Stereochemical aspects of parasympathomimetics and their antagonists: recent developments.

Progress in Medicinal Chemistry - Vol. 11, edited by G.P. Ellis and G.B. West 0 1975 - North-Holland Publishing Company

1 Stereochemical Aspects of Parasympathomimetics and their Antagonists: Recent Developments A.F. CASY,D.Sc., Ph.D., F.P.S., F.R.I.C.

School of Pharmacy, Liverpool Polytechnic, Liverpool L 3 3AF, England INTRODUCTION

1

X-RAY CRYSTALLOGRAPHY

3

NMR STUDIES O F CONFORMATION

1

METHY LACETY LCHOLINES

11

MOLECULAR ORBITAL CALCULATION OF CONFORMATION

12

CONFORMATIONALLY RESTRAINED ANALOGUES O F MUSCARINIC AGONISTS

13

ANALOGUES O F ACh

14

ANALOGUES O F MUSCARINE

24

SYNTHETIC AND STEREOCHEMICAL METHODOLOGY

21

DISCUSSION O F CONFORMATIONAL REQUIREMENTS

32

MUSCARINIC ANTAGONISTS

36

STEREOSPECIFICITY AND OPTICAL PURITY

41

DERIVATIVES OF 3QUINUCLIDINOL

55

RECEPTORS FOR ACh AGONISTS AND ANTAGONISTS

51

REFERENCES

60

1

2

STEREOCHEMICAL ASPECTS OF PARASYMPATHOMIMETICS

INTRODUCTION It is now almost 70 years since Cushny [ l ] reported a potency difference between hyoscyamine and the racemic mixture atropine, but interest in the stereochemical aspects of acetylcholine (ACh) and its congeners and antagonists did not really blossom until the 1960s. The work of this period has been summarized in a number of reviews and monographs 12-61 but since 1968 much novel work has been reported and this is presented here with a literature coverage extending t o mid-1973. During the last 5 years or so, much effort has been made to define the pharmacophoric or ‘active’ conformation of cholinergic molecules in the search for clues about the molecular nature of cholinergic receptors and the ways in which their agonists and antagonists (ligands) combine with the receptors [7]. Although there is promise that direct study of the receptors themselves may be possible eventually [8] information about them must still largely be inferred from the characteristics of their ligands. Questions to the forefront of the work reviewed are as follows: (1) Does the ‘active’ conformation of a cholinergic ligand correspond with its preferred stereochemistry or is an energetically less favoured form bound t o the receptor? ( 2 ) Is there a unique mode of ligand binding t o cholinergic receptors or do multiple modes exist? (3) May the dual effects (nicotinic and muscarinic properties) of ACh be explained in terms of conformational isomerism? [ 9 ] . (4) Do agonist and antagonist ligands occupy the same or different binding sites (with one‘or more features common to both)? Regarding (l), Portoghese has made the interesting suggestion that if the ligand-receptor association involves a higher energy conformer, the ligand may transfer its conformational free energy to the receptor component which, in a now activated state, is then capable of triggering a conformational perturbation of the receptor macromolecule which leads to the biological response [ 101 . In compounds with cholinergic properties, whether muscarinic or nicotinic, it is desirable that as much evidence as possible be gathered about their mode of action since meaningful structure-activity relationships may only be derived from a group of agents when it is established that they produce their pharmacological effects through interaction at a common receptor system. Ideally, the pharmacological evaluation of cholinergic agonists should involve the following: (1) Specific tests for muscarinic and nicotinic piopeities including the effects of appropriate antagonists upon responses e.g., hyoscine for muscarinic and hexamethonium for nicotinic blockade. ( 2 ) Tests for the presynaptic release of ACh, as occurs with indirectly acting agents. There is good evidence that nicotinic agents often function in this manner [11,12] and there are now several reports of muscarinic agents that act

3

A.F. CASY

via the release of the natural neurotransmitter, e.g., n-butyl acetate and related esters [ 131 , acetylcarbocholine [ 121 and scorpion venom [ 141 . Comparison of the potency of an agonist in the guinea pig ileum test measured with that obtained (a) on ileum stored at 2-4°C for 24 hr [15], (b) in the absence of calcium ions [12,16] and (c) in the presence of tetrodotoxin, a specific inhibitor of muscle responses elicited by nerve stimulation, [17], are some of the tests which distinguish between direct and indirect action [12] . (3) Evaluation of the agonist as a substrate for, or antagonist of, acetylcholinesterase (AChE) and other cholinesterases. T h s test is of special importance when a group of related compounds are being compared and guards against the possibility of activity differences being due to variations in resistance (for esters) to enzymatic hydrolysis or ability t o inhibit the action of AChE on endogenous ACh. Evidence may be gained by conducting cholinergic assays in the presence of a cholinesterase inhibitor such as eserine [17] provided problems resulting from the consequent build up of ACh may be overcome [16,18].

X-RAY CRYSTALLOGRAPHY During the past decade crystallographers have become increasingly interested in small molecules of biochemical and pharmacological importance. This development is well exemplified in the cholinergic field where several groups, notably that of Peter Pauling at University College London, have now made available information upon the solid state conformation of a wide range of agonists and some antagonists. The results of some of this work are summarized below. The torsion or dihedral angle parameter (7) most conveniently describes the 3-dimensional shape of a molecule. In the system X-C-C-Y, the torsion angle is defined as the angle between two planes, one containing the C-C and C-X bonds and the other the C-C and C-Y bonds, and is best depicted by means of a Newman projection (Figure 1.1). The torsion angle is considered positive or negative according to whether the bond to the front atom X requires rotation t o the right or left, respectively, in order that its direction may coincide with that of the bond to the rear atom Y; descriptions of various conformations are shown below [ 191.

r

00 synplanar

*60° synclinal

f 1200 anticlinal

(all ?300)

antiplanar

4


HA

HB

Figure 1.1. Representation of a dihedral (torsion) angle of 90" in a HACCHB fragment: one plane is defined by the C-Cand C-HA bonds and the other by the C-Cand C-HB bonds.

All cholinergic molecules studied contain the unit (1) (or a close variant) and the four T values required t o define its stereochemistry are: 71

72 73

74

CS-C4-N-C3 0 1-C5-C4-N C6-0 1-CS-C4 C7-C6-0 1-C5

7 1 5 4 C-0-C-C-N-C

6\'

OH

/"

\

2

c3

(1)

Of these, 71 and 74 usually fall close to 180" on account of a preference for the fully staggered conformation (2) and for a near-planar ester unit (3) [20]. Values of 72 and 73 are therefore of most interest, particularly 72 because this defines the relative dispositions of the acyl group and quaternary head, functions

::;:

5C, carbonyloxygen

H

/N

Me

in rear

c\

in rear

Go \

Me

I -

c7

Me

0-c ..J

\?=C, /O

c5

Me

(3)

(2)

both vital to pharmacological properties in ACh and its congeners [21]. Magnitudes of 72 and 73 observed in crystals of a variety of muscarinic agents are shown in Table 1.1. With the exception of ACh bromide [23] and the 1,3-dioxolan 73 values fall in the range 180" f 36"; this means that in the majority of cases the acetyl (Me-C=O) portion of the molecule is set well away from the quaternary head (4) [tN-C6 distance in pm (1 = 100 pm): ACh C1- 440 (73-167O); ACh Br-412 ( ~ 3 t 7 9 " ) ][22,24].

a

5

A.F. CASY

Table 1.1. CERTAIN TORSION ANGLES OBSERVED IN CRYSTALS OF MUSCARINIC AGENTS [22] ~

Compound

...

~~

72

.-

ACh bromide ACh chloride (+)-Muscarine iodide (+)-cis 2-methyl-4-trimethylammoniummethyl-l,3-dioxolan iodide 5-Methylfurmethide iodide b (+)$-Methyl ACh iodide (+)-a-Methyl ACh iodide a b erythro- cu,P-Dimethyl ACh iodide Carbachol (+)-trans 2-Acetoxycyclopropyl-1-trimethylammoniumiodide

Me

C6

':I

4I

1

(4)

+ 77 + 85 + 73 + 44 + 83 + 81 + 87 + 89

-150 + 76 +178 +137

73

-~

+ 79 -167 + 144 +103 +174 +176 -143 +167 -179 -155 -174 + 147

0

(5)

The torsion angle relating+NMe3 to OCOMe ( 7 2 ) commonly has a value in the range 73-94" so that the N and 0 functions are more or less synclinal (5). It turns out,+ in fact, that most compounds comprising the molecular feature 0-C-C-N where the charged group is quaternary nitrogen or a protonated base and the oxygen function is hydroxy or acyloxy, prefer the synclinal N/O arrangement in the solid state, e.g. L-a-glycerophosphorylcholine CdC12.3H20, choline chloride [25] and lactoylcholine iodide [26] . An electrostatic interaction between the charged nitrogen group and the ether oxygen of the acyloxy function is probably an important stabilizing factor which leads to a preference for the synclinal rather than the antiplanar (sterically favoured) conformation (see later). Some molecules of this type do, however, display a preferred antiplanar-anticlinal conformation. These include the potent agonists carbamyl chloride [27] (72 + 178", stabilized by several hydrogen bonds) and (+)-trans-2S-acetoxycyclo-

5


propyl-IS-trimethylammonium iodide [ 2 8 ] (72 + 137", fixed by the rigidity of the 3-membered ring), and the weakly active thio (72 171" for bromide) and seleno (72 175" for iodide) analogues of ACh in which ether oxygen is replaced by the bulkier and less electronegative sulphur or selenium atom [29]. In 5-membered cyclic analogues of ACh, synclinal N/O conformations are found for L-(t)-muscarine iodide (6) (72 -t 73") [30], (+)-cis-2S-methyl-4R-trimethylammoniummethy1-1,3-dioxolaniodide (7) (72 t 94") [3 I ] , and 5methylfurmethide (8) (72 t 81 or -+ 83") [32]. In the fury1 derivative an antiplanar N/O conformation is seriously destabilized by interactions between +NMe3 and 3-H of the heterocyclic a ring. Surprisingly, 72 in crystals of L(t)muscarone iodide (9) is t 162" [33] ; the shapes of the tetrahydrofuran rings of muscarone and muscarine also differ and this may lead to a reduction in steric interactions between +NMe3 and the 3-methylene group in the antiplanar N/O arrangement.

h?"

Me

I

Me

"G'

OH

940

O2 0

(6)

d

N

(7)

9

+

J81B830

,NMe3 CH2

'\

In addition to the muscarinic agents already described, the nicotinic agonists 1 , I -dimethyl-4-phenylpiperazine (DMPP) [34] , nicotine [35] , a-methyl ACh [36], and lactoyl choline [ 2 6 ] have also been examined by X-ray diffraction. Chotia and Pauling have compared the crystal structures of these molecules and noted several common features [34]. From these they propose that the conformation of ACh relevant to the nicotinic receptor is one with 72 approximately 75" and 73 near 180". The NCCN and NCCA,CA, torsion angles of DMPP and nicotine respectively are taken to be the equivalent of Ol-CS-C4-N(72) of the choline molecules. Arguments of this kind are of dubious value, however, since

1 M/c1

A.F. CASY

7

+?Meg y

methyl side

2

carbonyl side

(10)

there is good evidence that many nicotinic agents including nicotine itself and DMPP act indirectly through release of endogenous ACh (most muscarinic agents are believed to act directly at the receptor). From the evidence of solid state conformation, Chotia [37] considers that the conformations of ACh at muscarinic and nicotinic sites are similar and explains the differing actions of the neurotransmitter in terms of receptor interaction with either the methyl side (muscarinic effects) or carbonyl side (nicotinic effects) of the molecule (10). He argues that the carbonyl group is either blocked or absent in potent muscarinic agents such as ACTM, 0-methyl ACh, the dioxolan (7) and muscarine while the reverse obtains in nicotinic agents. Criticisms of these views are discussed later. The crystal structure of oxotremorine has also been reported [37a].

NMR (CHIEFLY PMR) STUDIES OF CONFORMATION

As a conformational tool, NMR spectroscopy has an advantage over X-ray diffraction in that it provides information about molecules in the solute condition; polar molecules can usually be examined in deuterium oxide (D20) or failing this another polar solvent such as deuterated dimethyl sulphoxide ((CD3)2 SO), hence conformations may be established for conditions that are close to physiological. However, while an X-ray analysis gives essentially the complete conformation of a molecule, information from NMR spectroscopy is usually limited to a few features. The ability of X-ray crystallography to establish absolute configuration (provided a suitable heavy atom can be incorporated into the molecule) [38] is now under challenge by the NMR technique through developments involving the use of chiral solvents [39a] and shift reagents [40]. The two techniques do not necessarily provide the same stereochemical answer, e.g., crystalline diprotonated histamine (as the diphosphate) is exclusively antiplanar with respect to the amino and imidazole features [41] while histamine as solute in 0.1 N D2S04 is populated by about 50% of the antiplanar and 50% of the two synclinal conformations [42]. However, in the

8

STEREOCIIEMICAL ASPECTS OF PARASYMPATHOMIMETICS

cholinergic field, most of the NMR evidence complements the results of the X-ray diffraction studies. This agreement may be a result of the molecules being stabilized by strong intramolecular interactions that are not seriously disturbed by solvents. NMR evidence of the conformation of compounds containing the unit (1) (see p. 4) is restricted to the torsion angle 01-CS-C4-N (72). In molecules like ACh the three possible staggered conformations relevant to r2 are (1 1-13). The results of NMR analyses establish the relative populations of the three forms and identify favoured conformations if any exist. All rotamers are freely interconverting because of the low barriers to rotation between the members and NMR provides a picture of the time-averaged conformation at room temperature 0

+N

(11)

+N (12)

+N (13)

(or higher or lower temperatures if desired). Conclusions reached are less precise than those of X-ray crystallography. Thus preferred conformations may usually be defined no closer than synclinal or antiplanar but on the other hand the NMR spectroscopist must take into account a mobile system rather than a futed one as presented by a crystal lattice. A brief account of specific cases will now be given. All analyses are based upon the fact that the extent of spin-spin coupling between two protons (3J) depends upon their stereochemical vicinally disposed as in H-C-C-H orientation as defined by the appropriate torsion angle, i.e. the 3J/cos2r relationship of Karplus [39b,43]. Estimates of 3J values associated with torsion angles of 60" and 180" may be made by the aid of data upon model compounds of fixed geometry and by taking into account the effects of the electronegativity of substituents attached to the H-C-C-H fragment [44]. Comparison of experimental and predicted coupling constant values then leads t o the conformational conclusion. The 4-proton bimethylene system of ACh and its analogues is described as AA'XX' or AA'BB' depending on whether the chemical shift difference between the methylene pairs is large or small [39c,45]. Rapid interconversion of conformers by rotation about the C-C bond renders each geminal pair of protons chemically equivalent but does not lead to a single averaged vicinal

9

A.F. CASY

coupling constant except when the conformer populations are equal. Hence two distinct 3J values arise because the extents of coupling between A and X (or B) and A' and X (or B) differ, and the values obtained are the population weighted average of contributions from the three staggered conformations (14-16) (the only ones likely to be significant) (see Scheme). Since the system is not Ifjt order.

H1

0

(a)

(b)

(15)

(16)

Fractional populations: 2a + b = I

J = J I ,4 = aJ; + aJg' + bJf g

J'

= J1,3 =

h J g " + bJ:

J t denotes a t r a n s coupling in an N/O synciinal conformation while g coupling in an N/O antiplanar conformation etc.

Je

denotes a gauche

J , , 3 and J , ,4 may not be derived directly from the spectrum. The line pattern does, however, provide estimates of the sum (N)and difference ( L ) of the two 3J values and these may be refined by means of an iterative calculation and computer programme [46] giving: N 9.49 Hz and L 4.43 Hz [47]. Evidence about the sign of L (unknown from the spectral analysis) is derived from Abraham and Pachlers' relationship

iN +iL

=

17.97 0.80 C E ~

where CE is the sum of the electronegativities of the substituents attached to the C-C fragment concerned [48]. Substitution of positive and negative values of L into this equation gives CB equal to 15.5 and 17.5 respectively. The CE value calculated using Huggins electronegativity values [49]is 15.3 hence L is probably positive. Since N t L = 2J it follows that J = 7.0 and J' = 2.5 Hz. These results are now compared with values anticipated if (i) antiplanar and (ii) Jf). synclinal conformers be favoured (and assuming Jk J: and

5 3'

10


(i) Antiplanar conformation favoured (see 16)

In this case the smaller coupling (2.5 Hz) could correspond with P , but the larger (7.0 Hz) is abnormally low for Jt even allowing for substituent effects. (ii) Synclinal conformations favoured (see 14 and 15)

J l , 3 = ;(Jg t J g ) = J g J , 4 = f(J' t J g ) Here the 2.5 Hz coupling is consistent with the J g value whereas J = 7.0 Hz approaches the expected value of 1/2 (J' +Jg),e.g., about (10 i- 3)/2 = 6.5 Hz. On this basisJ' has a value (1 1.5 Hz) within the normal range [47] . Cushley and Mautner [50] carried out similar analyses of the spectra of acetylthiocholine and acetylselenocholine in D2 0. The 3J values obtained (1 1.59 and 5.00 Hz for the thio and 12.62 and 4.83 Hz for the seleno analogue) were of magnitudes typical of J' and Jg coupling respectively, hence these analogues must exist almost exclusively in the antiplanar conformation in solution. The spectrum of acetylthiocholine has also been analysed by other groups [ 5 1,521 . Rotamer populations of ACh and its relatives may also be calculated by use of the magnitude of coupling between 1 4 N and a 0-proton (N-C-C-H) [52,53]. An advantage of the method is that these couplings may be measured directly from the spectrum e.g. from the OCH2 multiplet of choline bromide [52], the higher field NCH2 band is less complex because N-C-H coupling is near zero. If only two constants JdH and J d H are assumed, J N H for the ethyltrimethylammonium ion (17) (2.1 Hz) is made up as follows:

JNH =

i(Jh, i- 2 J i H ) = 2.1 Hz

+N

H

A.F. CASY

11

is close t o 0.7 Hz because values in this range are seen in spectra of choline derivatives shown to have strongly preferred N/X rotamers (e.g. thioacetylchot line IS), whence J N H = 4.9 Hz (if the NH coupling constants have the same

J$H

sign). If synclinal conformations are favoured for ACh, J N H will be made up of t the average of JN\ and JNHand the observed value of 2.5 Hz is close to that anticipated (0.7 + 4.9)/2 = 2.7 Hz.

METHY LACETYLCHOLINES Analysis of the 8-line multiplet due to the CH2 protons of 0-methyl ACh (18a)

by Bible's method [54] gave values of 9.7 and 1.5 Hz for the two 3J couplings that operate in the CH2CH fragment of the molecule [55]. The orders of magnitude of these values are typical of trans and gauche vicinal coupling respectively, hence either (19) or (20) is the preferred conformation. Cornpari-

0 (19)

Me

(20)

son of the methylene 3J values and the 0-methyl chemical shift with those of rigid models [55] and use of other arguments [52,53] leads to the selection of (20). First order analysis of the methine resonance of a-methyl ACh (CH2 CH) yields 3J values of similar magnitude (4.6 and 4.2 Hz); this fact shows that

12

STEREOCHEMICAL ASPECTS OF PARASY MPATHOMIMETICS

conformational preferences amongst a-methyl ACh conformers are low [55] . The 2 Hz coupling between the methylene protons and 14N supports this conclusion [52,53]. As must already be clear from the X-ray diffraction and NMR studies outlined so far, the solid state conformation of a cholinergic agonist is very similar to its preferred conformation as a solute in D 2 0 . This holds true for P-methyl ACh (72 + 85", 0-Me and 'N antiplanar, T - 152") [36] while the crystalline a-methyl isomer exists in two forms, a synclinal and a near-antiplanar N/O conformer (72 + 90" and - 148") [56]. Carbachol (Me,N'CH,CH,.OCO. NH2, synclinal as solute in D2 0) [ 5 2 , 5 7 ] is exceptional in being antiplanar in the solid state [27] but its unusual crystal form is considered to be stabilized by intermolecular hydrogen bonds that are unique to the carbamate. The prevalence of synclinal N/O conformers amongst ACh and its congeners is unexpected on steric grounds, but may be attributed to an electrostatic interaction between the positive charge of the onium group and the partial negative charge on the ether oxygen - an intramolecular interaction which operates most effectively when the two functions concerned are synclinal. IR evidence for such an interaction is provided by the fact that the carbonyl stretching mode of ACh is at a higher wave number than that of the corresponding tertiary amine and of ethyl acetate [55,58,59]. In thiocholines no significant electrostatic interaction can develop since sulphur is of low electronegativity, and the preferred conformation (antiplanar) is governed by Van der Waals repulsions. It is convenient at this point to note that nuclear magnetic relaxation data has been applied to studies of the molecular dynamics of ACh ( I 3 C , 1 4 N ) [60], and of the interaction between ACh and AChE ( I H) [61]. Studies of conformation and electron distribution in nicotine and ACh by I 3 C NMR have also been reported [61a].

MOLECULAR ORBITAL (M.O.) CALCULATION OF CONFORMATION By the methods of quantum mechanics it is possible, in principle, to establish the preferred conformation(s) of any molecule. The techniques involve computer aided calculations in which various molecular parameters such as bond angles and bond lengths, and the Coulomb integrals of electrons in specific atomic orbitals are employed. By these means the energies of a range of molecular conformations may be derived and conformers of lowest energy identified. Calculations are based on isolated molecules and do not take into account intermolecular interactions as may occur, for example, with solvent molecules. An account has been given of the principles of the M.O. approach [62].

A.F. CASY

13

Kier has been particularly active in applying M.O. calculations to pharmacologically active molecules 1631 including cholinergics, and he uses the Huckel molecular orbital (H.M.O.) method. His calculations for ACh lead t o energy minima for the torsion angles 72 (go"), 73 (180") and 74 (plateau between 120 and 240"), hence his model for the preferred conformation corresponds closely with the solid state molecule. Other groups, using different methods of calculation, arrived at a similar conclusion 165,661. The energy barrier t o rotation over the 72 range 80" (synclinal O/N) to 180" (antiplanar) is low; the antiplanar conformer is approximately 3 Kcal less stable than the synclinal arrangement [65] so conformer interconversions will be rapid down t o about 50°K [67]. The NCCO torsion angles for muscarine and muscarone were 60" and 120" respectively in lowest energy conformations [64] values which correlate reasonably well with X-ray diffraction data. Calculations have also been made upon /3-methyl ACh [68], oxotremorine [69] and nicotine [65,70] although it is recognized that neither of the last two agents is likely t o have direct cholinergic actions (see p. 2 and [71]). Pullman et al. [65] calculated the net total (u t n) electronic charges in ACh and muscarine and found that (i) the 'N atom of both compounds is in fact nearly neutral (0.06 eu), cf. data on 'NH3 of protonated histamine [72], (ii) most (-70%) of the formal positive charge is distributed among the three N-pethyl groups - more to the hydrogen than the carbon atoms, and (iii) the carbonyl and ester oxygens of ACh bear similar net total charges while the intermediate carbon carries an appreciable excess of positive charge viz: - 0.25 + 0.32 - 0.24 eu. These studies have been extended to other

0-c--0

ACh derivatives [73,73a]. CONFORMATIONALLY RESTRAINED ANALOGUES OF MUSCARINIC AGONISTS Although X-ray diffraction, NMR, and molecular orbital studies concur in identifying the preferred conformations of ACh and many of its active analogues as those with synclinal nitrogen and oxygen functions, there is no guarantee that such forms represent the conformation adopted by the agonist at the cholinergic receptor i.e., the 'active' conformation. Barriers to rotation in molecules such as ACh are low and easily overcome by energy derived from the thermal motion of the molecules or (in cases where barriers are higher) perhaps by that released on formation of the agonist-receptor complex itself [74] . Data upon conformationally restrained analogues of ACh, in which the dispositions of onium and acetate functions are more or less 'frozen' in relation t o those in the flexible

14


parent molecule therefore provide clearer information about the active conformation. In most molecules of this class barriers to conformational change are much higher than those of the acyclic forms hence the geometry of the molecule established in vitro, is unlikely to be altered significantly upon drug-receptor interaction. The drawback t o this approach is that the analogue must inevitably be larger than ACh itself and the skeleton used to restrict movement of the N and 0 functions will almost certainly impede binding of the pharmacodynamic groups to the receptor surface. Even a single methyl group may profoundly affect the activity of ACh, e.g., a-S, a-R, and 8-R methyls cause 232, 28, and 240 fold drops in the muscarinic potency of ACh respectively although methyl is not detrimental in this sense when present in the 0-S orientation [75J . Except for analogues based on cyclopropane, all rigid or semi-rigid derivatives of this type examined so far contain a far greater array of atoms than one additional carbon and potencies observed are generally of a much lower order than that of ACh. If the derivatives yield dose response curves parallel t o those of ACh and are able to reproduce the maximal response to this agonist when applied in high enough dose levels, the additional molecular features may be assumed to influence the affinity of the molecule for the receptor rather than its intrinsic activity. A modification of the intrinsic activity is also revealed if the derivative is shown t o have partial agonist or antagonist properties as is well illustrated by the results of altering the acyl feature of ACh [76]. Within a series of analogues of similar molecular dimensions and skeleton, however - and especially if a diastereoisomeric group is under study - activity variations may reasonably be assumed to have conformational significance even if the potencies recorded are of a low order, It must be emphasized that any conclusions drawn from the results about the active conformation of ACh rest upon the assumption that the analogues in question act directly at the muscarinic site (see p. 2). It is unfortunate that comprehensive evidence on this point is rarely presented. The review of conformationally restrained analogues which follows is subdivided into derivatives of ACh and muscarine. In each division isomer type and the relevant pharmacology are discussed followed by a brief summary of some of the synthetic and stereochemical methodology.

ANALOGUES OF ACh Schueler [77] first drew attention to the possibility of the muscarinic and nicotinic effects of ACh being mediated by different conformational isomers of

A.F. CASY

15

the flexible molecule. He examined the 3-piperidyl acetate (21) and the morpholine derivative (22) as models of antiplanar and synclinal N/O conformations of ACh respectively. Both were feebly active in muscarinic and nicotinic assays, with (21) the more potent. The validity of this comparison is doubtful, however,

/ ~

\ o

Me

“

o

Me ~

e

“7’ L;

. O CNO M e ~

/

Me

Me

M e ,I ; q o

\

Me

Me

(21 1

0

(221

because of the disparity of skeletal arrangements of atoms in the two molecules. More recently (21) was found to have 1/660 and its 4-piperidyl analogue 1/300 the muscarinic potency of ACh (effects on rat blood pressure, blocked by hyoscine HBr) [78], while ileum experiments also confirm the muscarinic properties of (21) [79,80]. The activity of the cis diacetate (22a) (Ill00 ACh) and inactivity of the trans isomer (22b) in muscarinic tests has been cited as

wo‘coMe

Me+, Me

M

e

-

.

~

~

~

~

e

o

M

e

0,COMe

(22al

(22b)

(unfavoured conformation)

evidence that synclinal N/O conformers are the receptor-bound species of ACh agonists [80]. The fact that the isomeric diacetates are far more likely to differ in their 4- rather than 3-substituent orientations (see p. 40) [78] invalidates this argument. The comparative pharmacology of (23) and (24) may reasonably be studied because these tropyl acetates differ only in their N/O geometry. It is reported M eMe ” ~ c o M e

Me’’w Me

OXOMe

[81] that the antiplanar form ( 2 3 ) (DL or L isomer) is 50-100 times less active than ACh while L (24) (synclinal) has no spasmogenic properties when tested at

16


the same concentration levels (rat sigmoid colon preparation). The synclinal analogue (24) was, however, more potent in nicotinic tests performed on the atropinized cat (rise in blood pressure: 0.5-1.0 mg/Kg (24) 1.O-2.0 mg/Kg (23); at same dose levels (24) caused contraction of the nictitating membrane while (23) was ineffective; 0.25 mg/Kg ACh produced positive responses in both tests). The decahydroquinolines (25) reported by Smissman and Chappell [82] are ACh analogues similar in type to the tropanes (23) and (24) all being related t o 3-piperidinol with the onium nitrogen forming part of the ring system. Only the synclinal form (23) had agonist properties (guinea pig ileum, molar potency = 0.02 with ACh = 1) but its affinity for the receptor appeared t o be less than

yLy mo'coMe = 0,COMe

~ ~ & o . c o M e

/N\ Me

(25)

/ Me

/

Me

Me

(26)

(27)

that of the antiplanar isomer (27) on the basis of study of the blockade of ACh-induced contractions ((27) caused 60% inhibition at 50 pg/ml while at least 100 pg/ml were required for blockade by (26)). Neither isomer was active in tests for nicotinic activity. True AChE (from eel) hydrolysed the equatorial isomer about a fifth as fast as ACh, while the axial derivative inhibited the enzyme [82,83]. Cyclic analogues of ACh containing the full trimethylammonium cationic head were first described in studies of cis and trans 2-trimethylammoniumcyclohexyl acetate (28) and the corresponding cyclopentyl derivatives (29) as substrates for AChE [84]. The 'muscarinic properties of the cyclohexyl pair has

recently been reported however 1851 ; RS-trans (28) was about 400-1000 times less potent than ACh while the cis racemic mixture was inactive at all concentrations used (Table 1.2). The 1R,2R-(-)-trans enantiomer was 4 times as active as the racemic mixture while the (+)-trans isomer was much weaker (8 X lop4

A.F. CASY

11

mol/l produced a contraction which was less that 50% of the ACh maximum). The superiority of trans (28) t o the cis racemic mixture has recently been confirmed [79,86]. A potency difference between cis and trans (28) is unexpected in terms of relative dispositions of nitrogen and oxygen functions in the isomeric pair. Their preferred conformations are (30) (cis) and (31a) (trans) [87,88] and 0-C-C-N

a)R=H

b) R = But

torsion angles must be close to 60°, i.e., the N and 0 substituents are synclinal in both isomers. However, inversion of the trans, but not the cis derivative, produces a conformation which is antiplanar with respect t o +NMe3 and OCOMe; hence, the active form of trans (28) may be the unfavoured NaOa conformer (see later). The cis form was inactive as a substrate for bovine AChE while the trans isomer was hydrolysed at a very slow rate [85] . Fusion of 2-trimethylammoniumcyclohexyl acetate to a second cyclohexyl ring prevents ring inversion provided the ring junction is trans. The decalins (32) are ACh analogues of this type and the 4 RS-isomers provide 3 synclinal (33-35) and one antiplanar N/O (36) disposition. Low orders of muscarinic activity were shown by the NaOa and NaOe isomers, the former being distinctly the more potent (Table 1.3) [ 8 9 ] . It may be argued that skew-boat conformations such as (37) are likely t o be favoured over the chair (36) in order that non-bonded interactions of the Table 1.2. MUSCARINIC POTENCY OF RACEMIC AND 1R, 2R-TRANS-2-TRIMETHYLAMMONIUMCYCLOHEXYLACETATE [85].

Compound

Effective conc (molll) inducing contraction of guinea pig ileum *

ACh RS trans (28) 1R, 2R trans (28)

1.3 X lo-' 6.1 X lop6 1.5 x 10-6

* Maximumcontraction produced

to to to

1.6 X

1.6 x 10-3 4.0 x 10-4

by ACh at 5.1 X lo-' mol/l. Atropine (1 X 10-9mal/l) inhibited all active compounds in similar degree. Hexamethonium (9.9 X mol/l) had no effect on the contraction produced by RS trans (28).

18


Table 1.3. PHARMACOLOGICAL DATA ON SOME DECALIN ANALOGUES OF ACha 1891 ~~~

Compound

Equipotent conc ( ~ g / m l ) ~

NaOa (36) NaOe (35) NeOe (33) NeOa (34) Erythro (39) Threo (40) ACh CI-

50 500-1000 Inactive a t 1500 Inactive at 1800 1.7 67 0.01 25

Relative molar potency (ACh = 100)' 0.052 0.0026-0.0052 -

1.2 0.029 100

a Relativeratesof hydrolysis by true AChE [ 8 2 , 8 3 ] (ACh = 100): NaOa (36) 14.6;all other isomers negligible with the NeOa form an inhibitor; erythro (39) negligible, threo (40) 9. Guinea-pig ileum test, all iodides except ACh. Corrected values (Smissman, private comm. J.

N

(36) NaOa

N

10 represents OXOMe and N, NMe31 0 R'

R = COMe, R' = H or COMe 2- and 3- substituent axial in chair conformation

OCOMe and +NMe3 groups be reduced (this is the case, in fact, for ring A of the steroid (38) in both the solid [9OJ and solute [88] condition), but NMR and

A.F. CASY

19

X-ray diffraction evidence show that the antiplanar conformation is maintained (see below). Pharmacological data upon diastereoisomers of aJ-dimethyl ACh (Me3N.CHMe.CHMe.0.COMe I-) were originally considered to correlate with those for the decalin isomers (Table 1.3) in that the more active erythro isomer would be expected to have a preferred N/O antiplanar conformation (39) on steric grounds while the corresponding threo conformer (40) would be unfa-

0

0

(39)

(40)

erythro (minimum gauche interactions between bulky groups)

threo (unfavoured conformation)

voured [89] . Subsequent X-ray diffraction analysis showed, however, that the erythro isomer adopted a synclinal N/O conformation in the solid state while that of the threo isomer was similar to the antiplanar form (40) [91]. No solute conformational studies have been reported. If antiplanar N/O conformers be in fact the active species, differences in the muscarinic potencies of these diastereoisomers are more probably determined, not by the population variations of the erythro and threo rotamers but by differences in the orientations of the two methyl substituents in the antiplanar forms. Subsequent results upon a-methyl e;+

e;4

+3;

(41) P D ~ 4.0

O.COMe

(42) 4.3

O.COMe

(43) 4.6

OCOMe

(guinea pig ileum: ACh pD2 = 6.3)

(41) and &methyl (42) (proposed models for erythro species) and a$-dimethyl (43) (model for threo species) derivatives failed to clarify this issue because the threo analogue proved the most active muscarinic agent and was not a substrate for AChE, as were (41), (42) and threo a&-dimethyl ACh (all slowly hydrolysed)

20


[92]. A further puzzle is the fact that methyl substitution raises the potency of the methyl-free derivative (36) (a direct comparison was not made), but it is significant that all the active decalins have antiplanar N/O conformations. A tertiary butyl substituent provides a conformational restraint in 2-trimethylammoniumcyclohexyl acetates that is an alternative to the use of the trans decalin skeleton. This approach was adopted in the hope of obtaining data complementary to that of Smissman and, perhaps, achieving higher orders of potency [88]. Although the last aim was not realized, the NaOa isomer (44) was O.COMe

found t o possess significant, albeit weak, muscarinic activity (guinea pig ileum ACh= 1, activity lost in the presence of assay: molar potency 1.27 X hyoscine but not affected by hexamethonium) [86]. The NaOe,NeOa,and NeOe analogues were all inactive. The muscarinic properties of trans 2-trimethylammoniumcyclohexyl acetate (3 1a) are lost when a t-butyl group is included in the molecule and this result may be due t o a detrimental effect of the extra substituent upon the affinity of the molecule for the receptor. An additional factor, however, may be the fact that the energy barrier for the inversion of (31 b) is substantially greater than that of (31a); hence significant populations of antiplanar N/O conformations may only be available in the disubstituted derivative. Criticism of the inverted form of (31a) as the active conformation has been made on the grounds of the free energy difference (AGO) between diaxial and diequatorial conformers probably being greater than the energy released on ligand-receptor binding [85]. However, if the free energy of binding of substrates and inhibitors to AChE (- 4.2 Kcal/mol) [ 1641 be taken as a realistic figure for the muscarinic receptor, AGO for chair conformers of (3 1a) may well approach this value if the diaxial form is deformed in the same manner as the P O a t-butyl analogue (44) (see p. 3 1). Precedent for the uptake of a ligand in an unfavoured conformation is provided by the case of N-acetylglucosamine residues which appear t o be distorted from chair to half-chair conformations on binding to the active site of lysozyme [93]. A similar argument applies to the P O e decalin (33). Cannon's group chose the cyclopropane ring as the smallest system capable of conferring conformational rigidity on an ACh analogue [94,951 and succeeded in obtaining an isomer that had a high muscarinic potency. This was (+)-trans-

A.F. CASY

21

Table 1.4. [94, 951 RELATIVE POTENCIES$ OF 2-ACETOXYCYCLOPROPYLTRIMETHYLAMMONIUM IODIDES Compound

Dog blood pressure*

Guinea pig' ileum

Frog rectus muscle

ACh (+)-trans ACTM (-)-trans ACTM (*)-cis ACTM

1.o 4.70 0.023

1.o 1.13 0.0022 0.0001

1.0 0.013 0.0028 0.0042

* Depressor effects of [95 I. +

*

-

(+) and (-)-ACTM and ACh blocked by atropine sulphate (2 mg/Kg)

Action of (+) and (-)-ACTM and ACh blocked by atropine stdphate but not by hexamethonium [94]. Relative rates of hydrolysis by AChE: (+)-trans 96, (-)-trans 59, ACh 100 1951.

2-acetoxycyclopropyltrimethylammoniumiodide (ACTM) (45) which equalled or surpassed ACh itself in two test systems; (-)ACTM was several hundred times weaker than the (+)-form while the racemic cis isomer (46) was virtually

0

inactive (Table 1.4). AU isomers were feeble as nicotinic agonists on the frog rectus preparation. X-ray diffraction analysis of (+)-trans-ACTM established the N-C-C-0 torsion angle as 137" in the crystalline state 1281 and this angle (within the anticlinal range of 120" f 30") is probably close t o that of the solute conformation because of the rigidity of the molecule. The (+)-isomer had a 1S,2S configuration, hence the arrangement of substituents about C-2 in (45) is the same as that about related asymmetric centres of the potent muscarinic agents (+)O-methyl ACh [96] and (+)-muscarine [ 1121 .

+

R

MeC0.C H 2

b

CH2.0.COMe

(47)

+

(48)

+

R = NMe, or CH2NMe3

OCOMe

(49)

22


The cyclopropyl and cyclobutyl derivatives (47-49) have also been examined [97] . These are analogues of acetyl y-homocholine or 4-acetoxybutyltrimethylammonium [98] and provide no useful data relevant to the active conformation of ACh. All isomers had feeble muscarinic effects but cis and trans (47) ( R = h e 3 ) had appreciable nicotinic activities (cis 1/18, trans 1/8 that of ACh, frog rectus preparation, responses blocked by curare). Cyclobutanes directly related to ACh have not yet been reported (due presumably to synthetic problems), but cyclopentane analogues have been described in a published dissertation [79] ; feeble spasmogenic properties are claimed for both cis and trans 2-trimethylammoniumcyclopentyl acetate (29) with the latter (near anticlinal +N/O conformer) the more potent and of higher intrinsic activity (rat ileum pD2 : cis 2.6, trans 3.4, carbachol 6.7). Dimethyl substitution of ACh either in the a,& or p,0 positions markedly reduces, but does not abolish, the muscarinic activity of the neurotransmitter (see 50) [99]. Analogues of the &fl-dimethyl derivative have also been examined +a

Me3NCH2.eH2 O C O M e

(50)

Potency (ACh = 1 ) (I? guinea pig ileum a-Me (RS)0.0339 p-Me (RS) 0.622 a,a-diMe 0.0025 0, p-diMe 0.001

in which the 0-methyls form part of a 6-membered ring (5 1 R = H, 2-Me, 3-Me, and 4-Me) [loo]. Of t h s set, only the r-1-acetoxy-c-3-methyl derivative (52) possessed muscarinic activity on guinea pig ileum (molar potency 8 X

-

Me3NCH, + d ; e M e

d

.

N

M

+e

3

OCOMe

(51)

a) R = H b) R = M e (where R = Me, cis and trans isomers separated)

(52)

Me

(preferred conformation)

ACh = 1, effects abolished by hyoscine but not by hexamethonium) and the (+)form was twice as active as the racemic mixture [86]. The weak, although signjficant, activity of (52) is in contrast to the complete inactivity of the 3-desmethyl derivative (5 1a) and suggests that an equatorial OCOMe function is pharmacologically advantageous in these derivatives (OCOMe is axial in the preferred conformation of 51a but equatorial in that of 52). The role of the methyl substituent could then be that of a conformationally holding group so placed that it does not impede binding to a receptor as may, for exapple, methyl in equatorial OCOMe isomers of the corresponding 2- and 4-substituted

A.F. CASY

23

derivatives (both inactive). It is argued that the low potencies or inactivity of /@dimethyl ACh and the cyclohexyl derivatives (51) may be due, in part at least, t o difficulty in attaining an antiplanar +N/O conformation. The populations of such species should be greater in the ap-cyclohexyl analogue (53) on account of the much lower steric demands of CH20COMe as compared with +NMe3, and it is of interest that (53) showed significant activity (molar potency 1.1 X lop4, ACh= 1) in the ileum test while (51a) was inactive [86]. Interpreta-

U (53)

tion of these results in terms of demands of the muscarinic receptor must be made cautiously, however, because there is evidence that (52) has an indirect mode of action [86]. The chemistry of diastereoisomeric aminobornanes (54) related to ACh has

(54) + a) A=?COMe, B=NMe3 b) A = NMe3,

B = O.COMe

been extensively reported [ 102-1031 , these derivatives providing NCCO torsion angles in the approximate range 0" to 120". None of the 3-amino isomers (54b) caused guinea pig ileum to contract at a bath concentration of lOOpM, or affected contractions produced by ACh; they did antagonize nicotine, however, and were shown to possess weak ganglion blocking activity which had only a small dependence on stereochemistry [ 1041 . In another report the compounds antagonized ACh on both guinea pig ileum and frog rectus preparations with cis more effective than trans isomers (Smail, private communication). Cis derivatives (54a) also proved more effective than trans forms as inhibitors of AChE but orders of activity were low [105]. The bicyclo[2,2,2]octane system has also been employed as a rigid support for ACh functionalities [106,107]. O n rabbit ileum, the anticlinal form (55b)/had significant muscarinic activity (370 times less than ACh on a molar basis, )docked by atropine but not by hexamethonium,

24


(55) a) A = O C O M e , B = H B = 0,COMe b) A = H,

no evidence for indirect action) while the synplanar analogue (55a) had no activity up to l o p 3 M; eel AChE hydrolysed (55b) about 40 times as fast as (55a) [106]. ANALOGUES OF MUSCARINE Interest in the chemistry of muscarine and its stereoisomers continues [ 1081 and convenient syntheses have been reported including that of the natural dextro isomer [109,110]. The (*)des-ether isostere of muscarine (56, cyclic 0 replaced by CH2) is claimed t o be 5-10 times as potent as the parent racemic mixture in guinea pig ileum [ 1 11] . Muscarine may be regarded as a cyclic analogue of ACh in which the C-6 and C-5 carbons are linked by a bimethylene bridge (57 + 56). It is not a particularly /

C H 2. NMe 3

(56)

(+) 2S,3R, 5s isomer

I - .

\

Me

(57)

good conformationally restrained model because it has rotational freedom about the C2-C6 bond (rC5-C4 of ACh) and the fact that 72 for crystalline muscarine is in the synclinal range (t 73") is of interest but not necessarily significant. The influence of changes in the configurations of the tetrahydrofuran substituents upon cholinergic activities are of greater interest and are well known (all configurations other than that shown in (56) lead to isomers that are at least 200 times less potent than dextro muscarine) [ 1121 . These stereochemical demands on the part of the receptor are also brought to light in the 1,3-dioxolan analogues of muscarine. It is now 10 years since Triggle and Belleau. [113] showed that Fourneau's compound F2268 was a mixture of geometrical isomers

A.F. CASY

and that the cis-member (58) was 5 tested as racemic mixtures). Belleau isomer and showed that the more related t o (+)-muscarine [48(58)100

25

times as active as the trans isomer (both and Puranen [ 1141 later resolved the cis active enantiomer was configurationally times as active as 4 R form]. 2,2-Dialkyl

(58) [4S isomer]

analogues of the quaternary (58) are much weaker agonists than the parent and the R/S potency ratio falls sharply with increasing size of the substituent (8.4 for Me, 3.2 for Et, 0.4 for Pr') [114a], The work on dioxolans has been extended t o analogues restricted about the C-4 to C-6 bond. These restrictions were achieved by (a) linking the cis 2-methyl and 4-methylamino substituents gving (59) and the homo analogue (60) [ 1151 ; (b) linking the methylamino

nitrogen to C5 via an additional carbon atom giving (61) [116] and (c) linking the methylamino nitrogen to C-4 via an extra carbon to yield the spirans (62) [ 1 17J . The NCCO torsion angles are synclinal in (59) and (60) and anticlinal in (61) and (62). None of the derivatives was significantly active in nicotinic tests. Muscarinic assays on the first series showed (59) to be inactive and (60) a weak agonist (0.012 X ACh) but with indirect action - it is caused the release of ACh from rat jejunum. Of the 4 analogues of type (61) examined, the most potent (61 R' = Me, R = H, 1/240 X ACh) was the one in which the methyl substituent was exo, i.e., directed away from the nitrogen containing ring. This result was

26

STEREOCHEMICAL ASPECTS OF PARASYMPATHOMIMETICS +

surprising because the 2-Me and 4-CH2NMe3 groups are cis in the parent dioxolan. The low orders of activity recorded are not unexpected since inclusion of a cis methyl substituent at C-5 itself reduces the potency of the dioxolan (58) by a factor of 300 (as confumed by a Dutch group [ 1181 who found 5-Pr and 5-Bu analogues to be weak antagonists). The spiran (62) proved to be reasonably potent, being about a tenth as active as the RS-dioxolan (58) (rat jejunum, intrinsic activity = 1, unaffected by hexamethonium). In contrast, the open form of the spiran (62) in which the 4-methyl group opposes an anticlinal NCCO torsion angle, was a feeble agonist with a low intrinsic activity (0.56). These results indicate that the active conformation of the dioxolan (58) at the muscarinic receptor is one in which the +NMe3 group is well extended from 0 3 ; the separation of these two features is evidently too great in the homo

analogue (63) which is over 1000 times less active than its parent [119] , (acetyl yhomocholine is likewise far less potent than ACh) [21,97] .Cholinergic antagonists related t o (58) are discussed elsewhere. Cyclic analogues of muscarine based on 1,3-dioxan in which 6-CH2 (see 58) is part of the ring system have been described but sharply divergent pharmacological data are reported. Tsatsas [ 1201 claimed remarkable levels of potency in cardiovascular tests for (64a) and (64b) in the dog (blood pressure fall: 64a 1 X,

(64)

a) R = H b) R = M e

64b 100 X ACh: arrest heart: both 50 X ACh) but found the two derivatives only a tenth as active as ACh in the rabbit intestine test. Recently these compounds have been resynthesized by a different route (a serious melting point discrepancy is reported for (64a), and (64b) isolated in cis and trans forms [121]. The dioxan (64a) proved to be inactive, while cis and trans (64b) were much weaker than furtrethonium (cis about 100 fold and trans about 30 fold) in both intestinal and cardiovascular tests. Furtrethonium (furmethide, 2-trimethylammoniummethylfuran) itself is at least 10 times less potent than ACh. No

A.F. CASY

21

nicotinic component to the actions of the isomers (64b) was detected. The 'NCCO dihedral angle in the preferred conformation of the more active trans isomer (65) is near 180" while that of the cis form is probably about 60" if

5(0x A

6

9

(65)

+

CH2"Me3

(66)

+NMe3 be axial (t-butyl, comparable in size t o +NMe3, is axial in the preferred conformation of cis 5-t-butyl-2-methyl-1,3-dioxan) [ 1221 . The 1,4-dioxan (66) with an unrestrained C H 2 h e 3 side chain is also reported to have cholinergic properties and is claimed t o be as potent asACh in several muscarinic tests [123]. If this compound is viewed as a muscarine analogue, then C-5 of the ring must be equivalent t o the side chain methyl of the natural product in order that the 5-atom rule be upheld (see p. 34). Nelson, Allen and Vincenzi [124] devised some 7-oxabicyclo [2,2,1] heptanes (67) as muscarine analogues that lacked the hydroxyl function of the

natural agonist. All four derivatives had weak muscarinic properties (guinea pig ileum, effects blocked by atropine but not by hexamethonium) but the more active (endo 67b 1/190 X ACh) was related t o a homomuscarine rather than muscarine itself (cf. 63). 5,6-Epoxy and 7-deoxy analogues of (67) had virtually no ACh-like activity [125]. The open chain analogue of desmethylmuscarine, 2-(2'-dimethylaminoethoxy)ethanol methiodide, has a muscarinic potency of 0.003 compared with ACh = 1 at guinea pig ileum sites [ 125al . SYNTHETIC AND STEREOCHEMICAL METHODOLOGY It is vital that the molecular geometry of all conformationally restrained derivatives used in these studies be well defined, and the following examples have been

28


chosen to illustrate some of the synthetic and configurational procedures used to satisfy this requirement. A variety of methods, utilizing reactions of established stereochemistry, were used to prepare the four decalin analogues (33-36) [89]. Treatment of transA*-octalin (68) with a peracid gave the 2,3-oxide (69) which was opened in a trans manner to form 3-(axial)-dimethylamino-2-(axial)-trans-decalol(70), the precursor of the NaOa ACh analogue (Scheme 1.1). Trans addition of silver

(70)

OH

NaOa

O.COMe

1. AgOCN-12

2. MeOH

f) HN-COMe

(72)

Scheme 1.1 (Partial structures)

NaOe

cyanate-iodine t o the alkene (68) gave the urethane (71) which on heating formed the cyclic analogue (72) with inversion of the C-2 centre. Subsequent reaction of (72) led t o the NaOe isomer (Scheme 2.Z). A Curtius rearrangement of the hydrazide derived from 3-(equational)-carboxy-trans-2-(equatorial)-decalo1 (73) led to the aminodecalol precursor (74) of the NeOe isomer (Scheme 1.2). Finally the NeOa isomer was obtained by a route involving the

A.F. CASY

=@(74)

29

GfGMe OCOMe

Scheme 1.2

Neoe

bromination of trans-2-decalone which gave the 3(axial)-bromo derivative (75) exclusively and the displacement of bromine with inversion by dimethylamine (Scheme 1.3).

moqo 7 pyridinium perbromide' HBr

Hq-Ptq

M :-e2

Br (cis add')

(75)

I

OCOMe

&Me?-

NeOa

Scheme 1.3

The aminoalcohol intermediates needed for making trans (28) and NaOa (44) were obtained from the appropriate epoxide (76) ( R = H or t-Bu) by the procedure of Scheme 1.1 with some modifications [88,89] . trans-2-Aminocyclohexanol was transformed to the cis isomer by an inversion process (thionyl

u & 0

R R = p.N02C6H4 or Me

R (76)

(77)

chloride converts N-acyl derivatives of the aminoalcohol to an oxazoline (77) whch gives the cis aminocyclohexanol on hydrolysis) [126] from whence cis (30) is derived. l h e NeOe derivative (80) was obtained from trans-4-t-butylcyclohexanol-trans-3-carboxylate(78) in a manner comparable r i t h the synthesis of the decalin analogue (Scheme 1.4). Inversion of (79) led to the precursor

30


Do (Et0)ZCO

BU'

c q Et

NaH

1. NaBH4 2. equilibrate with base But

several steps

But

But

Scheme 1.4

(79)

(80)

of the NeOa isomer while the NaOe form was obtained from 4-t-butylcyclohexene by the AgOCN-12 procedure of Smissman (Scheme 1.1) except that positional isomers required to be separated in this case. Information about the conformation of decalyl and cyclohcxyl ACh analogues was obtained from the dimensions of the N-C-H and 0-C-H methine PMR signals [88,89].The four combinations of vicinal couplings possible for the methine protons of 1,2-disubstituted cyclohexanols are shown below together with their ranking in terms of outer line separations; this order is based on typical 3J values for 6-membered alicycles [127]. Model values were obtained from the spectra of 6-membered derivatives which had clearly defined preferred Y

Y

2aa + lae > laa + 2ae (X) > 2ea +lee (Y)

4

2ee + lea

(a = axial, e = equatorial)

NMe3

1-H 2-H

(81) W1/,

7Hz

W x 18Hz

(82) 1-H bw" 24 Hz 2-H bw" 2 4 . 5 H z

(in DMSO.dg)

conformations such as (81) and (82) (in the first case the N-substituent is markedly larger than OH and so takes the equatorial orientation). Dependent on the particular combination of couplings involved, either the terminal line separation (bw*) or the width at half the maximum height ( W l / 2 ) of the signal provided the best guide to the conformation [88]. Combinations containing an aa component lead to the larger methine signal dimensions. In the t-butylcyclo-

A.F. CASY

31

hexyl derivatives (80) etc., the PMR evidence showed that deviations from preferred chair conformations only arose in quaternary salts which required an axial trimethylammonium group. In the NaOa analogue (83j of special interesr, the 1- and 2-methine signal dimensions, although clearly of equatorial rather than axial type, were larger than those seen in the spectra of analogues with smaller axial substituents such as (84) and this evidence together with chemical

shift data was taken t o indicate that a ilattenea ring was preferred with the onium group bent away from the alicyclic ring. The most active decalin methiodide (36) with similar methine resonance dimensions as (83) does in fact have a solid state conformation in which the 'NMe, group is bent away from the ring torsion angle of 14'7" [91]. to give a N-C-C-0 The decahydroquinolines (25) were derived from reduction products of 3-hydroxyquinoline [ 8 2 ] . Of the 3 isomers (85) isolated one was asigned a cis H

ring juncture since its PMK spectrum displayed a one proton signal at 62.82 (m,

W 1 l 2 9 Hz) typical of the C-10 methine resonance of cis-decahydroquinoline. The C-10 signal is obscured in the spectrum of trans-decahydroquinoline and in those of the remaining isomers (85) which must therefore be trans derivatives. The stereochemistry at C-3 followed from the dimensions of the C-3 proton signals as usual (63.7'0, W I j 221 Hz for equatorial OH and 63.7'2, W , j Z 7 Hz for axial OH isomer). 'NMe3

\

--;p

6' I

,C*O trans

(86)

cis

Me (87)

32


The copper-catalysed reaction between ethyl diazoacetate and 2-vinyltetrahydropyran led to a mixture of the cyclopropane isomers (86) (the tetrahydropyran group protects the cyclic structure from ring opening) which were separated by fractional distillation [128]. The major isomer was converted to 2-acetoxycyclopropyltrimethylammoniumiodide (ACTM) by a sequence involving the Hofmann hypohalite reaction (C0,Et + CONHz -+ NH,) and the racemic base so produced resolved with (-)-tartaric acid. The pharmacologically potent (+>isomer was shown to be the trans form (87) and t o have the 1S,2S absolute configuration by X-ray diffraction analysis [28]. The minor isomer, cis (86) did not yield pure products in the Hofmann sequence and this route t o cis ACTM was abandoned. No alternative procedure has yet been described although pharmacological data on both cis and trans ACTM are reported [94,95]. The cyclobutyl and additional cyclopropyl derivatives later reported [97] were all derived from the appropriate trans 1,2-dicarboxyIic acids or cis 1,2-dicarboxylic anhydrides.

DISCUSSION OF CONFORMATIONAL REQUIREMENTS Having reviewed evidence about the conformation of cholinergic agonists, some of the proposals that have been developed from the results will now be discussed. In general, there are two schools of thought upon the question of conformational requirements for cholinergic activity; one bases its conclusions upon comparisons of preferred conformation, experimentally or theoretically deduced [22,63] while the other favours interpretations based on multiple modes of ligand binding to the receptor [6,129,130] and does not accept that there is necessarily any correlation between preferred and 'active' conformation (as implied by the first school). Thus Pauling and his colleagues conclude that for potent muscarinic activity the following molecular parameters, in terms of 1 (p. 4), are required: 71 = 180" t o C3, 72 = + 73 to + 137", 73 = 180" ? 35", 74 = 180" or -137"; interatomic distances 'N-01 = 360, +N-C6 = 450, and +N-C7 = 540 pm. The low potency or inactivity of certain analogues of ACh are attributed to deviations from one or more of the above limits, e.g., (-)-3-acetoxyquinuclidine methiodide (p. 55) and the NaOa decalin derivative 36 are both feeble because their 7 3 values, +76 and -90" respectively, are outside the permitted range, while the low activities of acetylthio- and acetoselenocholine are attributed to their antiplanar 72 values. These arguments are advanced inspite of the fact that 73 for ACh bromide is + 79" while 72 for carbachol bromide is + 178",i.e., deviatlons in this sense are also observed for potent agonists. The conformations of ACh rele-

A.F. CASY

33

vant t o nicotinic and muscarinic sites are considered to be similar and Chotia’s concept of two ‘sides’ of ACh, the methyl side activating muscarinic and the carbonyl side activating nicotinic receptors, has already been outlined (p. 6 ) . Shefter and Triggle [I301 have criticised Chotia’s proposals; one of their points is that if high muscarinic activity requires that ‘the methyl side of ACh is preserved while the carbonyl is blocked’ [37] then threo ol,@-dimethylACh (88) should be more potent than the erythro isomer (89) when in fact the reverse is true. In his rebuttal [131], Chotia stresses that the 72 values of threo (88) and other weakly active ACh analogues deviate from the ‘active’ range. N+

N+

threo erythro 72 + 1430 r 2 + 760 (preferred solid state conformation shown)

Like Chotia, Beers and Reich [ 1331 also consider that dual receptor action is due t o interactions based on two different combinations of functional groups of the transmitter molecule, but the two proposals differ in detail. Essential elements of the Beers-Reich theory are as follows: nicotinic-quaternary nitrogen group or its equivalent (for coulombic interaction) plus a group which acts as a hydrogen bond acceptor, the bond t o be formed about 590 pm from the centre of the positive charge; muscarinic - the same but the H bond - ’-N distance is 440 pm. The requirements were arrived at from examination of Dreiding and space-filling models of fully or partially rigid compounds which affect nicotinic and muscarinic sites (both agonists and antagonists). In ACh itself the 590 pm distance required that the CO oxygen be the hydrogen bond acceptor for nicotinic activity while the ether oxygen of OCOMe must be the acceptor for muscarinic activity. The authors do not elaborate the confcrmations of ACh necessary to attain these dimensions, but the molecule is depicted as fully antiplanar in muscarinic and antiplanar apart from 74 in nicotinic figures. Beers (private communication) states that many conformations of ACh provide the described + N --- H bond distances, and that the illustrated conformations were chosen for reasons of graphic clarity, not to define a particular conformation. Emphasis is also placed on the need for a suitably placed alkyl residue, e.g., the acetyl methyl of ACh, for reinforcing binding of the agonist at

34


the receptor, this being a restatement of the well known 5-atom rule [134] (the apparent anomaly of carbachol (Me3fiCH2 C H 2 . 0 C O N H 2 )in this respect may be accounted for by the fact that it causes release of ACh from synaptic vesicles, i.e., it has an indirect action) [ 111. The authors take no account of mode of action (direct or indirect) in their choice of models, a factor which may particularly affect the validity of the nicotinic requirements (models include nicotine, cytisine, 0-erythroidine and strychnine). Some comment on drawbacks to the use of conformationally restrained analogues in providing evidence about ligand binding to the cholinergic receptor has already been made Cp. 14). Alteration of the binding mode of the parent agonist by the molecular skeleton used to restrict its functionalities has also been cited as a potential limitation t o the approach [6]. However, an impressive number of these studies concur in identifying anticlinal-antiplanar +N/O conformers as the ‘active’ muscarinic species, and clearly exclude synclinal forms. The low energy barrier between synclinal and antiplanar ACh rotamers [65] shows that there is no thermodynamic objection to the latter being the receptor-bound conformational forms. It has been pointed out that although preferred +N/O synclinal conformations are favoured both for ACh and local anaesthetics such as procaine, the fact that choline thiol (antiplanar) has greater depolarizing activity in the electroplax or muscarinic preparations than choline (synclinal) suggests that receptor uptake of cholinergic ligands involves an unfavoured conformation [74] . The conformational demands of the muscarinic receptor probably relates to a critical N to 0 separation for ligand uptake at the receptor, attainable in anticlinal-antiplanar but not in synclinal conformers of ACh and its congeners. In support of this view, the cyclic analogues of homo ACh (NCCCO) (89a) which

f8Ba)

pD2 4.0 N-0 distance (pm) 250-490

3.4 410

provide N t o 0 separations within or close t o the range of anticlind-antiplanar ACh conformers (350-370 pm) have muscarinic potencies comparable with cyclic derivatives directly related to ACh while trans isomers (N-0 distance > 430 pm) are much weaker or inactive [79] . Apart from the tropane derivative (23,24) conformationally restrained conge-

A.F. CASY

35

ners of ACh generally lack nicotinic properties so no evidence associating a particular +N/O disposition with nicotinic activity has been forthcoming. Most proposals upon the molecular basis of cholinergic ligand-receptor interactions rest on the assumption of a single unique neurotransmitter recognition site at which all ligands bind in greater or lesser degree. Moran and Triggle [129] however, point out that there is no reason t o believe that such is the case and that evidence from enzyme-substrate-inhibitionstudies supports the concept that multiple but overlapping binding sites are available. Similar proposals about narcotic analgesic-receptor interactions have been made [ 135,1361. Relevant evidence for cholinergic ligands is summarized as follows: (1) The characteristics of recovery of receptors in rat jejunum after blockade by the alkylating species (90) differ according t o whether polar (e.g. ACh) or non polar (e.g. C6H, 1kvle3) ligands are employed 1129,1371.

(2) The contribution of the methyl group (terminal carbon of the 5-atom sequence) t o the activity of muscarinic ligands becomes less pronounced and ultimately insignificant with decreasing polar character of the ligand [138]. If the terminal methyl group occupied a common site then the increment in activity might be expected to be essentially constant. (3) There are several examples of the failure of formally related chiral agonists to display the same dependence of activity upon configuration. This occurs for the pairs S-(+)-@-methyl ACh and R-(-)-3-acetoxyquinuclidine (p. 55) [ 1391 and S-(+)-muscarine and R-(-)muscarone [ 1121 . Different binding modes have been proposed for members of the latter pair [6,114] ; +NMe3 and methyl occupy common sites reinforced by the hydroxyl of S-muscarine (91) and the carbonyl of R-muscarone (92). Pauling and Petcher [33] advocate

similar binding modes for muscarone and muscarine and attribute the low potency ratio of R and S muscarone (3R: 1S compared with 200s: 1R approximately for muscarine) to the facts of a similar orientation of + M e 3 to 2-methyl in both enantiomers and a 'N-C-C-0 torsion angle (-162") outside the usual

36


range for crystalline muscarinic agonists. Beckett [ 1401 has also proposed similar binding modes for the two molecules in which an imidazole ring of a histadyl residue within the receptor surface binds either to muscarine (C-0-H ...N> K ,

s* =

YKS 1 Kw or-=--+ YKw+(l-Y)K, S* Ks

and Y + 1, ( 1 - 9 K, in Eq. 2 is negligible, hence

where S (true stereospecificity) = KJK,.

(1-Y) - L + ( l - Y ) Y S Y

A.F. CASY 3(

300

L=-1+ k . . Y 'S

s

49

y

S 20

S"

10

Y

Figure 1.3. The effect of the degree of resolution ( y ) on the observed stereospecific index (S*). S* is plotted against y for true values of the stereospecific index (S) of 30,100,300 and infinity (open circles). Values have been calculated on the assumption that both enantiomers are resolved to the same extent (y) [169]

An appreciation of the striking dependence of stereospecificity upon optical purity may be gained by study of a plot showing the relation between the degree of resolution Y and the observed stereospecificity S* for particular values of the true stereospecific index S (Figure 1.3).If the isomers are only 95% resolved, the highest S* value that could theoretically be observed (with one isomer complete-

50


ly inactive and S equal t o infinity) would be 19. If a value of 100 is obtained for S*, the degree of resolution should be better than 99.01% while a value of 1000 corresponds with material at least 99.9% resolved. Thus, if one is dealing with an object-mirror image pair that differs substantially in a type of pharmacological activity that can be measured accurately, the optical purity may be assessed more precisely by biological methods than by any physical techniques presently available. Of course the number of examples that arise in practice is limited but a case in point is that of enantiomers of procyclidine (122a) and benzhexol(122b) which have atropine-like properties. Optically active forms of these compounds obtained by resolution of the bases with (+)-tartaric acid [ 1721 had stereospecific indexes (-)/(+) in the guinea pig ileum test of 49 for the pyrrolidino and 9.8 for the piperidino derivatives. Barlow, using the same samples [ 1681 , confirmed that the (122a) pair had the higher index but found differences to be more extreme (375 for 122a, 5.5 for 12%). From the relationship ofFigure 1.3avalue S* = 375 shows that the pyrrolidino pair must be almost optically pure. The problem was then to decide whether the low S* value for the pair (122b) was due to a low degree of resolution or whether the stereospecificity of anticholinergic molecules was critically influenced by the structure of the basic moiety X . Barlow, Franks and Pierson [ 1691 showed the former explanation t o be correct when they obtained a stereospecific index of over 1000 for enantiomeric samples of benzhexol prepared by resolving the base with N-benzoyl-D-threonine and continuing the resolution until the biological activity of the weaker isomer did not decrease any further [173]. This work illustrates how misleading comparisons of optical specificities amongst related pairs of pharmacologically active enantiomers may be unless due consideration is given to optical purity. In other series, however, where care was taken t o ensure that optical purity was of a high or at least a constant order, substantial differences in antimuscarinic potency ratios were detected upon variation of the size of the amino function, e.g. (122c) [173a]. Thus, even though the asymmetric centre is at the other end of molecules such as (122c), changes in the composition of the onium X +NMe2H +NMe3 +NH(CH2)4 +NEt2H +NMe2Et +NH(CHds

* This

R/S ratio 19 251*

50 32 159 126

value sets lower limit of optical purity for the series at 99.36% allowing for errors in measuring the RS ratio and assuming no racemization during esterification.

A.F. CASY

51

group produce changes in stereospecificity chiefly as a result, so the authors conclude, of the binding of one enantiomer being disturbed more than that of the other. With a few exceptions, an increase in the size of the onium group caused a decrease in stereospecificity. Potency ratios for enantiomeric forms of [3.2 .I] octane hyoscyamine and 8-methyl-3~~-methyltropoyl-3,8-diazabicyclo bases were also markedly higher than those for the corresponding methiodides

[173b]. It is implicit in Eq. 1 (p. 48) that the biological activity of a mixture of enantiomers is the sum of the activities of its components. The assumption has been tested by measurement of the affinity constants of mixtures of R and S (1 18) of varying composition [169]. Virtually complete resolution of the two components could safely be assumed because a high stereospecific index (R/S > 300) was recorded. Plots of the apparent affinity constant K* against the percentage of the isomer Y , in the mixture, and log (K*-K,) against log Y s , were straight lines, confirming that both components of the mixture were competing with each other as well as the agonist at the receptor. The Porton group has also carried out a pains-taking study of some chiral anticholinergic drugs, namely the hexahydrobenzilates (123- 125), and drawn

Me2NCH2.CH2.0COR

OCOR

(1

Me2NCHMeCH2.0COR

(123)

some general conclusions from the results [ 1701 . Hexahydrobenzilates were chosen because the R and S acids may be obtained in high states of optical Table 1.7. R:S ENANTIOMERIC POTENCY RATIOS [ 1701

In vivo tests

In vitro test Structure

(123) (123) (124) (124)

Form

HCI Me1 HCl Me1

Affinity constant (Guinea pig ileum)

100 200 212 100

Mydriasis in mice

123 38 20 2.3

Antagonism of oxotremorine in mice Salivation

Tremor

>loo

>22

141 43 17.6

-

38 -

52


purity (see Scheme 1.6), hence meaningful comparisons of isomeric potency ratios could be made. Both in vitro and in vivo tests for activity were applied and R/S potency ratios for (123) hydrochloride were similar in whole animal and isolated tissue methods (Table 1.7). From this it was concluded that both in vitro and in uiuo differences in the potencies of R and S (123) HCl result from factors associated only with the drug-receptor interaction and further, that the anti-acetylcholine receptors in guinea pig ileum, mouse eye, and mouse salivary gland are essentially identical, Potency ratio agreement was less satisfactory for methiodides of (1 23) while in the case of the 4-piperidyl esters (124) the in viuo ratios were much lower than the in vitro values (Table 1.7). If the fact of receptor identity be accepted (cf. ref. [ 1741 )some other cause must be sought for these variations. The divergence between in vivo and in vitro ratios becomes greater the higher the affinity constant of the R-configurated ester: R( 123)HCl (logK 9.06); R(123)MeI (9.66); R(124)HCl (10.92); R(124)MeI (1 1.08). Furthermore, although the in vitro potency of R(124)MeI is greater than that of R(123)Mel, the in uiuo activities of the two salts are equivalent. From such comparisons it is proposed that there is a minimum dosage below which maximum anticholinergic effects cannot be obtained in v i m , and that the potencies of R(123) and R(124) methiodides approach this limit. The reason for a minimum dosage lies most probably in losses inherent in the transport of the drug to its site of action (non-specific absorption and to a lesser extent metabolism). With exceptionally potent agents such as (124) the advantages of a high affinity for the receptor are offset, therefore, by the need to administer enough drug to allow for its uptake at sites of loss. The authors also discuss the time-activity profiles of their esters and find some evidence that drugs with high affinity constants take longer to produce mydriatic effects and longer to reach equilibrium with the ileum than compounds with lower affinities, and also that drugs with h g h affinity have a more prolonged effect. Factors which control the time course of drug action do not appear to depend on stereochemistry since methiodides of S(124) and R(123) are alike in both in uiuo potency and time-activity profie. These studies emphasize the importance of giving due regard to the question of minimum dose requirement and time to onset of response in any conclusions drawn from activity comparisons between R, S, and RS forms of highly potent drugs, especially when in uivo testing procedures are employed. Substantiation and extension of these findings are made in a further paper [ 174al . Below a dose of about 0.03 pmol/kg- no anti-ACh drug of 18 examined produced maximum mydriatic effects and all drugs which had log K values > 9.49 produced maximal effects at approximately this dose. The authors believe that their results show that little is to be gained from further attempts to synthesize more potent

Scheme 1.6 [ 1 7 6 ] C6H11

cI = o

CHO

pa2

4

C6Hll

HO

Ph several steps

c

HllC6 CHpO

CH20

OCOMe configuration byPMR c

complete hydrolysis

C6H11

H:$'

NalO,

OH CH2.OH

'

d

7:;

HO

CHO

s-

(+)

C6Hl 1

2) 1) NaOH KOH-MeOH-12

i

HOtPh COpH

tiOF6:11

Ph

s- (+I

b

a Addition of the Grignard reagent is highly selective and the trace of minor isomer is readily removed. The R - ( - ) acid is made by reversing the sequence of the two Grignard reactions. Key signals: CMe, 6 0.44, 1.07; COMe 6 2.16. In isomer CMe2 signal is lower field (6 1.31, 1.61) and COMe higher field

(6 1.75) since acetate methyl is now shielded by the aromatic group while the isopropylidene methyls are not. For principles see ref. [ 39bj. Used t o make enantiomeric forms of the dioxolan. v,

w

54


anti-ACh drugs, or drugs that retain only certain specified antimuscarinic properties. Although the antimuscarinic receptors clearly differentiate between the R and S hexahydrobenzilate moiety it is of interest that the less active S isomer of (124) methiodide (log K 9.08) has a potency similar to that of atropine (PA;? 8.4-8.9) [75,175]. Hence even the binding contribution of two of the three substituents attached to the benzylic carbon of the ester (124) must add significantly to the affinity of the molecule for the receptor. The enantiomeric forms of hexahydrobenzilate esters feature prominently in the work of the Porton group; the method developed by Inch, Ley and Rich [176] for the synthesis of the parent acids from arabinose is of special interest and is outlined in Scheme 6 together with the procedure used to establish the stereochemistry. Benzetimide (126), an anticholinergic agent about as potent as atropine [177], also contains an asymmetric benzylic carbon atom (starred in 126).

(126) (one enantiomeric form shown)

The dextro isomer (dexetimide) is over 1000 times as active as the laevo form as judged by pAlo values measured on guinea pig ileum [178]. X-Ray analysis of dexetimide shows that it has an S configuration [ 1791 . A possible correlation Ph

(127)

Ph

(128) _____-

-

[molecules viewed from a direction remote from the substituent of lowest sequence rule ranking]

between the arrangements of substituents about C* in dexetimide (127) and an anticholinergic R hexahydrobenzilate (1 28) is that both isomers may present the same sequence of aromatic, hydrogen bonding donor, and C....fi features to the receptor (cf. [179a]).

55

A.F. C A W

DERIVATIVES OF 3-QUINUCLIDINOL A separate section of this review is devoted to derivatives of quinuclidine [180] because cholinergic agonists and antagonists based on this bicyclic skeleton display features that are strikingly at variance with ACh, muscarine, and their congeners. Thus the potent muscarinic agent 3-acetoxyquinuciidine (Aceclidine) (129) is a tertiary base hydrochloride (or salicylate) rather than a methohalide, @OCOMe

l

-

H X (129)

a O C O M e

";J

Me

i

Potency (ACh = 1) (-) 1/600 (+) 1/36,000

(130)

the latter salt being the weaker by a factor of about 200 [181]. This finding runs counter t o the generally far greater potency of quaternary salts over corresponding tertiary bases (protonated) in the cholinergic field [182]. The parent aminoalcohol was first resolved over 20 years ago [183] and a method for obtaining the dextro isomer in an optically pure state has been reported [184]. Robinson, Belleau and Cox [185] tested the (t)- and (-)-methiodides (130) as muscarinic agents on guinea pig ileum and potency differences were disclosed. No one has yet reported comparison of the tertiary base enantiomers (129). The more potent (but still feeble) laevo isomer (130) was initially thought to have the S configuration (comparable with S &methyl ACh) by application of an asymmetric sulphoxide synthesis, but was later reassigned to the R series [139]. This configuration has been confirmed by X-ray diffraction examination of both the acetate Me1 [ 1861 and benzilate HBr of (-)-3-quinuclidinol [ 1871 . In crystalline (-)-(130), 72 is 108" (anticlinal) while the C2-C3-09-C10 torsion angle of 77" is well outside the range of 180" k 30" observed for ACh and many of its congeners. The potency difference between R and S (130) is evidence that ACh and its congeners need to adopt an NCCO torsion angle that is both anticlinal and iositive for uptake at the receptor (see (130a) and p. 3 for definition); this

R-(-)-isomer

(,30a)


56

conclusion also follows from potency data on S, 2s and 1R,2R ACTM (p. 21). When S-(+)P-methyl ACh adopts a conformation of this form (130b), the &methyl substituent eclipses an &-hydrogen. The corresponding conformer of the C

\VC

(130b)

( 130c)

R-(-)-isomer (130c), however, requires eclipsed Me and ‘NMe, groups and in consequence has a much higher energy content. The superior potency of .Y$methyl ACh (of opposite configuration to the more potent quinuclidine isomer) may therefore lie in its smaller energy requirement for conversion t o the ‘active’ conformational species. Aceclidine is a bridged form of I-methyi-3-acetoxypiperidine, reported as a weak muscarinic agonist by several workers [78-801 both in methiodide (21) and hydrochloride form. Lambrecht [79] attributes the low potency of the 3-acetoxypiperidine to the fact that it must attain an energetically unfavoured boat conformation if it is to interact with the receptor in a manner analogous to that of the quinuclidine derivative. The potencies of anticholinergic esters derived from 0-methylcholine are little affected by the configuration of the aminoalcohol moiety (p. 42). This is not true, however, for esters of 3-quinuclidinol which block ACh as is clear from data upon diphenylacetates (131) [183] and benzilates [187]. It would be of interest to have information upon diastereoisomers formed from 3-quinuclidinol and chiral acids such as hexahydrobenzilic acid. (3R-quinuclidinyl 3R-hexahydrobenzilate, log K 11.67 for HC1, 11.28 for MeI, is the most potent anti-ACh agent yet reported, but no data on other isomers are available [ 174al). O.CO.CHPh, (-) 2 X atropine (+) (optically impure) v. low potency

57

A.F. CASY

x$Me

The isomeric pair of isoquinuclidine methiodides (132) had no muscarinic

Me I

a) R = OCOMe, R' = H b) R = H, R' = OCOMe

(132)

properties on rabbit ileum when tested at concentrations up to lo-'

M [188].

RECEPTORS FOR ACh AGONISTS AND ANTAGONISTS Atropine and most compounds with atropine-like actions are described as competitive antagonists of ACh and related agonists because they produce parallel shifts of the agonist concentration-reponse curves; behaviour of this kind can be quantitatively described by equations for competitive antagonism [6] . The simplest view of this situation is that of agonist and antagonist ligands competing for the same site with both agents sharing at least one common point of attachment. An early objection to this concept was made by Clark [189] who noted that the rate of offset of atropine antagonism of frog heart was slow and was unaffected by an increase in the ACh concentration. Chemical evidence on this question is provided by certain anomalies in the structure-activity relationships of ACh agonists and their antagonists, summarized below: (1) An increase in the size of the cationic head of ACh sharply reduces cholinergic activity [21] ; similar structural changes in an ACh antagonist have little effect o n potency, e.g., for +NR3 analogues of (123), logK 9.37 (--fiMed, 9.48 (-fiEtJ), 9.08 (Etfi(CH2)5) [76]. Furthermore, with a few notable exceptions, a quaternary nitrogen is mandatory for potent agonist activity but antagonists may have a tertiary or quaternary nitrogen. (2)The major influence of a- or 0-methyl substitution upon the agonist properties of ACh is in sharp contrast to the minor effects of such changes on the properties of choline esters with antagonistic actions; in such esters the stereochemistry of the acyl moiety (benzylic carbon centre) is of paramount importance (see p. 42). (3) In 1,3-dioxoIans with muscarinic properties, the configuration of the carbon centre 0-to nitrogen has a greater influence upon activity than that of carbon flanked by the two oxygen atoms. The stereochemistry of the 0-centre has, however, either a reduced or a negligible influence in 1,3-dioxolans which

58


block ACh, while in the case of antagonists that carry a benzylic substituent at C-2, potency is again chiefly governed by the configuration of the benzylic carbon (see p. 43). On this evidence it seems unlikely that ACh agonists and antagonists occupy a common receptor, and even the sharing of an identical or related anionic site appears improbable on the basis of item 1 above concerning the nature of the nitrogen function. Indeed an onium group is not essential for antagonistic activity [the hexahydrobenzilate of carbocholine (1 33) is reasonably potent with the R isomer 100 X as active as the S form] but may serve as a directing moiety [159,190]. It is true that acetylcarbocholine itself is a weak agonist but its action has been established as indirect [12]. Me3C.CH2.CH20.CO.CPh(CgH11 ).OH

pA2 7.3 ( R ) ;5.3 (S)

1133)

If the receptors for agonists and antagonists are topographically different yet chemically linked in such a way that the binding of one ligand species induces a conformational change at the site of the other, and vice versa, then the fact of competition may be better understood since ligand affinity would be affected at both receptors [6,191,192]. Allosteric mechanisms of this sort have been criticised, however, on the grounds of it being unlikely that the affinity of all agonists would be altered t o the same extent, or put another way, that all agonists would modify the affinity of an antagonist t o the same degree [160]. Thus, using different agonists and the same antagonist, different affinity constants for that antagonist might be anticipated. In fact, logK values for atropine measured in competition with ACh, carbachol, R and S @-methyl ACh, and oxotremorine, were identical. Derivatives of 3-quinuclidinol must be given separate consideration since a good case may be made for agonists and antagonists of this type acting at the same receptor. Points favouring this view are: (1) The more potent forms of 3-quinuclidinyl acetate, diphenylacetate and benzilate all have a tertiary nitrogen atom; corresponding methosalts of both the agonist and the antagonists are less active although potency drops are smaller for the blocking agents [ 1831 . (2) The potencies of the antagonists 3-quinuclidinyl benzilate and diphenyacetate are governed critically by the configuration of the carbon centre 0- to nitrogen, while the more active enantiomers of these compounds and the agonist (130) belong t o the same steric series. Further study of quinuclidinyl derivatives is clearly required if a better understanding of their unique behaviour is to be gained.

A.F. CASY

59

Findly, mention of a possible relationship between receptors for compounds which block ACh and those which antagonize histamine is made. Anticholinergic agents often have antihistaminic properties, and vice versa, and the more effective actions of such compounds are revealed by comparison of pA2 values measured against the two agonists, e.g., atropine has the pA2 values 8.91 (ACh) and 5.91 (histamine) [78]. Derivatives of diphenylhydrarnine (134) well illus-

trate this duality of action. The parent compound is more potent as an antihistaminic and this action is enhanced by a 4-methyl and diminished with concomitant rises in anticholinergic effects by a 2-methyl or 2-t-butyl substituent (Table 1.8). The potency ratios of enantiomeric forms differ but a rather erratic dependence of activity upon configuration is found. It is proposed that the anticholinergic and antihistaminic actions of the derivatives (1 34) are related Table 1.8. PHARMACOLOGICAL PROPERTIES OF SOME DIPHENYLHYDRAMINE DERIVATIVES IN GUINEA PIG ILEUM [I931 ~

Structure R In (134)

isomerC _ _ _

~

Against histamine

7.62 8.78 8.76 6.87 6.44 6.82 6.66 6.36 6.00

PA2

Against furtrethonium

6.68 6.14 6.14 5.86 6.66 7.05 6.55 6.03 8.12

a Benadryl Orphenadrine From ORD evidence, (+) 2-Me (134) has the same configuration as (-) 2-t-Bu and (-) 4-Me (134).

60


respectively t o high and low electron densities on the ether oxygen atom [aromatic (r)-oxygen (lone pair) orbital overlap, which reduces the electron density at oxygen, is promoted by a 4-methyl but sterically opposed by a 2-methyl substituent] and a complementary receptor model that accommodates all diphenylhydramines has been proposed on this basis [ 1931 .

REFERENCES 1. 2. 3. 4. 5. 6. 7. 8.

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