Junuary 1969
IHHEVEItSIBLE EXZYJIE IKHIBITOKS.
triazin-1-yl)phenoxyacetyl]sulfanilyl Fluoride Ethanesulfonate (2b) (Method D).-A mixtrire of 777 mg ( 2 mmoles) of 21a (Table 11), 6.5 mg of P t 0 2 , and 100 ml of EtOH was shaken with Ha a t 2-3 atm until reduction was complete. To the filtered mixture was added 223 mg of EtS03H then the solution was evaporated in vacuo. To the rehidual 20a.EtS03H were added 20 ml of reagent l\lerCO and 177 mg ( 2 . 2 mmoles) of cyanoguanidine.
CXIAI
101
After being refluxed with stirring for 18 hr, the cooled mixture was filtered and the product was washed (lte2CO). Recrystallization from EtOH-I-I,O gave 630 mg ( 5 3 : ; ) of white crystals, mp 224-226" dec. See Table I11 for additional data and compounds prepared by this method. Method E was the same except Raney nickel was used a:, a cata1yst.j
Irreversible Enzyme Inhibitors. CXLI. 1 , 2 Active- Site-Directed Irreversible Inhibitors of Dihyclrofolic Reductase Derived from 1-[p-(p-Fluorosulfonylphenylureidomethy1)phenyl]-4,6-diamino-1,2-dihydro-2,2-dimethyl-s-triazine
The title compouiid (1) showed a poor order of active-site-directed irreversible iiihibitioii of dihydrofulic reductase from mouse L1210 leukemia, liver, spleen, or intestine since too high a concentration of inhibitor had to be used to show good inactivation. Substitution of a methyl group meta t o the SOIF moiety of 1 gave compound 6 that was still a relatively poor irreversible inhibitor of the L1210 enzyme, but now showed tissue specificity by its failure to inactivate the liver enzyme. More effective irreversible inhibitors (5, 8, 9) were obtained by substitution of a chloro atom ineta to the junction of the 1-phenyl to the s-triazine ring; however, these compounds did not show specificity since the liver enzyme was still inactivated.
The title compound (1)4 was found to be a n activehite-directed irreversible inhibitor5 of the dihydrofolic reductase from L1210/0 and L1210/DE'8 mouse leul;emh6 However, 1 was not as good a n irreversible inhibitor as the prototype irreversible inhibitor +ice the total amount of inactivation by 1 was lower than 2 (Table I). Furthermore, neither 1 nor 2 showed hpecificity toward the L1210 enzyme with minimal effect on the enzyme from normal ti.;sues such as liver, bpleen, and intestine.6 Therefore, additional synthetics related to 1 were made and evaluated to see if a better
1, R = N H 2, R = CHI
and more selective irreversible inhibitor could be designed. The results are the subject of this paper. Enzyme Results.-& pointed out in t'he earlier summary paper,s assay of 1 for irreversible inhibition of dihydrofolic reductase n-as difficult due to a medium order of t'otal, but fast', irreversible inhibition that' sometimes gave low zero-time points. Similar difficulties n w e encountered wit'h 3,4which showed 0-30% irreversible inhibition of the L1210 enzyme depending upon ho\v lon. the zero-time point' for enzyme concen(1) Tliis uork was generously supported by Grant C.1-08695 from the National Cancer Institute, U . Y. Pulilic Health Service. (2) For the previous paper of this series see B . R . Baker and G. J . Lourens, J . .>fed. Chem., 12, 95 (1969), paper C X L of this series. (3) G . J. L. wishes to thank the Council of Scientific and Industrial Research, Republic of South .4frica, f o r a tuition fellowship. (4) B . R. Baker and G. J. Lourens, J . M e d . Chem., 11, 666 (1968), paper C S S V I I of this series. (51 B. R. Baker, "Design ol .%ctive-Site-Directed Irreversilile Enzyme Inliil~itors. The Organic Chemistry of the Enzymic .\ctive-Site," John Wiles and Sons, Inc., Xew l o r k . X. Y.,1967. (61 B. R . Baker, G . .J. Lourens. R.B . hleyer, J r . , and X. RI. J. Vermeulen, .I. .Met/. Cliem., 12, 67 (1Y69), pager C S X S I I I of this series. ( 7 ) 13. K. Baker &nilG . J . Lourens, ibiri.. 10, 1123 (19671, yaper C V of this aeries.
tration wa5; in repeated runs 3 failed to show an) irreversible inhibition of mouse liver dihydrofolic reductase. Thus, 3 showed selectivity, but a poor order of irreversible inhibition of the L1210 enzyme; the latter n-as most probably due to extensive enzymecatalyzed hydrolysis of the S02F When the side chain was moved to the mefa position (4), irreversible inhibition was lost . g The effect of substituents on either or both benzene rings of 1 on irreversible inhibition was then btudied. There is orily one position on the phenyl group next to the triazine that can be substituted without loss of binding, and that is the position meta to the triazine junction; ortho substitution leads to a large 1055 in reversible binding. lo,'' The 3-chloro atom was selected since this could have a beneficial effect on both reversible bindinglo and irreversible inhibition. The 3-chloro substituent 5 on 1 gave a fourfold increment in reverbible binding. Furthermore, 5 wa5 a better irreversible inhibitor of the 1,1210 enzyme than 1 when compared a t a similar concentration of reversible E1 complex;'* however, 5 still lacked specificity since poor irreversible inhibition of the mouse liver enzyme was still observed. Substitution of CH, meta (6) to the SOnF moiety of 1 also gave about a fourfold increment in reverbible binding. Xlthough 6 was not a better irreverqible inhibitor of the T,1210 enzyme a t equal E1 concentration,'* 6 wab more qecific than 1 since 6 did not inactivate the liver enzyme. Introduction of CHJ (7) ojtho to the SOJ? moiety of 1 gave about a twofold iricremerit in reversible binding; however, 7 was (81 B . R . Baker and ,J. A . IIilrIbut. i b z d , 11, 233 (lY68), paper C S I I I of this series. (91 B. R . Baker and G . .J. Lourens, ibid., 11, 39 (19681, paper C S I I of this series. (10) (a) B . R. Baker, ibid., 11, 183 (1968); (11) B . R . Baker and h l . .\. Johnson, i b i d . , 11, 486 (1868); (c) B. R . Baker and B . - T . Ho. J . Phurm. Sci..
63, 1137 (1964). (11) E. .J. Rlocieat, .I.
( J r y . C i i c m . , 21, 1 (195ti). ( 1 2 ) ].'or tlie kinetics of irreversiltle irlliiliitiun SUC rttf 3 . C~liiLI)1ur8 .
102
0 . uo4 1
Livei
0 01.5
0 002.5
Spleeti Iritebtiiie L1210 0
L1210 1)FX 1,1210 'Fl'L8' L12110 F I M m 1,12110 0 LlPlO,I)E'\ L1210 0
Liver Spleeu LlPlO 0 L1210 I)Fh
0 0'2s 0 066
il 012
0,0020
i) I112
0 Oo"0
0 019
0.00:: I
IJlvel
1,1210 I)FX 1,1210 0 Iivci L1210 0
0 011
ti0 Liver 'LIhc tecluiical nssistanw of I)iaiie Shea, Sharo Carolyii \ \ : ~ i l caitli tliese ' is :~c:liini~\I[:tl~etl. ' I 3 tho parent wild fitrain, while /&'It8 aiid 'DF8 are eve1 of dihydrofiilics r c d u t i t t.o :tmethopteriii with x sayed with 6 P M dihydrofolate and 30 pL,llT P N H in pH 7.4 Tris buffer containing 0.13 dl KCl as previously described.6 Incubated st 37" in p H 7.4 Tris buffer in the presence of 60 T P N H as previously described."' e I,,I = cOf:; inhibition. which is valid since [SI = 6 K , = 6 j d dihydrofolate; see ref 5, p 202. g Calcillated from [ E I ] Estimated from Ki = Km[l:~]/[S] Dabs from ref 6. Zero point determined = [Et],'(l Ki ' [ I ] )where [EI] = amount of total enzyme (St)reversibly complesed.12 the zeroby addition of inhibitor to assay ciivette.6.7 i From six-point time study.7 k Due to curvature i n the assay for 1 he zero time enzyme coiioentratiori was quite variable. 1 1)ata from ref 4. m Data from ref 9.
+
iicithclr L: better nor more specific irreversible inhibitor
1. The 3-chloro (9) and : h i e t h y l (8) derivatives of 7 were then synthesized for enzymic evaluation; little change in either reversible binding or irreversible inhibition was seen. From these studies it would appear that 5-8 mould he worthy of in ciiio testing, even though the criteria arbitrarily set for in viuo testing6 have not been met. 1'urt her in or(^, \iiiccx the C'HJ of 6 0 1 1 1 impal tilid the CI atom of 5 impurts better irrever5ible i i i l i i b i t i o i i , n combination of thew substituent5 (5a) would lw worthy of study where XI and R,are ;Lcombiliation t hun
1
IRREVERSIBLE ENZYME ISHIBITORS. CXLI
January 1969
requisite 0-phenylurethans (11) were prepared by reaction of the appropriately substituted sulfanilyl fluoride ( with phenyl chloroformate in boiling C,5H6.4 The urethans were then treated with 4-nitrobenzylamine (14)4 or its 2-chloro derivative (15)15 in DMF to give the mixed ureas (12, 13) (Scheme I).
103
single spot on tlc on Brinkmaim silica gel GF and had ir and uv spectra in agreement with its assigned structure; each gave combustion values for C, H, and N or F within 0.47, of theoretical. The physical properties of 5-9 are given in Table 11.
TABLE I1 PHYsIC.4L PROPERTIES O F
SCHEMEI
R,
1-
10
11 No.
14, R : = H
12, R I = H 13, R,=C1
15, R, = C1
I
J.
R2
VH,.EtSO,H
5 6 7 8 9
Ri C1 H H C1 C1
% LIP, O C Methodb yieldC dec D 3tjd 225-227 E 53d 165-167 E 46d 221-223 D 2 V 221-223 D 32d 213-216
RP
H 2-Me 3-hIe 3-hle 3-C1
Formula
CigH~iClFNrOaS.CzHsSOsH C?~HZIFN~O~S.C?H~SO~H CzoH~lFNi0aS.CzHsS0aH CtoH?aCIFPiirOaS.CzHsSOaH CisHtoClzFPi~OsS.CIHsS03H
,. Numbered from NH. See ref 2 for methods. c Yields of analytically pure material analyzed for C, H, F. d Recrystallized from EtOH-H20. e Recrystallized from MeOEtOH-HzO.
I
TABLEI11 PHYSICAL PROPERTIES OF A
7, R , = H 8, R,=C1 a, R > = H c, R z = 3-hIe b, R2=2-Me d, R2=3.C1
C~II,OCONH (Qjso, F e, R2
=
p-SO-C,H,CH-SHMe
21
-
-
R YO
The nitro group of 12 and 13 was reduced catalytically with a Raney Ni and PtOz catalyst, respectively; the resultant amines were condensed with cyanoguanidine and acetone by the method of AIodestlI to give the candidate irreversible inhibitors 5-9. The S-methyl-0-phenylurethans (19, 20) were prepared from S-methylsulfanilyl fluoride (18) ;I6 unfortunately, condensation of 19 with p-nitrobenzylamine (14) failed to take place and the more reactive 0-nitrophenylurethan (20)l7 gave mixtures. MeNHCGH,SO,F-p 18
I
2-OMe No.
yieldh
RC
Mp, 'C
FOImula
llb 2-Me 76 127-129 CirHinFN0,S llc 3-Me 83 120-121 CL~HI~FNO~S lld 3-C1 64 123-124 CiaH9CIFN0,S lle 2-OhIe 73 131-132 C:~HI~FNO,S 19 N-Me 58 70-71 CI,HI~FNO~S b Yield of analytically pure material a Numbered from XH. recrystallized from C6H6-petroleum ether (bp 60-110") and analyzed for C, H, N except lle.
TABLE ITPHYSKAL PROPER TIP^
p-RC,H,OCONSO,F-p
OF
I
Me 19,R = H 20,R = NO, %
p RC,H,CH,SCOSHC,H,SO,F-p I CH,
22,R = KO, 23, R = K H 2
n'-Methyl-4-nitrobenzylamine (21) condensed smoothly with l l a to give 22 which was reduced catalytically to 23 ; a crystalline triazine could not be prepared from 23 and cyanoguanidine.
Experimental Section Melting points were taken in capillary tubes on a Nel-Temp block and are uncorrected. Each analytical sample moved as a (14) B. R. Baker and G. J. Lourens, J . M e d . Chem., 11, 677 (1968), paper
CXXIX of this series. (15) Synthesized from 2-chloro-4-nitrotoluene b y W.Rsesaotarski in this laboratory, unpublislied. (16) B . R. Baker and G. J. Lourens. J . M e d . Chem., 11, 672 (19681, paper C S X V I I I of this series. (17) B . R. Baker and S . 11. J. Vermeulen, ibzd., 12, 74 (1969), paper C S X X I V of this series.
Ra H
No.
Ri
RP
12b 12e 12e
H H
%Me 3-hle 2-0hIe
13a
C1
H
13b 13c 13d 22e,d
C1
2-Me 3-Me 3-C1
H
H
Me
H
C1 Cl H
H
H H H H
yieldb 76 80 70 77 77 72 77 80
Mp, OC 224-225 205-206 172-173 dec 214-215 dec 250-252 dec 236-236 dec >235 166-167
Formula
CisHirFSaOsB CisHirFNaOsS ClsHirFN306S Ci4HiiClFNaOsS CisHiaClFNsOsS C~~HiaClFNaoss C~HioClzFPia0sS CisHirFSaOaS
Numbered from MI. Yield of analytically pure material that was recrystallized from lIeOEtOH-HnO and analyzed for C, H, K. c The starting 21 wa6 prepared by NaBH4 reduction of p-nitrobenzaldehyde and MeNH, in 40y0RIeOH, then isolated as its HC1 salt; aee C. Paal and H. Springer, Ber., 30, 61 (1897). d Catalytic reduction with a Raney Xi catalyst in EtOH gave 23 ~ ~H, ~ SS). in 727, yield, mp 155-157'. Anal. ( C I J ~ I ~ F N C, 4
p-Nitrophenyl-N-(p-fluorosulfonylpheny1)-N-methylurethan (20) (Method A).-A mixture of 3.78 g (20 mmoles) of 18,16 4.03 g (20 mmoles) of p-nitrophenyl chloroformate, and 50 ml of CsHs was refluxed with stirring for 2 hr. The mixture m-m spin evaporated in vacuo until the product began to separate, then cooled. The product was collected on a filter, washed with petroleum ether (bp 60-llOo), then recrystallized from C6H.s;
