YOHIMBINE AND ERGOT ALKALOIDS AS NATURALLY OCCURRING ANTIMETABOLITES OF SEROTONIN

BY ELLIOTT S&iw AND D. w. WOOLLEY*

(From the Laboratories of The Rockefeller Institute for Medical Research, New York, New York)

(Received for publication, February 6, 1953)

Recently it has been possible to produce structural analogues of sero- tonin, which behave as antimetabolites of this physiological constituent of animal tissues (1, 2). One of the pharmacological actions of these anti- metaboftes is to prevent the rise in the arterial blood pressure of dogs which would otherwise result when serotonin is administered (3). This property may be attributed to the demonstrated ability of these analogues to nullify in vitro the vasoconstriction which serotonin calls forth. Because these antimetabolites of serotonin were indole derivatives (as is serotonin itself), our attention was directed to naturahy occurring alkaloids which have, in the past, been used in the control of high blood pressure and which might be structural analogues of serotonin. Some of the pharmacological effects of these alkaloids might conceivably arise from their being anti- metabolites of serotonin. In this way attention was directed to yohimbine and to the ergot alkaloids, both of which are indole derivatives, and both of which have been used with varying success as drugs for the reduction of high blood pressure (4).

Experimentation readily showed that the constriction of segments of carotid arteries which is caused by the application of serotonin (2) was compIetely reversed by yohimbine. The antagonism of the metabolite and the drug was competitive over at least a IOO-fold range of concentra- tion. Furthermore, the order of addition of the two substances to the tissue was not crucial. Yohimbine would relax tissues previously made to contract by application of serotonin, or it would, when administered first, prevent the contraction. This was in marked contrast to the well known antagonism observed between yohimbine and adrenaline, because in this latter case the metabolite adrenaline is effective only when it is applied first (5). The antagonism by serotonin was thus somewhat clearer than was that by adrenaline. This was of interest because the prevailing opinion has been that yohimbine is an adrenaline antagonist.

The structural resemblance of yohimbine and serotonin may be seen in Fig, 1. The phenolic hydroxyl group of the metabolite has been replaced by a hydrogen atom. The aminocthyl side chain of the metabolite has

* With the technical assistauce of J. J. Gingell and G. Schaffner.

979

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980 ANTIMETABOLITES OF SEROTONIN

been incorporated into the third ring of yohimbine, and, in addition, two more six-membered rings have been fused to this one, Because there are several structural differences between the drug and the metabolite, many

o=c’ H(a,CWR @- cH3

H

R Ergotamine

Yohimbjne Semtonin (R= peptide residue)

FIG. 1. Structures of yohimbine, serotonin, and ergotamine

3-ethyl-5-aminoindole &amino-1,2,3,4- 6- aminoharman tetrahydroca&azole

3-(3-N-(l&3,4-tetrahydrolso- quinolyl) ethyl-5-aminoindole

Harman 3- (3.N-(1,2,3,4-tetrahydroiso- @nolyl) ethyl-indole

FIG. 2. Structural analogues of serotonin

investigators will be reluctant to consider yohimbine as an antimetabolite of serotonin. Certainly the case would be clearer if the two substances differed in only one respect. However, the following facts seem worthy of consideration. (a) Th e antagonism between the drug and the metabolite is freely reversible and competitive over a large range of concentration. (6) Both the drug and the metabolite are chemically related to tryptamine.

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E. SHAW AND D. W. WOOLLEY 981

The alterations in the .tryptamine structure have been more extensive in the case of yohiibine than with serotonin. (c) A number of synthetic antimetabolites of serotonin have been constructed. These form a series of related structures progressing by relatively small changes from the me- tabolite towards yohimbine. These are shown in Fig. 2 and Table III. Each of these analogues has been shown to function in the artery ring test as a reversible antagonist to serotonin. The original synthetic antimetab- olites of serotonin were 5-aminoindoles with alkyl side chains in position 3 (Compound I) or positions 2 and 3 (Compound II, Table III). These two side chains may be fused into a cyclohexyl ring, as in Compound III, without impairment of potency. The amino group must remain because Compound IV (Table III) was inactive. However, when the amino group was moved from the first ring and incorporated in a pyridine ring (which thus took the place of the cyclohexane ring), as in Compound VI, potency was enhanced. In fact, the retaining of the amino group attached to the first ring, as in Compound V, reduced potency. These facts made it seem that somewhere in the molecule a basic nitrogen was necessary for anti- metabolite activity. This could be an aromatic primary amine attached to the benzene portion of the indole system. On the other hand, the nitro- gen originally belonging to the aminoethyl side chain of the metabolite could be utilized for this purpose, provided, as the data in the preceding paper (2) indicated, that this nitrogen is not a primary amine but rather a tertiary base. In one of the instances tried in the present study, the possession of both kinds of basic groups, as in Compound V, was unde- sirable for highest potency, although in another case (Compound VII com- pared to Compound VIII) this was not so.

In considering whether yohimbine differs too much in chemical structure to be regarded as an antimetabolite of serotonin, one should take account of the classical case of the sulfonamide drugs. Little doubt seems to exist that sulfanilamide is a bona fide antimetabolite of p-aminobenzoic acid. Sulfathiazole likewise is felt to be securely in this class. The replacement of the 1 hydrogen atom in sulfanilamide by a thiazole ring is thus not con- sidered a change great enough to call in question the basic concept. In fact, this additional structural change, by conferring a more desirable dis- sociation constant, has resulted in a considerably more active compound. Similarly, phthalylsulfathiazole is regarded as just another antimetabolite of p-aminobenzoic acid, which has had a phthalyl radical added to it in order to give it a desirable solubility. Nevertheless, the structural re- semblance between yohimbine and serotonin seems as great as between phthalylsulfathiazole and p-aminobenzoic acid.

The facts presented in this paper should not be construed as arguments in favor of the view that yohimbine acts on animals solely as an antime-

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982 ANTIMETABOLTTES OF SEROTONIN

tabolite of serotonin. Quite probably, this drug has some properties which are not concerned with the action of this metabolite. This can readily be appreciated from a comparison of the pharmacological properties of harman and yohimbine. Both of these substances are structural analogues of sero- tonin, and both antagonize its action on artery rings. Yet, in whole ani- mals it is well known that they do not have identical effects. Clearly, the mode of action of the two drugs is not the same in all respects. They have some biological properties in common and they both are competitive antagonists of serotonin. The present study has shown only that some of the pharmacological properties of these drugs are probably the result of an antiserotonin action.

The ergot alkaloids are analogues of serotonin, but not in the same way that yohimbine is. The ring which has been formed from the aminoethyl side chain of serotonin has been fused to the benzene, rather than the pyrrole portion of the indole system. In the artery ring test, ergotoxine (which is a mixture of three closely related alkaloids) and ergotamine (a pure substance) antagonized the constriction caused by serotonin, and this effect was reversible by additional serotonin.

Shortly after our observations were made, a note by Reid and Rand (6) was published in which they mentioned that they had observed that yo- himbine antagonized the contracting action of serotonin on strips of carotid artery. A few months thereafter Erspamer (7) described experiments with kidneys and with uteri in which he had observed that ergot alkaloids inter- fered with the action of enteramine (serotonin). These studies apparently arose from consideration of the pharmacological properties of yohimbine, ergotamine, and serotonin, and the possibility was not considered that the first two substances were antimetabolites acting against the third. In other words, these studies recognized that serotonin caused contraction of blood vessels and other tissues and that, since yohimbine and the ergot alkaloids had previously been known to affect such tissues, an antagonism might exist. The present investigation was predicated upon the idea that serotonin was a metabolically essential substance or metabolite, with spe- cific receptor sites in diverse organs. Yohimbine and the ergot alkaloids, because of their structural resemblance to the metabolite, might thus com- bine with the serotonin sites and block the action of this metabolite. Their pharmacological effects might thus, in part, be attributed to their inter- ference with the action of serotonin. These additional independent factual reports by Reid and Rand and by Erspamer confirm t)he existence of the antagonism.

EXPERIMENTAL

Manner of Testing and Sources of Compounds-Tests mere conducted with ring-shaped segments of sheep carotid artery as described in the preceding

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E. SHAW AND D. W. WOOLLEY 983

papers (1, 2). The precautions previously outlined were carefully ob- served. All substances were dissolved in Ringer’s solution and adjusted to pH 7 before use. Serotonin was a synthetic sample of the creatinine sulfate double salt,l and all the weights of this metabolite refer to this double salt. Harman and 6-aminoharman2 were synthesized according to the directions of Snyder, Parmerter, and Katz (8). The other analogues of serotonin were prepared as described earlier (9). Ergotoxine was the ethanesulfonate obtained commercially. As is well known, this material is a mixture of substances which differ only in the amino acid composition of the peptide portion. Ergotamine was a pure specimen kindly supplied by the Sandoz Chemical Works, Inc.

S-p-N-(1 ,W,S,.&Tetrahydroisoquinolyl)ethyl-5-nitroindoZe Hydrochloride- 3-,&Chloroethyl-5-nitroindole (9) (1.25 gm.) and 1,2,3,4-tetrahydroiso- quinoline3 (3 ml.) in absolute ethanol (65 ml.) were refluxed for 20 hours, filtered, and concentrated at the water pump. The residue was converted to an insoluble hydrochloride by trituration with 3 N HCl (25 ml.). The supernatant acid was discarded, as were additional washings with similar portions of 3 N HCl and of water used to remove soluble impurities. The remaining gum was dried and crystallized from absolute alcohol. Two crops were collected totaling 410 mg., 20 per cent, m-p. 247-248”. Recrys- tallization did not alter the melting point.

CI~H~~OZN&I. Calculated, C 63.78, H 5.63; found, C 63.55, H 5.59

S-p-N-(1 ,W,S,&T t h d e ra y roisoquinolyl)ethyl-5-aminoindole Dipicrate- The nitroindole (0.30 gm.) was dissolved in warm ethanol (50 ml.) and reduced with alkaline hydrosulfite solution by the general method described elsewhere (9). On removal of the alcohol, the base separated as an oil, which was washed with water by decantation, and converted to the dipic- rate in ethanolic solution. Gradual addition of water provided 0.37 gm. of crystals melting with decomposition at 215-217”, a yield of 59 per cent. The melting point did not change on recrystallization.

C~~H&3.2C~H30~N3. Calculated, C 49.66, H 3.63; found, C 49.85, H 3.57

For biological testing, the dip&ate was converted to the dihydrochloride in the usual manner.

??-p-N-(1,2,5 ,~-TetrahydroisoquinolyZ)ethylindole-Indoleacetic acid (2.0 gm.) in dry ether (50 ml.) was treated at 0” with phosphorus pentachloride (2.7 gm.). Solution gradually took place. The volume was reduced to

1 Kindly supplied by the Abbott Laboratories. 2 This compound has occasionally been called 7-aminoharman because some in-

vestigators begin numbering the harman ring system at the indole nitrogen rather than at the carbon atom between the two nitrogens.

3 Kindly supplied by Dr. C. T. Bahner, Carson-Newman College, Jefferson City, Tennessee,

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984 ANTIMETABOLITES OF SEROTONIN

about 20 ml. by concentration under reduced pressure, and petroleum ether (200 ml.) was added. Lustrous flakes of the acid chloride formed and were collected by filtration after 2 hours. 1.45 gm. were obtained, m.p. 68”.

The acid chloride was dissolved in ethyl acetate (25 ml.) and mixed with a similar volume of ethyl acetate containing 1,2,3,4-tetrahydroisoquino- line (1.5 ml.) and N-ethylmorpholine (2 ml.). Heat was evolved and a precipitate formed. After 3 hours at room temperature, the suspension was filtered and the filtrate and washings were shaken several times with N HCl and with aqueous sodium carbonate to remove unchanged starting materials. The intermediate amide (1.75 gm.) was not crystallized but instead treated directly with lithium aluminum hydride.

TABLE I

Antagonism between Serotonin and Yohimbine in Segments of Sheep Carotid Artery

Serotonin Yohimbine hydrochloride

y per ml. y per ml.

0 0 0.2 0 0.2 0.06 0.2 0.2 0.2 1.0 0 4.0 2.0 0 2.0 0.2

Contraction in major axis

per ccnl

0 xl

28 6 4 0

26 26

The amide (1.1 gm.) in dry ether (200 ml.) was treated with an equal weight of lithium aluminum hydride slurried in ether. The mixture was stirred for 4 hours and decomposed cautiously with water, then 10 per cent NaOH (50 ml.). The base was extracted from the ether layer with several portions of 0.1 N HCl. The hydrochloride separated as an oil in the aque- ous phase and was converted to a picrate in aqueous acetone, yielding 1.45 gm., m.p. 167-169”, 76 per cent. The melting point did not change upon further recrystallization.

C, pHzoNz* CeH~NaO~. Calculated. C 59.40, H 4.59, N 13.86 Found. “ 59.32, ‘( 4.73, “ 13.71

Antagonism of Action of Xerotonin on Artery Rings by Yohimbine-The data of Table I will show that the vasoconstricting effect of serotonin was overcome by small amounts of yohimbine. This drug was more potent as an antimetabolite of serotonin than any of the synthetic analogues pre- viously examined, The inhibition index was approximately 1. In the usual test with artery rings, the serotonin was applied first, so that contrac-

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E. SHAW AND D. W. WOOLLEY 985

tion was initiated before the antagonist was introduced. This order of addition was followed in the present trials. However, several experiments were performed in which the order of addition was reversed. In these, the results were not distinguishable from those observed when serotonin pre- ceded the yohimbine. This was of interest in view of the crucial nature of the order of addition for the demonstration of antagonism between adrenaline and yohimbine (5).

Competitive Nature of Antagonism between Serotonin and Yohimbine- With each of three dilutions of serotonin ranging from 0.2 to 20 y per ml., a series of graded dilutions of yohimbine was examined. The point of half maximal inhibition was determined graphically as described previously (2). In this way the concentration of yohimbine was found which would nullify half maximally the constrictor effect of each concentration of sero-

TABLE II

Amounts of Yohimbine Hydrochloride Required to Antagonize Half Maximally Various Quantities of Serotonin

Serotonin Yohimbine required Inhibition index* -.

y per ml. y pw ml.

0.2 0.1 1 2.0 1.1 1.1

20 17 1.7

* The quantities of yohimbihe represent amounts necessary to overcome haZf the serotonin; consequently the inhibition index is 1 and not 0.5.

tonin. The results are summarized in Table II. The antagonism was competitive in character at least over a loo-fold range of concentration. An increase in serotonin required a corresponding increase in yohimbine in order to call forth equivalent responses in the artery rings.

Antiserotonin Activities of Analogues Intermediate between Yohimbine and Metabolite-The data of Table III will show the relative potencies of a number of structural analogues which may be considered intermediate in structure between serotonin and yohimbine. These were examined with artery rings as previously described (2). The activities of a few of these analogues have been reported earlier, but are included here along with the newly examined substances so that the complete series may be seen to- gether. With each of the analogues it was demonstrated that the inhibit- ing effect exhibited was reversible by increased amounts of serotonin.

Antagonism between Tryptamine and Yohimbine-Because it might be said that yohimbine resembles tryptamine more closely than it does sero- tonin, it was of interest to determine what, if any, antagonism might be

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986 ANTIMETABOLITES OF SEROTONIN

found to exist between tryptamine and yohimbine. The tests were made in the same way as was described with serotonin, except that contraction of the rings was produced with tryptamine (4 y per ml.) instead of with serotonin. The constricting effect of tryptamine was overcome by yohim- bine. Thus, just as with the synthetic antimetabolites of serotonin (2), yohimbine interfered with the constricting effect of either serotonin or tryp- tamine. Tryptamine was much less powerful than serotonin, and conse- quently the inhibition index was less. This point has been discussed in connection with the synthetic antimetabolites of serotonin (2). This an- tagonism between tryptamine and yohimbine correlated well with the prior findings of Raymond-Hamet in intact animals (10).

TABLE III

Amounts of Analogues Necessary do Inhibit Half Maximally Contraction of Artery

coYPd

I II

III IV

V VI

VII

VIII

IX

Rings Caused by 0.9 y of Serotonin per Ml.

NiUlle

3-Ethyl-5-aminoindole 2-Methyl-3-ethyl-5-aminoindole I, 2,3,4-Tetrahydro-6-aminocarbazole 1,2,3,4-Tetrahydrocarbazole 6-Aminoharman Harman 3-P-N-(1,2,3,4-Tetrahydroisoquinolyl)e~hyl-5-amino-

indole dihydrochloride 3-p-N-(1,2,3,4-Tetrahydroisoquinolyl)ethylindole

hydrochloride Yohimbine hydrochloride

y per ml.

30 9

11 Inactive at 30

15 1 5

4

0.1

Antagonism between Serotonin and Ergotoxine-When ergotoxine in graded amounts was examined with serotonin on artery rings, results such as those shown in Table IV were obtained. It can be seen that the alka- loids (ergotoxine is a mixture of three) interfered with the constricting effect of the metabolite. It was difficult to produce complete nullification of the serotonin effect, and the dose response curve differed in shape from that with yohimbine. However, the interference which did result from the ergotoxine could be overcome by increases in the amount of serotonin in the system. The low solubility of ergotoxine limited the range of con- centration which could be examined.

Antagonism between Serotonin and Ergotamine-The data in Table IV will also shorn that serotonin was antagonized by ergotamine, and that this pure ergot alkaloid was of the same order of activity as was ergotoxine.

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E. SHAW AND D. W. WOOLLEY 987

With ergotamine as with ergotoxine, the unusual shape of the dose response curve was still evident, but the antagonism was readily demonstrated.

Dihydroergotamine methanesulfonate was inactive when tested at 15 y per ml. This was in contrast to the activity of ergotamine. The satura- tion of the double bond thus changed the capacity of this alkaloid to act as an antagonist to serotonin. It is well known that in intact animals ergotamine and dihydroergotamine differ in pharmacological properties.

TABLE IV

Constrictions of Artery Rings Caused by Serotonin Plus Ergotoxine or Eygotamine

Serotonin Ergotoxine ethanesulfonate Ergotamine tartrate Contraction in major

axis

y per ml. y per d. y per ml. per cent 0 0 0 -3 0.2 0 0 17 0.2 2 0 14 0.2 5 0 18 0.2 10 0 8 0.2 15 0 6 0 15 0 -3

0 0 0 0 0.2 0 0 25 0.2 0 1 26 0.2 0 2 11 0.2 0 10 11 0.2 0 15 3 0 0 15 2

The trial with ergotoxine was on a different artery from that with ergotamine.

DISCUSSION

The data recorded in this paper indicate that yohimbine and the ergot alkaloids function as antimetabolites of serotonin in the segments of carotid arteries. Not only are both kinds of drugs seen to be structural relatives of the metabolite, derived from it in ways which might be expected to convert it into an antimetabolite, but also they are reversible antagonists of this metabolite. Furthermore, a third class of drugs, namely the harman alkaloids, as for example harman itself, has been shown to function in this fashion and thus to be implicated with serotonin in mode of action. It is of interest therefore to reflect upon the known pharmacological properties of these various drugs and to realize that they have in common certain kinds of effects which might be expected to arise from substances interfer- ing with the known physiological actions of serotonin. Witness, for ex-

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988 ANTIMETABOLITES OF SEROTONIN

ample, the effects upon various kinds of smooth muscle and upon a func- tion such as blood pressure.

Obviously, however, yohimbine, the ergot alkaloids, and the harman alkaloids are not pharmacologically equivalent. It is thus clear that their biological properties are not due solely to their capabilities of acting as antimetabolites of serotonin. The other features of their structures (aside from their kinship to serotonin) thus can be said to confer on each of them some biological properties which make each different from the others. Pos- sibly these additional attributes may be no more than enough to determine a different pattern of distribution to various organs. Possibly also they are such as to allow, let us say, yohimbine to interfere with the site of ac- tion of serotonin in tissue A, while ergotamine, let us say, is able to block serotonin sites in both tissues A and B. In any event, some of the biologi- cal effects of each of these drugs are clearly related to their being anti- metabolites of serotonin.

In recent years there have been many efforts made to produce syntheti- cally compounds which would mimic the pharmacological effects of yohim- bine, and especially of the ergot alkaloids. These attempts have pro- ceeded more or less empirically by various simplifications of the structures of the natural alkaloids. If the viewpoint of the present paper is adopted, then it may be possible to proceed more directly to the desired goal by deliberately setting out to make antimetabolites of serotonin. If sufficient attention is given in these efforts to the incorporation into the new drugs of features which would assure proper distribution to various organs, and which would protect the compounds from destruction en route to these organs, then it may prove possible to achieve simple drugs which will lack some of the undesirable effects of the natural alkaloids. Being only anti- serotonins properly aimed at certain functions of this metabolite, they would lack the additional attributes which are not concerned with sero- tonin. This is a working hypothesis which seems to have merit at least as great as the empirical approach nom being followed.

SUMMARY

Yohimbine was recognized to be a structural analogue of serotonin. In the test for antimetabolites of serotonin with segments of carotid arteries, yohimbine was found to be highly active. The antagonism was competi- tive over a large range of concentration. A graded series of compounds was synthesized which were all analogues of serotonin but which were pro- gressively more similar to yohimbine. Each substance was tested for its ability to act as an antimetabolite of serotonin. Most of these compounds proved to be active in this test. They thus formed a closely related series ranging from simple analogues of the metabolite up to the rather complex

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E. SHAW AND D. W. WOOLLEY 989

alkaloid. The fact that harman and aminoharman were members of this series, and were antimetabolites, suggested that these and other naturally occurring harman alkaloids might owe a portion of their pharmacological properties to interference with the action of serotonin. The ergot alkaloids were recognized as structural analogues of serotonin. Ergotamine and ergotoxine were shown to inhibit the action of this metabolite on segments of carotid artery, and to do this in a fashion reversible by it. Reasons were given for thinking that the entire pharmacological action of yohim- bine and of the ergot alkaloids was not due to their behavior as antimetab- olites of serotonin. Suggestions were made as to how the findings of this study might help in the realization of synthetic agents capable of taking the place of these natural drugs, and perhaps of improving upon them.

BIBLIOGRAPHY

1. Woolley, D. W., and Shaw, E., J. Am. Chem. Sot., 74,2948 (1952). 2. Woolley, D. W., and Shaw, E., J. Biol. Chem., 203, 69 (1953). 3. Woolley, D. W., and Shaw, E., J. Pharmacol. and Exp. Therap., 108,87 (1953). 4. American Chemical Society, Chemical factors in hypertension, Washington

(1950). 5. Chase, H. F., Yonkman, F. F., and Young, A. G., Proc. Sot. Exp. Biol. and Med.,

40,308 (1939). 6. Reid, G., and Rand, M., Nature, 169,801 (1952). 7. Erspamcr, V., Ricexa Scient., 22, 1568 (1952). 8. Snyder, H. R., Parmerter, S. M., and Katz, L., J. Am. Chem. Sot., 70,222 (1948). 9. Shaw, E., and Woolley, D. W., J. Am. Chem. Sot., 75, 1877 (1953).

10. Raymond-Hamet, Compt. rend. Sot. biol., 136, 1320 (1941).

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Elliott Shaw and D. W. WoolleyANTIMETABOLITES OF SEROTONIN

AS NATURALLY OCCURRING YOHIMBINE AND ERGOT ALKALOIDS

1953, 203:979-989.J. Biol. Chem. 

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