Inhibitors Table 11, R = CHa to R = CCH13 -log (I/S)o.s = 0 . 9 3 2 ( * 0 . 2 1 ) ~ - 0.483(%0.41) 6 0.987
0.157
Inhibitors Table 11, I1 = C7Ht; to 11 = C ~ H I ~ 3 0.996 0.012 -log (I/S)a.s = 0 . 1 9 ~ 1.66
+
TABLE I OF ADENOSINE DEAMINASE INHIBITION
(2)
BY SOME
(3)
judged either by r or s is good to excellent so that we can make comparisons of the two equations with some confidence. Because of the different stereoelectronic character of the two parent molecules me cannot compare intercepts, but we can compare the dependence of inhibitory activity on hydrophobic character ( x ) . The difference of slope for the two equations is quite striking and highlights the point previously made2 that the positioning of hydrophobic groups on this part of the inhibitor molecule is quite critical. The slope of essentially 1 of eq 2 indicates that the part of the enzyme into which these alkyl groups are fitting resembles the octanol-water reference system which defines5 T . This dependence of biochemical activity on n is similar to the highest found (-1.2) in investigation of many sysThe apolar region in which binding characterized by eq 2 occurs must be one of considerable fluidity. The slope of approximately 0.5 for eq 1 is close to that found for nonspecific binding by a wide variety of small molecules to various macromolecules.6~7 The difference in slope of these two equations must result from the different character of the area in which the R groups find themselves. To obtain evidence that the change in the slope of the two equations was not caused by some unexpected intramolecular bonding in the inhibitors which could cause a change in the partition coefficient of the compounds, the 1-octanol-water partition coefficients were measured for three sets of compounds (Table I and 11). Calculation of the contribution of the CH, group to log P within either series of compounds gave results which agreed well with the expected n value of 0.50. Comparison of series I11 with series I1 revealed a difference in log P values near -0.66, the calculated value for CH2OH. Because no unusual effects are observed in the partition coefficients in this series of compounds, we suggest that the difference in the slopes of eq 1 and 2 must be a result of the difference in the interaction with the enzyme of compounds of set I1 compared iTith compounds of set 111. Since the dimension of the hydrophobic region is the same for compounds of sets I1 and 111,it is probable that there is a single, large hydrophobic area on the enzyme and that both sets of compounds form complexes with this site. Previously we have c ~ m p a r e d ,for ~ , ~a series of compounds generalized by structures I1 and 111, the changes in free energy resulting from the addition of a single methylene group to the alkyl chain, and it was found that in I11 when R is lengthened from ethyl to propyl, the A F / C H ~was - 1.14 kcal. The magnitude of this change in free energy is clearly beyond simple hydrophobic transfer forces and probably reflects a conformational change in the enzyme. We suggest that the change in the slope of eq 1 and 2 from 0.45 to 0.93 supports the concept that compounds of general structure 11, where R = propyl through hexyl, induce a con( 5 ) C. Hansch, a n d 9. M. Anderson, J . Org. Chem., 82, 2583 (1967). (6) C. Hansoh i n “Drug Design,” Val. I, E. J. AriBns, Ed., Academic Press, New York, N. Y. (in press). (7) F. Helmer, K. Kiehs, a n d C. Hansch. Biochemistry, I , 2868 (1968).
-log (I/S)o.P
9-L4LI in the enzyme. Because of the quite differelit behavior uf tlie 3 ;uid 4 derivatives of I we have treated the two sets of isomers itidcpctndenf Iy. Using the t1:it:i in T:il)lc TIT, t h r mPfn
Experimental SectionIri
isomers :we correlated in eq 4 and 5 . Equation 4 shows th:lt the mefa isomers :we only poorlj. correlnted with
thc single paranirter T . Equat ioti 3 . employing both T arid u, gives a good correlation for nine mefa derivatives. The 3-COCH3 derivative is not included (see T:iblr 111). Some special intermolecular activity of this fuiiction causes unusually high activity when compared to the other 3 isomers. The coefficient with ir iu eq 3 is closer to that in eq 1 than thnt in eq 2 , indicatjig that substituents in the meta position of the bcnzj I moiety do not induce the niorc favorable binding site characterized by eq 2 . It, is of interest to compare the urisubstituted benzyladenine with the derivatives in set 11. This can be done hy adding 2.13 (T for CsHs) to 0.5 (T for the KCH, derivative) and substituting the value of 2.63 into eq 1. This yields a calculated value of 0.01. The experiment:d value is 0.20. This indicates that the aromatic ring of the benzyl moiety finds itself in the same enzymic environment as the alkyl groups of set 11. The slightly higher-than-calculated value is probably due to thc greater polarizability of the benzene ring. This import u t i c e of electron density 011 the aromatic ring is evident
Method A. 9- and i'-(n~-Cyanobenzyl)-6-~hloropurine.---A mixtiire of 6.72 g (31.2 mmol) of ni-cyaiiobeitzyl bromide, 4.92 g (31.9 niniol) of 6-chhroput,ine, and 4 . i 2 g (34.2 mmol) of R&O.I i i i 50 ml of 1)NF \vas red for 23 hr at, room temperatiire. T i ! t h e cooled mixture \ v : i ~ added 500 nil of HrO arid the mixture \vas kept at 0" for 1 hr. The solvent was decanted, aiid t h r rehidlie dissolved i t i 250 ml of CHCla, dried with hlgSO4, anti filtered. Evaporatioii of the filtrate in L'UCZIOgave 6.68 g ( 7 8 . 1 5 j. nip 105-123°. Additiori of .io0 nil of F l y 0 to the DMF-IIIO r ~dditional1.18 g of crude material; tolid X CIIC1, mliition of the crude matei.ial o i l a coliinin of neut>ralaluniina (210 a ) : !~-~~n-cyaiiobenzyl)-~i-chloi~opiiririe was eluted with CIICl, (900 rnl); yield, 4.60 g ~ . X l . i c , ~ ~tnp ; ) ; 1.Y~-lX0. One re tion (PhMe) gave 4.22 g 14X.6?; 1 of piire inaterial, nip .tnai. (c,,H,cIx~,) r,ri, CI, s. chlrirupiirine W:LS eluted with ari d d i tional 1.2 1. of C yield. 1.04 g (13.1Sb); nip 167-170". Two rec~ysta~lizat,ioii~ of the mide material (PhAIe) gave XO nig ( 6 . 8 8 ' $ )of t>hean:dyti(d in:iteri:tl, r r r i ) 170--177°. . l n n 7 . i ( ' 1 . t JI,('lX,) C. 13, ('1. f 1 Method B. 9-(iu-Ctanobenzvl -A mixture of YO; . ladenine. _ m g ( 1.14 ni~nolI of !)-f,,z-cvanobenzyl)-6-chloropuriiie in co. 15 mi of liquid XI13 LV:I- heated in :t steel bomb at 45" for 21 hi.. The volatile materials were evaporated at room t,eniperatiire. Two recryatallizatioi~sFrom NeOH gave 201 mg (72.65y0) ~f the a n a l y t i d sample, mp 2:34-2?6". Anal. ( C U H I O NC~,)H, ?;. 9-On-Carboxybenzy1)adenine Hydrochloride.-A solution IJf 250 mg (1.00 mnic.1) of !)-(ni-cyanohenzyl)aderlilie in 5 ml of ( x i t i cwitraled IIC1 \\-a? heated under reflux fur 21 hr. The prec.ii)itate was cdlecbted l)v filtration arid dried a t 100" to give 202 Method C. S-(iti-Methoxycarbonylbenzyl)adenine.-A mixt lire of 268 mg (0.876 mniol) of O-(/n-carboxyberizSl)aderiiiir
hvttrochloride in 1.5 ml of J ~ P O I 4I ~ iirated 1 with HCl was heated ..
~
~
~
~
I S ) 'I'. l:iijit:h, .J. I ~ a s a ani1 . L'. 119641.
Ilansr~ii..I. . I m e ~ .Cliem. S o c . , 86, 317.i
1s) C .
klansch. L.:. \V. Deutacli, and R. S . Smith, ihc!., 87, 2738 (1985). (101 T h e melting points. uniew noted otller\i-ise, were taken in 011en capillary tulles on R Mel-Temp block a n d are uncorrected. .All analxtiral sarniiles had ir s p w i r a coml)atilile n i t h their assigned striictiires and moved : i ~B single spot 011 t l c o n Hrinkman silica gel. Where analyses are indicated unly I)?nymhola of tile elements. analytical resulth obtained for ttiosr c.lenient,s were witliin &0.4$< of tlie theoretical values. The analysea \ \ w e T.erformm1 hy (;:ill)raitli 1Iicroanalytical I,ahoratories, Knoxville, Tenn.
ADENOSINEDEAMINASE INHIBITORS under reflux for 22 hr. The white precipilate was collected by filtration; yield, 280 mg ( l O O ~ , ) , mp 243-244.5" dec. The crude product was dissolved in 25 ml of HzO and filtered to remove insoluble material. To the cold filtrate wa5 added 10 ml of 5% aqueous NaHC03 and the white precipitate was collected by filtration: yield, 169 mg (68.67,); mp 198-199". Two recrystallizations from MeOH gave the analytical sample, mp 200-201 '. Anal. (C14Hi3?j502) C, H, K. 9-(mEthoxycarbonylbenzyl)adenine.-This compound was prepared by method C except EtOH saturated with HC1 was used, and the product was isolated as the HC1 salt, mp 246247" (EtOH). Anal. (CljH1&1N502) C, H, C1, N. Method D. 4-Chloro-5-amino-6-(m-hydroxymethylbenzylamino)pyrimidine.-A solution of 7.45 g (54.4 mmol) of m(hydroxymethyl)benzylamine, 9.85 g (60.0 mmol) of 4,6-dichloro5-aminopyrimidine, and 6.06 g (60.0 mmol) of (Et)& in 150 ml of n-PrOH was heated under reflux for 23 hr. After the volatile materials were removed in DUCUO, the residue was triturated with Hz0 to give 13.7 g (95.27,) of crude product, mp 145-149". Recrystallization from EtOAc gave 8.69 g (60.3Y0) of pure material, mp 152-154'. Anal. (C1~H13C1N40) C, H, C1, N. Method E. 9-(m-Hydroxymethylbenzyl)-6-chloropurine.-A solution of 2.65 g (10.0 mmol) of 4-chloro-5-amino-6-(m-hydroxymethylbenzy1amino)pyrimidine in 27 ml of triethyl orthoformate coiitaining 22 mg (0.20 mmol) of EtS03H was stirred a t room temperature for 112 hr. After evaporation of the volatile materials in vacuo, the residual oil was stirred at room temperature with 30 ml of hIeOH-C6Hla (1: 12) for 2 hr and the solid collected by filtration; yield, 2.57 g (93.87,); mp 110-118°. Recrystallization of the crude material from hIeOH gave 1.82 g (66.27,) of material, mp 124-125'. Anal. (Cl3Hl1C1NaO) C, H, C1, N. 9-(m-Hydroxymethylbenzy1)adenine.-Prepared by method B ; from 9-(m-hydroxymethylbenzyl)-6-chloropurine: yield, 717, mp 219-220" (MeOH). Anal. (C13H13Nj60) C, H, S . Method F. 9-(m-Bromomethylbenzyl)adenine.-Dry HBr was bubbled into a cold suspension of 511 mg (2.00 mmol) of 9-(in-hydroxymethylbenzyl)adenine in 25 ml of anhydrous RleOH over a period of 30 min. Evaporation of the clear solution in vacuo gave a thick oil. Addition of 5 ml of HzO to the oil, followed by addition of 25 ml of 5% S a H C 0 3 gave 601 mg (94.47,) of crude material. Recrystallization from hfeOH gave 357 mg (55.8% of pure material which softens with decomposition a t ca. 250". Anal. ( C I O H I ~ B TC, K ~H, ) Br, K. m-(a-Ethylenedioxoethy1)toluene.-A mixture of 23.8 g (17.8 mmol) of m-methylacetophenone in 200 ml of C6H6, 20 ml of ethylene glycol, and 133 mg (0.638 mmol) of p-toluenesulfonic acid was heated under reflux for 19 hr, and the HzO formed collected in a Dean-Stark trap. The cooled CsH6 solution was washed with 5% aqueous NazCOa (2 x 50 ml), then with H20 (2 X 50 ml). The organic phase was dried (hIgS04), filtered, and the filtrate evaporated in vcccuo to give 30.5 g (96.57,) of crude product. Fractional distillation of the product gave 17.4 g
Joicrnal o j Medicinal Chemistry, 1970, Vol. 13, No. 3 455 (70.6%) of analytically pure material, bp 98-98.5" (7.5 mm). Anal. (CllH1402) C, H A 9-(m-a-Ethylenedioxoethylbenzyl)-6-chloropurine.-To a mixture of 21.8 g (123 mmol) of N-bromosuccimide and 528 mg (2.18 mmol) of benzoyl peroxide in 25 ml of CClr was added dropwise 17.8 g (100 mmol) of m-(a-ethylenedioxoethy1)toluene in 200 ml of cc14. Upon heating, a vigorous exothermic reaction took place. After the reaction had subsided, the reaction mixture wm heated under reflux for 0.5 hr. The mixture was filtered and the filtrate evaporated in vacuo to give 34.4 g of crude m(a-ethylenedioxoethy1)benzylbromide which was used with 6chloropurine in a modification of method A: yield, 637,; mp 138-140" (toluene-hexane). Anal. ( C ~ ~ H & ~ N ~C,OH, Z )C1, N. 9-(m-Acetylbenzyl)adenine Hydrochloride.-9-(m-a-Ethylenedioxoethylbenzyl)-6-chloropurine was used in a modification of method B. The crude adenine derivative was heated with 1 ,V HC1 for 1 hr, cooled, and the product was collected by filtration. One recrystallization from Hz0 gave the analytical product, mp 245-24iO. Anal. (ClrH14ClK,O) C, H, C1, N. 6-Chloro-9- and 7-(p-~yanobenzyl)purines.-These compounds were prepared from 6-chloropurine and p-cyanobenzyl bromide by a modification of method A: yield of the 9 isomer, 50%, mp 200-201" (MeOH). Anal. (C13H8ClNS) C, H, C1, N. Yield of the 7 isomer, 13%, mp 197-199' (MeOH). Anal. (Cl13HsClS5) C, H, C1, S . 9-(p-Cyanobenzyl)adenine was prepared from 6-chloro-g-(pcyanoberizylpurine by method B: yield, 677,; mp 285-257" (i-PrOH). Anal. (C13H1006) C, H, S. 4-Chloro-5-amino-6-(p-hydroxymethylbenzylamino)pyrimidine was prepared from 5-amino-4,6-dichloropyrimidineand p-hydroxymethylbenzylamine .HC111 by a modification of method D: yield, 79%; mp 211-212" dec (HzO). Anal. ( C I ~ H ~ ~ C ~C,N ~ O ) H, C1, 3. 6-Chloro-9-(p-hydroxymethylbenzyl)purinewas prepared by method E: yield, 5970; mp 142-143" ( H z ~ ) . -4nUl. (ClIHliClX;40) . C., H., C1,, N. 9-(p-Hydroxymethylbenzyl)adeninewas prepared by method B from 6-chloro-9-ip-hydroxymethylbenzy1)piirine: yield, 667,; mp 248-250' dec (HzO). -4nal. (C13H13X50) C, H, N. 9- (p-Bromomethylbenzy1)adeninehydrobromide was prepared by a modification of method F from 9-(p-hydroxymethylbenzyl)adenine: yield, 377,; mp 247-249" dec (MeOH). Anal. ( G 3 Hl3BrZN5) C, H, Br, S . Reagents and Assay Procedures.-Adenosine deaminase (Type I, calf intestinal mucusa) was purchased from the Sigma Chemical Co. The assay procedure for the study of reversible inhibitors has previously been describeds and is a modification of the procedure of KaplanI2 based on the work of Kalckar.13 (11) R. Kuhn, a n d J. C. Jochims. Justus Liebiys A n n . Chem., 628, 172 (1959). (12) S . 0 . Kaplan, Methods Enzymol., 8 , 473 (1955). (13) H. M. Kalckar, J . Bzol. Chem., 161, 461 (1947).
