NOTES
Rlarch 1970
A Comparison of Some Stereochemical Requirements of the Acetylcholinesterase and Muscarinic Receptor Areas GEORGLH COCOL-\S, ELLEX C. ROBIVSOX,~ School of Pharmacy, L-nzverszty of Sorth Carolina, Chapel H d l , S o r t h Carolzna AND
WILLI~M L. DEWEY
Department of Pharmacology, School of Jfedzczne, rnzverszty of ,Vorth Cui olzna, Chapel Hz11, Xorth Carolina Received J u l y 9, 1969
The muscarinic receptor and acetylcholinesterase (AChE) demonstrate itereoselectivity in their interact ions mit h acet yl-a-met hylcholine, acet yl-p-met hylcholine,2 muscarine^,^ d i o ~ o l a n e s ,3-trimethylammo~ nium-2-acetoxy-tTans-decalins,s and 2-acetoxycyclopropy1trimethylammonium6 compounds. While acetylcholine (ACh) is regarded as a common cubstrate for tissue and enzyme, the lack of parallel activity to these receptorb by many optical isomers o ~ c u r s . These ~~~ events indicate that each receptor area may be presented with a different aqpect of ,lCh and that they are different. We have sought t o compare the effect of the eriaritiomeric form5 of acetyl-a-methylcholiiie and acetyl-p-methylcholine together with acetyl-ala-diniet hylcholine and acetyl-p,p-dimethylcholine to demonstrate how the respective receptor:, may interact with the choline fragment of -1Ch. From the data in Table I E(+)-acetyl-a-methylT;LIJLP: I 1lusc.iiuxrc AcmvITirx OF ACKTYLCHOLINE I O D I DOR' I : ~GUINF:.~ PIGILEUM Iodides
Xce:ylcholiiie
+
R I )-hcetyl-ol-methylcholiiie RS-Acet yl-a-methylcholiiie SI- )-Acetyl-a-methylcholine Acetyl-a,a-dimethylcholiiie
S!
Potency of ACh"
95y0 confidence limits
1.00 0.0240 0.0339 0.0045 0.002.i
0.0150-0,0370 0.0263-0.0437 0.0040-0.0031 0.0021-0.0031
+
)-Acetyl-p-methylcholiiie 0.802 0.661-0.973 RS-Acetyl-p-met.hylcholiiie 0.622 0.466-0.848 E:! - )-Acetyl-6-methylcholiiie 0,090 0.063-0.129 Aceiyl-~,&dimethylcholiiie 0.001 0.0007-0.0015 The A mean value for these assays is 0.123.
choline and S ( +)-acetyl-p-methylcholine show five and nine times, respectively, more muscarinic potency than their corresponding enantiomers. Clearly, a methyl group in the choline moiety does not appear to contribute in any way to activity since dimethylation of either the a or p carbons further reduces the potency of the chemical. Therefore, for optimal muscarinic ( 1 ) r a t i o n a l Science Foundation Undergraduate Research Participant, 1969 Lunsford-Richardson Pharmacy Awards Regional Kinner. (2) -1.H. Beckett, X. J. Harper, and J. W.Clitherow. J . Pitarm. Pharmacol.. 15, 362 (1963). (3) L. Gsermek a n d I XI11 > XT'III > XIX > XIV, XX > XXIII, XXT.'. With the triamides XVI-XXIII, where the alkyl substituent5 were N,N--lle2, N,N-Et2, N-Bu. N-hex, and N-cyclohexyl, hydrolytic activity decreased with increasing number of carbons. Loss of activity resulted when the alkyl chain was lengthened to eight carbons (XXI),when a double bond was introduced (XVII), and when the p carbon atoms of XT'III mere linked to give a pyrrolidine derivative (XXIV). Hydrolysis of the P-SH, bond is, however, restored in the piperidyl homolog, Acknowledgment.-The aut'hors wish to express apXXT.'. The high activity of XI11 is especially interestpreciation to the North Carolina Pharmaceutical ing since it contains a CICHzP(0) moiety, an analog of Research Foundation for their support' of t'he Underthe halomethylcarbonyl group which has been used a3 graduate Research Project' during the academic year a covalent bonder in the design of inhibitors.' arid to the Kational Science Foundation for their supSince the ultimate goal of this continuing study is the port during the summer period. We are also indebted design of phosphamidase inhibitors, several substrates to Dr. J a n Hermans and N r . Dave Pruett, of the Dewere screened for this property. Representative inpartment of Biochemistry for the polarimetric deactive compounds IV, V, X, XII, and XXIT' did not termiriations. affect phosphoramidate cleavage a t 12 m J I whereas XI and XV gave evidence of inhibition a t thii concentration, reducing S H 3liberation by 50%. Phosphorus-Nitrogen Compounds. XI. Although the P-KH, bond in XI was shown not to Phosphamidase Studies. I. be cleaved enzymatically in this study it is used as a histochemical substrate for the detection of phosphamiUnsubstituted Amides',* dase.8 This compound is probably cleaved preferenLIXDLET A4.C\,n.s tially a t the aryl S-P bond to phosphoramidic acid which, in turn, is converted into H,PO, and S H 3 . 17niversity o j Houston, College o j Pharmacy, Cnder conditions herein reported this acid, being formed Houston, Texas 77004 in low concentration, is not appreciably acted upon. Received September 3, 1969 Each of the triamides, as well as IX and XII, also contains substituted amido groups subject to phosThat enzymes capable of catalyzing reactions inphamidase activity, arid the short series of five Svolviiig P-S bonds occur in higher concentration in substituted phosphoramidic acids previously studied cancer than in normal cells has been well e ~ t a b l i s h e d . ~ with beef spleen preparation6 does riot permit the These may be the same as, or closely related to, relating of the effect of this substituent on enzymatic phosphamidase which has been isolat'ed from Escherichia activity. A future study in this series is expected to coZij4 hog eye,5 and beef spleen.6 Seither a natural yield more definite information concerning the effect substrat e nor a subst.rate-like inhibitor has been deof S-substitution of phosphoramidic acid on enzyme termined for this enzyme. In the design of the latter interaction. It may then be possible to devise a ft comn~oiipreliminary step is the selection of comsubstrate, i.e., R(R or H)P(O)S(CH,), where R is an pouiids with high affinity for the enzyme receptor site optimal iubstituent (s), possessing high enzyme affinity. for alteration, if iiecessarj-, to produce increased binding capacity. Experimental Section There have been only 12 different P-X compounds, Chemistry.-Previously unreported X I I I , XVII, and XXexcluding w few qualitative histo~hemicals,~ previously XXIV were prepared from the appropriate phosphorodichloriused to study phosphamidase preparations, including 2 dates according to the procedure of Goehring and Pl-iedenzu.9 phosphorodiamides and 5 substrates wit'h unsubstit,uted Their elemental analyses (C, H, ?;) were obtained with Coleman amido groups. This initial paper reports the examinaanalyzers with the results being within 4 ~ 0 . 4 7of~the theoretical values and spectra using a Beckman IR-8 were as expected. tion of 2 5 P-S substrates of type =P(O)SH,, P(0)(OH Melting points were determined on a Fisher-Johns apparatus and or OEt)SH2, and P ( O ) ( S H & with GO to 90 SAS beef are uncorrected. S H 3 (2.0-ml sample) was determined using spleen frwction. Twenty-one of these have not been Coriway microdiffusion dishesI0 with Obrink modification. (1) This investigation was supported hy G r a n t E-297 from t h e Robert A. Welrli Foundation. Houston, Texas, a n d G r a n t CA-08711 from t h e S a t i o n a l Cancer Institute. U. 9. Public Health Service, Rethesda, &Id. 12) For the previous paper in this series see L. A . Cates, J . .Wed. Chem., 11, 1075 (1968). (3) 31. S. Burstone. "Enzyme Histochemistry," Academic Press, S e n . York. S . Y..1962. p 231. (4) R . .I Smith . and D. J. Burro\\-. Biochim. Biophys. A c t a , 34, 274 (1953). ( 5 ) T. I\-onp. Comp. Biochem. Phyriol., 17, 139 (1966). (6) 11. F. Singer and J . S. F r u t o n , 3. B d . Chem., 229, 111 (1957).
Enzyme Studies.-Siibstrates I-XXV (0.012 M), 0.1 JI acetate buffer ( p H 6.0), enzyme preparation 60 to 90 SAS 1.0 (EU)P*/ml, and 0.02 -Ti' p-mercaptoethanol were incubated 10 mill a t 37" according to a previously described p r o c e d ~ i r e . ~Although this ( 7 ) B. R. Baker, J . Pharm. Sci., 63, 347 (1964). (8) G. Gomori Methods Enzymol., 4, 387 (1957). (9) See footnote o , Tahle I. (10) R . R. Johnston, 31, J. Mycek, a n d J. J. Frutrm. J . B i d . Chem.. 185, 629 (lY50).
