Neolignans of Saururus Cernuus L

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Title:
Neolignans of Saururus Cernuus L
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viii, 112 leaves : ; 29 cm.
Language:
English
Creator:
Alvarez, Francisco Martin, 1951-
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Subjects

Subjects / Keywords:
Antipsychotic Agents   ( mesh )
Pharmocognosy   ( mesh )
Plants, Medicinal   ( mesh )
Medicinal Chemistry thesis Ph.D   ( mesh )
Dissertations, Academic -- Medicinal Chemistry -- UF   ( mesh )
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bibliography   ( marcgt )
non-fiction   ( marcgt )

Notes

Thesis:
Thesis (Ph.D.)--University of Florida, 1981.
Bibliography:
Bibliography: leaves 108-111.
Statement of Responsibility:
by Francisco Martin Alvarez.
General Note:
Typescript.
General Note:
Vita.

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University of Florida
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All applicable rights reserved by the source institution and holding location.
Resource Identifier:
aleph - 000897856
oclc - 21363974
notis - AEK6546
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NEOLIGNANS OF SAURURUS CERNUUS L


BY

FRANCISCO MARTIN ALVAREZ





















A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL
OF THE UNIVERSITY OF FLORIDA IN
PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA


1981




































Digitized by the Internet Archive
in 2011 with funding from
University of Florida, George A. SmatherlTLibblritfamriIlysupport from LYRASIS and the Sloan Foundation


http://www.archive.org/details/neolignansofsaur00alva















ACKNOWLEDGMENTS

It would have been impossible for me to pursue these studies without

the help and encouragement of my family. I am deeply indebted to my

parents, Mr. and Mrs. Manuel Alvarez Diaz; to my brothers, Manuel and

Oswaldo; and to my sisters Mercedes and Celia for their continuing and

unfailing support during my graduate education.

I acknowledge my appreciation to Dr. K.V. Rao for his supervision and

guidance throughout the period of this investigation and during the writing

of the dissertation. I also would like to express my sincere appreciation

and gratitude to the past and present members of my supervisory committee:

Dr. W.M. Jones, Dr. R.H. Hammer, Dr. Lal C. Garg, Dr. P.F. Field, Dr. S.G.

Schulman and Dr. L.G. Gramling.

Finally, to all my colleagues and friends, I give my sincere grateful-

ness for the encouragement, faith and understanding which they invested in

me while attending the University of Florida.















TABLE OF CONTENTS

CHAPTER PAGE

ACKNOWLEDGMENTS........................................ inii
ABSTRACT................................................... vii

I INTRODUCTION .. ............................................ 1

II ISOLATION OF COMPONENTS FROM S. CERNUUS L.................. 7

Extraction and Fractionation............................ 7
Chromatography: Purification of SC-8..................... 11
Chromatography: Purification of SC-7.... ................ 19
Chromatography: Purification of SC-6..................... 19
Chromatography: Purification of SC-1..................... 22
Chromatography: Purification of SC-2 and 3................ 22
Chromatography: Purification of SC-10.................... 22

III STRUCTURAL ELUCIDATION OF COMPOUNDS SC-1, 2, 3 AND 10...... 23

Compound SC-1 ......................................... 23
Compound SC-2 .......................... ............... 26
Compound SC-3............ ............................ 26
Compound SC-10......................................... 30
Experimental................. ..... ........... 33

IV STRUCTURAL ELUCIDATION OF SC-6, 7 and 8.................... 42

Compound SC-8......................... ..... ............ 42
Compound SC-7................ ...... ............... .... ....... 54
Compound SC-6........................................ 55
Experimental...............................*............ 62

V BIOLOGICAL ACTIVITY..................................... 72

Toxicity and Behavioral Studies.......................... 72
Characterization of CNS Activity........................ 75
Potentiation of Pentobarbital........................... 79
Inhibition of Aggressiveness.. .......................... 79
Antagonism to Amphetamine............................... 81
SC-8 as a Neuroleptic Agent.............................. 82
Experimental......................................... 90

VI DISCUSSION................................................. 93

Lignoids............................................ 93
Neuroleptics............................................. 99










BIBLIOGRAPHY............. .............................. 108

BIOGRAPHICAL SKETCH.............................. .... 112














Abstract of Dissertation Presented to the Graduate Council
of the University of Florida in Partial Fulfillment of the
Requirements for the Degree of Doctor of Philosophy


NEOLIGNANS OF SAURURUS CERNUUS L

By

Francisco Martin Alvarez

June, 1981
Chairman: Koppaka V. Rao
Major Department: Medicinal Chemistry

Discovery of a pharmacologically active agent from a natural source

has several consequences: a) the compound itself may be therapeutically

useful, b) it may serve as a lead compound which can be modified to yield

useful drugs, c) its novel structure and/or mechanism of action may pro-

vide valuable tools in pharmacological and medical research. As an example,

reserpine, the active principle of Rauwolfia serpentina, is currently used

in the treatment of mild hypertension. At higher doses, reserpine acts as

a neuroleptic agent and because of its novel structure, it has been used

as a model compound for the synthesis of new antipsychotic drugs. In addi-

tion, the uniqueness of its mode of neuroleptic activity has provided the

pharmacologist with a powerful tool to study the central nervous system
and is partially responsible for the development of psychopharmacology.

In our laboratory, a program to develop natural products with drug poten-

tials from locally available plants has yielded many interesting compounds.

One such plant, Saururus cernuus L, is the subject of this dissertation.









Saururus cernuus L is a fresh water weed, with a rich history of medi-
cinal folklore. The extract of S. cernuus L showed toxicity in mice and

fractionation based on this toxicity led to the isolation of two active

principles, SC-7 and 8, plus five other nonactive components, SC-1, 2, 3,

6 and 10. All seven of these belong to the class of neolignans. Four out

of the seven compounds were found to have new structures, while SC-1, 3

and 10 were found to be identical with the known neolignans:austrobailig-

nan-5, veraguensin, and (+)guaiacin, respectively. Compound SC-2 repre-

sents a new member of the 2,5-diary1-3,4-dimethy1tetrahydrofuranoid type

neolignan and is isomeric with veraguensin. Compounds SC-7 and SC-8 repre-

sent a novel type of neolignan in which four phenylpropanoid units are

joined together and SC-6, likewise, with three such units. Examples of

a related class of compounds, lignans, with four and three phenylpropanoid

units have been reported in recent years. In accordance with the current

practice, SC-6 must be regarded as the first member of the class of ses-

quineolignans and SC-7 and SC-8 that of dineolignans.

The demonstration of neuroleptic activity in the dineolignan SC-8 of

Saururus is a highly significant discovery which will have far-reaching

effects in the area of neuroleptic drugs in particular and biomedical area

in general. Compound SC-8 is the first example of a lignoid to possess

neuroleptic activity, and the only natural product with this type of acti-

vity since the discovery of reserpine in the early 1950's. This compound

is not to be considered as merely another type of neuroleptic drug similar

to the many synthetic drugs that are being developed over the last twenty

years. Except for the butyrophenones and one or two other types, all of

the available neuroleptics are variants of the phenothiazine (chlorproma-

zine) skeleton. The dineolignan SC-8, on the other hand, represents a









total departure from all of the existing synthetic neuroleptics, in that

it is nonbasic and has no nitrogen. The impact of its entry into the

neuroleptic fieTd will be felt in many areas: medicinal chemistry (as a

new lead compound), in psychopharmacology (as a potentially useful pharma-

cological tool) and possibly, even in clinical application of SC-8 or one

of its analogues.


viii















CHAPTER I

INTRODUCTION

One of the most important objectives of research in natural products is

to isolate active principles from natural sources in order to evaluate their

drug potential. A famous example to illustrate this point is the alkaloid,

reserpine. The use of Rauwolfia serpentina to treat hypertension, insomnia

and insanity was known for many centuries in India. However, not until

the early 1950's was the active principle, reserpine, isolated and found

to possess antihypertensive activity and subsequently, neuroleptic activity

(1). Reserpine has since been used extensively as an antihypertensive and

as a tranquilizer, although it has been restricted in recent years to

combating mild hypertension. However, more important than being an effec-

tive therapeutic agent, this natural product has played a major role as a

tool in the development of psychopharmacology. The importance of this compound

can be equated with thatof such key drugs as nicotine and muscarine, which

have revolutionized the study of the nervous system. Not only has reserpine

provided enormous insight into the mode of action of other psychoactive

agents, but it has also been an invaluable tool for the study of the cen-

tral monoaminergic and peripheral adrenergic neuronal systems (2).

In general, two approaches are used in the discovery of biologically

active agents from plants: (a) the use of phytochemical tests and, (b) the

use of phytopharmacological assays to detect the presence of the active

principle and to assist in the fractionation. The former screening proce-

dures are generally adopted in laboratories in which there are no or only

limited biological testing facilities. In screenings of this type, groups

1









of natural products such as alkaloids, flavonoids, anthraquinones, sapo-

nins etc., are detected using specific color reactions. These are then

isolated on the basis of these tests and the purified compounds possibly

submitted for investigation of biological activity. Some examples are

Mayer's alkaloid precipitation test, Borntrager reaction for the detection

of anthraquinones, and the Liebermann-Burchard test for saponins and other

steroidal compounds. Such a phytochemical screening has always been the

most popular approach for plant screening. Unfortunately, the use of chem-

ical tests is not a good predictor of biological activity unless the active

principles happen to be alkaloids. An excellent survey of the phytochem-

ical screening of plants has been published by Farnsworth et al. (3).

The second approach, the use of pharmacological tests for the detec-

tion of biologically active agents at the earliest stage and fractionation

of the extract based on this activity, is by far the best method for the

isolation of active principles from plants. However, laboratories using

this approach are generally in the minority. For example, it is estimated

that approximately 500,000 species of plants exist and of these, perhaps

10% have been investigated phytochemically (4). Of this small minority

which has received chemical attention, only a tiny proportion has ever been

attacked with a systematic approach involving any type of biological assay.

In our laboratory, a program of this type was set up for the isolation of

active principles of locally available plants. A simple toxicity test was

employed as the bioassay to aid fractionation. This choice of toxicity

is justified because toxicity is an extreme form of biological activity

and, when the drug is given at nonlethal dose, it will show its most char-

acteristic activity which may be therapeutically significant. For exam-

ple, the therapeutically useful cardiotonic glycoside, digoxin, is one of









the toxic componentsinthe leaves of Digitalis lanata. Likewise, the poi-

sonous mayapple (Podophyllum peltatum) contains a series of lignans

responsible for its toxicity. The major toxin, podophyllotoxin, which

possesses anticancer activity,is used in the treatment of lipomas and

fibromas and analogs of it are presently being used for the treatment of

lymphomas and leukemias.

In the toxicity test, male Swiss mice (20-25 g, 6 per each sample)

were injected intraperitoneally (i.p.) with a solution of the sample (0.5

ml) once a day for five days. The minimum toxic dose (MTD) was defined

as the dose in mg/Kg required to kill four-sixths of the mice with three

or more doses being necessary. During this period, the animals were also

examined for any gross toxicity symptoms such as changes in respiration,

locomotion etc. This simple bioassay is by no means ideal; although, for

our purpose (i.e., the detection of new lead compounds with potentially

useful pharmacological activity) it satisfies many of the major require-

ments. For example: (a) the screen is "wide-spectrum,u that is, a large

variety of activities can be detected, with potential for detecting "new

types" of pharmacological activities, (b) once the MTD is established it

has been found to be very reproducible; this is of utmost importance since

the bioassay is used to monitor the isolation and purification procedure,

(c) the five day injection period allows the detection of activity having

a delayed onset; furthermore, during this period gross behavioral observa-

tions are made, thereby permitting a maintenance record of the "activity"

at different dose levels and times, (d) the method allows the use of crude

plant extracts, and by employing a single uniform dosing vehicle, one can

easily obtain reproducible results, (e) only 0.3 to 0.6 gram of crude

plant extract is needed to complete a five day test, (f) the procedure is

simple enough so that a full-time pharmacologist is not required for the









day-to-day operation of the program, (g) the procedure is relatively inex-

pensive, (h) the test is done in an in vivo system and the animals used

(mice) are easily obtainable, easily handled and resistant to infection,

Ci) the procedure is not too time consuming, therefore allowing large

numbers of plant extracts to be tested. In addition, when the toxicity

assay is coupled with gross behavioral observations, it is possible to

detect other pharmacological activities in plant extracts which were nega-

tive in the toxicity test e.g., the animal goes to sleep (loss of righting

reflex) or the animal becomes hyperactive (increase in motor activity)

etc. Still, it must be kept in mind that our major objective is to devel-

op new lead substances with potentially useful pharmacological activity,

and that the role of this primary screen is that of a universal detector,

one that can detect as many different types of activities as possible and

preferably, those which are unique and unexpected. Once the "active"

principle is isolated in its pure form, then, more sophisticated and ideal

pharmacological screening methods may be employed for the proper characteri-

zation of the activity (e.g., tranquilizer, sedative, etc.).

Of the several plants revealed as toxic by the above test, those

which had no prior description of such activity were selected for study,

and one of these is Saururus cernuus L, the subject of the dissertation.

Saururus cernuus L (N.O. Saururaceae), commonly known as lizard's tail,

is an aquatic weed native to North America, where it was once used in folk

medicine as a sedative,against tumors and for the treatment of irritations

and inflammations of the kidneys, bladder, prostate gland, urethra and epi-

didyim's (5,6). In fact, the family Saururaceae, consisting of five genera
and seven species, has a rich history of medical folklore (7). The clas-

sification of the Saururaceae, according to Wu (8), is presented inFigurel.1.







5

Lizard's tail grows to a height of 1 in, with drooping, curved spikes of

tiny white flowers. Instead of petals, the flowers are made of white

stamens along an elongated stem. The alternate cordate leaves have a

pleasant aromatic smell and are 8 to 10 cm in length. This perennial herb

is distributed from Florida and Texas to southeastern Canada and grows in

swamps, fresh water marshes, roadsides, or in moist soil.

Fong et al. characterized S. cernuus as a nonalkaloid-containing

plant (9). The aqueous extracts of the different plant parts (root, rhi-

zome, stem, leaf and fruit) were tested for alkaloid content using Mayer's

and Wagner's reagents and found to be negative. Isolation of terpenoid

compounds such as pinene, limonene and others has been reported by Tutu-

palli et al. (10). In their study, the steam-distilled essential oil from

the aerial parts of S. cernuus was shown to contain twenty-six components

by the use of gas chromatography (gc). Of the twenty-six components
detected, sixteen were identified using infrared spectrometry (ir), nuclear

magnetic resonance spectrometry (nmr), and the combination of gc and mass

spectrometry (gc-ms). Our work was initiatedon the basis of the repro-

ducible toxicity observed with the extract of the shoots. The "active

principles" were obtained using a series of purification techniques guided

by the mouse toxicity assay. Chemical and spectral methods were then

utilized to elucidate tneir structures and stereochemistry. Finally, a

variety of pharmacological procedures, some to detect multiple activities,

(e.g.,mouse behavioral assay) and others to detect a specific activity (e.g.,

mouse antiamphetamine assay),were used to characterize their biological

activity and explore their potential as neuroleptic agents.








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CHAPTER II

ISOLATION OF COMPONENTS FROM S. CERNUUS L

Extraction and Fractionation
The leaves and stems of S. cernuus L were dried and coarsely ground.

The plant material (2 Kg) was then extracted with ethanol at 250C for two

days. Three such extracts were combined and concentrated in a rotary

evaporator to a thick, dark green syrup (256 g). This green residue showed

a characteristic ultraviolet (uv) absorption maximum at 280 nm and a MTD

of 500 mg/Kg. Toxic symptoms included reduced locomotion and body tempera-

ture, unsteady gait and ataxia, followed by labored respiration and death.

Partition of the concentrate between ethyl acetate (1 Z) and water (1 Z)

at pH 9 gave three fractions: (a) the aqueous layer, (b) an insoluble frac-

tion and, (c) the solvent layer, the latter containing all of the toxicity,

Figure 2.1. Thin-layer chromatography (20% acetone-benzene) of the extract

showed a complex mixture of substances of widely varying degrees of polar-

ity. A preliminary separation through solvent partition appeared to be

beneficial. Thus, concentration of the ethyl acetate layer, followed by

partition of the residue (104 g) between 80% aqueous methanol (500 ml)
and hexane (500 ml), allowed highly nonpolar impurities to be extracted
into the hexane layer (FRACTION I, Figure 2.1) while relatively polar

components stayedin the 80% aqueous methanol layer, together with all of

the toxicity. Concentration of this (58 g, brown gum) and partition

between 50% aqueous methanol (300 ml) and benzene (300 ml) gave the activ-

ity in the solvent layer while leaving behind the relatively polar impuri-
ties in the aqueous methanol. At this point, it was found to be convenient

7












S. cernuus L, shoot, 2 Kg


Concentrated EtOH


Extract,
I


256 g, 3.2 x 106 AU


HOH, pH 9, 112 g, 4.2 x 105 AU


EtOAc,


104 g, 2.2 x 106 AU


Insoluble, 4.0 g,
3.0 x 104 AU


MeOH-HOH (80%), 58 g,
1.6 x 106 AU


MeOH-HOH, 26 g,
3.4 x 104 AU


Hexane, 38 g, 4.8 x 105 AU
"FRACTION I"


SC-1
Benzene, 44 g,
1.4 x 106 AU


0.1 N NaOH


EtOAc, 6.0 g
6.8 x 104 AU
"FRACTION II"


HOH, pH 4


Ethyl Ether, 34 g,
1.1 x 106 AU



CCD-7
withdrawal


MeOH-HOH (3:1), 18.8 g
"FRACTION III"


CCl14-Hexane
(2:1)


SC-2, 3, 6, 7, 8, 9


Figure 2.1. Fractionation


SC-10









to do a further partition between 0.1 N sodium hydroxide (250 ml) and

ethyl ether (250 ml) in order to remove additional impurities (e.g., phe-

nolic compounds). The active fraction remained in the ether layer (34 g)

and showed a MTD of 39 mg/Kg. Acidification of the aqueous layer and

extraction with equal volumes of ethyl acetate yielded the phenolic extract

(FRACTION II) which was not active.

Although the residue from the ethyl ether layer represented a pro-

duct whose purity has been increased manyfold (13 times more active than

the crude ethanol extract), it was still a very complex mixture and needed

further fractionation, such as column chromatography, preferably of the

type partition chromatography. However, the quantity of the sample at this
stage was somewhat large and, hence, would require repeated runs. For

example, fractionation was started with 2 Kg of plant material which gave

about 256 g of crude ethanol extract residue. After the series of solvent

partitions, this was reduced to 34 g. If this would be reduced by half

with some additional purification, the chromatographic procedure can be

much more efficient. One of the best methods to achieve such an objective

is through the use of countercurrent distribution (11). In the present

case, the withdrawal method was used with seven separatory funnels. The

solvent was carbon tetrachloride, hexane, methanol, and water (2:1:3:1).

Results of the countercurrent distribution are shown in Table 2.1. Every

two or three fractions from the distribution were combined and tested for

toxicity in mice. All of the aqueous layers were shown to be toxic at

various dose levels, while none of the solvent layers had any activity at

a dose > 44 mg/Kg, i.e., higher than tnat of tne sample used for the experi-

ment. A bell-shaped distribution curve was observed when the funnel num-

ber was plotted against the degree of toxicity, with most of the toxicity










Table 2.1. Countercurrent Distribution


Fraction Solvent Mouse Dose
number phase assay (mg/Kg)



0-2 A Toxic 22

3-4 A Toxic 11

5-6 A Toxic 22

0 S Not Toxic 44

1-2 S Not Toxic 44

3-5 S Not Toxic 44


A = MeOH: H20, 3:1, 240 ml

S = CC14: P.E., 2:1, 240 ml










being present between fractions 3 and 4 (MTD: 11 mg/Kg). Nevertheless,
all the aqueous layers were combined to yield 18.8 g of a yellowish oil

(FRACTION III) having MTD of 22 mg/Kg. Thus, reducing the weight of the

toxic fraction by one-half and at the same time increasing the specific

activity by a factor of two was achieved. At this point, the sample was

ready for the partition chromatography.

Thin-layer chromatography of this sample in benzene/acetone mixtures

showed a series of closely related components, all of which appeared as

bright red spots when sprayed with H2S04/HoAc and heated gently. Based

on the decreasing Rf these were designated as SC-1, SC-2... SC-9 (Figure

2.3). Of these, homogeneous preparations of SC-1, SC-2, SC-3, SC-6, SC-7,

SC-8 and SC-9 were obtained through the use of partition or adsorption

chromatography. SC-4 and SC-5 appeared to be distinct but have not been

completely purified. The phenolic fraction, designated as SC-10, was also

obtained pure by chromatography of Fraction II.

Chromatography: Purification of SC-8

The "active" fraction (FRACTION III) was concentrated (18.8 g) and

chromatographed using a partition column chromatography technique. The

column was prepared from Sephadex LH-20 as the support (Pharmacia Fine

Chemicals; bed size 3.6 x 65 cm) with the system 80% methanol-water as

the stationary phase and petroleum ether with varying concentrations of

benzene as the mobile phase. The elution profile of "FRACTION III" (5.6 g

aliquot) on a column of this type is shown in Figure 2.2. The dotted

lines correspond to the elution solvent gradient: 10 to 75% benzene in

petroleum ether. The column was followed using uv absorption at 280 nm,

tlc (15% acetone-benzene) and the mouse toxicity assay. Data for the uv

profile are expressed as absorption units (AU) which is defined as: AU =









(Absorbance)(Volume)(Dilution factor). Most of the activity (toxicity)

applied to the column was eluted between fractions 420-440 and corresponded

to the major active component, designated as compound SC-8 (742 mg).

This was followed by two smaller symmetrical peaks which were identified

as compound SC-6 (fractions 450-475; 401 mg) and compound SC-9 (fractions

510-540; 302 mg). A very broad and less defined peak, fractions 215 to

350, was also found to be toxic and corresponded to a mixture of three

compounds: a nontoxic fluorescent compound (F), and two other toxic agents,

compounds SC-5 and SC-7. The two major components from the 10% benzene-

petroleum ether eluate (2 a) were assigned as compounds SC-2 and SC-3.

They possessed the characteristic 280 nm uv absorbance as in compound SC-8;

however, theywere devoidof toxicity.

Although separation of the various components was evident in the

above procedure, when the fractions corresponding to compound SC-8 were

combined, tlc showed the presence of some unresolved compound SC-6 plus

other minor impurities. Hence, the mixture (2.5 g) was rechromatographed

using the identical chromatographic system (bed size 3.6 x 65 cm),the

results of which are shown in Figure 2.4. The second partition column

chromatography yielded compound SC-8 (1.0 g) as the major peak (fractions
375-415) followed by a minor peak, which corresponded to compound SC-6

(fractions 435-465). Further purification of compound SC-8 (1 g) using

a tlc-grade silica gel (50 g of silica gel containing 4% water; EM Reagents,

Type 60) afforded the pure toxic agent (SC-8, 773 mg; 0.04% overall yield).
The complete fractionation scheme is shown in Figure 2.1.

Compound SC-8 chromatographed as one band on tlc in two different sol-

vent systems and on high performance liquid chromatography (hplc) with a

regular phase analytical column, Table 2.2. The progress of purification

during the isolation of compound SC-8 is shown in Table 2.3. The initial














Solvent Gradient
% benz. in P.E.


120 90 60 30
AU(X4), 280 nm.


160


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Mixt SC-1 SC-2 SC-3 SC-4 SC-10SC-5 F SC-6 SC-7 SC-8 SC-9
SC Compounds
Figure 2.3. Thin-layer chromatography of SC-compounds.
























Solvent Gradient
% benz. in P.E.
25


1000 800


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Figure 2.5. Silica gel column chromatography of SC-8 fraction









Table 2.2. Purity of compound SC-8


Chromatographic Chromatographic number of components
methods systems


tic, Sio2 20% acetone-benzene one, Rf 0.36

tic, SiO2 6% methanol-chloroform one, Rf 0.78

hplc 1% MeOH-CHC13 1.6 ml/min one, Tp 5.8 min.

TLC detection: acid spray (2% H2S04 in acetic acid) plus heat.









Table 2.3. Toxicity of Various Fractions and Extracts during
of S. cernuus L


Fractionation


Fraction or MTD No. of
Extract (mq/Kg) Injections Results

concentrate of 500 3 Toxic
EtOH extract

EtOAc layer 109 3 Toxic

Ethyl ether 39 3 Toxic
layer

CCD, MeOH: HOH 22 4 Toxic
fraction

Partition column 11 5 Toxic
chrom., SC-6,8"
fraction

SC-8 3 3 Toxic









series of solvent partitions and countercurrent distribution yielded a

toxic fraction having a MTD of 22 mg/Kg. After column chromatography on

Sephadex and silica gel, the pure agent was obtained with a MTD of 3 mg/

Kg. This represents an increase in specific activity of about 167-fold

with respect to the starting material (concentrated ethanol extract).

Chromatography: Purification of SC-7

Compound SC-7, the second toxic component, was obtained from the

active mixture corresponding to fractions 215 to 350 of the above parti-

tion column chromatography, Figure2.2. Concentration of the combined frac-

tions (2.1 g, from three such columns) followed by chromatography using

a silica gel (60 g, mesh) column with 2-20% acetone-benzene step gradient

elution, afforded the active agent (700 mg) from the 10% acetone-benzene

eluate. Final purification of SC-7 was achieved using a partition column

chromatography similar to that in Figure 2.2, where pure SC-7 (505 mg,

0.025%) was obtained from the 25% benzene-hexane eluate. Toxin SC-7 had

a MTD (5 mg/Kg) similar to that of SC-8 and chromatographed as a single

band on tlc and hplc, Figure 2.3 and Table 2.4, respectively.

Chromatography: Purification of SC-6

The peak corresponding to compound SC-6 (Figure 2.2, 1.4 g from three

such columns) was rechromatographed using the same partition column system.

The elution profile of the second partition column is shown in Figure 2.6.

The 50% benzene-petroleum ether eluate, fractions 250 to 310, contained com-

pound SC-6. At this point it was noticed that SC-6 was weakly acidic (phe-

nolic) in nature, hence, final purification was possible using a solvent/

base partition method. However, because of its high lipophilicity, a sys-

tem consisting of 50% benzene-petroleum ether (200 ml) and 50% methanol-

0.5 N sodium hydroxide (200 ml) was required. The aqueous layer was then









Table 2.4. Hplc of SC-1, 7 and 8


Chromatographic Number of Components
Compound system and Retention Time, Minutes

SC-1 40% hexane-CHzC12 one, TR 3.80

SC-7 1% methanol-CHC13 one, TR 5.00

SC-8 1% MeOH-CHC13 one, TR 5.80
1.6 ml/min

















Solvent Gradient
% benz. in petroleum ether
50 25







0
4-)

(a
C-,
I-
o ko
0


4-,

Cd,
0
_-. ____ ---oC) W





N E
0




1L

I0
C-)
I \ o 5






s-





0 0

SL-
i I.


AU, 280 nm


600 500 400 300 200 100









acidified, diluted with an equal volume of water and extracted with chloro-

form (200 ml). Concentration of the chloroform layer yielded SC-6 as a

colorless amorphous powder (585 mg, 0.03% overall yield).

Chromatography: Purification of SC-1

"FRACTION I" from the partition scheme (Figure 2.1) was processed by

adsorption chromatography. The sample (25 g aliquot) was taken up in

hexane (125 ml) and chromatographed on silica gel (500 g) via a stepwise

gradient elution of 0, 5 and 50%benzene in hexane. During development,

column chromatography was monitored using uv absorbance at 280 nm and tlc

behavior. The major uv absorbing component eluted with 50% hexane-benzene

and was isolated as an oil (SC-1, 0.1-0.2%). SC-1 gave a single band on

tlc and hplc, Figure 2.3 and Table 2.4, respectively.

Chromatography: Purification of SC-2 and 3

The 10% benzene-hexane eluate from the partition column, Figure 2.2,
was concentrated (1.1 g) and rechromatographed using adsorption chromato-

graphy, silica gel (40 g, column grade) being the stationary phase and

benzene as the mobile phase (2 z). Two major uv absorbing components

eluted with benzene and were isolated as crystalline solids: SC-2, 0.04%,

elution volume 0.80 Z, and SC-3, 0.01%, elution volume 1.3 Z.

Chromatography: Purification of SC-10

"FRACTION II", 6.0 g, from the partition sequence (Figure2.1)was ad-

sorbed to a silica gel (150 g, column grade) column and eluted with benzene

containing a gradient of acetone (0-10%). During development column chroma-

tography was monitored using tic behavior (SiO2, 10% acetone-benzene, Rf

0.4). When the acetone concentration reached 10%, a major band eluted and

was isolated as a colorless crystalline solid (419 mg) from ethyl ether

(SC-10, 0.021% yield).















CHAPTER III

STRUCTURAL ELUCIDATION OF
COMPOUNDS SC-1, 2, 3 AND 10
In Chapter II is described the isolation and purification of seven

components from S. cernuus L. These compounds have been divided into two

groups, depending on the toxicity and their proton magnetic resonance

(pmr) spectral characteristics. The first group consists of compounds

which possess biological activity and/or whose pmr spectra are very simi-

lar to that of the bioactive substance, SC-8. Group two consists of com-

pounds which lack toxicity and whose pmr spectra are quite different from

that of SC-8. Accordingly, the first group is represented by compounds

SC-6 and 7, and will be discussed in Chapter IV along with SC-8, while

the structural elucidation of the latter group, consisting of SC-1, 2, 3

and 10 will be the subject of this chapter.

Compound SC-1
20
Compound SC-1 is a colorless oil, with an optical rotation of [a] -

27.20 and uv absorbance maxima at 230 and 288 nm. There is no carbonyl

or hydroxyl absorption in the infrared spectrum (see experimental), but

it contains a strong band at 930 cm 1 characteristic of a methylenedioxy

group along with other bands at 1435, 1240, 1185 and 1035 cm 1. The pmr

spectrum also suggested the presence of the methylenedioxy groups (4 H,

T 4.15, s) and the lack of other oxygen functionalities (e.g.,OCH3,

Ar-CH-0, etc.). Other groups found in the pmr spectrum were two equivalent

methyl groups of the type CH3CH (Tr 9.33, 9.22, d), two benzylic methylene

groups (r 7.58) and six aromatic protons (T 3.33-3.57). The spectrum
23









suggested a symmetrical molecule, possibly consisting of two C6-C3 units
joined together as in lignoids. (Lignoids are discussed in Chapter VI;
they are natural products made of two or more phenylpropanoid units).
The mass spectral behavior of SC-1 was characteristic of that of the di-
arylbutanes (12). The mass peak (M+ 326) had a medium-low intensity of
25% as observed with similar methylenedioxy lignoids. Indeed, the methy-
lenedioxy benzyl or its equivalent was responsible for the base peak at
m/e 135. Other fragmentations were of very low abundance, with m/e 191
(MW-135, 2.3%) and m/e 163 (cleavage at the 8-8' bond, 3.1%) being the
most significant with respect to structural information, Figure 3.1. A
search of the literature showed possible identity of compound SC-1 with
austrobailignan-5 (13) with the structure 3.1. The agreement between SC-1
and austrobailignan-5 in the sign and magnitude of rotation indicated their
stereochemical identity.
Reaction of SC-1 with DDQ gave crystalline 3.2, C20H1604, with uv
absorbance at 239, 284, 315, 329 nm and pmr: T 2.29-3.50 (6 aromatic pro-
tons); T 4.04, 4.17 (2 methylenedioxy groups); T 7.62 and 7.94 (2 methyl
groups). These resembled the data given for the known 6,7-dimethoxy-4-
(3,4-dimethoxy)phenyl-2,3-dimethylnaphthalene (dehydrodimethyl guaiaretic
acid,3.3), (14) except for the differences of methoxyl versus methylene-
dioxy groups. Such 4-arylnaphthalenes are common dehydrogenation products
of various neolignans, although3.2 itself has not been previously described.


0 00 CH3ON0
yC CH.O S


3.2


3.3

















m/e 326 (M ,


+


7+


m/e 135 (100%)


m/e 191 (1.1%)


m/e 1


Mass spectral fragmentations of SC-1.


Figure 3.1.









Compound SC-2
This is a colorless crystalline compound, C22H2805 (M+ 372), with uv
absorbance at 239 and 280 nm, and an optical rotation of [a]J20 + 48. The

pmr spectrum (see experimental) was relatively simple and characteristic
of a symmetrical molecule: six aromatic protons (T 3.17), two Ar-CH-0
(T 4.32 and 4.64), four methoxyl groups (r 6.14), and two CH-CH3 groups
(r 7.33, m; T 9.25, 9.33, d). The above information suggested a lignoid
structure,possibly of the tetrahydrofuran type. Since the ir did not
indicate the presence of hydroxyl or carbonyl groups, the fifth oxygen
might be considered as part of an ether function (1135 and 1160 cmn1).
Mass spectrometry was very useful for providing the overall structure of
SC-2. It has been amply demonstrated that the tetrahydrofuran function
can strongly direct fragmentation of lignoids possessing this nucleus.
This allows positional isomers of the tetrahydrofuran lignoids to be dif-
ferentiated via a well-defined fragmentation pattern (15). Spectral
analysis showed base at m/e 206, together with major peaks at 191, 175,
165, 151 and 138, all of which are characteristic of 2,5-bisaryltetrahy-
drofuran-type lignoids (Figure 3.2), and leading to 3.4 as the structure
for SC-2, with the stereochemistry to be decided.

H3 CH3

CH30- 0 0 OCH3

CH3 3.4 OCH3
Compound SC-3
SC-3 was obtained as a crystalline solid, C22Hzg2805 (M+ 372) with uv
absorption (Xmax 239 and 280 nm) and mass spectral fragmentations (base
peak m/e 206 and other peaks at 191, 175, 165, 151 and 138) identical to
compound SC-2. However, the pmr spectrum was rather more complex than that











7t


m/e 206 (100%)


m/e 165 (16%)







m/e 138 (11%)


m/e 191 (39%)


HCH3
HC-CH2
m/e 175 (37%)


m/e 151 (12%)


Figure 3.2. Major mass spectral fragments of SC-2.









of SC-2 (see experimental): a broad multiple T 2.89-3.13 due to six aro-

matic protons; two doublets T 4.87 and 5.56, corresponding to two benzylic

protons; four aromatic methoxyl groups, T 6.10-6.15; a very broad multi-

plet formed by two protons of the type CH-CH3; and, unlike SC-2, there

was a doublet for each methyl group, one at r 8.93 and the other at T 9.33.

The above data suggested a bisaryltetrahydrofuran structure for compound
SC-3 although the spectral differences could be ascribed to stereochemical

differences. Search of the literature and comparison of physical (mp 122-

1230, and [a]20 + 34) and spectral properties revealed SC-3 to be identi-

cal to the known lignoid veraguensin (16) with the structure 3.5.



CH30 0 0-- CH3

CH3 3.5 OCH3


Lignoids of the tetrahydrofuran type undergo an acid-catalyzed rear-
rangement to a 4-phenyltetralin system. Both 3.4 and 3.5 gave the same
product 3.6, C22H2604, when treated with p-toluenesulfonic acid; pmr:

T 3.37-3.50, m, five aromatic protons; T 3.90, s, one olefinic proton;

T 6.15, 6.25, s, four methoxyls; T 7.67, m, one proton of the type CHCH3;

T 8.20, s, a methyl group of the type =C-CH3; and T 8.87, 9.00, d, three

protons (CH-CH3). The spectrum no longer showed the doublet (Ar-CH-0)
characteristic of 3.4 and 3.5.

Treatment of 3.6 with DDQ gave a fully aromatic product 3.7, C22H2205,
ir: 1690 cm'1; pmr: -r 1.84, s, 1H; T 2.67-3.27, m, 6 H (arom H); T 6.0,

6.14, 6.27, s, 12 H (OCH3); T 7.5, s, 3 H (CH3). These,and a positive

2,4-dinitrophenyl hydrazine test, indicated that 3.7 had an aldehyde group,

presumably derived from the oxidation of one of the methyl groups. Cata-
lytic hydrogenation of 3.7 gave 3.3.










3.4 or 3.5 --3 3 )
CH30O CH3O-

0o 0o
3.6 OCH3 3.7 OCH3
CH3 CH3
With regard to the stereochemistry of SC-2, there are six possible
lignoids of this structure. Of these, two have the meso structure: gal-
gravin 3.8 (17) and tetrahydrofuroguaiacin 3.9 (18) and SC-2 differs from
both because of its optical activity. It differs also from two other
optically active isomers: galbelgin 3.10 (17) and veraguensin 3.5 (16).
Of the remaining hitherto unknown isomers 3.4 and 3.11, the pmr spectrum
of 3.11 must have signals due to nonequivalent aromatic protons and methyl
groups. Since SC-2 gave signals to indicate equivalent methyl (r 9.25,
9.33, d) and aromatic protons (T 3.17, s), and a generally symmetrical
structure, it must have the structure 3.4. SC-2 is thus a new compound.


A-^T-A0 A. A)^A
A 3 'Ar A 0r Ar Ar Ar
3.8 3. A 10 3.9




Ar 0 '"'Ar 3.1 Ar Ar 0 Ar
3.5 3.1_3.


Ar = 3,4-dimethoxy-benzene








Compound SC-10
The phenolic compound which was obtained as a crystalline solid (mp
204') did not show toxicity in mice. Elemental analysis and mass spectro-
metry indicated that the compound had a molecular weight of 328 correspond-
ing to C20H2404. It showed uv absorption maximum at 280 nm and upon addi-
tion of base, there was a shift of the maximum to higher wavelength, 295
nm. The ir spectrum (see experimental) indicated that it was aromatic and
it showed an intense hydroxyl band at 3440 cm 1. The pmr spectrum indicated
the presence of two active hydrogens (presumably phenolic groups) at T 2.35,
Figure 3.3. In the presence of D20, this signal disappeared, Figure 3.4.
The presence of the two phenolic hydroxyl groups was confirmed by the forma-
tion of a diacetate, C2H2806g, M+ 412; pmr: T 7.70, s, 3 H, T 7.80, s, 3 H.
Other groups indicated by the pmr spectrum were: five aromatic protons
(r 3.8-3-1), two aromatic o-methyl groups (Tr 6.2), three benzylic protons
(T 7.6, 2 H and r 6.67, 1 H) and two -CH-CH3 groups (r 8.45, m; T 9.17 and
8.95, two doublets). The mass spectral fragmentation of SC-10 showed im-
portant peaks at m/e 328 (Mi, base peak), 272, 241, 204, 189, 164 and 137,
which are characteristic of the 1-phenyl-l1,2,3,4-tetrahydronaphthalene lig-
noids (19). The molecular ion forms the base peak (m/e 328) as observed
in various phenyl-tetrahydronaphthalene lignoids such as isogalactin (19),
phyllanthin (20) and the podophyllotoxins (19). The m/e 164 can be attri-
buted to the structure 3.12, which is also consistent with the aromatic
substitution in the pmr spectrum. However, the most characteristic


0
HOX
OCH3
3.12


























0 -- .-\ -., <__ -.. '
I-'






I,
--n
as

3 *)
.0
4-


0.


^~ ~~~ 1 -Ji
(-)

0~
2:- C,,?
:i 4



. g as ; (o


Ig : 2 = "
E4 a



4:L
11'~~ 6 .: j 5 .



Im
E g- l

"~~ ~~ f :{ii i































I

~i" I
ii
:Iii~









fragmentation is that,due to the reverse Diels-Alder reaction on the
parent compound (m/e 272) followed by loss of a methoxy group, m/e 241,
Figure 3.5.

Methylation of SC-10 and reaction with DDQ gave a crystalline solid,

C22H220s which was identical with 3.7 and gave the known 3.3 on catalytic
hydrogenation. These data and an optical rotation value of [a]20 + 42
indicated structure 3.13 for SC-10, identical with (+) guaiacin (21) in

structure and stereochemistry.

0 > > 3.7 > 3.3
H

3.13

T CH3
OH Experimental
General
Melting points were taken on a Fisher-Johns melting point apparatus.
All melting points are reported as uncorrected. Elemental analyses were
performed by Atlantic Microlab, Inc., Atlanta, GA. Ultraviolet spectra
were recorded on a Beckman Model 25 spectrophotometer. Infrared spectra
were recorded on a Beckman Acculab 3 infrared spectrophotometer. Samples
were examined as potassium bromide pellets (unless otherwise specified),

usually with a concentration of 1 mg of sample per 100 mg of potassium
bromide. All pellets were prepared with the aid of a minipress made by
Wilks Scientific Corporation.
Proton magnetic resonance spectra were recorded in deuteriochloroform
(CDC13) with tetramethylsilane (TMS) as the internal standard. Spectra
were obtained using a Varian T60A spectrometer: s = singlet, d = doublet,
t = triplet, q = quartet and m = multiple. Coupling constants (J) were
expressed in herz (Hz).
















m/e 328


+\


n +


7 +


+
itc c.
m/e 272 (36%)
*+ 2 H
:XCX
.. +



+ 4

m/e 241 (89%)


om/e 164 (4.4%)
m/e 164 (4.4%)


~H2
m/e 137 (16%)


Figure 3.5. Mass spectral fragmentation of SC-10.







35

Optical rotations were measured with a Perkin-Elmer 141 polarime-

ter.

Mass spectra were obtained on a DuPont 491 chemical ionization spec-

trometer and Hitachi-Perkin-Elmer high resolution mass spectrometer, model

RM U6 E.

Qualitative thin-layer chromatography (tic) was performed on micro-

slides, 3 x 1 inches or 3 x 2 inches, coated by hand with Merck silica gel

HF 254 + 366 acc. to Stahl, and the plates were allowed to dry at room
temperature. Preparative plate chromatography (preparative tic) was per-
formed on glass plates 8 inches x 8 inches, coated by hand with a slurry con-

sisting of 18 gm of Merck silica gel HF 254 + 366 acc. to Stahl in 45 ml

of distilled water. After coating, the plates were allowed to air dry at

room temperature for a minimum of 48 hours before use. A maximum of 200 mg

of material could usually be applied to these plates.

High performance liquid chromatography (hplc) was carried out in a

Spectra-Physics SP 3500B liquid chromatograph with uv detector (280 and 254

nm). A Whatman analytical column, Partisil P x S 5/25 Col. No. 1A1799 was

used during the analyses.

Preparative scale adsorption column chromatography was carried out with

a 1:1 mixture of silicic acid (Mallinckrodt 275-325 mesh) and cellulose

(Brown and Co., tic grade).

Compound SC-1, Austrobailignan-5 (3.1)
20 E t0H
This is a colorless oil, [a]20 27.2 (C = 1, CHC13). Uv:?xEt0H 230,
D max
and 288 nm (log E 3.91, 3.85); ir (film on a NaCi cell), v: 2950-3000, 2900,
1480, 1410, 1435, 1240, 1185, 1035, 930, 855 and 805 cm 1. Mass peaks: 326

(M 25%), 191 (1.1), 190 (2.3), 163 (3.1), 149 (2,5), 136 (33), 135 (100),
105 (6.3) and 77 (14); pmr, T: 3.33-3.57, m,6 H (arom H); 4.15, s, 4 H (0-CH2-
0); 7.58, t, J 5, 4 H (Ar-CH2); 8.3, q, J 6, 2 H (CH-CH3); 9.22, d, J 6, 6 H,
(CHCH ).









Reaction of SC-1 with DDQ

Compound SC-1 (100 mg) was dissolved in dioxane (15 ml) and 2.1 equi-

valents of DDQ (2,3-dichloro-5,6-dicyano-1,4-benzoquinone)were added. The
reaction mixture was refluxed at 1050C for thirty minutes. At the end of

the reaction, tic analysis (10% benzene-hexane, uv lamp as detection sys-

tem) indicated complete conversion to a single, lower Rf-component. The

reaction mixture was cooled and the reduced reagent (2,3-dichloro-5,6-

dicyanohydroquinone) filtered. The filtrate was diluted with benzene and

extracted with aqueous sodium hydroxide solution. The unreacted DDQ and

the hydroquinone were extracted into the aqueous layer, while the product

from SC-1 remained in the benzene layer. The solvent layer was concen-

trated under reduced pressure and chromatographed using a silica gel (15 g)

column in 75% benzene-hexane, 1 ml/min. Fractionsofthe major band yielded

a white crystalline solid 3.2, mp 175-176 from ethyl ether (70 mg). Com-
poun 3.2 20 04~ v: Et0H
pound 3.2, C20Hig04, uv: maxt, 239, 284, 315, 329 nm; pmr (T), 2.29-3.50,
m, 6 H (arom H); 4.04, 4.17, s, each 2 H (0-CH2-0); 7.62, 7.94,-s, each
3-H (=C-CH3); mass spectrum: 320 (M 100%), 275 (4.3), 247 (3.7) 202 (6),

189 (8.8), 145 (3.2), 116 (4.2), 101 (6.5).

Compound SC-2 (3.4)

This is a colorless crystalline solid, C22H2805, M + 372, mp 80-81
(ether), [a]20+ 48 (C = 1, CHC13); uv XEtOH 239, 280 nm (log e 4.10,3.69);
D max
ir, v:1590, 1510, 1450, 1410, 1375, 1355, 1340, 1255, 1230, 1160, 1135,

1025, 960, 850, 810 and 760 cm 1. Mass peaks: 372 (M 13%), 287 (2.2),
207 (12), 206 (100), 192 (6.2), 191 (39), 178 (29), 176 (6.2), 175 (37),

166 (5), 165 (16), 151 (12), 145 (4.2), and 138 (11). Pmr (CDC13, T):

3.17, s, 6 H (arom H); 4.32, 4.64, d, J 5, 2 H (Ar-CH-0); 6.14, s, 12 H,

(OCH3), 7.33, m, 2 H (CH-CH3) and 9.25, 9.33, d, J 5, 6 H (CH-CH3),Figure3.6.









Compound SC-3, Veraguensin (3.5)

This is a colorless crystalline compound, C22H2805s M t 372, mp 122-

1230 (ether), fa]23 + 34 (C = 1, CHC13). Uv: XE0H 239 and 280 nm, ir,
D max
v: 1600, 1520, 1465, 1420, 1385, 1370, 1335, 1260, 1240, 1170, 1140, 1060,

1030, 970, 870, 820 and 770 cm 1. Mass peaks: 372 (M 26%), 287 (1.6),
207 (13), 206 (100), 192 (6), 191 (33), 178 (9), 176 (7), 175 (30), 166

(2.7), 165 (5.3), 151 (5.5), 145 (2.3), and 138 (6.7). Pmr (CDC13, -r):

2.89-3.13, m, 6 H (arom H); 4.87, d, J 8, 5.56, d, J 8, 2 H (Ar-CH-0); 6.10,

6.12, 6.13, 6.15, s, 12 H (OCH3); 7.5-8.4, m, 2 H (CH-CH3); 8.93, d, J 7,

9.33, d, J 7, 6 H (CH-CH3), Figure 3.7.

Acid-catalyzed Rearrangement of SC-2 and SC-3

A solution of the lignoid (SC-2 or SC-3, 0.2 g) in benzene (5 ml) was

refluxed with a saturated solution of p-toluenesulfonic acid (10 ml) for

30 minutes. Tic (10% acetone-benzene) showed formation of a single com-

ponent with higher Rf than the starting material. The cooled mixture was

washed with aqueous bicarbonate and the solvent layer concentrated to dry-

ness. The product was subjected to a silica gel (20 g) column chromato-

graphy in benzene. The product (3.6),eluted in the 2% acetone-benzene

fraction, was obtained as a crystalline solid from ether (mp 1020;

0.154 g); [a]23 + 127; lit. mp 100-101; [a]27 + 135.50 (16).

Alternatively, the neolignan (0.2 g) was dissolved in trifluoroacetic

acid (2 ml) at 50C and the solution kept at that temperature for thirty

minutes at which time the reaction was complete. The mixture was diluted

with water, neutralized with sodium bicarbonate and extracted with benzene.

The solvent extract was concentrated and purified by column chromatography

(as above), to obtain 3.6, 80% yield. Compound 3.6, C22Hz204, has uv:

Smax : 240 and 280 nm (log e 4.52, 4.11); pmr, T:3.37-3.50, m, 5 H (arom H);
ma~x









3.90, s, 1 H (olefinic H); 6.15, 6.25, s, 12 H (OCH3); 7.67, m, 1 H (CH-

CH3); 8.20, s, 3 H (=CH-CH3); and 8.87, 9.00, d, 3 H (CH-CH).

Reaction of 3.6 with DDQ

Compound3.6 (0.lg) was dissolved in dioxane (15 ml) and treated with

2.1 equivalents of DDQ. The reaction mixture was refluxed at 1000C for one

hour and the product treated as before (see above, reaction of SC-1 with

DDQ). A solid was obtained, compound 3.7 (0.08 g): ir, v:1690 cmn1; uv:
NEt0H 260, and 310; pmr, T: 1.84, s, 1 H (?-H); 2.67-3.27, m, 6 H (arom H);
max
6.0, 6.14, 6.27, s, 12 H (OCH3); 7.5, s, 3 H (CH3).

Catalytic Hydrogenation of 3.7

A solution of 3.7 (0.08 g) in ethanol (10 ml) was hydrogenated in a

Parr hydrogenator in the presence of 5% Pd/C (0.1 g) for twenty minutes

at 50 psi. Tic (5% acetone-benzene) showed that the reaction was complete.

The mixture was filtered through a bed of celite, the filtrate diluted with

water and extracted with benzene. The solvent layer was concentrated to

dryness and a solid 3.3 crystallized from ether/hexane, 47 mg, mp 1800;

(lit. mp 178-179)(4) uv, A EtOH:239, 284, 315, 329 nm; ir: 1600, 1575,
max
1500, 1455, 1425, 1395, 1370, 1315, 1245, 1160, 1135, 1025, 1005, 885 and

755 cm"1; pmr, T: 2.53-3.4, m, 6 H (arom H); 6.0, 6.14, 6.33, s, 12 H

(OCH3); 7.59, 7.9, s, each 3 H (CH3).
Compound SC-10, (+) guaiacin (3.13)

This is a colorless crystalline compound, mp 204-206 (benzene),
[a]20 + 42 (C = 1, CHC13). Found: C, 72.92; H, 7.40; C20H2404 requires:
D
C, 73.14; H, 7.36. Uv, AEtQH:230 and 280 nm (log c 4.12,3.80); ir:
max
3540, 3420, 1610, 1510, 1450, 1445, 1270, 1255, 1225, 1210, 1030, 885, 865

and 795 cm'1. Mass peaks: 328 (M 100%), 272 (36), 241 (89), 204 (34),
189 (29), 164 (4.4) and 137 (16). Pmr,(CDC13, T): 2.35, broad, 2 H (OH);









3.1-3.8, m, 5 H (arom H); 6.2, s, 6 H (OCH3); 6.67, m, 1 H (Ar-CH-Ar);

7.6, d, J ~8, 2 H (Ar-CH2); 8.45, m, 2 H (CH-CH3) and 8.95, d, J -8, 9.17,

d, J 5, 6 H (CH-CH3).

Acetylation of SC-10

To 0.10 g of SC-10 were added 3 ml of acetic anhydride and 0.5 ml of

pyridine. The mixture was heated at 1000 for fifteen minutes, and allowed

to cool to room temperature, after which water was added and the mixture

shaken vigorously. After fifteen minutes, the solid was filtered and crys-

tallized from aqueous methanol, 0.093 g; mp 116-117; C24H2806, (M+ 412);

pmr, (T): 3.0-3.7, m, 5 H (arom H); 6.3, s, 6 H (OCH3); 6.5, d, J -9, 1 H

(Ar-CH-Ar); 7.75, d, J -5, 2 H (Ar-CH2); 7.70, 7.80, s, each 3 H (OAc);

8.4, m (CH-CH3); 8.95, 9.17, d, J 5, each 3 H (CH-CH3).

Methylation of SC-10

A mixture of SC-10 and 200 mg of anhydrous potassium carbonate in

20 ml of acetone was treated with 0.4 ml of dimethyl sulfate and stirred

under reflux (900) for two hours. After completion of the reaction (ac-

cording to t1c), the mixture was allowed to cool to room temperature and

filtered. The filtrate was concentrated to one-third the volume and the

residue partitioned between ethyl ether and water. The ethyl ether

extract was concentrated to almost dryness, at which time the methylated

product was obtained as a crystalline solid, 85% yield; mp 130-132; (lit.

mp 1300). Found: C, 73.91; H, 7.86. C22H2804 requires C, 74.13; H, 7.91.

3.7fromSC-10 Methyl Ether

SC-10 methyl ether (above) was dissolved in dioxane (12 ml) and 2.1

equivalents of DDQ were added. The reaction mixture was refluxed at 1050C

for one hour and the product treated as before (see experimental: reaction

of SC-1 with DDQ). A solid was obtained, 65 mg, identical to 3.7 (tlc,

pmr).








40












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z

I /



r l i C






S --







CL CL
1 8 -
i~ (jo








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M 0{ I:
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to 8
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E





















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0 0 V *- I

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Z '"- -

s~ i E
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*~0 ^ __- _C' *)^*
'~~~~z ^ ^^ ^ -s ; :.1 1

< i^ : ^J=!^
_________a_______________ 3 .s uS _














CHAPTER IV


STRUCTURAL ELUCIDATION OF SC-6, 7 and 8

The toxic principles SC-7 and SC-8 of the leaves and stems of S. cer-

nuus L were obtained using a series of purification steps guided by the

mouse toxicity assay, described in Chapter II. Compound SC-6, although

devoid of toxicity, accompanies SC-8 closely and was also isolated and

found to have a proton magnetic resonance spectrum very similar to that of

toxin SC-8. For this reason, these compounds will be treated as a group

and the elucidation of their structures is presented below.

Compound SC-8

The major active principle SC-8, obtained as a colorless solid,

was optically active, [a] 100 and showed uv absorption maxima at 235

nm (log e 4.62) and 280 nm (log e 4.16), unchanged by base and character-

istic of a 3,4-dimethoxybenzyl system. The ir spectrum (see experimental)

indicated that it was an alcohol (3500 cm"1) and aromatic (1510 cm 1).

The proton magnetic resonance spectrum (Figure 4.1) showed the following

signals with their probable assignments, although the number of protons

of each category will be discussed later: T 2.95-3.30, m, aromatic H;

T 4.50, 4.60, d, benzylic protons of the type Ar-CH-0; T 5.30, 5.43, d,

benzylic protons of the type Ar-CH-O; T 5.90, m, CH3-CH-0; T 6.14, s,

methoxyl groups; T 7.70, m, CH-CH3; -r 8.80, 8.90, d, CH-CH3 and -r 9.22,

9.32, d, CH-CH.. The spectrum showed general characteristics of that of

a lignoid and specifically those of 2,5-bisaryltetrahydrofuranoid lig-

noids such as veraguensin and SC-2. The presence of a hydroxyl groups)

was confirmed by conversion to an acetate which showed ir band 1735 cm 1
42

































0
z



I
U
00

(/)
1 '4-

SE

Iz u




E^ i








OJ a
ILI
0















zy

wzC )
0 cc
CLC
-3 r' S.-
"53-' -
S ~y~i : 0
SS'-' : C
Se ^ ff




0.

. j~ ~s G



'- S OS a)C
zs =g
wu^S z l a








and pmr signal T 7.80, s, characteristic of an acetate of an alcoholic
hydroxyl. The acetate also showed significant downfield shifts of the
doublet T 5.30, 5.40 to T 3.90, 4.00 and the multiple Tr 5.90 to T 5.47
thus suggesting that the hydroxyl is benzylic and was located in the vicin-
ity of the group 0-CH-CH3. The mass spectrum of SC-8 was not very useful;
it showed no molecular ion and no significant peaks above m/e 192. The
major peak was at m/e 165 which can be assigned to a dimethoxybenzoyl
fragment. Elemental analysis pointed to an empirical formula C21H2605-6
which, while showing relationship to a diaryltetrahydrofuranoid lignoid,
could not be relied upon for any further structural information.
In spite of the many points of similarity between SC-8 and a diaryl-
tetrahydrofuranoid lignoid 4.1, there are some discrepancies which cannot
be explained with such a structure. The most important one is the presence
of a benzylic hydroxyl. It cannot be located at either 2 or 5 positions
because: a) there will be no room for a "benzylic" proton and, b) the com-
pound would behave as a potential ketone similar to the lignoid magnolenin
C 4.2 described recently from these laboratories (24). The hydroxyl also
cannot be located at 3 or 4 or on the methyl groups because, according to
the pmr spectrum, there are two methyl groups, each attached to a -CH unit.
H3C CH3 CH3 CH30 O

CHCH
HOD O CH
Ar r C
4.1 CH3 0CH Ou
4.2 4Glu T OCH
42 CH3 43 CH3
To confuse the issue further, preliminary experiments showed that,
like the 2,5-diaryltetrahydrofuranoid neolignans, SC-8 readily underwent
acid-catalyzed transformation to a product with pmr spectral characteris-
tics very similar to those of cyclogalbelgin 4.3 obtained from galbelgin,









veraguensin and SC-2 (see Chapter III) and still retained the hydroxyl
groupss. Also, the ratio between the aromatic protons and the methoxyl
protons in SC-8 could not be clearly defined and was in the range of 5:12
and 6:9.

Because of this, several other possibilities were considered for a
hypothetical structure to serve as a basis for further work. One of these,
4.4, is shown below. It has two different CH-CH3 groups, two benzylic pro-
tons, one next to a hydroxyl and another next to an ether and is, in gener-

al,in conformity with biogenetic considerations in the lignoid field. Such
a compound must generate on oxidation a methoxylated phthalic acid such as
m-hemipinic acid 4.5. Consequently, alkaline permanganate oxidation of
SC-8 was studied. Noneof the products recognized by their thin-layer
chromatographic or uv/pmr spectral behavior corresponded to either the
acid 4.5 or its isomer 4.6. Instead, the major products were found to be
veratric acid 4.7, 3,4-dimethoxyphenylglyoxalic acid 4.8 and an acid, the
methyl ester 4.9 of which had the molecular formula C20H2207 with the spec-
tral properties: uv, 285 and 307 nm; ir bands at 1700 and 1670 cm"1, char-
acteristic of a conjugated/aromatic ester and a ketone and pmr signals at:
T 2.33-3.17, m, 6 aromatic H; T 4.47, q, X-CH-CH3; T 6.14, s, 4 methoxyls
and T 8.19, 8.30, d, CH-CH3. The mass spectrum gave a molecular ion at m/e
374 and showed important peaks at m/e 343 (M-OCH3), 209 (M-165) and 165
(base peak, M-209). The base peak can be assigned to a dimethoxybenzoyl
fragment 4.10 and the peak at m/e 209 to the fragment 4.11, thus repre-
senting the fission of the molecule into two units.

OCH3 C H30 COOH
H3C O CH30 COOH COOH
H4.5 CH30 0 5I

H30 'OH H-OCH3 rCHO











CHH R 10 6OCH3
CH3V CH30O0 OCH

CH3 OCH3
4.7, R COOH3 H O 3
4.8, R CO-COOH O OCH3
The structure of 4.9 was established by synthesis through 0-alkylation
of methyl vanillate 4.12 with 2'-bromo-3,4-dimethoxypropiophenone 4.13.


CH3 OCH3
HO 4.9
CH3 0 4.12 0 > 49
CH30 0 COOCH3
Formation of 4.9 from SC-8 gave a basis for altering the hypothetical
structure 4.4 to 4.14 which contains many of the features known so far,
although the acid-catalyzed formation of cyclogalbelgin type of product
might be difficult to explain. In order to establish the nature of this
acid-transformation product, a more detailed study was undertaken of this
reaction. Treatment of SC-8 with p-toluenesulfonic acid in benzene gave
two products which were very difficult to separate from each other, in
contrast to veraguensin or SC-2 which smoothly gave cyclogalbelgin 4.3 as
the sole product. Separation of the two products was possible when it
was recognized that one of them was phenolic. The neutral fraction was
obtained as a colorless oil, C11H1403, with uv: 280 nm; ir: 1685 cm'1 and
pmr: T 3.25, m, 3 aromatic H; T 6.17, s, 2 OCH3; T 6.40, s, Ar-CH2 and
T 7.87, s, CH3-CO. These properties pointed to the structure of 3,4-
dimethoxyphenylacetone 4.15, which was confirmed by comparison with an








authentic sample prepared from 0-methylisoeugenol 4.16 by the action of
boron trifluoride.
OCH3


4.14 OH 4.16CH3 CH30
CH 0 CH30 CH3 4
OCH, CH-CH3
The phenolic fraction from the acid-transformation reaction was a
colorless glassy solid 4.17 with uv, Amax 282 and 312 nm which, on addi-

tion of base, shifted to 29C and 330 nm respectively. Proton magnetic
resonance indicated the presence of two exchangeable hydrogens (phenolic
groups) through a signal at T 4.67; five aromatic protons (r 3.43); one
olefinic proton Ar-CH=C (r 3.97); two methoxyls (C 6.27) and two methyl
groups, one of type CH3-C= (T 8.27) and the other of type CH3-CH (T 8.90,
9.00, d). Acetylation of 4.17 gave a diacetate 4.18 (T 7.74, s, 6H).
Methylation of 4.17 yielded a crystalline product 4.19, C22H2604, found to
be identical with cyclogalbelgin 4.3. Dehydrogenation of 4.19 with Pd/C
afforded a crystalline product 4.20, uv: 282, 314, 329 nm; pmr: T 1.95,
s, 1 H; T 2.50, s, 1 H; T 3.04, m, 4 H; T 6.07, s, 3 H; 6.17, s, 3 H;
T 6.33, s, 6 H; T 7.53, s, 3 H; T 7.90, s, 3 H which was identical with
the known 6,7-dimethoxy-4-(3,4-dimethoxy)phenyl-2,3-dimethyl naphthalene
(dimethyldehydroguaiaretic acid). It was also observed that if SC-8 is
heated with p-toluenesulfonic acid for a longer time, it was converted
to 4.21, C20H2004, (M t 324) which on methylation yielded 4.20.









CH 0 CH3 O0

R RO

0 CH3 0 0 O
4.17, R:H CH3 4.20, R= CH3 OC
4.18, R COCH3 4.21, R=H R
4.19, R:CH3
Formation of aryl dihydronaphthalenes such as 4.17 is very character-
istic of the 2,5-diaryltetrahydrofuranoid neolignans and structures such
as 4.14 cannot satisfactorily explain this reaction. However, other struc-
tures might generate compounds such as 4.15 and formation of 4.17 alone
cannot be used as a proof for the possible existence of a 2,5-diaryltetra-
hydrofuran system in SC-8. To establish this point, SC-8 was subjected
to catalytic dehydrogenation with Pd/C reaction which is known to generate
a 2,5-diarylfuran. Two products were isolated from the reaction mixture:
a neutral compound and a phenolic compound. The former was a colorless
oil, C11H16,0, which was identical with an authentic sample of 3,4-dime-
thoxyphenylpropane 4.22 obtained from the catalytic hydrogenation of
0-methyl isoeugenol 4.16. The phenolic product 4.23 which showed pmr
spectral characteristics of a 2,5-diaryltetrahydrofuran on methylation
yielded a crystalline methyl ether 4.24, C22H,280,, pmr: T 3.17, 6 aromatic
H; T 5.32, 5.44, d, 2 Ar-CHO; T 6.17, s, 4 OCH3; T 8.17, m, 2 CH-CH3 and
T 8.90, 9.00, d, 2 CH-CH3. Compound 4.24 was found to be identical with
the known neolignan galbelgin.
4.23, R=H
SH4.24, R:CH3
CH30 OCH3 CH30O *i*
4.22 OCH3 OR
OCH3









Although the Pd/C reaction of SC-8 gave a diaryltetrahydrofuran deri-
vative instead of a diarylfuran derivative, the latter was obtained by
reaction of SC-8 with dichlorodicyanoquinone (DDQ). The product 4.25
showed uv-maxima at 283 and 325 nm and pmr spectrum in which the signals
due to the benzylic proton (AR-CH-0, T 4.50, 4.60) were absent and a
methyl signal (CH3 C =) appeared at T 7.70, thus indicating the presence
of a diarylfuran system. The remaining groups were intact: the aromatic
protons (T 2.80-3.17, m); Ar-CH-OH (r 5.27-5.44, d); methoxyl (T 6.04,
6.14, s) and CH-CHj (T 8.77, 8.87, d). Compound 4.25 was then subjected
to catalytic dehydrogenation (Pd/C) followed by methylation to yield a
crystalline solid 4.26 with uv maxima 252 and 326 nm and pmr: T 2.74-3.05,
m, 6 H; T 6.19, s, 12 H; T 7.80, s, 6 H, identical with the known 2,5-bis-
(3,4-dimethoxy)phenyl-3,4-dimethylfuran (14).

4.25, RC,,H,,1503


XO O OCH3 4.26, R-CH3
OR

From the foregoing data, it is clear that SC-8 does have a 2,5-diaryl-
tetrahydrofuran system, as indicated by the isolation of 4.24 and 4.26
and even 4.19 through acid-catalyzed transformation, which is characteris-
tic of such a system. However, the formation of the acid 4.9 as well as the
pmr spectrum with signals for Ar-CH-OH and 0-CH-CH3 clearly indicate the
presence of other functionalities not normally seen in a diaryltetrahydro-
furan system. Also, each of the reactions described above, dehydrogenation
with Pd/C and acid-catalyzed transformation, gave a neutral product and a
phenolic product, either a diaryltetrahydrofuran or an aryltetralin, thus
suggesting that the neutral fragments were attached to the phenolic hydroxyl









of each of the aryl groups and were cleaved in the process. All these
observations can be reconciled by a structure such as 4.27i.in which a cen-

tral diaryltetrahydrofuran system with a phenolic group on each of its
aryl groups is attached to a phenylpropanoid unit (C9). A proof for such
a structure was obtained by a careful analysis of the products of the acid-
catalyzed transformation. It was found that the ratio between the ketone
4.15 and the phenol 4.17 was 2:1, thus showing that two C9 units (ketone)
were joined to the aryl tetralin system and hence, a diaryltetrahydrofuran
system. OCH

4.27a / OCH3
CHO30 0 0 O

CH3 OCH3

Additional support for this structure was sought through 13C nmr

spectral studies. The completely decoupled spectrum of SC-8 indicated
the presence of eighteen unique carbon types (Figure 4.2). Furthermore,
the off-resonance-decoupled spectrum (Figure 4.3) showed that, out of the
eighteen unique carbon types, six'were 40 carbons, nine were 30 carbons

and three were methyl carbons. This information, coupled with the chemical
shifts and the pmr data indicating three methoxyls and possibly six aro-
matic protons, allowed the following assignment for the 13C nmr spectrum of
SC-8: 6 150.4, 148.9, 148.7, 146.3, 136.2 and 132.6 (6 40 aromatic car-
bons); 6 83.6 and 83.2 (2 Ar-CH-0); 6 78.1 (CH3-CH-O), 6 55.7 (3-OCH3);
6 44.0 (CH-CH3); 6 16.8 and 14.7 (2 CH-CH ).
The 13C nmr spectrum agreed very well with information obtained from
the pmr spectrum and suggested the probability of at least one element of
symmetry in the compound, since there are fewer observable carbon-types








































C)O






co










Table 4.4. 13C-nmr spectrum of SC-8


t fihS ( ppm )


14.7
16.8
44.0
55.7
78.1
83.2
83.6
110.1
111.8
118.4
118.6
119.8
132.6
136.2
146.3
148.7
148.9
150.4


Carbon TvyDe


-CH3
-CH3
-CH
-OCH3
-CH
-CH
-CH
-CH
-CH
-CH
-CH
-CH
-6-
-t-
-C-
-C-

-p-
4-9


DescriDtion


CH CH3
CH CH3
CH CH3
OCH3 (3?)
0-CH-CH3
0 CH Ar
0 CH Ar
CH (Arom) (2?)
CH (Arom)
CH (Arom)
CH (Arom)
CH (Arom)
C (Arom)
C (Arom)
C (Arom)
C (Arom)
C (Arom)
C (Arom)


Peak #


I ,









53































co

I,

4-
0







^ S.-
U/)








E
0=
U,

co




1.--



0
r-

CL






0
U
u







0

0
U,
~C


O




U)




o,
cu



0,

L.
&* lr
___ u


S SS =S S
giiSSili' = 5S SSSl33S IiE
-

---------------









(eighteen carbon types) than the total number of carbons present (at least

twenty-one carbons present).

The proposed structure agrees well with the elemental analysis,

giving a molecular formula of C42H52011 and is consistent with all the

spectral and degradative data presented. A dimeric structure with an ele-

ment of symmetry accounts for the existence of magnetically equivalent

functionalities.

Compound SC-7
The second active principle SC-7 is a colorless, optically active

fa]D 99 solid with elemental analysis in agreement with the formula

C41H48011. It showed uv and ir spectra very similar to those of SC-8.
Its pmr spectrum was nearly identical to that of SC-8 except that a two

proton singlet was formed at T 4.12 which may be assignable to a methy-

lenedioxy group and other minor differences in the integral ratios. The

ratio of aromatic protons vs. methoxyl protons was found to be 6:6 in

SC-7 in contrast to 6:9 in SC-8. These differences indicated that SC-7

might differ from SC-8 only by replacement of two of the methoxyls by one

methylenedioxy group. The existence of two alcoholic hydroxyls was con-

firmed by the formation of a diacetate, ir: 1735 cm'1 and pmr: T 8.0, 6 H.

As seen with SC-8, action of p-toluenesulfonic acid on SC-7 yielded trans-

formation products except that three were formed in a ratio of 1:1:1 in-

stead of two in the ratio 2:1. Two of these products were neutral and,

when separated, found to be identical with 3,4-dimethoxyphenylacetone 4.15

and 3,4-methylenedioxyphenylacetone 4.28. The third product, a phenolic

compound, was identical with the aryl tetralin derivative 4.17 obtained

from SC-8. These considerations led to the structure 4.29 for SC-7.









4.29 -
CH3O O


4.28 Q C C

Compound SC-6
This is an amorphous solid, C31H3804, optically active [a]D 52.3
with uv: x 206, 226, 278 nm and ir, v:3440 (OH), 1600, 1650, 1260,

1230 and 1030 cml1. It showed no M in the mass spectrum and very little

or no fragments above m/e 192. Its pmr spectrum: T 3.00 3.27, m, 9 H

(arom H); 4.52 4.62, d, 2 H (Ar-CH-0); 5.29, 5.42, d, 1 H (Ar-CH-OH);

5.84, m, 1 H (CH3CH-0); 6.10, s, 12 H (OCH3); 7.74, m, 2 H (CH3CH); 8.87,

8.97, d, 3 H (CH3CH-0); 9.25, 9.35, d, 6 H (CHCH) was very similar to

that of SC-8, the only differences being due to the integration values.

For example, the CHCH, doublets as well as Ar-CH-0/Ar-CH-OH showed a ratio

of 2:1 instead of the 1:1 observed with compound SC-8. Similarly, the
ratio of aromatic H/methoxyl H was 1:1.33 instead of 1:1.5. The presence

of hydroxyl groups was confirmed by acetylation to a diacetate C35H401,o,

1735, 1760 cm'1 and T 7.70, s, 3 H, T 8.00, s, 3 H, in which one alcoholic
(benzylic) and one phenolic hydroxyl were involved. SC-6 underwent acid-
catalyzed rearrangement with formation of the same components seen with
SC-8, 4.15 and 4.17 but in a 1:1 molar ratio. Thus, SC-6 is assigned

structure 4.30, identical to SC-8 except for the absence of one of the
3,4-dimethoxyphenylpropanoid units. Furthermore, confirmation of the
structures 4.30 for SC-6 and 4.27a for SC-8 was obtained via conversion

of SC-6 to SC-8. This was accomplished by the alkylation of SC-6 with
3,4-dimethoxy-2'-bromopropiophenone to yield 4.31. The ketone 4.31 was
then reduced with sodium borohydride whereby a mixture of the two dia-
stereomers 4.27e,b was obtained.








56




































o
C,


o












in
i E











ac

I.3






o
W U) 4J
0 0









C-)




,L M ) E


~ za
^- -








SZz.S =
^ ^ ^ss
S5 32S
Ij^ sij .












4.30 HO- ) U CH




CHH3 OCH
C H 30 3]O OCH3

4.30 --0> 9` 431 X ---- > 4.27a,b





Stereochemistry of SC-6, 7 and 8
Compounds SC-6, 7 and 8 contain six to eight asymmetric carbons,
corresponding to as many as sixty-four to two hundred and fifty-six pos-
sible stereoisomers. To simplify the task of elucidating the stereochem-
istry of these compounds it was found convenient to divide the molecule in-
to two units: a) the 2,5-diaryltetrahydrofuran system and, b) the phenyl-
propanoid-0-aryl system.
The tetrahydrofuran system was discussed in detail under the stereo-
chemical elucidation of SC-2, at which time it was observed that the pmr
seemed to be a very useful tool for the analysis of the six possible repre-
sentations of the 2,5-diaryltetrahydrofuran system: galbelgin 4.24 (17),
galgravin 4.32 (17), veraguensin 4.33 (16), tetrahydrofuroguaiacin 4.34
(18), SC-2 4.35, and an unknown lignoid 4.36. In the pmr analysis of these
compounds, the benzylic protons (Ar-CH-0) are clearly sensitive to the
orientation of the aryl groups, and their chemical shifts can be used to
differentiate the various isomers. Furthermore, structures with equivalent









methyl groups (one doublet) are differentiated from dissimilar methyl-
containing isomers (two doublets as in veraguensin).






Ar 'Ar Ar 'Ar ,Ar Ar
4.32 4.33 4.34






A`O Ar A0 Ar
4.35 4.36

Ar = 3,4-dimethoxybenzene

This type of analysis allows us now to study the stereochemistry of
the SC-lignoids with respect to the tetrahydrofuran system. The pmr spec-

tra of SC-6, 7 and 8 were found to resemble that of SC-2 closely (Table 4.1)

and were very different from that of the other possible isomers, thus giving

a clear indication of a 2:3-cis/3:4-trans/4:5 cis arrangement for the four

substituents on the tetrahydrofuran ring, identical to that seen in SC-2.

Further evidence for this type of system was obtained from the 13C-nnr spectrum.

Here, a pattern can be seen, in which the signal due to the benzylic carbon

C-2 (or C-5) is located at 6 82-83 ppm if the aryl substituent is cis to

the methyl group at C-3 (or C-4), and at 6 87-88 ppm if the groups are trans

(23). Comparison of the 6 values of the methines at C-2 and C-5 (Table 4.2)

with the reported values of the known isomers, indicated that SC-8 corre-
sponded to a 2-3 cis/4-5 cis system. In support of this, we observed ear-
lier the ready isomerization of SC-8 to the more stable, all-trans, galbel-
gin 4.24 system when heated with Pd/C.









Table 4.1. Comparison of pmr data (c) of SC-6, 7 and 8 with that of
related lignoids


Lignoids CH3-CH Ar-CH-0


8.95 (d), 3 H 5.52 (d), 1 H
9.35 (d), 3 H 4.90 (d), 1 H

Galbelgin 4.24 8.95 (d), 6 H 5.40 (m), 2 H

Galgravin 4.32 8.95 (d), 6 H 5.47 (m), 2 H

SC-2 4.35 9.30 (d), 6 H 4.48 (d), 2 H

SC-8 9.27 (d), 6 H 4.55 (d), 2 H

SC-7 9.28 (d), 6 H 4.57 (d), 2 H

C-6 9.30 (d), 6 H 4.57 (d), 2 H









Table 4.2. Comparison of the '3C-nmr of SC-8with that of related lignoids


Chemical Shift (ppm) Stereochemistry
Lignoids C-2 C-5 at positions
2:3 4:5



galgravin 4.32 87.1 87.1 trans trans


veraguensin 4.33 87.1 82.8 trans cis


tetrahydrofuroguaiacin 4.34 82.4 82.4 cis cis


SC-8 83.2 83.2 cis cis








Finally, we should consider the stereochemistry of SC-6, 7 and 8 at
the phenylpropanoid-0-aryl system. Compounds which incorporate this sys-
tem were originally obtained from the oxidative coupling of isoeugenol
(25) (4.37, 4.38) and since then, two natural products of similar structure,
virolin 4.39 and surinamensin 4.40, have been isolated (26). Of the two
possible isomers, the erythro isomer 4.37 was obtained from the corre-
sponding ketone 4.41 by reduction with sodium borohydride while the threo


RI HO HO
0 OH 0 H O
OCH3 CH3 H

H OCH3 CH3O H CH3


4.38 4.41
4.37, R1- R- H

4.39, R CHH R2 H

4.40, RI-CH3 R2= OCH3

form was formed exclusively through the enzyme-H202 catalyzed coupling
reaction (25) which formed the dimer. The two isomers can be differentiated
by comparing their respective chemical shift of the 7-methine and the cou-
pling constant (erythro: T 5.15, J = 3; threo: T 5.38, J = 8). The pmr
spectra of SC-6, 7 and 8 showed doublets at T 5.35 with J value of 8,
clearly indicating the threo configuration as reported for virolin and
surinamensin (26). The unperturbed doublet structure also indicates that
both phenylpropanoid chains have the same threo configuration. In con-
trast, the semisynthetic SC-8 4.27b shows signals due to both isomers:








r 5.15, J = 3 (erythro) and T 5.38, J = 8 Hz (threo), each equal to one
proton. The structure and stereochemistry of SC-6 4.42, SC-7 4.43 and SC-8
4.44 can therefore be represented as shown in Figure 4.5.
CHC3 CH3

CH30 H

CH30, O0 .O 0 OH


4.27b HC3 OCH3 H
Experimental
General

Physical measurements and other experimental material, when applicable,
are described on page 33.
Compound SC-8, 4.44
This is an amorphous powder, [a]20 100 (C = 1, CHC13). Found:
C, 68.66; H, 7.30; C42H52011 requires: C, 68.83; H, 7.15; uv XEtOH 212,
max
235 and 280 nm (log e 4.82, 4.62, 4.16);ir,v:3490, 1600, 1585, 1500, 1405,
1370, 1255, 1130, and 1020 cm-1. Mass peak, m/e: no M+, 370 (0.72%), 324

(3.8), 220 (1.3), 203 (0.81), 192 (47), 178 (28), 167 (27), 165 (100), 151

(38), 145 (34), 107 (31), 91 (41), 77 (46). Pmr, r:2.95-3.30, m, 12 H
(arom H); 4.50, 4.60, d, J 6 (2 Ar-CH-0); 5.30, 5.43, d, J 8 (2 Ar-CH-OH);
5.90, m (2 CH3CH-0); 6.14, s (6 OCH ); 7.70, m (2 CH-CH3); 8.80, 8.90, d,
J 6 (2 O-CHCH3); 9.22, 9.32, d,J 6 (2 CH-CH3).
Acetylation of SC-8
To 0.20 g of SC-8 were added 4 ml of acetic anhydride and 0.5 ml of
pyridine. The mixture was heated at 1000 for twenty minutes, and allowed
to cool to room temperature, after which water was added and the mixture
shaken vigorously. After fifteen minutes, the solid was filtered and an
amorphous powder obtained from aqueous methanol, 0.17 g. Found: C, 67.50;










.ICH3


4.44


CH3


4.43


,.CH3


HO'


4.42


Figure 4.5. Structure of compounds SC-8, 7 and 6.









H, 6.96; C46,Hs603 requires: C, 67.62; H, 6.91; ir, v: 1735 cm-1 (C=0).

Pmr, T: 3.02-3.27, m, 12 H (arom H); 4.03, 4.15, d, J 7 (2 Ar-CH-OAc);

4.53, 4.62, d, J 6 (2 Ar-CH-0); 5.42, m (2 CH3CH-O); 6.18, s (6 OCH3);

7.73, m (2 CH-CH3); 8.00, s (2 OAc); 8.78, 8.88, d, J 6 (2 OCH-CH); 9.25,

9.35, d, J 6 (2 CH-CH3); Figure 4.1.

Oxidation of SC-8 with Alkaline Potassium Permanganate

To a solution of SC-8 (0.30 g) in pyridine (5 ml) was added 5% aque-

ous permanganate dropwise while the mixture was being stirred, and then

heated under reflux for one hour. The reaction mixture was monitored for

the disappearance of SC-8 (tic, 25% acetone-benzene). The cooled mixture

was then treated with 6 N H2SO4 and sodium bisulfite until a clear solu-

tion resulted. It was extracted with ether and the ether extract washed

with aqueous sodium bicarbonate to separate the neutral from acidic com-

ponents. The mixture of the acidic components was treated with ethereal

diazomethane and purified by using a preparative tic plate. The three

major bands were isolated and identified. Band 1: methyl veratrate, 33 mg,

pmr, T: 2.33, 3.13, m (3 H, ABX, arom H); 6.08, s (9 H OCH3); homogeneous in

tic with a reference sample and with identical spectral data. Band 2: com-

pound 4.9, 15 mg. This is a crystalline solid, mp 139-140, uv X
max
285, 307 nm; ir, v: 2965, 1700, 1670, 1580, 1505, 1445, 1412, 1375, 1295,

1260, 1225, 1158, 1145, 1120, 1015 and 760 cm'1; pmr, T: 2.33-3.17, m (6 H,

arom H); 4.47, q, J 7 (1 H, CH3 -CH); 6.14, s (12 H, OCH3); 8.19, 8.30, d,

J 7 (3 H, CH3i-H), mass peak: m/e, 374 (M ), 343, 209, and 165 (base peak);
identical by ir and pmr spectral comparison with a standard sample. Band 3:

(methyl-3,4-dimethoxyphenylglyoxalate 4.8, 8 mg) pmr: T 2.4, 3.13, ABX

pattern (3 H, arom H); 6.05, s (9 H, OCH3); ir, v: 2915, 2820, 1680, 1590,
1515, 1465, 1425, 1300, 1270, 1235, 1140, 1025 and 760 cm"1; identical by

tic, pmr and ir spectral comparison with the standard compound.









Catalytic Dehydrogenation of SC-8 with Pd/C and Heat
A mixture of SC-8 (0.60 g) and Pd/C (0.2 g) in 2,2'-oxydiethanol

(10 ml) was boiled under reflux for two hours. When the tic (15% acetone-

benzene) showed the absence of the starting material, the cooled mixture

was filtered, the filtrate diluted with water, pH 2 (30 ml), and extracted

with ethyl ether (40 ml). The ethyl ether separated into the neutral and

acidic components. The neutral extract was concentrated to dryness and

the product purified by preparative tic (10% acetone-benzene). The major
EtOH
band was isolated as an oil (0.12 g): uv x 280 nm; pmr, T: 3.3, s (3 H,
max
arom H); 6.17, s (6 H, OCH3); 7.47, t, J 6 (2H, Ar-CH2-CH2); 8.45, sextet,

J 6.5 (2 H, Ar-CH2CH2-CH3), 9.06, t, J 6.5 (3 H, Ar-CH2CH2-CH3), 9.06, t,

J 6.5 (3 H, Ar-CH2CH2CHO); tic, and pmr characteristics identical with

those of a reference sample: 4.22.

The acidic component was methylated ((CH3)2S04/anhydrous K2C03/acetone,

reflux, 1 hr) and the product purified by a preparative tic (7% acetone-

benzene). The major band yielded a crystalline solid, 80 mg; mp 134-1350;

ir,v: 2900, 1585, 1500, 1445, 1415, 1385, 1255, 1225, 1135, 1010, 960, 865,

825, 795, 730 cm 1; pmr, T:3.00-3.33, m (6 H, arom H); 5.4, d (2 H, Ar-CH-

0); 6.11, s, 6.16, s (12 H, OCH3); 8.25, m (2 H, CH3CH); 8.97, d, J 6

(CH3CH); mass peak, m/e: 372 (M ), 206 (base peak), 191, 175, 165, 151 and
138; found: C, 70.62; H, 7.66; C22H2805 requires C, 70.94; H, 7.58; 4.24.

Reaction of SC-8 (or SC-6) with p-Toluenesulfonic Acid

A solution of SC-8 (or SC-6), 0.20 g, in benzene (5 ml) was refluxed

with a saturated solution of p-toluenesulfonic acid (5 ml) for two hours.
When the tic (15% acetone-benzene) showed the absence of SC-8 and forma-
tion of two higher Rf band, the cooled mixture was partitioned with hexane-

benzene-methanol-0.5 N NaOH (1:1:1:1) to separate the neutral from acidic









components. The neutral fraction was purified via preparative tic (12%
acetone-benzene) and obtained as an oil, 4.15, 45 mg; ir: 2920, 1680,

1595, 1515, 1465, 1425, 1300, 1270, 1235, 1140, 1025 and 760 cm"1; pmr,

T:3.25, m (3 H, arom H); 6.17, s (6 H, OCH3); 6.40, s (2 H, Ar-CH2);

and 7.87, s (3 H, CH3CO); it was identical with a synthetic sample of

3,4-dimethoxyphenylacetone by tic, ir and pmr spectral comparison.

The phenolic fraction was purified by preparative tic (12% acetone-
benzene). Two components were isolated; the higher Rf component was

obtained as a crystalline solid 4.21 (21 mg); mp 248-9; C20H2004 (M+

324), uv XEtOH 239, 284, 315, 329 nm. Methylation of 4.21 ((CH3)2S04/
max
anhydrous K2C03, acetone, refluxed for 1 hr) gave a crystalline solid with

mp and ir identical with 4.20 previously obtained, Chapter III.

The lower Rf phenolic component was obtained as an oil 4.17 (77 mg);

uv m 282, 312 nm (shift in base to 290, 330 nm); C20H2204 (M+ 326),
max
pmr, T:3.43, m (5 H, arom H); 3.97, s (1 H, Ar-CH=C); 4.67, broad (2 H,

Ar-OH); 6.27, s (6 H, OCH3); 7.6, broad (1 H, CH-CH3); 8.25, s (3 H,

CH3C=); and 8.90, 9.00, d J 6 (3 H, CH-CH3). Diacetate of 4.17 (acetic

anhydride/pyridine 80; 10 min.); pmr: T 3.2-3.4, m (5 H, arom H); 3.93, s,

(1 H, Ar-CH=C); 6.30, s, (6 H, OCH3); 7.6, broad (1 H, CH3CH); 7.74, s,

(6 H, OCOCH3); 8.23, s (3 H, CH3C=); and 8.85, 8.97, d (3 H, CH3CH).
Methylation of 4.17 ((CH3)2SO4/anhyd. K2CO3, acetone, reflux, 1 hr.):

gave a crystalline solid, mp 980; found: C, 74.55; H, 7.39; C22HzOb4 requires:

C, 74.32; H, 7.65, identical to 4.3 by tic, ir, pmr spectral comparison.

Dehydrogenation of 4.19

A mixture of 4.19 (50 mg) and Pd/C (100 mg) in oxydiethanol (5 ml)

was boiled under reflux for 1-2 hours. The cooled mixture was filtered,

the filtrate diluted with water, pH 2 (10 ml) and extracted with ethyl

ether (20 ml). A crystalline solid (25 mg) was obtained from the









concentrated ether extract, mp, ir, pmr identical with 4.20 previously

obtained (see Chapter III).

The reaction of SC-8 with p-toluene sulfonic acid was carried out as

described above and the reaction mixture worked up so as to obtain a quan-

titative recovery of the ketonic and phenolic components. The extinction

of each of these components at their appropriate A was determined and,

based on the extinction values reported in the literature, the relative

concentrations were obtained to provide the ratio: [ketone] The ratios
Lphenol]
of the components from SC-6, 7 and 8 are shown in Table 4.3.

Reaction of SC-8 with DDQ and Degradation to 4.26

A solution of SC-8 (150 mg) in toluene (3 ml) was stirred at 250 with

1.5 equivalents of DDQ until absorbance ratio, Abs at 325 nmreached a
Abs at 280 nm
constant value. After filtration, the filtrate was extracted with sodium

bicarbonate solution. The solvent layer was concentrated and the major

component 4.25 purified on preparative tlc (20% acetone-benzene). Com-

pound 4.25 is an amorphous powder, 127 mg; uv XEt0H 283 and 325 nm; pmr,
max
r:280 3.17, m (12 H, arom H); 5.27, 5.44, d, J 8 (2 H, Ar-CHOH); 5.85,

m (2 H, CH3CH-0); 6.04, s (6 H, OCH3); 6.14,s (12 H, OCH3); 7.7, s (6 H,

CH3-C=); 8.75, 8.87, d, J 6 (CH3CH).

Compound 4.25 (127 mg) was dissolved in oxydiethanol (5 ml) and

refluxed for two hours with Pd/C (100 mg), cooled, diluted with water,

pH 4 (25 ml) and extracted twice with ether. The ethyl ether fraction was
separated into the neutral and the phenolic components. The neutral

extract was purified using preparative t1c, from which an oil identical

to 4.22,(tlc, pmr) was obtained.

The phenolic component was methylated ((CH3)2S04/anhydrous K2C03,

acetone, reflux, 1 hr.) and the mixture purified via preparative t1ce. A










Table 4.3.


Determination of molar ratios from the acid-catalyzed
rearrangement of SC-6, 7, 8 with P-TSA


Sample Experi- Product Absorbance Concentra- Mole Ratio
ment # 280 nm tion (M) 4.15 4.28
.TT_7 4.17
4.15 251 0.0806
1 ----- 1.8 -
4.17 591 0.0458
SC-8
4.15 170 0.0545
2 1.7 -
4.17 406 0.0316

4.15 206 0.066
1 0.93
SC-6 4.17 914 0.071
4.15 195 0.062
2 0.8 -
4.17 110 0.081

4.15 276 0.088
1 1.0 1.0
4.17 1140 0.088
4.28 386 0.088
SC-7
4.15 238 0.076
2 0.97 1.0
4.17 1004 0.078
4.28 388









pale yellow crystalline solid 4.26 (24 mg) was obtained mp 1720; uv H
max
326, 252 (log E 4.42, 4.12); ir: v 2930, 2830, 1595, 1575, 1505, 1453,

1380 (sh), 1315, 1245, 1215, 1167, 1120, 1020, 825, 795, 760 cm-1; pmr:

T 2.74 3.05, m (6 H, arom H); 6.19, s (12 H, OCH3); 7.80, s (6 H, CH3);

identical with 2,5-bis-(3,4-dimethoxy)phenyl-3,4-dimethy1furan (derived

from SC-2 oxidation).

Compound SC-7, 4.43

This is an amorphous powder, [a]20 99 (C = 1, CHC13). Found: C,

68.48; H, 6.87; C41H48011 requires: C, 68.70; H, 6.75; uv hx 212, 235,
max
280; ir: v 3440, 1600, 1590, 1505, 1450, 1375, 1260, 1140, 1030, 920, 850,

805 cm 1; pmr: T 2.90 3.33, m (12 H, arom H); 4.12, s (2 H, 0-CH2-0);

4.52, 4.62, d, J 6 (2 H, Ar-CH-0); 5.30, 5.47, d, J 8 (2 H, Ar-CH-OH);

5.83, m (2 H, CH CH-0); 6.10, s (12 H, OCHO); 7.73, m (2 H, CH3CH); 8.80,

8.90,d,J 6 (6 H, CH3CH-0); 9.23, 9.33, d, J 6 (6 H, CH3CH).

Diacetate (acetic anhydride/pyridine, 1000C, 15 min): amorphous pow-

der, found: C, 67.84; H, 6..74; C45H,20,3-H20 requires: C, 67.48; H, 6.54;

ir, v:2960, 2930, 1735, 1595, 1505, 1445, 1230, 1130, 1025 and 805 cm-1;

pmr, T:3.00 3.33, m (12 H, arom H); 4.12, s (2 H, O-CH2-0); 4.12, s,

(2 H, Ar-CH-OAc); 4.57, 4.67, d, J 6 (2 H, Ar-CH-0); 5.5, m (2 H, CH3CH-

OAc); 6.17, s (12 H, OCH,); 7.8, m (2 H, CH3CH); 8.0, s (6 H, OAc); 8.8,

8.9, d, J 6 (6 H, CH3CH-0); 9.25, 9.37, d, J 6 (CH3CH).

Reaction of SC-7 with P-toluenesulfonic Acid

The reaction of SC-7 with p-toluenesulfonic acid was carried out as

described for SC-8 (page65). The acidic fraction was worked up as before

and a crystalline solid 4.19 obtained, identical to cyclogalbelgin (mp,

ir, pmr).

The neutral component was purified using preparative tic (12% acetone-

benzene) and two major components isolated; an oil 4.15, identical to









previously identified 3,4-dimethoxyphenylacetone; and a second oil 4.28,

pmr, T:3.1 3.6, m (3 H, arom H); 4.12, s (2 H, O-CH2-0); 6.43, s, (2 H,

Ar-CH-CO); 7.88, s (CH3C=0); tic and ir identical to a reference sample

of 3,4-methylenedioxyphenylacetone.

Compound SC-6, 4.42

This is an amorphous powder, [a]2D 52.3 (C = 1.05, CHC13). Found:
C, 67.92; H, 7.23; C31H38083- H20 requires: C, 68.01; H, 6.95; uv XEt0H
max
212, 235, 280 ir: v 3440, 1600, 1650, 1510, 1455, 1380, 1260, 1230, 1135,

1030, 930, 855, and 810 cm 1; pmr: T 3.00 3.27, m (9 H, arom H); 4.52 -

4.62, d (2 H, Ar-CH-0), 5.29, 5.42, d (1 H, Ar-CH-OH; 5.84, m (1 H, CH3CH-

0); 6.10, s (12 H, OCH3); 7.74, m (2 H, CHUCH); 8.97, 8.89, d (3 H, CH3CH-

0); 9.25, 9.35, d (6 H, CH3CH), Figure 4.4.

Diacetate of SC-6 (acetic anhydride/pyridine, 1000/15 min.): amor-
phous powder; found:C, 65.60; H, 6.91; C35H42010H20 requires: C, 65.62;

H, 6.56; ir, v 2945, 1760, 1735, 1595, 1505, 1365, 1260, 1230, 1145, and

1025 cm-1; pmr, T:3.07-3.30, m (9 H, arom); 4.05, 4.17, d, J 6 (1 H,

Ar-CH-OAc); 4.53, 4.63, d, J 6 (2 H, Ar-CH-0); 5.43, m (1 H, CH3CH-0);

6.33, s (12 H, OCH3); 7.70, 3 (3 H, OAc); 8.00, s (3 H, OAc); 8.77, 8.88,

d, J 6 (3 H, CHCHO) and 9.23, 9.35, d, J 6 (6 H, CHUCH).

Partial Synthesis of SC-8

A mixture of SC-6 (0.2 g), DMF (10 ml), 3,4-dimethoxy-2'-bromopro-

piophenone, and K2CO3 (1 g) was stirred at 250 for eight hours. It was
diluted with water (15 ml) and extracted with benzene (20 ml). The sol-

vent extract was purified on a preparative tic yielding a major band, which

was taken up in methanol (5 ml) and reacted with sodium borohydride (0.1 g).

After ten minutes, the reaction mixture was diluted with water and the

colorless solid filtered, semisynthetic SC-8 4.27b: [a]25 = -36.3
D






71

(C = 1, CHC13); ir, v:3490, 1600, 1585, 1500, 1405, 1370, 1255, 1130 and
1020 cm 1; pmr, T:3.13, m (12 H, arom H); 4.52, 4.62, d, J 6 (2 H, Ar-CH-0);
5.15, 5.70, d, J 3 (1 H (erythro), Ar-CH-OH); 5.30, 5.43, d, J 8 (1 H-
(threo), Ar-CH-OH); 5.83, m (2 H, CH3CH-O); 6.13, s (18 H, OCH3); 7.73,
m (2 H, CH-CH3); 8.78, 8.89, d J 6 (6 H, CH3CH-O); 9.23, 9.33, d, J 6

(6 H, CH3CH).














CHAPTER V

BIOLOGICAL ACTIVITY
Of the lignoids described in Chapters III and IV, only SC-7 and SC-8

have biological activity. Interest in these compounds was generated

because of their toxicity and lack of any prior data on this aspect. The

following studies were conducted to characterize the type of biological

activity possessed by the SC-lignoids, and to assess their potentials as

useful therapeutic agents. Nearly all of the work described here has

been conducted using SC-8, the major active principle.

Toxicity and Behavioral Studies

SC-8 has an LD50, the lethal dose for 50% of a test group, of 5.4

(r = 0.93) mg/Kg, as determined from the dose-response curve, Figure 5.1.

Each experimental point of the dose-response curve corresponds to the

result from ten mice at the particular dose level. After twenty-four

hours, the percent dead was recorded and plotted against the logarithm

of the dose. The sigmoidal curve so obtained was transformed into a linear

plot using the probit method of analysis (27). In this method, instead

of showing "percent responding" on the y-axis, each percent response is

converted to probability units or "probits," as obtained from a table of

probits. The resulting probit values from 3-7 cover values of percent

response from 2-98. By plotting probit values versus logarithm of dose

for the SC-8 toxicity experiment, Figure 5.2 was obtained, which made the

determination of the LD50 value easier and more accurate.

Animals receiving lethal doses of compound SC-8 usually die within

twelve hours. Initial signs of toxicity include hypothermia, central nervous
72








73
















o
















0-
o
oo




























0.4 0.6 0.8 1.0
o 0






0-







0.4 0.6 0.8 1.0

Log dose mg/Kg

Figure 5.1. Toxicity (LD50) determination of SC-8.









74



























0
119










- *r-
00




C C
E0




0 '






u



0
*r
-J
C C











r-
Lt
4-










*f-


IS
mc>

Eo
C )
~~CT

U,0


C)
-J


7 6 5 4 3 2


Probit









system depression, and an unsteady gait. The animal then goes into a

state of catalepsy where the mouse tends to remain in any position in

which it is placed for an unusually long period of time. A terminal period

then follows, in which respiration is slow and labored with death resulting

via respiratory failure.

The central nervous system (CNS) toxicity was detected using the

mouse behavioral assay. This assay is a modification of a procedure

reported by Campbell and Richter in 1967 (34). The method involves injec-

tion of six mice with the test substance and observing for the presence of

sixteen signs (Table 5.1) after thirty minutes of treatment. Depending

on the type of activity, a certain pattern is observed as shown in Table

5.2. For example, a parasympathomimetic agent will show salivation,

lachrymation, mydriasis and piloerection. On the other hand, a neurolep-

tic substance will show increase in paw temperature, decrease in rectal

temperature, ptosis, abolished righting reflex, head drop with righting

reflex present, decreased motor activity, abduced hindlegs with righting

reflex present and an unsteady gait. Interestingly, a pattern similar to

that shown by neuroleptic agents was observed when sublethal dose of com-

pound SC-8 was administered to mice, Table 5.3. The following signs were

observed: decrease in body temperature, head drop, decrease in locomotion

activity, abduced hindlegs, and an unsteady gait.

Characterization of CNS Activity
The above behavioral studies showed SC-8 to have CNS depressant

activity with the presence of catalepsy at high dose levels. These results

are suggestive of possible neuroleptic activity. According to Turner (28),

neuroleptic agents show the following characteristics in mice: potentiation

of barbiturates; suppression of combativeness when animals are sensitized

by solitary confinement; decrease in spontaneous motor activity; reduction










Table 5.1. Signs observed in behavioral assay


1. Paw temperature

2.,3. Rectal temperature + +

4. Ptosis

5. Salivation

6. Lachrymation

7. Mydriasis/miosis

8. Piloerection

9. Locomotion activity +

10. Straub tail phenomenon

11. Righting reflex (r.r) abolished

12. Head drop with r.r present

13. Positive Haffner with r.r present

14. Locomotion activity +

15. Abduced hindlegs with r.r present

16. Unsteady gait































+ + +















V) 0
I- + + +















S+ + +
+ ++






















+ + +
4+


4i- -W +


W 0 + ( +1


-) + +1 + + +

































(o to ( c m
uI +1 +




















S C )I I C. I I
r (n) U N
U E 4-1 In In .
*,- In *,- > In +J +J ,-

S +' -C: -= O I0 In c
U u 4 4 ) *I I

o *r4 > C+ 4 V) U) c c
r- O O In Q I S- S- S- o^


U) 0. 0. IO era I *.- o r

f L 0 / 0.. CL L L 0 0:


4-)

0
>1

S4.)
4- S- *r 3 -
Ua) U 4.)
5- C U '1- 0
I. 4- co .-C
7 0 4-
S- S- "a
*r -0 o 0 U) co
4-) 4-) (u U)
-C -o 0 4-+
0n M In0 Vn
W- U 0 .0 C



i- i i -o


C
4> 0


4- C
U 0)
0.




E n 0)
> 0 (0 S- 0 -Q
Si- U
- 0 0 m
o *0 ,- ...j S-
u >) 4-)
-i -M- )


us ~ o CT o
r-


+4-






4-) 0M (a
S a




3 U U
* *~










Table 5.3. Behavioral assay of Saururus extract


Body temperature

Ptosis

Salivative

Lachrymation

Mydriasis, miosis

Piloerection

Locomotion activity +

Straub tail phenomenon

Righting reflex abolished

Head drop with r.r

Positive Haffner with r.r

Locomotion activity +

Abduced hindlegs with r.r

Unsteady gait


37.00 -* 30.80

+









of body temperature; potentiation of depressant drugs, such as analgesics,

and antagonism to stimulant drugs, such as amphetamine, morphine and mesca-

line. To provide support for the presumptive activity seen in the behav-

ioral assay, some of the above tests were performed. These include:

a) potentiation of pentobarbital; b) inhibition of aggressive response in

mice and, c) antagonism to amphetamine-induced stereotypy.

Potentiation of Pentobarbital

A common characteristic of neuroleptic agents is that they potentiate

the action of hypnotics. However, this is a nonspecific test which indi-

cates general CNS-depressant activity. Sedatives, hypnotics, tranqui-

lizers (minor and major) etc. show this type of activity which is defined

as at least a 200% prolongation of sleeping time of a barbiturate. The

procedure employed was that described by Turner (28). The experiment

involves the injection of one group of animals with the test substance.

After twenty minutes the treated animals, plus controls, are injected with

pentobarbital. The sleeping time is then recorded for both groups.

Results (Table 5.4) showed prolongation of pentobarbital-sleeping time

of 200 to 300% in the dose range of 0.05 to 1.0 mg/Kg, which is far below

the toxic doses (LD50: 5.4 mg/Kg, LD10, 4 mg/Kg). Furthermore, at 4.0 mg/

Kg, a percent increase of 875 was observed. It was therefore concluded

that SC-8 does potentiate the action of pentobarbital in mice.

Inhibition of Aggressiveness

Suppression of isolation-induced aggressive behavior in mice by

neuroleptic drugs has been carefully studied by Yen et al. (29). His

laboratory found that reserpine and chlorpromazine suppressed aggressive-

ness in 80 to 90% of the animals at dose levels of 3 and 10 mg/Kg, respec-

tively. It appears that isolation produces an increase in the turnover










Table 5.4. Potentiation of pentobarbital sleeping time by SC-8


Pentobarbital
mg/Kg

42

42


Sleeping Time
Control Treated

16 140

16 44.8


% increase of
sleeping time

875

280


1.0 40 10.8 32.4 300

0.5 40 10.8 38.6 357

0.25 40 10.8 32.8 304

0.125 40 12.0 19.0 158

0.06 40 12.0 26.0 217

0.01 40 12.0 21.0 175


Drug
mg/Kg

4.0

0.4









of brain dopamine (30),thereby providing a possible model for the detec-

tion of neuroleptic activity.

The method employed in our laboratory was based on that described

recently by Weinstalk and Weiss (31). Male mice at least four weeks old

were isolated in cages for three weeks, during which period they did not

see other mice. They were not disturbed except for replacement of food.

Under these conditions, about two-thirds of the mice became aggressive

as manifested by their vicious attacks within one minute on a nonisolated

male mouse placed in their cage. Such combative mice were divided into

two groups and, with one group held as control, the other was injected

with the drug. The animals were tested for aggressiveness before the

injection and eighty minutes after the injections. If no fighting took

place with the nonisolated mouse introduced into the cage, this was

regarded as inhibition of aggressiveness, Table 5.8. In the first experi-

ment SC-8 was tested at three levels: 2.0, 1.0 and 0.2 mg/Kg with three

animals per dose level. Only the top dose showed marked inhibition of

combativeness. To confirm this, the 2.0 mg/Kg dose was again tested using

five animals and was likewise active. Fielding and Lal have reported 67-

100% suppression of isolation-induced aggression in mice by 1.25 2.5

mg/Kg of haloperidol (32).

Antagonism to Amphetamine

A very specific experiment which was used to recognize and/or confirm

neuroleptic activity is the antiamphetamine assay. Amphetamine is a CNS-

stimulant which causes agitation manifested by a certain stereotypical

behavior in mice. Suppression of this behavior is one of the most impor-

tant criteria for the recognition of a neuroleptic agent. The method used

here was that described recently by Weissman and Koe (33).









Eighty mice were kept in individual observation chambers (inverted

1 a beakers) in eight groups of ten mice each. Groups 2-8 were injected

ip with a solution of SC-8 at doses 1.5, 1.2, 1.0, 0.5, 0.3, 0.1 and 0.03

mg/Kg. Thirty minutes later, all the mice were injected with () ampheta-

mine sulfate (12 mg/Kg). After the injections were completed, the mice

were observed for agitation and stereotypy using the following grading

scale: 0 = sleeping; 1 = alert but not moving; 2 = moving around chamber;

3 = sniffing; 4 = licking; 5 = biting or gnawing. The mice were also

examined for hydration (sweating) resulting from the amphetamine and

scored for the degree of inhibition by the drug. The results are shown

in Table 5.5. In a second experiment the above procedure was performed

with haloperidol as the test substance. These results are shown in Table

5.6. The composite results from the two experiments are shown in Table

5.7. Using the probit method as described earlier, a log dose-probit

response plot was obtained from which an ED50 (effective dose for 50% of

a group) for SC-8 and haloperidol were determined, Figure 5.3. The results

are quite impressive since they show SC-8 (ED50 = 0.21 mg/Kg) to be twice

as potent as the standard reference drug, haloperidol (ED50 = 0.50 mg/Kg).

The LD50 of SC-8 was obtained earlier as 5.4 mg/Kg, thus giving a thera-

peutic index of 26.

SC-8 as a Neuroleptic Agent

Today, no single in vitro or in vivo test is able to provide absolute

proof for an experimental agent as having neuroleptic activity. However,

a series of tests may provide more reliable conclusions with respect to

the agent possessing potential neuroleptic activity. Known neuroleptic

agents show several activities in rats and mice and other species and

the following presumptive activities in mice (28): potentiation of barbi-

turates; suppression of aggressiveness; decrease in motor activity; reduction

















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Table 5.7. Inhibition of
haloperidol


amphetamine-induced stereotypy by SC-8 and


Sample Dose Log Dose % Inhib. Probit
SC-8 1.5 0.176 80 5.84
1.2 0.079 80 5.84
1.0 0 70 5.52
0.5 -0.30 60 5.25
0.3 -0.52 60 5.25
0.1 -1 40 4.75
0.03 -1.5 20 4.16

Haloperidol 1.8 0.25 90 6.28
1.3 0.11 70 5.52
1.0 0 60 5.25
0.75 -0.12 50 5.00
0.5 -0.3 40 4.75
0.1 -1 20 4.16
0.05 -1.3 10 3.72





































SC-8 ED50, 0.21 mg/Kg (r = 0.986)
Haloperidol ED50, 0.50 mg/Kg (r = 0.947)


-1.5 -1.0 -0.5 0 + 0.5


Log dose (mg/Kg)


Figure 5.3.


Inhibition of amphetamine-induced stereotypy
by SC-8 0 and haloperiddl A .









Table 5.8. Suppression of aggressiveness by SC-8


Number of Mice


% Inhibition of
Acgressiveness


2.0 3 Pronounced (>75%)

1.0 3 none

0.2 3 none

2.0 5 pronounced (>75%)


Dose









of body temperature, and antagonism to amphetamine. In this chapter we

have seen that SC-8 possesses all of these activities and at levels well

below the toxic dose (e.g. LD50 = 5.4 mg/Kg and antiamphetamine ED50 =

0.21 mg/Kg). Also, this relatively low value for ED50 places SC-8 among

the "high-potency" neuroleptic drugs.

Experimental

General

Unfasted albino mice of either sex (unless otherwise indicated),

weighing 20-30 g, from the ICR strain, were used. Body temperature was

measured by placing a regular thermometer against the body of the animal

and folding its skin over it. Estimation of pupil size was made with the

aid of an achromatic lens with a magnification of twenty times. Drugs

were administered intraperitoneally either as solution in water or as sus-

pension in water containing 0.3% "Tween-20" (v/v). The volume administered

varied between 0.17 to 0.25 ml per 10 g body weight. In the case of plant

extracts (Chapter I), the crude extracts were administered intraperitoneal-

ly as suspension in water containing no more than 4% flour (w/v) to act as

a dispersing agent, 0.3% "Tween 20" (v/v), and 0.8% saturated sodium bi-

carbonate (v/v).

Toxicity-Behavioral Assay

The method presented is a modification of a procedure reported by Camp-

bell and Richter (34). It is applicable to both pure drugs and crude plant

extracts. On the day of testing, animals in groups of six were trans-

ferred from their housing containers into observing containers without

access to food or water. The animals were weighed and body temperature

in 00C measured. The test substance was first injected into six mice at

dose level of 500 mg/Kg. Within fifteen-thirty minutes after injection,









each animal was observed within its container for the following signs:

ptosis, piloerection, Straub tail phenomenon, decreased or increased motor

activity, and any other unusual signs. Thereafter, each animal was suc-

cessively taken out of the container. While keeping the mouse immobilized

with one hand, it was examined for the following signs i.e. salivation,

lachrymation, mydriasis and miosis. It was then placed on a smooth sur-

face and observed for the following signs: abduced hindlegs, head drop

and ataxia. Loss of the righting reflex was considered to be positive when

the animal, placed on its back, remained there for at least thirty seconds.

Using Haffner's technique (35) a small artery clip was then applied to

the tail one cm from the base. The test was considered to be positive

when the animal did not bite the clip within thirty seconds after appli-

cation. Thirty minutes after drug injection, body temperature was mea-

sured again. An increase or decrease of 2.00 or more indicated drug

action. Each mouse was regarded either as a reactor or a nonreactor to

each of the listed signs. A reactor is a mouse showing the presence of

a sign which is unambiguously observed by a trained worker and which is

not present in a control animal. Any group of mice with four, five and

six positive reactors was considered a reactive group.

After all observations were recorded the animals were transferred

back to their housing containers with access to food and water. Twenty-

four hours later, the number of dead mice was recorded as a fraction i.e.,

four-sixths means four mice died out of six that were treated. If all six

mice were dead, the test solution was diluted by one-half (500, 250, 125,

62.5, etc. mg/Kg) and injected into another set of mice. Again, observa-

tions were recorded during the first thirty minutes and toxicity was









noticed after twenty-four hours. On the other hand, if less than six mice

were recorded dead after the first injection, the survivors were injected

a second time with the same solution and observations recorded. This was

continued for a total of five days (five injections). The minimum toxic

dose (MTD) was defined as the dose in mg/Kg required to kill four-sixths

of the mice with three or more doses being necessary.

The signs observed with the test substance may occur either singly

or in combination, forming more or less typical patterns as described by

Campbell and Richter (34). Table 5.2 shows the drug categories and their

sign patterns. Results for SC-8 are shown in Table 5.3.

Potentiation of Pentobarbital

Mice were injected ip with SC-8 (4, 1, 0.5, 0.4, 0.25, 0.125, 0.060

and 0.01 mg/Kg) twenty minutes before a similar injection of 40 mg/Kg

pentobarbital. Control groups received the vehicle of the test compound

and the barbiturate. Sleeping time was measured as the interval between

the loss and the recovery of the righting reflex. Results from three dif-

ferent experiments are given in Table 5.4. A test substance is active if

it shows at least a 200% prolongation of sleeping time of pentobarbital.




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