IRREVEHSIBLE EXZYXE IXHIBITOI~S. CS
qJ:tiiuary 196s TABLEI ISHIBI,~TOK OF DIHYDROFOLIC"'~
~ n u u c ~ a s Bn Ys
NH? NAY*
HJ~~~?(CH,)_ R
----
-----
Pieeon
I.1 .lI \Valker
liver
256
0,024d 0.29
0.19
0.22
150,
n
Nu. 3 4
m-CH:CIIKel€j m-CH?CHCoHj
~i21oj
1
6 6
CHdHCOCHL3r Tn-(CH?)rC6Hs
0 . 002Te
m-CH(Ckf?)&kk
0.96
0.43
3.1
0.010e 1.6
o,30
o,3:5
0.088f 5 8
0 78
0.50
I i 8
C liuN11COC11:13r p-(CHt)iCslls p-C11(CHr)L!611s
!
Y
10
C I€Xt€COCII?l3r m-O(CH>)3CaIIs I,L-SHCoCti?Ilr-p-O(C~l?)rCEIIi
The techilical a&stalice of Barbara Bailie arid Jean Reeder with these assays is acknowledged. Bssayed with 6 PM dihydrofolate at p H 7.4 as previouslg described;Ya 12 ~ 3 T1 P S H was used with the pigeon liver enzyme and 30 p X T P S H with the other two. c Ijo = concentration of inhibitor required for 50yG ~ illhihitioll. d ]lata from B. R.~~k~~ a,ld B,-T. H ~ )J, , ~ c y d i c Chem., 2, i % (1963). e Data from B. R. Baker, B.-T. Ho, a ~ (;. ~ J. d Loureiia, J . Pharm. Sci., 56, 737 (1967). f Data from ref 8. 11
met'hods previously described. 3a Even though the reversible iiihibitioii data indicated that the bromoacetamido groups of the candidate compounds were in contact with the enzyme surface, it' is probable that
-
02NQ
,YEtSOiH
irzeta isomer (12). These studies, o n 11 and 12, although completed after those on the bromoacet'amide leaving group reported here arid earlier,2were published first in order to est'ablish that, the methodology of the enzyme assays was correct. Chemistry.-The key reaction for synthesis of compounds of type 2 was t'he condensation of a substituted l)henylacetonitrile such as 13 or 25 with ti substit'uted benzaldehyde or cinnamaldehyde. l1 Condeiisatioii of ciiinamaldehyde with 77%-nit'rophenylacetonitrile(13) in the presence of methanolic KOH afforded the diene (15) in 24% yield (Scheme I); the p a m isomer (16)ll'' was prepared similarly in 75yc yield. Hydrogenation of 16 with a Pd catalyst reduced the nitro group arid the butadiene system, but riot' t'he nitrile group as anticipated, to afford 20 isolated its its crystalline ethanesulfonate salt, in 8G% yield; the ,mela isomer (19) v a s prepared similarly but was obtained as an oil. Coridensatiori of 19 or 20 with cyarioguanidine and BIe2C0 by the three-component method of Modest l 2 afforded the crystalline s-triazines 17 and 18. Hydrogenation of the cx group of 17 aIld 18 J+-itha pt, catalyst proceeded in the presence of ethariesulforiic acid to give the t aminomethyl ~ ~ ~derivatives 21 and 22; these were not further purified, but were treated13 with bromoD l I F in the preserlce of triethylacetic anhydride amine to give the crystalline bromoacetamides 6 and 8. condensation of ?ti-nitrobenzaldehyde (23) with phenylacetonitrile (25) in methanolic KOH afforded the stilbene (26) in 56% yield (Scheme 11). A similar condelisation of 25 with tti-iiitrociiirianialdeh\-de(24) afforded the butadiene (27)"' as B mixture of geo-
SCHEMEI
02N@
CHLCN 13,meta isomer 14, para isomer
H J
d
C=CHCH=CHCGH,
I
N/ N - - Q C H ( C H ; ) d c G H I H,NG~/](CHJ~
I CY
EtSodH
-H~NG~/~ NAN-
H , N ~ ~ ) I cCH(CHJG& HJ, I CH2NHiEtS03H 21, meta isomer
18, para isomer
22, para isomer
no amino acid on the surface in contact' with the bromoacetamide has t'he proper type of nucleophilic group required for reaction with the leaving group. The results from this and the previous study2 011 compounds of type 1 bearing the halomethyl carbonyl type of leaving group suggested that a leaving group be explored that could a,ttack a serine or threonine hydroxJ.1. Such a compound is 11 which rapidly in-
IIIUNG:~N:;;ISQ'(CH~~~CO~H
I
NHI,EtSOdH
17, meta isomer
/Y
+
CH(CH&C6H5
CN 19, meta isomer 20, para isomer
r
N/N*
Q
.EtSO,H
CN 15, meta isomer 16, para isomer
--+
33
0
so,F
11, para isomer 12,meta isomer
act'ivates the dihydrofolic reductase from Walker 256 rat, tunlor, 1,1210/1>~s leukemia, and pigeoIl liver; o d y the latter enzyme was inactivated by the
(cH,),
CH(CH&Hj
I
CH,NHCOCH,Br
6, meta isomer 8, para isomer
metric isomers. Hydrogenation of 27 with a l'd catalyst reduced the nitro group and the double bonds to give 28 which was directly converted to the crystalline s-triazine ethanesulfonate (30). In contrast, hydrogenation of the stilbene 26 with a I'd catalyst reduced the nit" group but, riot the double bond; the presence of the latter in 29 was showi by ti uv maximuni at 315 mk characteristic of its conjugated sjxtem. Conversion of 29 to the s-triazine h>.drochloridc (31) proceeded smoot'hly. Hydrogenation of 31 with a PtO, catalyst iii the presence of HC1 reduced both the cyano aiid the stilbene double bond t o give 32. Bromoacetylation of 32 (11) (a) H. L e t t 6 TV. Haede. and L. S c h i i t ~ r 2 , . P h y , i o l . Chem.. 289, 298 (1952); (b) P. Remse, B e r . , 23, 3133 ( 1 8 Y O ) . ( c ) .J. Colonge a n d 1'. F r a n k , B U E ISOC. . Chim. Friince, 3090 (1964). (12) E. %J.lIodest, J . O v . Cliem., 2 1 , 1 (1956). (18) I{. R . I3aker, 11. V. Santi, J . li. Coward. H. S.Jhapiro, ant1 .J. H . Jordaan, J . Heteroc!/clic c / , ~ ?3,~425 . . (IYBB).
23. ! i 24. r i
26, n 27. n
=0 =
I
=0 =!
i '
HCI NH\CH=('C,II
I
32. n 33. / i
= =
1; H S = H('I 3: H S = E r S 0 , H
38. K = H IO ri = R~CH.CO
4.tt = 1: HX = Hc'1 34. 11 = 3 : fHX = EtSO
H
Jant1ar.y 100s TamE 11 PHYSIC.4L PROPERTIES' O F
R, $&{-Rj
R,
CN %
so.
RI
Ra
R?
Metliod
yield
llp,
oc
--Calcd. C
-1
I
h
'
I'ound. YG---
C
H
P
.
j
..
73.9 4 . 3 7 1 0 . 1 73.9 4.60 10.1 C61%CH=CHCH= 24h 196-198 4 p-XOn .. CsH;CH=CHCH= -4 72 208-209' m-NHad H C,H,(CH,), B Oile P-NH~~ H CaH,(CH2)3 B 861 184-183 63.3d 6.68 7.74 6 3 . 1 6.82 7.77 H .. m-?JOrCeH,CH= A 568 133-134 72.0 4.02 11.2 7 2 . 2 4.18 1 1 . 5 II .. TTL-?~'OZC~HICH=CHCH= A 42b 142-16ih.; 73.9 4.37 1 0 . 1 74.0 4.30 1 0 . 3 28 IT H m-NH2C6H4(CH2)qd B Oile 29 H .. ~-NHZC~H~CH=' B 49',k 182-185dec 7 0 . 2 j 5.10 10.9 70.3 5 . 2 7 11.0 a All analytical samples had ir and uv spectra in agreement, with their assigiied structiires; each moved as a single spot on tlc unless otherwise indicated. Recrystallized from 2-methoxyethanol. c Lit.11b mp 203-206". d Ethanesulfonate. e Directly converted to striazine (see Table 111). f Recrystallized from i-PrOH. 0 Recrystallized from EtOH. h Softens at 100' ; a mixture of geometric isomers. Lit.11cmp 172". Tlc with 1: 1 CJ16-petroleum ether (bp 30-60") showed t,wo spots. j Hydrochloride. Amax 233, 31.5 mp. 15 16 19 20 26 27
m-30,
70 P;o.
HS
17 18
EtSOBH EtSOaH
6
EtSO3TI
R
~~-C~H,(CH~)ZCH(C~) D - C ~ H ; ( C H ~ ~ C )H ( C ~
LIetliod
yield
C
42* c 7id
C
lip,
oc dec
17.5-176 196-197
---Calcd, C
%--H
.59,.5 6.6,; 59.5 6.6.; .59,.i 6.6.: 6 3 . 1 .i..X t j . 5 . t j ,j.8%
N
17.3 17.3 17.3 22.0 19.4
-Found, %C H N
Xl.6 ,59..5 *59.2 65.0 55.2
6.72 6.72 6.76 3.46 5.71
17.3 17.3 17.1 21.8 19.2
6.11
13.9
. i l . 2 6.11 1 3 . 8 31.4 6.20
13.6
.51,'2 6 11 1 3 . 8 50 9
10 HC1 ,50 4 3 38 1 6 . 0 .50.6 .5.46 1.5.8 a All analyt,ical samples had ir and iiv spectra in agreement' with their assigned structures: each moved as a single spot on tlc on polyamide-MX. Over-all yield for two steps from nitro precursor. Recrystallized from I.'-PrOH-EtOAc. d Recryst,allized from iPrOH. e Recrystallized from absolute EtOH-petroleum ether (bp 3 0 4 0 " ) . f Xmnl 243, 313 mp. 0 Recrystallized from H,O-Et.OII. * Amax 244, 320 mp. Reprecipitated from lleOH-EtrO. Gave positive 4-(p-l1itrobenzyl)pyridinetest13 and moved as a single spot on polyamide-Nh' biit, contained about 20c', solvent after drying at 30'. The compound decomposed at higher t,emperatiires. Recrystallized from i-PrOH-petroleum ether (bp 60-110O). So extra acid added for hydrogeiiation and no Et3N added for bromoacetylation
3-Nitro-4-(phenylpropoxy)aniline Hydrochloride (37).--4 mixture of 1.57 g ( 5 mmoles) of 36, 10 ml of EtOH, and 10 ml of 12 S HC1 was refluxed with stirring for 1 hr, during which time the product separated. The mixture was cooled, and the product was collect,ed o n a filter aiid washed with 1: 1 i-PrOH-petroleirm ether. Recrystallization from EtOH-HI0 gave 1.19 g (75yc)of light yellow crystals, which gradiially decomposed over 183' : umbr 2800, 2530 (KH+), 1610, 1590 (C=C, NH), 1323, 1330 (NO,), 1260 (C-0-C), 840, 830, 740, 693 rm-' (phenyl CH). Anal. Calcd for Cl:HlGN20q.HC1: C, 3X.3; H, 5.34; N, 9.(17. Found: C, 58.2; H, 5.70; N, 8.83. 1-[ p - (I-Cyano-4-phenylbutyI)phenyl] -4,6-diamino-1,2-dihydro2,2-dimethyl-s-triazineEthanesulfonate (18). Method C.-A mixture of 2.71 g (7..i mmoles) of 20, 0.63 g (7.7mmoles) of cyanoguanidine, and 50 ml of reagent 31erC0 was refluxed with stirring for 20 hr. The cooled mixture was filtered aiid the product, was washed with hIepCO. Recrystallization from 1'-PrOH gave 246 mp; 2.81 g ( 7 7 7 , ) of white crystals: mp 196-197'; A,, umsx 3370, 3:323, 3130 (NH), 2230 ( C z S ) , 1660 (C=NH+), 16.50, 1.560, 1,510 (NH, C=C, C = S ) , 1190, 1040 (So3-), 750, 7 0 0 rm-l (phenyl CH). See Table I11 for additional dat,a.
-
1 [ p - ( 1-BromoacetamidomethyI-4-phenylbutyl)phenyl] -4,6diamin0-1,2-dihydro-2,2-dimethyl-s-triazine Ethanesulfonate ( 8 ) . Method D.--.4 mixture of 069 mg ( 2 mmoles) of 18, 100 mg of PtO,, 100 ml of EtOH, and 220 mg ( 2 mmoles) of EtS03H was shaken with H2 at 2-3 atm until 4 mmoles of Hr were consumed (about 3 hr). The filtered soliitioii war spin-evaporated in U U C U O leaving 1.13 g (94';;) of 22 as a hygroscopic white solid, Amax 244 mp. To a holiitiun of 449 mg (0.75 mmole) of 22 in 0.30 ml of DXF, stirred i n a11 ice bath, was added 0.:38 ml of 2 .lf Et& in NeOH (0.76 mmole) followed by 288 mg (1.18 mmole-:) of bromoacetic anhydride. After 20 min in the ice bath, the mixtiire was diluted with 10 ml of EtsO. The solution was decanted from the sepa, rated grim. The latter was tritiirated several times with fresk Et'yO (six 5-ml portions). Crystallization, then recrystallization from i-PrOH gave 200 mg ( 4 4 5 ) of white ( The comporind gave a positive 4-(p-1iitrobenzyl)pyridinetest13 and had A,,, 245 mp; uInax 3300, 3130 ( S H ) , 1680-1630 (amide C=O, C=?iH+), 1600, 1550, 1320 (NH, C=C, C=S), 1180, 1040 (SO,-), 740, 700 cm-l (phenyl CH). See Table I11 for additional data.
