Sovember 1967
IRREVERSIBLE ENZYME INHIBITORS. CVII
1129
Irreversible Enzyme Inhibitors. CVII. Proteolytic Enzymes. 11. Bulk Tolerance within Chymotrypsin-Inhibitor Coniglexes B. R . RAKERA N I ) .IEFF.REJ DepartmPnt of Chemistry, Lrnzaersit!/ of California (it S‘anta
L4. H I T R L R I - T ~ h
i
ha/ u , Santa Barbara, California 93106
Received July 18, 1967 Fifty-eight compounds related t o phenoxyacetone and phenosyacetamide have been measured as inhibitors of a-chymotrypsin. Substitution of 3-chloro, 2,3-dichloro, or 3,4-dichloro groups on phenoxyacetone increased hinding of phenoxyacetone 11-23-fold; bulky groups such as methyl or ethyl were detrimental to binding, brit, flat hydrocarbon substituents such as o-phenyl or benzo were beneficial to binding. Ariilides and E-benzylamides of phenoxyacetic acid showed enhanced binding over N-methylphenoxyacetamide; this N-phenyl hinding to the enzyme was also enhanced by nitro or chloro groups. N-(p-Chloropheriyl) derivatives of 3,4-dichlorophenoxyacetamide (59) and 3-chlorophenoxyacetamide (58) and N-(m-chlorobenzyl) (61) and N-(p-chlorobenzyl) (60) derivatives of 3,4-dichlorophenoxyacetamidewere good inhibitors of chymotrypsin; 5841 were complexed one to two times better than the substrate, N-glutaryl-L-phenylalanine-p-nitroanilide,and 55-110fold better than phenoxyacetone, the compound with which this study was started. Since there is bulk tolerance for these N-phenyl moieties within the enzyme-inhibitor complex, placement of appropriately dimensioned leaving groups on these N-phenyl moieties make likely candidates for active-site-directed irreversible inhibitors of chymotrypsin that can attack outside of the active site.
A series of proteolytic enzymes in blood serum are involved in blood clot,ting,blood clot solut’ion,histamine release, and antibody-augmented foreign-cell rejection; such areas of research4 are important in the cardiovascular and organ transplant problems. At least t’wo clear classes of these enzymes exist: (a) those that are ‘Lchymotrypt’ic”and prefer to hydrolyze the polypept’ide bonds of arylamino acids, and (b) those that are “tryptic” and prefer to hydrolyze the polypeptide bonds of basic amino acids. Selective reversible inhibition of one of these myriad of serum proteolytic enzymes for a given disease state is not apt to be a ~ h i e v e d ;also ~ irreversible inhibitors of the endo type5s6 that attack within the act,ive site are not sufficiently elective.^ I n contrast, active-site-directed irreversible inhibit’orsjfj of the ex0 type that operate by complexing to the active site, but covalently link to the enzyme outside of the active site,7have an extra dimension of specificity8 that could lead to selective inhibition of only one of t’hese many serum prot’eases. The successful design of an active-site-directed irreversible inhibitor that operates by the exo mechanism is considerably more complicated than the design of a reversible inhibitor or an endo-type irreversible inhibitor. Success has so far only been achieved when a definite modus operandi is fo1lowed:j (a) t’he binding points necessary for reversible inhibition are determined (phase I), (b) bulky groups on the inhibitor t’hat are tolerated by the enzyme (phase 11),and (c) placement of a proper leaving group in the bulk tolerance area of the inhibitor (phase 111). Since to our knowledge no exo-type irreversible inhibitor for a chymotryptic enzyme has yet been designed, we have initiated work on chymotrypsin toward this object’ive; the results of (1) This work was supported in part by Grant C.l-08695 from t h e National Cancer Institute, U. S. Public Health Service. (2) For t h e previous paper of this series see B. R. Baker and E. H. Erickson, J . Med. Chem., 10, 1123 (1967). (3) N D E A predoctoral fellow. (4) For a discussion and key references see ref 2. ( 5 ) B. R. Baker, “Design of Active-Site-Directed Irreversible Enzyme Inhibitors. T h e Organic Chemistry of t h e Enzymic Active-Site,” John \Viley and Sons, Inc., Nexv York, N. Y , , 1967. (6) B. R. Baker, J . Pharm. Sci., 5 3 , 347 (1964), a review. (7) T h e active site is defined as t h a t part of t,he enzyme necessary for complexing t h e substrate plus t h a t part necessary for the catalytic action. (8) See ref 5, Chapter IX.
phase I and I1 studies with chymotrypsin are the subject of this paper. Even though chymotrypsin has been one of the most extensively studied enzyme^,^ these studies did not have the continuity necessary for the design of an exotype irreversible inhibitor.lo The work described here is based on the following o b ~ e r v a t i o n s . ~ (a) ~ ’ ~Methyl hydrocinnamate ( 1 ) l 1 binds to chymotrypsin as well as methyl S-benzoyl-L-phenylalaninate (2),12 but the latter has a much superior velocity as a substrate. (b) Both 1-chloro-3-phenoxyacetone(4) l 3 and L-l-chloro-4phenyl-3-tosylamido-2-butanone(3) (TPCK)l 4 inactivate chymotrypsin by the endo type of active-siteC,H,CH,CHCOOCH,
I R
CsHjCH2CHCOCH2CI
I
NHSOZCBHdCH3-p
1, R = H 2, R = CsHjCONH
3
C,H,OCH,COCH,CI 4
directed mechanism, each attacking histidine-57, but with 3 reacting considerably faster. Therefore, com-
I R, 5
6
pounds of types 5 and 6 were considered for phase I and I1 studies. The first consideration was the nature of the bridge b e h e e n the benzene ring and the carbonyl function. I n addition to the -OCH,- bridge of 5 and the -CH&H,- bridge of 6, the -CHz?rrH-, -CH=CH-, -SCH2-, and -CH,- bridges were investigated (Table I ) ; in addition to optimum binding, it would be of (9) For a review of t h e literature on reversible binding t o chymotrypsin pertinent to t h e design of active-sitedirected irreversible inhibitors, see ref 5 , Chapter 111. (10) Reference 5 , Chapter VIII. (11) J. E. Snoke and H. Keurath, Areh. Biochem., 11, 351 (1949). (12) S.Kaufrnan and H. Neurath, ibid., 21, 437 (1949). (13) K. J. Stevenson and L. B. Smillie, J . M o l . Biol., 12, 937 (1965). (14) (a) G. Schoellrnan and E. Shsw, Biochemistry, 1, 252 (1963); (h) E. 13. Ong, E. Shaw, and G. Schoellman, J. Biol. Chem.. 240, 694 (1965).
'' Assayed with 0.2 m X N-glutaryl-LpheiiylalaiIiiie p-nitroanilide (GPYA) in 0.iLi .If Tris buffer ( p l l 7.4j contailling JO', I )AlSO. I:,c = m X concentration for 50% inhibition. Ratio of concentration of inhibitor t o 0.2 mill GPSA giving 5056 inhibition. d llasi,5872 (1955). Prepared according iniini solubilit,y. e Prepared according to C. W.Whitehead and J. T. Tr to S . Maxim, Buli. SOC.Chim. France, 39, 1@24(1926). 0 Prepared a c Sci. Food -lg/,.,10, ,777 f l % 9 j . h Prepared by Johnson in this laboratory. Prepared vekhgeiiner, I z v . -lkciri. .\.u7ik SSSIZ, Old. Khim hztk, 2061 (1960). ' Prepared according to Prcyareti : ~ c ~ c ~ i i d i r i p Prepared acwirdirig to J. E. Ranfield, W.navies, X . IT-.Gamble, and S. .\liddleton. .I. f " h m . Soi,.. 4701 f 1!)>6). i o ref 1%. "
vulue to find the most easily synthesized bridge for the i'iirther structural variants of 5 and 6. Since hydrocinriamic esters are slm- substrates, they c;tn be measured as inhibitors in the presence of a fast siihstrate. Ethyl hydrocinnamate (7) (Table I) w a s complexed l / Z 0 as well as the substrate, N-glutaryl-rJpl-ienylalanine-p-nitroanilide (GP ) . I 5 Since GPKAr\ lias KIT,= 6 X M, 2 has K , = 16 X X , and 1 has Kn, = 39 X lou4;ll,llthe obseived binding of 7 is iii reasonably good agreenieiit. Since ethyl cinnaniatc (8) binds the same as 7, it is clear that the --CH-CHhritlge is as good ab the -CH2C",- bridge; furthermure, the latter bridge has the staggered conformation when wmplexed to the enzyme. In contrast, the -CH,SH-hridge of 9 was only one-quarter as effective as the -CH,CH2- bridge of 7; this result can be attributed to (lither the decreased binding of the carbonyl group of 9 or that the KH group of 9 is forced into a hydrophobic region with resultant repulsiou. That tertiary amides of hydrocinnanlate can complex to the enzyme is showii with 10 and 11. Therefore, identical tertiary amide groups can be used for cornp r i s o n of the bridges. S o t e that in the comparison of the pair, 11 and 12. the -OCH,- bridge is about twice as effective as the -CH2CH2- bridge; however, i i t comparison of the methyl ketones 13 and 14 or 16 :md 18 the bridges are essentially equivalent. Three other points emerge from Table I. The -CH,-bridge of 15 is about as effectivc as the -CH,CH,bridge of 13. The secund point is that the aldehyde carbonyl of 17 is about twice as effective as the ketone carbonyl of 16; however, the synthesis of aldehydes of type 17 is less convenient than synthesis of methyl ketones of type 16. The third point is that the -SCH2 bridge of 19 is about twice as effective as the -0CH2hriclge of 14. The most convenient bridge for study of binding arid I)ulli tolerance is the --OCH2- bridge of compounds of types 12 and 14 since structural variants can be obtained merely by using one of many commercially availablr Yribstituted phenols. 15) I < E Lrlanaer, F. Edel, a n d A. G . Cooper, Arrh. Blochem. B z o p h ~ s . . 115. LOR 11966)
(16) For a review on t h e possible modes of complexin& bet\yeen inliikjilora and enzymes, see ref 5 . Chapter 11.
IRREVERSIBLE ESZYME INHIBITORS. CVII
Sovcniber 1967 TABLE I11 Tlririsr
41
R,
1x2
H I1
CHs
pN02 p-302
CHI C6HS CHI C6HS CHB C6Hs CHs C6Hs CoHs
p-COOH p-COOH P-CHBO p-CHzO p-CI p-CI HOOC(CH?),
C6t1Sd
m.lI ronrn 12 1.0 4.0 0.2e 10
4.0 17 0.75' 3.5 O.le
(;
inhil) 50 15 50 0 0 0 50 29 50 15
I:stSO( >20f 17 1.5 3.9 -0.5
1[11
[~~I)~I.s" 60 -25
20 >5 >250
>loo 85 9.5 19 -2.5
'a A-sayed with 0.2 m J l GPK'A in 0.03 M Triv buffer (pH 7.4) rontaining lOV0 DAISO. * Ijo = m X concentrat,ion for SOYc inhibition. Ratio of eoncentration of inhibitor to 0.2 mM subst,rate giving 30?c inhibition. d Prepared according to ref 19a. e llasirniim solitbilitp. f Since 15yc inhibition is readily detected, the Lo is greater than five times the concentration measiired.
and p-methyl (27) substituents. l$7hen the til-methyl (28) group was increased to m-ethyl (33), a loss in binding occurred ; similarly, the two nz-methyl substituents of 34 caused a loss in binding. Thus bulky alkyl groups at the meta or para position are detrimental to binding, but flat planar phenyl groups of o-phenylphenyl (31j, 6-naphthyl (35), or a-naphthyl (36) give inereabed binding. Whether or not there is some charge-transfer interaction16 between these aryl groups and the enzyme is difficult to ascertain with the data in Table 11, but it does not appear likely. The interpretation is clouded by the fact that the benzene ring is interacting with a hydrophobic area on the enzyme, but the substituents with varying Hammett u constants (electronegativity) also have varying Harisch K constants (polarity). Perhaps a Hansch T-u computer analysis of the data could resolve thib issue, li although interpretation may be difficult due to steric hindrance of proper interaction of the benzene ring with the enzyme by such groups as p-acetamido. Polar groups such as cyano (22 and 23) and carboxylate (24) are detriniental to binding. The better binding caused by the nitro substituent of 18, 20, and 21, but slight loss in binding by p-methoxy1 (26), suggested that the nonpolar, but electronn-ithdrawing, halogens be investigated. The p-chloro substi tuent of 29 gave a threefold increment in binding, but the nz-chloro of 30 was even more effective and gave an ll-fold increment in binding. The effects of the p-chloro and ni-chloro were not completely additive, but the 3,4-dichloro derivative (37) was only slightly more effective than the ni-chloro derivative (30). Even more effective was the 2,3-dichloro derivative (38) which coniplexed 24 times more effectively than the parent phenoxyacetone (14). Thus maximum binding on Table I1 was seen with the 3-chlorophenyl (30), 3,4-dichlorophenyl (37), and 2,3-dichlorophenyl (38) moieties, these three groups differing only twofold in binding. The next area studied was the replacemerit of the methyl group of phenoxyacetone (14) by phenyl (39) (Table 111). Five pairs of compounds were synthesized for comparison; unfortunately, the w-phenoxyacetophenones with p-nitro (40) and p-carboxy (41) groups (17) T. Fujita. J. Iwasa, and C. Hansch, J . Am. Chem. Soc., 86, 5176 (1'364).
I\'
r I o s i L o i ? C ~ r i ~ a r o ~ r r i Y r sBY I l r I~,C~T140CIInCOTl~
('UIll~,d
14 :39 18 40 24 41 26 42 29 43
T.\BLE
1131
Ljb mX Estd concn inhib Iso' NRIR? 3.5 50 3.5 N(CHa)CsHs 2.0d 42 2.6 SHC6Hs NHCHGHs 1.1 50 1.1 1.0d 30 2.3 H NH(CHz)zCeHs H 0.030~ o >0.15' NHCsHiNOrp 0 60d 33 1.2 H iY(CH3)CsHaNOrp O.lXd 31 0.30 H NHCeHaC1-p H 0.20d 36 0.35 NHC6H4Br-p 0.30d :ji 0.53 H "C6HiCI-m 0.20d H 0 >1.0' NHCeHiCI-o 28 0.44 H X H C H Z C B H ~ C I - ~ 0 ad .?lo 0.24 H NHCH~C~HICI-m 0.24 50 0.65 0.65 H NHCHzCsHaCI-o 0.19 50 0.19 m-C1 SHCHiCeHa 0.11 50 0.11 m-CI NHC6HiCI-p 0.05Od 20 0.21 3,4-C11 NHCeHaC1-p 27 0.14 3,4-C1z ? ~ " C H ? C ~ H I C I - ~ O . O R O d 28 0.13 3,4-CI? X H C H ~ C ~ H I C I - ~ 0.060d 50 0.35 H N H C H ~ C ~ H I N O J - O~. : < R 25 50 2.5 XHCHa H
Compd R3 12 H 45 H 46 H 47
48 49 50 51 52
53 54 55 56 57 58
59 60 61 62 63
([I]/ [S])o.sC
50 13 5.5 11
>o.i 6.0 1.5 1.7 2.i >5 2.2 1 2 13.2 0.95 0.55 1.1 0.70 0.65 1.8 125
Assayed wit,h 0.2 m;M GPSA in 0.03 JJ Tris hiiffer (pH 7.4) containing 10% DMSO. * 15, = mM cBoiicentration for 50C, inhibition. c Ratio of concent,ration of inhibitor to 0 . 2 mill siihstrate giving 50yo inhibition. AIasimum solubility. e Since 15% inhibition is readily detectable, the LOis greater than five times th'e concentration measured.
could r o t be compaied due to insolubility. The three remainmg comparisons were subject to large error since at maximum solubility only 15% inhibition was seen; nevertheless, comparison of 14 and 39, 26 and 42, and 29 and 43 indicated that the phenyl group gave a three- to eightfold increment in binding. Hoxever. this series was considered t o be too insoluble t o be of further utility although it is possible that compounds such as 44 or its isomers could be useful for this study. Attention was turned to the more soluble anilides, since N-methylphenoxyacetanilide (12) was as good an inhibitm as phenoxyacetone (14) (Table I). Replacement of the S-methyl group of 12 by H (45) gave a fourfold increment in binding (Table IT'). Therefore, the higher analogs, 46 and 47, were synthesized; 1,he S-benzyl substituent of 46 was more than twice as effective as the S-phenyl substituent of 45, but the K-phenyl and S-phenethyl (47) were about equivalent. Therefore, 45 and 46 were selected for study of substituent effects on the benzene ring. Introduction of the p-nitro group (48) on 45 gave a compound too insoluble to measure. However, the more soluble S-methyl analogs, 12 and 49, could be compared showing that the p-nitro group gave a ninefold increment in binding. Introduction of a pchloro (SO), p-bromo (51), or in-chloro (52) group gave a five- to tenfold increment in binding over the parent 45; however, the o-chloro substituent of 53 did not give as ;good an iiicrement in binding. If these effects by the electron-withdrawing nitro and halogen groups are identical in mechanism, they could be due to the effect on the polarizability of the carbonyl group (a binding point),g or could efftct charge-transfer character if the benzene ring were binding partially t o the enzyme as an electron acceptor, or both. These two effects are separable with the ?;-benzyl analogs where the methylene group insulates the carbonyl group against electronic effects of the benzene ring.
64
65,K-= CH, 66. R-= C, H-
presencta ot .odiuni carbonate aiid socliuni iodidr : I' t h e u-phenoxy:tcct ophenones (66) WPIC prep:ircd iiinilarly froin p1ien:icyl chloride. l o The only difficultic.\ encountered were in synthesis of the p-carbosyphenosy lietoncs. The chloro ltetones smoothly alliylatcd ethyl p-hydroxybenzoate. but saponification o f 65 01 66 with hot base led t o resinification; :it room tcmpcraturc the hydrolysis was slowJ but the appioprintc p-c'ar1)oxypheriosy lietoncz could hc iholatd in lo\\ yields. The miidcs v\-ere~n:tdchy rcnction of the, :ippropi i x k acid chloridch and arriiries in chloroforni. Pyridine ii:L\ used ab an :icid acceptor with the rtroinatic amine\, t)ut used with the inore lxibic d i p h : t t :tmincs; in three c a w s :in cxcesh of u n i i i ~i\:i\ ~ i i w l :I\ t h c T : i d arccptoi.. i c b
(18) (a) I). i. T a r l d i J . Ory. ( ' h e m , , 7 , 2 5 1 (1942): ( 1 ) ) .I. AI. I)o!v\.eil. .lr,, H. S. 3IcCiillougli. and P. K. Callon'ay. .I. A m . C h e m . S o c . . 70, 22ti (1948); ( e ) 11, Suter and H, Z u t t r r . Ann., 576, 215 (1952); ((1) C'. D. 1Iiirrl a n d .'E I'erlerz, J . 4 m . CIIWL.S o u . , 68, 38 (1946). (19) !a) 1'. Tares. I,. I'armun. a n d G . Stout. ibid.. 80, 106 (1928): I'. liroelinke, G. Iirrwhnkc, iincl C;. \iirenholL, J . Pra1:t. Chum., 11, 2::!4 (1960). (20) .\I1 a n a i y t i i d sainl~leshail infrared spectra compatible u i t l i ~ l i r i r assigned str11ctures ani1 inorril as a single sliot o n tlc on Urinkinann r i h l gel G F x i t h ethyl ac~iatepetrriiP111r1ether (bp 60-110°); s p o t e n w t ' ilri w t e d b y risual examination under 1 1 v light. Melting point,s were dpIerrtiined in capillary t ~ ~ b on e s a hlel-Temp hlork and are corrected. 121) 13. R. Baker, \\., I\.Lee, \\, .1.Skinner, .i. P. Martinez, and E. 'r(>nc, . I . N e d . Pharm. C h ~ m , .2,. 633 (1960)
IRREVERSIBLE ENZYME INHIBITORS. CVII
Kovember 1007
1133
TABLEV PHYSICAL CONSTAXTS OF Compd
RI
Rz
Method
JQOCH,COR, Ri
'A
R P (mm) or
yield
mp, OC
----Calcd, C
70H
K
--Found, C
%--H
N
S (CH3)CfiHb 12 H D 715 S0-93 74.7 6 . 2 7 5 . 8 1 74.6 6.22 5.72 B 33' X2-84C 20 m-XO2 CH3 B 67d 63-68e 21 0-NO* CH3 B gdsa 61-62 68.6 5.18 8.00 68.4 5 . 2 5 8 . 1 5 22 p-cs CH3 23 m-CN CH, B 17" 68-70 6 8 . 6 5.18 8.00 6 8 . 3 5.32 8.:30 F 59 158-171 dec 61.8 5.19 6 1 . 7 5.08 24 p-COOH CH3 B 36" 88-90 (0.18), 4 7 4 9 66.6 6 . 7 1 66.5 6.57 26 p-CHaO CH3 27 p-CH3 CH3 B 41 61-63 (0.15)h B 43 30 m-C1 CH3 94-96 (0.6) 58.5 4 . 9 1 58.4 5.10 31 o-C~H~ CH3 24 33' 120-130 (0.3), 65-67 79.6 6.24 7 9 . j 6.22 A 31b 103-105 32 p-C& CH, 79.6 6.24 79.4 6.23 B 22 61-64 (0.05) 74.1 7 . 9 2 33 VZ-C~H: CH3 73.9 7.86 B 34 78-80 (0.3), 37-41 74.1 7.92 7 4 . 3 8.09 34 3,5-(CH3)2 CH, 37 3,4-Cl2 CH3 B 41" 104-108 (0.3), 49-51 49.3 3.68 4 9 . 5 3.70 38 2,3-C1* CH3 A 57" 114-115 (0.4), 58-59 49.3 3.68 4 9 . 4 3.00 40 p-NO1 CsH5 B 4Ti
