Design, synthesis, pharmacokinetic, and pharmacodynamic evaluation of a new class of soft anticholinergics


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Design, synthesis, pharmacokinetic, and pharmacodynamic evaluation of a new class of soft anticholinergics
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xiii, 124 leaves : ill. ; 29 cm.
Huang, Fenglei, 1965-
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Subjects / Keywords:
Research   ( mesh )
Cholinergic Antagonists -- pharmacology   ( mesh )
Cholinergic Antagonists -- pharmacokinetics   ( mesh )
Cholinergic Antagonists -- chemical synthesis   ( mesh )
Drug Design   ( mesh )
Drug Evaluation   ( mesh )
Department of Pharmaceutics thesis Ph.D   ( mesh )
Dissertations, Academic -- College of Pharmacy -- Department of Pharmaceutics -- UF   ( mesh )
bibliography   ( marcgt )
non-fiction   ( marcgt )


Thesis (Ph.D.)--University of Florida, 1999.
Bibliography: leaves 113-123.
General Note:
General Note:
Statement of Responsibility:
by Fenglei Huang.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
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Table of Contents
    Title Page
        Page i
        Page ii
        Page iii
        Page iv
    Table of Contents
        Page v
        Page vi
    List of Tables
        Page vii
    List of Figures
        Page viii
        Page ix
    Key to symbols and abbreviations
        Page x
        Page xi
        Page xii
        Page xiii
    Chapter 1. Introduction
        Page 1
        Page 2
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    Chapter 2. Research design and specific aims
        Page 39
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    Chapter 3. Materials and methods
        Page 43
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    Chapter 4. Results and discussion
        Page 60
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    Chapter 6. Conclusions
        Page 109
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    Biographical sketch
        Page 124
Full Text







To my parents

Their love makes the impossible possible!


I would like to express my sincere gratitude to Dr. Nicholas Bodor for his invaluable support, encouragement, and guidance throughout this research. Of the professors with which I have worked, Dr. Bodor is certainly the finest. I was allowed much intellectual freedom in pursuing my research under his guidance and found great joy in doing pharmaceutical research. To him I am deeply indebted.

I thank the members of my supervisory committee: Drs. Guenther Hochhaus, Laszlo Prokai, Jeffery Hughes, and Cheng-I Wei for their advice and assistance throughout. Special gratitude goes to Dr. Hochhaus for teaching me how to do receptor binding studies and allowing me work in his laboratory. Many thanks go to Dr. Wei for his advice personally and professionally since early 1994. I can always count on him. I would like to thank Dr. Simpkins for allowing me using equipment in his laboratory. I would also like to thank Drs. Laszlo and Katalin Prokai for their advice and friendship. They made my tenure in the Center enjoyable.

Special gratitude goes to Dr. Whei Mei (Emy) Wu and Mr. Fubo Ji. They taught me how to do pharmacological studies and chemistry. Without their help, I would not have been able to finish my research. I would also like to thank Drs. Eugene Browne, Hassen Farag, Peter Buchwald, Attila Juhasz, Ming-Ju Huang, and Jiaxiang Wu for their


advice and assistance at the various stages of my program. The timely help from Elvie Guy, Kathy Eberst, and Julie Berger deserves special mention.

I would like to thank all friends and colleagues at the Center for Drug Dicovery and Department of Pharmaceutics who made this tenure pleasant and enjoyable. Special thanks go to Jeff Stark, Julie Berger, and Emy Wu for proofreading my writing.

Most importantly, I would like to thank my parents and brother for their love, encouragement, and moral support. They deserve credit for every achievement I have made.



ACKNOWLEDGMENTS...................................................... ii

LIST OF TABLES.......................................................... vi

LIST OF FIGURES........................................................... viii

ABSTRACT.................................................................. xii

I. INTRODUCTION...................................................... I

Drug Design ........................................................
The Antonomic Nervous System ....................................... 7
Radioligand Binding as a Tool in Drug Discovery .................... 19
Muscarinic receptors ................................................... 26
Pharmacokinetics of Anticholinergics................................... 28
Anticholinergics as Mydriatic Agents................................... 37

2. RESEARCH DESIGN AND SPECIFIC AIMS............................. 39

Objectives ............................................................. 39
Design of Soft Anticholinergics Based on N-Alkyl-Nortropine
Esters of 2-Phenyl-2-Cyclohexenecarboxylic Acid....................... 39
Specific Aims........................................................... 42

,. MATERILS AND METHODS ............................................. 43

Materials............................................................... 43
Methods.............................................................. 4

4. RESULTS AND DISCUSSION ................................................ 60

Synthesis................................................................... 60

Pharmacodynamnic Evaluation......................................... 65
Phannacokinetic Studies .............................................. 99

5. CONCLUSIONS........................................................... 109

REFERENCES ................................................................ 113

BIOGRAPHICAL SKETCH .................................................. 124




4-1 Binding parameters of reference compounds at four muscarinic receptor
su bty pes .............................................................................. 66

4-2 Binding parameters of soft anticholinergics at m, m,. m, and m4 receptors... 68 4-3 Receptor binding values for 9(a-b) and 13(a-b) .................................... 81

4-4 Receptor binding values fbr 15(a-b) and 18(a-b) ..................................... 87

4-5 In vitro stability of compounds 9a, 9b, 13a. and 13b in biological media ........ 102 4-6 In vitro stability of compounds 15a, 15b, 18a, and l8b in biological media .... 103

4-7 Pharmacokinetics of 13a after intravenous bolus administration in rats ......... 105




1-1 Two typical structures of classical anticholinergics ............................... 9

1-2 Structures of atropine and scopolamine .............................................. 9

1-3 Structures of some anticholinergics currently in the market ..................... 14

1-4 Hydrolysis of soft analogs of conventional anticholinergics ...................... 17

2-1 Design of a new class of soft anticholinergics based on inactive metabolite
A pproach ...................................... .............................. 40

3-1 Synthesis of 2-pheny-cyclohexenic acid ............................................... 45

3-2 Synthesis of 9a, 9b, 13a, and 13b ..................................................... 46

3-3 Synthesis of I5a. 15b, 18a, and 18b ................................................. 52

4-I Binding isotherms of classical anticholinergics .................................... 67

4-2 Structures of soft anticholinergics for the receptor binding studies ................. 71

4-3 Correlation between pki (M3) and pA ............................................... 76

4-4 Calculated versus experimentally measured m3 pKi data for inactive
metabolite-type soft compounds containing a tropine moiety ............ 79

4-5 Binding isotherms of 9a displacement of specific [3H] NMS binding
to MI InM2, M 3, m4 muscarinic receptor ....................................... 84


4-6 Binding isotherms of 9a displacement of specific [3H] NMS binding
to mI, M2, MI3, m4 muscarinic receptor .................................... 85

4-7 Comparison of AUC,4hr of soft anticholinergics with AUC4hr of
atropine and tropicam ide .................................................. 90

4-8 Time course of mydriatic response (treated eye) for atropine sulfate.
tropicam ide, 9a, 13a, and 13b ............................................. 91

4-9 Time course of mydriatic response (treated eye) for atropine sulfate,
tropicam ide. 15a. 18a, and 18b ........................................... 92

4-10 Time course of mydriatic response (control eye) for atropine sulfate,
tropicamide. 9a, 13a, and 13b after unilateral administration ......... 94

4-11 Time course of mydriatic response (control eye) for atropine sulfate,
tropicamide. 15a. 18a, and 18b after unilateral administration ......... 95

4-12 Brandycardia protective effects of 9a, 13a. and atropine MeBr as
illustrated by percentage change in heart rate ............................. 97

4-13 Brandycardia protective effects of 15a and atropine MeBr as
illustrated by percentage change in heart rate ............................. 98

4-14 Pharmacokinetic studies of 13a by intravenous injection at 5mg/kg, 10mg/kg,
and 15m g/kg dosage ...................................................... 106



AUC area under the curve

0C degree centigrade

CDC1, deuterated chloroform

cm centimeter

g gram

hr hour

HPLC high performance liquid chromatography

i.v. intravenous

m,4 cloned human muscarinic subtype receptor (1-4).

M,3 muscarinic subtype receptor 1-3 from animal tissue (1-3)

mg milligram

mmin minutes

mmol millimoles

mp melting point

NMR nuclear magnetic resonance


pA, an empirical parameter defining the negative logarithm of the
molar concentration of the antagonist which produces a two-fold
shift to the right of a concentration-response curve PPM parts per million

QSAR quantitative structure activity relationship

r I regression coefficient

SEM standard error of mean

t 1 halff life

TLC thin layer chromatography

vs versus

w/v weight/volume

P9 microgram

PI microliter


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


Fenglei Huang

December 1999

Chairman: Nicholas Bodor
Major Department: Pharmaceutics

The present research involves design, synthesis, and evaluation of a new class of soft anticholinergics with muscarinic subtype receptor selectivity. N-Alkyl-nortropine esters of 2-phenyl-2-cyclohexenecarboxylic acid, potent anticholinergics with muscarinic subtype receptor selectivity, were chosen as lead compounds. Soft drug design approach based on inactive metabolite were applied to the design.


A receptor binding method was developed for the evaluation of the potency and subtype selectivity of soft anticholinergics using cloned human muscarinic receptors. The soft anticholinergics previously made in our laboratory were evaluated by this method.

Eight compounds were synthesized. The in vitro receptor binding studies demonstrated that this new class of soft anticholinergics were able to attain the potency of the lead compound and two of compounds showed muscarnic receptor subtype selectivity. In vivo activity of the newly synthesized soft anticholinergics was evaluated by mydriatic studies in rabbits. Two of the compounds were found to have shorter duration of action than that of tropicamide after unilateral administration of equieffective doses. The untreated eyes were found to dilate in tropicamide and other classical anticholinergics treated animals, but not in soft drug treated animals, which indicated a lack of systemic activity of the topically administrated soft drugs. The cardiac activities of the soft anticholinergics were evaluated by the ability of protecting the charbachol induced bradycardia effects. Three soft drugs evaluated possessed much shorter protective effects (about 15 to 30 minutes) than that of atropine (at least 2 hours).

In vitro biotransformation studies and in vivo pharmacokinetic studies of the soft anticholinergics demonstrated that the newly synthesized soft anticholnergics were hydrolyzed to inactive metabolites and the metabolites were rapidly eliminated from the systemic circulation.

In summary, the soft nature of the compounds, in conjunction with the subtype receptor selectivity, increased the therapeutic index of this new class of soft anticholinergics.



Drug Design

Drugs were discovered by one of the following paths: serendipity, random screening, extraction of active principals from natural sources, molecular modification of known drugs, rational drug design (Korolkovas, 1988), and combinatorial chemistry.

Retrometabolism Approaches of Drug Design

Despite attempts to introduce rational and logical processes in drug discovery, the success ratio is alarmingly low. Very few compounds with maximal or optimal activity will become clinically useful drugs; the main and most frequent reason is the toxicity of these compounds. In conventional drug design, the sole goal of the drug design is to maximize pharmacological activity of the existing compounds. Unfortunately, in most cases, the side effects are related to the intrinsic receptor affinity responsible for the desired activity. It is not surprised that with the increasing of the activity during the drug discovery process, the toxicity of the compounds increase accordingly. The therapeutic index, defined as TD5o/ED5o, is unchanged. Thus, the main objective of drug design


should not be the activity of the drug but its therapeutic index, the ratio of pharmacological activity to undesired side effects (ED,0/TD,0). How toxicity are generated during the disposition process?

Toxicity results from a combination of many processes and factors. including not only the other pharmacological effects of the drug itself, but also the various effects of its

Drug (D)

kO Delivery
Direct Elimination Process
of Unchanged
or Conjugated D Il* ...I*m
Reactive Intermediat

______ j k9...kv

D 1...D n 1 M 1...M i kj...kq /. Metabolite of
Active Analogous Activity different IC 1... .ICn
to Parent Drug from Parent Toxic Metabolites or
/ k7 krProducts

Elimination Mj ...Mq

Scheme 1. The metabolic fate of a conventional drug after administration.

metabolites, reactive intermediates, and various compounds resulting from direct interactions with cell components. Bodor (1984) has summarized in a scheme (Scheme 1). The toxic effects of a drug as resulting from a combination of factors which include all the other pharmacological effects of the drug (D) itself, the effects of the direct and indirect metabolites (Dl .. Dn. Ml I ... Mj and Mj .. Mq, respectively), reactive intermediates (1*1 I .. .l*m), and the various compounds (iC 1 ..ICn) resulting from the interactions of these intermediates with cellular components. The overall toxicity of a drug could be described as the summation of toxicity due to the drug itself. which essentially is its lack of selectivity, and the toxicity due to its various metabolic products.

Since the toxicity is generated from the metabolism of the compounds in the body, it is necessary to include metabolic consideration into the drug design process.

The incorporation of metabolic consideration. or, structure metabolism relationship into the drug design process is the fundamental principle of the retrometabolic drug design.

Retrometabolic drug design approaches include two major methods to improve the therapeutic index of a drug. One is the general concept of chemical delivery systems (CDS). A CDS is defined as a biologically inert molecule that requires several steps in its metabolic conversion to the active drug and that enhances drug delivery to a particular organ or site. This requires multiple enzymatic and/or chemical transformation.

Chemical delivery systems can be divided into several classes as follows:

(1) enzymatic physical-chemical-based CDS exploit site-specific traffic properties by sequential metabolic conversions resulting in changed transport properties; and (2) site-


specific enzyme-activated CDS exploit specific enzymes found primarily, exclusively, or at higher activity at the site of action.

Soft Drugs

Another ma or method of retrometabolic drug design is the soft drug approach. The basic principle of soft drug design is to control and direct metabolism by drug design rather than avoid it. Soft drugs are defined as biologically active, therapeutically useful chemical compounds (drugs) characterized by a predictable and controllable in vivo destruction (metabolism) to nontoxic moieties after achieving their therapeutic role.

From the earlier discussions of the metabolic fate of drug design after they enter the body, it is obvious that the main purpose of the design of soft drugs is to avoid oxidative metabolism as much as possible. To avoid oxidative metabolism, the soft drug concept advocates the use of hydrolytic enzymes to achieve predictable, controllable, and direct drug metabolism. As shown in Scheme 2. the soft drug (SD) that replaces D simply eliminates the majority of the unwanted process and deliberately simplifies the disposition of the drug. Ideally, the soft drug is inactivated in one step. It is possible for the inactive MI metabolites to be further metabolized or conjugated again to other inactive products, all of which are eliminated. Thus, the formation of active metabolites and reactive intermediates is avoided.

Approaches to Soft Drug Design

There are several potential strategies to design soft drugs. Based on the current knowledge, there are five distinct approaches for designing soft drugs. 1) Soft analogs

A soft analog is a close structure analog of a known drug, in which a specific metabolically sensitive spot is incorporated, which makes the modified drug undergo a one step detoxification.

Direct Elimination Delivery S
Processes \ k3

P e M 2 ...M k

inaciv metabolit


W k5 .. k m

Scheme 2. The soft drug concept (Bodor, 1984).


2) Activated soft compvounds

In this method of soft drug design, an inactive. nontoxic compound is activated to certain pharmacological function by introducing a pharmacophore group into its structure. The activated soft compound will release the pharmacophoric moiety in situ and reverts back to the original nontoxic compound. Thus it serves as a carrier for the pharmacophore.

3) Active metabolites

An active metabolite. preferably in the highest oxidized state, of a known drug is considered to be a soft drug, since it avoids the metabolic pathway which are highly variable among individuals and are subject to modulation by enzyme inducers and inhibitors.

4) Endogenous compounds as natural soft drugs

The human body has efficient metabolic pathways for deactivation of endogenous compounds such as steroids and neurotransmitters. Hence these compounds can be considered as natural soft drugs.

5) Inactive metabolites

In this approach, an inactive metabolite of a known drug is reactivated by structural modification (isosteric and! or isoelectronic ) to resemble the parent compound. The new compound is designed in such a way that in vivo it is metabolized in a predictable one-step degradation to the original inactive metabolite.

The Autonomic Nervous System

The nervous system in the human hody consists of two major parts: (I) the central nervous system, and (2) the peripheral nervous system. The peripheral nervous system includes the somatic nervous system, or voluntary system. which we are able to consciously control and the autonomic nervous system (ANS). which we are not able to consciously control. The autonomic nervous system coordinates activities of organs that function at the subconscious level, such as respiration. circulation. digestion. metabolism and endocrine gland secretion. fhe autonomic nervous system is divided into sympathetic and parasympathetic division. chased on where the pregranglionc nerve originates: sympathetic from thoracic and lumbar region: parasympathetic from cervical and sacral regions. The sympathetic fibers ramify to a much greater extent than the parasympathetic fibers. This diffuse discharge of the sympathetic nervous system can prepare an organism for the "'fight or flight" reaction. The parasympathetic nervous system, however, generally displays a 1:1 ratio between preganglionic and postganglionic fibers. It is prepared for the discrete. local discharge of nCurotransmitters and is primarily involved in the conservation and restoration of energy needed for everyday bodily functions. Any blocking of its transmission will have the opposite effect as activation of the ANS. In the most common situation, sympathetic division enhances the activity (e.g.. heart rate). whereas the parasympathetic system decreases the activity.

Anticholineroic Auents

Anticholinergic agents competitively inhibit the actions of acetylcholine by blocking the interaction of this endogenous neurotransmitter with its receptor. This process can occur at autonomic effectors innervated by postganglionic nerves, as well as on smooth muscles that lack cholinergic innervation. Atropine is the prototype for drugs which antagonize the muscarinic activities of acetvlcholine. Atropine and its analogs have very little effect at nicotinic acetylcholine receptor sites and thus this type of compound is referred specifically as antimuscarinic.

The structural elements of cholinergic antagonists (Wess, 1990) are

(I) a cationic "head group" which is either a tertiary base which is protonated at physiological pH or a quaternary ammonium moiety: (2) some "heavy blocking moieties." e.g.:, alicvclic or aromatic rings, for hydrophobic interaction with the receptor:

(3) an interconnecting structural element (ester or amide) of definite length: and (4) an "anchoring group." e.g., hydroxyl group(s) are often present at key positions.

Figure 1-I showed two typical structures of classical anticholinergics. where the ester oxygen and the quaternary head are separated by 2 or 3 carbon atoms, respectively. It is generally believed that muscarinic receptors required at least two carbon atoms separating the quaternary head and the ester oxygen in order to have significant binding and activity.


R-) C


Figure I -1. Two typical structures of'classical anticholinergics.

/CR3 /CH3


0- 0

ANtropi ne Scopolamine

Figure 1-2. Structure of atropine and scopolamnine.

Figure 1-2 shows the structures I atropine and scopolamine. the two most frequently used anticholinergics. They belong to heladonna drugs. .\tropine and scopolamine are organic esters formed by combination of an aromatic acid, tropic acid. and a complex organic base. either tropine or scopine. Scopine differs from tropine only in having an epoxyl group in tropine structure (Figure I-2). The intact ester of tropine and tropic acid is essential for the antimuscarinic action of atropine, since neither the free acid nor the base exhibit significant anticholinergic activity. The presence of a free OH group in the acid portion of the ester also is important for activity. Substitution of other aromatic acids for tropic acid modifies hut does not necessarily abolish the anticholinergic activity.

Pharmacology of Anticholinergic Agients

In the eve. the pupillary constrictor muscle is dependent on muscarinic cholinoceptor activation. This activation is efflectively blocked by topical atropine and other tertiary antimuscarinic drugs and results in mvdriasis. The second important ocular effect of antimuscarinic drugs is weakening of contraction of the ciliary muscle, or cycloplegia. In the Cardiovascular system, in the presence of low lose atropine (antimuscarinic agents) causes parasympathetic stimulation and results in bradycardia. Moderate to high therapeutic doses of atropine cause tachycardia. In the respiratory system, both smooth muscle and secretory glands of the airway receive vagal innervation and contain muscarinic receptors. The antimuscarinic agents cause bronchodilation and reduction of secretion. In the gastrointestinal tract. antimuscarinics reduce the volume of saliva and produce dry mouth. They also reduce both the volume and the total acid

content of gastric secretions. A\tropine-like drugs inhibit the activity of sweat glands. causing anhvdrosis.

Therapeutic Use of Anticholinergics

Antimuscarinic compounds have been used primarily in the treatment of peptic ulcer disease and irritable bowel syndrome. However, they also have been utilized effectively as bronchodilators, antiperspirants. preoperative medications to inhibit secretions of the respiratory tract. and prophylactics in the prevention of motion sickness. :\ntimuscarinics have also been employed in the management of Parkinson's disease and as ophthalmic aids.

Development of Anticholinergic Agents

The most common toxicities associated with the use of anticholinergic compounds are due to the indiscriminate binding of these drugs to all muscarinic receptors. thereby eliciting both wanted and unwanted pharmacological effects. Muscarinic receptors have four subtypes. Each subtype receptor has specific action and particular locations in the human body (For review see a later part of this chapter). Cholinergic antagonists will reduce the tone and motility of the stomach as well as decrease gastric secretion. and also produce dry mouth, blurred vision and tachycardia (Brown and Taylor. 1996). The frequency and severity of adverse effects are generally dose-related. Therefore, a reduction in dosage may attenuate unwanted side effects, but may also reduce any potential therapeutic effect. Infants, geriatrics, and patients with Down's syndrome are particularly sensitive to the actions of this class of drugs. In

addition. patients with open-angle glaucoma may he especially susceptible to increased intraocular pressure when exposed to these compounds (Greenstein et al.. 1984).

In view of the toxicities related to the anticholinergics, more potent and safer anticholinergics are needed. To address the satfetv issue. the obvious and direct approach would be searching anticholinergic agents that specifically bind to the particular receptor which elucidate the desired effects. This is a pharmacodynamic approach. The strategy to achieve this goal is to study the molecular structure of the muscarnic subtype receptor. in conjunction with studying of quantitative structure activity relationship (QSAR) of the existing anticholinergic agents in order to design new class of anticholinergic agents with muscarinic subtype slectivity. Another approach is to confine the desired anticholinergic action in the ideal location. This is a pharmacokinetic approach. The strategy to achieve this goal is through retrometabolic drug design (soft drug design).

Searching for Subtype Selective Anticholinereic Agents

A search for compounds that only hind to the receptors which are responsible for the desired effects was initiated decades ago (Korolkovas. 1988). In an effort to reach this goal. numerous compounds have been synthesized based on atropine as a lead. These are the first group of synthetic anticholinergic agents. This pioneer work has led to realization of the quaternization of the nitrogen atom of atropine changing the pharmacokinetic characteristics of the drug. This alternation allowed for the reduction or elimination of the CNS effects. However, the new quaternary ammonium compounds displayed nicotinic as well as muscarinic receptor blocking activities (Kirsner etal. 1957). In order to further increase pharmacological selectivity and decrease toxicity, many

amino alcohol esters of substituted acetic acids. not based on atropine. were synthesized. The compounds were later tested for biological activity. particularly for the clinical useful antisecretory properties.

The gastric antisecretory effects of 35 of these quaternary ammonium compounds were tested for potency and for occurrence and severity of side reactions. Parallel trends in activity and toxicity were apparent throughout the study. The drugs which displayed good antisecretory efficacy also produced moderate to severe side effects. In this category were compounds such as methylscopolamine. propantheline bromide and hexocyclium methylsufate. I unfortunately. thle use of antisecretory compounds which produced adequate symptomatic relief often had to be discontinued due to the development of undesirable side action.

In recent years. some progress has been made in the development of anticholinergics with subtype selectivity. A lifew compounds have reached clinical trials: some of them have been approved for clinical treatment for related disease (Eglen and Watson. 1996).

Pirenzepine. an antagonist with relatively high affinity for the muscarinic M, and modest affinity for the muscarinic M, receptor, is approved for clinical use in the treatment of peptic ulcer disease (Carmine and Brogden. 1985: Hirschowitz et al.. 1995). Structurally related compounds in clinical development include telenzepine and nuvenzepine (Eglen and Watson. 1996). BIBN 99. a lipophilic. centrally acting

0 N-C
(D /0
COOCIIC[IN(Cl 1,,)(I'So(' 1117)l N

Propantheline N


N-C C11,

;jo HIC-N

0-; /I-- 0 o
N //'- N I I
U o-C7C

AF-DX 116 t/;

FiLlure 1-3. Structures of'some anticholinergics currently in the market.

muscarinic M, receptor antagonist. may he uLseul in the treatment of .\lzheimer's disease, since it could reverse the auinhibitorv control of acetylcholine release (Doods et al.. 1993). Conversely, peripherally acting muscarinic M, receptor antagonists, such as AF-DX 116 (Otenzepad) may be useful in the treatment of bradycardia (Schulte et al.. 1991). Selective blockade of muscarnic M, receptors may be therapeutically useful in the treatment of respiratory disorders, such as chronic obstructive airway disease. gastrointestinal disorders. such as irritable bowel syndrome (Wallis, 1995). and urinary tract disorders, such as urge incontinence (Taira. 1972: Andersson. 1993). In terms of obstructive airway disease. stimulation ofl cholinergic nerves provides the major bronchoconstriction control of animal and human airways (Morley. 1994). Tiotropium is a novel antagonist with a preferential slow off from muscarnic M, receptor with respect to muscamrnic M, receptors (Maesen et al. 1993: 1faddad et al. 1994). Newer compounds, with selectivity for the M, and M, receptors over M, or M, receptors include zamiftenacin and the structurally related, darifenacin (Houghtm et al.. 1993: Wallis. 1995: Wallis et al.. 1995). Darifenacin is a selective muscarinic receptor antagonist in phase II clinical trial for both urinary incontinence and irritable howl syndrome (Wallis ct al., 1995). Vamicamide is also a novel compound under development for the treatment of urinary incontinence (Oyasu et al., 1994). It is selective toward muscarinic M, and M, receptors over M, receptor in vitro. In vivo, vamicamide dose-dependently inhibits spontaneous bladder contractions caused by elevation in the intravesical volume. At a 3-10 fold higher dose, no effect was seen on the contractions of stomach or colon, response also mediated via activation of muscarinic receptors. In summary, several advances have been made in the identification of antagonist that can discriminate between muscarinic receptor

subtypes. Most research have centered upon selective muscarinic M antagonist, by virtue of their therapeutic potential in the treatment of smooth muscle pathophysiology. Fo date. most of these compounds discriminate well between M, and M, receptors. and less so between M, and M, receptors.

Soft Anticholinerics

Besides through searching for anticholinergics with subtype selectivity to improve therapeutic index of the anticholinergics, soft drug design concept has been successfully applied to design sater anticholinergics (Kumar and Bodor. 1996).

Of the live basic methods for the design of soft drugs (see Soft Drug section). soft analogue and inactive metabolite approaches were the two adopted to design soft anticholinergics. Bodor et al. (1980) reported design and synthesis of a series of "soft" anticholinergics based on soft analog approach. It was generally believed that the muscarinic receptors required at the least two carbon atoms separating the quaternary head and the ester oxygen in order to have significant binding (Figure 1-1). However. Bodor et al. shortened two carbon bridge of the conventional anticholnergic agents to one carbon to incorporate a metabolic sensitive spot into the structure of "soft anticholinergics". This new series of soft analogs of conventional anticholinergics were proved to be the potent anticholinergic agents which were hydrolyzed in ivo in a predictable time frame to an acid, an aldehyde. and a tertiary amine acid (Figure 1-4).

RC / G t O OC/0 IF -, R 'C -- oii" N .....

Figure 1-4. Hydrolysis ot'f soft analogs of conventional anticholinergics.

The soft analog concept was also applied to the design of soft propantheline (Brouillette et al.. 1996). The ethylene bridge of propantheline (Figure 1-3) was shortened by one carbon to produce a new series of compounds retaining of anticholinergic activity and decreased hydrolytic stability.

Inactive mrnetabolite approach has been applied to the design of several series of soft anticholinergics. Hammer et al. (1988) used inactive metabolite soft drug design principle to design soft anticholinergics based on atropine. Hammer chose a hypothetical metabolite of atropine, an oxidation product of the primary hydroxyl group, as the lead compound. This lead compound was reactivated by esteritication with aliphatic and cycloaliphatic alcohol of varying chain length. [he resultant compounds exhibited similar activity as atropine but with less duration in the biological media. One of the resultant compounds, tematropium methyl sulfate, has actually been able to reach phase 11 human trial as short-acting mydriatic diagnostic agent. Tematropium is also currently undergoing human development as a safe antiperspirant.

The inactive metabolite principle for the design of soft drugs was also applied to design soft drugs based on methscopolamine (Kumar et al.. 1993a). Hypothetical carboxylic metabolite of methscopolamine and scopolamine were chosen as the lead

compounds. The lead compound was reactivated by esteritication with various alcohols to produce a series of soft drugs. Soft drugs of methylscopolamine were found to be potent anticholinergics in both in vitro and in vivo. All soft drugs examined were metabolically more unstable than methscopolamine in all the biological media tested, indicating that soft drugs will be cleared from systemic circulation at faster rates than methscopolamine. thereby minimizing the systemic side effects.

As a general principle. I lammer et al. and Kumar et al.'s findings indicated that the introduction of the metabolically sensitive ester function in the atropine or scopolamine analogs, resulted in soft anticholinergics with somewhat reduced intrinsic activity. For example, tematropium showed a pA, of about 0.6 to 0.8 log unit lower than that of its parent compound. Based on the observation that in the soft analog class, such as (+)-[(ct-cyclopentylphenylacetoxy)methyl]triethylammonium chloride the introduction of a cyclopentyl group in the acidic component did enhance activity (Bodor et al., 1980), our laboratory tried to introduce a cyclopentyl group into the tematropium molecule (Juhasz et al.. 1998). H owever, the resulting ethoxycarbonylphenyicyclopentylacetyl.V.N-dimethyltropinium methyl sulfate (PCMS- l) and

methoxycarbonylphenylcyclopentylacetyl-N..N-dimethyltropinium methyl sulfate (PCMS-2) were shown somewhat less potent than temaptropium. A possible reason for the resulting compounds being less potent than expected is that the introduction of the bulkier group, e.g.. cyclopentyl group. enhanced the activity of the anticholinergics, but such enhancement was compromised by the molecular volume increase: The molecular

volume is one of the major factors contributing to the activity of soft anticholinergics (Kumar et al.. 1994).

Radiolisand Binding As a Fool in Drug Discovery

The ultimate goal of' drug discovery is to develop therapeutic agents which prevent. alleviate, or cure human disease states. In order to meet this goal. an efficient and reliable pharmacological method must be establish to test the newly synthesized compounds. Historically, animal models, or systemic screening tests were heavily employed as the valid tools to predict the potential therapeutic utility. But. the systemic test has two major limitations: (1) It is not efficient. Generally speaking, it takes days or weeks to accomplish a test which is becoming a rate-limiting point in the drug discovery process. (2) It does not provide information of molecular mechanism ot drug action.

Conceptually. it is not difficult to understand why animal model basis of pharmacological tests is difficult to work with. It is the nature ofanimal models that they are complex and the dynamic arrangement of molecular mechanisms limits their ability to selectively target one mechanism of action.

Mass screening tests, most of which are based on radioligand binding, have been proved to be fast and dependable methods for screening They also provide very valuable information on the mechanism of action of the particular drug during the process of evaluation of pharmacological activity. Thus it has produced a shifting of priorities and attitudes from "systems" screening towards mass ligand screening strategies. which offer

a rapid. efficient. and reliable means or identilving compounds on the basis of mechanism of action information (Sweetam et al.. 1995).

Principles of Drug-Receptor Interactions

Our current concepts of receptors have their origins in the work of Paul Ehrlich (1845-1912) and J.N. Langley ( 1852-1926). Both Ehrlich and Langley drew attention to a most important feature of drug action chemical specificity, or mutual recognition of drug and receptors.

Not all sites with which drugs are able to hind are necessarily receptors. The current concept of a receptor is of a macromolecule with which a drug interacts, leading to a change in cellular function. Thus the concept of a receptor pharmacologically includes both the capacity to bind to or react with a drug and to mediate both the positive and negative biologic alteration in function. The connection of receptor to the inner cellular function is the integral part of this important concept. The old term acceptor has been used to describe sites to which a drug can combine but not cause a biologic change.

Affinity is used to describe the propensity of a drug to bind at a given receptor site: intrinsic activity describes its ability to initiate biologic activity as a result of' a binding. Presumably because of the complexity of the binding process, a drug may possess affinity, that is. be able to bind to a binding site. yet not initiate specific activity. But, in the current receptor binding studies, we assume that the drug with high affinity toward a particular receptor will show a high specific pharmacological activity. Two chemically similar drugs that initiate the same selective activity probably do so by acting

on the same population of receptors. If one is effective at the lower molar concentration

than the other it is said to be more potent.
Ligands for a receptor can be specified as agonists and antagonists. An agonist is a drug. hormone or neurotransmitter substance that elicits a cellular response when it combines with a receptor. .,\ full agonist is capable. at sufficient high concentration, of producing a maximal cellular response whereas a partial agonist's maximum effect is less than the maximal response of which the tissue is capable. An antagonist is a drug which prevents the effect of an agonist by combining with the same receptor without causing activation. Antagonist can he reversible or irreversible, and competitive or noncompetitive. Ligands not only differ in their ability to produce an effect upon drugreceptor complexation. but full agonists have an intrinsic activity of one and antagonist have an intrinsic activity close to zero. but also differ in their affinity for a receptor.

Kinetics of Drug-Receptor Interactions

Numerous mathematical. thermodynamic, and biochemical models have been put forth to describe the interactions of drugs with their receptors. The preeminent theory from the point of view of attempting to describe drug-receptor interactions has been the occupation theory. in which a response is thought to emanate from a receptor only when it is occupied by an appropriate drug molecule. This model is the first proposed and its historical development traces the essential elements of drug-receptor interactions. Another model termed the rate-theory, equates drug-receptor activation with the kinetic rate of the offset of drugs and describes activation in terms of kinetics rather than binding. A model that bridges these two approaches is called the inactivation model. The two-state model

suggest that the binding of a drug molecules to a receptor is not an independent process in the sense that the binding to the receptor by one drug molecule is believed to affect subsequent binding of another drug (Kenakin. 1993).

When a ligand (L) binds to a single class otf noninteracting binding sites (R). the following equilibrium exists:
KI A -1
R + L ------" RL R + L :- RL (1)

where K, is the rate constant for the association and K, is that for the dissociation of the receptor-ligand complex. The equilibrium dissociation constant for this reaction is given by.

Kd (2)

The affinity constant is defined as

Ka (3)

At the equilibrium. l- j[L K. RL]= 0 (4)

[R][L] K
which means Kd (5)

The proportion of occupied receptors can be expressed as [R][L]
[RL] [RL] Kd [L]/ Kd [L]
[Rtot] [RL]+[R] [R][L]+[R] [L]/Kd+l [L]+Kd

This means that Kd represents the ligand concentration at which 50% of the

receptor is occupied.

In order to determine the affinity of a compound for its isolated receptor, one can design an in vitro competition type of binding experiment. The receptor preparation is incubated with increasing concentrations of a nonradioactvative drug (D) and a fixed low concentration of radioactive ligand (L*). The advantage of this method is that no radioactivity need to be used in the synthesis of the compound of interested (D). In a system with saturable, reversible binding. hound radioactivity will decrease as nonradioactive drug competes with L* for a fixed number of binding sites (Rtot). For a radioactive ligand concentration, low enough to act as a tracer for unoccupied sites ([L* J
Recall. R + L *-- RL* R+ L RL

where KdKi

and the proportion of occupied receptors equals [RL*l [L*1
[Riotl IL*1+ Kd

In the presence of D. however, one additional equilibrium has to be taken into account:

R+D 2.+ RD R + D RD where Ki =K2

and Rtot= [R]+[RL*] =[RD]= [R]+IIL* [R][D] (7)
Kd Ki


So that the proportion of' receptors occupied by L* becomes

[RL*I [L*]
[RtotI [L*]+Kd(+D/Ki)

If the assay is set up under such conditions so that the receptor concentartion is

low compared to that of the ligand. then the lignd concentration [L*] remains essentially unchanged in the presence or absence of drug and/or receptor ([L*] in equation (6) and (8) become identical ). At a drug concentration of IC, where the ratio of the proportion of [RL*I in the presence of D to the proportion of [RL*I in the

absence of'D is half the following equation can be set up:

[L* I
[L*I + Kd(1 + I10 / Ki) (9
[L*] -2()

[L*]+ Kd

From this equation, we can have : Ki = I l 0)
l+IL*]! Kd (0

Thus, if we know the IC,. of the second drug (D) at the competition and the dissociation constant and concentration of the radioactive ligand. it is easv tor us to

derive the Ki (dissociation constant) of the competitive agent (D).

Basic Methodology of Receptor Binding

We have two basic methods for receptor binding studies: (1) saturation receptor inding, which requires the tested ligands to be radio-labeled, and (2) competition receptor binding. The compounds need to be tested are not radio-labeled. They compete with the known radioligand for the same binding sites of the receptors. When reaching the

equilibrium. the percentage replacement of the known radiolignd will be used to calculate the Kd (dissociation constant) ofthe tested ligand.

(1) Saturation experiment ---- finding the dissociation constant of the radioligand By definition, a saturation experiment adds increasing amounts of radioligand to a fixed amount of tissue preparation and measure of the resulting binding. The goal is to measure the dissociation constant.

B,,x *X"
K, + Pi


a: specifically bound of radioligand. [ 11] NMS. Blax: maximum binding site X: concentration of radioligand. [H]NMS K,,: dissociation constant of radioligand.

(2) Competition experiment

The most common type of binding experiment is the addition of increasing concentration of nonradioactive drug to a fixed low concentration of radioactive ligand and tissue. By measuring the displacement of the radioligand (filtration). we are able to make a curve of displacement.

%A(bound) = 00 B"
IC) + B"



A: radioligand

Kd: dissociation constant of radioligand and receptor complex. Ki: affinity constant of antagonist and receptor complex.

Muscarinic Receptors

In 1914, Sir Henry Dale discovered two different actions of acetylcholine. One was selectively mimicked by muscarine and blocked by atropine. The other was mimicked by nicotine and blocked by d-tubocurarine. This lead to the concept of different subtypes of cholinergic receptors. referred to as muscarinic and nicotinic receptors. Muscarinic receptors mediate most of the inhibitory and excitatory effects of acetylcholine (Ach) on central neurons and the majority effects of Ach in the periphery (see Buckley and Caulfield. 1992 for review). By 1980. it was realized that the muscarinic receptor-mediating action could not be accounted by a single receptor subtype. In particular, radioligand experiment demonstrated that tissue-specific differences in the affinity of the antiulcer drug pirenzinpine. suggested the presence of at least two receptor subtypes. MI and M, (Hammer et al., 1980). However, when taking account for the discrimination observed with 4-diphenyl acetoxy-methyl piperidine methiodide (4-DAMP) between the "M," receptors in heart and ileum (Barlow et al.,

1976), it was apparent that at least three muscarinic-receptor subtypes were needed to account for the data. New compounds hexahvdro-sila-difenidol (HHSiD) and its parafluoro analogue (p-F-HHSiD) were developed by Lambrecht. Mutschler, and Tacke (Mutschler and Lambrecht. 1984). The compounds exhibited a 70-fold higher affinity for smooth muscle and glandular muscarinic receptors than for cardiac muscarinic receptors in functional in vitro experiments and in radioligand binding studies. Further evidence for the heterogeneity of muscarinic receptors with a low affinity for pirenzepine was obtained from studies on potent at the inhibitory autoreceptors in the guinea-pig ileum (M_) rather than the inhibitory heteroreceptors in the rat hear (M,).

In summary, the antagonists selectivity for Mr. M,. M, and M4 are listed as follows.

M, receptors have been defined as those with high affinity for pirenzepine and low affinity for compounds such as AF-DX 116: this is not an adequate definition, as there is significant overlap between affinities of pirenzepine for M, and M, receptors. Hlimbacine is currently the only antagonist that can differentiate between M, and M, receptors. (Lazareno et al.. 1990: Caulfield and Brown, 1991: Bernheim et al.. 1992).

M, receptors are usefully defined by high affinity for methoctramine (7.9-8.3) and low affinity for pirenzepine (6.3-6.7). 4-DAMP (8.2-8.4) and Para-Fluorohexahydrosiladifenidol (p-FHHSiD :6-6.9).

M. receptors have high affinity for 4-DAMP and p-FHHSiD (7.8-7.9) but low affinity for pirenzepine (6.7 -7.1).

M4 receptors can now be defined as having moderate (binding experiments : 7.27.6) to high affinity (functional experiments: 7.7. Caulfield and Brown. 1991: Bernheim

et al.. 1992) for pirenzepine and high affinity fI r himbacine (8-8.5).

Molecular cloning of muscarinic receptors has established the presence of five receptors as ml, m2. m3. m4. and m5 (Bonner et al.. 1987: Peralta et al.. 1987: Bonner et al.. 1988). Most likely that the functional classification of muscarinic receptor (M,- M, maybe M4) is corresponded well with cloned receptors (m, -inm) (Hulmet et al. 1990: Dorje et al.. 1991: Caulfield. 1993).

M, receptor predominantly exists in the brain, which is involved in behavioral and cognitive functions (Hammer and Giachetti. 1982: Watson et al.. 1983). The heart is one of the rare tissues where only one of the subtype muscarinic receptor presents: M,. (Caulfield. 1993: Waelbroeck et al, 1989). In secretory glands the muscarinic receptors mediating enhancement of secretion are the M, subtype. M, also occurs in the smooth muscles of airways, the gastro-intestinal tract and the urinary bladder (Mutschler and Lambrecht. 1984, Doods et al., 1987). The physiological function of M, has not been elucidated yet even though the protein of M, receptor has been fotbund in the peripheral lung strip of the rabbit (Dorje et al., 1991: Lazareno et al.. 1990).

Pharmacokinetics of Anticholinervics

Although anticholinergic drugs have been used in clinical anesthetic practice for many decades, their detailed pharmacokinetics have been evaluated only during the last few years due mainly to the latest development of new analytical methods for drug determination.

Determination of Anticholinerzics in Bioloizical Fluid.

Several methods have been used to measure antichoinergics in humans (AliMelkkila et al.. 1993). Regarding atropine. a few of radioimmunoassays (RIA) with sufficient sensitivity for clinical pharmacokinetic studies have been published (Wurzburger et al.. 1977: Berghem et al.. 1980: Virtanen et al.. 1980: Ellinwood et al.. 1990). The sensitivity of such assay is to be able to reach I ng/ml. However, RIA method may have different cross reactivity to D-hyoscyamine and L-hyoscyamine from lot to lot. This could explain why the C x value of atropine with the same dose varies so much from experiment to experiment (Xu et al..1995).

Radioreceptor assay (RRA) can be applied to the bioanalysis of all anticholinergic drugs normally used in clinical practice. The principle of RRA is based on the competition between drug and a radiolabelled ligand for binding to a certain receptor. When a competitive drug is added to the mixture containing fixed amount of receptors and a radiolabelled ligand. the drug will displace a certain amount of the labelled ligand. depending on the contrentration and dissociation constant. The actual concentration of the drug can be calculated from the logit-log transformation standard line. The RRA monitors the drugs reacting with the cholinergic receptor in vitro at the muscarinic binding site and therefore, only the biologically active components of an anticholinergic agents will be measured. Thus, the concentration measured by RRA are more likely to correlate with the pharmacological effect compared with chemical methods. On the other hand, RRA has its own limitation. It measures all drugs and their components that are capable to bind to muscarinic receptors. Consequently, it can not discriminate between the parent drug


and the possible active metabolite, and the other drugs with anticholinergic activity can interfere with the assay.

Radioreceptor assay (RRA) has been successfully applied to the studies of pharmacokinetics of atropine (Metcalfe, 1981, Aaltonen et al., 1984), scopolamine (Cintron and Chen. 1987), ipratropium (Ensinger et al., 1987), oxyphenonium (Ensing et al., 1984), and tropicamide (Vuori et al., 1994). Iratropium and oxyphenonium are quaternary ammonium muscarinic antagonists.

Okuda et al. (1991) developed a high performed liquid chromatography (HPLC) method for the determination of atropine in biological specimens. The samples were extracted by methylene chloride then reconstituted with mobile phase. The retention time of atropine could be varied by either changing the acetonitrile-water ratio in the mobile phase or the pH of the mobile phase. The required sample volume was 2 ml. The detection limit was 8.5 ng/ml. It is the most successful HPLC method development for the determination of atropine in plasma. GC/MS methods has also been published (Hinderling et al..1985a: Kehe et al.. 1992) fotbr the detection of anticholinergics in biological fluid. Hinderling's methods were to extract atropine by chloroform then hydrolyze it into tropine. Subsequently, tropine was converted into its heptafluorobutyryl derivative which was measured by GC-MS. The sensitivity was 0.5 ng/ml. The methods were very tedious due to the complicated extraction and derivation methods. This method has been successfully applied to the studies of PK/PD modeling of atropine. Kehe's method has a limit ofquantitation of 1.0 ng/ml with 1 ml sample. A simple liquid chromatagraphy/tandem mass spetrometric (LC/MS/MS) method (Xu et al., 1995) was developed and validated to facilitate the pharmacokinetic studies of L-hyscyamine. This


method utilized a methylene liquid/liquid extraction and gave a limit of quantitation 20 pg/mi with 1.0 ml of human plasma. However, an expensive instrument, tandem MS. was needed.

Pharmacokinetics of Anticholinergics

Most available data of pharmacokinetics of anticholinergics are derived from human studies.

Absorption, elimination, and metabolism

The tertiary amines, atropine and scopolamine, are absorbed relatively well from the gastro-intestinal tract. An oral dose of atropine is rapidly absorbed from the mucosal surfaces and from the intestine, but not from the stomach (Beerman et al., 1971). The gastrointestinal absorption of the quaternary amines, like glycopyrrolate, appears to be slow and erratic (Ali-Melkkila et al.. 1991). It is likely due to the fact that it is difficult for the positive charged quaternary amine to cross the biological membrane.

The various chemical analytical methods used have resulted in a highly variable picture of the metabolism and excretion of atropine (Wurzburger et al, 1977). Atropine and glycopyrrolate are excreted to a great extent as unmetabolized parent agent and/or as pharmacologically active metabolites capable of binding to muscarinic receptors in vitro (Ali-Mekkila et al., 1990; Ensing et al., 1984: Kentala et al., 1989).

Fifty-seven percent of atropine (gas chromatographic-mass spectrometric assay) excreted in the urine as unchanged drug, and 29% was excreted as an inactive metabolite, tropine (Hinderling et al., 1985a and Hinderling et al., 1985b). Kaiser and McLain

(1970) tbund that thirty percent of atropine was metabolized (tropine-labelled as ["C]atropine).

Intravenous administration

Atropine has a short distribution half-life of approximate I min and there is a rapid decline in concentration within the first 8-10 min. It has been calculated that the amount of atropine remaining in the circulation at 10 min after iv. injection corresponds less than 5% of the administered dose (Berghem et al., 1980). Atropine is widely distributed in tissue, the apparent volume of distribution (VP) is over 1 I/kg (Adams et al, 1982: Aaltonen et al., 1984: Hinderling et al., 1985a: Hinderling et al.. 1985b: Thiermann et al., 1996). The total clearance is 10 to 15 ml/mim/kg. The half life is about 2 hours (Ali-Melkkila et al.. 1993). However, the different method for the determination of atropine in plasma resulted in significant difference in the pharmacokinetic parameter (Aaltonen et al., 1984: Kentala et al., 1990: Thiemann et al., 1996). The radioreceptor assay (RRA) only detects the active 1-hyoscyamine. on the other hand, the radioimmnoassay (RIA) and other chemical chromatography methods determined both dand I- hyoscyamine. thus, the plasma concentration determined by RIA is significantly higher than that of RRA. So is AUC. As a result, the clearance and volume distribution calculated from RRA are significantly higher than that of RIA. So, one should be cautious when comparing the pharmacokinetic parameters determined by one method to another method. This is particularly important when new sensitive chemical analysis (HPLC-MS/MS) is replacing the traditional bioassay. Due to the different distribution behavior of d-and 1-hyocyamine, only two distinct phases were seen in the serum curve as

determined by RIA where the levels measured by RRA were well fitted by the 3comartment open model (Aaltonen et al., 1984).

A few of pharmacokinetic studies of atropine in other species were reported. Urso et al. (1991) reported the studies of pharmacokintics of atropine after i.v. in rats with RRA. They found that the clearance was 58 ml/min/kg, and the volume of distribution of central compartment was 3 1/kg. Thiermann et al. (1996) studied pharmacokinetics of atropine in dogs with RRA method to determine the concentration of atropine in plasma. The clearance was found to be 44 ml/min/kg, which is much higher than the value of 13.5 ml/min/kg reported by Wurzburger et al. (1977). It was believed (Thiermann et al., 1996) that the difference is due to the different methods for the determination of plasma concentration of atropine. Wurzburger et al. utilized RIA method to detect plasma concentration of atropine. Moore et al. (1991) has studied pharmacokinetics in sheep. Unfortunately, they did not report the weight normalized clearance and other pharmacokinetic parameters.

Integration of Pharmacokinetics and Pharmacodvnamics.

Despite the clinical use of antimuscarinic agents for many years. relatively little data is available on the relationship between their pharmacokinetic and pharmacologic activity. It is well known that antimuscaric agents have numerous pharmacological activity. Integration of heart rate, saliva flow, and CNS activity with atropine pharmacokinetics have been attempted, but not a solid PK/PD model related to these group of agents have been established.

Hinderling et al. (1985a; 1985b) applied a data analysis approach, which


simultaneously fitted the pharmacokinetics and pharmacodynamic data to an integrated kinetic-dynamic model employing the digital computer program TOPFIT. It was assumed that atropine was pharmacologically active, but any of its metabolite was inactive.

The kinetic part of the model consists of a linear intravenous infusion twocompartment model with central (P) and peripheral (tissue) compartment (T), with elimination from P. The dynamic part of the model comprised two effect compartments (Thr and Tsf) which are kinetically undistinguished from the peripheral compartment (T). The intensity of the heart rate response (increase), Ehr, and the saliva response (decrease), Ef, are proportional to the amount of drug in the peripheral compartment, T, in accordance with the empirical equation:

Eh, = (E max- Eo)h, ( r)h, + Eo, hr

= Eo,..t *( ),

where Ea=,, T(501, and Eo correspond. respectively, to the true maximum effect. the amount of drug in the peripheral compartment evoking a one-half maximum response, and the baseline effect. y is the Hill coefficient.

The results demonstrated that the drug effects were proportional to the amounts of atropine in the peripheral compartment. The author found that a statistically significant positive linear correlation between the mean heart rate and the mean amounts of drug in the peripheral compartment.

Ellinwood et al. (1990) designed two experiments to examine the pharmacokinetic-pharmacodynamic relationship for the central nervous system and

peripheral effect of atropine. The central nervous system tests included wheel tracking, a coordination task, digit symbol substitution. a memory-psychomotor speed task, the physiological variable was the heart rate. They found that changes in plasma atropine levels and heart rate showed positive correlation. In contrast, the CNS activity were not correlated with plasma concentration of atropine.

Scheinin et al. (1998) developed a new PK-PD modeling to elucidate the time course and concentration-effect relationship of parasympatholytic effects of three anticholinergic drug using spectral analysis of heart rate (HR) variability. The concentration of the anticholinergic agents in plasma were determined by radioreceptor assay (RRA), and the pharmacokinetics of three agents were adequately described by a two-compartment open model. A hypothetical effect compartment body model was proposed to link the pharmacokinetic and pharmacodynamic part the models (Scheme 13). The classical parametric approach based on a hypothetical effect compartment linked to the central compartment by a first-order process was used. The concentration of the drug in effect compartment was fitted to a sigmoid inhibitory effect model with baseline effect using the individual PK parameters as constants. Equation I (effect site concentration) and equation 2 (effect) were fitted together using the nonlinear regression program PCNOLIN.


P E~- B --: Effect

-- - - - -----PK PD

Scheme 1-3. A PK/PD model linked by a hypothetical effect compartment (E). T: tissue compartment, P: central compartment.

-ai -JlkI -ke0t

C Dko, (k,,-a)ea (k21-f)e (k2,-keo)e
C *[+ + ] (1)
Vc (-a)(ko-a) (a -)(k, -) (a-ko)(R-ko)

Ema C(2
E = EO '" (2)
EC5o + C

where C, is the drug concentration at the effect-site. D is the dose. K,) is the equilibration rate constant. V, K,, c.c, and 3 are the modeled PK parameters, t is time after injection, E is the effect, Eo is the baseline. E,, is the maximal effect. ECs0 is the concentration at the 50% of E., and y is the sigmoidicity (Hill) factor.

Anticholinerzics as Mvdriatc Aents

Autonomic Systems in Eye

The iris-ciliary body is composed of smooth muscles which are innervated by the autonomic nervous system. The iris is a forward extension of the choroid and arises from the anterior face of the ciliary body. The pupil forms a central aperture in the iris. Two different types of muscle layers are contained in the iris. These are dilator and sphincter muscles. The dilator muscles receive sympathetic innervation. An increase in activity of these muscles produces mvdriasis. The sphincter muscles receive parasympathetic innervation. An increase in activity of these muscles produces miosis. The ciliary muscle is composed of smooth muscle units which receive parasympathetic innervation. Stimulation of these muscles causes contraction, which in turn results in a lessening of the tension on the suspensory ligaments which causes lens to change shape so that it becomes more convex and allows near objects to be brought into focus at the retina.

Clinical uses of mydriatics include their diagnostic use for accurate examination of retina and optic disc and in the treatment of acute iritis, iridocyclitis. and keratitis (Moroi and Lichter, 1996). Complete cycloplegia may be useful in certain clinical use.

Anticholinergics as Mydriatic Agents

Atropine, scopolamine, homatropine, cyclopentolate and tropicaminde represent the anticholinergic drugs currently marketed as mydriatic-cycloplegic agents. These agents are used in ophthalmic procedures for the examination of the interior of the eyes.

However, when these agents applied as eye drops into the eye, side effects resulted from drainage of the agents into systemic circulation could be serious, sometimes dangerous. At least 6 deaths have been attributed to the ocular administration of atropine (Frunfelder, 1989). Various psychic disturbances have been reported to the application of atropine (Wright, 1992), scopolamine (Birkhimer et al., 1984), and cyclopentolate (Khurana et al., 1988) as mydriatic/cycloplegic agents. Even for the relatively safe agent tropicamide, a myasthenia gravis-like syndrome has been described after topical application (Meyer et al., 1992). Anticholinergic eve drops have long been regarded as important household poison because accidental ingestion of small quantities can produce a severe atropinepsychosis and are particularly dangerous in children (Westfall, 1983). Another drawback of these agents are their long duration of action. In clinical setting, the time for the patients to recover from the myriatic/cycloplegic action of this agents range from six hours (tropicamide) and several days (atropine) (Brown and Taylor. 1996). Because of the prolonged recovery time from the effects of diagnostic mydriasis, many patients are reluctant to undergo diagnostic mydriasis during an office visit (Steinmann et al.. 1987).

It is imperative that a new class of mydriatic agents, which are safer and shortacting, are needed to be developed.



The main objective of the work described in this dissertation was to develop a novel soft anticholinergics. This new class of soft anticholinergics are expected to fulfill the requirement of the soft drug: potent agents with controllable and predictable metabolism. This new class of agents are expected to be locally active but systemically inactive. They could be useful as mydriatic agents or safe antiperspirants.

Design of soft anticholinergics based on N-alkvl-nortropine esters of 2-phenvl-2cyclohexenecarboxylic acid

N-alkyl-nortropine esters of 2-phenyl-2-cyclohexenecarboxvlic acid have the general structure as I (Figure 2-1). Originally, they are synthesized as bronchodilator agents (Turbanti et al., 1992). Pharmacological studies have demonstrated that some of the compounds showing high anticholinergic activity (Turbanti et al., 1992). Receptor binding and functional studies indicated that one of the compounds, ()-3[[(2-phenylcycloexene-1 -yl)carbonyl]oxy]j-8,8-diethyl8-azoniabicyclo[3.2.1 ]octane iodide, is the potent and selective antagonist for the M3-receptor subtype (D' Agostino et al., 1994).



Sytithetic rcacti,,atimi 11 (Hypothetical Macti e metabolite) III (Soft Druus)

HOOCII,)C, N "Cl-li f poclic-, C, I I

SvIlthetic reactivatiOll In vim mciabollsill Ila (Hypothetical Inactive iiietabolitc) Ilia (Sok DrUUS)

Rj= (- CA-1, Figure 2-1. Design ot'a new class ot'soft anticholinci-gics based on inactive metabolite.

Ihese characteristics prompt us to investigate the possibility to design safe antiperspirant and/or mnvdriatic agents based on N-alkyl-nortropine esters of 2-phenyl-cyclohexenic acids.

It is expected that. like all other anticholinergics. prolonged utilization of Nalkyl-nor-tropine esters of 2-phenyl-cyclohexenic acids as antiperspirants will result in unwanted systemic side effects (see Introduction).

It is desirable to design soft anticholinergics based on N-alkyl-nortropine esters of 2-phenyl-2-cyclohexenecarbaoxylic acids. 'he inactive metabolite approach for the design of softi drugs advanced by lBodor ( 184) and adopted by Hammer et al.(1988), Kumar et al. (1993a; 1993b) is applicable to the design of soft drug based on N-alkvlnortropine esters of 2-phenyl-2-cyclohexenecarboxylic acids. A hypothetical metabolite II (Figure 2-1) is chosen as lead metabolite for the design of the soft drugs. Even though II has not been detected as the metabolite ofI N-alkyvl-nor-tropine esters of 2-phenyl-2cvclohexenic acids. it is a logical choice as a lead compound. since it is the highest oxidized state of the axial group of N-alkvl substituent. which will ensure to "avoid oxidation during the in vivo metabolism as much as possible" (Bodor. 1990). The designed soft drugs are expected to hydrolyze rapidly to III in vivo. Another hypothetical metabolite Ila is an isomer of II. Hla has the axial instead of equatorial position of the R group. The smaller the size at equatorial N-substituent of N-alkyl-nortropine esters of 2phenyl-2-cyclohexenecarboxylic acids, the more potent the compound is (Turbanti et al.. 1992). It is expected the soft drug based on Hla as the lead will be less potent than II The hypothetical metabolites are expected to be highly polar and ionized at physiological pH. and thus be subject to facile elimination from the systemic circulation either directly

or after coniugation. Strong nucleophilic groups are shown to he present at the muscarinic receptor site (Sokolovsky. 1984). Here is a possibility that the carboxviate metabolites (11 or fla) that results from the hydrolysis of the soft drugs will have an unfavorable interaction with the receptor site are less active than the original soft drug. Fhe hydrolysis is expected to be facile in the skin due to the abundant presence of nonspecific esterase in skin. Ihlius a shorter local action with potentially reduced systemic side effects can be visualized with these soft drugs.

Specific .\ims

The aim of these studies is the development of a new class of soft anticholinergics. This new class of sott anticholinergics are expected to be active locally at the site of application but are hydrolyzed in a facile manner in the systemic circulation to an inactive polar metabolite. Fhe therapeutic index of this new class of soft anticholinergics should be increased. The experimental protocol is as follows:

1. Synthesis of two series of soft anticholinergic agents.

2. Development of a suitable analysis system to evaluate the stability and the metabolic

pathways of the soft drugs synthesized.

3. Evaluation of in vitro stability in various biological media.

4. Evaluation of in vitro pharmacodynamic activity by receptor binding studies. Evaluation of in vivo activity in suitable animal models.

6. In vivo pharmacokinetic studies of selected soft anticholinergics.



All chemicals used were reagent grade. Pirenzipine and (-)-p-fluoro-hexahvdrosila-difenidol hydrochloride (p-F-lIHSiD) were obtained from Research Biochemical International (Natick, Massachusetts). N-['H]-Methyl- scopolamine was obtained from Dupont NEN Research (Boston, MA). Atropine, scopolamine, and propantheline were from Sigma Chemicals Co. (St. Louis, MO). All other chemicals were from Aldrich Chemical Company (Milwaukee, Wisconsin). Scintiverse BD and other solvents were from Fisher Scientitic Co. (Pittsburgh, PA). .A\ll melting points were recorded using Fisher-Johns melting point apparatus and are uncorrected. NMR data were recorded with Varian 300 NMR spectrometer and are reported in parts per million (6) relative to tetramethylsilane. Akll compounds were dissolved in CDChI The elemental analysis were carried out at Atlantic Microlab. Inc.(Atlanta, Ga). Thin layer chromatography was carried out using EM Science DC-Plastic foil plate coated to a thickness of 0.2 mm with silica gel 60 containing florescent (254) indicator. Column chromatography was performed with silica gel (70-230) with appropriate mobile phase. All the animal studies were conducted in accordance with the guidelines set forth in the Declaration of Helsinki



and The Guiding Principles in the Care and (Ise oI Anilals (DHEW Publication. NIHi 80-23). The following strains of animals were used in the studies: ( I1) male New Zealand albino rabbit weight 3 kg and (2) male Sprague Dawlev rats weighting 250-300 gms.


Synthesis of soft Anticholinertics Based on l'ropvl 2-Phenvl-2-Cvclohexen- I ('arboxvlate (9a. 9b. 13a, and 13b) (Figure 3-1 and Figure 3-2)

Protection ofethyl 2-oxo-cvclohexanecarboxviate ( I)

A mixture of ethyl 2-oxocvclohexanecarboxvlate (12 g 0.0705 mole), 1,2 diethanol (10.9 g, 0.178 mole), PTSA (205mg 1.06 mole) in 300 ml of benzene was retfluxed with stirring for 20 hours. The mixture was diluted with Et,O. the organic layer was washed with saturated NaHCO, (2x 100 ml.). brine (2x 100 ml.) and dried over Na,SO, Ether was removed under reduced pressure to afford an oily crude product. which was Jistilled at 7("-75"C (0.35 mm I 1) to give ethyl 2-ketalcyclohexanecarboxvy late.

Reduction of ethyl 2-ketalcvclohexanecarboxv late (2)

A mixture ot'f 200 ml of absolute ether and 100 ml of 1.0 M LiALH, in THF was stirred under argon in ice bath for 30 minutes. To this mixture a solution of 2 (17.5g,

0.0816 mole) in Et,O (100 ml) was added dropwise with stirring. When the addition was



PSTA benzene
00 C

Sio, oxalic acid CHCl-,

COOH Na-,CO; I k0l i C6H MgB, CHOH


('6H KMn04 R.T.


85:15/acetic acid:li-)SO4

" OOH 611

Figure 3- 1. Synthesis of 2-phenyl-cyclohexenic acid.

c 00If
tropm\ 1-h ','I
7 8
CI I iCI IC'K)C'( Al CI N Clcf Icl [-,(,I


c R 11,

%leol I h: CA 1,


N c 11, c o o O-C, fircl Icool ,


,,IeSo, ('111" N 'CHICOOR,

a: CI 1; clilso,- h: CA 1,


Figure 3-2. Synthesis of9a, 9b, 13a, and 13b.


ended. the ice bath w\as removed, the mixture \\5as aillowcd to warm to r.t. and stirred for 17 hour. Fo the stirred mixture was added slowly \ater 13.8 mil). 15%o NaOH (14mi). water (14 ml), and anhydrous NaSO, (60 g) in that order. The mixture was filtered and the solvent removed under reduced pressure to give colorless liquid, which was distilled at 65 70C (0.2 mm lHg) to afford 2-hvdroxmethlvlcvclohexane ethylene ketal.

Deketalization of 2-hivdroxvmethvlcvclohexane ethylene ketal (3)

To 105 g of SiO, in 700 ml of CI ,C('L, was added dropwise 1.5 g of oxalic acid in 14 ml of 1,() with continuous stirring until it mixed in about 15 minutes. To the above mixture, 16 g ot" 2-hydroxymethylcyclohcxane ethylene acetal (3) was added with continuous stirring at r.t. for 3 days. Distillation of the product at 70"C (0.3mm Hg) to give 2-hydroxymethylcyclohexanone (4)

(irinard reaction with 2-hvdroxvmethvlcvclohexenone (4)

To a well-stirred cold mixture of 2.0 moles of anhydrous phenyvlmagnesium bromide in 750 ml of anhydrous ether under argon., was added dropwise (4.09 g (0.5 mole) of 2-hydroxymethylcyclohexenone (4) in 100 ml of dry absolute ether with stirring on ice bath. The addition required one hour: stirred with cooling in ice was continued for an additional one-half hour. During this time a crystalline complex separated. The remaining mixture was then decomposed by pouring onto ice-cold saturated ammonium chloride solution. The ether extracts (crystalline complex) were dried over sodium sulfate and concentrated under reduced pressure to give crude product. which was distilled at 126-1280C (0.1-0.12 mm Hg) to afford a thick oil. The oily product was brought to

crystallize in hexane t45 mil) and ether (5 mi) to generate I-phenyi-2-hydroxvmethylcyclohyxanol.

()xidizing 1-phenvl-2-hvdroxvmethvlcvclohvxanol (5)

To a solution of potassium permanganate (10 g, 0.032 mole) and dry sodium carbonate (5 g) in 500 ml of water. was added 5 g (0.024 mole) of finely powered 1phenyl-2-hyroxymethylcyclohexanol. The suspension was stirred at the room temperature for 21.5 hours. The manganese dioxide was filtered off and the filtrate was decolorized with sodium hisutite and then acidified with concentrated hydrochloric acid. The above mixture was filtered to yield 2-phenyl-2-hydroxycyclohexane- I -carboxvlic acid (6)

Dehydration of 2-hvdroxv-2-phenvlcvclohexane-1 -carbarboxvlic acid (6)

2-iHydroxy-2-phenylcyclohexane-l-carboxvlic acid (6,l g. 4.5 m mole) was added to a mixture of sulfuric acid (1.5 ml) and acetic acid (8.5 ml ) with stirring at room temperature. Fhe mixture was stirred for another 15 minutes to dissolve all solids then poured into 100 ml of ice water with stirring. .\After white crystallized solids appeared. the mixture was continuously stirred for another 30 minutes. After stored in -20C refrigerator overnight, the above mixture was filtered to give 2-phenyl-2-cyclohexen-lcarboxylic acid (7)

Synthesis of 2-phenvi-2-cvclohexene- I -carboxvlic chloride

To 2-phenyl-2-cyclohexen-l- carboxylic acid (7, Ig .4.93 m mole) in 15 ml of absolute ethyl ether with one drop of DMF. thionvl chloride (0.65g, 5.43 m mole) was


added. Ifhe above solution was refluxed LIndcr alr.on fr 2 hours. Solvent and excess ether w\as removed under reduced pressure. Agree portion o) 10 mi of dry henzene were added and removed under reduced pressure to give reddish brown oily 2-phenyl-2cvclohexene- I -carboxvlic chloride.

Synthesis of tropyl 2-phenvl-2-cvclohexen- I -carhoxvlate (8)

To tropine (1.32 9.38 m mole) in 10 ml of anhydrous FHF. was added by syringe butyl lithium (4.69 ml of 2 M hexane solution. 9.38 m mole) at OT'C under argon atmosphere. Then the mixture w\as stirred at room temperature Ifor 30 minutes. Fo the above solution was added 2-phenyl-2-cyclohexene- I -carboxylic chloride in THF at 0 C. The mixture was stirred at room temperature for I 9 hours. The above residue from evaporation of solvent was acidified by adding I N HCI until pH-I 2 and extracted with ether twice. Aqueous phase was basified with NaHCO, until pH 8 and extracted with ether again. ()rganic layer was separated. 1L\aporated to give yellowish oil. which was purified by flash chromatography on a silica gel (methanol: NH,01=100_2.5) to yield pure 8

Synthesis of 2-phenvl-2-cvclohexen- I -carb-N(-methoxycarbonvlmethvltropinium bromide (9a) and 2-phenvl-2-cvclohexen- I -carboxvv-N3ethoxvcarbonvlmethyltropinium bromide (9h).

To 2 g (6.14 m mole) of tropyl 2-phenyl-2-cyclohexen-l-carboxvlate (8) in 20 ml anhydrous acetonitrile. 15.36 m mole of methyl bromoacetate or ethyl bromoacetate was added. The above mixture was stirred under argon for 19 hours. Evaporation of


.icetonitrile to iienerate ain oily substance. v% which \xxas Curther purified by precipitate to jive pure 9a or 9h,

Synithesis ot'nortropvl 2-phenvl-2-cyclohexen- I-carhox\'late ( II)

Fo I g (3.07 mmole) of' 8 in 10 ml ol' 1.2-dichloroethane at 0 T. 1.09g (7.63 il mole) of' l-choroethvl chiorotormate in 5 ml of I .2-dichoroethan was added dropwiselv. Fhe mixture then refluxed for I hour. Residue From evaporation of' solvent ot' the reaction mixture wvas then retluxed in methanol for 45 minutes. Removal of methanol Lender reduced pressure gave I]

Synthesis of methoxvcarbonvlmethylniortropvI 2-rhenvl -2-cvclohexen- I -carboxylate (12a),and ethoxycarbonvlmrethylnortropyl 2-phenvi -2-cvclohexen-1 -carboxvlate ( 12b)

Fo well stirred compound I I ( I ,, 3.2 1 mnmole) in 20 ml of N.N-dimethvl t'ormamide (DMF) wVith I g of' K.CO,. was Lidded 3,.21 ml mole of methyl bromoacetate or ethyl bromoacetate. The mixture was stirred under argon for 20 hours. Then the DMvF wvas removed Under reduced pressure. The residlue waLs added with 5 ml1 saturated Nai-CO, solution. which was then extracted 3, times with absolute ethyl ether to give crude 1 2. 1 2 was further purified by flash chromatography onl ethyl acetate to give pure I 2a or 12b

Synthesis of 2-Phenvl -2-cvclohexen- I-carboxvl -Nx-methoxvcarbonvlmethvltropinium mnethvlsulfate (13a) and 2-phenvi -2-cvclohexen- I-carboxyl -Naxethoxycarbonvlmethyltroviniuum methvlsul fate (1 3b).


Fo compound I'2 (a or h. 2.50 11moi III () m i ol' anhvdrOUS acetonitrile was added dimethvl sultate (0.788 25 mi nole). !'he miiXture w as stirred at room temperature tbr 15 hours and then acetonitrile wa S removed tinder reduced pressure. The residue was purified by adding methylene chloride-dissolved mixture dropwise into ethvl ether to give precipitated I ',a and I 3 h.

Synithesis of' Phenvlcvclorentvl Acid Series ol'New (Class of* Soft Anticholinenztics I 5a, I la~and I18b) (liiure 3-3).

Synthesis of tropvi (t-plhenvlcvclorpentzineacciaite ( 14)

To (x-phenvlcyclopeniianeacetic acid _ '79mmol ) in 15 ml of' absolute ethyl ether. I drop of'N. N-dimethyl formamide and thionyl chloride (I .28g. 10. 77 mnmol) were added at room temperature. The mixture was refluxed for 2 hours. Then the ethyl ether wvas removed under reduced pressure to live (III> (i-phenvlcyclopentaneacetyl chloride. Fo tropinec hydrochloride salt ( 10.77 mmol 1) 9 1 in I5 mnl of' itromethane was

dddthe above oily (-phienvIcv-clopenitancct% l chloride In 5 nil of'itrornethane. I-hen the mixture retluxed f'or 214 hours. Removal of itrorrethane to give oily Substance. which was basitied with NaHICQ.The mixture was extracted 3 times with ethyl ether to give crude tropyl (x-phenylcyclopentaneacetate. which was further purified by flash silica

chroatoraphy on silica gel (Methanol -.Nl14,0-1= 100:2.5) to gv uetoy x

phenylcyclopentaneacetate (14)

Synthesis of Phenvicvclopentvl-N13-methoxvcarbonvlmethvlItropinum bromide (1 5a) and Phenvlcvclop~entvl-N13-ethoxvcarbonvlmethvltropinum bromide (I 5b).



H --c

If,CCII()C- lir 14
RO ()('C'l 1,, 11;



o-C b:

('H,\., ,lcll,cool )

Figure 3-3. Svnthesis ot'l 5a, 15b. 18a. and 18b.


Fo 2 c (6.10) mmob) tropyl (t-phenvic clopentaneacetatc ( 14) in 2) iii anhydrous acetonitrile. 15.26 mmol of' methyl bromoacetate or ethyl bromoacetate wvas added. Thie above mixture was stirred under argon pressure thOr 19 hours. L'vaporation of acetonitrile io generate an oily substance. \Tilch w as I'Lrthier purified hy precipitate (methylene chiloride/ethyl ether) to give pure 15Sa or I 5h.

Synthesis of'rnortropvl ut-phienvlcvclopenitanca~cetate (1 6)

lo I 03.05 m1molc ) ol trophy Lt-phienvc\vcioperitanieacetate In I () nil, of' 1.2dichioroethane at 0 TC. 1 .09 g(7.63 in mole) of' I-chioroethlv ciorof'ormate in 5 mlf of' I .2-dichoroethan was added dropwisely. 'Fhe mixture then retluxed Cor I hour.

FvNaporation of the reaction mixture in vacuum to Live oily residue, wich was retluxed in miethanol for 45 minutes. Removal of' methanol under reduced pressure gave 16.

\lethoxvcarbonvmiethvlniortropvlI-NUt-pheni' Ic\vclonenitanecacetate i! 7a) and AhLloxv-carihon\vlmieth\vlnortropvl-N(t-phieni,cvclonenitaneacetate ( 17h1

1'o well stirred Compound 10 (1(-. 3 19 mole) in 20ni1 of' N.N-dimethyl lormarilde ( DMF) with KCO, g., was added 319 nmmdc of meithyl bronioacctate or ethyl bromoacetate. The mixture was stirred under argon pressure for 20 hours. Mhen the DMF was removed under reduced pressure. Thie residue was extracted 3 times with absolute ethvl ether to give 1.12 gof' crude 17. which was further purified by flash chromatography on silica gel with ethyl acetate to give pure I17a ( 0.98 g, 79.7% ) or I 7b (1.0 1 g.79.2%)


IhenvIcvclopentvl-Nct-rnethoxvcarbonvlmethvItropium nethvlsulfate I Sa) and Ilhenvicvclopentvl-Nc-ethoxvcarbonvlmethvlitropium methvlsulfhte ( 18b)

Fo compound 17 (a or bh. 2.50 m mole) in I () mi of anhydrous acetonitrile was added dimethyl sulfate (0.788 g. 6.25 m mole). l'he mixture was stirred at room temperature for 15 hours and then acetonitrile was removed under reduced pressure. The residue was purified by adding dropwise methvlene-dissloved mixture into ethyl ether to give precipitated I a18 or I b .

In, vitro Pharmacodvnamic Evaluation of the A\ctivitv of the Soft AnticholinergicsReceptor Binding

Methods for receptor binding studies of soft anticholinergics

Bindingi studies were performed with 1'Ill-methyl-scopolamine following the protocol from RBI Co. (Natick, Massachusetts). Binding buffer (Phosphate Buffered Saline-PBS. pH 7.4) consisted of )0.15 M Nati. 1.5 imM KI IP,() and 2.7 mM Na.IPO, 10) mM NaF was added into the buffer as an esterase inhibitor. The assay mixture (I ml contained 100 pl diluted membranes (receptor proteins, final concentration: ml. 25 ptg/ml: m2.42 ptg/ml: m3.15.9 pg/ml: m4. 20 tLg/ml). The final concentrations of NMS for the m2-m4 binding studies were 0.5 nM. and for m I was I nM. Specific binding was defined as the difference in ['-I] NMS binding in the absence and presence of 1 pM atropine. Incubation was carried out at room temperature for 60 minutes. The assay was terminated by filtration through a Whatman GF/B filter (presoaked with 0.5%


povlyethvleneimine). IThe filter was then washed three times with 10 ml ice cold binding suffer, transferred to vials and 10 ml of scintiverse liquid were added. Finally. detection was performed on a Packard 31800 liquid scintillation analyzer (Packard Instrument Inc.. Downer (irove. IL).

Data analysis

Data from the binding experiment were fitted the following equation: 4"i1['H] NMS bound = 100- 1100x' .'k/( 1 --x" /k) to obtain I fill coefficient n. IThen to:%[H] NMS bound = 100-1100x" IC, ( I+" 'IC,)j to obtain iC.(,, where x = concentration of the tested compound (in a series concentration). Ki was derived by the method of Cheng and Prusoff (1973): Ki =IC,,/( I +L/Kj) ). where L is the concentration of the radioligand. IC, is the concentration of drug causing 50% inhibition of specific radioligand binding and K, is the dissociation constant of radioligand receptor complex. Data were analyzed by a nonlinear least-squares curve fitting procedure using the program Scientist MicroMath Inc.. Salt Lake City. i ').

In vivo Pharmacodvnamic Evaluation of the ('ompounds Mvdriatic Study

Using atropine-MeBr and tropicamide as reference compounds. the mydriatic activities of the newly synthesized soft anticholinergics were evaluated. Tropicamide ophthalmic solution (1%) was purchased from Schein Pharmaceutical Inc. (Florham Park, NJ). Healthy male New-Zealand White rabbits, each weighing about 3.0 kg were used in

dhe experiments. For studying mydriatic activities, a dose oI 100 ti of each drug in water at various concentrations was administered in one eve. and the other eve was used as a control. Experiments were carried out in a light and temperature controlled room. At appropriate time intervals, pupil diameters for both eves were recorded.

In vito Pharmacodvnamic EFvaluation: C 'ardiac Studies of Selected Solt Anticholinerics

Fhe following procedures were used: .\ntagonistic effect on carbachol induced bradvcardia.

Male Sprague-D)awicy rats (I arlan Sprague i)awlev Inc. Indianapolis. IN). each weighing 300 30 g, were anesthetized with 50 mg/kg i.p. Na pentobarbital. Baseline electrocardiograph (LCG) recordings and all drug administrations were performed after I5 minutes stabilization periods. Needle electrodes were inserted s.c. into the limbs of the anesthetized rats and were joined to a GO(I .) 2000 recorder ((GO I.D Inc.. Cleveland. )l-). Standard leads i. II. and Ill were recorded at a paper speed of 25 mmisec. Recordings were taken before, during, and aftlier the administration of any of the compounds until all basic ECG parameters returned to that of the baseline. ECG recordings were evaluated for the following parameters: PP cycle length (msec), RR cycle length (msec). heart rate (1/mrin) by the equation of 60000/RR cycle length, and presence of Mobitz II type atrio-ventricular (A-V) block (2:1. 3:1. etc.). Cholinomimnletics such as carbachol have four primary effects on the cardiovascular system: vasodilation, a decrease in cardiac rate (negative chronothropic effect), a decrease in the rate of

conduction in the sinoatrial (SA) and atrioventricular (.\V) nodes (negative dromotropic ecfect). and a decrease in the force of cardiac contraction (negative inotropic effect) (Higgins et al., 1973). To evaluate the effects of carbachol. only the negative chronotropic and dromotropic effects were analyzed here. Fhese effects of carbachol were manifested on the surface ECG as sinus bradvcardia (lengthening of the PP cycle) and as a development of Mobitz II type A-V block. After analyzing the ECG recordings, both heart rate and percent changes of heart rate. as compared to that of the baseline, were plotted against time. and the effects of the different drugs on the heart rate and on the percent changes of the heart rate were characterized. Fach point on the figures represents the mean + S.D. of three experiments. All drugs were dissolved in 0.9%0 NaCl (vehicle), and solutions were administered by direct injections into the jugular veins on either side ofthe rats. Anticholinergic drugs (such as 9a and I 3a) and atropine (0.2 and 2 ptmol/kg = 0.102 and 1.02 mg/kg. in -0.3 ml volume) or vehicle (-0.3 ml volume) were administered at (0 time. while carbachol (5 8 pIg'kg 27 44 pmolikg in -)0.06-0.1 ml volume according to the initial individual l((i response of each rat) was injected at -5. 1, ;. 5 10. I 20. 30. 45, and 60 minutes (with some exceptions). .A\nalvsis of variance followed by Duncans test was used for statistical evaluation.

In vitro Pharmacokinetic Evaluation of the Soft Anticholinercics Stability Studies

A.\nalytical Method

A high performance liquid chromatographic method has been developed to assay the soft drug The system consisted of a Spectra Physics (San Jose. CA) SP 8810 isocratic

pump. SP 8450 uvivis detector with detection set to 254 nm. and an SP 4290 integrator. A Supericosil LC ABZ column (Supelco, B3ellofonte. PA) was used, and the mobile phase Slow rate of 1.5 ml/min/ ) consisted of acetonitrile :water (40:60), with final concentration of 0.1% octanesultonic acid. 0.2% acetic acid, and 0.1% THF.

Stability in BioloLical Media

The following biological media will be used in the study: rat plasma. rat blood. and rat liver homogenate (25%). Procedure: lo the biological medium (2mil). was added 10 il of the stock solution of the compound. and the sample was mixed. Ihe mixture was kept at 37"C while being shaken. Samples ( 100 (.l) were withdrawn at the appropriate time intervals and immediately diluted with 200 pl of ice-cold acetonitrile to stop enzymatic reaction and vortexed. The supernatant after centrifugation was analyzed by I IPLC fbr both the original compound and degradation products.

In vivo Pharmacokinetic evaluation of the Soft A\nticholineroics- In Vivo Pharmacokinetic Studies

Pharmacokinetic studies of softt anticholinergics

Once the assay was validated. The pharmacokinctics and metabolism studies of the compounds can be performed.

Rats were anesthetized by intraperitoneal injection of sodium pentobarbital (30 mg/kg). The soft anticholinergics were injected into tail vein, or jugular vein, over one minute. at a dose of 5, 10, 15 mg /kg and a dosing volume of 8 ml/kg. For the data

treatment (as bolus injection), the mild-timrne ot the infection was used as U time. The tail vein injections were conducted very carefully to assure that no leakage occurred during the injection. Blood samples. U.1 ml. were collected through the jugular vein at appropriate time intervals fbr 150 minutes. Subsequently. the urine samples were taken and animals were sacrificed by over dose ot pentobarbital.

Noncompartmental and compartmental pharmacokinetic analysis

For noncompartmental analysis. the area under the curve. AUC. of the blood concentration versus time were calculated using the trapezoidal rule, and the area from the last measurement. C,. to infinity was calculated as C,/lp, where 0 was the terminal disposition rate constant. The total body clearance. CL,, was calculated as Dose/AUC. Mean resident time. MRT. was calculated as \AUMC/AUC. where AUJMC. the area under the first moment curve, was calculated using the trapezoidal rule from the curve of"blood concentration x time vs time", and the area trom the last time point. t. to intinitv was calculated as (,li+ C,,3. The volume of distribution at the steady state. Vd,. was calculated as CI,, multiplied by MNIRT. For compartmental analysis, a pharmacokinetics analysis program. PK-Analvst (Micromath. Salt Lake City. UT) was used to assist analyses.



The physical and spectral characteristics of the compounds synthesized are given below

Ethyl 2-ketal-cvciohexanecarboxylate (2, 3g, 95 0,' ). Viscous liquid. 'H4 NMR (CDCL3): 1.21(3H, t, CHCH2), 1.4-2.0 (8H, overlapping, cyclohexyl H) 2.61-2.70 (1 H, dd, CHCO2 ), 3.90-3.98 (4H-, m, OCH2CH2O), 4 25 (2H, q, CH3CH2 ) ppm. 2-Hydroxymethvicvclohexane ethylene ketal (!, I13.2g, 0.07Gmole. Q93 %). Liquid. H NMR (CDCIh). 1. 28-1.90 (8H, overlapping, cyclohexanyl), 2.38 (I H, br, CHCOA) 3.51-3.56 (111 H. m CILOH1-), 3 66 (IH-. in. CllOH ), -3.98-4,01 (41-1 in, OCH2CH0'.)


2-Hydroxymethvicvclohexanone (4, 5.45g, 0.4 18 mole, 45 %.Liquid. 'H- NM/R (CDCI4,) 1.45-2.55 (8H, m, cyclohexanyl), 2.79 (1H, b, CHCOA) 3.57-3.90 (2H, 2xm, CH2OH) ppm.

I -Phenyl-2-hydroxymethyl-cyclohyxanoI (5, 27. 0 g, 25 q/). 'H NMR (CDC13): 1.40-2. 10 (9H,m, Cyclohexanyl), 3.40-3.55 (2H, mn CH2OH), 7.20-7,50 ( 5H, m, ph) ppm.



-Plien%,I--- -cvciohexen-1-carbON%'ilC Md (7_I (). 7 8 1 11 N M R (('D ('1,): 1. 71 -2. -5 6

ioH. in. 'Cfi,, cvc1ohexanyl), 3.09 (111. b. (11CO,). 0.22 (Il-i. t c oh x n 37.20-7.33 (51-1. ill. ph) ppm.

1'ropyi 2-pheny I -I -cvclohexen- I -carboxvi ate (S. 0,49 g. 30%). Liquid. I-FNMR

(CDCI,,): 1.20-2.27 (141-1. ill. 3CII, cyclohexcnyl. 4CII,. atropine 22.26 (31-1. S. NC140
.98-3. 10 (214. brd. trop ne's 1.5-1 1) 33,70 ( I 11. s. Cl IM), 4,87
I ( I 11, CHO), 6.22 (1 H.

cvc1ohexene"s 3-1-1). 7-25-7-34 (51-1. ill. ph) ppm.

'-I)heii, ,1-'-cvcioliexcii-i-carhx%-i-N, -iiietiiox-,,cirhotiviniettiNItror)ipium I)romide (9a) ind '--tihenvi-2-cNcloliexeii-l-clrhON- 'IV-N',i-cthoxNcarbotivii netlivitropinium bromide


Qa (2.4 81(),0 ). M.P. 170-171"('. 11 NMIZ(('DC'I;) : 1.26-2.28. 2.03-2.69 (1414. ill.

X'l 1, cyclolicxenyl, 4CI 1% Li-Opine), 3.57 (311. s. NCI lo 3.78 (41-1. s. OCI 13 and 1 -11 of cyclohexenyl), 4.59-4.091-4.02 (-'11. hr ct, tropyl's 1.5-1 h 4.84 (2 11. NCI 1,). 5.05 (1 I-L t
CI 10). o-s ( Ilt t' -'-I I ot, C%'Cloilexenyl ), 7."--."
I -_) / -,-, ( l 1. 111, 1)11) jjpjjl. FICIllelital analysis: calculated/fout-id: C, 00.25,160.06: 11. 0.74/6.S I : N. 2.9')/-87. Qh (2.2 g, 73%). M.P.186-187"(. 'If NMR(C'D(.'I;): 1.26-2.60 (1411. ill. 3CI-12

cvciohexenvi. 4CH,. atropine 1.3 1 (3H. t. Cll-)CH3). 3.37 (-')H. S. NCII,,) 3.76 (1 H. b.

1-1-1 ofcvc1ohexenyl). 4.22 (211, q, CHCl-l,,), 4.58. 4.67 (211. hr d. tropyl's 4.82

(2H. NCH,), 4.95 (1 H. t. CI-10), 6.22 (Ill. t. 2-14 of cyclohexenvi). 7.20-7.31 (51-1, m. Ph)

ppm. Elemental analysis: calculated/found. C, 60.97/61.10. 14. 6.55/6.96: N. 2.84/2.79.


Nortropyl 2-phenvi-2-cyclohexen- I -carboxvIaLe ( I I

11 (0.93. 97-/,). I-INMR (CDCI;): 1.46-2.25. 2.66-2.90 (14H. in. 3CH, cyclohexenvi.

4C'I-i,, tropine). 3.71 ( 11-1. s. I-If ofcyciohexeiiNIh. 3.80.-3.85 (2H. br d. Lropine's 1.5-1-1).

4.95 (111. t (A40). 0.2) (1 H. t. cyclohexene's 3-11). 7.23-7.36 (51-1. m. Ph) ppm.

\,IethoxvcarbonvimetiivliiortropN'I 2-pheiivl -2-cvc1ohexen-I-Carboxviate (12a) and ()-ethox ,carbonvlmethvlnortropNi 2-phenvi -'-cvclohexen-I-carboxvIate


12a (0.95. 750/0). '11 NMR (('D('I-,) 141L in. 3CH-) cyclolicxenyl. 4CFI,.

tropine),3.01-3.06('-Il.brd.tropyi"s L5-11), ',M6(-'l-l-s.Nl-l,)'.63(4H.s. 11; an

I -H ofcyclohexenyl), 4.82 (114, t, OCK 7.12-7.28 (5 H. in. Ph) ppm. 12b (0.97, 76%). 'H NMR (CDC13): 1.23-2.27 ( t4l-l. in, 3CH, cyclohexenvi, 4CH,. tropinc). 1.25 OH, t, Cli,), 3-01-3-15 (211. hr d. tropyl's 1.5-H). 3.72 HH. t. I-H of

cvclohexenvi). 4. 15-4.17 (21-1, (-1. ('H,('[ k), 4.89 (11-1. in, OCH). 7.25-7.15 (51-1. in. Ph)


'-PhenNA -2-cvc1ohexen-1-carboxvi -N(x-inethov, carbogy I methyltropini LIM inethvisultate 13a) and 2-phenvi-2-cycighexen- I -carbox -i-Not-etliox ,carbonvii-nettivitropiniuum methvIsulfate ( I 3b).

13a (1.15g, 90%). M.P.150-151"C. 'H NMR (CDCI,): 1.25-2.70 (1414. in. 3CH-,

cyclohexenyl. 4CH,, tropine). 3.18 (31-1. s, WHO. 3.66 (3H. s. CH.-S04), 3.71(114. b, IH of cyclohexenyl). 3.77 OR s. OCI-l-), 4.35-4.45 (21-1. br d, tropyl's 4.70 (211. s,

NCH,), 5.03 (IH. L OCH), 7.26-731 (51-1. in. Ph) ppm. Elemental analysis: calculated/found. C. 58.92/58.97, 1-1, 6.92/6.86. N. 2.75/2.45.


i 'b 0 A Qg. "I "I T. 109-1 70''C. 11 \\IR (Cl)(1) 1.25-2.oo ( 1411. Ill. 3CH.,

c-vclohexenvI. 4'11, LI-OPHIC). 3_401-1. N('11,). -',.7-" (41i.s. ('HSO,4 and 1-14 of

cyclohexenyl), 4.24 (211. LI. ('H,,CIi,_()), 4.40-4.54 (211, hr d. tropyi's 1.5-1-1). 4.60 (214.s.

NCH,), 5.01 (Ili. m. OCII). 0.30 (11-4. t. CVCIOIICNCn 'l 1-1-1) -17.21-7.40 (51-1. m. Ph)

ppm. Elemental analysis: calculated/flound. C. 58.82/58.77. H. 5. 15/7.08. N, 2.63/2.60.

Fropyl (-t-pheiiNIc 'cloneiitaneacetate ( 14)

14. 2.5 (-,. 78%) .'11 NMR (CD('I;): 0.9-2.1 11611, 4xiii. ((_'HICII))2C'lJ and tropyl's -111. s. Cll,,N). 2-5-2.64(111. m. (A. Pentyl 1-14).

tropyl's 1.5- 10,3.25HILd. I'h(A1).4.Q0( 111, t.('(),C1h. 7.'9-7.,6(5l-l.rn.Ph)ppm.

Phenvlcvcior)eiit,,,I-Nf -iiietiioxNcarbonNItlICLfl\'ItropilILlilI bromide ( 15a) and VhenvlcvclopeiitNI-Ni -ethox 'carboiivinieth\,Itl-OpillLlllI hromide( 15b). 15a (2.2 80% ). M.P. 178-1 79'C. 'IINMR(CDCI,;): 1,00, 1.90-2.37

116H. m, (CIiC11+Cl I and tropyl's 2. 4.6 .7 -Ill. 2.71. 2.78[l 1-1. 'xbr. (Cl-lCll))2cHj,

).2401-L d. PhCH), ,.00011. s, NCI 10, '.78(311. s. COCI 10. 4.62. 4.84(')l 1. 'xbr.,

tropyl's 1,5-11), 4.80. 4.()2('-l 1. 2d. C'I 1,0 ), 12( 111. t 1) 7.24-7.30(511.

Ar-I 1) ppm. Elemental analysis: calculated/ I'OLInd.15b 82 ') ) ). M.P. I S I I 82"C'. 'I i NM 1.00, L-10. 1,40- 1.82. 1.90-2.37

11 6H. 111. (Cll_'ClJI)2Cl I and tropyl's 2. 4. 6 .7 -111. L2S (3H. t. CIII-,Cl+,). 2,71. 2.78[l 11.

_Xbr. .(CIJCll,)2CII]. 3.24(11-1. d. PhCH). 3.60(31-1. s, NCIII,,). 4.2201-1. q, Cll,,CH-)CO,),

4.62. 4.84(214. 'xbr. tropyl's 1.5-H), 4.7. 4.82(1-11. -1d. C'OCHN ), 5. 120 14. t.

COICH), 7.24-7.30(511. ni. Ph) ppm. Elemental analysis: cal c elated/ fo Lind: C. 60.73/60.47. H. 7.34/7.36. N. 2.83/2.86.

Nortropyl cx-phenvicNclopentaneacetate (16)

0-95 9.z 09). 111 NNIR ((D(1VI; U-9-2.I 1611. i ~.l~1 and trophy's L4 ..7 -1 1]. 2. 51 2.-53 I 1-1 2b r. (Al1. Pentyl 1 -11). ,.5( I 11. d. PhCH1). 3.80. ,.95(21-1. br d.

tropyl's 1,5- 14). 5. 12 (114. t. CO-,CI 1). 7.29-7.36 (51-1. in. P1h) ppm-.

\lIethoxvcarbonvlm-ethvlinortropvl, (t-phenvIcN-cloperitaneacetate (1I 7a) and ethoxvcarbonvimethylnortropyi ut-phenvlcv-clopenitaincetate ( I 7b ) I 7a (0.98 g., 79.7") 'I1 INMR (('D(13)- U-9-2. 1 l16Of 5xi-.
NCI 12). 3.23 1II. d. PhCI )3.70 3 '11. s. ()CLI 1,). 4.95 ( 114. m. COC1 1). 7.20-7.40

(5SR. Ill. Ph1).

I 7b ( 1.0 1 g 7().2% ).11 NMIR (('DC'I;): 0.9-2. 1 1111 5xm.(H2H )C I and tropy1~s 2A6.7-H]1, 1.25 (311. s. CRO,). 5 2 7 [11- im. (CR2ICI I1,)CH11, 3. 1-3.2(211. br cf. tropyl~s

1.5-H '). 315 (2141. s. NC Fl,. 3. 23 ( 114. d. PhCH- 4.18 (211. q, OCH2CI 11) 5.-0 1 (1I

(JCIf1). 7.20-7.40 (5 11. in. Ph) ppm.

()P~hcnvlcvclopentvi-Nou-iniethox'carbon hncthvltrop un-O i inthll n1dVS tnC Ii Sa) and pt-icnvicyc opentvi -N U-CtOx NCarhon I methy It trOPI U I'leth\'l. SUI te ( I 8a ( 1.13g, 88.3%) ). M.P. 159-1 60.5C. 'If NNMR ICDCI ): 0.9-2.1j1161H. i.

(CHCH,)2CI1 and tropyI's 1. 2.5-2.7[1I1-. im.
NCH,), 3.23 (11-1. d. PhCH ) .3.64 (3141. S, (ilI;S04). 3.80 s31 ()CII;,). 4.25-4.57(21H.

hr d. tropyl's 1.5- 1-1. 4.81 (2141. brd. NCHJ,) -5.15 (1 H. t. COCH), 7.20-7.40 (5H-. m.

Ph)ppm. Elemental analysis: calculated/found: C, 5 8.68/ 58.85: It 7.28/7.29. N. 2.73/ 2'.7 1.

18b (1. 17g, 89.0% ).M.P. 1-53-1540C. '14JNMR (CDCI,): 0.9-2.1 [16H. 5Xm.

(CH)CH,)2CH and tropyl~s 2.4,6.7-14], 1.20 (3H. s. CH-3), 2.5-2.7[IH. in.

(CI-1CI )2CHI,. 3.19(3H. s. NCII). 3.23 (III. dL. PhCH ) 3.70 (31. s. CH SO4),4.22

(2H1. q. OCH2CI ). brd. tropyls 1.5- H). 4.65 t2H. m. NCII,). 5.15 (1H.

t. CO&CH), 7.20-7.40 (51-1. m. Ph) ppm. Elemental analysis: calculated/ found: C. 59.41/59.39. H. 7.48/7.54. N, 2.67/2.67.

Pharmacodvnamic valuation

In Vitro Activity--- Receptor Binding Studies.

1. Receptor binding studies of reference compounds and existing soft anticholinerzics.

One goal of this research was to eCaluate the soft anticholinergics made in the ('enter for Drug Discovery at the I niversitv of Florida during the past '0 years for the development of safer antiperspirants and mvdriatics. In order to establish and validate the receptor binding methods. several reference compounds were tested. The pKi values of these compounds were listed in TFable 4-I. The binding data of existing and some newly synthesized soft anticholinergics were listed in Table 4-2. For the comparison. PA2 values were also listed in the same tables.

Table 4-I. Binding parameters of reference compounds at four muscarinic receptor subtypes. Fhe affinity estimates were derived from I I1NMNIS displacement experiments and represented the mean (+S.E.M. n=3-5) for the negative logarithm of Ki. The Hill coefficients are given in parentheses.

Reference Muscarinic Subtypes Receptor pA2
Compound m I m2 m3 m4
Atropine 9.44 0.05 8.96 0.06 9.14 0.04 8.960.10
( 1.00 0.04) 0.97 0.03 1.I 1 +0.02 1.00+0.03 8.95" Scopolamine 8.96 0.036 8.66 0.05 9.470.07 9.470.07
(0.990.03) ( 1.020.02) (1I.02 0.01) (1.1 0.01) 9.50 Pirenzepine 8.35 0.04 6.02 0.04 6.4 10.08 7.740.06 ---(1.080.03) (0.95 0.02) (0.950.02) (0.980.03)
p-F-I IHSiD 7.76 0.08 6.61 0.11 7.90+0.01 7.64+0.04
_ _ (0.850.03) (0.93+0.02) (1.0 1 0.05) (1.070.01) Propantheline 9.68 0.07 9.47 0.08 10.100.10 10.11 0.13
____ (1.02 0.04) L (0.95 + 0.01) (1.050.01) (I1.07 0.02) 8.93
Data was adapted from Bodor et al. (1980). Data was adapted from Kumar and Bodor (1996).

ml receptor binding m2 receptor binding
120 - ~----- t-* 20

00 1~ [~ ,'00,J) 80 804~ .......

6 0 4N 6 0

MA 405 40 -N..

20 'U 0

0 0
101 100 1 9 1) 0 0 0 1l l 0 0 1 914 1 5 J)

concentration iM) concentration (M)

m3 receptor binding m4 receptor binding
S. .,..,..- 120 .- ____1 103


60, 2\

51 q
~40. 51 m


0'' 10 1T 10" 0 10' 10" 10" '10''1 10' 0 .01 o 10' 100 101

concentration (M) concentration (M)

F-igure 4-1. Binding2 isotherms of classical anticholinergics: atropine (1I). scopolamine (2'). F-p-HIHSiD. pirenzepine (4), and propantheline (5) for the displacement of fH]-NMS binding to four clone muscarinic receptors.

Fable 4- 2. Binding parameters of soft anticholinergics at m l, m2. m3. and m4 receptors. Ehe affinity estimates were derived from t' HINMS displacement experiments and represented the mean (S.E.M. =3-5) for the negative logarithm of Ki. The Hill coefficients are given in parentheses.

Compound Muscarnic Subtype Receptor Binding PA,
mI m2 m3 m4
DMPC 9.250.04 8.760.08 9.140.06 9.42+0.08 9.3a
(0.950.02) (1.050.04) (0.770.03) (0.940.01)
MPC 8.48 0.08 8.08 0.53 8.58 0.07 8.71 0.07 8.4"
(0. 88 0.04) (0.79 0. 05) (0.98 0.03) (0.98 0.01) AQC 9.07 0.02 9. 16 0.03 9.20 0.05 8.76 0.18
(0.96 0.07) (0.88 0.02) (1.05 0.01) (0.04 0.02) 8.55,
MDP 8,60 0.03 8.71 0.01 8.57 0.08 8.25 0.05
0.98 0.05 0.91 0.07 0.87 0.05 0.97 0.02 7.95a
PMTRet 7.35 0.18 7.390.06 8.100.10 7.780.20 7.85b
(0.80 0.10) (0.660.05) (0.720.03) (0.970.03)
PMTRCh 8.050.12 7.26 0.15 7.400.04 7.450.09 7.35"
(0.900.01) (0.780,05) (1.060.02) (0.950.02)
PMTRHx 7.3 90.03 6.920.02 6.820.02 6.860.02
(0.940.01) (0.860,03) (0.940.05) (0.760.03) 6.40 PMSCet 7.560.06 7.130.04 7.080.03 7.280.03
(0.640.03) (0.640.03) (0.740.05) (0.910.03) 7.40b
PCMS-I 7.95 0.02 7.82 0.06 8.18 0.07 8.2 0.05
(0.98 0.03) (0.98 0.03) (0.89 0.02) (0.88 0.02) 7.19'
PCMS-II 7.240.05 7.370.08 7.340. 03 7.410.04
(0.93+0.04) (0.930.04) (0.89 0.12) (0.95 0.01) 7.02' PMMSO 7.25 0.04 6.90 0.10 7.02 -0.09 6.91 0.18 7.2
(0.59 0.01) (0,59 0.01) (0.67 0.01) (0.960.01) PSDT 6.50 0.13 6.76 0.03 7.12 0.05 N/A
(0.56 0.21) (0.58 0.04) (0.50 -0.03) 5.88
PMDT 7.18 0.02 7.79 0.02 7+79 0.20 7.72 0.10
(0.57 0.04) (0.65 0.10) (0.71 t 0.01) (0.78 021) 7.29 52-19 8.00 0.05 7.82 0.13 8.05 0.05 8. 18 0.21 N/A
(0.96 +0.01 ) (0.85 0.34) (0.68 -0.04) (0.79 0.05)
52-21 7.57 t_0.08 7.29 0.22 7.73 0.08 7.40 0.01 N/A
(0.85 U 0.06) ( 0.94 0.02) (0.68 0.02) ( 0.76 0.03)
544 6.64 0.03 6.45 0.10 6.46 0.05 6.90 0.09 --(0.94 0.01) (0.89 0.04) (0.89 0.01) (0.91 0.03) ......
548 7.54 0.05 6.94 0.04 7.81 0.01 8.02 0.04 ---(0.98 0. I1) (0.86 0.04) (1.03 0. 04) (0.88 0.02)
Data was adapted from Bodor et al., 1980
Data was adapted From Kumar and Bodor, 1996. Data was adapted from Juhasz et al., 1998.


CH3 9 CH, CHCli,, PCMS-l
0 -?<]


CH3 (D CH3
-R Compound

ethvi PMTRet

COOR cyclohexyl PMTRCh
0 hexvi PMTRHx

CH3 (D CHi

0 PMsCet




161-0 Za Nk PMDT



PMMSO A 10 0 "1---s-I I


s Isopropy,
52 19

C (D CH-3SO j- 548

Ey -OCH3 H3C CH,

0 @ H3 544


R Compowid


-- N] MPC



Figure 4-2. Structure of soft anticholinergics for the receptor binding studies.

Table 4-1 and Table 4-2 shows the mean pKi +S.E.M. values obtained for each compound in receptor binding studies. Our pKi values of atropine. scopolamine, p-FHHSiD (m3 selective agent), and pirenzepine (m selective agents). were in agreement with published data (Buckely et al., 1989: Dorje et al., 1991, Wess et al.. 1991; Bolden et al.. 1992). The Hill coefficients. n. for the above compounds were not significantly different from unity. indicating that the drug-receptor interactions obeyed the law of action and that binding was only to one site. This further validates the method we used to evaluate the binding of soft anticholinergics. However. Hill coefficients for soft anticholinergics, are significantly different from unity. Theoretically. n is an integer reflecting the number of molecules that bind to a specific drug receptor. Normally the binding of classical antagonists to muscarinic receptors is well described by the simple Langmuir isotherm, indicating a Hill coefficient close to unity (Hulme et al., 1978). It is believed that low Hill coefficients are often attributed to either recognition by the antagonist of more than one receptor site or receptor conformation, or to interaction of the antagonist with a second binding site on the receptor molecule. causing a negative cooperative effect on the first site (Nathanson. 1987: Hume et al., 1981; Barbier et al.. 1995).

The cause of Hill coefficient significantly differ from unity might be related to the partial hydrolysis of the soft drugs generating structural similar inactive metabolites during incubation. The rate for the hydrolysis of soft drug in biological media is concentration dependent (Bodor et al., 1995: Yang et al., 1995). Normally, the lower the concentration, the faster the hydrolysis is. Because the hydrolysis of soft drug is mediated by esterase enzyme. Like all other enzyme involving mediating chemical and biochemical


reaction, esterase has the its capacity limitation. [-his Is due to the fact that certain number of the active sites are existing in a fixed amount of the enzyme. When large amount of the substrates (in this case. soft drugs) are introduced into the reaction system. The active sites of the esterase enzyme are saturated. Thus. only fraction of the substrates are involving the hydrolysis reaction at one time. However. in the receptor binding studies. soft drugs were diluted to extremely low concentration (l10 to 10-li M). The ubiquitous esterase enzyme in receptor system might be able to reach its full capacity to hydrolyze the relative tiny amount of the soft drugs. At such condition, even fixed amount of esterase enzyme inhibitor is not able to completely prevented the hydrolysis of the soft drugs. The slightly decomposed soft drug is the reason to cause Hill coefficent smaller than 1. The metabolite resulted from hydrolysis may interfere the existing equilibrium between the receptor, the radioligand. and tested compound. Preliminary experimental data showed that the Hill coefficient (n) of AQC M3 receptor binding was 0.4 to 0.5 when no enzyme inhibitor was added into the buffer. N increased close to unity when enzyme inhibitor (NaF) was added. The exact reason as to why n was significant different to unity is under further investigation. Searchingz for more effective enzyme inhibitor may improve the receptor binding methods. Our results showed that the results (pKi) were reliable (compared with PA,) even though the Hill coefficient did not reach unity.

It is believed that M3 mediated smooth muscle in airway and GI tract contraction (Grimm et al.,1994). pA2 value for guinea pig ileum contraction has been a classic functional study for the determination of anticholinergics affinity toward the M3 receptor. The pA2 values for soft anticholinergics generated from guinea pig ileum


contraction studies are generally comparable to pKi value from m3 binding studies. even though in most cases the pA2 values are somewhat lower than the pKi value of m3 binding. The relative value of individual compounds tested by either method was essentially the same. In our lab, the pA2 value of guinea pig ileum contraction was the method of choice for the screening of relative potency of soft anticholinergics (Kumar and Bodor, 1996). Because of the faster screen nature. receptor binding has an advantage over pA2 value for providing information as to the relative potency of soft antichoinergics. The correlation of m3 binding value and PA2 were performed. The results were shown in Figure 4-3. Correlation coefficient was determined as R2=0.82. PMTRet. PMTRCh. and PMTRHx are soft analogs of atropine (Hammer et al., 1988: Kumar et al., 1993a). PMSCet is the soft analog of scopolamine. The design of these compounds is based on the inactive metabolite approach of soft drug design (Bodor, 1984). These compounds are expected to metabolize into hypothetical metabolites in vivo. Since atropine and scopolamine do not show muscarinic subtype selectivity, it is expected that these compounds will not exhibit subtype selectivity. The rank of order of potency is generally. ml>=m3>>m2. From these three compounds, we may conclude that the substituent of the ester group has a significant impact on the binding of the soft anticholinergic to muscarinic receptors. Wess (1990) has proposed the following structural elements for cholinergic antagonists. (1) a cationic "head group" which is either a tertiary base protonated at physiological pH or quaternary ammonium moiety; (2) some "heavy blocking moieties," e.g., alicyclic or aromatic rings, for hydrophobic interaction with the receptor; (3) an interconnecting structural element (ester or amide) of definite length; (4) an "anchoring group,"e.g., hydroxyl group(s) is often present at key

positions (Barlow and Ramtoola. 1980). Obviously, the differences in the structures of the above three compounds are located at the "'anchor positions." Originally atropine has a hydroxyl group at the anchor position. It has been shown that replacement of the hydroxyl group with an ester group significantly decreases binding of an antagonist to muscarinic receptors. It is confirmed that the hydroxy group forms a hydrogen bond at the binding site, which is a critical element fotbr the pharmacophore (Waelbroeck et al., 1990). It is also demonstrated that the size of the substituent has a great influence on the binding. As the size of the substituents increases, the potency decreases (Kumar et al., 1994). But there is an exception. Compared with a cyclohexyl substituent, an n-hentyl substituent has a smaller volume than cyclohexyl substituen. But n-hentyl sustituent is much less potent than cyclohexyl sustitutent (Kumar, 1993a). It is believed that the steric hindrance of the "anchor group" (in this case, a long chain n-hentyl group) has decreased the activity. Similar results were found by Banerjee and Lien (1990), when they studied amino esters of (a-substituted phenyl acetic acid, ct-methyl tropic acid, and related compounds. DMPC, MPC. AQC, and MDP are soft drugs, where design is based on the soft analog approach (Bodor et al., 1980: Bodor, 1984: Brouillette et al, 1996). The soft analog approach requires a specific metabolically sensitive spot incorporated into the structure, leading to a close structural analog of known active anticholinergic drugs. The soft analogs were designed to hydrolyze in vivo in a predictable time-frame to an acid, an aldehyde and a tertiary amine, all inactive metabolites (Bodor, 1984). We did not find any muscarinic receptor subtype selectivity for this group of compounds.

y=0.878x+ 1.316
9.0 R2 = 0.8223
E 8.5
CL7.5 ,

6.0 .
5 6 7 8 9 10


Figure 4-3. Correlation between pki (m3) and pA2.

The general order of anticholinergic potency relative to the nitrogen head is. 1.2dimethylpyrrolidine > 3-acetoxyquinuclidine> 1-methylpyrrolidine. This is in agreement with the findings of Bodor et al. (1980) and Brouilette et al.(1996).

PCMS-I and PCMS-II comprised of a group of new anticholinergics. They are quite similar to atropine analog PMTREt, except a cyclophenyl- group was incorporated into the structure. Again, this group of compounds do not exhibit subtype selectivity. Interestingly, the ethyl ester of this group is shown to be 5 times more potent than the methyl ester. Because we did not make the methyl ester soft analog of methatropine (Kumar, 1993c), we don't know this (in PCMNS-II) is a unique case, where the methyl ester is less potent than the larger substituent, ethyl ester. (in PCMS-I) or it only happened when a cyclopentyl group was incorporated into the parent compound methatropine. Generally, the potency of the anticholinergics based on methatropine are inversely proportional to the molecular volume of compounds (Kumar et al., 1994).

The utilization of anticholinergics as antiperspirants has been evaluated for a long time. Scopolamine has been shown to be very effective in the inhibition of eccrine sweating (Shelley and Horvath. 1951). Other muscarinic antogonists also have been found to effectively suppress sweating (Stoughton et al.. 1964; Macmillan et al., 1964; Oroshnik and Soldati. 1978). However, due to the tremendous risk of systemic toxicity when overdose, it was suggested these agents should be used under direct physician supervision and not be available for over-the-counter sale (Lasser, 1967). On the other hand, the availability of soft anticholinergics should warrant the safe use of these agents as antiperspirants, since they do not show systemic toxicity (Bodor. 1984). Obviously,

the effectiveness of topically applied antiperspirant is related to the intrinsic activity of these agents and the ability to penetrate the skin. Binding and functional studies on muscarinic receptors of secretory cells have demonstrated that all muscarinic receptors in granular cells appear to be of the M3 subtype (Goyal. 1989). The results of our data have shown that PMTRET. AQC, and DMPC have very high affinity toward m3 receptor. Kumar (1993c) has found a linear correlation between the log partition coefficients (log Kp) and log permeability coefficients (log p) of the soft anticholinergics tested. The log KP can be readily estimated by the a semiempirical quantum chemical method (AM1) (Bodor et al., 1989: Bodor et al.. 1992). It should be easy to screen the most promising candidates for a safe antiperspirant by combining the m3 binding data and log Kp values obtained from computer estimations. Our data showed the m3 binding values are comparable to functional studies on guinea pig ileum. It has long been regarded muscarinic receptor on smooth muscles are of M3 subtype. The latest research have demonstrated that the muscarinic receptors in guinea-pig ileum are heterogeneous, with a major M2 receptor population (-80%) and a minor M3 population (-20%). The function of the minor M3 population is clearly related to contraction, but the function of the predominate M2 population is unclear. It may be related to the inhibition of relaxation of the muscle (Eglen et al., 1994). Therefore, utilization of receptor binding data of m3, for the in vitro estimate of inhibition of eccrine sweating should be more accurate than that of functional studies based on smooth muscles contraction.

Quantitative structure activity relationship (QSAR) studies were carried out to investigate the relationship of physicochemical parameters with receptor binding values. It was found the following formula to describe the QSAR:

pK (m3) = 22.747(+2.700) ).728(1.357)O 0.104(0.025)D n = 20. r = 0.843. a= 0.426. F = 20.90 It showed that for soft anticholinergic agents containing a tropine moiety, pKj

(m3) values correlated well with molecular ovality (O) and dipole moment (D). Consequently, receptor binding for this group of compounds in addition to overall shape and size (Oe) is determined by electronic properties (D) as well. The correlation between predicted and experimental values is shown in Figure 4-4.





6 7 8 9 10
m, pK, exp.

Figure 4-4. Calculated versus experimentally measured m3 pK data for inactive metabolite-type soft compounds containing a tropine moiety.

In conclusion, the binding experiment performed on cloned muscarinic receptors provided valuable affinity information about soft anticholinergics toward individual muscarinic subtype. The information will assist in the study of the structure activity relationship and screening the ideal candidates for the development of soft antiperspirants and soft mydriatics. This information will also assist us in the development of soft anticholinergics with muscarinic subtype selectivity. This should further increase the therapeutic index of soft anticholinergics.

Receptor Binding Data for the New Class of Soft Anticholinergics

Receptor binding studies were performed on the newly synthesized soft anticholinergics 9(a-b) and 13 (a-b). The results were listed in Table 4-3. The newly synthesized soft anticholinergics are able to attain the potency of the lead compound. It is also demonstrated that the newly synthesized compound 9a and 13a showing muscarnic subtype selectivity (m3/m2). In addition, compound 9a also had m3/mI selectivity.

From the QSAR studies, Turbanti et al. (1992) proposed that the smaller the size at equatorial N-substituent of N-alkyl-nortropine esters of 2-phenyl-2cyclohexenecarboxylic acids, the more potent the compound. According to their proposal, 13a of our series should be much more potent than other soft anticholinergics. Actually, this was not case. There is not a significant difference in the potency

Table 4-3. Receptor binding values for 9(a-b) and 13 (a-b). The affinity estimates were derived from [3 H]NMS displacement experiments and represented the mean (S.E.M, =3-5) for the negative logarithm of Ki. The H-ill coefficients are given in parentheses. To ensure the experimental conditions are consistent. the receptor binding values of atropine were determined simultaneously with soft anticholinergics at each experiment.

Comp ound ml m2 m3 m4
atropine 9.08 0.12 9.04 0.20 9.28 0.07 9.500.04
_______(0.98 0.03) (1.01 0.02) (0.96 0.02) (1.01 0.04)
9a 7.86 0.03 7.73 0.10 8.99 0.01 8.43 0.07
_______(0.78 0.05) (0.91 0.10) (0.81 0.02) (0.90 0.02)
9b 7.93 0. 04 7.97 0.03 8.64 0.05 8.20 0.06
(0.86 0.01 ) (0.88 0.03) (0.87 0.06) (0.91 0.07)
1 3a 7.89 0.07 7.38 0.07 8.490.02 8.11 0.06
________(0.83 0.05) (1.07 0.07) (0.81 0.04) (1.07 0.02)
13b 7.98 0.04 7.70 0.06 8.620.05 8.17 0.03
(0.91 0.05) (0.80 0.06) (0.87 0.05) (0.8 005) lead 8.20 7.47 8.64 N/A
a~ data were adapted from D'Agosting et al.. 1994.

between methly- or ethly- ester soft anticholinergics. There is not a significant difference in the potency between cx and 3 isomers.

Muscarinic receptors are involved in the control of functions of many organs in the body. Three major muscarinic receptor subtypes: M1, M2, and M3 mediate a verity of basic function of the body (for review, see Introduction). These receptors have a specific location and control a particular physiological activity. However. most of the currently available anticholinergic agents are not subtype selective agents. Such anticholinergics should equally stimulate all muscarinic subtype receptors in the body once they are bought into systemic circulation. Thus. a therapeutics for an intended symptom usually result in many undesired effect. The usefulness of the anticholinergic agents are limited due to their relatively low selectivity. It is not difficult to understand why the available anticholinergic agents have very low subtype selectivity once we examine the genetic structure the muscarinic receptors. The human genes which encode five muscarinic subtype receptor have 70% similarity in their sequence (Bonner et al.. 1987). The three dimensional structures of the receptor. critical tactor determining the selectivity of the receptor. are based on the sequence of the genes. In developing a receptor to perfectly fit into a subtype receptor, there is a very good chance it will fit into other subtype receptors. However, the need to develop muscarinc subtype selective agents is great. The Miselective antimuscarinic agents can be used to inhibit gastric secretion and is applied in the treatment of peptic ulcer disease (Goyal. 1989). Cardiac M2 receptor mediate bradycardia. M2-selective antagonist could be used to the therapy of brandyarrhythmias. The M3 receptor selective anticholinergic agents could be used as bronchodilators in the treatment of chronic obstructive pulmonary diseases. They can also be used as safer

premeditative agents for the reduction of the secretary effects of anesthesia. at the same time minimizing the cardiac effects of the traditional premeditative agents. such as. atropine and glycopyrrolate (Brown and Taylor. 1996). For soft anticholinergics with subtype selectivity (M3/M2), they possess the safer feature of the soft drugs, that is. locally active but systemicaly inactive. This is the pharmacokinetic and metabolism approach to increase the therapeutic index. Besides the safer feature from pharmacokinetic consideration. this new class of soft anticholinergics holds unique properties: subtype selectivity. It is certainly a pharmacodynamic approach to enhance the therapeutic index. As our earlier discussion. such an approach would not be able to completely ensure the safety of the agents because of genetic similarity of the subtype muscarinic receptors. It is obvious, if the goal of the design of new therapeutics is to maximize the therapeutic index, the inclusion of consideration of both pharmacodynamic and pharmacokinetic properties in the drug design would greatly speed up the drug discovery process and produce much safer and effective agents. The possible clinical use of the soft anticholinergics with subtype selectivity (M3/M2) are as follows. (1) Mydriatic agents: we have mydritic studies to demonstrate such usefulness. (2) Antiperspirants. Anticholinergics have been explored as antiperspirants for a long time (MacMillan et al.. 1964; Stoughton et al.. 1964). However, the potential side effects prevented their use for this purpose. The development of the subtype selective quaternary soft anticholinergics would certainly reduce the risk of the cardiac effects and CNS toxicity. (3) Anticholinergics, such as atropine and glycopyrrolate. are frequently used for premedication to reduce oral and respiratory secretions and prevent bradycardia (Brown and Taylor, 1996).

9a binding curve on (ml-m4) receptor

.4- 3


- - 6 0- --* - - - - I- - -
C0 20 ------9-----------0

10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

concentration (M)

Figure 4-5. Binding isotherms of 9a for the displacement of specific (3H] NMS binding to ml (1), m2 (2), m3 (3), and m4 (4) muscarinic receptors.

13a binding curve on ml-m4 receptor

10 0 - - - - -- . . - -



10-11 10-10 10-9 10-8 10-7 10-6 10-5 10-4

concentration (M)

Figure 4-6. Binding isotherms of 1 3a for the displacement of specific [3 H] NMS binding
to ml (1), m2 (2), m3 (3), and m4 (4) muscarinic receptors.

They are also administered with neostigmine tfor the reversal of non-deploarizing neuromuscular blockage (Wettersiew et al.. 1991. Mirakhur et al.. 1977: Takkunen et al.. 1984). However, such application has been complicated by the cardiac side effects of the anticholinergics, even though glycopyrrolate causes less cardiac side effects (Gomez et al.. 1995; Wetterslew et al.. 1991. Mirakhur et al.. 1977: Takkunen et al.. 1984). This is particularly dangerous to the patients with pre-existing cardiac disease (Mostafa and Vucevic. 1984). The development of soft anticholinergics with subtype selectivity (M3/M2) will assist the safer administration in anesthesia: either as premeditation for reducing excessive salivation and secretions of the respiratory tract induced by administration of general anesthetic agents or as an agent to reverse the non-deplorizing neromuscular blockage. The occasional serious arrhythmias effects associated with agents (Brown and Taylor, 1996) should be greatly reduced due to subtype selectivity (M3/M2). The soft nature of the compounds will allow us to add the exact amounts of anticholinergics needed during the anesthesia practice by titration.

Receptor binding studies of soft anticholinergics based on tropvl a-phenyl cvclopeneactate

Receptor binding studies were performed on the newly synthesized soft anticholinergics based on tropyl-ct-phenylcyclopeneacetate. The results were listed in Table 4-4. The resulted showed that the Pki's of methyl soft drugs (15a and 18a) are higher than that of ethyl soft drugs, indicating the methyl soft drug were relatively potent than ethyl counterpart. It is in agreement with the previous finding from our laboratory (Kumar et al., 1994; Juhasz et al., 1998) that the smaller the molecule size, the more

Table 4-4. Receptor binding studies: The numbers of the table stand for pKi (Ki: dissociation constant, data were the mean of three determinations). The higher the pKi. the more potent the compounds.

Compounds ml m2 m3
15a 7.65 0.01 7.540.18 7.750.10
(0.83 0.04) (0.780.02) (0.730.01)
15b 7.42 0.04 7.200.03 7.570.08
(0.87 0.02) (0.83 0.04) (0.750.01)
18a 7.330.10 7.140.06 7.510.15
(0.760.03) (0.850.07) (0.800.06)
18b 7.000.08 6.940.09 7.21 0.2
(0.880.05) (0.850.07) (1.010.03)