Irreversible enzyme inhibitors. CLXXIII. Cure of Walker 256 ascites by

Cure of Walker 256 ascites by reversible and irreversible inhibitors of dihydrofolic reductase derived from 1-(substituted-phenyl)-4,6-diamino-1,2-dih...
0 downloads 0 Views 873KB Size
TABLEI I n Vitro INHIBITION' OF DIHYDROFOLIC REDUCTASE WITH

Rat

I50,b

Inhih,

No.

tlSSllP

p. \I

p211

Time, min

lf

w230 1112 Liver Spleeir Kidney

0.01:

0.06.i 0.063 0.06.i 0.065 0.00.5 0.00.;

60 20 00 20 20 20

0.18 0.18 0.18 0.18 0.18 0.18

F0 20

0,030 0.030 0.0.50

0.012

Tritesliire

5Q

80

WL50 11Lk Liver Spleen Kidney Intestine

0 , 0.57

W230 1lLk Liver Spleen Kidney Intestine

0.012

W256 D L' Liver Spleen Kidney Intestine

0.014

W2X D Lk Liver Spleen Kidney Intestine

0.02.5

W256 DLk Liver Spleen Kidney Inteqtirie 100

110

W23G DLk Liver Spleen Kidney Intestine

W256 DLk Liver Spleen Kidney Intestine

0.0.i2

0.014

II

0 , mi

8+

04 98 .2i GG 38 70

0.003

5

60 20 00 20 20 20

77

0,003

7+

83

0 , 0002

8+

0.050 0.050 0.060

60 20 60 20 20 20

0.074 0,074 0.11 0.11 0.11 0.11

60 20 60 20 20 20

64 72 30 70 11 6.i

0.4

2+

0.050 0,050 0.050 0.020 0.050 0.030

60 20 60 20 20 20

86 34 86 GI 43 9

0.0::

6+

0.0.50 0.030 0.060 0.060 0.060 0.060

60 20 60 20 20 20

62 5 12 36 8 5

0.2

4+

0.050 0.050 0.050 0.050 0.0.50 0.050

60 20 60 20 20 20

86 70 16 36 2.i 8

0.001

8+

,

0.050 0.0.50

0.027

0 . 008

0.018

0.014 0.022

0,015 0.01;

EDso,d

in0

o o:,n

0.018

7c i naetvn'

0.050 0.050 0.0.iO

00 20 20 20

Ratinge

78 28

27 7 ;>, * I)

+

0 S

10 28

21 03

7 15 0

0.0002 0 W256 0.0029 0,010 Liver a The technical assistance of Diane Shea with the enzyme assays is acknowledged. 160 = concn for 50y0 inhibn when assayed with 6 p M dihydrofolate in pH 7.4 Tris buffer contg 0.15 M KC1 as previously described." c A 45-90y0 (NHI)*SOIfraction of enzyme incubated with inhibitor at 37" in pH 7.4 Tris buffer contg 60 pM TPNH, then the remaining enzyme assayed as previously described," the 20-min runs were done at 24" due to the thermal instab lity of the enzyme. d Concn for 50% inhibn of growth of L1210 cell culture. e Arbitrary rating for selectivity of enzyme inhibn. Irreversible inhibn of W256 enzyme: 5 + , 85-100; 3+, 70-84; l+, 60-69. 0 S y n t h e ~ i s . ~ ~ Synthesis.I2b b a SyntheSpecificity: 1+ for each normal tissue showing
3 'I;

,-i

i-

1 6 2 fi

I fi :1 ti

I (i I1 1;

64

50 25 12.5 6.2.5 3 .13 1.56

12.6 6.25 3.13 1.56 0 78 0.:j9 0 1')

v

Toxic 3 16,

lI.ir(

25

-Survivors---1)ay 6n

7+

IRREVERSIBLE ENZYME INHIRITORS. CLXSIII

Journal of Medicinal Chemistry, 1970,Vol. 13, No. 6 1133 TABLEI11 (Continued)

R

N O

8

3.CI.4.0CH2CONH

0

Test group'

Ae A Ge G Eh E E

SOIF

'4 A G p

:c

Eh E E

E E ID

Ae A Ge

G G Eh E E E 11

3CI.4-(CH2),C0NH

0

Ac Ge

SO,F

Day 80

50 25

Ratingd

16

2 f

2

G+

1

4+

>64

8+

C,,2.i 3.13 1,56

n,m 0,no

100 . 0i 2.i 12 -5 6.28 3,13 1 ,56 0,78 0,39 0.19 I

50 25 12.5 6.25 3.13 1.56 0.78 0.39 0.19

F F

25 12.5 6,25 3.13 1.56 0.78 0.39

F

0.19

G Fe F

Chemother index'

12,5

l? Ae

.

--Survivors

w/kg

per dsyb

1 /G 1 '6 4 6

5/ G 2 /6

0 0 25 12.5 6.25 3.13 Dh 1.56 D 0.78 D 0.39 n 0.19 Test group indicates which compds were run simultaneously with the same control group. * Single ip doses on days 1-9 after injection of lo5 ascites cells on day 0.Ien c The chemotherapeutic index was arbitrarily selected as the dose range giving 4/6 30-day surviAnimals sacrificed In vitro rating of selectivity of enzyme inhibn from Table I. e Controls survived a median of 8 days. vors. after 30 days and longer survival data not obtd. Median survival time was 13.5 days; T / C = 170. Controls survived median of Median survival time was 14 days; T / C = 186. 1 Median survival time was 12.5 days; T / C = 166. 7.5 days. 12

Ge G G G

3,4-Ch

Q

achieved, the results with some of the more selective compounds indicated that an insufficient amount of the inhibitor was reaching the target enzyme. Part of the difficulty was traced to poor transport through the L1210 cell membrane by use of L1210 cell culture.ll This difficulty was soon overcome by use of appropriate structures that allowed good transport.12 A second complication was found by Folsch and Bertino. l 3 They observed that irreversible inhibitors (12) (a) B. R . Baker and R . B. Meyer, Jr., ibid., 12, 668 (1969), paper CLIV. (b) B. R. Baker and E. E. Janson, i b i d . , 12, 672 (1969), paper CLV. (c) B . R . Baker and N. M . J. Vermeulen, i b i d . , 12, 680, 687 (1969). papers CLVII, CLVIII. (d) B . R . Baker and W. T. Ashton, ibid., 12, 894 (1969), paper CLIX. (e) B . R . Baker, E. E. Janson, and N. M . J. Vermeulen, i b i d . , 12, 898 (1969), paper CLX. (f) B. R . Baker and N. hl. J. Vermeiilen, i b i d . , I S , 82 (1870), paper CLXVI. (13) E . Folsch and J. R. Bertino, MOL, Pha.rrnncol., 0, 93 (1970).

of dihydrofolic reductase of the sulfonyl fluoride type, such as 1 and 3, were rapidly converted by mouse

l,X=F 2,X=OH

4,X=OH

(Ill,

I

+-I( 1

I

2 .-,

2 (i 4 (i

2 (i 1 ti

1'11

wrum into metabolic product. which LWTC moit probtliv 1,1210 ccll mcmbr:itir; :I niow watt :tpprc)xini:tt i o i i :tbly the corresponding sulfonic x i & , 2 :tiid 4, row t i h c h obtained b) taking into :tccourit both thct r('sl)cctivcllj ; this nwtabolic conversion was much Irs. vcriihl(Jatid irrcvcmiblc effect of tlic compound. on t l i o III r:Lt ieruni :ml unobserv:ible with human serum. target insid(>t h c c ~ l l l .iixrirly, dill? di-ofolic rcduct:i.(. I ' Since it mas clear from their results that the mouse The reversible Isu'h of the h compound* with the R ; t l l ~ c ~ r :in utiiatisfactory test aniinal, we turned to the 2% enzyme differed only seveiifoid from 9 to 5. lo-^ I-mring Walker 2SC, in the ascite form in the prritorie:il of the-r inhibitor howed little difierciicc i t I r w c i siblcs c:ivit\. Eight irreversible irih bitors of dihydrofolic inhibition of W:Llker 236 11s.liver wzynic, the n i : i \ i m i i i i i tw1uct:Lw of the dihydro-s-triazine type such as 1 were rpre:id for :t singlr cornpoutid being twofold. 11.wlcctecl for test C J ~ oJn the basis that they showed good seversible inhibition of the tumor enzyme varicd froin t o fair tr:in5port characteristics; one 2,&diamino100yLCor 1 down to E!ycfor 10. X potent revoriit I(% IJJ rimidinc w:iq alia selected even though transport WL. inhibitor (12) is iticlutied for cornpiirison; 12 is thc mc'-t 1e.h effective. The results are the subject of this paper. potent compound i n Table I :IS :L reversible iiihibitol / I ( l'itro Assays.----4ssay of t h e S dihydro-s-triazines of the Walker 3.50 c ~ r i zmr ~ : ~ n d:i,\ :HI inhibitor of 1,1201 n i t h thti dihydrofolic reductase from Walker 256 rat cell culture. tumor :tnd 4 normal tiwurq are collated i n Tablr I. In order to t r j to rorrrlate e m ) me activit), i n 'l':lbl(x TIIP El),l, i n 1,1210 cell culturet4representb a first apI with the c4fec.t of t h e compounds o n W d l t e ~2.3; I)roYim:itiori of thr ahilitv of the compoiind to penetmtr :tsritck. i l l 13ioo ('l':~ble I I I ) , w c h compound n:is :I+ 111 I I O I I V K C \ \ I I ~ T P c B f ( l i p C ( ' h s ( for IIIPW i!iia .igirc(l :L r;ititig ~ ' ( I I , wlc.rtivit\. by their effective tie.^ 01 i 1 1 , 1 1' I I ~ m m r l f ~ i i ~ l i it hd i 1 1 I> l,i(ik I n (

TABLE VI In Vivo INHIBITION OF WALKER 256 ASCITES BY REVERSIBLE INHIBITORS

---__I n vivo

Ko.

I5

1 (i

R

Rat tissue

~ - ( C F T ~ ) ~ C O N H C R H I C H ~ - ~W2.56 . liver

.?-CI-4-O( C H ~ ) ~ O C R H B

W25O 1,iver

~ ~ r.ll

n , n22 n ,ox0

~

,

a

Test

grollgc

Ke K K K K

n. 04s n. 04.1

Kp K K K K

n.02G

Fe F F F F L!

n .n4s

1,

I, W256 Liver

0,0086 0.021

Fe F F F F Lf L

L L L W2.56 Liver

0,007s

Fe

n.

F F F F Lf L

mnkx per d a y d

. 0i 2.5 12.*5

6.25 3.12

.io 23 12.5 6.25 3.12 in0 .3 0 2.5 12.5 6.25 3.12 1.56 0 78 25 12.5 6.25 3.12 1.56 6.25 3.12 1.56 0.78 0.39

25 12.5 6.25 3.13 1 ..i6 6.2.3

3.12 1.56 L 0.78 L 0.39 The technical assist>anceof Diane Shea with the enzyme assays is acknowledged. = concn for SOY0 inhibn of dihydrofolic rediiotase when assayed with 6 &' dihydrofolate in pH 7.4 Tris buffer contg 0.15 M KC1 as previously described.'' * Concn for 50% Test, group indicates which compds were run sirnultaneoiisly with the same control group. inhibn of growth of 1,1210 Fell culture. d Single ip dose on days 1-9 aft,er injection of 1W ascites cells on day 0. e Median survival of controls = 8 days.16:I f hledian survival of rontrols = 8 days.I6b L

irreversible inhibition on the tumor enzyme and lack of effectiveness on the enzyme from 4 normal tissues. This rating system varied from 0 to 9f as described in footnote e of Table I. The compounds with the highest rating ( 8 f ) were 1, 7, and 11 ; the irreversible inhibitors with lowest rating were 8 (2+) and 10 (4-k). Of course, a reversible inhibitor such as 12 would have a rating of zero since 12 has no selectivity in inhibition at the target level. Corresponding data with a reversible and a n irreversible inhibitor of the 2,4-diaminopyrimidine type are shown in Table 11. In Vivo Inhibition of Walker 256 Ascites.-When the

compounds in Tables I and I1 were assayed against peritoneal Walker 256 ascites in the rat ip,16all showed some cures except the potent reversible inhibitor 14 (Tables I11 and IV). 16a The irreversible inhibitors were clearly more effective in vivo than the reversible inhibitors, 12 and 14, even though the latter 2 compounds were more potent in vitro against L1210 cell culture. The further superiority of one irreversible inhibitor (1) over the excellent reversible inhibitor (12) (15) CCNSC, Cancer Chemother. R e p . , % l ,1 (1962).

(113) We wish to thank Dr. Florence White of the CCNSC for these d a t a obtained by (a) I . Wodinsky of Arthur D. Little, I n r . (h) 4nn Smith of Soiirhem Reqenrrh Institute, (r) Ha7elton J.&horntorier.

No.

!I

NJ

K S

IO

1;.

w:w clearly demonstrated by delay of treatment until 2 tl LJ before death (Table I'); I h a , l > a number of cures by 1 \t ere still obtained, demon*tr:iting the reni:irkable :\ctivity of 1 in this test system. Attempts to correlate the chemotherapeutic indicc'i with the i ) i vitro ratings of the irreversible inhibitors in 'l':tble I I1 were poor; other arbitrary assignments foi, the chemotherapeutic index : i d it/ ~ i f r orating gavc. ( ~ c ' i ipoorer correlation. Thii lacL of correlation indicated that one or more other unl\non.n factors than those presented in Tables I and 111 were playing a rolr I I I tlie helectivitj-. This lacli of correlation between the irreverhible inhibitors also ca e doubt on the conclusion that irrevcrsible inhib uch as 1 were more effective t h : ~ ;in) rcvcrsible inhibitor; for example, it was possible--though seemingly improbable-t hat the irreversible inhibitors, 1 and 5-11, were more effective than the reversible inhibitor 12 due to the more complex side ch t i m of the irreversible inhibitors. Therefore, 5 ievcrsible inhibitors (15-19) having the structure of 1 , 5, 8, 9, and 10 (Table 111), respectively, where oril~ the SOzIi' group was removed, were synthesized for i l l i ' i w evaluation. Both in v i t w and in vivo data are presented in Table Comparisons of some of the rcwmible and irreversible inhibitors in delayed treatinctit re coll:ited in Table VII;lflb a number of tlic' compouiiclh were reillarkably effective, particularly the

reversible inhibitor.. A s could be expected, thc conipounds wcre less effective by ip injection against Walltet, ?.'Xi i n the leg muscle. but still showed a positive rv-E)OMC (T:lble 1,111). The Ihnning leulmriia in asciteh form i- rc.spoiisivcb to :imethopterin treatment, (iO-7OC; extenhioil of lifv being obsriwd, but thcrr i r e r ~no 3O-d:ij Yurvivors (Table I S ) . 8c~ver:ilof tlie revcr-iblc arid irrevcr4bl(b inhibitorh were tar mor(' effective than arnethopteriii, the m o h t effrrtive being the reverbiblcl inhibitori 17-19

Discussion A *erirs 01 9 :Ictive-~ite-dirrcted irrrvcrsibl~~ inhibitors of d~h~-tlrofolic rfductase were selected for extenhive cvaluation iri the rat against Walltci 236 ascitei. This zelectiori \ \ a b based mainly on the> ability of the compounds to be transported through a mammalian cell n all as approximated by inhibition of L1210 cell culture, regardlei- of 11hether :L compound shoved good tissue specificitj. in inhibition of dihydrofolic reductase (Tables I and 11). Some of these irreversible inhibitors were remarkably effective i n curing the Walker 250 ascites (Table 111)even when treatment n a s delayed to 2 days before death (Table

1.). 'I'litl Furt that irrwersible inhibitors such as 1 arid 5 11 n ere niuc*li m o i effective ~ i r i r / w than the potent

5

0

3-CI-4-WCH2),0

SOzF

Test groupa

me/k dayb

---Tumor Test

w--t Control

P P P

100 50 25 25 12.5 6.25

0.9 0.5 1.6 4.4 5.6 7.1

8.5 8.5 8.5 7.2 7.2 7.2

18 61

1.2 2.0 2.6 3.7 4.7

3.9 3.9 4.9 4.9 4.9

30 51 53 75 95

-6 -9 -8 +2

0.9 1.6 1.3 2 .R 3.5

3.9 3.9 4.9 4.9 4.9

23 41 26 31 71

0.7 1.4 1.7 2.9 3.5

3.9 3.9 4.9 4.9 4.9

17 3.5 34 59 71

Q Q Q R

R R S S S

R

77 98

016 6/6 6/6 6 16 6/6 6 /6

- 26

- 10 -9 -3 -1

- 19

30 26 25 12.5 6.2:

4 /6 6 /ti

6 /6 6 /6

- 19 -3 -6 0 +2

8 S

100 50 25 25 12.6 6.25

6 is 616 6 /6 6 /6 6/6 6 /6

- 30 - 15 -2 0 +3 +1

1.2 2.1 2.5 3.2 2.8 3.1

3.9 3.9 3.9 4.9 4.9 4.9

30 53 64 65 57 63

8 8 8

50 25 12.5

6/6 6/43 6 /6

- 16

0.4 0.9 "8

4.!) 4.9 4.0

18 t57

8 8

2.i 12. .i 6.25

6 /6 616 616

-3

1.0 1 . !J 3.2

4.9 4 . !) 4.9

12.5 6.25 3.13

6/6 6 /6 616

- 19 -7 -2

0.6 1.9 2.1

4.9 4.9 4.9

42

-2 -4 -4

0.1 1.7 2.6

4.9 4.9 4.9

2 34 53

3.9 3.9 4.9 4.9 4.0 day> 3-6.

28 84 26

11

lt S S S

11 11 11

8

8

S S 8

6/6

616 616

s/s

s

100

616

S

50 25

6 /6 6 /6

S It 11

25 12.5 8 12.5 6.2d 8 d 3.13 a Teht group iudicate, which compds were r u n simultaiieoudy with the bame controls. d Day 7 .

reversible inhibitors (Tables I11 and IV) with a 3,4dichlorophenyl side chain (12, 14) was initially exciting since it supported our concept that good irreversible inhibitors should be better agents than reversible inhibitors;' this concept was later formalized in the bridge principle of ~ p e c i f i c i t y . ~ However, *~~ it (17) Reference 6, pp 173-184.

3

6 /6 6 16

s

~-(CHZ)ZCOX"CGH~CH~-WL

10

100 50 60 25 12.5

R 8 8

15

100 50 25 25 12.5 6.25

TIC

-4 -1 1 0

6 /6 - 14 1.1 6 16 -a 3.3 6/6 -7 1.3 6 16 +1 2.9 616 +4 4.8 coiitrul gruup.16~ b Single ip dube 011

x

20 38

65 12

38

59 97 c Froin

was disturbing that good correlation of in vitro irreversible specificity (Table I) with in vivo cures was not seen; therefore a series of reversible inhibitors (15-19) differing only from the irreversible inhibitors by replacement of the SOzF group Fvith H were synthesized for comparison. The reversible inhibitors (15-19) n i t h more complex

res1

Illy;,'kg

A 0.

proiiiln

tier dah'

1

'I' 1' 1

'I' 'I' 'I' T

'r

'I' '1' T ti

*:o.seL

ti ,

:i.I:;

I . .ili

v

I 2 . .i

\' \

t i , 2Tr

IT 1'

\v 11:

:;, 1:; I 2 , .-I ti . 2.i : j . I:) 1 . .i(i ,-It I 2.i I2 ,.-I

li . ;;. 1 : i

2 .-I l2,.i

r \v

li.2.-i

\'

I 2 , *-I li . 2.; :i. 1:;

\\

v kv

LV

iv

:i. 1:; 1 ..io

1 ..i(i ( I . 7h

I1

.io

I'

2.i I 2 .-I

IT Il

.

ti 2,i

iv

:i. 1:i

1'

I2 . ,-I ti .2 .-I :i, I::

IT I'

M.

iv r U

u [ Amethopt eriri]

7s

1 1 . 7h

U

20

I2 , .-I li . 2.i :;. 1:t I ..ili

'I' '1'

u

4-(CH2)rC'6€Ia

2.i

2 .-I 12.5

-I'I' 'I'

\I'

1!Id

l).iS

'1'

IT

17

:i,I:$ I . Tili

IJ.

u

II

12,ti li.2.-)

'I'

I'

10

?.-I

I .!-Ai

U.7K

12 , .-I ti, 2.i :3 . 1:3

T T

rr

I ..5 0.75 0.38

1'

0.1H

w

)

side chains were much more effective in vivo (Table VI) than the 3,4-dichlorophenyl analog 12, even though the latter was more effective as a reversible enzyme inhibitor and in inhibition of L1210 cell culture (Table I). When the reversible inhibitor-irreversible inhibitor pairs (1 us. 15, 5 us. 16, 8 us. 17, 9 us. 18, and 10 vs. 19) were compared in vivo against Walker 256 ascites (Tables 111, VI, VII) the reversible inhibitors were not only as effective cures, but in some cases were better than the corresponding irreversible inhibitors. That a potent reversible inhibitor of dihydrofolic reductase could be effective against Walker 256 was anticipated;lXWalker 256 cells are dependent upori folic acid-folic reductase for growth, but are deficient in the active transport of folic acidlg and therefore would be inhibited by a reversible inhibitor that enters the cell by passive diff usion.lE However, this borderline folic acid deficiency would not explain the difference in i t i vivo activity between the simpler reversible inhibitors (12, 14) and the more complex reversible inhibitors (15-19). Some of the compounds were then assayed by ip injection against Walker 256 in the leg muscle (Table VIII). A number showed inhibition, but were much less effective than ip treatment of peritoneal Walker 256 ascites as might be expected; first, the local ip concentration would be much higher initially than the concentration achievable in the leg by ip injection and, second, a compound would have to go through the blood stream from the ip cavity to the leg with the possibility for destruction of the compound. Again the reversible inhibitors were as good as the corresponding irreversible inhibitors. Since Walker 256 cells are on the verge of folic acid deficiency due to impaired transport a folic acid, Walker 256 does not respond t o amethopterin which presumably must enter a cell by the folic acid transport mechanism.lS l 9 Therefore a rat tumor with an adequate active transport system for folic acid that! was resporisive to amethopteriri-namely the Dunning leukemia ascites-was selected for evaluation of the folic reductase inhibitors (Table IX). There was no correlation between irreversible inhibition of the Dunning leukemia dihj.drofolic reductase (Table I) arid iri vivo activity; in fact some of the poorest irreversible inhibitors (9, 10) were the most effective. Surprisingly, reversible inhibitors were definitely more effective than the corresponding irreversible inhibitors. Even though some of these inhibitors are sufficiently active in vivo to warrant pharmacologic studies prior t o study in man, the lack of correlation of in vivo and it! aitro data is indeed disconcerting for a so-called program in rational design of enzyme inhibitors for cancer chemotherapy. Therefore, an ancillary study 011 the metabolic fate of l was undertaken.2u The only metabolic product detectable in the urine arid feces was the sulfonic acid 2 which accounted for 77% of the radioactivity injected; no sulfonyl fluoride (1) was excreted. Since rabbit liver, kidney, intestine, and plasma contain (18) B. R. Baker, D. V. Santi, P. I. Almaula, and \V. C . Werkheiser, J . M e d . Chem., 7 , 24 (1964). (19) (a) W.C. Werkheiser, Proc. Amer. A s s . Cancel. Res., 3, 371 (1962). (b) W.C. Werkheiser, L. W.Law, R . A. Roosa, and C . A . Nichol, ibid., 4, 7 1 (1963). (20) A . J. Ryan, N . M . J. Verr~reulen,a n d 1J. H. Esker, J . M e d . Cham., 13, 1140 (1970), paper CLXXIV.

an enzyme that can hydrolyze DFP to diisopropyl hydrogen phosphate21 and since mouse serum contains an enzyme that can hydrolyze 1 to the sulfonic acid ZJ13 rat liver and serum were investigated for hydrolysis of 1 to 2. Hydrolysis by rat serum was slow,13requiring 3 hr for 65% completion. Hydrolysis by a rat liver extract (either 0-45% or 43-90% (SH&SOI fractions) was nearly complete in 1hr; it was clear that 1 was being rapidly detoxified to 2.20 Although 2 was a good reversible inhibitor, this zwitterion penetrated the membrane cell wall very poorly (EDSO= 18 pJ1; compare 1) and was therefore a detoxification product. The occurrence of this “fluoridase” in liver brought up the possibility that the tissue specificity of inactivation of dihydrofolic reductase by a number of inhibitors was riot due to differences in enzyme structure from tissue to tissue, but was due to the relative rate of hydrolysis of sulfonyl fluorides by this “fluoridase.” In another ancillary study, dihj drofolic reductase from mouse arid rat liver was purified by an affinity column, then the irreversible inhibition reinvestigated. 2 2 All of the tumor specific dihydrofolic reduct ase inhibitors now showed good irreversible inhibition of the purified liver enzyme. Even though these sulfonyl fluorides effective on 1,1210 or Walker 256 dihydrofolic reductase were ineffective o n the liver enzyme due to the presence of the “fluoridase” in liver, the tissue specificity is still real and still should be usable in chemotherapy in the same manner as isozyme specificity, a possibility previously demonstrated.8 With this new insight on a major biochemical difference between at least some tumors arid a normal tissue such as liver-namely, the presence of the detoxifying “fluoridase” in liver and its absence in several tumors-furt her investigations on irreversible enzyme inhibitors of the sulfonyl fluoride type will he on w firmer basis. I t is now less likely that tumor and liver dihydrofolic reductase are isozymes, since no isozyme specificity has been seen with at least 100 active-sitedirected irreversible inhibitors of the sulfonyl fluoride type. However, it is still possible that other enzymes will have isozyme forms-particularly those of large molecular weight that are dissociable into subunits; selective irreversible inhibition of lactic dehydrogenase, which has 2 different subunits, v a s achieved early in this program.8 With enzymes of lower molecular weight such as dihydrofolic reductase (20,000), or even dissociable enzymes of higher molecular weight, the desired tumor specificity for chemotherapy still may be achievable by use of selective detoxification of sulfonj.1 fluorides by “sulfonyl fluoridase” in normal tissue. Finally, the question can be aslied why are the more complex reversible inhibitors (15-19) more effective against Walker 236 in vivo than the simpler reversible inhibitor 12; if and when this question is answered, perhaps anot her new arid usable concept for specificity will arise.

Experimeiital Section The reversible inhibitors (15-19) in Table VI were synthesized by the same routes used for the corresponding irreversible (21) .L Rlaeur, J . Bid. Chem.. 164, 271 (1946). (22) B, R . Baker and N. M. J. Verxneulen, J . .Wed C‘hem., 13,1143 (1970), LJayer CLYXV.

lrreversible Enzyme Inhibitors. CLXXIV. Metabolisrii of 4-[p-(4,6-Diamino-l,2-dihydro-2,2-dimeth?-l-s-triazin-l-~ l)hvdrociniiaiiiitlo]-otoluenesulfonyl Fluoride (NSC-113423),an liclive- Site-Directed Irreversible Inhibitor of Dihj,drofolic Reductase

The IC-labeled title coinpound (1) was synthesized frum [1+CJcyaiioguaiiidine, acetolie, and p-(p-;triiiuohydliJcinnamido)-o-toluenesulfonyl fluoride (4). The sulfoiiic acid 2 corresponding to hydrolysis of the SOZFgroup W H Y the only radioactive excretion product, 20yc of the radioactivity appearing in the urine and 58Yc in the feces. Investigation of serum and liver extract, of the rat showed that the liver contained a "sulfonyl fluoridase" that rapidly converted 1 into 2 ; hydrolysis by serum was rniich slower, biit c*ornpletein 20 hr t % t37".

In the previous paper of this series,2 9 active-site-directed irreversible inhibitors of dihydrofolic reductase of the S02F type were tested against Walker 256 ascite. and Dunning leukemia ascites in the rat ; although cures could be achieved, no correlation between tissue specificity of irreversible inhibition of the enzyme and in vivo effectiveness was apparent. Furthermore, 5 reversible inhibitors-where the S0Jp group had been replaced ( 1 ) This work was generously supported by Grant S o . CA-08695 from t h e National Cancer Institute, U. S. Public Health Service. 12) For t h e previous paper in t,his series see B. R . Baker, N . M. J. Vermeillen, W.-1.-4shton. and .%. .J. Ryan. J . .\led. Chem.. 13, 1130 (1970). ,:$I On sabhaticsl leave from t h e University of Sydney, Australia. t $1 (a) N . 11. .J. Yermeulen wishes t o thank the Council of Scientific and Industrial Research, Republic of South .\frica, for a tuition fello\r.alli~~. ( I > ) TI, whorn correspondence should he addressed

1 ) ) H- -were ab effective 111 uivo ab the irreversible inhit)itors. These results indicated that other factors sucli transport and metabolism might be playing a r o l ( b

which negated correlation. Therefore one of the coiiipounds (1) wab labeled with I4C in the triazine ring :iiitl subjected t o a metabolic study in the rat. The rcsultc are the subject of this paper. Chemistry.-The *4C-ld~led1 I\ ab 73 Iithchized h i condensation of [14C]cysn~guanidlne,the arylamiiici precursor 4,J and acetone according to the general method of RIodest.6 Two obvious possibilities for metttbolic change of the d e chain of 1 were hydrolysis of ii) (

13 I< l$dhel a n d