Hemicholinium-3 and Acetyl-seco-hemicholinium-3
Journal of Medicinal Chemistry, 1977, Vol. 20, N o . 1 59
Effect of Sulfur Substitution for the Noncarbonyl Oxygen in Hemicholinium-3 and Acetyl-seco-hemicholinium-3. Synthesis, Biological Activity, and Structure-Toxicity Relationships. 2l Floyd R. Domer,* David M. Chihal, H. Cecil Charles, and Arvind B. Rege Department of Pharmacology, Tulane University School of Medicine, N e w Orleans, Louisiana 70112. Received January 12, 1976 As a continuation of our efforts to develop and study inhibitors which act presynaptically on neuromuscular function, sulfur analogues of hemicholinium-3 (HC-3, 1) and acetyl-seco-hemicholinium-3 (AcHC-3, 3) were prepared. In each case sulfur is substituted for the noncarbonyl oxygen in HC-3 (1) and AcHC-3 (3). As expected on the basis of conformational differences between acetylcholine and acetylthiocholine both of the thio analogues are produced in the seco form and do not cyclize spontaneously or when subjected to aqueous, acidic conditions up to 100 "C. Both compounds are stable in aqueous pH 7.4 solutions a t 37 "C and in slightly acidic D20 solutions for more than 24 h. While thio-seco-hemicholinium-3(11) is stable in the presence of acetylcholinesterase and butyrylcholinesterase in HzO a t pH 7.4, acetylthio-seco-hemicholinium-3(12) reacts within seconds to form the hemiacetal form of thiohemicholinium-3 (16). Mouse toxicity studies (LDm) indicate that while 12 is approximately as toxic as HC-3 (1) and AcHC-3 (3), 11 is 226 times less toxic. As in the studies with 1 and 3, mice were protected from 11 by choline and slightly by neostigmine. I t is of interest, however, that almost equal and intermediate protection against 12 was afforded by choline and neostigmine. Structure-toxicity relationships of 1, 3, 11, 12, and 16 are discussed.
In 1954 Long and Schueler reported the synthesis and initial pharmacological investigations of hemicholinium-3 (HC-3, l),a prototypical agent, which causes prejunctional inhibition in neuromuscular preparatiom2 HC-3 (1) is initially synthesized as the seco (open ring) form 2. However, on solution in water 2 rapidly undergoes intramolecular cyclization to the biologically active hemiacetal (closed ring) form l.2*3In order to evaluate the
+N--j 2
, + '
Br-
~ r -
,I
1
significance of cyclization on pharmacological activity, the acetate of the seco form of HC-3 (I), acetyl-seco-hemicholinium-3 (AcHC-3, 3) was ~ynthesized.~Both 1 and
3
3 produce a slow depression of neuromuscular function and also inhibit cholinesterase at high concentration^.^^^ However, unlike HC-3 (l), AcHC-3 (3)has been shown to be a potent inhibitor of choline acetylase both in vitro6>I and in vivo: a parasympathomimetic, and an inhibitor of reuptake of neuronally released catecholamine^.^ AcHC-3 (3) slowly undergoes hydrolysis with subsequent cyclization to form HC-3 (1) in water at pH 7.4. Thus it was thought possible that a portion of the pharmacological action of 3 could derive from HC-3 (1) produced in solution. Therefore, three stable seco analogues of AcHC-3 (3)-the ether 4a, the ketone 4b, and the alkane IC-were synthesizedl and were found to be 10-40 times less toxic than HC-3 (1) or AcHC-3 (3). Each was also found to have some HC-3-like activity, i.e., slowly developing neuromuscular blockade which could be reversed by choline. This indicated that cyclization is not necessary for this type
4a, R = o
n
0 1 I
b, R = A c, R=-
of activity as had previously been suggested by the work of DiAugustine and H a a r ~ t a d .Recently ~ an investigation of norphenyl-HC-3 (5) and a seco derivative of norphenyl-HC-3 (6) was reported which likewise indicates that cyclization is not a prerequisite to HC-3-like activity.1° The mechanism of action of 4a-c was also found to vary to some extent with the type of substituent. It was interesting that AcHC-3 (3) was found to bind irreversibly to acetylcholinesterase or butyrylcholinesterase without deesterification in vitro in buffered aqueous solution at pH 7.4. None of the other seco derivatives 4a-c studied showed this characteristic and were markedly less toxic (10-40 times) than AcHC-3 (3).'>5 I t has been shown in studies of acetylcholine (7) and several related esters that the -NCCO- grouping is in the gauche conformation both in the crystal" and in solution.12 Substitution of the acyloxy oxygen of acetylcholine with either S or Se, however, leads to the trans conformation for the -NCCB- grouping in the acetylcholine derivatives 8a and 8b (a, B = S; b, B = Se).13-15 Although the depolarizing abilities of 8a and 8b are greatly altered in various pharmacologicalpreparations,16-18the molecules' roles as substrates of acetylcholinesterase are not altered.lg Similar conformational differences exist for choline (9)20 and thiocholine ( These conformational differences should also carry over to HC-3 (1) and AcHC-3 (3)and their acyloxy sulfur-substituted analogues. Thus, as a logical continuation of our interest in both the configurational and conformational structure-activity relationships of cholinergic compounds, an extensive investigation of thio-seco-hemicholinium-3 (11) and acetylthio-secohemicholinium-3(12) annd their relationships to HC-3 (1) and AcHC-3 (3)was initiated. Herein we describe the synthesis, chemistry, and preliminary biological activity of 11 and 12 and compare them to the parent compounds 1 and 3. Structure-toxicity relationships of these compounds are also discussed. Further biological evaluation of 11 and 12 for cardiovascular activity, inhibition of
Domer, Chihal, Charles, Rege
60 Journal of Medicinal Chemistry. 1977, Vol. 20, No. 1
11
esterase without a subsequent deesterification. Under identical conditions acetylthio-seco-hemocholinium-3 (12) also undergoes a rapid interaction with acetylcholinesterase and butyrylcholinesterase. This interaction, however, does not appear to be irreversible. Instead, 12 appears to undergo immediate deesterification followed by cyclization to the hemiacetal form of thiohemicholinium-3 (16). This proposed mechanism is supported in two major ways.
12
neuromuscular function, and cholinesterase and choline acetylase inhibition is in progress and will be reported subsequently. Chemistry. Both thio-seco-hemicholinium-3 (11)and acetylthio-seco-hemicholinium-3 (12)were synthesized as the dibromide salts by the reaction of a,a'-dibromo4,4'-biacetophenone (13),prepared by the method of Long and Schueler,2 with the appropriate amines. The 2thioacetylethyldimethylamine (14)required for the synthesis of 12 was obtained from 2-dimethylaminoethanethiol (15) by acetylation with acetic anhydride using the method of T a m m e h Z 2The 15 required for this synthesis as well as the synthesis of 11 was obtained initially by the method of H a n ~ e nfrom ~ ~ the reaction of ethyl sulfide with dimethylamine. The amine 15 was subsequently obtained commercially from ICN Pharmaceuticals, Inc. The structures of 1 1 and 12 were confirmed by the method of synthesis, satisfactory combustion elemental analytical data, and lH NMR, ir, and uv spectral data. That 1 1 is, in fact, the stable, nonpolymerized thio-seco derivative of the oxy-hemiacetal 1 is evident from the presence in its ir spectrum of a carbonyl signal at 1674 cm-' as well as a sulfur hydrogen signal at 2663 cm-'. The presence of a thiol group in 11 is further confirmed by the 'H NMR spectral data which, when obtained in MezSO-ds, include a triplet at 6 2.08 attributable to the thio hydrogen. This signal is not observed in D20 due to hydrogendeuterium exchange. Further support for the presence and stability of a thiol group in 11 comes from the studies of stability in D20 or H20. Interaction with DTNB confirms this. Additional support of the seco designation of 11 is derived from the uv spectrum of 11 in which the compound exhibits a A, (H20) of 307 nm with an t max of 32 000, values which support retention of the carbonyl adjacent to the phenyl rings. It should be noted that loss of this group by formation of the hemiacetal structure shifts the A,, (H20) to shorter wavelength, e.g., as occurs in 1 which shifts the Amax (HzO) to 262 nm. The four compounds of interest here, HC-3 (l),AcHC-3 (3), thio-seco-hemicholinium-3 (1l), and acetylthioseco-hemicholinium-3 (12)are stable in DzO or H20 solutions under slightly acidic conditions or at pH 7.4 for extended periods of time. For example, slightly acidic D20 solutions of 1 and 11 have been shown by lH NMR methods to be free of decomposition products within the limits (
