R. B. Barlow

Some studies on cytisine and its methylated derivatives

1 In mice cytisine hydrochloride is less toxic intravenously than nicotine hydrogen tartrate, but more toxic by intraperitoneal or oral administration. Compared with cytisine, caulophylline hydrogen iodide is one‐fifth to one‐tenth as toxic and caulophylline methiodide is less than one‐thirtieth as toxic. 2 The surprising low oral toxicity of cytisine and nicotine may be ascribed to the method of administration; if the drug is placed directly in the stomach there is no possibility of absorption through buccal mucous membranes. 3 The peripheral effects of nicotine, cytisine and caulophylline are similar, though on some preparations those of nicotine last longer. In most tests cytisine is active in doses from a quarter to three‐quarters of those of nicotine, caulophylline in doses from 10 to 20 times those of cytisine. Caulophylline methiodide is virtually inactive. 4 Cytisine and caulophylline may differ from nicotine in their central effects. 5 Cytisine and caulophylline are active as the cations. The pKa of cytisine is 7.92 and that of caulophylline is 7.04; the difference accounts, in part, for the weaker activity of caulophylline. The caulophylline ion is generally one‐sixth to one‐third as active as the cytisine ion. 6 The introduction of the second methyl group to form the quaternary salt does not appear to cause a dramatic change in the conformation of the molecule. Caulophylline methiodide appears to be feebly active because it has feeble affinity.

Relationships between chemical structure and affinity for acetylcholine receptors

1 . Series of analogues of acetylcholine have been prepared in which the acetyl group was replaced by phenylacetyl, cyclohexylacetyl, diphenylacetyl, dicyclohexylacetyl, (±)‐phenylcyclohexylacetyl, benziloyl and (±)‐phenyl‐cyclohexylhydroxyacetyl groups and the trimethylammonium group was replaced by Me2EtN+, MeEt2N+, Et3N+, Further series were prepared in which the acetoxyethyl group was replaced by ethoxyethyl, phenylethoxyethyl, cyclohexylethoxyethyl, diphenylethoxyethyl, and dicyclohexylethoxyethyl groups, and by n‐pentyl, 5‐phenylpentyl, 5‐cyclohexylpentyl and 5:5‐diphenylpentyl groups. 2 . The ethoxyethyl and n‐pentyl series contain some compounds which are agonists or partial agonists when tested on the isolated guinea‐pig ileum, but all the other compounds are antagonists. 3 . The affinity of the compounds for the postganglionic (“muscarine‐sensitive”) acetylcholine receptors has been measured in conditions in which the antagonists have been shown to be acting competitively. There were considerable differences between their affinities, the most active (log K, 9.8) having one million times the affinity of the least active (log K, 3.7). 4 . The changes in affinity as the onium group was modified were not entirely independent of changes in the rest of the molecule. Increasing the size of the onium group, as judged from conductivity measurements on simpler onium salts, increased affinity in the series containing one large group (phenyl or cyclohexyl) but, in the series with two large groups, affinity declined when the size was increased beyond ‐N+MeEt2. 5 . In general, the effects of changes in the rest of the molecule on affinity were bigger than the effects of changes in the onium group and there were bigger interactions. Affinity was increased to a greater extent by introducing one phenyl and one cyclohexyl group together than by introducing either two phenyl or two cyclohexyl groups; the increment was greater than the separate contributions made by one phenyl and one cyclohexyl group. 6 . The factors which influence the binding of molecules to receptors are discussed. There is no evidence that the separation between the onium group and the group in the receptor with which it interacts is greater in compounds with high affinity nor is there any evidence, from the study of the series which contain agonists and partial agonists, that ability to activate receptors depends upon the onium group being able to come close to this charged group in the receptors.

THE IONIZATION OF PHENOLIC AMINES, INCLUDING APOMORPHINE, DOPAMINE AND CATECHOLAMINES AND AN ASSESSMENT OF ZWITTERION CONSTANTS

1 The dissociation constants of many phenolic amines, including benzylamines, phenethylamines, phenylethanolamines, phenylpropylamines, catecholamines, and apomorphine have been measured by potentiometric titration at 25°C. Measurements have also been made with many of their methoxy derivatives and with series of phenolic quaternary ammonium salts. Some compounds were also studied at 37°C. 2 Usually at least five titrations were made with each compound and Debye—Hückel theory was applied to convert concentrations to activities but the estimates of pKa were not constant and found to increase with increasing concentration. The range studied was usually 5–15 mm and a least‐squares line‐fit, based on the empirical assumption that pKa varies with (concentration)1/2, has been used to calculate values for 10 mm solutions and to extrapolate to infinite dilution and to 100 mm. The dependence of pKa on concentration was much less at 37°C than at 25°C. 3 At 37°C the pKa values of many biologically interesting compounds in the group, dopamine, noradrenaline, adrenaline and isoprenaline, coryneine (the trimethylammonium derivative of dopamine) and apomorphine are within 1 log unit of physiological pH, indicating the presence of a significant proportion of either the zwitterion or of the uncharged phenolic amine. 4 Zwitterion constants have been estimated from the pKa values of the phenolic amines and those of their methoxy and quaternary trimethylammonium analogues. Zwitterion formation does not appear to be associated with activity at α‐adrenoceptors and probably not with activity at β‐receptors. The active species seems likely to contain the unionised phenolic group but at dopamine receptors this may be in the uncharged phenolic amine rather than in the phenolic ammonium salt.

A comparison of the affinities of antagonists for acetylcholine receptors in the ileum, bronchial muscle and iris of the guinea-pig

1 . Isolated preparations of bronchial strip and of intact iris from the guinea‐pig have been adapted for the measurement of affinity constants of substances which block post‐ganglionic acetylcholine receptors. 2 . The affinity constants of 28 compounds on bronchial muscle and of 8 compounds on the iris have been compared with values measured on the guinea‐pig ileum. 3 . Although the compounds differed up to a million‐fold in affinity, most of the estimates of log affinity constant for the bronchial muscle and iris differed only slightly from those on the ileum. 4 . Some of the differences could be attributed to the actions of hexamethonium, used in the tests on the ileum but not, initially, in the tests with the bronchial strip and iris. Hexamethonium reduced most of the estimates of log K for the receptors in the bronchial strip by a variable but significant amount, which could be due, at least in part, to a weak post‐ganglionic blocking (atropine‐like) action. On average, hexamethonium had little effect on the estimates made with the ileum, appreciably decreasing estimates with some compounds and increasing those with others. 5 . The results indicate that, although there may be differences between the acetylcholine receptors in the three types of tissue, there is no conclusive evidence, because the differences in affinity which we have observed could have arisen from differences in the experimental conditions. This is illustrated by results obtained with the guinea‐pig ileum recorded with the same technique as was used for the bronchial strip, which are presented as an appendix. 6 . Such differences as may exist between these three types of acetylcholine receptor are likely to be limited to the replacement of one aminoacid in the receptor protein by a homologue.

Specificity of some ganglion stimulants

1 The specificity of several ganglion stimulants has been tested on the isolated guinea‐pig ileum by measuring the dose ratios produced by concentrations of hexamethonium. 2 Most ganglion stimulants are also active at postganglionic receptors, some as blocking agents (for example, lobeline and dimethylphenylpiperazinium), others as agonists (for example, o‐aminophenethyltrimethylammonium and, to a lesser extent, nicotine). The most specific ganglion stimulant, with the least activity at postganglionic receptors, was p‐aminophenethyltrimethylammonium. 3 The affinity constants of lobeline and dimethylphenylpiperazinium for the muscarine sensitive receptors in the guinea‐pig ileum are 1·05 × 106 and 3·71 × 104, respectively. 4 The antagonism of p‐aminophenethyltrimethylammonium by hexamethonium gave results consistent with competition up to dose ratios of about 20. Such results could also be obtained if the antagonism were non‐competitive, however, provided large responses could be obtained with less than about 5% of the receptors in the ganglia activated. The affinity constant of hexamethonium is about 2·6 × 105. 5 It is suggested that the affinity of hexamethonium can largely be ascribed to hydrophobic bonding.

The relation between biological activity and the degree of resolution of optical isomers.

Previous reports of the low stereospecificity of benzhexol can be ascribed to inadequate resolution of the samples tested and a report of much higher stereospecificity has been confirmed. The two enantiomers have been found to differ over 1000‐fold in their affinity for the postganglionic acetylcholine receptors of the guinea‐pig ileum. Mixtures of the enantiomeric forms of phenylcyclohexyl‐glycolloylcholine and of benzhexol have been tested on this preparation and the dose‐ratios used to calculate apparent affinity constants. With both pairs the results indicate that the two enantiomers compete with the agonist and with each other and justify the use of the stereo‐specific index to set limits to the degree of resolution. For compounds such as these, in which one enantiomer has appreciably higher biological activity than the other, this biological method for assessing stereochemical purity is likely to be at least as satisfactory as any nuclear magnetic resonance method currently in use, because of the very great sensitivity of the stereospecific index to the degree of resolution.

Relationships between chemical structure and affinity for postganglionic acetylcholine receptors of the guinea-pig ileum

1 Some phenylacetyl, diphenylacetyl, benziloyl and (±)‐cyclohexylphenylglycolloyl esters have been made with 2‐ and 3‐hydroxymethylpyrrolidines, 3‐hydroxymethyl‐N‐methylpiperidine, piperidin‐3‐ols, piperidin‐4‐ols, 2,2,6,6‐tetramethyl‐N‐methylpiperidin‐4‐ol, tropine, pseudotropine and quinuclidin‐3‐ol, and the affinity of these compounds and of their metho‐ and etho‐ derivatives has been measured for postganglionic acetylcholine receptors of the guinea‐pig isolated ileum. 2 Some of the compounds were very active indeed; the benziloyl esters of N‐methylpiperidin‐4‐ol methiodide, tropine methiodide, and quinculidin‐3‐ol, and the (±)‐cyclohexylphenylglycolloyl esters of N‐methylpiperidin‐4‐ol and its methiodide had affinity constants greater than 1010. 3 The effects of inserting an additional methylene group onto the nitrogen were extremely variable, ranging from a decrease in log K of 1.64 units to an increase of 0.97 units. The effects of replacing hydrogen by phenyl in the acid portion ranged from an increase of 1.04 units to an increase of 3.06 units and of replacing hydrogen by hydroxyl from a decrease of 0.09 units to an increase of 1.94 units. 4 The extent of the variation in the effects of a particular change in structure on affinity does not appear to be any different in these relatively rigid compounds from that observed with the same changes in open‐chain aminoalcohols. 5 Reasons for the variable effects of groups on affinity are discussed. If differences in effects on preferred conformations of these particular compounds in solution are of secondary importance, the effect of a group on affinity will be the net result of what it could contribute to binding, offset by the disturbance it causes to existing binding. The maximum effect observed in a large number of comparisons may indicate the contribution in the absence of disturbance and for groups containing only carbon and hydrogen it appears to be related to size, assessed from the increments in apparent molal volume at infinite dilution. The variation in the effects of these groups also appears to be related to size. Changes involving groups containing oxygen can produce bigger contributions to binding, and a bigger variation in contribution, than would be expected from their size. 6 It is difficult to predict the extent to which groups may fail to produce their maximum effects. Variation is greatest with groups which could produce the biggest changes and so are of the greatest interest. 7 The relevance of the results to the successful prediction of biological activity is discussed.

The affinity and activity of compounds related to nicotine on the rectus abdominis muscle of the frog (Rana pipiens)

1 . Series of pyridylalkyl‐ and substituted phenylalkyl‐trimethylammonium salts, triethylammonium salts, diethylamines and di‐n‐propylamines have been made. The substituents in the benzene ring were nitro, chloro, bromo, methoxy, hydroxy and amino groups and the alkyl residues had one, two, or three methylene groups separating the aromatic nucleus from the cationic head. 2 . Most of the trimethylammonium compounds caused a contracture of the frog rectus muscle, but some were partial agonists and a few were antagonists. The di‐n‐propylamines were all antagonists, as were most of the diethylamines and triethylammonium compounds, though some of these were partial agonists and a few triethylammonium compounds were agonists. The affinities of the antagonists and partial agonists for the receptors stimulated by β‐pyridylmethyltrimethylammonium (and by nicotine) were measured. The equipotent molar ratios of all the agonists were measured relative to β‐pyridylmethyltrimethylammonium. 3 . The dissociation constants of the pyridylmethyldiethylamines and substituted benzyldiethylamines were measured. The effects of substituents on the pKa of benzyldiethylamine were similar to their effects on the pKa of aniline, though there were differences with some of the o‐substituted compounds, which could be attributed to internal hydrogen‐bond formation. 4 . There is no obvious correlation between the effects of a substituent on the pKa of benzyldiethylamine and its effects on affinity. Although increasing the size of the cationic group usually increased affinity, it did not always do so. The compounds with the highest affinity, p‐hydroxybenzyldiethylamine (log K, 5·90) had about half the affinity of (+)‐tubocurarine (log K, 6·11), but the triethylammonium analogue (log K, 4·17) had only about one‐fiftieth of the affinity of the tertiary base. The binding of the drug to the receptor appears to involve many factors which include the size of the groups as well as their electron‐releasing or withdrawing nature and other properties, such as their polar and lipophilic or lipophobic character. 5 . There is no obvious correlation between the effects of a substituent on the affinity of the diethylamino or triethylammonium compounds and its effects on the activity of the trimethylammonium analogue. The most active compounds contain hydroxy‐ and amino‐, phenyl or β‐pyridyl groups, m‐hydroxyphenyl‐propyltrimethylammonium being about 50 times as active as nicotine, but the corresponding diethylamino or triethylammonium compounds do not have high affinity. There does not seem necessarily to be an inverse relationship between activity and affinity, however, because some m‐nitro and m‐chloro trimethylammonium compounds have considerable activity and the analogous triethylammonium compounds have considerable affinity. 6 . It is suggested that ability to activate these receptors is associated with the presence of substituents which can interact with water molecules which may be involved in the action of the drug at the receptor.

Antagonist inhibition curves and the measurement of dissociation constants

Experiments carried out on guinea‐pig isolated ileum with carbachol as agonist and diphenyl‐ acetoxyethyl‐ dimethyl‐ethyl‐ ammonium (DADMEA) bromide as antagonist gave results which fit the theoretical relation between fractional inhibition (Q) of the effects of an agonist ([A]) and the concentration of a competitive antagonist ([B]): this also involves the Hill coefficient (logistic slope factor, P) for the agonist concentration‐response curve and the degree of agonist stimulation, [A]/[A]50, where [A]50 produces a half‐maximum response. Values of IC50 and an exponent, P′, can be obtained by fitting Q to [B] using a logistic approximation to the relation. Both P′ and IC50 should be greater with higher agonist stimulation but the increase in P′ may be masked by errors in extreme values of Q. Estimates of IC50, however, invariably increased with higher agonist stimulation but with a steep concentration‐response curve (P>1) and low agonist stimulation ([A]/[A]50 <1), IC50 can be less than KD. K D was calculated from the results in three ways: (i) by a least‐squares fit of Q to [B] using the values of P and [A]/[A]50 calculated from the control concentration‐response curve; (ii) from the value of IC50 for each line and the values of P and [A]/[A]50 and (iii) by using the agonist concentration‐response curve to calculate the dose‐ratio and estimate of KD for each response in the presence of the antagonist. The methods gave similar results (nm: 11 experiments), 12.4±1.1 (i), 11.7±0.9 (ii), 14.8±1.6 (iii) but there are advantages in using methods (i) or (ii) rather than (iii). The method by which KD is calculated is less important than the experimental design: the plan used in this work, with alternative small and large responses from the tissue, is very suitable for estimating KD with low concentrations of antagonists and small dose‐ratios. Although it is not a sensitive test for competitive behaviour because only a small range of concentrations of antagonist is tested, the estimate of affinity should be free from complications involved in the use of higher concentrations of antagonist (and agonist) and the nature of the antagonism can always be checked by doing further experiments in the presence of a known competitive antagonist.

Actions of some esters of 3,3-dimethylbutan-1-ol (the carbon analogue of choline) on the guinea-pig ileum.

1 Estimates were made of the affinity constants for postganglionic acetylcholine receptors of the guinea‐pig ileum of the esters of 3,3‐dimethylbutan‐1‐ol with benzilic, (±)‐cyclohexylphenylglycollic, (±)‐mandelic, and diphenylacetic acids. 2 Attempts were made to check the competitive nature of the antagonism by using as wide a range of concentrations of antagonist as possible, consistent with their limited solubility, and by testing some of the compunds in the presence of a known competitive antagonist. 3 By comparing the affinities with those of the corresponding quaternary nitrogen compounds, the contribution made by the positive charge in the onium group to the binding by receptors may be assessed and has been found to be variable. The carbon analogue of benziloylcholine has about one‐tenth of its affinity, that of (±)‐cyclohexylphenylglycolloylcholine has only about one‐sixtieth of its affinity, but that of (±)‐mandelylcholine has slightly higher affinity than that of (±)‐mandelylcholine itself. 4 3,3‐Dimethylbutylacetate appeared to be a partial agonist with an affinity constant of about 2.6 × 103. The contribution made by the positive charge to the binding of acetylcholine at these receptors therefore seems likely to lie within the range observed with antagonists and there is no reason to believe that there is necessarily greater intimacy of association by agonists than by antagonists. 5 Although the C—C and N—C bonds in —CMe3 and —NMe3 are similar in length, the groups do not occupy the same volume in solution in water.