays a primary role in the extraneuronal inactivation of endogeneous catecholamines (dopamine, norepinephrine, epinephrine) as well as the detoxification of catechol drugs(isoproterenol, L-dopa). OH AdoMet AdoHcy rOCH, COMT, MQ2+ R ~OH y R Figure 2-1 COMT reaction of catechol. Recently, Patrick et al.53 reported their metabolic studies in dogs, focusing on the fate of dobutamine after intraveneous dosing. When they examined dobutamine as a substrate of COMT, they found that dobutamine has a K01 and Vm:ix similar to that of dopamine, the natural substrate. They also reported that metabolites are excreted mostly in the urine and feces and the major urinary metabolites are the glucuronide conjugates of dobutamine and meta-0-methyldobutamine. The

PAGE 34

1 9 relative amount of dobutamine glucuronide in the urine was estimated to be 7 ~ 0 whereas the amount of meta-0-methyldobutamine was estimated as 82% at 24 hours post i.v. administration. Like most of the intraveneous sympathomimetic amines, the slow steady-state plasma levels of dobutamine are increased in proportion to the infusion rate. The elimination half-life of dobutamine is approximately two minutes.5 4 Adverse Effects The most serious adverse effect of all the sympathomimetic amines is the precipitation of arrhythmias. The electrophysiologic properties of dobutamine are similar to those of isoproterenol, and ventricular arrhythmias have been associated with the use of both drugs. However, it is claimed that dobutamine causes a lower incidence of arrhythmias as compared with isopreterenol" and dopamine5 6 Dobutamine may cause a marked increase in heart rate or systolic blood pressure at high dosage, but reduction of dosage usually reverses these effects promptly. Other minor side effects reported include nausea, headache, anginal pain, palpitation and shortness of breath. Design of Dobutamine Analogues As Selective 81-Agonists Design of 61-Substituted Dobutamine Analogues Since the pharmacologic profile of the action of dobutamine is particularly desirable, especially due to better selectivity of ~1 over ~2 and a receptors, a number of studies have been done to modify the chemical structure of dobutamine

PAGE 35

20 to make it orally effective conserving in the same time its pharmacological profile. Recently, Tuttle et al. 57 reported that replacement of the para hydroxyl group in dobutamine with carboxyamide at the end of the molecule increased inotropic potency threefold, but it introduced presser activity that detracted from the inotropic selective profile of dobutamine. However, shifting the carboxyamide to the meta position avoided presser activity and further enhanced inotropic potency to nine times that of dobutamine as measured in the anesthetized dog. In contrast to dobutamine, this compound(?), KM-13, produces a sustained (7) increase in left ventricular dP/dt with only immediate change in heart rate when administered orally to conscious dogs. It was also reported that the isobutyl bridge between the amine and phenyl ring bearing the carboxyamide is required to achieve the cardiac potency observed and blood pressure criteria. Based on the above report, it became of interest to study the substituent effects on the phenyl ring and to investigate

PAGE 36

21 the further structural requirements needed to meet the potency and blood pressure criteria as a part of structure-activity studies. Therefore, we have synthesized two dobutamine analogues which are substituted with R(=OCH3 at 6~position and have examined their pharmacological activity. Design of 5-(2'-Aminoethyl)carbostyril System R= OCH3 ( 8) CH3 ( 9) The intraveneous route of administration greatly limits the number of patients who can get benefit from inotropes because this mode of administration requires the close supervision of medical personnel and usually requires hospitalization. Clearly, the development of oral agents for chronic maintenance therapy of CHF is a high priority goal. Although a number of the symphatomimetic amines are available in an oral form,~ a new class of dobutamine analogue which would have longer durations of action and oral bioavailability are needed because of their potentially beneficial pharmacological profile. According to metabolism of dobutamin~,B the major reason of short duration of action of dobutamine is its fast

PAGE 37

22 elimination from the body by transformation in the liver to inactive glucuronide conjugates of the meta-0-methyldobutamine. A number of different approaches have been suggested to prolong the duration of similar catecholamines. The first approach was based on esterificatioh of the vulnerable phenolic OH groups, which might reduce the inactivation Bretschneider5 9 rate and result synthesized the in prolonged activity. 3,4-diacetate and 3,4-dipropionate esters of isoproterenol and other catecholamines but did not report their pharmacological studies. The synthesis and biological testing of various esters were initiated in 1966.w~i In 1975, Tullar et al.~ reported that aromatic esters of N-tert-butylarterenol resulted in a long acting derivative and bronchodilator-cardiovascular significant activity. separation Similarily, of the replacement of them-OH group of catecholamines by various substituents such as a methanesulfonamide, 63-65 a 66 CH20H, a ureido group67 68 or the aromatic N atom of 8-hydroxyquinoline was also investigeted.~ In 1976, Yoshizaki et al.m reported that sympathomimetic amines containing 8-hydroxycarbostyril moeity probably exist as resonance hybrids having two acidic hydrogen atoms in a configuration approximating the hydroxyl groups of catecholcontaining adrenergic agents. When this carbostyril system was applied to salbutamol(lO), it was found that the resulting procaterol(ll) possesses more potent bronchorelaxing activities than isoproterenol and better selectivity for ~2 -

PAGE 38

23 receptors than salbutamol(lO}. Recently, Kaiser et al.71 reported that similar modification of the catechol ring on dopamine produced measurable activation of dopamine-sensitiv e adenylate cyclase and the potency of carbostyrils was enhanced by 8-hydroxylation and appropriate substitution of amino group of ethylamine side chain. OH Y = CH10H; R = t-Bu (10) (11) Since sympathomimetic amines with an 8-hydroxycarbostyril moeity exist as resonance hybrids which possess two weekly acidic hydrogen atoms in about the same general vicinity as those in catecholamine but are not good substrates for COMT(see Figure 2-2), it is evident that one of the most effective hydroxyl replacement group in catecholamines is the NH of a carbostyril derivative to produce longer duration o f action. The recent results of Kaiser et al.7 1 have reinforced our strategies to improve dobutamine and to develop longer acting, orally effective cardiotonic agents.

PAGE 39

24 -0 OH OH Figure 2-2 Tautomers of 8-hydroxycarbostyril. In order to determine if the better potency, high ~1 selectivity and prolonged effectiveness accompanied isosteric modification of m-hydroxyl of dobutamine with an amide type carbostyril system, we have synthesized some modified 5-(2'aminoethyl)carbostyril derivatives(l2,13,14) and examined their pharmacological effects. R=CH3(12), R=H(l3) (14) Design of (2'-Aminoethyl)-1-hydroxy-2-pyridone System as Dopamine Receptor Agonists Structure-activity relationship studies among dopamine receptor agonists are ambiguous regarding the significance of the catechol system for binding to, and activation of dopamine receptors. Both hydroxyl groups apparently are important for

PAGE 40

25 stimulation of the D-172 or DA/3 subpopulations of dopamine receptors that are involved in activation of dopaminesensitive adenylate cyclase and initiation of smooth muscle relaxation. However, there are notable exceptions to this generalization. Thus, some 2-aminotetralins that bear only a single hydroxy group in a position meta to the embedded ethylamine side chain, retain a marked degree of D-1 agonist activity. Although selective noncatechol D-1 receptor agonists have not been identified, stimulation of this receptor subtype is also dependent upon the pattern of substitution of the amine t 74 n1 rogen. Clearly, the catecholic system is not required for activation of D-272(not associated, or negatively linked with cyclic-AMP) and DA/3(located on sympathetic nerve endings and subserving inhibition of norepinephrine release) receptor. Thus, the monohydroxylated tyramine derivative RU 24213(N-n-propyl-N-(2phenylethyl)-tyramine) is a selective D2 receptor agonist.TI However, some of the D-2 agonists are also capable of stimulating dopamine-sensitive adenylate cyclase. Miller's group~ also found that the nitrogen atom of dopamine is not essential for dopaminergic agonist activity and sulfonium analogues of dopamine possess significant activity in both dopamine binding and behavior studies. As a generalization, the presence of a hydroxyl group or a moiety that can imitate this functionality in a position meta to ethylamine is considered to constitute the dopaminergic pharmacophore. 7 6

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2 6 From the previous SAR studies among dopamine receptor agonists and metabolic pathway of dopamine by COMT, it can be speculated that drugs should have the ethylamine unit if the y are to show dopaminergic activity, but a hydroxyl group meta to the ethylamine chain catechol is not necessary to achieve long duration and/or oral effectiveness. On the basis of the above hypothesis, the l-hydroxy-2-pyridone system attached to the ethylamine side chain was considered as a new class of potential dopaminergic agonists as the l-hydroxy-2-pyridone moiety can tautomerize to the 2-hydroxypyridine-1-oxide form as shown in Figure 2-3. This system has isosteric structural similarity with dopamine without having the vulnerable m-OH u N 0 u N 0 I I I O--H -o---H Figure 2-3 Tautomers of l-hydroxy-2-pyridone. group which is a substrate to COMT. This may give long duration of activity and/or oral effectiveness to the dopamine analogues. Accordingly, the synthesis and pharmacological evaluation of 5-(2'-aminoethyl)-l-hydroxy-2-pyridone(l5) and 4-(2'-aminoethyl)-l-hydroxy-2-pyridone(l6) were undertaken.

PAGE 42

H0~2,HC1 (15) ~ 2 .HCl OH.-,~ (16) 27

PAGE 43

CHAPTER III EXPERIMENTAL Materials and Methods Salts and nondeutrated solvents were obtained from Aldrich Chemical Co. and Fischer Scientific Co., unless otherwise noted. All salts were reagent grade. Solvents used for high pressure liquid chromatography were of spectral grade. All other nondeutrated solvents were of either reagent or spectral grade. Deutrated sol vents were obtained from Aldrich Chemical Co. Melting points, given in degree Celsius, were determined on a Fischer-Johns melting point apparatus and were uncorrected. Ultraviolet spectroscopy was performed on a Varian Cary 210 spectrophotometer. Proton nuclear magnetic resonance spectra were recorded on a Varian EM 390 spectrophotometer. Chemical shifts are reported in parts per million units(ppm) on the 6 scale downfield from tetramethylsilane which was used as an internal standard. The solvent used are given in parentheses for each spectrum reported. Multiplicities of proton are designated as singlet(s), doublet(d), triplet(t), quartet(q) or multiplet(m). Infrared(IR) spectra were recorded on PerkinElmer 281 spectrophotometer. Solid samples were run as either 28

PAGE 44

29 a KBr pellet or a nujol mull; liquid samples were analyzed neat as a thin film between NaCl plates. Mass spectra were performed by Department of Medicinal Chemistry, University of Florida, Gainesville, Florida. Elemental analyses were performed by Atlantic Microlab. Inc., Atlanta, Georgia. High pressure liquid chromatography was carried out on a modular system composed of a Autochrom M 500 pump a Rheodyne 7125 injector with a 20 ul loop and a Spectroflow 757 variable-wavelength detector. Chromatograms were recorded on a Fischer Scientific Series 5000 chart recorder. A Bio-Sil ODS-5, c1 8 10 um particle size, reversed phase silica gel column 30 cm x 4.1 mm internal diameter was used. The Bio-Sil column was protected with a guard column packed with Whatman pell icular ODS-C1 8 media. A Dynamic centrifuge having a ma x imum spin rate of 3,000 rpm was used to spin down tissue homogenates. Chromatographic samples in vitro were centrifuged using a Beckman Microfuge 11 capable of a 13,000 rpm spin rate. Synthesis Synthesis of 61-Substituted Dobutamine Analogues(8,9) Preparation of 3-iodo-4-methoxybenzamide(18) To a solution of 3.00 g(0.0108 mol) of 3-iodo-4-methoxybenzoic acid in 150 ml of benzene was added 1.54 g(l.2 eq) of thionyl chloride and one drop of pyridine at room temperature. The reaction mixture was heated for 2 hrs at 65 C and then cooled in ice-water bath before the reaction was

PAGE 45

30 quenched with an excess of 28% saturated ammonium hydroxide solution. The white precipitate which formed immediately was collected and recrystallized from 50% aqueous ethanol to give 2.12 g of the desired product(70.6% yield). m.p. : 151-152.5C. IR(nujol mull) 3370, 3170, 2930, 2860, 1650, 1620, 15 9 o 14 7 O 14 4 0 12 7 5 1 O 5 O 1 O 2 0 cm-1 1H NMR{DMSO-dd : 3.96(3H,s,-OCR_,) ,7.18(1H,d,JAB=9Hz), 8.10(1H,m,JAI3=9Hz,JAc=2Hz), 8.45{1H,d,JAc=2Hz), 7.1-7.5(2H, br.). Preparation of 4-methoxy-3-(3'-oxybut-2'-enyl)benzamide(19) A solution of 0.276 g(lO mmol) of compound(l8), a catalytic amount of 5% Pd-C, 0.081 ml of methyl vinyl ketone (1.1 eq) and 0.15 ml of triethyl amine in 10 ml of acetonitrile was refluxed for 2.5 hrs. Next, the solution was cooled to room temperature, and another 0.073 ml of methyl vinyl ketone(l.O eq) and 0.115 ml of triethyl amine(l.O eq) was added. After being refluxed again for 2 hrs, the solution was filtered to remove the catalyst and was concentrated to give a crude product which was identified as starting material with a small amount of desired product. The product was collected by column chromatography on silica-gel with ethyl acetate as an eluent to give a trace amount of product which was identified only by NMR. 1H NMR{CDC13 ) 2.32{3H,s,-COCH3), 3.95{3H,s,-OCH3), 6.83{1H,d,J=16Hz,vinylic fl), 7.16(1H,d,J=9Hz), 7.25{2H,br), 7.80{1H,d,J=16Hz,Vinylic fl), 8.lO{lH,m,JAB=9Hz,JAc=2Hz),

PAGE 46

31 8.45 (1H,d,JAc=2Hz). Preparation of 5-nitrosalicylaldehyde(21) Nitric acid (8.0 g) was added dropwise to the solution of 10 g(0.081 mol) of commercially available salicylaldehyde (20) in 40 ml of acetic acid while stirring in an ice-water bath. The temperature was kept below 15 C during addition of first portion(l/3) of nitric acid. The resulting mixture was stirred at 15 C for 25 min., followed by for 2 hrs at 45C, and then poured immediately into 100 g of ice-water. The yellow precipitates which were identified as a mixture of 3-and 5-nitro isomers were collected by filtration and dried to give a 12.03 g of product(89.0% yield). Separation of 5-nitro isomer from mixture was performed as follow: The mixture of isomers was dissolved in 60 ml 0.83 N NaOH while warming. On standing overnight, the 5-ni tro isomer precipitated and was filtered, washed with cold 0.83 N NaOH and redissolved in water. Finally, the resulting sodium salt solution of 5-nitro isomer was neutralized with dilute HCl solution to give a yellow precipitate which was dried to give 4.72 g of 5-nitrosalicylaldehyde (34.89% yield from salicylaldehyde). m.p: 127-128.5 C ( refn : 126C). IR(KBr) : 3450, 3030, 2940, 1665, 1630, 1590, 1530, 1480, 1340, 1290, 1100, 930, 780 cm -1 1H NMR(CDCl 3 + DMS0-d 6 ) : 7.14(1H,d,JA B = 9Hz), 8.10-8.62 (2H,m), 10.33(1H,s,-CHO).

PAGE 47

32 Preparation of 2-methoxy-5-nitrobenzaldehyde(22) A solution of 2.47 g(0.0148 mol) of 5-nitrosalicylalderhyde(21) and 2.65 g(l.3 eq) of K2C03 in 30 ml of DMF was stirred at 60 C for 1 hr and was treated with 1.10 ml(l.2 eq) of methyl iodide dropwise at room temperature. The resulting mixture was stirred for 2 hrs at 60 C again. When reaction was completed, the reaction mixture was poured into 50 ml of ice-water. The yellow precipitate was collected, washed with water and dried to give 2.52 g of crude product. Recrystallization from ethanol product(86.3% yield). m.p : 85.5-87.5C. gave 2.31 g of pure IR(KBr) : 3045, 1680, 1590, 1520, 1485, 1340, 1275, 1170, 1 1030, 1010, 970, 840 cm. 1H NMR(CDCl:,): 4.13(1H,s,-OCH:,), 7.20(1H,d,J/\13=9Hz), 8. 40-8.75(2H,m), 10.57(1H,s,-CHO). Preparation of 2-methoxy-5-nitro-3-(3-oxobut-l-enyl)benzene illl A mixture of 9.47 g(0.0523 mol) of compound(22) and 20.0 g(l.2 eq) of l-triphenylphosphoanylidene-2-propanone in 250 ml of benzene was heated between 50 and 60 C degree overnight. After all solvent was removed in vacuo, the remained crude product was purified by column chromatography on silica-gel with ethyl acetate-hexane(l:2) as an eluent to give a 9.13 g of pure product(79.0% yield). m.p. : 139.5-141.5C.

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3 3 IR(KBr) : 3050, 1690, 1610, 1580, 1470, 1340, 1270, 1090, 1020, 830, 750 cm -1 1H NMR(CDCl3 ) 2.43(3H,s,-COCH3), 4.05(3H,s,-OCH3), 6.85(1H,d,J=l6.6Hz,vinylic fl), 7.04(1H,d,JAn=9.2Hz}, 7.81(1H, d,J=l6.6Hz,vinylic fl), 8.26(1H,dd,JAn=9.2Hz,JAc=3.0Hz), 8.42 (lH,d,JAc=3.0Hz). Elemental Analysis for C11H11N02 Cald. C:59.72, H:5.01, N:6.33 Found C:59.80, H:5.06, N:6.30. Preparation of 5-arnino-2-methoxy-3-(3'-oxobutyl)benzene(24) A mixture of 2.00 g(9.04 mrnol) of compound(23) and 65 mg of 10% Pd-C in 90 ml of ethyl acetate was hydrogenated at 25 C and initial pressure of 15 psi for 80 min. After the reaction was completed as indicated by TLC, the reaction mixture was filtered and concentrated to give the desired product, which was used in the next reaction without further purification. 1 IR(KBr) : 3330(br.), 2960, 1705, 1510, 1330 cm 1 H NMR(CDC13 ) ; 2 .12 (3H, s, -COCH:,), 2, 75 (4H, s), 3. 40 (2H, s, -NH2), 3.73(3H,s,-OCH3), 6.40-6.85(3H,m,aromatic fl). Preparation of 5-cyano-2-methoxy-3-(31-oxobutyl)benzene(25) A mixture of 2.40 g of the crude amine(24) in 5 ml of cHCl and 5 ml of water was chilled to a slurry in ice-water bath. To this mixture, 1.02 g(l.2 eq) of sodium nitrite in 4 ml of water was added dropwise and stirred for 20 min. at o-5 0C. After addition of a few crystal of urea to remove excess of sodium nitrite, the resulting mixture was carefully neutralized with an excess amount of calcium carbonate and

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3 4 filtered into a warm solution of mixture of 1.73 g of CuC N and 2.31 g of NaCN which was prepared in 25 ml of water and 2 5 ml of benzene. The resulting mixture was stirred for 1 hr at room temperature, and then extracted with 100 ml of ether. Combined organic layers were washed with 5% NaOH solution, 5 % HCl solution, water and brine continuously. Finally, evaporation of solvent in vacuo gave a brown colored crude product, which was purified on silica-gel column chromatography with ethyl acetate-hexane ( 1: 3) to give 84 O mg of pure product ( 3 3. 3 % yield). m.p. : 64.5-66 C. IR(nujol mull) 2920, 2860, 2230, 1710, 1605, 1500, I 1430, 1380, 1260, 1030, 820 cm. l H NMR(CDC13 ) : 2.12(3H,s,-COCH3), 2.80(4H,m), 3.88(3H,s,-OCH3), 6.90(1H,d,JAn=9Hz), 7.40-7.65(2H,m). Elemantal Analysis for C1 2H13NO Cald. C:70.91, H:6.44, N:6.89 Found C:70.84, H:6.47, N:6.86. Preparation of 4-methoxy-3-(3'-oxobutyl)benzamide(26) A mixture of 300 mg(l.40 mmol) of compound(25), 170 mg of potassium bicarbonate and 1.00 ml of 30% hydrogen peroxide in 3.0 ml of methanol was stirred overnight at room temperature. After the reaction was completed, 25 ml of water was added to the reaction mixture and the resulting solution was extracted with 100 ml of ethyl acetate. The combined organic layers were washed with water and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give

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3 5 287 mg of crude product which was recrystallized from EtOHEtOAc(l:1) to give 265 mg of pure product(85.6% yield). m.p. : 128.5-130 C. IR(KBr) : 3420, 3365, 3180, 1720, 1660, 1605, 1505, 1440, 1390, 1260, 1170, 1030 cm1 1H NMR(CDC13 + DMSO-d d : 2.15(3H,s,-COCH3), 2.83(4H,m), 3.90(3H,s,-OCH3 ) ,6.85(1H,d,JAB=9Hz) ,6.00-7.50(2H,br.,-CONH2), 7.65-7.85(2H,m). Cald. Found C:65.14, H:6.83, N:6.33 C:65.02, H:6.89, N:6.28. Preparation of 3,4-dihydroxy-N-(3-(2'-methoxy-5'carbamoylphenyl)-1-methyl-n-propyl)-B-phenethylamine hydrochloride(8) A mixture of 900 mg(4.07 mmol) of compound(26), 580 mg of dopamine(3.79 mmol), 10 mg of 10% Pt02 and 200 mg of 10% Pd-C in 2 5 ml of methanol and 8 ml of acetic acid was hydrogenated for 72 hrs at an initial pressure of 30 psi. After the reaction was completed as indicated by TLC, the reaction mixture was filtered and added 1 ml of c-HCl was added. The solution was concentrated to give a white colored foam product, which was subjected to column chromatography on silica-gel with CHC13-MeOH-Acetic acid(6:2:0.5) and treated with 20% hydrogen chloride-methanol to afford 1.21 g of the desired hydrogen chloride salt of compound(8) as a powder(80.9% yield). 1H NMR(CD30D) 1.75(3H,d,-CH3), 1.70-2.40(3H,m), 2.67-3.37(8H,m), 3.40(1H,m), 3.95(3H,s,-OCH3), 6.55-6.87(3H,m),

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36 7.05-7.lO(lH,d), 7.90(2H,m). Preparntion of 2-methyl-5-nitrobenzyl alcohol(28) To a solution of 24.50 g(0.135 mol) of acid(27) in 140 ml of anhydrous THF cooled in an ice bath was added 145 ml of 1. 0 M BH3-THF solution dropwise via a syringe. After the resulting solution was stirred for 14 hrs at room temperature, it was carefully quenched with 10 ml of water and the solvent evaporated under reduced pressure. The residue was dissolved in 50 ml of ethyl acetate and extracted with 5% NaOH solution and water, dried over anhydrous magnesium sulfate and concentrated in vacuo to give 20.70 g of benzyl alcohol(91.8% yield). m.p. : 73.5-75 C. IR(KBr) -I : 3250(-0H), 1600, 1530, 1340, 1040, 740 cm 1 H NMR(CDC13 ) : 2.37(3H,s,-CH3), 3.20(1H,s,-OH), 4.73(2H, s,-CH2-0H), 7.27(1H,d,JAil=5Hz), 7.97(1H,d,JAil=5Hz), 8.21(1H,s). Preparation of 2-methyl-5-nitrobenzylaldehyde(29} Chromium trioxide, 18.00 g(0.180 mol) was added to a magnetically stirred solution of 28.5 g(0.360 mol) pyridine in 450 ml of methylene chloride for 15 min. at room temperature to give a deep burgundy solution. To this mixture, a solution of 7.00 g(0.042 mol) of alcohol(28) in 10 ml of methylene chloride was added in one portion. A tarry, black deposit separated immediately. After stirring an additional 20 min., the solution was passed through a 5-cm thick silica gel colummn to remove inorganic salt and residue and then was washed with 5% NaOH solution, 5% HCl solution, saturated

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37 NaHC03 solution and brine and dried over anhydrous magnesium sulfate Finally, evaporation of solvent afforded 6.35 g of the aldehyde. Recrystallization from ethyl acetate-hexane ( 1: 2) gave a 6.30 g of pure product(91.3% yield). m.p. : 54.5-55.5 C (ref.7 8 : 55C). IR(KBr) : 3035, 2860, 2750, 1700, 1605, 1580, 1505, 1340, I 1180, 1100, 830, cm. 1H NMR(CDC13 ) : 2.83(3H,s,-CH3), 7.50(1H,d,JAB=5Hz), 8.35 (1H,dd,JAJ1=5Hz,JAC=l.5Hz), 8.65(1H,d,JAc=l.5Hz), 10.40(1H,s,CHO). Preparation of 2-methyl-5-nitro-3-(3-oxobut-l-enyl)benzene(30) A mixture of 10.43 g(0.0632 mol) of aldehyde(29) and 24.14 g(l.2 eq.) of l-triphenylphosponylidene-2-propanone in 300 ml of benzene was heated between 50 and 60C for 7 hrs. After all solvent was removed in vacuo, the remaining crude product was dissolved in minimum amount of chloroformand was transferred into column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent to give 12.30 g of crude product, which was recrystallized from ethyl acetatehexane mixture to give 11.65 g of pure product(90.0% yield). m.p. : 95.5-96.5c. IR(KBr) : 1705, 1630, 1510, 1355, 1280, 980 cm -1 1H NMR(CDC13): 2.43(3H,s,-COCH3), 2.57(3H,s,-CH3), 6.80(1H,d,vinyl fi,J=l6.5Hz), 7.42(1H,d,JA13=5Hz), 7.80(1H, d, vinyl fi,J=l6.5Hz), 8.15(1H,d,JAl3=5Hz), 8.40(1H,s). Elemental Analysis for C11H11N03 Cald. : C:64.38, H:5.40, N:6.82

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38 Found: C:64.42, H:5.41, N:6.79. Preparation of 5-amino-2-methyl-3-(3'-oxobutyl)benzene(31) A mixture of 5.00 g(0.0239 mol) of compound(30), 1.00 g of 10% Pd-C in 110 ml of ethyl acetate was hydrogenated at room temperature and an initial pressure of 15 psi for 80 min. After reaction was completed, the reaction mixture was filtered and concentrated to give the desired product quantatively. This material was directly used in the next reaction without further purification. I H NMR(CDC13 ) : 2.10(3H,s,-COCH3), 2.18(3H,s,-CH3), 2.70 (4H,m,-CH2-CH2-), 3.53(2H,s,-NH2), 6.45(2H,m), 7.92(1H,d, J Al3=5Hz) Preparation of 5-cyano-2-methyl-3-(3'-oxobutyl)benzene(32) A solution of 4.85 g of the crude amino compound(31) in 10 ml of c-HCl and 10 ml of distilled water was chilled to a slurry in an ice bath. To this solution, 1.97 g(l.2 eq.) of sodium nitrite in 10 ml of water was added dropwise and stirred for 1 hr at 0-5 C. After addition of few crystals of urea to remove excess sodium nitrite, the solution was carefully neutralized by adding sodium carbonate while vigrously stirring and was filtered into a solution of 3.46 g(2.0 eq.) of cuprous cyanide and 4.62 g(2.5 eq.) of sodium cyanide in 50 ml of water and 50 ml of benzene at room temperature. The resulting dark solution was stirred for 2 more hrs at room temperature and was extracted with 200 ml of ether. The combined organic layer was washed with 5% NaOH solution, 5% HCl solution, saturated NaHC03 solution and

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39 brine, dried over anhydrous magnesium sulfate and concentrated to give a dark brown colored crude oil, which was purified by Krugnonal distillation, followed by column chromatography on silica-gel with ethyl acetate-hexane(l:2) to give 920 mg of white colored product(20.1% yield from compound(30)). m.p. : 52.5-53.5C. IR(KBr) : 2900, 2050(-CN), 1700, 1340, 1270 cm -1 I H NMR(CDC13 ) : 2.20(3H,s,-COCH3), 2.40(3H,s,-CH3), 2.65-2.95(4H,m), 7.20-7.45(3H,m). Cald. Found Elemental Analysis for C1 2H13NO C:76.97, H:7.00, N:7.48 C:76.84, H:7.01, N:7.42. Preparation of 4-methyl-3-(3'-oxobutyl)benzamide(33) A mixture of 900 mg(4.71 mmol) of the cyano compound(32), 1. 50 g of potassium bicarbonate and 3. 0 ml of 30% hydrogen peroxide in 6 ml of methanol was stirred for 2 days at room temperature. After the reaction was completed, the solution was added to 20 ml of water and then extracted with 50 ml of ethyl acetate. The combined organic layer was washed with 20 ml of water, dried over anhydrous magnesium sulfate, filtered and concentrated to give 960 mg of the desired product, which was recrystallized from ethyl acetate-hexane(l:1) mixture to give 945 mg of an analytically pure compound(97.8% yield). m.p. : 127-128 C. I IR(KBr): 3370, 3180, 1705, 1640, 1615, 1420, 1340, cm 1H NMR ( DMSO-d d : 2. 13 ( 3H, s, -COCH3 ) 2. 3 0 ( 3H, s, -CH3 ) 2.80(4H,s,-CH2-CH2-), 7.22(2H,d,J=5Hz), 7.65(1H,d), 7.60-7.95

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( 2H, br. -CONH2 ) Cald. Found Elemental Analysis for C12H1 5N02 C:70.22, H:7.37, N:6.82 C:69.66, H:7.41, N:6.76. Preparation of 3,4-dihydroxy-N-(3-{2'-methyl-5'carbamoylphenyl)-1-methyl-n-propyl)-B-phenethylamine. hydrochloride(9) 40 A mixture of 1.14 g(0.0056 mol) of the keto compound(34), 0.772 g(0.9 eq.) of dopamine and 20 mg of Pt02 and 250 mg of Pd-C in 31 ml of methanol and 10 ml of acetic acid was hydrogenated for 48 hrs with an initial pressure of 32 psi. After reaction was completed, the mixture was filtered. 5 ml of 20% HCl-methanol was added to the filterate and the resultant solution was concentrated under reduced pressure to give white foam, which was subjected into column chromatography on silica-gel with chloroform-methanol-acetic acid(30:10:0.1) to give 1.45 g of the product. (75.9% yield) m.p. : 165-169 C. I H NMR(CD30D) : l.75(3H,d,-CH-CH3), 1.90-2.40(3H,m), 2.70 (3H,s,-CH3), 2.90-3.30(4H,m), 3.40-3.60(2H,m), 6.95(3H,m), 7.55(1H,d), 7.95(2H,m). Synthesis of 5-(2'-Aminoethyl)carbostyril Derivatives(12,13, ill Preparation of 5-chloromethyl-8-hydroxyguinoline{35) To a 14.6 g(0.10 mol) of 8-hydroxyquinoline(34) in 100 ml two necked round bottom flask were added 16 ml of c-HCl slowly. The reaction pot was cooled in an ice-water bath and

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41 16 ml of 37% formaldehyde was added. Hydrogen chloride gas was passed into the reaction mixture over a period of 3 hrs. The mixture was stirred overnight at room temperature. The yellow crystals which formed were filtered, washed with ether and dried in vacuo to give 20.7 g of product(90.0% yield). m.p. : 281-283 C (ref.7 9 : 283C dee., ref.80 : 250C). Preparation of 5-cyanomethyl-8-hydroxyguinoline(36) To a solution of sodium cyanide(lO.O g, 0.20 mol) in 150 ml of DMSO at 80-90C was slowly added a hot solution of 9.2 g(0.04 mol) of compound(35} in 100 ml of DMSO. The mixture was stirred at 80-90C for 45 min. It was allowed to cool and was carefully acidified with 15 ml of c-HCl in order to decompose excess sodium cyanide. The resulting brownish solution was then poured into 500-700 g of crushed ice and neutralized with 10% aqueous NaOH. The precipitate was filtered, washed well with water, and dried. Crystallization from benzene gave 1.20 g of product(l6.3% yield). m.p. : 179-182 C (ref.80 : 178-180C, ref.8 1 : 300C). IR(nujol mull) : 3350, 2970, 2230(CN), 1510, 1470, 1425 1380, 1280, 1195, 835, 795 cm -1 1H NMR(CDCl 3 + DMSO-d d : 4.20(2H,s,-CH2-CN), 6.95-7.60 (3H,m), 8.35(1H,dd,JAB=9Hz,JAc=2Hz), 8.85(1H,dd,JAn=9Hz, J Ac=2Hz) Preparation of 5-cyanomethyl-8-methoxyguinoline(37) To 0.866 g(l.4 eq.) of a 60% dispersion of sodium hydride in mineral oil was added 2.70 g(l5 mmol) of compound(36} in 25 ml of anhydrous DMF dropwise over 5 min. at room

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42 at room temperature. After the addition was completed, the soluti0n was stirred at 50C for 2 hrs. Next, the solution was cooled to room temperature and 1.16 ml (1.2 eq.) of methyl iodide in 3. o ml of DMF was added dropwise. The resulting mixture was stirred at 50 C for 30 min. again, and then, it was poured into 30 ml of ice water. The mixture was extracted with 160 ml of ethyl acetate. After being washed with water, the extracts were dried over anhydrous magnesium sulfate, decolorized with charcoal and concentrated to give a deep orange colored semisolid. This material was washed with nhexane to give 0.79 g of solid product(27.2% yield). m.p. : 152.5-155 C (ref.7 1 153-155 C} IR(nujol mull) : 2940, 2860, 2260(CN), 1620,-1580, 1510, -1 1470, 1380, 1270, 1110, 800 cm. 1H NMR(CDCl 3 ) : 4.05(3H,s,-OCH3), 4.20(2H,s,-CHz-}, 7.10 (1H,d,J=9Hz), 7.40-7.65(2H,m), 8.40(1H,dd,JAn=9Hz,JAc=2Hz), 8.85-9.05(1H,m}. Preparation of 8-hydroxy-5-methylguinoline(42) To a suspension of 10.0 g(0.0434 mol) of compound(35) in 200 ml of ethyl alcohol was added 1.0 g of 10% Pd-C. The resulting mixture was hydrogenated at room temperature and an initial pressure of 50 psi for 3 hrs. After the reaction was completed, the mixture was filtered and washed with water. The combined aqueous alcoholic solution was concentrated in vacuo to give a yellow residue, which was dissolved in 100 ml of water then neutralized with dilute ammonium hydroxide to give a yellowish precipitate. The resulting precipitate was

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4 3 collected, washed with water and dried to give the desired product(6.42 g, 93.0% yield). Recrystallization from ethyl acetate gave the pure compound. m.p. : 123-124.5 C (ref.80 : 122-123C). IR(KBr) : 3220(br.), 1580, 1470, 1410, 1270, 1180, 960, 7 80 cm -1 1H NMR(CDCl 3 ) : 2.55(3H,s,-CH3), 7.05-7.50(3H,m), 8.27 (lH,dd,JAl3=6Hz,JA c=l.5Hz), 8.80(1H,dd,J=3Hz,J=1Hz). Preparation of 8-hydroxy-5-methylguinoline-N-oxide(43) A solution of 12.5 g(0.0786 mol) of compound(42) and 29.5 g(0.170 mol) of 80% m-chloroperbenzoic acid in 270 ml of chloroform was stirred at room temperature for 8 hrs. The resulting solution was filtered through 5-cm layer of neutral alumina(Activity grade I). Concentration of the filtrate gave a crude product, which was purified by column chromatography on silica-gel with chloroform as an eluent to give 9.78 g of pure product(70.1% yield). m.p. : 131-132.5C. IR(KBr) : 3430(br.), 1605, 1530, 1480, 1430, 1395, 1310, ] 1270, 1135, 1050, 880, 820, 735 cm. 1H NMR(CDCl 3 ) : 2.53(3H,s,-CH3), 7.02(1H,d,J=6Hz), 7.25-7.50(2H,m), 7.95(1H,d,J=6Hz), 8.40(1H,d,J=4Hz). Preparation of 8-acethoxy-5-methylcarbostyril(44) A mixture of 9.78 g(0.0558 mol) of compound(43) and 60 ml of acetic anhydride was heated between 80-90 C for 5 hrs. The white solid which had formed was filtered and filtrate was poured into 100 ml of water, neutralized with dilute ammonium

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4 4 hydroxide to give another white solid, which was subsquently collected. The combined solid was washed with water again and dried to give 8.50 g of product, which was used in the next reaction without further purification(70.2% yield). IR(KBr) : 2950(br.), 1750, 1645, 1490, 1435, 1360, 1180, 1145, 1040, 885, 835 cm-1 1H NMR ( DMS0-d6 ) : 2. 3 7 ( 3H, s, -COCH3 ) 2. 52 ( 3H, s, -CH3 ) 6.60(1H,d,J=6Hz), 7.17(1H,d,J=6Hz), 7.65(1H,d,J=6Hz), 8.08(1H, d,J=6Hz). Preparation of 8-hydroxy-5-methylcarbostyril(45) A suspension of 8.50 g(0.0392 mol) of compound(44) in 100 ml of c-HCl was heated at steam bath temperature for 7 hrs. Following dilution with 100 ml of water and cooling in ice-bath, a pink colored precipitate was collected, washed with water and dried to give 4.05 g of product(59.2% yield). Recrystallization from ethanol gave an analytically purre product. IR(KBr) 3100(br.), 1630, 1610, 1545, 1390, 1285 1160, 1060, 84 Ocm-1 1H NMR(DMS0-d6 ) : 2.40(3H,s,-CH:d, 6.57(1H,d,J=6Hz), 6.90(2H,s), 8.00(1H,d,J=6Hz). Preparation of 8-methoxy-5-methylcarbostyril(46) To a stirred suspension of 4.00 g(0.0228 mol) of compound(45) in a solution of 3.76 g(l.O eq.) of K2co3.1/2H20 in 6.5 ml of water and 32 ml of acetone were added in one portion 2.16 ml(l.O eq.) of dimethylsulfate. The resulting mixture was refluxed for 1 hr and then concentrated. The

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4 5 residue was extracted with chloroform. The chloroform layer was washed with water, dried over anhydrous magnesium sulfate, filtered and concentrated to give 3. 89 g of the desired product ( 89. 7 % yield) Recrystallization from chloroform-hexane mixture gave an analytically pure product. m.p. : 188-191 C. 1H NMR(CDCl 3 ) : 2.50(3H,s,-CH3), 3.93(3H,s,-OCH3), 6.70 (1H,d,J=6Hz), 6.90(2H,m), 7.92(1H,d,J=6Hz) Elemental Analysis for C11H11N02 Cald. C:69.82, H:5.86, N:7.40 Found C:69.58, H:5.91, N:7.34. Preparation of 5-formyl-8-methoxycarbostyril(49) To a cold solution of 2.55 g(0.0134 mol) of 8-methoxy-5-methylcarbostyril(46) in 15 ml of acetic anhydride were slowly added 3.0 ml of concentrated sulfuric acid. After the mixture was cooled in an ice-salt bath(about -5 -0 C), a solution of 3.60 g(0.036 mol) of chromium trioxide in 25 ml of acetic anhydride was added, with stirring at such a rate that did not cause the temperature to exceed l0 C and stirring was continued for 3 hrs at 5-10 C in an ice-water bath. The contents of the flask were poured into 200 ml of ice-water, which was allowed to stand overnight. The resulting dark solution was extracted with 200 ml of chloroform and organic layer was washed with water, saturated sodium bicarbonate and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a crude product, which was purified by

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46 column chromatography on silica gel to give 320 mg of product(l2.0% yield). m.p. : 227.5-229 C. 1H NMR(DMSO-d d : 4.10(3H,s,-OCH3), 6,85(1H,d,J=6Hz), 7.10(1H,d,J=5Hz), 7.60(1H,d,J=5Hz), 9.15(1H,d,J=6Hz), 10.50 (lH,s,CHO). IR(nujol mull) : 3170(br.), 1690, 1670, 1605, 1555, 1290, 1 1270, 1215, 1090, 1070, 840 cm. Cald. Found Elemental analysis for C11H9N03 C:65.03, H:4.46, N:6.98 C:64.14, H:4.50, N:6.72. Preparation of 8-hydroxyguinoline-N-oxide(51) To a solution of 11.14 g of 80% m-chloroperbenzoic acid in 80 ml of chloroformwas added 5.00 g(0.0344 mol) of 8-hydroxyquinoline(34 ) in 40 ml of chloroform at room temperature. The resulting solution was stirred for 16 hrs at room temperature and filtered through 5-cm layer of neutral alumina(activity grade I). The filtrate was evaporated to give a yellow colored crude product which was recrystallized from ethyl actate-hexane mixture to give 2. 72 g of the pure product(49.1% yield). m.p. : 141-143 C. IR(KBr) : 3410(br.), 1600, 1530, 1460, 1405, 1280, 1155, 1 O 5 O 8 2 o cm-1 1H NMR ( CDCl1) 8.24(1H,d,J=4Hz). 7.05-7.50(4H,m), 7.78(1H,d,J=5.5Hz), Preparation of 8-acetoxycarbostyril(52)

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47 A mixture of 1.15 g(7.14 mmol) of compound(51) and 15 ml of acetic anhydride was heated in a steam bath for 5 hrs. The precipitate which had formed was filtered, washed with water and dried. The brown filtrate was neutralized with dilute ammonium hydroxide to yield another precipitate, which was also collected by filtration, washed with water and dried. The combined white precipitates were used in the next reaction without further purification. m.p. : 252-255"C (ref.~ : 251-154"C, ref.~ : 250"C). IR(KBr) -1 : 1 7 6 0 16 6 0 16 4 0 16 0 0 12 7 0 118 5 cm 1H NMR(DMS0-d6): 2.40(3H,s,-COCH3), 6.60(1H,d,J=6Hz), 7.10-7.40(2H,m), 7.65(1H,dd,JAil=5Hz,JAc=l.5Hz), 8.00(lH,d, J=6Hz) Preparation of 8-hydroxycarbostyril(53) A suspension of 22.15 g(0.108 mol) of compound(52) in 2 00 ml of c-HCl was heated in a steam bath for 5 hrs. Following dilution with 200 ml of water and cooling in an ice bath, the tan solid which formed was collected(l4.8 g, 84.7% yield). Recrystallization from EtOH gave an analytically pure sample as light tan needles. m.p. : 289-295" C (ref.~ : 297-299" C, ref.~ : 287-288"C). IR(KBr) : 3100(br.), 1635, 1600, 1545, 1285, 830 cm -1 1H NMR(DMS0-d6 ) : 6.58(1H,d,J=6Hz), 7 .02-7.30(3H,m), 7.93 ( lH, d, J=6Hz) Preparation of 8-methoxycarbostyril(54) To a solution of 8.0 g(0.050 mol) of compound(53) in a solution of 4.3 g(0.027 mol) of potassium carbonate, 12.5 ml

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4 8 of water and 62.5 ml of acetone were added dropwise 6.3 g(0.05 mol) of dimethyl sulfate during stirring and refluxing. Refluxing was continued for another 1 hr. The reaction mixture was concentrated and extracted with chloroform. The chloroform layer was evaporated to dryness and the residue was recrystallized from ethyl acetate-hexane mixture to give 7.45 g of pure product(85.1% yield). 7 0 m.p. : 108-109.5 C (ref. : 108-109C}. IR(KBr) : 1645 1600, 1465, 1270, 1090, 830 -1 cm. 1H NMR(CDC13 ) : 3.97(3H,s,-OCH3), 6.65(1H,d,J=6Hz), 6.95-7.20(3H,m), 7.73(1H,d,J=6Hz). Preparation of 5-chloroacethyl-8-methoxycarbostyril(55) To a suspension of 1.61 g(0.01 mol) of compound(54) and 2.82 ml(2.5 eq. ) of chloroacethyl chloride in 25 ml of carbon disulfide were added 4.40 g(3.3 eq.) of aluminum chloride in small portions while stirring and cooling in an ice-water bath. The reaction mixture was refluxed for 2 hrs. After the reaction was completed, the reaction mixture was cooled and the carbon disulfide layer was decanted. The residue was mixed with chipped ice to give a crude solid product which was collected and washed with water and methanol to give the desired product(l.3 6 g, 54.0% yield). The product was used in the next reaction without further purification. 1H NMR(CDC13 ) : 4.01(3H,s,-OCH3), 5.20(2H,s,-CH2-Cl), 6.70(1H,d,J=6Hz}, 7.08(1H,d,J=5Hz}, 7.90(1H,d,J=5Hz), 8.55(1H, d,J=6Hz). Preparation of 5-(2'-chloroethyl)-8-hydroxycarbostyril(57)

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4 9 To a suspension of 100 mg(0.42 mmol) of compound(55) in 4 ml of trifluoroacetic acid were added 2.0 ml of triethylsilane dropwise via a syringe under nitrogen atmosphere. The resulting suspension was stirred overnight to give a yellow solution, which was diluted with water to produce a white precipitate. The precipitate was collected and dried to give 62 mg of product(65.8% yield). 1H NMR(DMS0-d6 ) : 3.00(2H,m,-CH2-), 3.55(2H,t,-CH2-Cl), 6.42(1H,d,J=6Hz), 6.80(2H,s,aromatic ti), 7.90(1H,d,J=6Hz). Preparation of N-(2-(4'-methoxyphenyl)ethyl)-2,2,2-trifluoroacetamide(60) To a cold solution of 36.0 g(0.238 mol) of p-methoxyphenethylamine ( 59) in 360 ml of methylene chloride under nitrogen atmosphere was added dropwise with stirring a solution of 100 g(0.476 mol) of trifluoroacetic anhydride in 50 ml of methylene chloride. After the mixture was stirred for 1.5 hr at room temperature, the volatile material was removed in vacuo, toluene was added and removed, and residue was crystallized from 600 ml of mixture of ethyl ether-petroleum ether(l:l) to give a 48.70 g of white crystal(82.8% yield). m p : 8 4 8 5 C ( ref. 85 : 8 4 c) 1H NMR(CDC13 ) : 2.80(2H,m,-CH2-), 3.60(2H,m,-CH2-NH-), 3.80(3H,s,-OCH3), 6.95(2H,d,JAD=6Hz), 7.20(2H,d,JAD=6Hz). Preparation of N-(2-(4'-methoxy-3'-nitrophenyl)ethyl)-2,2,2-trifluoroacetamide(61) To a cold solution of 15.0 g(0.0605 mol) of compound(60) in 127 ml of trifluoroacetic acid under nitrogen atmosphere was added dropwise while stirring 3.9 ml (1.2 eq.) of

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5 0 concentrated nitric acid. After the mixture was stirred for 3 hrs at room temperature, the solvents were removed and the residue was dissolved in 150 ml of ethyl acetate, which was successively washed with 5% HCl solution, dilute sodium bicarbonate solution and brine and dried over anhydrous magnesium sulfate-activated carbon. The mixture was filtered and the filtrate was concentrated. The resulting crude reddish solid was crystallized from ethyl acetate-hexane(l:1) to give 15.0 g of product(85.2% yield). m.p. : 92-93C(ref.85 : 92.5-93C). 2.93(2H,t,-CH2-), 3.62(2H,m,-CH2-NH-), 7.15(1H,d,JAil=5Hz), 7.47(1H,dd,JAil=5Hz,JAc=l.5Hz), 7.77(1H,d, J Ac=l. 5Hz) Preparation of N-(2-(3'-amino-4'-methoxyphenyl)ethyl)-2,2,2-trifluoroacetamide(62) A solution of 12.10 g(0.0414 mol) of compound(61), 1.20 g of 10% Pd-C in 160 ml of ethanol-ethyl acetate(l:1) was hydrogenated at room temperature and an initial pressure of 50 psi for 1 hr. After reaction completion, the reaction mixture was filtered and concentrated to give a crude product, which was crystallized from ethyl ether-hexane mixture to give 10.27 g of product(94.5% yield). m.p. : 87-88C(ref.85 : 87-88C). 1H NMR(CDC13 ) : 2.70(2H,t,-CH2-), 3.47(2H,m,-CH2-NH-), 3.83(5H,s,-OCH3 & -NH2), 6.50-6.85(3H,m,aromatic H), 7.lO(lH, br.,-NH-). Preparation of N-(2-(3'-(N-acetoacetyl)amino-4'methoxyphenyl)ethyl)-2,2,2-trifluoroacetamide(63)

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51 The following reaction was modified from the reported method(ref 13). To a solution fo 5.82 g(0.0222 mol) of amine(62) in 20 ml of anhydrous THF were added 2.05 ml(l.2 eq.) of diketene dropwise via a syringe under nitrogen atmosphere. The reaction mixture was refluxed for 3.5 hrs and solvent was removed in vacuo. The resulting reddish thick oil was subjected to column chromatography on silica-gel with ethyl acetate-hexane(l:l) to give 6.12 g of product(79.6% yield). m.p. : 104-105 C. 1H NMR(CDC13 ) : 2.32(3H,s,-COCH3), 2.75(2H,t,-CH2-), 3.60 (2H,-COCH2CO-), 3.50-3.70(2H,m,-CH2-NH-), 3.90(3H,s,-OCH3), 6.75(1H,br.,-NH-CO-), 6.90(2H,s,aromatic tl), 8.23(1H,s, aromatic tl), 9.27(1H,br.,-NH-CO-). IR(nujol mull) : 3290(amide), 1720(C=O), 1675(C=O), 1600, 1465, 1380, 1185, 1145, 1035 cm-1 Cald. Found C:52.20, H:4.95, N:8.09 C:51.98, H:4.96, N:8.05. Preparation of 5-(2'-trifluoroadetamido)ethyl-8-methoxy-4-methylcarbostyril(64) A solution of 720 mg(2.08 mmol) of compound(63) in 25 ml of concentrated sulfuric acid was heated between 80 to 90 C overnight. After the reaction mixture was cooled to room temperature, it was carefully poured into crushed ice. The resulting precipitate was filtered, washed with cold water and dried to give 292 mg of a grey colored compound which was

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5 2 insoluble in most solvents except organic acids such as trifluoroacetic acid(45% yield). m.p.: 224-227 C. lH NMR(TFA) : 2.90(3H,s,-CH3), 3.25-3.80(4H,m,-CH2-CH2-), 3 .95(3H,s,-OCH3), 7 1 7 ( lH, s =CH -) 7.15-7.45(2H,m,aromatic ti), 7.70(1H,br.,-NH-CO-). IR(nujol mull) : 3245(br., amide), 1715(C=O), 1645(C=O), 1605, 1545, 1210, 865 Mass spec. ( 7 OeV) 1 cm Preparation of 5-(2'-aminoethyl}-8-methoxy-4-methylcarbostyril hydrogen chloride(65) A solution of 10.50 g(0.0320 mol) of compound(64) in 31.6 ml of ethanol and 72 ml of water containing 36 ml of concentrated HCl was refluxed for 10 hrs under nitrogen atmosphere. After the solution was cooled, the resultant white precipitate was collected and the filtrate was concentrated to give another brownish solid. This solid was dissolved in a small amount of methanol and diluted with ether to give a white precipitate. The combined white material was reprecipitated from MeOH-ether solution to give 8.20 g of HCl salt(95.43% yield). The free base of compound(65) was obtained as follows: 3.00 g(0.0112 mol) of compound(65) were dissolved in a small amount of water. The resultant solution was made basic with dilute NH40H, and then extracted with 200 ml of methylene chloride. The methylene chloride layer was dried over anhydrous magnesium sulfate, filtered and concentrated to qive 2.34 g of

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53 a white product(90.2% yield). m.p. (HCl salt) 235-245C(dec.). m.p. (Free base) 159-160c. 1 H NMR(DMS0-d 6 ;HC1 salt) : 2.70(3H,s,-CH3), 2.90-3.15(2H, br.-CHz-), 3.20-3.55(2H,br.-CHz-), 3.92(3H,s,-OCH3), 6.53(1H, s), 7.03-7.27(2H,m,aromatic fl), 8.40(3H,br.-NH3+). Elementary Analysis for C 1 3 H 1 6 N202 (free base) Cald. C:67.22, H:6.94, N:12.06 Found C:67.07, H:7.00, N:12.00. Mass spec. (70eV,free base) Preparation of 3-(3'-oxobutyl)benzamide(66) 38.2 g of ethyl 2-(3'-cyanophenylmethyl)-3-oxobutyrate, which was prepared by heating m-cyanobenzyl bromide with acetoacetate for 2 hrs under reflux in 500 ml of concentrated hydrochloric acid. To the reaction mixture were added 500 ml of water, followed by extraction with ethyl acetate(J x 500 ml). The organic phase was washed with water and dried over anhydrous sodium sulfate after which the solvent was removed. The resultant 3-(3'-oxobutyl)benzoic acid was mixed with 500 ml of benzene and 17 ml of thionyl chloride and heated for 2 hrs under reflux. The reaction solution was poured into an ice-cooled concentrated aqueous ammonia solution and the resulting amide extracted three times with 500 ml portions of ethyl acetate. The organic layer was washed with brine and dried over anhydrous sodium sulfate. The solvent was removed and the residue was recrystallized from ethyl acetate-hexane mixture to give 23.1 g of the product(59.8% yield).

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54 m.p. : 122-125C. 1H NMR(CDCl3) : 2.10(3H,s,-COCH3), 2.80(4H,s,-CHz-CHz-), 7.32(2H,d,aromatic fl), 7.75(2H,m,aromatic fl), 7.90(2H,br. ,CONH 2 ) Preparation of N-(2-(3'-(N-cinnamoyl)amino-4'methoxyphenyl)ethyl)-2,2,2-trifluoroacetamide(67) To an ice-cooled solution of 2.62 g(0.010 mol) of compound(62) in 20 ml of benzene and 20 ml of anhydrous THF containing 1.5 ml of pyridine were added 1.83 g(l.1 eq.) of cinnamoyl chloride in 20 ml of anhydrous THF dropwise while stirring. After stirring in an ice-cooled water bath for 1 hr and subsquently at room temperature overnight, the reaction mixture was filtered to remove the formed white precipitate which was pyridine salt, and then it was concentrated. The resulting mixture was dissolved in 100 ml of ethyl acetate and organic layer was washed with 5% HCl solution, saturated sodium bicarbonate solution and brine. The dried organic layer was concentrated to give 3.47 g of product(88.5% yield). m.p. : l39-140 C. 1H NMR(CDCl :d: 2.83(2H,t,-OCH2-), 3.65(2H,m,-CH2-NH), 3.90(3H,s,-OCH3), 6.68(1H,d,J=10Hz,-OCH=), 6.95(1H,s,aromatic H), 7.00(2H,br.,-NH-), 7.38-7.70(5H,m,phenyl H), 7.78(1H,d, J=lOHz, =CH-NH-) 8. 12 ( lH, s, aromatic fl) 8. 4 5 ( lH, s, aromatic fl) IR(nujol mull) : 3380, 3200-3060(br.), 1720, 1665, 1630, 1595, 1545, 1490, 1355, 1265, 1225, 1195, 1180, 1145, 1030, 805 cm-1

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Cald. Found C:61.22, H:4.88, N:7.14 C:61.17, H:4.91, N:7.11. Synthetic attempts of ring-closure of compound(67) 55 To a solution of 1.40 g(3.57 mmol) of compound(67) in 30 ml of chlorobenzene were added portionwise under stirring 2.38 g(5.0 eq.) of aluminum chloride. The reaction mixture was heated 80 to 90 C for 3 hrs. The resulting dark solution was carefully poured into ice and was extracted with ethyl acetate(2 x 100ml). The combined organic layers were washed with 5% HCl solution, saturated sodium bicarbonate solution and brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a reddish oil, which was identified as a mixture of products. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-methoxy-4-methylcarbostyril(l2) Reductive Amination Method : To a suspension of 1.34 g(0.0050 mol) of HCl salt of compound(65) and 0.95 g(0.0050 mol) of keto compound(66) in 35 ml of methanol and 25 ml of ethanol were added 250 mg of sodium cyanoborohydride, portionwise at room temperature. The resulting solution was stirred for 24 hrs at which time another 150 mg of sodium cyanoborohydride was added and stirred for additional 24 hrs. After the reaction was completed, the solution was concentrated in vacuo to give a white solid which was subjected to chromatography on silica-gel with methanolchloroform(3:l) as an eluent to give 450 mg of product and 430

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5 6 mg of starting material(22.0% yield). The acetate salt as a white powder was obtained from an acetic acid-methanol solution, followed by dilution with ether. Hydrogenation Method: A mixture of 800 mg(0.00343 mol) of the free base of compound(65), 0.72 g(l.l eq.) of the keto compound(66), 300 mg of 10% Pd-C and 20 mg of Pt02 in 30 ml of MeOH and 10 ml of acetic acid was hydrogenated for 24 hrs at an initial pressure of 32 psi. After the reaction was completed as indicated by TLC, the mixture was filtered and concentrated to give a residue which was subjected to column chromatography on silica-gel with chloroform-methanol to yield a white solid following evaporation of the solvents .. The acetate salt of the product was obtained from an acetic acidmethanol solution, followed by dilution with ether. m.p. (AcOH salt) : 141-145C. 1H NMR(AcOH salt in DMS0-d 6 ) : l.35(3H,d,-CH3), 1.60-2.20 (3H,br.-CH-CHz -CH z-), 2.00(3H,s,-CH3 COOH), 2.80(5H,s + br.-CH3 &-CH2-CH 2-), 3.10-3.35(2H,br.), 3.50-3.75(2H,br.), 3.98(3H,s,OCH3), 6.63(1H,s,), 7.20(2H,m), 7.57(2H,m), 7.95(2H,m). Cald. Found U.V. (MeOH) : 258 nm(max.). Elementary Analysis for C26H33N 3 05 (AcOH salt) C:66.79, H:7.11, N:8.98 C:66.53, H:7.18, N:8.90. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-hydroxy-4-methylcarbostyril(l3) A solution of 200 mg of compound(l2) in 7 ml of 48% HBr was refluxed for 20 hrs. After the solution was cooled to room

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5 7 temperature, a white precipitate was formed which was filtered while the brownish filtrate was stored in a refrigerator. The filtrate gave a pale brownish solid, which was filtered, washed with acetone and dried. m.p. : 153-155C. 1HNMR(CDC13+DMS0-d6): l.50(3H,d,-CH3), l.80-2.20(3H,br.), 2.80(3H,s,-CH3), 2.75-2.85(2H,br.), 3.10-3.70(4H,br.), 6.67 (lH,s,), 7.20(2H,m,carbostyril ring fl), 7.55(2H,m,o-Phenyl H to amide), 7.97(2H,m,phenyl fl). Cald. Found C: 4 O O 2 H: 5 2 5, N: 6. O 8 Br: 3 4 7 2 c: 4 o. 6 o, H: 5. 2 6, N: 6. 16, Br: 3 4 9 8. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-hydroxy-4-methylcarbostyril(lJ) I) Generation of the free amine of compound(65) : 1.00 g of acetate salt of compound(65) was dissolved in minimum amount of water and made basic with dilute ammonium hydroxide The resulting basic aqueous solution was extracted with chloroform(2 X 50ml). The chloroform layers were washed with 10ml of water, dried over anhydrous magnesium sulfate. filtered and concentrated to give 570 mg of a white solid. II) Reaction with BBr3 : A solution of 570 mg(0.00140 mol) of free amine(65) in 50 ml of dry dichloromethane was added to 30 ml of 1.0 M BBr3 solution dropwise via a syringe in an icewater bath under nitrogen atmosphere. After stirring the resulting suspension overnight at room temperature, it was carefully quenched with methanol and concentrated in vacuo to

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5 8 give a yellowish foam, which was directly subjected into column chromatography on silica-gel with chloroform-methanol(l:3) as an eluent to yield a pale yellowish product. This product was purified by reprecipitation from a methanolether mixture to obtain 263 mg of pure product. (48.1% yield). 1H NMR(CD3 0D) : l.50(3H,d,-CH-CH3), l.80-2.20(3H,br.-CHCH2-), 2.80(3H,s,-CH3), 2.75-2.85(2H,br,-CHz-NH-), 3.10-3.70 (4H,br.,2 of -CH 2-ring), 6.67(1H,s), 7.20(2H,m), 7.55(2H,m), 7.97(2H,m). Cald. Found Elemental Analysis for C23H27Np3 1. 6HBr. 1. 3Hz0 C:50.48, H:5.72, N:7.69, Br:23.40 C:50.43, H:5.55, N:7.52, Br:23.25. Preparation of 5-(2'-(N-(l-methyl-3-(3'-carbamylphenyl)-npropyl))aminoethyl)-8-methoxy-4-methyl-3,4-dihydrocarbostyril(14) A solution of 400 mg of the free base form of compound ( 12) in 100 ml of methanol and 4 00 mg of 10% Pd-C was hydrogenated at an initial pressure of 58 psi for 3 days. After the solution was filtered, the filtrate was concentrated to give a white solid which was dissolved in chloroform and filtered. The filtrate was diluted with ether to give a pale yellow precipitate. 1375 IR(nujol mull) 1 cm 3170-3450(br.-CONH2), 1660(C=O), 1460, l.15(3H,d,-CH3), l.45(3H,d,-CH3), l.75-2.40(4H,br.), 2.60-3.45(8H,br.), 3.85(3H,s,-OCH3), 6.90(2H,m), 7.43(2H,m), 7.72(2H,m).

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Cald. : C:63.39, H:6.84, N:9.07, Cl:10.32 Found C:63.39, H:7.17, N:9.12, Cl:10.14. 59 Synthesis of (2'-Arninoethyl)-1-hydroxy-2-pyridone Analogues (15,16) Preparation of 6-hydroxypyridine-3-ethylcarboxylate(71) A suspension of 5.00 g(0.0359 mol) of 6-Hydroxynicotinic acid(69) and 7 ml of c-H2S04 in 60 ml of absolute ethanol was refluxed for 12 hrs. After the reaction mixture became homogeneous, it was concentrated and then diluted with 50% of ammonia solution. The resultant white precipitate was filtered and dried to give 2. 70 g of product. The filtrate was extracted with 200 ml of chloroform, the chloroform layer was dried over anhydrous magnesium sulfate filtered and concentrated to give 1.97 g of product. Recrystallization from ethyl acetate gave 4.67g of a white product(77.9% yield). m.p. : 151-152C(ref.~; 150C). 1H NMR(CDC13 ) : 1.40(3H,t,-CH3), 4.37(2H,m,-OCH2-), 6.65 (1H,d,JAB=7Hz,5-py-H), 8.10(1H,dd,JAB=7Hz,JAc=l.5Hz,4-py-H), 8.30(1H,d,J8c=l.5Hz,2-py-H). Preparation of 2-methoxy-5-carbethoxy pyridine(72) Silver carbonate(5.95 g, 0.02 mol) and 2-hydroxy-5-carbethoxypyridine(3.35 g, 0.02 mol) were reacted with methyl iodide(20.5 g, 0.14 mol) for 24 hrs in 30 ml of benzene at room temperature in the dark. The reaction mixture was filtered and the filtrate was concentrated in vacuo to give a sticky oil which was directly subjected to column

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60 chromatogrphy on silica gel with ethyl acetate-hexane(l:4 ) a s an eluent to yield 2.72 g of the product(75.1% yield). IR(neat) : 3050, 1710(C=O), 1600, 1490, 1370, 1260, 1110, 1 1020, 840, 780 cm. l.45(3H,t,-CH2-CH3), 4.00(3H,s,-OCH3), 4.45(2H,m,-OCH2-), 6.80(1H,d,JAB=6Hz,3-py-H), 8.25(1H,dd, JAB=6Hz,JAc=l.5Hz,4-py-H), 8.87(1H,d,JAc=l.5Hz,6-py-H). Preparation of 2-methoxy-5-hydroxymethylpyridine(73) To a suspension of 0.85 g of LiAlH4 in 3 ml of anhydrous THF were added 1.38 g (0.00762 mol) of compound(72) in 7 ml of anhydrous THF dropwise in an ice-water bath under nitrogen atmosphere. The reaction mixture was stirred for 1 hr at room temperature and quenched with 0.85 ml of water, 0.85 ml of 15% NaOH solution and 3 x 0.85 ml of water. After the resulting solid was filtered and washed with ether(2 x 15 ml), the combined ether fractions were concentrated, the residue was redissolved in ethyl acetate, washed with brine and dried over anhydrous magnesium sulfate. This suspension was filtered and concentrated to give a pale yellowish liquid which was purified by silica-gel column chromatography with an ethy l acetate-hexane mixture to give 870 mg of the pure produc t (82.1% yield). IR(neat) : 3360(-0H), 2960, 1610, 1570, 1490, 1390, 1285, 1210, 1150, 1015 cm -1 1H NMR(CDCl3 ) : 3.90(3H,s,-OCH3), 4.35(1H,br. ,-OH), 4.55 (2H,s,-CH2-), 6.72(1H,d,3-py-H), 7.67(1H,dd,4-py-H), 8.07(1H, d,6-py-H).

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61 Preparation of 2-rnethoxy-5-chlorornethylpyridine(74 ) To a solution of 780 mg(0.00561 mol) of cornpound(73) in 5 ml of chloroform were added 0.80 ml(2.0 eq.) of thiony l chloride dropwise at room temperature under nitrogen atmosphere. After the resulting mixture was stirred for 4 day s at room temperature, it was concentrated to give a sticky yellowish oil, which was dissolved in 20 ml of water, made basic with dilute ammonia solution and then extracted with chloroform ( 2 x 3 0 ml) The combined chloroform layer was washed with brine, dried over anhydrous rnagnes i urn sulfate. filtered and concentrated to give a yellow liquid which was used in the next reaction without further purification. IR(neat) 2960, 1605, 1570, 1490, 1385, 1285, 1255, 1120, 1020, 825, 750 crn1 I H NMR(CDCl 3 ) : 3.93(3H,s,-OCH3), 4.55(2H,s,-CH2-Cl), 6.80 (lH,d,3-py-H), 7.68(1H,dd,4-py-H), 8.20(1H,d,6-py-H). Preparation of 2-methoxy-5-cyanornethylpyridine(75) Method 1: The mixture of 760 mg(0.00501 mol) of compound (74), a catalytic amount of sodium iodide and 0.50 g of sodium cyanide in 8 ml of methanol and 1.5 ml of water were refluxed for 1.5 hr. The resulting brownish solution was cooled and concentrated. The residue was dissolved in 30 ml of water and extracted with chloroform(2 x 20 ml). The combined chloroform layers were washed with water and brine, dried over anhydrous magnesium sulfate filtered and concentrated to give a yellowish oil, which was subjected to column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent to

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62 give a white solid after evaporation of the solvents. An analytically pure product was obtained from recrystallization from ether-petroleum ether mixture(400 mg, 50.5% yield). Method 2: A mixture of 440 mg of compound(74), an excess of sodium cyanide and a catalytic amount of sodium iodide in 15 ml of dry acetone were stirred for 2 days under nitrogen atmosphere. After the reaction was completed as indicated by TLC, the reaction mixture was concentrated and redissolved in chloroform. The chloroform layer was washed with water and brine, dried and filtered. The filtrate was concentrated to give a reddish residue, which was purified by silica-gel column chromatography with ethyl acetate-hexane(2:3) to give 220 mg of product(78.5% yield). m.p. : 53-54C. IR(nujol mull) 2250(-CN) I cm. 1H NMR(CDC13 ) : 3.67(2H,s,-CH2-CN), 3.95(3H,s,-OCH3), 6.80 (lH,d,3-py-H), 7.60(1H,dd,4-py-H), 8.17(1H,d,6-py-H). Cald. Found Elemental Analysis for C8H8N20 C:64.85, H:5.44, N:18.90 C:64.92, H:5.48, N:18.85. Preparation of 2-methoxy-5-(2'-aminoethyl)pyridine(76) To a solution of 220 mg(l.39 mmol) of compound(75) in 8 ml of anhydrous THF in an ice bath were added 7.0 ml(5.0 eq.) of 1.0 M BH3-THF complex dropwise by syringe under nitrogen atmosphere. After reaction mixture stirred for 24 hrs, it was quenched with methanol and concentrated in vacuo to give a residue, which was subsquently dissolved in 30 ml of 5 % HCl

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63 solution. The acidic aqueous layer was washed with ethyl acetate, and then made basic with 10% NaOH solution and finally extracted with chloroform(2 x 30 ml). The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give the yellow oil, which was used directly in the next reaction. 1H NMR(CDCl 3 ) : l.23(2H,s,-NH2), 2.60-3.00(4H,m.-CH2 -CH 2 ), 6.70(1H,d,3-py-H), 7.40(1H,dd,4-py-H), 8.03(1H,d,6-py-H). Preparation of 2-methoxy-5-(2'-acetaminoethyl)pyridine(77) A mixture of crude compound(76), 0.5 ml of acetic anhydride and a catalytic amount of pyridine in 5 ml of dichloromethane was stirred for 40 min. at room temperature under nitrogen atmosphere. The reaction mixture was concentrated in vacuo and stripped with toluene once. The resulting oil was dissolved in 10 ml of water and made basic with 10% NaOH solution, and then extracted with chloroform(2 x 3 O ml) The combined chloroform layers were washed with brine, dried, filtered and concentrated to give a crude product, which was purified by column chromatography on silica-gel with ethyl acetate to give a white solid. (151 mg, 52.7% yield from compound(74)). m.p. : 54.5-55.5 C. 1H NMR(CDC13 ) : l.93(3H,s,-COCH3), 2.70(2H,t,-CH2-), 3.45 (2H,m,-.Q:h-NH-), 3.90(3H,s,-OCH3), 6.20(1H,br.,-NH-), 6.78(1H, d,3-py-H), 7.45(1H,dd,4-py-H), 7.97(1H,d,6-py-H). Elemental Analysis for C10H14N 2 0 2 :

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Cald. Found C:61.83, H:7.26, N:14.42 C:61.87, H:7.31, N:14.37. Preparartion of 2-methoxy-5-(2'-acetarninoethyl)pyridine-loxide (78) 64 A solution of 300 mg(0.00154 mol) of compound(77) and 0.47 g(l.5 eq.) of 85% m-chloroperbenzoic acid in 5 ml of dichloromethane was stirred for 24 hrs at room temperature under nitrogen atmosphere. After the reaction was completed as indicated by TLC, the reaction mixture was concentrated in vacuo to give a residue, which was directly subjected to column chromatogrphy on silica-gel with ethyl acetatemethanol (2: 1) as an eluent to give 180 mg of the desired product(55.6% yield). m.p. : 128-129 C. 1H NMR(CDC13): l.93(3H,s,-COCH3), 2.78(2H,t,-CH2-), 3.45 (2H,m,-CH2-NH-), 4.05(3H,s,-OCH3), 6.95(1H,d,3-py-H), 7.30(1H, dd,4-py-H), 7.62(1H,br.,-NH-), 8.15(1H,d,6-py-H). Mass spec. : 2ll(M+). Preparation of 5-(2'-acetaminoethyl)-l-hydroxy-2-pyridone(79 ) To 410 mg(0.00195 mol) of a white solid compound(78) were added 6. o ml of acetyl chloride dropwise under nitrogen atmosphere. The resulting mixture was refluxed for 1 hr and the excess acetyl chloride was evaported under reduced pressure. The resulting sticky yellowish residue was identified as 1-acetoxypyridone from NMR, which was converted to 1-hydroxypyridone without further purification.

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65 Hydrolysis of 1-acetoxypyridone : The yellowish residue was dissolved in 10 ml of water and was stirred overnight and concentrated in vacuo to give a sticky residue. This was stripped with methanol-toluene twice and extracted with hot chloroform. The combined chloroform layers were concentrated to give a white solid, which was crystallized from chloroformether mixture to give the product{l95 mg, 51.2% yield). m.p. : 124.5-125.5 C. NMR(CD30D): l.98{3H,s,-COCH3), 2.70(2H,t,-CH2-), 3.37{2H, m,-CH2-NH-), 6.82{1H,d,3-py-H.), 7.15(1H,dd,4-py-H.), 7.95{1H,d, 6-py-H.). IR(nujol mull) 3320(N-OH), 3100(NH-C=O), 1695(NH-C=O), .] 1580, 1365, 910 cm. Cald. Found C:52.67, H:6.36, N:13.65 C:52.93, H:6.06, N:13.62. Preparation of 5-(2'-aminoethyl)-1-hydroxy-2-pyridone hydrogen chloride(15) A solution of 1.10 g of compound(79) in 8 ml of MeOH-H ~O c-HCl{l:1:2) mixture was refluxed for 10 hrs under nitrogen atmosphere. After the reaction was completed, the solution was concentrated to give a crude solid product, which was recrystallized from ethanol-water mixture to give the desired compound(740 mg, 76.0% yield). m.p. : 242-244 C. IR(nujol mull) : 3150-2700(br,OH & NH3+), 1650(C=O) -1. cm

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6 6 I H NMR(DMS0-d6 ) : 2.65-2.72(2H,m,-CH2-), 2,85(2H,br. ,-CH2 -NH /), 6.50(1H,d,3-py-J:!), 7.37(1H,dd,4-py-J:!), 7.80(1H,d,-6-py-+ J:!), 8.25(3H,br. ,-NH3 ) Cald. Found Mass spec. : 155(M+). C:44.10, H:5.81, N:14.69, Cl:18.59 C:44.19, H:5.82, N:14.64, Cl:18.52. Preparation of 4-(2'-{N-benzoyl)aminoethyl)pyridine(81) To a solution of 12.20 g(0.10 mol) of 4-(2' aminoethyl)pyridine, 15.03 ml(l.1 eq.) of triethylamine in 70 ml of chloroform was added dropwise benzoyl chloride(12.78 ml, 1.1 eq. in 20 ml of chloroform) in an ice-water bath under nitrogen atmosphere. The resulting dark solution was stirred for 1.5 hrs at room temperature and then poured into 100ml of ice-water. The solution was made basic with aqueous ammonia solution and extracted with 200 ml of chloroform. The combined chloroform layers were washed with water, dried over anhydrous magnesium sulfate and concentrated to give a yellow solid, which was recrystallized from acetone to give 15.5 g of white product(70.6% yield). m.p. :119-120C. 1 HNMR(CDCl :d: 2.90(2H,m,-CH2-), 3 .65(2H,m,-CH2-NH-), 7.10 (2H,m,pyridine J:!), 7.42(3H,m,phenyl J:!), 7.35-7.70 (lH,br. ,NH), 7.78(2H,m,phenyl J:!), 8.37(2H,m,pyridine H). Preparation of 4-(2'-(N-benzoyl)aminoethyl)pyridine-1-oxide Dill

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67 Method A: A mixture of 1.10 g(0.0050 mol) of compound (81) and 1.21 g(l.2 eq.) of 80 -85% m-chloroperbenzoic acid in 25 ml chloroform was stirred for 2.5 hrs at room temperature under nitrogen atmosphere. After the reaction was complete, the mixture was concentrated and then chromatographed on silica-gel with ethyl acetate-methanol(3:l) mixture as an eluent. The combined fractions were concentrated to give a white product(76.3% yield). Method B A solution of 1.10 g(0.0050 mol) of compound(81) and 3 ml of 30% hydrogen peroxide in 15 ml of acetic acid was heated to between 70 and 80 C for 24 hrs under nitrogen atmosphere. After the mixture had cooled down, it was concentrated in vacuo and made basic with dilute ammonia. The aqueous solution was extracted with 100 ml of chloroform The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated to give a viscous oil. The oil was subjected to column chromatography on silicagel with ethyl acetate-methanol mixture to give 285 mg of the white product(24.1% yield). m.p. : 139-140C. 1 HNMR(CDCl 3): 2.91(2H,m,-CHz-), 3.63(2H,m,-CHz-NH-), 7.10 (2H,d,J=6.5Hz,pyridine tl), 7.40(3H,m,phenyl tl), 7.80-7.95(4H, m,phenyl H & pyridine tl), 8.23(1H, br.-NH-). Mass spec. (70eV) : 243 (M+l). Synthetic attempts for the rearrangement of N-oxide(82) A solution of 500 mg(2.12 mmol) of N~oxide(82) in 10 ml of acetic anhydride was heated to between 90 and 100c for 40

PAGE 83

68 min. under nitrogen atmosphere. After excess acetic anhydride was removed, the resulting black tarry residue was subjected to column chromatography on silica-gel with ethyl acetatemethanol(3:1) to give yellowish oil, which was identified as 4-(2'-(N-benzoyl)aminoetheneyl)pyridine by NMR data. 1H NMR(CDCl 3 ) : 3.93(1H,dd,JAB=9Hz,JAX=5Hz), 4.50(1H,dd,JAB=9Hz,JAx=5Hz), 5.70(1H,dd,Jsx=7Hz,J.AX=5Hz), 7.30 (2H,m,pyridine H), 7.50(3H,m,phenyl H), 8.15(3H,m,phenyl H), 8.65(2H,m,pyridine H). General synthetic attempts for 4-(2'-(N-benzoyl)aminoethyl)-2-chloropyridine with POC13 A mixture of 540 mg(2.28 mmol) of N-oxide and 3.0 ml of neat phosphorous trichloride(or alternately, dissolved in 1 5 ml of methylene chloride) was refluxed for 4 hrs under nitrogen atmosphere. The resulting yellow solution was poured onto 20 ml of ice-water and made basic with 10% NaOH. The pink colored suspension was extracted with chloroform(2 x 50 ml) and the combined chloroform layers were washed with water, dried over anhydrous magnesium sulfate, f i 1 tered and concentrated to give a yellow oil which was composed of many products. Preparation of 4-carbomethoxypyridine-1-oxide(88) Method 1 isonicotinate(87) A solution of 10.0 g(0.0729 mol) of methyl and 17.3 g ( 1.1 eq.) of 85% m -chloroperbenzoic acid in 150 ml of dichloromethane was stirred overnight at room temperature under nitrogen atmosphere. After the resulting white precipitate was filtered, the filterate

PAGE 84

69 was diluted with 100 ml of chloroform. The organic layer was washed with saturated sodium bicarbonate solution(200 ml) and aqueous layer was re-extracted with chloroform(2 x 50 ml). The combined organic layers were dried over anhydrous magnesium sulfate filtered and concentrated to give 8.94 g of white product(B0.1% yield). Method 2 : A solution of 10.0 g(0.0729 mol) of methyl isonicotinate, 20 ml of 30% hydrogen peroxide and 50 ml of acetic acid were refluxed for 6 hrs under nitrogen atmosphere. The solution was poured into 50 g of ice and made basic with sodium carbonate. The basic aqueous solution was exhaustively extracted with chloroform(3 x 50 ml). The combined chloroform layers were dried, filtered and concentrated to give the product(72.3% yield). m.p. : 122-123C. 1H NMR(CDC13 ) : 3.97(3H,s,-OCH3), 7.97(2H,d,3 & 5-py-!::!), 8.36(2H,d,2 & 6-py-!::!). Preparation of 4-carbomethoxy-2-pyridone(89) A solution of 5.4 g(0.0352 mol) of carbomethoxypyridineN-oxide(88) in 50 ml of acetic anhydride was refluxed for 1 8 hrs under nitrogen atmosphere. After removal of excess acetic anhydride in vacuo, the resulting black tarry residue was taken up in hot methanol, treated with charcoal and filtered, cooled to give a brown solid. This was recrystallized from methanol to give a pale yellowish product(767 mg, 14.3% yield). m.p. 213-215 C (ref.~ 211-213 C).

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7 0 1H NMR(CDC13+CD3 0D) : 3.98(3H,s,-OCH3), 6.98(1H,dd,5-py H), 7.23(1H,s,6-py-H), 7.56(1H,d,3-py-H). Preparation of 4-carbomethoxy-2-ethoxypyridine(90) Silver carbonate(5.76 g, 1.0 eq.), compound(89, 3.20 g, 0.0209 mol) and 3.3 ml(2.0 eq.) of ethyl iodide in 40 ml of benzene were stirred for 48 hrs at ambient temperature under nitrogen atmosphere in the dark. The reaction mixture was filtered and washed with 50 ml of benzene. The filtrate was washed with 10 % sodium bicarbonate solution and brine successively, dried and concentrated to give a crude liquid product, which was purified by column chromatography on silica-gel with ethyl acetate-hexane mixture as an eluent to give 3.14 g of the pure product(82.9% yield). IR(neat) : 3000, 1735(C=O), 1600, 1560, 1100 -) cm 1H NMR(CDCl 3 ) : l.33(3H,t,-CHz -CH 3), 3.90(3H,s,-OCH3), 4.35(2H,m,-OCH2-), 7.27(1H,s,3-py-H), 7.35(1H,dd,5-py-H), 8.22(1H,d,6-py-H). Preparation of 2-ethoxy-4-hydroxymethylpyridine(91) To a suspension of 0.66 g(2.0 eq.) of LiAlH4 in 10 ml of anhydrous THF cooled in an ice bath were added 3.20 g(0.0176 mol) of compound(90) in 15 ml of anhydrous THF dropwise under nitrogen atmosphere. The mixture was stirred for 20 min. and then quenched with O. 66 ml of water, 0. 66 ml of 15% NaOH solution and 3 x 0.66 ml of water successively. The resulting precipitate was filtered and washed with 2 5 ml of ethyl acetate. The combined filtrate was washed with brine, dried,

PAGE 86

71 filtered and concentrated to give 2.61 g of the product(96.1% yield) IR(neat) : 3470(-0H), 1610, 1555, 1145, 805 1 cm. 1H NMR(CDC13): l.37(3H,t,-CH2-CH3), 3.65(1H,br.,-OH), 4.30(2H,m,-OCH2-CH3), 4.65(2H,s,-CH2-0H), 6.70(1H,s,3-py-H), 6.78(1H,dd,5-py-H), 8.00(lH,d,6-py-H). Preparation of 4-chloromethyl-2-ethoxypyridine(92) The mixture of 2.60 g(0.169 mol) of compound(91) and 1.86 ml(l.5 eq.) of thionyl chloride in 30 ml of chloroform was stirred overnight at room temperature under nitrogen atmosphere. After the reaction mixture was concentrated in vacuo, the residue was dissolved in 30 ml of water and made basic with dilute ammonium hydroxide and extracted with chloroform(2 x 50 ml). The chloroform layer was washed with water and brine, dried over anhydrous magnesium sulfate filtered and concentrated to give the desired product(2.83 g, 97.1% yield). IR(neat) -1 : 3020, 1615, 1570, 1170, 1050, 725 cm. 1H NMR ( CDCl 3 ) 1.42(3H,t,-CH2-CH :d, 4.35(2H,m,-OCH2-), 4.48(2H,s,-CH2-Cl), 6.72(1H,s,3-py-H), 6.85(1H,dd,5-py-H), 8.14(1H,d,6-py-H). Preparation of 4-cyanomethyl-2-ethoxypyridine(93) A mixture of 0.53 g(0.00309 mol) of compound(92) and excess amount of potassium cyanide in 4 ml of ethanol and 0.8 ml of water was refluxed for 2 hrs under nitrogen atmosphere. The reaction mixture was cooled, diluted with chloroform and filtered. The filtrate was washed with water and brine, dried,

PAGE 87

7 2 filtered and concentrated to give reddish oil, which was subjected into column chromatography on silica-gel with ethyl acetate-hexane(l:3) as an eluent and 313 mg of product was obtained(64.0% yield). m.p. : 55.5-56C. : 2270(CN) 1 cm. IR(nujol mull) 1H NMR (CDC13 ) l.40(3H,t,-CH2-CH3), 3.72(2H,s,-CH2-CN), 4.35(2H,m,-OCH2-), 6.70(1H,s,3-py-H), 6.87(1H,s,5-py-fi), 8.17 (lH,d,6-py-H). Cald. Found Elemental Analysis for C9H10N20 C:66.64, H:6.21, N:17.27 C:66.53, H:6.22, N:17.24. Mass spec. : 163 (M+ ) Preparation of 4-(2'-aminoethyl)-2-ethoxypyridine(94) To a solution of 440 mg(0.00271 mol) of compound(93) in 3.0 ml of anhydrous THF were added 6.0 ml(2.2 eq.) of 1.0 M diborane solution via a syringe at ice-water temperature under nitrogen atmosphere. The suspension was stirred overnight and resulting solution was quenched with methanol, and then concentrated to give a reddish oil. The oil was dissolved in 30 ml of 5% HCl solution and washed with 20 ml of ethyl acetate. The acidic aqueous layer was basified with 10% NaOH solution and extracted with chloroform The combined chloroform layer was washed with brine, dried over anhydrous magnesium sulfate filtered and concentrated to give an orange colored oil, which was directly used to next reaction.

PAGE 88

73 1H NMR ( CDCl 3 ) : 1. 4 0 ( 3H, t, -OCH z -CH 3 ) 2. 7 3 ( 2H, m, -CH 2 -CH 2 -NH2), 2.93(2H,m,-CH2 -CH 2 -NH 2), 4.32(3H,qt,-OCH2 -CH 3), 6.70(1H, s,3-py-H), 6.87(1H,dd,5-py-H), 8.17(1H,d,6-py-H). Preparation of 4-(2'-acetaminoethyl)-2-ethoxypyridine(95) A mixture of the crude amine(94), 0.5 ml of acetic anhydride and two drops of pyridine in 5 ml of methylene chloride were stirred for 1 hr at room temperature under nitrogen atmosphere. The mixture was concentrated to give a reddish oil, which was dissolved in water, made basic with 10% NaOH and extracted twice with 20 ml of chloroform. The chloroform layer was washed with brine, dried over anhydrous magnesium sulfate. filtered and concentrated to give a crude product, which was purified by column chromatogrphy on silicagel with ethyl acetate-methanol mixture. m.p. : 84.5-85.5C. 1H NMR(CDC13 ) : l.37(3H,t,-OCH2-CH 3), l,93(3H,s,-COCH3), 2.70(2H,t,-CH2 -CH 2-NH-), 3.40(2H,m,-CH2-NH-), 4.30(3H,qt,-OCH2 -CH3), 6 .45(1H,br. ,-NH-), 6.52(1H,s,3-py-tl), 6.70(1H,dd,5-py tl), 8.00(lH,d,6-py-H). Cald. Found Mass spec. (70 eV) : 208 (M+). C:63.44, H:7.74, N:13.45 C:63.51, H:7.75, N:13.44. Preparation of 4-(2'-acetaminoethyl)-2-ethoxypyridine-l-oxide l.2... A solution of 500 mg(0.00240 mol) of compound(95) and O. 62 g of m-chloroperbenzoic acid ( 1. 2 eq.) in 7. o ml o f

PAGE 89

74 dichlorometane was stirred overnight at room temperature under nitrogen atmosphere. The reaction mixture was concentrated in vacuo to give a yellowish residue, which was directly subjected to column chromatography on silica-gel with ethyl acetate-methanol mixture to give 380 mg of product (70. 6 % yield) m.p. : 133.5-135C. 1H NMR(CDC13+CD30D(3:1)) : 1.40(3H,t,-OCHz -CH 3), 1.97(3H, s,-COCH3), 2.60(2H,t,-CH2-CH2-NH-), 3.25(2H,m,-CH2-NH-), 4.17(2H,m,-OCH2-), 6.77(1H,dd,5-py-fi), 6.82(1H,s,3-py-fi), 7.97(1H,d,6-py-H). Mass spec. : 225(M+l). Preparationof 4-(2'-acetaminoethyl)-1-hydroxy-2-pyridone(86) A suspension of 300 mg of compound(85) in 5.0 ml of acetyl chloride was stirred for 20 min. at room temperature and refluxed for 2 hrs under nitrogen atmosphere. Excess acetyl chloride was removed in vacuo and resulting residue was dissolved in 10 ml of water. The aqueous solution was stirred overnight and concentrated to give a yellow sticky oil. This oil was stripped with methanol twice to give a very hygroscopic yellow colored amorphous type solid. ) 3.40(2H,m,-CH2-NH-), 6.60(1H,dd,5-py-fl), 6.70(1H,s,3-py fl), 7.97(1H,d,6-py-fi). Mass spec. : 225(M+l). Preparation of 4-(2'-aminoethyl)-1-hydroxy-2-pyridone hydrogen chloride(16)

PAGE 90

7 5 A solution of crude compound(86) in 8.0 ml of methanolwater-c-HCl(l:1:2) mixture was refluxed overnight under nitrogen atmosphere. After the reaction mixture was concentrated in vacuo, the resulting reddish sticky residue was dried under reduced pressure, and then washed with ethanol to give the crude solid product, which was recrystallized from ethanol-water mixture to give a yellow product(630 mg: 41.1% from N-oxide). m.p. : 224-226C. IR(nujol mull) : 2600-3300(br. ,-OH & -NH /), 1650(C=O) -1 cm 1 H NMR(DMS0-d 6 ) : 2.62-3.25(4H,m,-CH2 -CH 2-), 6.17(1H,dd,5py-H), 6.40(1H,s,3-py-H), 7.83(1H,d,6-py-H), 8.25(3H,br., NH/). Cald. Found U.V. (MeOH) Mass spec. 2 2 8 3 O 3 nm 155 (M+). Elemental analysis for C7H11N202Cl : C:44.10, H:5.81, N:14.69, Cl:18.59 C:44.18, H:5.86, N:14.62, Cl:18.51. High Pressure Liquid Chromatography System A mobile phase system was developed in order to analyze compounds produced in the in vitro hydrolysis experiments. The composition of the mobile phase for the carbostyril compounds consisted of acetonitrile:water:acetic acid:hexanesulfonic acid sodium salt(40:59:l:O.l). The carbostyril compounds(l2, 13 and 14) had retention times between 2.6 and 4.2 min when

PAGE 91

76 the flow rate was 1.5 ml/min. using a Bio-Sil-ODS 5 column. The metabolites of the carbostyril compounds in 80% human plasma had retention time between 3.2 min. and 5.6 min. The chart recorder was set at 0.25 cm/min. All carbostyril compounds were analyzed using ultraviolet detection at 258 nm. A BiO-Sil-ODS 5 reversed phase silica column was used in all analyses. Chemical Stability stability of the Carbostyril Compounds{l2, 13 and 14) in pH 7.40 Phosphate Buffer Solutions of monobasic potassium phosphate(0.2 N) and dibasic potassium phosphate ( o. 2 N) were made and used to prepare a pH 7.40 phosphate buffer by mixing together. Buffer solutions(4.9 ml) were equilibriated at 37 C. At time zero, 50 ul of a 5.3 x 10~ M stock solution of test compound in methanol were added to a buffer solution. At designated time points, 100 ul samples were removed and added to 900 ul of ice-cold 40 % acetonitril-water. The final concentration of test compound would be 5.3 x 10~ Mat time zero. Samples were stored at 0 C until analyzed by HPLC. In Vitro Studies Stability of the Carbostvril Compounds(12. 13 and 14) in 100% Whole Human Blood Freshly collected heparinized blood was obtained from the Civitan Regional Blood Center, Inc. (Gainesville, FL). The blood was stored in a refrigerator and used the next day.

PAGE 92

77 Thirty microliters of a freshly prepared 0.061 M solution of test compound in methanol was added to 3. O ml of blood, previouly equilibrated to 37 C in a water bath and mixed throughly to result an initial concentration of 6.1 x 10~ M. At designated time intervals, 100 ul aliquots were withdrawn from the test medium, added immediatly to 900 ul of ice-cold acetonitrile, shaken vigorously and placed in a freezer. The final test compound concentration was 6 .1 x 105 M at time zero. When all samples had been collected, they were centrifuged at 13000 rpm for 5 min. The samples were kept at 0 C until analyzed by HPLC. Stability of the Carbostyril Compounds(l2, 13 and 14) in 80% Human Plasma Freshly collected plasma used was obtained from the Civitan Regional Blood Center, Inc. (Gainesville,FL) and contained about 80% plasma diluted with anticoagulant citrate phosphate-dextrose solution U.S.P. The plasma was stored in a refrigerator and used the next day. Thirty microliters of a freshly prepared O. 061 M solution of test compound in methanol was added to 3.0 ml of plasma, previously equilibrated to 37 C in a water bath and mixed throughly to result an initial concentration of 6.1 x 10~ M. At designated time intervals, 100 ul aliquots were withdrawn from test medium, added immediately to 900 ul of ice-cold acetonitrile, shaken vigorously and placed in a freezer. The final test compound concentration was 6.1 x 10~ Mat time zero. When all samples had been collected, they were centrifuged at 13,000

PAGE 93

7 8 rpm for 5 min. The samples were kept at 0 C until they were analyzed by HPLC. Stability of the Carbostyril Compounds(12, 13 and 14) in 20% Rat Liver Homogenate The liver homogenate was prepared by the following method. One Sprague-Dawley rat was killed by decapitation, and liver was removed, weighed and homogenated in a tissue homogenizer in 0.05 M aqueous phosphate buffer(pH 7.4) to make 20% 1 i ver homogenate. Thirty microliters of 5. 3 X solution of test compound in methanol were added to 3.0 ml of the homogenate, previously equilibrated to 37 C in a water bath, to result in an initial concentration of 5.3 x 10~ M. At various time points, 100 ul of samples were withdrawn from the test medium, added immediately to 400 ml of ice-cold acetonitrile, shaken vigrously and placed in a freezer. The final test sample concentration was 1. 0 x 105 M. When all samples had been collected, they were centrifuged at 13,000 rpm for 5 min. and were stored at 0 C until analyzed by HPLC. In Vitro Evaluation of the Prolactin Inhibitory Effects of the Pyridones(15,16) Adult female rats(Charles Rivers Labs), weighing 220 -250 mg, were maintained on food and water ad libitum. Animals were sacrificed by decapitation; their pituitary glands were quickly removed from the cranium. The anterior pituitary(AP) of each animal was dissected into two equal halves and placed into incubation media. (media 199 supplied by Grand Island Biological Co.) The incubation was conducted at 37 C, under continuous aeration(95% Oz/5% CO2); the pH was 7.61. After 30

PAGE 94

79 min., the media was discarded and replaced with fresh media. After one-half hour additional preincubation, the media were discarded and replaced with fresh media containing either compound(15, 1 x 10~ M) or compound(16, 1 x 10~ M). In all cases, one-half of AP received the test drug; the other, the media 199 control. After 30 minutes, samples were taken from the media, and the remaining media were discarded. Fresh media containing compound(15, 1 x 10~ M), compound(l6, 1 x 10~ M), respectively, were then added. Thirty minutes later, the second samples were taken. Same procedure was continued through the 1 x 10~ M doses of these compounds. At end of experiment, each half of the AP was weighed. The samples were diluted 1:50 with phosphate-buffered saline and then assayed in triplicate by the radioimmunoassay method described. The data are given as nanograms of prolactin released per milligram of wet tissue weight. Paired Student's test was used to evaluate the significance of the inhibitory effects of the test drugs on prolactin secretion. The control AP half and the drug-treated half were employed in each paired comparision. In Vivo Studies Cardiovascular Effects of the Compound(8) in Dogs This test was followed by the reported method.57 Adult mongrel dogs were anethestized with sodium pentobarbital, 30 mg/kg i.v., and given supplemental doses(i.m.) as needed. The femoral artery was cannulated to the level of the abdominal aorta for measurement of arterial blood pressure. Both femoral

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8 0 veins were cannulated for the infusion of drug. AT-shaped tube was inserted into the trachea and the dog was placed on the respirator at 25 ml/kg tidal volume at 10-12 breaths/min. In some dogs, the chest was opened via a mid-sternum incision. The pericardium was removed and any fat on the ascending aorta was dissected away. A snug-fitting, calibrated electromagnetic flow probe was placed on the aorta and connected to a Carolina flowmeter. A Walton-Brodie strain gauge was used to measure cardiac contractile force. It was sewn on the surface of the right ventricle and stretched until a maxmimum contr~ction occured(the top of Starling's curve). Lead II ECG was recorded and was used to trigger a cardiotachometer. Total peripheral vascular resistance was estimated as mean arterial blood pressure/aortic blood flow. In other dogs, anesthetized as above but with closed chest and unsupported respiration, cardiac contractility was assessed as the maximum change in left ventricular (LV) blood pressure per unit time (LV /dP/dtnn J This parameter was computed with a calibrated Grass differentiator(model 7P20) from a LV pressure signal obtained from a Millar transducer-tip catheter after introduction through the left carotid artery. Drug was introduced as an intravenous infusion for five minutes. For intravenous infusion, compound(8) was dissolved in saline. Infusions of drug were given in random order and doses were maintained for 5 minutes or until a steady state of cardiovascular responses occured at volume rates of 0.5 -2.0 ml/min. The volume of vehicle did not affect cardiovascular variables.

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81 Electrophysiological Studies of the Compounds(l3,14) Adult mongrel dogs were anethestized with sodium pentobarbital(30 mg/kg, i.v.), intubated and ventilated with room air. Bipolare and hexapolare catheters(French 5 and 6) were introduced percutaneously and positioned under X-ray and ECG control in four locations; atriocaval junction, right artrial appendage, right ventricular apex and aortic root. Three standard surface ECG leads were recorded simultaneously with intracardiac electrograms. After insertion of catheters, the following basic parameters of conduction intervals were recorded and measured; intraatrial conduction(PA) which is the interval from oneset of the P wave on surface leads to oneset of the low atrial activity in the His bundle recording, atrial and atrioventricular/AV-node conduction(AH) which is the interval from oneset of low atrial activity to oneset of His potential, His potential which is the interval from oneset to conclusion of the His potential in the His bundle recording, and His-Purkinje conduction(HV) which is the interval from oneset of His potential in the His bundle recording to the earliest oneset of ventricular activation in any intracardiac or surface leads. In order to measure the programmed electrical stimulation, atrial pacing was accomplished using a battery powered programmable stimulator, delivering pulses of 2 ms duration at twice diastolic threshold. The following two parameters of sinus node function were determined by atrial pacing and programmed atrial stimulation; sinus node recovery time(SNRT) which is the interval from the last paced

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82 complex to the oneset of the first sinus beat as measured from the right atrial electrogram, and sinuatrial conduction time(SACT) which is the interval from the last paced beat to the first return sinus beat as recorded in the high right atrium. At same time, by using programmed electrical stimulation of the right atrium, the following parameters of AV node His-Purkinje function were determined; atrial effective refractory period (AERP), atrial fuctional refractory period (AFRP), effective refractory period of the atrioventricular node(AVNERP), functional refractory period of the atrioventricular node(AVNFRP) and effective refractory period of His bundle(HisERP). Drugs were dissolved in saline and introduced as an intrveneous infusion through the femoral vein. Pharmacokinetic and Metabolism Studies of Compound(l2) in Rat An adult female Sprague-Dawley rat(255 g) was anethestized with sodium pentobarbital. 200 ul of solution of compound ( 12, lOmg/ml) in 20% DMSO-water were administered intraveneously through the femoral vein. At selected time points after drug administration, 100 ul of blood were withdrawn from the juglar vein, added immediately to 200 ul of 5 % DMSO-acetonitrile and vortexed. When all samples had been collected, they were centrifuged at 12,000 rpm for 5 min. The samples were kept at o c until they were analyzed by HPLC.

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CHAPTER IV RESULTS AND DISCUSSION Synthesis Synthesis of 3,4-Dihydroxy-N-(6'-methoxy-3'-carbamylphenyl)l-methyl-n-phenyl-B-phenethylamine Hydrochloride(B) The synthesis of compound ( 8) was initially attempted according to Figure 4-1. 0' i)S0C l 2 ~: MVK Pd-C/H 2 (8) ii)NH 3 10% Pd-C C OOR CONH2 (17) (18) (19) Figure 4-1 Synthetic attempts of compound(B). The required starting material(l7) in Figure 4-1 was prepared from commercially available 4-methoxy-3-nitrobenzoic acid via reduction of nitre group under hydrogenation condition followed by diazotization and the Sandmeyer reaction. Conversion of the carboxylic acid to amide was successful after generation of the corresponding benzoyl chloride using thionyl chloride in the presence of catalytic amounts of pyridine, followed by treatment with saturated ammonia. However, substitution of the iodine atom by methyl vinyl 83

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84 ketone(MVK) in the presence of catalytic amounts of 10% Pd-C and triethylamine was not successful although similar reaction with compounds which do not have the methoxy group in the ortho position to iodine gave moderate yields. In order to find the right conditions, the above reaction was further studied. The results of attempts were summerized in Table 4-1. Table 4-1 Substitution reaction of compound ( 18) Rxn # Ratio (IV: MVK: NEt3 ) Rxn Time Yield 1 1 1.1 1.1 2 hrs trace 2 1 1.1 1.1 overnight trace 3 1 1. 5 1. 5 6 hrs trace 4 1 1. 5 1. 5 24 hrs intractable 5 1 2.0 2.0 48 hrs intractable From the results of Table 4-1, it might be concluded that electronic and steric effects of the methoxy group is very important in the above reaction as formation of a palladium complex with iodine is the rate controlling step in the abov e reaction and the methoxy group may inhibit formation of palladium complex. After the unsuccessful results shown in Figure 4-1, another sequence outlined in Figure 4-2 was employed to synthesize the compound(6). Salicylaldehyde(20) was utilized as starting material. This was nitrated with c-HNO~acetic acid to give a mixture of 3-and 5-nitrosalicylaldehyde(21),

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O H &CHO (20) (26) Figure 4 2 OH c-HNOJ o2'6rCHO AcOH i)NaN0 2 ii) CuCN Dopami ne (24) OH qHO 2 c 21) K2co3 C H 3 I (8) N y CHO (22) tlCHictt3 Synthetic reaction sequence for compound( 8 ) 85

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8 6 77 which was separated by using reported methods Accordingly the mixture was dissolved in 0.83 N of NaOH solution(lO g/300 ml) while warming. On standing overnight in a refrigerator, the 5-nitro isomer was precipitated. 0-Methylation of compound ( 21) with methyl iodide under basic condi ton was successful. Application of the Wittig reaction to compound ( 2 2) using l-triphenylphosphoranylidine-2-propanone in benzene afforded compound(23) in good yield. Hydrogenation(l5 psi) of compound(23) in ethyl acetate reduced the nitre group and the double bond to afford the corresponding amine ( 24) without formation of the secondary alcohol. However, when same hydrogenation reaction was attempted in ethanol at high pressure(50 psi), the resulting secondary alcohol was formed in significant amounts. Conversion of the amino group to the cyano group was completed by diazotization of compound ( 2 4 ) adding a sodium nitrite in HCl solution to form diazonium HCl salt, followed by addition of the neutralized diazonium solution to the cuprous cyanide solution. The resulting cyano compound(25) was oxidized by using 30% hydrogen peroxide in basic condition to give the desired keto product(26) in a good yield. Finally, the reaction of compound(26) with dopamine under hydrogenation condition(Pt02,10% Pd-C/H2 ) gave the desired dobutamine analogue ( 8) with small amounts of unreacted starting materials. Separation of the product from the reaction mixture was first attempted using column chromatography. Silica-gel was used with chloroform-methanol mixture as an eluent. Although separation was achieved using

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87 the above eluting system, the product was also partially decomposed. To reduce the decomposition during the column chromatography, the eluent was slighty modified to chloroformmethanol-acetic acid(6:2:0.5) and this eluting system resulted in better yield. After the product was collected by column chromatography, it was converted to the hydrochloric acid salt by adding HCl in methanol solution and then concentrating the solution to give a foam product, which was repurified by dissolving it into a minimum amount of methanol followed by dilution with ether to give gray colored product. The final product was very sensitive to moisture and oxygen and easily decomposed under normal atmosphere condition. The proton NMR spectrum of compound(8) is seen in Figure 4-3. Synthesis of 3,4-Dihydroxy-N-{6'-methoxy-3'-carbamylphenyl)l-methyl-n-phenyl-E-phenethylamine Hydrochloride{9) The synthesis of the keto compound which will be used toward the dobutamine analogue(9) was accompolished according to Figure 4-4. The commercially available 2-methyl-5-nitrobenzoic acid(27) was used as a starting material in this scheme. First, the starting material was reduced by 1.0 M BH3 -THF solution to give the corresponding alcohol(28) in very good yield. Next, partial oxidation of alcohol group gave the aldehyde group using Collin's reagent78 This reagent was prepared by addition of 1.0 equivalent of chromium oxide(VI) to a magnetically stirred solution of 2. O equivalents of pyridine in methylene chloride. Compound(28) was then added to the reaction vessel, resulting in the formation of the

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I ; 111 I"l !JVijj; ~ !111/!l 1 11 \ : 1 Pr. : I I I j t) :. I ,.,. I : I : I I i i 1 I 'i : I I I 0 N co '"d i::; ;::l 0 p F.: 0 u i::; 0 0 p (1) ..c: E--t CX) M 0 .... I ""' 88

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89 Qr OOH BH3-THF r'OH Py 2 .Cro3 &CHO N02 N02 N02 (2 7) (28) (29) 1~3PCHgCH3 i)NaN0 2 Pd-C/H 2 ii)CuCN CN N02 (32) ( 31) (30) 1H20/KHCO J Dopamine ::~~ Pd-C,Pt02/H2 CONH2 CONH2 (33) (9) Figure 4-4 Synthetic reaction sequence for compound(9).

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90 aldehyde(29) in 92% yield. From this aldehyde(29), the 6-methyl keto compound was prepared in a manner similar to the synthesis of the 6-methoxy keto compound. Thus, the Wittig reaction was performed on the aldehyde compound(29) with the triphenylphosporylidene-2-propanone to produce compound(30). Catalytic hydrogenation of compound(30) gave the amino compound(31), which was converted to the cyano compound(32) via diazotization, followed by cyano group replacement. Oxidation of compound(32) using 30% hydrogen peroxide in basic condition gave the desired benzamide(33). Finally, coupling reaction between dopamine and the keto compound ( 3 3) under hydrogenation condition, which was applied to synthesize the compound(8), to give the desired compound(9). The proton NMR spectrum of compound(9) is seen in the Figure 4-5. Synthesis of the Carbostyril Derivatives(l2,13,14) The first attempts to synthesize compound(l2) were made by following a previously published method71 (Figure 4-6). Based on this reported method~ the commercially available hydroxyquinoline(34) was first chloromethylated at 5-position in very good yield by dissolving the starting material in a 37% formaldehyde solution and passing dry HCl gas through the reaction vessel Next, replacement of the chloro group by the cyano group to produce the ethyl amine unit by using warm NaCN/DMSO condition was performed. However, the yield of this reaction was poor even though the reported yield was moderate(62% yield). In order to achieve the expected higher yield, several reaction conditions including changing reaction

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IT ; F ::,WE:!' .-~~ -------------------~~-----------JOOYz -50 -;co G = ~ ) i _L ____ --.. 1------, -.. ---iO 9 8 7 6 5 4 J Figure 4-5 The proton NMR spectrum of compound(9). T 11 .... ::'IC .:F :f'" 1------------____ .__ _____ 2 0 I.O f-'

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QO (34) NHCR 00 OCH 0 3 (39) l Ac 20 CJ NHCR (40) Figure 4-6 92 HCHO 00 NaCN HCl(g) DMSO OH ~Cl (35) (36) li)NaH ii)CH3r mCPBA i)Raney-Ni/H2 6o ii)RCOCl (R~,CH3 ) OCH3 (38) (37) 48% HBr OH ( 41) Attempts of synthetic reaction sequence for compound(41).

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9 3 time, temperature and ratio of NaCN/sample were employed. But, there were no significant changes in the actual yield even though a 78% yield of benzyl alcohol in ethanolic water condition was obtained. The results of various attempts are shown in Table 4-2. Table 4-2 : Replacement reaction of compound ( 3 5) Solvent Temp. Rxn Time Ratio(NaCN/Sample) %Yield DMSO so-go c 45min. 5.0 16.3 DMSO so-goc 2 Hrs 5.0 13.2 EtOH/H20 reflux 2 Hrs 2.0 78.0 DMSO so-go c 12 Hrs 3.5 12.5 In addition to poor yield of this step, the synthetic procedure was also undesirable because the use of excess(5.0 eq.) NaCN, followed by decomposition of the excess NaCN by cHCl to generate toxic HCN. The next reaction was 0-methylation using a NaH/MeI method. Unfortunately, the yield of this reaction was also fairly low although the reported yield was 42%. As the reported method7 1 was proven to give poor yields of most steps and inappropiate reaction condition in the second step, another synthetic approach was considered to avoid these problems. The modifications, described in Figure 4-7, included the use of 8-hydroxy-5-methylhydroxyquinoline as a new intermediate because of its facile synthesis without using

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QO HC!I O Pd-C/H 99 HCI (g) ;l (34) (35) (4r mCPBA Q w c -HCl Ac2 0 0 Ac O (45) (44) ( 43) l K2C03/ (CH3 ) 2so 4 NBS Q? \\ \( OCH u O OCH3 3 ( 46) (4 7) (4r JcrO/AcOH N02 CH~NO~ OCH3 (49) (50) ( 4 1 ) Figure 4-7 Attempts of synthetic reactio n sequence for c ornpound(41). 94

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9 5 toxic conditions and the probability of its convertion to the ethyl amine unit in a later step. 8-Hydroxy-5-methylhydroxyquinoline(42) was easily prepared from 8-hydroxy-5-chloromethylquinoline.HCl by using a reported method~ Thus, compound(35) was dissolved in ethanol and hydrogenated at initial pressure of 50 psi for 3 hrs to give the compound(42) in 93% yield. After preparation of the N-oxide(43) by oxidation of compound(42) with m-chloroperbenzoic acid, it was rearranged to the carbostyril compoundby refluxing it with acetic anhydride. The resulting 8-acetoxy compound(44) was hydrolyzed under acidic condition to the 8-hydroxy compound(45). 0-Methylation of compound(45) was carried out by using dimethylsulfate under basic condition to give 8-methoxy-5-methylcarbostyril (46) in 62% of the total yield from compound(42). The next reaction was the conversion of the 5 -methyl group to the ethylamine side chain. In order to put the amine functional group at the benzylic position of compound ( 4 6) bromination and subs ti tut ion by cyano group were considered. Thus, N-bromosuccinimide(NBS) in carbon tetrachloride was initially tried. However, the poor solubility of compound(46) in CC14 resulted in only trace amounts of the product ( 4 7) with mostly unreacted starting material present. After failure of the above bromination in several reaction conditions, conversion of 8-methoxy-5-methylcarbostyril(46) into 8-methoxy 5-formyl carbostyril(49) under Cr03-AcOH condition was considered and attempted. Although partial oxidation of compound(46) was successful with

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96 12% yield, the addition reaction with nitromethane was unsuccessful and gave gummy residues. From the Figure 4-7, it was realized that synthesis of 5-(2'-aminoethyl)carbostyril compound from 5-methyl-8-methoxycarbostyril is difficult because of the poor solubility and limited reaction pathways of 5-methyl-8-methoxycarbostyril. To overcome these problems, another modification using a Fridel-Craft acylation to attach the ethylamine unit to 8-hydroxycarbostyril was considered(Figure 4-8). According to Figure 4-8, the 8-hydroxyquinoline(34) was first transformed to 8-hydroxycarbostyril ( 53) by following the previously reported method. 87 0-Methylation of the phenolic group using dimethyl sulfate under basic condition gave a compound ( 54) in a fairly good yield. In order to put the required ethyl amine functional group at 5-position, FriedelCraft acylation was used by following Yashizaki's method.70 In this reported procedure, 8-hydroxycarbostyril and chloroacetyl chloride were suspended in the solvent and an excess amount of aluminum chloride was added to the suspension. The resulting mixture was stirred in an ice-water bath and was subsquently refluxed for 12 hrs to give crude 5-chloroacetyl-8-hydroxycarbostyril, which was used in the next reaction without further purification because of instability of the compound. The same reaction was applied to 8-methoxycarbostyril, but yields in this case were lower than with the 8-hydroxycarbostyril reaction. To improve the yields,

PAGE 112

00 mCPBA 00 Ac2o OH (34) 0 (55) t~CH2"H2 (56) Figure 4-8 0 OH 0 OAc (51) (52) lc-HCl ClCHlOCl ~o Kl03 w AlC13 (CH3 ) z80 4 u OCH3 (54) (53) ~H "Gabriel Rxn" \ u 0 CH3 (58) l ( 41) Attempts of synthetic reaction sequence for compound(41). 97

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98 different reaction conditions were tried and the results of various attempts were summarized in Table 4-3. Table 4-3 The results of Fridel-Craft acylation reaction of compound(53,54). Compound Solvent Ratio(Comp./Acyl Chlo. /A1Cl3 ) yield 53 Dichloroethane 1 : 2.0 3.0 No Rxn 53 CS2 II 14 54 Dichloroethane II Trace 54 cs 1 2.0 3.0 54 Replacement of chloro group by an amino functional group was first attempted using benzyl amine by following the reported method7 0 but resulted in the formation of a viscous gummy polymer. To reduce the reactivity of the primary amine, dibenzylamine was also tried, but gave the same results. After the unsuccessful results from the above reaction conditions, it was considered that the chloroacetyl group is too reactive and it needs to be changed to a less reactive group like a chloroethyl group. Thus, after reduction of chloroacetyl group was achievied with triethylsilane and trifluoroacetic acid yielding compound(57), a Gabriel synthesis~ was applied to convert the chloro group into amino group. However, this reaction did not take place as expected. As previously discussed, it is difficult to attach the ethylene amine functional group to either 8-methoxycarbostyril or 8-methoxy-5-methylcarbostyril by following known method70 because of poor solubility and/or instability of the

PAGE 114

9 9 intermediates. Therefore, another approach which started with the ethylene amine moiety already in the molecule, was considered based on retrosynthetic analysis of the desired compound(Figure 4-9). Q 9 0 NHCCH=CHCR O C H 3 Figure 4-9 Retrosynthesis of carbostyril compound Based on the illustrated retrosynthesis, p-methoxyphenethylamine(59) was chosen as the starting material because of the presence of the ethylene amine functional group and its commercial availability. According to Figure 4-10, the amine group of p-methoxyphenethylamine (59) was first protected with trifluoroacyl group using trifluoroacetic anhydride in methylene chloride. The resulting compound(60) was nitrated under c-HN03/CF3COOH condition and then the nitro group was reduced under hydrogenation to afford compound(62) in 67% of the total yield from starting material. The first attempt to close the ring was based on a ~eported method8 9 in which the carbostyril ring was successfully produced from the

PAGE 115

OCH 3 ( 60) QOO (R=CF3CO) 0-Cl OCH 3 (68) 100 NHR 0 NH 2 OCHJ (61 ) / OCH3 (62) HR ~CH=CHCOC11 OCH 3 (6 7) 0 H~CH=CH0 diketene ~Br CH)~CJIZ~OC 2 HS, OCH3 0 0 .:Htc H }ctt3 (63) Jc-H2so4 l H 2o2/KHC03 ~ ) CONH2 ~Y'1QH2 HCl .....-'O CH 3 (12) Figure 4-10 ~H2 (65) (64) Synthetic reaction sequence for compound(l2), compound(13) and compound(14).

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1 01 corresponding cinnamoyl amide on methylbenzene under aluminum chloride-chlorobenzene condition at 90 C. 78 % CH 3 AlC1 3 The reaction of compound ( 62) with cinnamoyl chloride in anhydrous THF gave a corresponding amide compound(67) in 88 % yield. By following the reported procedure, the cinnamoyl amide compound was dissolved in chlorobenzene and AlC13 was added to the mixture. The reaction mixture was then heated to 130 C for 3 hrs but, following work up, gave only a dark viscous polymer. Several different reaction conditions wer e tried, but all failed. The results of these attempts were summerized in Table 4-4. Table 4-4 : Cyclization reaction of compound(67). Ratio(A1Cl3/Com ) Rxn Time Rxn Temp. Result 5.0 3hrs 90-100 C mess 3.0 4hrs 90-100 c mess 2.5 12hrs 60-70 C polymer&s.m Since A1Cl3-chlorobenzene ring closure route was unsuccessful presumably due to steric hinderance of ethylene unit next to reaction site, another ring closure was also attempted with an anilide compound under acidic condition.

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1 02 Although the original target compound does not have a methyl group at the 4-position of the carbostyril ring, this compound was decided to pursue for activity because it can be easily synthesized from compound(62) and because the methyl group at 4-position may not affect the activity of original compound. Thus, reaction of compound(62) with commercially available diketene gave an anilide compound(63) in good yield. Ringclosure of compound(63) in various acidic conditions(c-H2S04 c-HCl, PPA) was tried and best results were obtained in concentrated sulfuric acid ( 45% yield). The cyclization of compound ( 63) in polyphosphoric acid (PPA) was unsuccessful. Deprotection of the trifluoro group of compound(64) was carried out under methanolic HCl condition to give compound(65) in good yield. Finally, the coupling reaction of compound(65) with the counterpart of keto compound(66), which was synthesized from m-cyanobenzyl bromide via three steps, was achieved under either reductive amination condition(NaBH3CN/MeOH) or hydrogenation ( PtOz/Pd-C/MeOH) condition in fairly good yield. After the desired free amine(12) was synthesized, it was converted to the more stable salt form. Several salt forms were made and it was found that the acetic acid salt of compound(12) was most stable under atmospheric conditions. The proton NMR spectrum of compound(12) is seen in Figure 4-11. Compound(13), the a-hydroxy derivative, was obtained by the demethylation of compound(12). Demethylation was first tried with 48% HBr which gave a white precipitate after work

PAGE 118

103 up, but the elemental analysis was not consistent with the structure in spite of appropriate proton NMR spectrum. Demethylation of the material with 1.0 M BBr3 solution gave the desired free hydroxy compound ( 13) in 38% yield with consistent elemental analysis. The proton NMR spectrum of compound(l3) is seen in Figure 4-12. Compound(l4), the 3,4-dihydro-8-methoxy derivative, was also synthesized by reduction of compound ( 12) The hydrogenation was performed in methanol with an initial pressure of 50 psi for 3 days to yield a pale yellowish product, which was first purified by column chromatography on silica-gel with a chloroform-methanol mixture as an eluent. Collected fractions were concentrated to give a white residue. The analytically pure material was obtained by dissolving the white precipitate in a minimum amount of chloroform, followed by dilution with ether. The proton NMR spectrum of compound(l4) is seen in Figure 4-13. Synthesis of 5-(2'-Aminoethyl)-l-hydroxy-2-pyridone{l5) As shown in Figure 4-14, 6-hydroxynicotinic acid(69) was chosen as a starting material. The attempted direct reduction of the carboxyl ic group with 1. 0 M .diborane solution was unsuccessful because of poor solubility of the borane complex of the starting material during the reaction. Reduction trials of compound(69) with LiAlH4 were also unsuccessful because of its poor solubility in either THF or ether. Therefore, the starting material was converted into the corresponding ethyl ester(71) to increase its solubility in organic solvents.

PAGE 119

I I r t it I -' I :r I ;\ 6 10 8 6 4 Pigure 4 11 The proton NMR spectrum of compound(l2). i l l I I I 2 0 f-' 0 .i,.

PAGE 120

. -1r 1 ;T:; I. ) i! ,,, I i ': ': : ,f) ... 1 ....... I I ~rr,t"'~ : I !~ :.-:' 1 l l .. I ::.: .1 J --<'f'" ,. j 1 I j I I ~/ I I ; i I : 1--"' I ,.I, I 'f:i. '!j ; L ; ~ : f _ti;~ 0 _N co 0 "O s:: ::l 0 0 E 0 u lH 0 E ::l H u (lJ 0, Ul s:: 0 0 H 0, (lJ .c E-< N ...-4 I 'Sl' 105

PAGE 121

--'.. L ~::~:~:~{ ; -~l=~=-i0~~1~~==,~"" cS 10 8 6 4 .,. ; r. I: I 1 : i i '3 ~ t +~ !;' --;-, ; Hi 2 Figure 4 -13 The proton K~R s pectrum of compound(14). If: I : 0 t--' 0 O'I

PAGE 122

HO ~OOH (69) RO gCN (7 5) 1BH3-THF (76) Figure 4-14 ~oM02Et > M02Et c-HlOl Agco3 EtO I RI RO N ( 7 2) (71) (R=CH3,c2 H 5 ) I HO_..~H (70) KCN ROffl MeOH ( N (74) ) ~HAc RoA\() (77) c-HCl (15) S0Cl2 mCPBA lLAH ~H RO N ( 7 3) ~HAc RO~\() d (78) li)AcCl ii)H2 0 ~HAc I OH (79) 107 Synthetic reaction sequence for compound(15).

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1 08 The ethyl ester compound(71) was easily obtained in good yield from the starting material using ethanol with catalytic amount of c-H2S04 Reduction of the ester(71) with LiAlH4 in THF resulted compound (70), which was very hygroscopic and poorly soluble in most of organic solvents. In order to increase the solubility of this compound in THF and to protect the hydroxy group at 2-position, the hydroxy group of compound(71) was masked with methyl or ethyl group using the AgC03/MeI(EtI) method~ which is well known for selective oalkylation. Next, reduction of compound(72) under normal LiAlH4 condition converted the material to the hydroxymethyl compound(73) within 20 min. in 96% yield. However, when above reaction was carried out for overnight, the pyridine ring was also reduced and resulted in a messy complex product. The chloromethyl compound ( 7 4) was made by substitution of the hydroxy group with chlorine using thionyl chloride and the resulting chloromethyl compound ( 7 4) was converted into the cyanomethyl compound(75) with either sodium cyanide-methanolic water or anhydrous sodium iodide-sodium cyanide-acetone condition in fairly good yield. Reduction of the cyano group in compound(75) was achieved using an excess of 1.0 M diborane-THF solution and the resulting amino group(76) was acylated with acetic anhydride to give a more stable acylated compound(77) in 48% yield. Oxidation of compound(77) with mCPBA gave the N-oxide(78), which was converted to the desired N-hydroxy-2-pyridone compound(79) using the reported methods9 1(AcCl, then hydrolysis) in 38% yield. Finally,

PAGE 124

109 deprotection of the acyl group was achieved in refluxing MeOHH20-c-HC1 to give compound(l5). Recrystallization from ethanol-water mixture gave an analytically pure pale pinkcolored product. The proton NMR spectrum of compound(l5) is seen in Figure 4-15. Synthesis of 4-(2'-Aminoethyl)-l-hydroxy-2-pyridone(16) First synthetic attempts were made according to Figure 4-16. 4-(2'-Aminoethyl)pyridine(80) was chosen as the starting material. The first step of protection of the amino group with benzoyl chloride was successful. N-Oxidation of pyridine b y either mCPBA or with H20iJAcOH gave a hygroscopic N-oxide derivative(82). Rearrangement of the N-oxide into 2-pyridone was performed by following the reported methods92 Accordingly, the N-oxide(82) was dissolved in acetic anhydride and refluxed for 40 min., but resulted the unexpected compound(83) instead of the 2-pyridone(85) in 72 % yield. From the spectroscopic data, the product was identified as the pyridine enamine compound(83). This reaction could be explained based on the following mechanism;i.e. NHAc NHAc + 4 ( 83)

PAGE 125

6 10 8 6 4 Figure 4-15 The proton NMR spectrum of compound(l5). ..,. _~ L .. p -;l. -"' C"Vul '*r 1',. 1 2 0 I-' I-' 0

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0 ~"2 ~t-c1 6HR mCPBA 6R (80) NHR 6 N 0 H (85) l 0 OH (86) Figure 4-16 0 (81) I (82) (R=
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112 after the N-acetyl compound was formed, the acetate ion acted as a base rather than nucleophile toward N-acetyl compound and generated the dihydro anion, which was rearranged to the pyridine enamine type compound(83). The proton NMR spectrum of pyridine enamine compound(83) is seen in Figure 4-17. When the rearrangement of compound ( 82) in hot acetic anhydride resulted the pyridine enamine compound, conversion of N-oxide compound(82) into 2-chloropyridine(84) was considered because the 2-chloropyridine compound(84) could be converted into the 2-pyridone derivative(85) under aqueous basic condition. Phosphorus oxychloride was first used to obtain the 2-chloro compound under several different reaction conditions, but only gave a trace amount of the expected product and tarry black residue.s. Phosphorous trichloride was also tried for this purpose, but was also unsuccessful. The results of synthetic attempts to produce the compound(84) were summerized in table 4-5. Table 4-5 : Synthetic attempts for compound(84). Rxn # Amount of POC13 (eg.) Rxn Time Temp. ( C) Result 1 2.2 4 hr 110 Mess 2 2.2 in CH2Cl2 14 hr reflux No Rxn 3 2.2 1. 5 hr 80-90 Trace 4 PC13(2. 2eg) 5 hr 80-90 Mess After various attempts of converting aminoethyl)pyridine-l-oxide(82) to 4-(2'-4 ( 2 I -

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"' 0 -N Jus i -ii .... : .. : l: co 0 .... -113 M ex:> 'O i::: ::i 0 p E 0 u i::: 0 .. 0 H 0. Q) ...c:: E-< r--,...; I "-1' Q) H ::i tr, ..-I i:,...

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114 aminoethyl)pyridone(85) by rearrangement of the N-oxide in hot acetic anhydride were unsuccessful, other approaches using methyl isonicotinate as a starting material were employed to make compound(l6) as described in Figure 4-18. Commercially available methyl isonicotinate(87) was easily oxidized by using either mCPBA or H20z!AcOH in good yield. Rearrangement of the N-oxide(88) to the 2-pyridone(89) was carried out following the reported method~ giving 12 14% yield although the reported yield was 54%. In order to obtain the reported yield, the above reaction was carried out under several differnt conditions and it was found that the different reaction conditions used did not improve the actual yield. Results at different conditions were summerized in Table 4-6. Table 4-6 : Rearrangement reaction of N-oxide compound(88). Rxn # solvent rxn time rxn temp. yield 1 acetic anhydride 6hrs reflux product & s. m. 2 II 18hrs reflux 14.3% 3 II 48hrs 110 c 12.2% 4 Ac20LNaOAc ( 1. 0 eg.} 18hrs reflux 12.0% As applied to synthesis of 5-(2'-aminoethyl)-l-hydroxy-2-pyridone, similar synthetic pathways were applied to make a 5-(2'-aminoethyl)-l-hydroxy-2-pyridone. Thus, compound(89) was converted to the ethoxypyridine(90) to increase its solubility in organic solvents. Reduction of compound(90) with

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115 c&Me H202 t>JMe Ac2 o 0 AgC03 ~OEt .. AcOH Etl N N N I H 0 (8 7) (88) (89) (90) lLAH NH2 r6i ~113 -TIIF &Et Cl &Et KCN (soc12 N 'OEt N MeOH N Et (r) (93) (92) ( 91) Ac2 o NHAc NHAc lAc NHAc i)AcCI 6 mCPBA N Et N OEt N H 6 ii) H 2 0 I (95) (96) (97) OH (86) r-HCl ~NH, HCl ~. OH,... (16) Figure 4-18 Synthetic reaction sequence for compound(16).

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116 LiAlH4 afforded compound(91), which was converted to the chloromethyl compound(92) with thionyl chloride in good yield. Replacement of the chloro group by the cyano group was first tried using NaCN/NaI/Acetone but gave an unexpected product which did not show a CN absorbance in the IR. The expected material was obtained using refluxing KCN/MeOH-H20. Reduction of the cyano group(93) to the amino group(94) was achieved by using an excess of 1.0 M BH3-THF solution, and the resulting amino group was acylated to give compound(95) in 38.3% total yield from cyano compound. Oxidation of compound(95) with mCPBA gave the N-oxide(96), which rearranged to l-hydroxy-2-pyridone ( 86) using the reported method9 1 Finally, deprotection of the acyl group was achieved using MeOH-water-HCl to give the desired product(l6) in 41.1% yield. Recrystallization from methanol-water mixture gave analytically pure 4-(2'aminoethyl)-l-hydroxy-2-pyridone hydrochloride(l6). The proton NMR spectrum of compound(l6) is seen in Figure 4-19. High Pressure Liquid Chromatography(HPLC) System The development of an appropriate mobile phase for a HPLC system was necessary before the analysis of in vitro stability studies of compounds which had been previously synthesized. Initial attempts to develop a suitable analytical method for the carbostyril compounds were unproductive using a C-8 reversed phase lOum column. Various mobile phases with d ifferent proportions of acetonitrile-water, or acetonitrilewater-acetic acid, or acetonitrile-water-acetic acid-hexane

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cS 10 8 6 4 Figure 4-19 The proton NMR spectrum of cornpound(l6). --~ 2 0 I-' I-' -..J

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118 sulfonic acid sodium salt as an ion-pairing reagent were preparP.d and tested. Unfortunately, while these mobile phases gave a reasonable retention time, they gave broad chromatographic peaks. Therefore, an alternate column was required. An improved HPLC system was developed using a C-18 reversed phase lOum column. A mobile phase composed of 40% acetonitrile-59% water-1% acetic acid and 0.1% hexanesulfonic acid sodium salt as an ion-pairing reagent was first used to examine the stability of compounds in pH 7.4 buffer. At a flow rate of 1.0 ml/min., the carbostyril compounds had retention times between 3.0 min. and 4.5 min., but in biological media interfering peaks appeared. The adjustments in the proportions of the aqueous and organic phases resulted in similar pattern of interference, which required to change another column. An appropriate HPLC system was finally developed using Bio-Sil-ODS 5 10 um silica packing column. The final mobile phase was composed of acetonitrile-water-acetic acidhexanesul fonic acid sodium salt ( 4 0: 59: 1: 0. 1) This mobile phase was capable of separating all the carbostyril compounds with retention times between 2.4 and 4.2 min., as well as metabolites of the carbostyril compounds which occured in 80% human plasma(see Figure 4-20). At a flow rate of 1.5 ml/min., the carbostyril compounds(12,13 and 14) had retention times of 3. 8 min. 2. 4 min. 4. 2 min. respectively and peaks associated with the biological media appeared at 1.6 min.

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I II Il I (A) (B) Figure 4-20 The HPLC chromatogram of compound(12,I) and its ~etabolite(II) in 80 % human plasma at 23 hrs(A) and 72 hrs(B). 119

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120 Chemical Stability The testing of the chemical stability of the carbostyril compounds in pH=7.40 phosphate buffer solution was the first step in evaluating the relative stability of each compound and the potential usefulness of these compounds as a drug before in vitro biological stability test. Rates of disappearance for each carbostyril derivatives was measured at 37 C in 0.05 M phosphate buffer at pH=7.40 by using the above described HPLC method. As seen in Table 4-7, all of the carbostyril compounds(12,13 and 14) were extremely stable at pH=7.40, and no observed decomposition occured over a 21 day period. This result was not unexpected since the carbostyril compounds do not possess obvious chemically labile bonds in the structure s uch as the ester linkages or catechol groups. In Vitro Studies Stability in Whole Human Blood and 80% Human Plasma The stability of a drug in human blood is usually ah important factor in evaluating its potential success as a useful therapheutic agent since human blood has numerous enzyme activities and can mimic in vivo metabolism. Therfore, a l l carbostyril compounds were tested and their rate of d isappearance were measured in both whole human blood and human plasma. Fresh human blood which was obtained through the Civitan Regional Blood Center was used for this experiment. The

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Table 4-7. Half-life(hr) of disappearance and correlation coefficient for carbostyril compounds in pH=7.40, in 100% human blood, in 80% human plasma and in 20% rat liver homogenate. Com:gd. :gH=7.40 100% 80% 20% 1 2 1 Human Blood3 Human Plasmab Liver-Homogenatec -0Me(12) d 189e 94.4 (0.999) (0.995) -OH(13) d g 345e (0.957) Dihydro. d g 544e (14) (0.999) a. Whole human blood. b. Plasma from one individual. c. Liver from one rat. d No decomposition observed over 21 day period. e Experiment followed for< one half-life. f No decomposition observed over 80 minutes. g No decomposition observed over 6.6 hrs. f f f

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122 studies were continued for 6.6 hrs at which point the blood has denatured. As expected from the result of chemical stability test, all carbostyril compounds were very stable in blood and plasma and no significant decomposition was observed over the 6.6 hour period. As seen in Table 4-7, the half life of compound(12), calculated from extrapolation was found to be 189 hr and compounds ( 13 and 14) were stable over the prescribed time course. In an attempt to more accurately determine the stability the carbostyril derivatives, stability in 80% human plasma were also examined. Human plasma was obtained from Civitan Regional Blood Center. The rates of disappearance for the carbostyril compounds were measured as previously described. Interestingly, metabolite peaks of compound(12) were clearly observed after 23 hrs with a retention time of 4.8 min. This peak increased as a function of time(see Figure 4-20). Similar patterns was also observed with compound(13,14). As shown in Figure 4-21, compound(12) was the least stable compound tested yielding a 94.4 hr half-life. The other compounds(13 and 14) displayed 345 and 545 hr half-lives. This trend was consistant with the rate of decomposition in human blood as compound(12) had the least stability in human blood as well as plasma. The overall results of these two experiments indicated that the carbostyril compounds had few if any enzymatically labile bond or group and the enzymes involved in the formation of metabolite were present in plasma portion because the rate

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20 15 ,...._ 13 u '-" .J 10 ..c:: tlO H (lJ :i:: 5 0 Figure 4-21 24 48 72 Time (hr) In vitro results of compound(12,o), compound(13,6) and compound(14,D) in 80% human plasma. 123

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1 24 of disappearance of compound(l2) was faster in human plasma than in human blood. Stability in 20% Rat Liver Homogenate The testing of the stability of compounds in liver homogenate was important since hepatic tissue is normally the major site of drug biotransformation. This metabolic pathway can be especially significant for drugs administered orally. Any drug given by the oral route may be subject to the firstpass effect. This common occurance is the result of hepatic enzymatic activity on a drug which can prevent the apprearance of significant levels of the drug in the circulation after oral administration. 93 The results of the in vitro liver homogenate assay indicate that hepatic biotransformation may perform a key role in the inactivation of drugs under in vivo condition. In order to perform the desired stability testing in liver homogenate, fresh rat tissue was used. Liver from one rat was homogenized in pH=7. 40 phosphate buffer and the resulting 20% liver homogenate was briefly stored in ice until the experiment began, at which time the homogenate was incubated in a 37 C water bath. Again, all carbostyil compounds were extremely stable in 20% liver homogenate and no decomposition was observed over 80 min., at which time the homogenate has denatured. These results were consistent with the previous stability information in pH=7.40, in 100% human blood and in 80% human plasma and demonstrate that the carbostyril ring system is

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125 metabolically insensitive to hepatic enzymes wh i l e the catechol ring system is a good substrate of hepatic enzym e s especially COMT. From the overall results of the in vit r o stability studies(see Table 4-7), it was predicted that all of carbostyril compounds may have oral bioavailability In Vitro Evaluation of the Prolactin Inhibitory Effects of the Pyridones(15,16) In order to assess the dopaminergic activity of the 1-hydroxy-2-pyridone compounds(15,16) in comparision t o dopamine, inhibitory effects of prolactin secretion from anterior pituitary in rats were performed. Fresh anterior pituitaries obtained from female rats were incubated with various concentrations of compound(9) and compound(lO), respectively, and their effects on the rate of release of prolactin were measured and compared with t h e control anterior pituitary. As seen in Table 4-8 and T a b l e 4 -9, the compound(lO) caused a continuous reduction of prolactin secretion at 10-9 10-6 M concentration ranges while t h e compound(9) showed its activity at 10~ M concentration. In comparision with dopamine, dopamine caused a 57% reduction of the prolactin secretion at a 2 x 10~ M concentration.~ Th e s e results indicated that both compounds(9,10) have dopamine r g i c activities and the activity of each compounds is less tha n that of dopamine. However, it is interesting to point out tha t compound(lO) showed the dopaminergic activity at even low e r concentration than the concentration at which dopami n e revealed its inhibitory effect on prolactin secretion. From

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Table 4-8 In vitro activity of the compound(15) .3 prolactin, (ng/mg)/mg protein concentration(M) controlc compound ( 15) c 1 X 10 127.73 + 20.70 135.19 + 19.64 1 X 10-8 88.75 + 13.72 104.94 + 13.74 1 X 10-7 76.23 + 9.80 92.51 + 12.05 1 X 10-6 74.00 + 16.70 39.13 + 5.03 a: On freshly obtained anterior pituitary(AP) at All values are average of seven separate AP-S. b: Prolactin release of the incubated AP-S. c: Weight of the AP-S: control, 7.75 + 0.61 mg ; (15) treated, 7 .97 + 0.30 mg. % 37 126 change + 6 + 18 + 21 -47 oc.

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Table 4-9 In vitro activity of the compound(16) .3 prolactin, (ng/mg)/mg protein concentration(M) controlc compound ( 16) c % change 1 X 10-9 91. 80 + 17.94 82.84 + 11. 91 -9 1 X 10-8 89.59 + 12.70 63.33 + 7.06 -29 1 X 10-7 80.17 + 9.15 68.79 + 5.22 -13 1 X 10-6 37.49 + 4.59 26.90 + 2.23 -28 a: On freshly obtained anterior pituitary(AP) at 37 C. All values are average of seven separate AP-S. b: Prolactin release of the incubated AP-S. c: Weight of the AP-S: control, 6.68 + 1.02 mg ; (16) treated, 6.85 + 0.98 mg. 127

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128 these results, it was predicted that this system may be used as new starting material for new dopaminergic agents and/or new cardiotonic agents which are not good substrates for COMT. In Vivo Studies Cardiovascular Effects of Compound(8) in Dogs The hemodynamic responses to compound(8) were evaluated following intravenous administration in acutely instrumented anesthetized dogs as described in the E xperimental. Myocardial contractility(dP/dtm u), heart rate, mean arterial blood pressure and peripheral vascular resistance were recorded. A s shown in Figure 4-22, for a 100% increase in cardiac contractile force, compound(8) was about 5 times as potent as dobutamine and half time as potent as KM-13. Compound(8) also raised heart rate in a dose dependent manner like KM-13 but less potent than KM-13. However, compound(8) did not cha n g e mean arterial blood pressure and peripheral vascula r resistance while dobutamine and KM-13 increased mean arter i a l pressure in a dose-related manner. These results indicate tha t the inotropic profile of the compound(8) is very similar to dobutamine and KM-13 although less changes in mean arterial blood pressure and peripheral vascular resistance were achieved. Electrophysiological Studies of the Compound(l3,14) In order to evaluate in vivo cardiotonic effects of the carbostyril system, the changes in cardiac electrophysiological parameters of compound(l3) and

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2so I f 2001 U 150 11 100 I SOI 01 !'0 1 10 o I A Car d iac Contra c tile Force j ... KM-13 C 201 Dobutamine 0 Mean Ar teri al Blood Pressur e r E 10 () ---Compound (8.) E o 0 I ... ./T / ~-- ... / .. 0 I I I B Hearl Rate / 0. ....... 0 0 5 0 1 O ~ Figure 4 22 ~
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compound ( 14) in dogs were observed. Percutaneous technique which is employed in human electrophysiological studies were used. After the dogs were anesthetized with 130 catheter clinical sodium pentabarbital, all basic parameters were first determined. The drugs dissolved in 0.9% saline were administered intravenously (infusion rate : 10 ug/kg/min.) after a bolus of 1 mg/kg was given, and the electrophysiological parameters were determined after 15 min of drug administration. As seen in Table 4-10 and 4-11, there were no significant changes in all parameters by compound(l3) and compound(l4). These results indicate that these compounds have no significant electrophysiologica l effects on the dog's heart and that there is no direct stimulation of the adrenergic receptors located in the heart. Therefore, these compounds may be considered as having good potential as novel cardiotonic agents. Pharmacokinetic and Metabolism Studies of Compound(l2) in Rat In order to understand the fate of carbostyril compounds in the body, the in vivo kinetic study of compound(l2) was performed. Thus, after one injection of the solution o f compound(l2) in 20% DMSO-water to rat, the amount of compound in rat blood was determined at selected time periods by an HPLC method. As seen in Figure 4-23, compound(l2) was quickly distributed to organs with a half-life of 7.1 min(k3 = 9.7 x 10-2 min-1 ) while elimination process was rather slow with a half-life of 103.9 min(kc = 6.6 x 10-3 min-1). These results clearly show that the new carbostyril compound is much more

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1 3 1 Table 4-10 Electrophysiological data of the compound( l 3) Basic parameters 15 min after drug adm i n (msec) (msec) PA 10 10 AH 40 40 H 10 10 HV 30 30 SCL 360 375 SNRT 420 455 CSNRT 60 70 SACT 40 45 AERP 150 170 AFRP 160 170 AVNERP 160 170 AVNFRP 240 250 HPSERP 260 260 a: Dose of drug administration(i.v.) was 10 ug/kg /min. after bolus(l mg/kg).

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132 Table 4-11 Electrophysiological data of the compound(14) a Basic parameters 15 min after drug admin. (msec) (msec) PA 10 10 AH 60 60 H 10 10 HV 40 40 SCL 370 380 SNRT 450 460 CSNRT 80 80 SACT 40 40 AERP 110 110 AFRP 160 160 AVNERP 160 160 AVNFRP 200 200 HPSERP 200 210 a: Dose of drug administration(i.v.) was 10 ug/kg/min. after bolus(l mg/kg).

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5x10-6 :z s:: 0 rl m s:: Q) u s:: 0 u lxl0-6 Figure 4-23 phase(k ) a 30 60 90 Time(min.) The semilog diagram of in vivo pharmacokinetic result of compound(l2) in rat. 1 2 0 f-' w w

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134 stable than dobutamine in the body since the half-life of dobutamine in plasma is approximately 2 minutes.93 AM-1 Calculation of (2'-Aminoethyl)-1-hydroxy-2-pyridone Systems The development of a quantitative quantum mechanical molecular model is very useful for chemists in studies o f chemical reactions and reaction mechanisms. To be useful in this connection, such a procedure must be not only sufficiently accurate but also applicable to the molecules in which chemists are directly interested rather than confined to simple models. Two effective models, MIND0/3~ and MND0% are being widely used for calculation of various physiochemical parameters, but these methods were replaced by more 9 7 accurate approximation. Thus, recently, Dewar et al developed a new parametric quantum mechanical model, AM-l(Austin Model 1), based on the NDDO approximation. In this model, the major weakness of MNDO(in particular failure to reproduce hydroge n bonds) have been overcome without any increase in computing time. In order-to understand the structural and electro nic features, semiempirical MO calculations(at AM-1 level) we r e performed on two pyridone compounds ( 15, 16) as composed to dopamine. As shown in Table 4-10, it is clear that these two pyridone compounds(l5,16) are somewhat less lipophilic than dopamine as the log P values of these two compounds are less than that of dopamine. However, comparing to each other, the

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compound ( 15) compound(l6) compound ( 15) 135 is more lipophilic than the compound(l6} while is more stable by about 1 Kcal/mol than The geometrical structures between the N-hydroxy group in the pyridone and the corresponding hydroxy group in dopamine were also compared from the AM-1 calculation results(Table 4-11, 4-12 and 4-13) since l-hydroxy-2-pyridone is tautomerized to 2-hydroxy-1-oxide as reported.9 8 The bond length between the hydrogen and oxygen atoms in the N-hydroxy group was slightly longer(0.04 angstroms) than that in the hydroxy group of dopamine and the bond angle and the bond twist of the hydrogen atom in the N-hydroxy group were more shifted toward the adjacent carbonyl oxygen as compared to the catechol system in dopamine. These trends are consistant with the reported tautomerization in the pyridone model because, if there is any interaction between the hydrogen atom of the N-hydroxy group and the carbonyl oxygen atom in carbonyl group, this interaction should result longer bond length and more twisted bond angle of the hydrogen atom. Therefore, these AM-1 calculations indicate that there is a weak interaction between the hydrogen atom in the N-hydroxy group and the adjacent oxygen atom, and therefore, the hydroxy group in the pyridone compounds should not be a good substrate of COMT.

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136 Table 4-12 Physio-chemical data of compound(l5), compound(l6) and dopamine from AM-1 calculation.a compound (15) (16) dopamine Hr(Kcal) dipole moment(Debye) -18.48 3.04 -19.50 4.43 -75.59 1.31 a: Calculation was performed using a VAX computer. 0.48 0 .27 0.97 b: Estimated value was derived from the equation developed by Bodor et al. c c: N.Bodor, unpublished results.

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137 Table 4-13 A!1-l calculation results of cornpound(15). ATOM C HF.11 l c : /\I. LII. INll I .. FN1: I H HI INII ANGlE lWlST l'INIJL.1:. CHM CF NUHJ.lER SYMBOL < AJ~1;r, rrw~,~ -> ( Dl (!flH''. I < DC:Gn Et.'.3 > NI> I NB NI\. J NC : NU: NA I C -0. l t;Q "j .. C I. "() i (' J .. o. o :~05 <' :1 tJ 311;, I\ ... 1 ; : (171l::1 ~ .. -n. 1 :_1 I "/ ,, C :._~ ,i r ,r-. ~ .-, ... 1 I 7 "1 ~'7'; 0 1 )()()0() .. U 31 4, !l C i. '1 ( I f i .. ;:;:: rt... 1 r-p 0 (,'f)l)("() .. -0. 21\:l 1 f:, H 1 O''l'i :,:, t ; l o 0(100<) -180. 00()(11) ... 0 1~9() 7 r. :1c:1, t 11 I Ul ,1;11~ n 0 .' O(H)()(' -o. 0 ~ ''.''1 {1 It I 0'1'711 ( 1 ... I ~'t' ()()()()() -180. 0000(1 () 147<, 9 (I I. 1011 I '.I ... I I 'i 'i' t .. I R(). ()()()()<) -o. ~1740 to C I 511000 l.~O. 00000 -180.00000 "' -0. l '1411 i 1 C 1. 53'.'l/.,I, ... 11 I 0:3~86 "' 180. 1)(1(.lf)Q -o. ()l~23 12 H 1. I 11\l,E! ... t 11. o : v,o? If 59. 97376 0 09 I\ :i 13 H 1. 1 I '1 AE! 111. 03609 -~9.97371, n 094? i 'l N 1 s ;:wo o 111. .. I 0 7 f,:::'flf1'1 ., .. A{,OJ'l 0 J 112', '.)/ 16 H I. r.11 ;-in; .. I 'J7. t .~~fJRI\ -57. 6bo3q "' 0 11l:i2 17 H l. 1 l I\(, ~ 107 fll,0'/ t -~m. 1303'i ... 0. 081\~ 1 !'I If l I l 1\/,? .. I 07. f l/.,() / 1 .. 5R. 1 '.10 ~~'.1 0 0(:1/l II SI~ I( l I ()~I' !'I .. I 1 IV, '.i 7 J ,, 1 no nooor, .If 0. I 71 I ;!Cl CJ l. ~i(IOO(l 120 00001) .. 180. 0000(' ... -o. 20:~1 a1 H l ()(l()(J(l 1011. 'l',''?'79 ti -1 7?. "(,2'7'..1 0. ;:?533

PAGE 153

138 Table 4-14 AM-1 calculation results of compound(16). ATOH <:111:MlCAL 111.11,JO 1.F~m IH ROt~D MIGl.F.. TWt!H I\NGLE 1.Hlll?GI: NUMBER SYMBOL (ANGS1HlJM!3) !IJE6 0 09ftl 16 H \ I 1 '11,'J I Oi'. IJ(J t /() ,, -I; I ~ 16926 0 O'J71~ 1 ., n 1 101\ 1 S I IS.

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139 Table 4-15 AM-1 calculation results of dopamine. ATOM CHEMICAL. BONl) LENGTH BOND ANGLE TWIST ANGLE CHI\RGE NUMBER SYMBOL (ANGSTROMS> l~A: I NU: NI\: 1 NC: NU: NA: I C 0 0672 ;, C l :J9~,);j l> -o. l 340 8 C 1 :i95U'l ... 1 ~O llOO O O -o. 0680 II C 1 3950'.1 120 00000 0 0000!) -o. 125;:, 5 C :J95C. 3 120. 00000 0 00000 -o 1677 6 C 1 39504 120. 00000 0 .00000 -0.0076 7 () 1 50000 120 00000 -180. 00000 -o. 2711 8 II 0 96113 ... 103. 5:J664 -0.00003 0 2277 9 H 1 09',65 120. 00000 -180.00000 0. t351 10 H 1. 09965 120. 00000 180.00000 0 1885 I 1 0 l 50000 120. 00000 1BO,:~gg -o. 2498 .i+ ,Q,,, 0 1 '3'J:!CJU '! \, 0 .2355 1 8 ll 1 09',6!.i 1 ;?O. 00000 180. 00000 0 1518 1 '\ C 1 5400() 120. 00000 180. 00000 -o. 12:w l H 1 111S6 .. 109. '\7 1 0 ) 180.00000 0 IOOA 1 6 H 1 11156 109. 4 i' I 00 60.00000 0 08G''1 p C I S4000 109. '\i'IOO ii -60. 00002 -o 07b3 I A H I 111 56 109. ,11100 -100.89330 0 0934 19 H \ 111 5 6 109. 47100 19. 10670 0 0461 ;!O N 1 52000 109. 4710 0 139. 10674 -0.3506 21 H 1. 01282 107. 62882 -53. 61377 0 1408 2=.! H 1 01;;!82 107. 6288;1 -169.35513 0. 1479 H 13 ~s /H18 /21 C N 12 14 ""cv 20 "-H 22 I ~9 H0 16 8 ft9

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CHAPTER V SUMMARY AND CONCLUSIONS Three different types of chemical manipulations of dobutamine were investigated as new novel cardiotonic drugs. The designed two 6 '-substituted analogues of dobutamine, three analogues of carbostyril and two analogues of (2'-aminoethyl)-1-hydroxy-2-pyridone were synthesized by a variety of synthetic procedures. The cardiovascular effects of the 6-methoxy analogue of dobutamine were evaluated in dogs in vivo. This compound showed to be five times more potent than dobutamine in cardiac contractility and half as potent as KM-13. However, there were no significant changes in heart rate, mean arterial blood pressure and peripheral vascular resistance. Once the syntheses of the three analogues of carbostyril were completed, analytical methods were developed to test their stabilites in chemical and biological media. A high pressure liquid chromatography system was used to follow their stability in chemical and biological media. All carbostyril analogues were found to be extremely stable in pH = 7. 40 phosphate buffer, whole human blood, 80% human plasma and 20% rat liver homogenate. The in vivo cardiac electrophysiological studies in dog were performed to evaluate the effects of the carbostyril analogues on the function of sinus node and the 140

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141 cardiac conduction system. The 8-hydroxycarbostyril analogue and the 3,4-dihydro-8-methoxycarbostyril analogue showed no significant changes in the electrophysiological parameters. The overall results of the in vitro stability studies and in vivo cardiac electrophysiological studies indicate that all carbostyril analogues may have oral bioavailability and do not stimulate adrenergic receptors. Two analogues of (2'-aminoethyl)-1-hydroxy-2-pyridone were tested for their potential dopaminergic activities by measuring the inhibitory effects of prolactin secretion from the anterior puituitary in rats. The activities of these compounds were compared to the activity of dopamine. Since these compounds show significant inhibitory effects on prolactin secretion, it was predicted that these compounds may be used as starting materials for either new dopaminergic agonists and/or new cardiotonic drugs. Before the syntheses of the two analogues of (2'aminoethyl)-1-hydroxy-2-pyridone, semiempirical MO calculations(at the AM-1 level) were performed in order to understand the structural and electronic features as compared to dopamine. Based on these calculations, it was concluded that both new compounds are less lipophilic than dopamine, but that they are true isosteric-isoelectronic analogs. This project was designed to develop novel cardiotonic drugs which have long duration of action and oral bioavailabili ty. Since the carbostyril system and the 1-hydroxy-2-pyridone system showed the desired pharmacological

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1 42 activities, further chemical manipulations of these systems sould be performed to optimize the features toward safer cardiotonic drugs.

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BIOGRAPHICAL SKETCH The author was born in Masan, Korea, on March 4, 1955. He received his B.S. and M.S. degree in the Department of Chemistry, Seoul National University, Seoul, Korea, in 1978 and 1980, respectively. After he served his military duty in the Korea Military Academy as a full-time chemistry instructor for three years, he came to the United States in 1983 and enrolled in the Department of Chemistry, College of Liberal Arts and Sciences at the University of Florida. In 1986, the author transfered to the Department of Medicinal Chemistry, College of Pharmacy, University of Florida, and continued his education to obtain a Ph.D. degree. The author is a member of the American Chemical Society. He married Saun-Joo in 1983 and they have a lovely daughter, Alyssa, born in 1987. 149

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I certify that I have read this study and that i n my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality a s a dissertation for the degree of Doctor of Philosophy. Nicholas S. Bodor, Chairman Graduate Research Professor of Medicinal Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. ,,~ ~ w ~ MardH.Hammer Professor of Medicinal Chemistry I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. 'J : ,..._.., -'. I{', ..,-' ) --- I :'.:L,, Margaret o. James Associa'te Professor of Medicinal Chemistry I certify that I have read this study and that in m y opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. J'ames W. S irnpJdns/ Professor of Pharrnacodynamic

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I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentntion and is fully adequate, in scope and quality, as a dissertation for the degree of Doctor of Philosophy. Alan R. Katritzky Kenan Professor of Organic Chemistry This dissertation was submitted to the Graduate Faculty of the College of Pharmacy and to the Graduate School and was accepted as partial fulfillment of the requirements for the degree of Doctor of Philosophy. May, 1989 Dean, College of Pharmacy Dean, Graduate School