Studies on Latent Derivatives of Aniinoethanethiols as Potentially Selective Cytoprotectants. V. Syntheses of S-(2-Aniinoethyl) Ifethyl Hydrogen Phosphorothioate'
I, 1; 11, 1: 111, 1:
= = =
11
CIl, C(. therapeut i r agents with corisequent incw:ise i l l t1icr:ipcwtic. effect in rmcer. T i i t , sodiuni s:ilt of the parent compound cysteapiiic 8-phosp1i:ttc (I) ivas prepared by the iiiethod of ,lker-
fcltlt, involving the rewtion of 2-bronioethyl:tmiii(~ hytlrobroriiidc with trisodiurii phosphorothioate, the 1:ittc.r tieing obtained by the rcnc%iori of sulfur wit 11 1Y"l followrtl hy treatincrit of tlic proiluct with sodimii Iiyilrouid~. h
I Y Y 1--+ ~ l'SC1, \ICli
XaOlf
-+PSiOS:r)1
I3rCilL!II+Ilf~~I W r
+I
\\-(a origiii:illy tried t o syntlicizc 111by an :malogou> route. Thiophosphoryl chloride c t h:u io 1 t o y i cld 0 - e t hy 1 p l i o q h ) Hydrolysi5 o f the latter with rotliurii hydroxide, lion-ever, tailed to yield the desired disodiurii salt (IVaj. T h c 0-methyl :tiinlog (11%)had been prepared? in 1911
PSCl,
(RO)PSCl, +-
(RO)PS(ONa),
IVa, R=C2Hj 11, R=CH11) Slipuorted by Research contract PII43-62-170, Cancer Chemotllerapy Kational Service C e n t e r , Xational Caiicer Institute, Kational Institute- o f Healtli. 13etliesda, lid.
( 2 ) 3. .\kerfcldt. A d a C h u m . Scand., 13, 1479 (1969). [:$I H. K. Yauiida and J . I,. Lambert, .I. .Am. C h e m . Soc.. 76, 5326 (1515.%,. i 1) \ \ . C . l.:mmrtt a n d 11. 0. Jones, .I. Chpnz. Soc., 99, 71.5 11911).
antiacetylcholinesterase activity of esters of 0,Odiethyl-S-(acetylpheny1urea)dithiophosphoric acid was investigated using rat brain homogenate as the source of the enzyme.
mide in 10 ml of dimethylformamide and the mixture was stirred for 4 hr. At the end of this time the solvents were removed under reduced pressure a t room temperature and the residue was extracted with methanol. Evaporation of the methanol gave a yellowish oil which was treated with ethanol and filtered, and the filtrate was evaporated to give a white powder. This was extracted with chloroform, and the CHCL extracts were dried (X&SO4), treated with charcoal, and evaporated to 1.0 g of a yellow oil. Anal. Calcd for C~HL~NOJPS: C, 21.05; H, 5.85; N, 8.18. Found: C, 21.35; H, 5.67; N,8.25.
Experimental Section'
Antiacetylcholinesterase Activity of 0,O-DiethylS-(acetylpheny1urea)dithiophosphoric Acid Esters' A . K.
SES
623
NOTES
July 1966
GL-PTA,R. c. SRIVASTAVA, A S D SCREXDRA s. P A R M A R
Department of Chemistry, Lucknow University, and Department of Pharmacology and Therapeutics, K . G. Meclical College, Lucknow, India Received February 23, 1986
A search for new insecticides led us to synthesize esters of 0,O-diethyldithiophosphoric acid by condensation of an alkali metal salt of the acid with ehloroacetyl-substituted phenylureas. The insecticidal, rodenticidal, and fungicidal properties of a-chloroacetylphenylureas have been reported by Hoegberg, et aLZa
Subst,ituted phenylureas were synthesized by treating a solution of potassium cyanate with aniline, toluidines, and anisidine. a-Chloroacetylphenylureas were prepared according to the method of Jacobs, et ~ l . by , ~refluxing 0.1 mole of the respective phenylurea and 0.11 mole of chloroacetyl chloride in dry benzene for 2-3 hr. On cooling, water was added and the solid mass which separated out was filtered. The a-chloroacetylphenylureas after washing with cold water were recrystallized before further use. 0,O-Diethyldithiophosphoric acid was prepared according to the method of Hoegberg, et al.6 Esters of 0,0-diethyldithiophosphoric acid &-ereprepared by condensation of a-chloroacetylphenylnrea (1 mole) n i t h 0,O-diethyldithiophosphoric acid ( 1 mole) in the presence of anhydrous Na2C03 (1 mole). The mixture was reflused in dry acetone for 15-18 hr, cooled, and filtered to remove NaHC03 and KaC1. After distilling the solvent the residue was recrystallized from ether. The characterization of esters of 0,O-diethyldithiophosphoric acid was done by their sharp melting points and also by analysis. Determination of Acetylcholinesterase Activity.-Adult rats weighing approximately 150 g were killed by decapitation. Brains were quickly removed, weighed, and homogenized in icecold 0.25 J!I sucrose. The final concentration of the homogenate, without further purification, used throughout these studies was 1Oyo w/v. Acetylcholinesterase activity was determined colorimetrically using acetylthiocholine as substrate' The inhibitory effect of esters of 0,O-diethyldithiophosphoric acid on rat brain acetylcholiiiesterase during the hydrolysis of
TABLE I
ANTI.WETYLCHOLINESTERASE ACTIVITY" OF ESTERS OF ~,~-DIETHYLDITHIOPHOSPHORIC ACID R
S
Yield,
R
Mp, o c
%
Formula
-Carbon. Calcd
%-Found
-Hydrogen, 0Calcd Found
-Nitrogen, %Calcd Found
Antiacetylcholinesterase activity Inhibition, 160, % 1 x 10-4 M
84-86 55 Ci3Hi&Vz04PS2 43.0 42.88 5.2 5.82 ... ... 60.5 =t 0.50 2.30 5.6 5.49 ... ... 22.1 f 0.32 8.10 2-CH3 94-95 70 C14H219204PS~ 44.6 45.4 3-CH3 94-96 60 C14H21N204PS2 44.6 45.1 5.6 5.46 7.4 7.62 6 0 . 5 i. 0 . 5 0 2.34 5.6 6.0 7.4 7.51 1 8 . 7 =k 0.97 10.0 4-CH3 112-115 60 C I ~ H ~ ~ N ~ O ~44.6 P S ~ 44.8 3-OCH3 68-70 60 ClaH21NzOjPS2 42.8 43.1 5.3 5.G .. . ... 50.7 f 0.54 3.0 a Enzyme activity was determined as change in extinction per 100 mg of wet tissue during 10 min of incubation. Each tissue sample was done in triplicate. Suitable controls for tissue and substrate blanks were taken. Per cent inhibition was calculated on the basis of decrease in the ehzlyme activity using esters a t a final concentration of 3 X M. X e a n values with standard error are recorded. Iw values indicate the concentration required to produce 5070 enzyme inhibition. Eserine was used as a standard acetylcholinesterase ' under identical conditions. The reactjion mixture in a volume of 2 ml contained inhibitor. The value for eserine was 5.49 X 10-7 h p H 7.4, NaCl (350 m M ) , acetylthiocholine (1.5 m X ) , and 0.3 ml of 10% brain homogenate. Tris biiffer (43.7 a),
Introduction of an alkyl chain in the benzene nucleus has been shown to increase lipoid solubility and enhance insecticidal activity.2b Furthermore, esters of 0,O-dialkyldithiophosphoric acid have been shown to possess insecticidal propert'ies. I n the present study (1) The authors wisli t o express their thanks t o Drs. hl. L. Dhar and N. Nand of Central Drug Research Institute, Lucknow, for providing facilities for microanalysis. Grateful acknowledgment is made t o the Council of Scientific and Industrial Research, Kew Delhi, for providing financial assistance t o R. C. Srivastava. (2) E. I. Hoegberg, U. S. Patent 2,562,863(1961); Chem. Abstr., 46, 1585 (1952); (b) A. B. Sen and A. K. Sen Gupta, J . I n d i a n Chen. Soc., 32, 616 (1855). (3) R. D. O'Brien, G. D. Thorn, and R. W. Fisher, J . Econ. Entomol., 51, 714 (1958).
acetylthiocholine is shown in Table I. 811 of the esters inhibited the enzyme activity. Substitution on the phenyl nucleus influenced their enzyme inhibitory properties; the degree of inhibition observed with unsubstituted derivatives was greatly reduced by substituting methyl groups in the ortho and para position. Substitution of the methyl group in the meta position did not alter their inhibitory properties. However, a slight decrease in the inhibition was observed with the methoxy derivative as compared to unsubstituted or m-methyl-substituted esters. ( 4 ) Melting points were taken in capillary tubes and are corrected.
( 5 ) W. A. Jacobs, 31. Heidelberger, and I . P. Rolf. J . A m . Chem. Soc., 41, 458 (1919). (6) E.I. Hoegberg and J. T. Cassaday, ibid., 73, 557 (1951). (7) 5. S. Parmar, &I. Suter, and hl. Kickerson, Can. J . Biochem. Physiol.. 39, 1335 (1961).
