.January 1909
IRREVERSIBLE
ENZYLIE ISHIBITORS.
then much less reactive toward a neutral nucleophile such as thioureaS8 Therefore it was essential that a neutral nitrogen nucleophile be used with 5 under aprotic conditions so that the N-acetyl group would not be cleaved with resultant deactivation of the 4-chloro group; such a nucleophile is azide ion. When 5 was heated with S a s s in D l I S O at S5-90°, reaction was complete in 2 hr as Shown by tlc. A crystalline azide (7) was isolated in 9370 yield, but showed two spots on tlc that had identical uv spectra; whether or not these two hpot5 were due t o equilibration with the isomeric tetrazole (8)1°was not ascertained, since both 7 and 8 would be reducible to the .ame amine (6). The crude azide (7) was catalytically reduced with a Raney S i catalyst t o 2,4-diaminopyrimidine (6) which was isolated as its sulfate in 61% yield. Due to its insolubility, attempts to convert the sulfate salt of 6 to 2 with vi-fluorosulfonylbenzoyl chloride in DAIF or DMSO in the presence of E t 3 S were unsuccessful. The sulfate salt of 6 was converted to the noncrystalline base with aqueous S a O H , which was iholated by CHC13 extraction. Acylation then proceeded in DAIF when the 2,4-diaminopyrimidine moiety of 6 was allowed to act as the acid acceptor; the yield of pure 2 from the sulfate salt was only 14%. It was found more convenient to treat the more soluble hydrochloride salt of 6 with acid chlorides where 1,5diazabicyclo [4.3.0]non-5-ene (DBS) was used as the acid acceptor; this method was used for preparation of 9 arid 10. The urea derivative (11) was prepared similarly from 6 'HC1 by reaction with p-nitrophenyl S-(3-fluorosulfonyl-4-methylphenyl)carbamate.
Experimental Section i\Ielting points were taken in capillary tubes on a Nel-Temp block and are uncorrected. Each analytical sample moved as a single spot on tlc on Brinkmann silica gel GF, and had ir and uv spectra in agreement with their assigned structures; each gave combuhtion values for C, H, and N or F within 0.47, of theoretical. 2-Acetamido-6-methyl-5-( p-nitrophenoxypropyl)-4-pyrimidino1 (4).-A stirred mixture of 3.50 g (11.5 mmoles) of 3' and 40 ml of A c 2 0 was heated at 80-85" for 4 hr, solution not taking place. The mixture was spin evaporated zn vacuo. The residue was suspended in 30 ml of EtOH and the evaporation was repeated; the evaporation was repeated with two 30-ml portions of EtOH. Recrystallization from MeOEtOH gave 3.10 g (78%) of product, mp 232-233". Anal. (ClsHl,N40:) C, H, N. 2-Acetamido-4-chloro-6-methyl-5-( p-nitrophenoxypropy1)pyrimidine (5).-A stirred mixture of 5.0 g (14.5 mmoles) of 4 and 25 ml of POc13 was protected from moisture in a bath at 75' for 1 hr, solution being complete in 15 min. The cooled solution was poured into 200 ml of petroleum ether (bp 60-110°), then allowed to stand until the solution was no longer turbid (1 hr). The petroleum ether was decanted from the gum. the latter was washed with an additional 100 ml of petroleum ether (bp 60-110'). To the residual gum was added a mixture of 50 g of ice and 250 ml of 1 0 5 aqueous NaOAc. The mixture was rtirred for 30 min when the gum had changed to a solid. The latter was collected on a filter and washed with H 2 0 . Recrystallization from 1\IeOEtOH-H20 gave 2.7 g ( 4 9 7 3 of off-white crystals, nip li3O. Anal. (ClSH1,ClN,04) C, H, N. 2-Acetamido-4-azido-6-methyl-5-( p4trophenoxypropyl)pyrimidine (7).-'4 stirred mixture of 5.0 g (13.7 mmoles) of 5 , 3.3 g (54 mmoles) of S a x 3 , and 30 ml of reagent grade DJISO WAS (10) (a) C . Temple, Jr.. and ,J. A. Montgomery, J . Org. Chem., 30, 826 (196.5); (13) C. Temple, .Jr., R. L. MrKee. a n d .I. A . Rlontyomery, i h i d . , SO,
829 (196.5). ( 1 1 ) B. R.Baker a n d N . X I . ,J. Vermeiilen, .I. . I f ~ d .Chem., 12, i 4 (1969), paiier C S S S I V of t h i n series.
cxrm
109
heated in a bath at 85-90" for 2 hr when tlc showed 5 had been consumed. The cooled reaction mixture was diluted with 100 ml of H20and 200 ml of CHC18. The separated CHC13 layer was washed wit,h 100 ml of HzO, then dried with MgSO4 and evaporated in uacuo to about 20 ml. Petroleum ether was added to turbidity, when the mixture was chilled at 5'. The crystalline product was collected on a filter and washed with petroleum ether; yield 4.1 g (83%); mp 141-144" dec; "2:: 1680 (amide I), 2100 (N3), 1520, 1350 cm-1 (YO,); this was suitable for the next step. Tlc in EtOAc showed two major spots and one minor Spot. A small-scale t.lc ,separation of two major spots showed they had identical iiv spectra: Xmal 256, 314 mp (pH 1 ) ; 300 mp (pH 13). Since the minor product may have resulted from deacetylation, but, is also convertible to 6, no further purification waS considered necessary. 5-(p-Aminophenoxypropyl)-2,4-diamino-6-methylpyrimidine (6) Sesquisu1fate.-A solution of 1.3 g ( 3 . 5 mmoles) of crude 7 in 200 ml of MeOEtOH was shaken with H2 at 2-3 atm in the presence of 1 g of Raney S i for 1 hr. To the filtered solution was added 20 ml of 6 S HCl, then the solution was heated on a steam bath for 30 min to remove the N-acetyl group, which was shown by the shift in Amax at pH 1 from 264 to 2 i 3 mp. Evaporation to dryness in vacuo gave a glassy hydrochloride that could be precipitated as a white powder from a small volume of hot H2O by addition of i-PrOH to turbidity; yield 8 i % (calculated as 3HC1), mp >200" dec, sufficiently pure for further transformation. For analysis a sample was dissolved in 20 ml of hot 2 -V H2SO4, then the solution was clarified with carbon. The hot solution was diluted with i-PrOH t,o turbidity, then cooled; yield 6 l % , mp > 2 X 0 dec. $nul. (C2,Hz1N:0.1.5H2S04) C, H, N. Preparatively, it was more convenient t o use the HCI salt of 6 for further transformation. 2,4-Diamino-5- [ p ( m fluorosulfony1benzamido)phenoxypropyl] -6-methylpyrimidine (2) Ethanesu1fonate.-A mixture of 0.90 g (2.1 mmoles) of 6 sulfate, 30 ml of CHCl3, and 100 ml of 1 S NaOH was vigorously shaken until solution was complete. The separated H2O layer was extracted with three 30-ml portions of CHC1,. The combined extracts were dried with MgS04, then of 6 base as a gum. The evaporated in vacuo leaving 0.50 g (85yo) latter (1.8 mmoles) was dissolved in a mixture of 3 ml of DMF, 5 ml of CHCl,, and 200 mg ( 2 mmoles) of Et&. To this solution cooled in an ice-salt bath and protected from moisture was added 0.33 g (1.5 mmoles) of nz-fluorosulfonylbenzoyl chloride. After being stirred Id min, the solution was treated with 0.60 g ( 5 mmoles) of EtSOsH, t,hen the CHC13 was removed by evaporation in vacuo. The evaporation was repeated with three 20-ml portions of o-PrOH. The residual D l I F solution was dillited with about 30 ml of r5070 aqueous i-PrOH, then cooled. The product that separated on standing was collected on a filter and washed with 50% aqueous i-PrOH; yield 0.12 g (145: based on the acid chloride) of white needles, which gradually softened over 145', but showed no definite melting point. -$nul. (C,,H??FN5OiS.C2HjSO3H) C. H, F. 2,4-Diamino-5- [p-(p -fluorosulfonylbenzamido)phenoxypropyl] 6-methylpyrimidine (9).-To a ztirred mixture of 250 mg (0.65 mmole) of 6.3HC1 and 145 mg (0.65 mmole) of p-fluorosulfonylbenzoyl chloride in 2 ml of DMF protected from moiature and cooled in an ice bath v,-as added 200 mg (1.6 mmoles) of 1,5diazabicyclo[4.3.0]non-5-ene(DBN). After 30 min the solution was poured into 50 ml of saturated aqueous NaHC& The product was collected on a filter and washed with H,O. Recrystallization from EtOH-H20 gave 100 m?: (3370) of white crystals, mp 229-231", that moved as; single spot on tlc in 1:4 EtOHCHCI3. Anal. (CZ1Hz2FN504S)C, H. F. 2,4-Diamino-5- [p-(3-fluorosulfonyl-4-methylbenzamido)phenoxypropyl] -6-methylpyrimidine (10) Hemisulfate.-React ion of 400 mg (1.04 mmoles) of 6.3HC1 with the acid chloride from 240 mg (1 mmole) of 3-fluorosulfony1-4-methylbenz~icacidz for 1 hr as described for 9 gave a solution that was poured into 20 ml of 1 1V HzSO,. The product was collected on a filter, wa*hed w i t ' i H20, then recrystallized three times from EtOH-H20 wheii i t moved as one spot, on tlc; yield 60 mg (l16z)of white needles, mp >18O0 dec. Anal. (C,?H,,FN:O,S,O.5H?SO,.H~~)(", €1, F. 2,4-Diamino-5- [p-(3-fluorosuifonyl-4-methy1phenylureido)phenoxypropyl] -6-methylpyrimidine (11) Hemisulfate.-Eeaction of 250 mg (0.65 mmole) of 6.3HC1 with 220 mg (0.62 mmole) of p-nitrophenyl ?u'-(3-fliiorosrilfonyl-4-methylphenyl)carbamate11 for 1 hr as described for 9 gave a soltition that was poared into a stirred mixture of 20 ml of 1 A- HVSO, and 20 ml of CHC13.
- -
-
The product was collected on a filter arid recrystallized three times from MeOEtOH-Hz0 when it moved as single spot on tlc; yield 90 mg ( 2 6 5 ) , mp >lY4' dec. Anal. (C?2H?jFr6O1S.0.3H1SO4. Ir?o)c, TI, F.
Enzyme Results and Discussion The following arbitrary criteria had been choseri t o determine whether a candidate irreversible inhibitor of dihydrofolic reductase i3 ivortliy of in 2 iz'o aqbay: tht. conipoLl1id (a) should htive Ijo N (iKl _< 0.1 pAlr, (h) h u l d give >iOyc inactivation of the tumor eiizy~iic~ :it :I K , concentratioii, :iiid (c) sliould give 12K1concentration. ('onipound 2 meet., two of the three criteria but shon CI only cj0-(j5yL itinctivutiori of the tumor enzyme at :i concentration of 0.016 p J 1 (about OK,) arid in:ictivation at 0.003 p J I ( : h u t 2K,) concentration. When the SOLI' group of 2 w;i- moved to the pala position, the reiultant 9 \\'as not :ii good i i t i irrever-ible inhibitor as 2: :I concentration of 31so of 9 -1iun-etl only mictivatioii of the 1,12lO//DlS eiizynie compurtd :31311 conceiitratiori of 2 which gave 100ci iiiacti1.at ioii. This difference can he rxtionnlized CJII thc, ba I
of the S021' group1? to rate' ot iiuctivation by
9
1s
highw than with 2. dlight. but perhap$ iriii i r r e v e r 4 l e inhibition of thc livrr enzyme T with 9. Insertion of ;i Ale oitho t o t h e S:OL1.' of 2 gave 10 that showed little chaiige i i i reversible binding or bpecificity of irreversible inhibition. When the COKH bridge of 10 n a s Iengtheiied t o XHCOSH ( l l ) , I 3 revcrsith. iiihibitiori of dihydrofolic reductuw did not cliaiige; lio.iwver, 11 \\a\ both :t 1 effectivc and le-\elective irreversible inhibitor t l i a r , sirice 11 showed - o i w irreveriible inhibition of the iiiouhe liver enzyinc. The best compoundq 111 Tiible I tire 2 and 10. Hon tivtr, 2 .;till had two faultq, the -econd oi which \\a\ major (a) 2 failed to give good irreveryible iiihibitioii of dihydrofolic at :t K , concentration. ~ i n d(h) 2 failed to . t ~ o ~111 LYKI activity or toxicity i r i mice licaririg 1,1210 0, ti- reported previouhly 111 :i -unimniy papel ' ?'hc f:iilurc. of 2 t o iho\\ i lt'o :tctivity c.oult1 bt. :lecountecl for by the fact that 2 ihowed poor periehtion of the 1,1210,O cell n-all, a- ~ 1 i o ~ v -by n t i s u r culturo studic>. The EDsoof 2 againit 1,1210 0 111 cc~I1culture \\ :ii 2 . 2 p.1/;'4 since 0.05 pLnl of 2 -1io~v- coIiiplrtc> iri:wtiv:ition of the 1,1210'0 crizynie in :L hroLeii cell nil the increment betn eeii I,o :md effectivci iutrncellular conccntmtion i- a t lea-t 40-fold. Thew results with 2 -houlti be contrahted n ith thoie of t h c diliydrotrinzinc~ (12). The dihydrotriazirir (12) showed :L 70% life extension ~f Inice mith 1,1210 0 NH
.Jnnunrv 1069
IRREVERSIBLE RSZlME ISHIBITORS. CXT,III
Enzyme source
----ReversiblebL0,d p Jl
Inhib,
x
IrreversibleC Enzyme, Time, p Jl inin
w
/o
inactvn
0,030 50, 84, 100u.h 0.024 2 , 8, 60 0.016 0.024 2 , 8, 60 24, 48, 488.)1 0,0030 0,027 60 10 0.00~0 0,0020~ 60 ,j2u,i 0.016 0.0027 0,050 0.023 60 94 L1210 '0 0.016 0.023 60 !50 Liver 0.019 0.0032 0.60 0.024 60 6 Spleen 0.20 0.025 60 0 Intestine 0.20 0.023 60 0 0.069 0.027 8, 13, 30 9 CbH4SOiF-p L1210/DF8 17, 28, 28" Liver 0.10 0,024 60 9 0.020 0.0033 0.040 0,023 60 52 10 CbH1-4-l\Ie-3-SO~F L1210 0 0.040 0.021 60 82 L1210 'DF8 Liver 0.10 0.024 60 0 0.01,j 0.0023 11 NHC6H3-431e-3-SO~F L1210,'DF8 0.021 60 44 0,045 0,029 Liver 0.10 60 19 Assayed with 6 p M dihydrofolate 5 The technical assist,ance of Sharon Lafler and Diane Shea with these assays is acknowledged. and 30 p M TPSH in pH 7.4 Tris buffer containing 0.15 JP KC1 as previously described.j Unless otherwise indicated, the incubation was performed a t 37" in p H 7.4 Tris buffer in the presence of 60 pill TPNH, then the remaining enzyme was determined by dilution of an aliquot 1 : 10 with buffer containing 0.17 JP KC1 and assayed with 12 p J P dihydrofolate and 30 pJP TPSH;6 the zero point was deter1 6 0 = concentration for 30% inhibition. e Unless otherwise indicated, the Ki mined by adding the inhibitor to the assay cuvette. was estimated from Ki = K,[Iso]/[S] which is valid since [SI = 6 K , = 6 p M dihydrofolate; see ref 3, Chapter 10. Data from ref 5 From six-point time s t ~ 1 d y . l ~ Determined by Dixon plot of 1 / V us. [I] using 6 and 12 unless otherwise indicated. 0 New data. p M dihydrofolate, which showed competitive kinetics. j Incubated a t 25" with one-tenth the usual amount of enzyme by assaying t,he aliquot without dilution.
1,1210 ITIF8
0 016
K,,$ p .I1
111
these conditions with 0.005 p3I 2 and 0.002 pi71 enzyme, 52y0 inactivation \vas seen even though the ratio of excess inhibitor to enzyme could theoretically inactivate all the enzyme. Enzyme-catalyzed hydrolysis of the SOzF is still a pertinent factor; this is further supported by the fact that the para isomer (9) in 2.6-fold excess over the enzyme gave only 28y0 total inactivation in 15 min, then no further inactivation occurred. A posteriori, the irreversible inhibition data reported in the accompanying papers and in previous papers were, in general, run with an enzyme concentration of 0.02-0.04 p 3 I . I n only a few cases was the inhibitor concentration lower than the enzyme concentration; these cases are now subject to reinvestigation. For example, 0.01 pL31 12 gave only 10% inactivation of 0.023 p L l l enzyme, considerably beloxv the theoretically achievable 430j0; however, when 0.01 p J 1 12 was incubated with 0.002 p X enzyme, 6S70 inactivation occurred, which is comparable to the 73% inactivation observed with 0.07 p N 12 and about 0.020 pLdI enzyme. From the above discussion it is clear that care must be taken with the first of the three criteria for in vivo evaluation; with irreversible inhibitors that have a Ki < 0.03 p M , the irreversible inhibition experiment
0 0027 0 00311
must take into consideration the enzyme concentration. Thus, if the proper ratio of 0.003 pLJI = Ki concentration of 2 to enzyme is used, >70% inactivation of the enzyme should be achievable. I t follows that 2 can meet all three criteria for in vivo evaluation. It also follows that a fourth criterion should also be met before whole animal testing is performed; the compound should show good transport through the L1210 cell wall as determined in cell culture. It can be stated that 2 meets only the first three of these four criteria, that is, 2 penetrates the L1210 cell mall poorly. The dihydrotriazine (12) meets a different three of these four criteria; 12 can penetrate the L1210 cell wall effectively, but fails the first criterion in that it does not show selective inhibition of the L1210 dihydrofolic reductase with no inactivation of this enzyme in normal cells. By further manipulation of the structure of 2, 12, or 6-substituted 5-aryl-2,4-diaminopyrimidines5 it is reasonably certain that a compound mill emerge that meets all four criteria; such a compound should therefore be more effective than 12 against 1,1210/0 in mice. Studies on further modification of 2, 12, and 6-substituted 2,4-diamino-5-(3,4-dichlorophenyl)pyrimidines5 with the four criteria follow-up are being vigorously pursued.
