'
TABLE I DIHYDROFOLIC I I E D U C T LIES
1 ~ E V E I l b I I j L EI S H I U I ~ I O N OF
13Y
NH2 I
Ii0,".L
NU.
1
6 7 8 !J
10 11 1% 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 298 300 318
Pigeon liver
K
nl-(c~12 )4c6H5 m-(CH2)4CsH4SHCOCII2Br-p n p ( CH2)4C6H4NHCOCH2Br-nz m- (CH, )?CsHi WL-(CH~)~C~H~NHCOCH~BI.-?, ?n C6HQ-n m-CHZKHCOCH2Br nl-O(CH~)YOCGH: i~2-O(CH2)2OC6Hi?iHCOCI-IIB1.-p ~)L-O(CH~)~OC~,H~XHCOCIIIBP~~~ ~~-O(CHI)IOC~H~SHCOCI-T,B~-~ m-O (CH,)3OC,H, VL-O(CH~)~OC~H~SHCOCH?B~-~ v~-O(CH~)~OC~,H&HCOCH~B~-)YL m-OCH2C6NHCOCH2Br-nz n2-OCsH4SHCOCH2Br-p ~-O(CHX)ZOC~HJXHCOCH~BY-~ p-COCHzCl p-(CHz),COCH?CI m-COCHzC1 nt-(CHz)?COCH?Cl m-(CH2)aCOCH2Cl
0 .0 0 2 i d 0 , 00!)% 0.013 0 . 024d 0.0091 0.030d 0.41 0 . 07.jc 0.0.53 0.04.3
pAII--p
\\-alker 2.X
_______1.1210FR8
0.0042 0,0039
0.011 0.044
0.0054
0.0043
0.26
0.26
0.0073 0.022 0.022
0.0:39 0 .O i B 0.14
0.11e 0.19 0.028 0.090 0.12 0,023 0.11 0.22 0.12 0.11 0,075 0.012 0.032 0.053 0.020 0,057 1.2/ 0.33 0.90 0.025.f 0.012 0.036 1.7f 0.61 1.3 0.083 0.080 0.035 0.025 0.080 0.027 VZ-O(CH?)IOC~H~NHCOCH=CHCOOH-JJ 0,078 0.00i.i 0.022 VZ-O(CH,),OC6H,XHCOCH=CHCOOH-p 0,076 0,0080 0,025 0 . 070 0,020 0.080 p - (CH,)2COXHC6H4SO,F-p 0.10 0.064 0.078 VL-(CH~)~COKHCCGH~SO~F-~ 0.31 0.046 0.30 ~L-CHICOXHC~H~SO~F-~ I:, = co~iceiitratioiiof inhibitor ,tance of Barbara Bailie and Jeali Reeder with these assayi is ackiiou-ledged. necessary for XCi, inhibition. Assays were run in 0.05 W Tris buffer with 6 p M dihydrofolate as previously described:"% 12 p . l l T P X H was used with the pigeon liver enzyme aiid 30 p.21 T P X H with the two tumor enzymes. d Data from ref 13. e Data from ref 14. f Data from ref 3b. n Data from ref 3a.
inhibitors of dihydrofolic reductase can be summarized by 4; if (a) the proper bridge length, B1, between the phenyl groups and, Bz, between the outside phenyl group and the leaving group, I,, exists and (b) if L is a leaving group capable of reacting with a juxtaposed nucleophilic center on the enzyme, then an active-sitedirected irreversible inhibitor should emerge. Initial
work \\-as focused on the chloromethyl ketone and bromoacetamido groups for the I, group of 4, since these two groups have the electrophilic ability to react with any one of seven out of a total of fifteen different proteinic amino acids containing a third functional group. Sineteen compounds with these two leaving groups and the maleanilic group, but n i t h varying BI and B2 groups (see 4), were synthesized and evaluated enzymatically. The results are the subject of this paper.
Enzyme Results.-Reversible inhibition with these nineteen candidate irreversible inhibitors on dihydrofolic reductase from pigeon liver, Walker 266 rat tumor, and L1210 FR8 mouse leukemia are presented in Table I ; for comparison purposes some related reversible inhibitors are also collated. The following information on reversible complexiiig to dihydrofolic reductase can be gleaned. (1) Introduction of the bromoacetaniido group (6, 7) on the nz-phenylbutylphenyl-s-triazine( 5 ) leads to a 3-4-fold 105s in binding; this indicates that the bromoacetamido group is in contact with the enzyme, but is not complexed. ( 2 ) The m-bromoacetamidomethylphenyl-s-triazine (11) is a 13-fold poorer inhibitor than the nz-butylphenyls-triazine (10). Since the latter shows a threefold better inhibition than the parent phenyl-s-triazine (Ijo = 0.11 p M ) , l d the n-butyl group has some hydrophobic interaction with the enzyme ; thii hydrocarbon area on the enzyme could repulse the polar carboxamide group of 11. (3) Introduction of the bronioacetamido group (9) 011 the 772-phenethylphenyl-s-triazine (8) gives a threefold increased binding; this result also indicate5 the bromoacetamido group of 9 is n ealdy coniplexed n ith the enzyme and therefore is in contact with the enzyme. (4) When an amide group is iubitituted on the ter-
29 SCHEME I
37,n=O
Cl
C1 32
41,n=l 42,n=2
The four amine hydrochlorides (39, 40) were converted to the dihydro-s-triazines (41 42) by reaction with cyanoguanidine in l l e 2 C 0 , the three-component method of ;\lodest.21 Conversion of 41 aiid 42 to caiididate irreversible inhibitors (6, 7, 9) is discussed later. The second and largest group of candidate irreversible inhibitors were derived from phenoxyalkyloxypheiiyls-triazines, of which 13 is a member. The key intermediates were the dipherioxj-alkanes substituted with an amino group on one phenyl arid a nitro oil the other (47). These mere readily synthesized by alkylation of nz-acetamidopheiiol (43) with the appropriate bromoalkoxybenzene bearing a nitro group (44, 45) to 46 followed by aqueous alcoholic HCI hydrolysis to compounds 47 which ivere isolated as their hydrochlorides (Scheme 11). The amines 46 were converted to the ?
SCHEMEI1
OH 43
44, n = 2 45, n = 3
O(CH,), 0
P NO2
46, R Ac 47,R=H SH,.HCl
48
6H 43
49,n=l 50,n-0
51 (21) E. J. Modest.
J. Ore. Chem., 21, 1 (1966).
nitrophenj luted dihydro-s-triazine (48) by the threecomponent method.?’ The third group of candidate irreversible inhibitors corisists of 19 aiid 20 (Table I). The niethod of synthesis was closely related to the second group in that a nitrobeiizyl halide (49) or .l-chloroiiitrobeiizeIie (50) was used to alkylate 3-acetamidophenol (43). The fourth class consisted of carididate irreversible inhibitors synthesized by rcduction of the terminal c) a ~ i ogroup followed bj. bromoacetylation a. represented by 11 (Table I). Three-component condensatioris?’ of I,i-amiIiobenzoiiitrile ethanesulforiate (52) with cyanoguanidine aiid ,\IesCO afforded the dihydros-triazine (53) with a terminal cyano group (Scheme 111). Hydrogenation of the c j ano group of 53 with l’t02 in EtOH coritairiirig 1 equiv oi EtS0,H afforded the aminomethyl-s-triazirle as itq bisethaiiesulforiate (54).2 L Similarlj , alkylation of i w n i t r o p h e d with w-haloalkylnitriles afforded 55, which were catalytically reduced to 56, then converted to the dihydro-s-triazines (57) with a terminal cyano group. Reduction of the termiiial cyano group of 57 to itmiiio appeared satisfactory, but their hromoacetiinlido derivatives could riot be suitably purified. The fifth class of candidate irreversible inhibitors consisted of chloromethyl ketones 22-26; synthesis of 22-24 has been previously r e p ~ r t e d . ~ ”The key intermediates mere the anilines (63, 64) wbstituted with a termirial chloromethyl ketone function. These were syntheqized by Wittig coiideiisatio1i~482~of m-nitrobenzaldeh) de (37) or Ill-nitrocinnamaldehyde (38) with the phosphorarie (58)” to give the unsaturated ketones (59, 60) (Scheme IT). Iii order t o reduce the double bonds and the nitro group to 63 arid 64 without concomitant reduction of the halogen or ketone funct i o n ~ the , ~ ~ketone function Jvas converted to a dioxolane (61, 62), then reduced to 65 and 66; the dioxolarie blocking group was removed by hydrolysis with HC1 in SO% aqueous i-PrOH. Although the resultant amine hydrochlorides (63, 64) could not be crystallized, (22) similar (2.3) (24) (25)
The corresponding pnrrr isomer lias been previously prepared IIY a process.?3 13. R. I3aker a n d U . - T . Ho. J . Heteroryclir Chem., 2, 385 (1966). 13. R . Baker a n d J. H . J o r d a a n , J . Jleii. Chem., 8 , 35 (1965). R . F. Hudson a n d P. A. Chopard, J . Ore. Chem., 28, 2446 (1863).
('N
;"H
I
EtSO H.NH, I
37.r1 = o 38. / I = I
61, / I = 1 6 2 . 11 = 2
NH. HCI
65.11= 2 66, ri 1
63.11 2 64.11 1
I
.-
70
31 T-inLE 11 PHYSICAL PROPERTIES" OF
N C C H 311, OC 5.28 8.90 9.09 70.3 7 0 .1 .i.2:3 190-191 5.17 5.98 9.09 1.52-1.53 70.0 70.1 5.23 h 9.90 9.92 3.03 68.0 1,53-156 68.1 .5.00 A 9.78 3.04 67.9 9.92 182- 186 A 68.1 3.00 8.61 4.98 60.6 8.8.; 60.8 5.10 149-130 B 8.,j1 60. .i 5.16 8.8.5 60.8 .i.10 118-122 B 8.74 8.83 5.04 60.5 60.8 3.10 102-10.i B o-NO?~ 7.98 8.23 5.66 129-130 60.1 60.2' 3.64 p-NO,d B 8.58 61.3 5.39 118-119 8.48 61.2 5.49 7n-?;Opd B 8 .!I3 5.19 8.8.i 60.6 160-161 p-SO,d 60.8 -7.10 B 9.72 4.99 62.8 210 p-XOp C 63.3 9.40 9.23 3.10 63.3 4.99 dec >220 ni-S02 C 9.94 4.i3 10.1 60.6 60.8 4.73 dec >213 //l-NO, C 4.72 10.2 10.1 60.7 dec >203 60.8 4.73 p-NO, C .i4, .i 5.00 8.82 9.01 54.1 4.86 dec >195 p-SO1 C 9.20 9.01 +53,9 4 , 9 2 181-182 34.1 4.86 C nt-SOr 8.88 4.8i 9.01 .i4.0 130- 153 54.1 4.86 C 0-NO, 8.41 8.62 -53. 3 .i .26 172-174 .i.5. .5 5 , 27 p-SO, C 8.31 3,; , 3 .5. 27 8.62 ,j.j,.5 5.27 160-161 mSO? C 8.78 52 ,.5 4.98 8.76 .52.6/ .j.04 dec >'LOO C p-NOr C der > 1 7 3 p-NOz -9,90 9.98 4.57 .I.?. I .).I. 6 4.66 dec >190 /iL-?;02 C _ 9.97 9.98 a).6 4.68 .).I. 6 4.66 dec >200 p-XO2 C Recrystallized from z'-Pi,OH. Recrystallized a All compounds had ir and uv spectra compatible with their assigned striictures. from EtOH. Starting bromide prepared by method AI. e Ptartiiig bromide prepared by method L. Hemihydrate. RecrystalStarting acetamido derivative, mp 137-138", prepared according to H. E. Recrystallized from EtOH-H20. lized from MeiCO. Ungiiade and E. Haiisbiirg, J . Org. Chent., 17, 742 (1952), except NaOXIe was used iii place of K. i Rerryatallized from EtOH-EtrO. K. Ikawa [Yak7rgaka Zasshi, 79, 760 (1959); Chern. =1bstr., 53, 21761 (1959)l reported nip 195'.
K?
p-NO? ?/t-NO? in-N02 p-NO2 p-SO*d ,pl-NOnd
Slethorl
A
_--
L
Q
1
Experimental Section rri-Acetamidobenzyltriphenylphosphonium Chloride (34).--4 ,wlution of 22.7 g (50 mmoles) of 3229iii 200 ml of HOAc was nhakeii with H2 at 2-3 atm in the presence of 0.2 g of PtOs for 12 hr duritig which time 0.15 mole of H, was consumed. The filtered solution was spin-evaporated in rucuo leaving 33 as an oil. Y o attempt was made to crystallize the oil, but it was dissolved i n 40 ml of Ac,O and stirred at ambient temperature for 25 min: the prodiict began t o separate in aboirt 10 min. The prodlict, was collected oii a filter and \+-auhed with T H F : yield 16..i g (7:iS), mp 284-286". Recrystallizatioii from lIeOII-Et~O gave 11.2 g i50c;) of product, mp 290-292". tallized oiire more to give white ~ . ~ ~ 3340, 3240 (NH), 16i5 290-292" : Y , ~ 3380, (amide C=O), 1600 (C=C), 1550 (amide 11), 1435 (P-pheiiyl), 810, 7.50, i 2 . i j 700, 690 cm-' (phenyl CH). ;Inal. Calcd foi, C? C, 72.7; H , 5.63; iY,3.14. Foiurd: C, 72.5; H, 5. The ortho isomer as the bromide salt was prepared similarly in 5OYc yield, mp 285-286" dec. Anal. Calcd for Cp,H2aBrNOP: C, 66.1; H, 5.14; S , 2.86. Foiiiid: C, 66.1: H, 5.14; N, 2.89, 3-Acetamido-4'-nitrostilbene(35). Method A.-To a stirled mixture of 1.31 g (10 mmoles) of p-iiitrobenzaldehyde and 4.90 g (11 mmoles) of 34 iii 10 ml of AIeOH cooled in ai1 ice bath and protected from moisture was added a solution of 0.57 g (10.5 mmoles) of S a O l I e i i i 5 ml of lIeOH. Solution took place, theu (28) Melting points were taken in capillary tubes on a Mel-Temp block a n d are uncorrected. .ill analytical samples gave ir (Kl3r pellet) a n d u v spectra ( E t O H ) compatible with their assigned structures and each moved as a single spot on tlc. Hrinkmann silica pel G F was used for tlc on all compounds except the diliyilro-s-triazine salts \vilere I3rinkmann pol>-amide.\IN u a s employed: spots \rere detected by visilal examination under u,. Light. (29) Pi. N. Mel'nikov, .I. E . Iiretov, and 13. I. lIel'tzer, J . G e i ~Chrm. C S S R . 7, 4631 ( 1 Y ; j i ) .
the product begaii to separate ill about 10 miii. After beilig stirred a t 0" for 1 hr and 3 hr at ambient, temperature, the mixtiire was chilled, t,heii filtered. The product was washed with cold XleOH (two 10-ml portioiis), 20 ml of .iOC;, aqiieoiis XIeOH, arid filially 25 ml of HpO; yield 1.36 g (48%) of product, mp 181183", v,-hich moved as a single spot on tlc in EtOAc. liecrystallizatioii from absoliite EtOH gave 1.21 g (43%) of yellov crystals: mp 182-186": Y , . , ~ 32.50 (XH), 16.iO (amide I), 1600, 1 . W l.iO0 (KH, C=C, SOU), 13:30 (NO,), 960 (trans C=C), 690 245, 355 mp. See Table I1 for analytical cm-I (CGH;); A,, data and additional compolinds prepared by this roiite. l-(~n-Acetamidophenoxy)-3-(p-nitrophenoxy)propane(46). Method B.--A mixture of 5.20 g ( 2 0 mmoles) of p-(3-bromopropy1osy)iiitlohenzeiie (45), :3.40 g (23 mmoles) of 43, 2.76 g (20 mmoleh) of anhydrous KPC03,aiid 30 nil of reageiit XIe&O was refluxed with htirring for 40-4.5 hr. Solvelit wac removed by spill-evaporatioii in i'acuo. The residue was extracted with .iO ml of hot absolute EtOH, theu the filtered solution was cooled; yield 4.85 g ( 7 1 5 ) , mp 129-130". The compound moved as a single spot on tlc in EtOAc and had umRx 3400 (NH), 1635 (amide C=O), 1600, 1545, 1500 (C=C, T H , KO?), 1340 (XO?), 1265 (C-0-C), 840 (n1-C6H4),690 cm-' (p-C6H4). See Table 11 for analytical data and additional compounds prepared by this method. 1-( n~-Aminophenoxy)-3-(p-nitrophenoxy)propane Hydrochloride (47). Method C.-A mixture of 6.78 g ( 2 0 mmo1e.i) of l-(nzacetamidophenoxy)-3-( p-iiitropheiioxg)propaiie (46), 60 ml of EtOH, and 60 ml of 12 LV aqueous HC1 was refluxed with stirriiig for 1 hr. After being cooled to U", the mixture was filtered and the product was washed with cold 50% aqueous EtOH. Recrystallizatioii from i-PrOH gave nearly white crystals, mp 172174', yield 3.5 g (5457), that moved as a single spot on tlc in LIeOH and had vmnr 2860 (broad), 2550 ( T H &), 1600, 1,590, 152.5 (XH, C=C, NO?), 1340 (NO,), 1242, 1040 (C-0-C), 84.5 (,nCsHk), 685 cm-I (p-C6H4). See Table I1 for analytical data and for additional compomids prepared by this route. 4-(tr~-Aminophenoxy)butyronitrileHydrochloride (56, n = 3).
\'Ill,
T.\flLE
I1
I11
" Each compoiind had i r a i d I I V spectra i i i agreemeiit wiih its assigned ,,(ructiire. Each moved a> a single spot o 1 i ilc o i i polyariiidt?3lX. Recrystallized from absoliite EtOH. e Recrystallized from EtOH-H,O. d Hemihydrate. e Recrystallized from 2-met'hoq.elhaiiol-EtOAc. f Recrystallized from i-PrOH-H,O. 9 Recrystallized from absolute EtOH-petrolelim ether (bp 60-110"). " Itetallieed from IILIF-EtOH. Ethaneaulfonaie salt. j Reduction of t,he C r S to CT12NI12followed hy bromoacetylat ioii I)?. method G gave a mixture. iimorphoiis product moved as a single spot on tlc, gave a positive 4-(p-iiitroberizyl)pyridi~ictest for actiw d hdogeri, and gave a negative Bratton-llarshall test for aromatic amine. This material contains about l.icc solvent which c ~ d i1o1 Run u-ith triethylamine equivalent to amine d t . I
