Jourrml of J f d i c a r ~ u lChettiiatry, 1970, Vol. 12, .Vu. 6
1155
3.5% inactivation of the crude mouse liver enzyme. normal tissue that can rapidly convert the SOzF group With purified mouse liver enzyme, the inactivation \vas to SOaH; this “fluoridase” is apparently absent in about the same, 23%; thus 17 is not attacked by the Walker 256 and L1210 cells. We therefore embarked “sulfonyl fluoridase” and 17 still shows a difference beon design and synthesis of analogs of 1 and 2 in a search tween inactivation of the L1210 and purified mouse for more tissue-specific irreversible inhibitors that still liver enzyme-indicating that the enzymes from these \\ ould show good cell n all transport ; 3 , 8 , 9 these inhibitors two tissues are structurally different. Similar results were measured for inactivation of both crude and puriwere observed when a 4-C1 atom (18) n a s introduced on fied dihydrofolate reductase in order to determine 17; 17 is considered superior to 18 since 17 is 10-times whether the specificity was due to a structural difference as effective against L1210 cell culture, indicating better in the enzyme or due to rapid hydrolysis of the SOzF transport of 17. group by the “sulfonyl fluoridase.” The results are the When the SOsF group of 2 was moved to the 2 posisubject of this paper. tion, the resultant 19 was a poor irreversible inhibitor of Biological Results.-The compounds in Table I can the enzyme from Walker 256 or L1210. Introduction be divided into 4 subclasses, namely, analogs of the 4of a 5-C1 (20) or 4-C1 (22) atom gave compounds with phenylbutyl series (5, 12-14), analogs of the 3-chloro-4excellent irreversible inhibition of the enzyme from phenylbutyl series (2, 15-22), analogs of the 3-chloro-4Walker 256 and L1210; unfortunately tissue specificity phenethyl series (4, 6-11), and analogs of the nzwas poor. I n contrast, introduction of a 3-C1 gave a phenylbutyl series (1,23-28). compound (21) that was even less effective than 19. The parent p-phenylbutylphenyl-s-triazine with il The parent 3-chloro-4-phenethylphenyl-s-triazine (4) terminal S02F group on the para position ( 5 ) was prewas an excellent irreversible inhibitor of the enzyme viously shown to be a good active-site-directed irreverfrom Walker 256‘j and L1210.3 There was poor tissue sible inhibitor of the dihydrofolate reductase from specificity in the rat,6 but good tissue specificity beL1210 mouse leukemia3 and a fair inhibitor of this entween L1210 and mouse liver;3the latter was due to the zyme from Walker 256 rat turnorf it was also active in action of the “sulfonyl fluoridase” as shown by excellent vivo.6 Tissue specificity was shown with crude enzyme irreversible inhibition of the purified mouse liver enfrom mouse liver3 and several rat tissues;6 this poor inzyme.’ I n addition, 4 showed in vico cures of Wallier hibition of the enzyme from normal tissues was shown 256 ascites.6 Introduction of a 2-C1 (6) or 3-CI (7) did t o be due to rapid hydrolybis of the SO2F group of 5 to not change the specificity pattern; 7 was about as effecS03H.’ Introduction of 2-C1 (12) on the benzenesultive as 4 against L1210 cell culture, but 6 had greatly fonyl fluoride moiety resulted in loss of tissue specificity, L e . , 12 mas an excellent irreversible inhibitor of the impaired cell wall transport. crude enzyme from both tumor and liver. When the SOzFmoiety of 4 was moved to the 2 position and a 4-C1 (8) or 5-C1 (9) substituent placed on the When the bridge was changed from Bu (5) to BuO (13) in order to increase the coriformational flexibility benzenesulfonyl fluoride moiety, the inactivation of the of the terminal phenyl group, irreversible inhibition of enzyme from Walker 256 or L1210 was somewhat imthe Walker 256 enzyme was not changed; honever, the paired. However, the 4-C1-2-S02F (8) showed an ineffectiveness of 13 as an irreversible inhibitor on the teresting irreversible inhibition pattern. Whereas the L1210 enzyme \\-as considerably impaired. Good tissue parent 4 showed no tissue specificity between Walker specificity v a s seen with 13 between Walker 256 and rat 256 arid rat liver enzymes, 8 did; that this tissue specifiliver or intestine; in the case of the rat liver enzyme the city was due to the riction of the liver “sulfonyl fluoridase” was shown by the extensive inactivation of the specificity was due to the action of the “fluoridase” as shown b j the good inactivation of purified rat liver dipurified rat liver enzyme by 8. I n contrast, the tissue hydrofolate reductase. The conformational flexibility specificity between L E 1 0 and mouse liver seen with 8 of 13 compared with 5 apparently aided transport was unchanged when the purified mouse liver enzyme through the L1210 cell wall since 13 \\-as 200-times as was inactivated by 8 ; thus 8 apparently is not attacked effective as 5 against L1210 cell culture by comparison by the “sulfonyl fluoridase” and the results indicate a of EDbo/I50 ratios.$ When the bridge of 5 was increased difference in the primary structure of dihydrofolate refrom Bu to Hex (14), little change in irreversible inductase from L1210 and mouse liver. Unfortunately 8 hibition, specificity, or inhibition of L1210 cell culture was 35-fold less effective than 4 against L1210 cell culoccurred. ture. The parent 3-chloro-4-phenylbutylphenyl-s-triazine The 4-C1-3-SOLl:analog (11) of 4 showed tissue speci(2) showed excellent irreversible inhibition of the dihyficity between L1210 and mouse liver, the specificity drofolate reductase from both L1210 arid Walker 256, being due to the hydrolysis of 11 by the “sulfonyl fluobut tissue specificity was poor to fair. Insertion of t~ ridase.” 2 4 1 group (15) on the benzenesulfonyl moiety of 2 led I n the fourth subclass, the parent m-phenylbutyl-sto decreased selectivity between mouse tissues, but triazine (1) showed good irreversible inhibition of the little apparent change in specificity between rat tissues. enzyme from L1210 and Walker 256 with poor tissue A 3-C1 group (16) was detrimental to irreversible inspecificity (Table I ) ; 1 was effective zn vzvo against hibition of the enzyme from the two tumors. Walker 256.G Introduction of 4-C1 on the inside Ph When the SO,F moiety of 2 was moved to the 3 posi(23) on 1 practically destroyed the irreversible inhibition, the resultant 17 still showed strong inactivation of tion. However, introduction of 2-C1 on the benzenethe enzyme from Walker 256, rat liver, and L1210, and sulfonyl fluoride moiety gave 25 which still showed excellent irreversible inhibition of the L1210 and Walker (8) B. R . Baker, G. J. Lourens, R. B. Meyer, Jr., and K.M. J. Vermeulen, J Med Chem., 11, 67 (1969j, paper CXXXIII of this series. 256 enzymes. Although 25 showed no enzyme specifi(9) l3 R. Baker and R. B. Rlejer, J r . , tbtd., 12, 068 (19OYj, paper CLIV c i t j between Walker 256 and rat liver, 25 did show g o d of this series.
1166
\
IRI~EVEIWBLE ENZYME ISHIBI~DRS.CLXXVII
Journal of Medicinal Chemistry, 1970, Vol. 12, N o . 6 1157
TABLE I (Continued) S O
Enzyme source'
%
ED^,^
1M.d
Inhib,
lrM
PM
Time, min
inactvnE
PM
EDm/Iao
11
L1210/DF8 (A) Mouse liver (A) Mouse liver (C) W256 (A) Rat liver (A) Rat intestine (A)
0.0054
0.05 0.05 0.05 0.05 0.05 0.05
60 60 60 60 60 20
76 5 60 (34 53 38
0.16
30
12
L1210/DF8 ( A ) Mouse live1 (A) W256 (A) Rat liver (A) Rat intestine (A)
0.011
0.05 0.03 0.05 0.05 0.05
60 60 60 60 20
69 98 93 99 60
0.4
40
13
L1210/DF8 (A) Mouse liver (A) W256 (A) Hat liver (A) Itat liver (C) Kat intestine (A)
0.05 0.05 0.05 0.05 0.05 0.05
20 20 60 60 60 20
45 0 66 0 79
0.0003
0.04
0.0073
4
14
L1210/DF8 (A) RIouse liver (A) Mouse liver (C) W256 (A) Rat liver (A) R a t liver (C)
0.075
0.15 0.15 0.15 0.15 0.15 0.15
60 60 60 60 60 60
66 10 84 84 23 83
0.1
1
1.i
L1210,DFS (A) Mouse liver (A) W256 (A) Rat liver (A) Rat spleen (A) Rat intestine (A)
0.017
0.050 0.050 0.050 0.050 0,050 0.050
60 60 60 60 20 20
94 95 97 100 5 22
0.01
0.6
16
L1210/DF8 (A) W256 (A)
0.020
0.050 0.050
60 60
0,007
0.4
17
L1210/DF8 (A) Mouse liver (A) Mouse liver (C) W256 (A) Rat liver (A) Rat spleen (A) Rat intestine (A)
0.0094
0.0.50 0.050 0.050 0.050 0.050 0.050 0.050
60 60 60 60 60 20 20
100 35 23 96 97 25 0
0.02
2
18
L1210/DF8 (A) llouse liver (A) Mouse liver (C) W256 (A) Rat liver (A) Kat intestine (-4)
0.0078
0.050 0. 050 0.050 0.050 0.050 0.050
60 60 60 60 60 20
97 37 33 94 98 46
0.2
20
19
L1210/DF8 (A) W256 (A)
0.017
0.050 0.050
60 60
46 27
0.0004
0.02
20
L1210/DF8 (4) Mouse liver (A) W256 (A) Rat liver (A) Rat intestine (A)
0.088
0.18 0.18 0.18 0.18 0.18
60 60 60 60 20
99 41 100 82 85
0.001
0.01
21
L1210/DF8 (A) W256 (A)
0.0063
0.0*5 0.05
60 60
0 30
0.0003
0.05
22
L1210/DF8 (A) Mouse liver (A) W256 (A) Rat liver (A) Rat spleen (A) Rat intestine (A)
0.015
0.050 0.050 0.050 0.050 0.050 0.050
60 60 60 60 20 20
93 56 100 97 7 0
23
L1210/I)F8 (A) W256 (A)
1.5
3.1 3.1
60 60
13 8
6
4
3.i 31
il)ecilicit> htbtivwn 1,1210 arid niou-tl livcr; this cifitit). w:i. apparently duc to L: diffweiict>in t'nzyme structurc, siriw the purified liver cnzyme \viis inactivated, 1 25 about t h c h a i n ( ' extent a s the crude preparation. Sot 1' that 25 \v:ib traiisport(d through thc I,1210 cell \\ :ill niuch li,ss c#t~ctivc~lj~ than 1, hut still suficierit. Iiit roduction of 3-Cl (26) \v:w detrimmt:il t o inacti\'at 1011 of tlic c.uxymt' from L1210 :tnd Walker 256. Siniilarl>,w l i c ~the SOLE'group it-: hifted to thv ortho I)ohition, the resultant 27 w a s ii ver oor irreversible inhibitor. When the p-S02Fgroup of 1 was moved t o tho meta position (3)? inactivation specificity between 1'1 210 and mouse liver was see11that was apparently duv to thtl ctiffercnci. i n of thrl cmzj-rne since thci purified mouw 1ivc.r e n z j mt' I\ riot inactivated apI)rt&bly inore. Unfortunnt e1y 3 is transported about 1000-fold less rffectively than 1. Introduction of 4-C1 (111 3 g t t w 28 \vith :thout the s;i~n(' bpecificit). and tntiis1)ort p:i t t c>rri.
li ,b
1'
I'usitiod of ( C H=C H)
,,
Alp, o c
R2C
3-Cl 341 3-C1 3-c1 3-C1 3-C1
4 1 1SY-13ti 2-Cl-4-SO2E: 1 4 3-Cl-4-SOzF 136-138 4 1 149-153 4-Cl-2-SOzF 1 4 119-122 S-Cl-2-SOzF 1 4 112-1 33 3-Cl-2-SOZF 1 4 130-1 36 4-CI-3-SOzF H 223-224 2 4 2-Cl-4-SOpF' II 4 137-160 Y 4-S02Fm :3-c1 2 4 274-276 dec 2-C1-4-S02F' > 3-C1 4 240-243 3-Cl-4-SO2F' 3-Cl 2 4 3-S0zFO.P 208-210 3-C1 2 4 224-230 4-Cl-3-SOzF' 2 3-e1 4 2-SOzFo 216-21 8 3-Cl 2 4 228-230 3-C1-2-S02Fo 3-C1 2 4 2 12-2 14 Y-CI-2-SO2P 3-Cl 2 4 24.5-247 4-C1-2-S02F0 4-C1 1 190-1 9 1 3 4-SOnFn 2 4-c1 158-196' 3 4-SOzF 2 ;I H 175-180 2-C1-4-S02Fk H 2 201-20.5 3 S-Cl-4-SOzF' > H 218-220 3 d-C1-2-S02Fk H 2 3 4-CI-3-SOzF' 184-186 '* All compouiids prepared by method A ill ref 14. b Numbered from l-NOr. c Numbered from 1-viiiyl group. A4nnlgticallypure material. e Anal., C, H , K unless otherwise indicated. f ltecrgstd from EtOH-THF. liecrystd from EtOH-H20. * Ilecryst,d from Et,OH. Iiecrystd from Et,OH-THF-HsO. 1 Two spots of cis-trans mixture 011 tlc. k For starting aldehyde see C. F. Giihriiig, l h . , 18, 720 (188.5). A4nal.,C, H, F. For starting aldehyde see ref 19. 7L For the starliiig Wittig reagent see ref 12. 0 For starting aldehyde see ref 3. 2' For the starting Wit,tsigceagent see ref 3. I
-
TABLE IV: PHYSICAL Pitomitins VH,.HX
01-
iield,L No
"
HX
%
N p , OC dec
EtSOjH 52 228-229 EtSOsH 4% 2 16-2 19 EtSOjH X 44 209-2 11 EtSO3H 0 32 210-213 EtSO3H 10 40 224-226 11 EtSO2H .i2 2 1e?-2 16 12 EtSOaH 32 204-20.5 39 223-223 HCI 13' 14 EtSOaH 34 20.5-207 15 EtSO3H 49 201-203 EtSO3H 16 44 2 14-21,j EtSO3H 17 34 183-187 EtSOaH 18 44 198-200 1'3 EtSO3H 42 194-197 20 EtSO3H 45 198-194 21 EtSO3H 21 >%l.i 22 EtSO3H 29 200-202 23 HCl 37 218-220 24 EtYOaH 24 205-206 25 EtSO3H 32 168-170 EtSO3H 26 43 185-187 27 HCl 16s 190-192 28 EtSO3H 28 176-178 Prepared by catalytic reduction of compounds in Table I11 in MeOEtOH with a PtO, ca.talyst in the presence of 1 equiv of HX, followed by condensation with ~yanoguanidine,~ unless otherwise indicated. * Numbered from triazine a t position 1. c Analytically pure material recrystallized from i-PrOH-HtO, unless otherwise indicated. d Anal., C, H, F unless otherwise indicated. e Anal., C, H, N. f Reduction performed with Raney Ni catalj st, then HC1 added. 0 liecrystd from hIe2CO. liecrystd oiice from EtOH and twice from ?IIe&O. ti 7
33
U
34
1
35
1
38
39
Table 1, tlxcept 13, \\ ere prepared bj thc. previousl!. described general methods used for 1-5.3r12-14 The :Ippropriate fluorosulfonylbenzyl bromide (31) way converted into the Wittig reagents (30) with PhJ'; the chloro derivatives of 30 were :tvailable from another study.15 Wittig condensation of 29 and 30 afforded 32; theac were catalytically reduced in thP pfesence of lXKl,jH and PtO? t o 33 which w r e condensed n ith woguanidine arid acetoiie by the method of Modest to givtl the rcquisite dihydro-s-tri:tziries (35). l lation of /,-1i? drox?beiizriiesulfori! 1 fluorido" ufforded 39; thts l a t t t ~I\ curiverted into 13 h! r 0 1 1 it Mel-Temp t)lork tuid are uncorrected. .ill arialyticd 5amples had proper ir spectra and moved single spot on tlc; Brinkmanii silica gel ( ; F WB? lined for ompounds except 35 where Brinkmami !I21 1 4 . I+. I3aker and G . J. I.ourenc, . I . J I e d . ChPm., 11, 666 (19681, p a i w ( ' X Y V I I r > f tliis w r i e s . , I : $ ) 1 4 . I < . I3aker a n ( 1 G, .J. L,ui~resns, i l i i d . . 12, 95 Cl96!1), paper (!XI, of t l i i s serie,..
1 11. It. I3aker a n d
N. 11. J . Vermeiilen, ibid., 12, 89
(lues), paper
( 1 3 ) 11. K. Baker a n d J. A . Hurlbiu, i h i d . , 12, 902 (19OQ), paper C L S I of tliis series. (16) I:. J . hlodest, J . Orp. Chern., 21, I (1936). (17) 1%. H . Baker a n d pi. .\I. .J. T'erineiilen, .I. .Ired. Chem., 13,82 (IHTO), I,al,er (.I,Y\-I of this series.
-
IJL)lyaIIlideJLx \Vas eIllpllJyed. .ill a l l d bustion values for C, H, S , or F within
2-Chloro-5-nitrocinnamaldehyde (ZSa).--Condensatioii of 2c~hloro-5-riitrobenzaldehyde18 with hIeCHO, as describedl8 for Ihe cvndensation of 4-riitrocinnanialdehyde with hleC130, ii m t d e product that was recryatd from C6Hs; yield, 10 imalytically pure material, mp 141--143'. ,4nol. (Cg13&'1
r, H,s.
3-Chloro-4-fluorosulfonylbenzyltriphenylphosphonium Bromide (30a). Method A:---i roln uf 2.0 g (7 niinoles) of 3-c.1iltri.oI-fliiorosiilfoiiylbenzyl bromide (31a)'j and 1.9 g (7.:< mnu)Ie3-i of PhiP i n 100 ml of C6Hswas refluxed 16 hr; dririiig t h i h tinw eparated. The cooled reaction mist lire was filtered; the solid \vas washed Lvith C6F16 and recrystd from E;tOII-('6116 1 0 give white crystals, nip 268-270". See Table 11 for additiotitil data and other compounds prepared by this method. 4-(p-Nitrophenylbutoxy)benzenesulfonylFluoride (39). I
