"2(N
N&uu - 0 0mco NH-
9 SO,F
35
of nil-rninialian dihydrofolic reductases, but s h o m d selective irreversible inhibition; a 1 &lI concentr,d t io11 ' of 35 could rapidly inactivate the Walker 2-56 rat turnor ciizyme :ind tlic 1.1210/FKS mouse leukemia ctizynie biit did not illactivate the rat or mouse liver enLynie.: at thiq conceutr:itioii.lj
Irreversible Enzyme Inhibitors. CXl*III.l' Hydrophobic Bonding to Dihydrofolic Reductase. XII.' Further Comparisons with the Enzynie from Walker 256 Rat Tumor and Rat Liver
I~ti\irtceiioi?ho-diit)stitiitet1 1-plien~-l-4,8-dinniiiio-l~~-dih~~~o-s-t riiieiiiej h:tve kieeii nmisiired as iiihibiturs of the diliylrofolic reductase from Walker 256 rat tumor aiid rat liver: i i o appreciable tiifferelice iii t~iiidiiigt o the enzyme from the two soiirces was foriiid. T h e 600-fold lo>, i i i biiidiiig nheii 4,6-diamiiio-2,2-dimethq-l-l,2tiih?.dro-l-pheii)l-s-triazirie ( 4 ) is srihtitriteti with ail o-chloro groiip ( 5 ) raii he recoiiped ti>- frirther substittitioil of a 3-chloro groiip (16) or 4-pheiiylbLityl group (20).
In the previous paper of this series a comparison of the binding to the hydrophobic bonding region of the dihydrofolic reductase from Walker 256 rat tumor a d rat liver were made with 31 substituted 4,6-diarnino-1.2dihydro-s-triazines. The largest difference in binding between the two eiizynies was observed3 with 1 and 2, the tuinor enzyme being inhibited better by 100- and 40-fold, respectively. Even though o d i o substituent.: 011the 1-phenyl group of dihydro-s-triazines give a great loss in biological a ~ t i v i t y this , ~ ~ area ~ has no\v been further studied particularly since it was possible that
x2
IVY N-R
NH~~,,-~cH~ CH,
the lowered activity could be recouped by additional substituents on the benzene ring5 arid it was possible that t'issue specificity might be uncovered. The result,s of this study on binding of 3 and related coinpounds to Walker 2 3 and liver dihydrofolic reduct'ase of the rat is the subject of this paper. The inhibition of dihydrofolic reductase froin two sources with 14 ortlio-substituted 1-phenyl-s-triazines aiid ihree related compounds are collated in Table I. S o specificity in binding to the two enzymes n-as observed; nevertheless, some intoresting correlat'ions on (1) This work ,vas penerorialy sripporteti 1)s G r a n t C.1-0869d from the S a t i o n a l Cancer I n s t i t u t e , U. S . Public Iiealtli Service. (2) F o r t h e previous paper of this series, PPC B. R . Baker, .l. Iferl. Ctiern., 11, 48:+ (19681. . I:. I'olpy, l ' r o c . Ani. .lisnc. C O r g . C/ie?n., 2 1 , 1 (1956). m. S c i . , 53, 1137 (1864). (r)) P,. R. Baker, B.-'l. 1-10, a n d (;. J. Loiirens, zbid.. 56, 737 (19671, l ~ a l i f ~ i 1.XS.Y V I of tliis serira.
the ortho effects caii be made. For discussion purposes only the Walker 236 data will be used. The o-chloro sub*tituciit of 5 causes 3 GOO-fold loss in hindiiig t o t he c~izynic; Ihis loss corresponds to 1 he t otiil increment iii 1-phenyl binding compared to 1iiietli) I,? indicating that the 1)henyl ring of 5 is now out-
h l t y 196s
IRHEVEI~S IBLE ENZYME IKHIBITOHS. CXVIII
of-plane with the s-triazine ring, as previously p r o p ~ s e d . ~ This lack of coplanarity with 5 is further supported by the observation with the 2-phenyl-s-triazines 11 and 12. When phenyl groups are present at both the 1 and 2 positions of the s-triazine ( l l ) ,a 37-fold loss in binding occurs compared to 4; this loss in binding can also be attributed to some restricted rotation where one of two phenyl groups must be out-of-plane. If both the o-chloro substituent of 5 arid the "phenyl substituent of 11 forces the 1-phenyl out-of-plane, then no further loss iri binding should occur if both structural changes are made in the same molecule as in 12; note that 12 and 5 differ only tn-ofold in binding. Holvever, 11 is a 15-fold better inhibitor than 5 , indicating that other factors are also involved. One of the uninterpretable factors that may be involved is electronic, but such electronic factors as the Hammett cr value are unreliable with ortho substituents. For example, the o-fluoro substitution on 8 causes a 150fold loss in binding compared to 4; since the fluorine atom is not much larger than hydrogen, this result cannot be due to restricted rotation forcing the 1-phenyl group out-of-plane as in the case of 5 , but must be electronic in nature. That the fluorine has some electronic effect is also indicated by comparison of 11-13; 12 and 13 are complexed equally. both being about 30-fold less effective than the corresponding compound without an o-halogen (11). That restricted rotation is not the complete story is also indicated by comparison of 5-10. If the size of the ortho atom and its resultant effect on copolanarity of the 1-phenyl group were the only effect, then the order of effective binding should parallel decreasing size, that is, F > C1 > Br, CH3, CH,O > I. The observed order is F > I > Br > C1 > CH3 > CH30. At least two more effects should be considered, namely, electronic effects and hydrophobic interaction of the group with the enzyme.6 If it is assumed that C1, Br, and I have similar electronic effects, then the order of binding can be accounted for by the order of hydrophobicity of I > Br > Cl.7 I t has been previously observed that the in-chloro substituent of 15, compared to 4, gives a 7-l5-fold increment to binding to the e n z y m e . ? ~ Recently, ~ evidence has been accumulated to indicate that the interaction of this 772-chloro with the enzyme is direct, perhaps through a dipole donor to the enzyme, and not due to a hydrophobic interaction since m-methyl gives a poorer i n t e r a c t i ~ n . ~ A , ~ pleasant surprise was the observation that the m-chloro substituent of the 2,3dichloro derivative (16) gives a 240-fold increment in binding compared to the o-chloro derivative ( 5 ) ; that the In-chloro group of 16 most probably directly interacted with the enzyme is indicated by the fact that the 4- and 5-chloro of 17-18 did not give a gain but a two- to fivefold loss in binding. If the in-chloro group has a point interaction with the enzyme, then one must reconcile the probability that the phenyl group of 5 and 16 should be equally out-of-plane in the ground state. One possibility is that the phenyl group of 16 is in-plane when com(6) B. R. Baher, B.-T. Ho, a n d D. V. Santi, .I. Pliarm. S ' C ~ .54, , 1415 (1965). ( 7 ) T. rulita, J. I v a s a , a n d C. IIanscll, J . A m . Chrm. Soc.. 8 6 , 5 l i 5 (1964). (8) B R. Baker a n d G. J. Lourens, J . I'harm. Sen., 56, 871 (1967), paper L X X S V I I of this series. (9) H R. Baker a n d B.-T. Ho, J . Heterocycl. C h e n . , 2, 335 (1965).
4Si
plexed to the enzyme and that energy required to be inplane is derived from binding energy. This explanation would then require that the m-chloro of 16 be complexed to the enzyme considerably stronger than the in-chloro of 15; this might be feasible if the 2-chloro group could augment the supposed dipole interaction of the 3-chloro group with the enzyme. Regardless of mechanism, the important point remains that almost all of the binding lost when an o-chloro group ( 5 ) is introduced on 4 is regained with the second chloro of 16. I t was previously notedj that introduction of a p phenylbutyl substituent on the 1-phenyl group of 4 gave about a 30-fold increment in binding to the enzyme from pigeon liver. Introduction of the p-phenylbutyl substituent (20) on the o-chlorophenyl-s-triazine 5 gave an even larger increment in binding, namely 240fold. This greater effect 011introduction of the phenylbutyl group on 5 than 011 4 is difficult to reconcile, but may be due to the stronger hydrophobic interaction of a phenylbutyl group to the rat tumor enzyme2 than the pigeon liver enzyme;: nevertheless, the fact remains that nearly all of the binding lost in substituting an ochloro group ( 5 ) on 4 is regained by further substitution of a p-phenylbutyl group to give 20. Since some species seem to be able to bind an out-of-plane phenyl group to their dihydrofolic reductases,'O the observations xvith 16 and 20 may be useful to increase potency. Although none of the compounds in Table I showed appreciably greater inhibition of the rat tumor enzyme than the rat liver enzyme, it is still theoretically possible, though unlikely, that compounds with the 50,000-fold difference in binding needed for chemotherapy'l can be found However, it is clear that active-site-directed irreversible inhibitors with tissue specificity'? for dih)-drofolic r e d ~ c t a s e ' may ~ ~ ' ~be easier to find than specific reversible inhibitors. Chemistry.-Alll of the dihydro-s-triazines (22) in Table I were prepared by the t hree-component met hod of with R 2 = R3 = CH3, l\IenCO was the reactant and solvent, but with R1 = H and R3 = C6Hh, benzaldehyde was the reactant and EtOH was the solvent. -411 of the arylamines 21 except 26 were commercially available. This amine (26) was prepared by TTittig condensation of ciiirianiyltripheriylphosphoriium chloride'j with the commercially available aldehyde, 24, followed by hydrogenation with a PtO, catalyst (Scheme I). Experimental Section All aiialyt,ical samples had ir and iv spectra and combustion analyses ( 1 0 . 3 ) in agreement with their assigned structures; each moved as a single spot on tlc o n Bririkmann polyamide 11K except. for 25 and 26 where silica gel G F was employed. 1Ielting poiiit.s were determined in capillary tubes o n a MelTemp block and those below 230" are corrected. (101 B. R o t h , R.B . Burro\!% a n d G . H. Hitchings, J . .If& Chem., 6 , 370 (1963). (11) (a1 G. H. Hitchings a n d J. J. Burchall, Adran. Enzymol.. 27, 417 (1965); (11) J. J. Burchall a n d G. H. Hitchings, M o l . P h a r n a c o l . , 1, 126 (1965). (12) B. R. Baker, "Design of .Ictive-Site-Directed Irreversible E n z y m e Inhibitors. T h e Organic Chemistry of t h e Active-Bite," J o h n Wiley and Sons, Inc.. Ne!\- T o r k , N. Y., 1967, Chapter I S . (13) B. R . Baker a n d G. J. Lourens, J. .Ired. Chem., 10, 1113 (1907), paper CV of this series. (14) B. R. Baker a n d R. B . illeyer, Jr., ibid., 11, 48Y (1968), paper C S I S of this series. (15) R. N. RIcDonald a n d T. W. Campbell, J . Ory. Chem.. 24, l96Y (1959).
22
C',,H CH=CHCH,P( C, H I )
(:I
CI I
I
25
26
1-(3-Chloro-4-nitrophenyl)-4-phenylbutadiene(25j.-To :i stirred iriisliirc ( i f 2.6.1; g (14 nimoles) of 24 (Aldrieh) oiiti 6.40 g ( 13 mmoles) i i f 2315 i n 40 nil of NeOH was added a ~~oliitioii I J ~ I . . i i g ( 2 : ; ninioles) of XaOAle iii AIeOII. =\fter being htirreti :{I :inibient temperatiire proterted from moisttire f o i , 20 hr, the, mixtiire wa,? filtered. The yellow prodiict was washed siic~c~e*sivel.~\\.itti 2 - i nil of ciild AIeOF-I, 2.5 ml of c ~ i l t i50' 2.5 nil of cold AIeOlI; yield 1.75 g, nip 1200.1 g (totit1 46;;) \vas isolated froni the wnibiiiecl filtrate i i i i i i
