>larch 1970
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lated giiiiiea pig ileum. The ileiim was suspended in KrebsHeiiseleit wlution, aerated x i t h 95% 02 and 5y0 COZ,and maintained at 37". AcCh and ruiknown drug were tested a t minimal, medirim, and submaximal concentrations as determined prior to the actiial away. The compounds were run on separate days using different guinea pigs for each experiment. The data were subjected t o variance ana ivhich showed that none of the compoiiiids altered the sen i t ? of the tissue to AcCh. Enzymology.-Enzyme-catalyzed hydrolysis of the compounds and iithihitiori of ACh hydrolyhis ivere determined a t pH 7 . 5 by titratioii of the liberated AcOH with S a O H solution (0.0065-V) wing a Saygent pH-Stat. Coilcentrations of sitbstrate varying from 1.0 to 5.0 X 111 were used in a medium consisting of 0.01 -11 lIgCl?, 0.1 M S a C1, and 0.01 mg 'ml of AChE for the K , determinatioris. Inhibitor concentrations were 3.0 X JI. The reaction rate3 were measured at 2.5' and were linear. ,1 1' z's. S provided K , and Ki values. Comparirates were carried out at 5.0 x 10-4 J I substrate concentration.
investigated previously with phosphamidase and i are new chemical entities. The only substrates subject to P-SHZ hydrolysis by the beef spleen preparation are certain triamides having two unsubstituted amido groups and two phosphonic diamides (XIII, XIV). As shown in Table I, the order of degree of hydrolysis is XVI > 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 Grant E-297 from the Robert A. Welrli Foundation. Houston, Texas, and Grant CA-08711 from the Sational 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. Fruton, 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, and J. J. Frutrm. J . B i d . Chem.. 185, 629 (lY50).
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