J . Med. Chem. 1989,32, 569-575
569
Quinazoline Antifolate Thymidylate Synthase Inhibitors: Nitrogen, Oxygen, Sulfur, and Chlorine Substituents in the C2 Position Peter R. Marsham,*)' Paula Chambers,' Anthony J. Hayter,' Leslie R. Hughes,+ Ann L. Jackman,* B r e d a M. O'Connor,t Joel A. M. Bishop,* and A. Hilary C a l v e d I C I Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SKlO 4TG, England, and Institute of Cancer Research, 15 Cotswold Road, Sutton, Surrey SM2 5NG, England. Received J u n e 27, 1988 The synthesis of 16 new pO-propargylquinazolineantifolates with methylamino, ethylamino, (2-aminoethyl)amino, [2-(dimethylamino)ethyl]amino,(2-hydroxyethyl)amino, (carboxymethyl)amino, dimethylamino, imidazol-1-yl, methoxy, ethoxy, phenoxy, 2-methoxyethoxy, 2-hydroxyethoxy, mercapto, methylthio, and chloro substituents at C2 is described. In general, the synthetic route involved the coupling of diethyl N-[4-(prop-2-ynylamino)benzoyl]-~-glutamate (5a) with 6 4bromomethyl)-2-chloro-3,4-dihydro-4-oxoquinazoline in N,N-dimethylformamide with calcium carbonate as the base, displacement of the C2-chloro substituent with nitrogen and sulfur nucleophiles, and deprotecton using mild alkali. The C2-ether analogues were most conveniently prepared by coupling 5a with 6-(bromomethyl)-2,4diakoxy(or diphenoxy)quinazolines. In this series the final deprotection step with aqueous alkali gave simultaneous selective hydrolysis of the CCalkoxy or CCphenoxy substituent. The compounds were tested as inhibitors of partially purified L1210 thymidylate synthase (TS). As a measure of cytotoxicity, they were examined for their inhibition of the growth of L1210 cells in culture. The C2-methoxy analogue l l a was equivalent to the previously described tight binding T S inhibitor W0-propargyl-5,8-dideazafolicacid (CB3717, IC1 155387, l a ) against the TS enzyme and exhibited enhanced potency in culture. The C2-methoxy substituent also gave a 110-fold enhancement in aqueous solubility relative to the C2-amine. These results suggest that lla will be an interesting compound for further study as a potential antitumor agent in vivo. A further series of 2-methoxyquinazoline antifolates with modified alkyl substituents a t NO ' is also described. None of these analogues equalled the activity of l l a . Thus the propargyl group appears to be the optimum NIO substituent in both 2-amino- and 2-methoxyquinazoline antifolates.
The quinazoline-based antifolate W0-propargyl-5,8-dideazafolic acid (la)'P2is a potent inhibitor of the enzyme thymidylate synthase ( T S , EC 2.1.1.45). Its antitumor
iti
b C H , Y " C O
N H COzH
xlN YECH
gument a major advance was made when the highly water soluble 2-desamino analogue l b was ~ y n t h e s i z e d . ' ~Despite only an 8-fold loss in TS inhibitory activity, a 10-fold improvement i n LE10 cytotoxicity was observed. Of
CH2CH2C02H
/
la, X = NH,
lb, X = H
activity in vitro and in vivo has been shown to result from inhibition of TS alone with no complicating action at a n y other 1 0 c u s . ~ - ~On the basis of these properties la was selected for clinical study. In phase 1/11studies responses were observed in patients with a variety of tumor types, particularly i n breast,6 o ~ a r i a n , and ~ , ~ hepatocellular8 carcinomas. Despite this initial promise, the use of the compound in the clinic is likely to be limited by renal and hepatic t o ~ i c i t y . ~In* ~mice ~ ~ i t has been demonstratedgJO that la accumulates in both the liver and kidney, and this accumulation, in the latter at least, is probably responsible for the toxicity observed. Indeed the solubility in urine is only 0.04 m g / m L at pH 7.9 Thus modifications to the molecule that lead to increased aqueous solubility could be expected to give compounds having lower toxicity to these key organs. Providing the a n t i t u m o r potency can be maintained, t h i s could give rise to a more acceptable clinical agent. A search for an analogue of la having an improved therapeutic ratio was therefore undertaken. Modifications to the Nlo-substituent," the benzoyl ring,12 and the amino moiety13 have failed t o produce compounds more active than la as inhibitors of TS or cell growth. Attention was turned t o the C2 region of the molecule, for despite the large number of folic acid analogues made as potential anticancer agents there has been little s t u d y of the requirement for the C2-amino group. T h i s is a potential weak hydrogen bond donor and as s u c h m a y contribute to the lack of solubility of la t h r o u g h intermolecular forces i n the solid state. In following t h i s art IC1 Pharmaceuticals.
* Institute of Cancer Research. 0022-2623/89/1832-0569$01.50/0
Jones, T. R.; Calvert, A. H.; Jackman, A. L.; Brown, S. J.; Jones, M.; Harrap, K. R. Eur. J. Cancer 1981, 17, 11. Synonyms: IC1 155387; CB3717; NSC 327182; N-[4-[N-[(2amino-4-hydroxy-6-quinazolinyl)methyl] -N-prop-2-ynylamino]benzoyl] glutamic acid. Jackson, R. C.; Jackman, A. L.; Calvert, A. H. Biochem. Pharmacol. 1983, 32, 3783. Jackman, A. L.; Calvert, A. H.; Taylor, G. A.; Harrap, K. R. In The Control of Tumour Growth and its Biological Bases; Davis, w., Maltoni, C., Tanneberger, S., Eds.; Akademie-Verlag: Berlin, 1983; p 404. Jackman, A. L.; Taylor, G. A.; Calvert, A. H.; Harrap, K. R. Biochem. Pharmacol. 1984, 33, 3269. Calvert, A. H.; Alison, D. L.; Harland, S. J.; Robinson, B. A.; Jackman, A. L.; Jones, T. R.; Newell, D. R.; Siddik, Z. H.; Wiltshaw, E.; McElwain, T. J.; Smith, I. E.; Harrap, K. R. J. Clin. Oncol. 1986, 4, 1245. Calvert, A. H.; Newell, D. R.; Jackman, A. L.; Gumbrell, L. A.; Sikora, E.; Grzelakowska-Sztabert, B.; Bishop, J. A. M.; Judson, I. R.; Harland, S. J.; Harrap, K. R. NCZ Monogr. 1987,5, 213. Bassendine, M. F.; Curtin, N. J.; Loose, H.; Harris, A. L.; James, D. F. J . Hepatol. 1987,4, 349. Newell, D. R.; Siddik, Z. H.; Calvert, A. H.; Jackman, A. L.; Alison, D. L.; McGhee, K. G.; Harrap, K. R. Proc. Am. Assoc. Cancer Res. 1982,23, 181. Newell, D. R.; Alison, D. L.; Calvert, A. H.; Harrap, K. R.; Jarman, M.; Jones, T. R.; Manteuffel-Cymborowska, M.; 0'Connor, P. Cancer Treatment Rep. 1986, 70, 971. Jones, T. R.; Calvert, A. H.; Jackman, A. L.; Eakin, M. A,; Smithers, M. J.; Betteridge, R. F.; Newell, D. R.; Hayter, A. J.; Stocker, A.; Harland, S. J.; Davies, L. C.; Harrap, K. R. J. Med. Chem. 1985,28, 1468. (12) Jones, T. R.; Smithers, M. J.; Taylor, M. A.; Jackman, A. L.; Calvert, A. H.; Harland, S. J.; Harrap, K. R. J . Med. Chem. 1986, 29, 468. (13) Jones, T. R.;Smithers, M. J.; Betteridge, R. F.; Taylor, M. A.; Jackman, A. L.; Calvert, A. H.; Davies, L. C.; Harrap, K. R. J. Med. Chem. 1986,29, 1114. (14) Jones, T. R.; Jackman, A. L.; Thornton, T. J.; Flinn, A.; 0'Connor, B. Proc. Am. Assoc. Cancer Res. 1987,28, 276. 0 1989 American Chemical Society
Marsham et al.
570 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3 Table I. Preparation of Antifolate Diesters 7 (Method A)
%
compd
starting amine 7a methylamine (33% in EtOH) 7b ethylamine 7c ethylenediamine 7d N,N-dimethylethylenediamine 7e ethanolamine 7f glycine ethyl ester hydrochloride 7g dimethylamine (33% in EtOH) 7h imidazole "The product was purified by trituration with 25% MeCN in HzO.
C2-substituent NHCH3 NHCH2 CH3 NHCHZCHZNHZ NHCHZCHZN(CHJ2 NHCHZCHZOH NHCHzCOzEt N(CH3)z imidazol-1-yl
yield 83 61 38 56 82O 68 85" 96
Table 11. Preparation of Antifolate Diacids 2, 17, and 18 (Method B)
mass spectra, m / z [M - HI
%
compd
C2-substituent NH2 NHCH3 NHCHzCH3 NHCHZCHZNHZ NHCHzCHZN(CH3)z NHCHzCHzOH NHCHZCOZH N(CH3)z imidazol-1-yl SH SCH3
yield
inhibn of TS (IRP)* 1.0 9.2 15.0 23.0 260.9 9.5 60.0 115.4 217.4 26.3 5.8
inhibn of L1210 cell growth in culture: ICm, FM 3.4 24.0
mp, "C formulan la' 81 232-235 C24H23N506 Cz~H~5N506*2.5HzOf 490 2a 95 208-211 2b 72 174-188 C26Hz7N,06*2.2HzO 504 >loo 2c C26HZ8N606'4HZ08 519 97.0 79 >240 2d 27 192-195 C Z ~ H ~ Z N ~ O ~ * ~ . ~ H547 ~O >100 2e 62 182-185 Cz&7N507*2.5H2Oh 520 47.0 2f 23d 191-196 Cz&5N508.2Hz0' 534 >loo C26Hz7N506.1.5H20 504 >loo 23g 61 185-187.5 2h 527 >100 C27H24N606'H20 6gd 155e 17a Cz4HzzN406s.Hzo 493 60.0 92d 161-166 17b 94 157-163 CZ,H~,N~O~S*O.~~H~O 507 13.0 18 c1 95 150' C~4H~~C1N~O~"l.33H~O495 10.0 67.0 Anal. C, H, N except where stated otherwise. * IRP = inverse relative potency, defined as Zm (compound)/Zm (la) determined in the same test. CSeeref 1. dReaction carried out at 40 "C. eSinters above this temperature but does not give a discrete melting point. fH: calcd, 5.6; found, 4.9. gH: calcd, 6.1; found, 5.5. "H: calcd, 5.7; found, 5.0. 'H: calcd, 5.0; found, 4.5.
particular note was the lack of renal and hepatic toxicity in mice.15 This observation of improved cytotoxicity in the C2-desamino derivatives stimulated us to undertake a systematic study of new substituents in the C2 position of the quinazoline ring with the aim of finding analogues of la that combine similar or greater potency against the TS enzyme with increased aqueous solubility in anticipation of lower toxicity. In this paper we describe the synthesis and biological activity of a novel series of analogues of la containing nitrogen, oxygen, sulfur, and chlorine substituents in this position of the quinazoline ring.
Chemistry The series of compounds (2a-h) having secondary and tertiary amino functionality at C2 were synthesized by the procedure outlined in Scheme I. The strategy involved the key 2-chloroquinazoline folate diester 6, which readily reacted with a variety of amine nucleophiles to insert the required 2-alkylamino groups. This 2-chloro intermediate was conveniently prepared in 50% overall yield by the bromination of 2-chloro-3,4-dihydro-4-oxo-6-methylquinazoline (3)16 using N-bromosuccinimide (NBS) followed by alkylation of diethyl N-[p-(N-propargy1amino)benzoyllglutamate (5a)' with the resulting bromo derivative 4. Treatment of 6 with the amines listed in Table I afforded the desired antifolate diesters 7a-h. In this reaction high yields were achieved and unwanted amide byproducts were avoided when 1-methyl-2-pyrrolidinone at 100 O C was used as solvent. Of the amines tried in this reaction, only glycine failed to react with 6. However, glycine ethyl ester hydrochloride reacted smoothly under the standard conditions to yield the triethyl ester precursor 7f to the 2- [ (carboxymethyl)amino]quinazolineantifolate (15) Jackman, A. L.; Newell, D. R.; Taylor, G. A.; O'Connor, B.; Hughes, L. R.; Calvert, A. H. Proc. Am. Assoc. Cancer Res. 1987, 28, 271. (16) Bindra, J. S. U.S. Patent 4085213, 1985.
Scheme In
3
'r
5a
6 R'R~NH 0
N aq NaOH, EIOH b
C
H
HN
2
Y
O
c\H2
R j p N A N
/
C
O NH it!C0,H
CH,CH2C02H
ClCH
2 a
See Table I1 for values of R1R2.
2f. This observation suggests that protonation at N1 promotes the nucleophilic displacement of the CZchlorine atom. Deprotection of the diesters was accomplished in the final step by treatment with aqueous alkali (Table 11). In the preparation of the series of analogues bearing a C2-methoxy substituent, it was decided to elaborate the molecule as a 2,4-dimethoxyquinazolineto give the suitably protected diethyl ester precursors loa-h (Scheme 11). Bromination of 817 (NBS) was followed by amination of
Quinazoline Antifolate Thymidylate Synthase Inhibitors
Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3 571 OR
Scheme I1
12, X = H ;
13, X.Br
8
14 H
N
~
c
H
z
y
~ NH+C02H c o
CH
I
N 15 a, b. C, d,
R=-CH,CH, A=-Ph R -CH,CH,OCH, R = -CH,CH,OH e. R = -CHZCH20COPh
CO2H
I
R
CH,CHzCO,H
H3C0 11
-
-
CZCH
N aq NaOH, ElOH. 60°C
HN
a, R = CHpC ICH b, R=.H C. R=-CH, d, R = -CH,CH, e, R = -CH,CH=CH, 1, R -CH,CH,OH g, R = -CH,CH,CH,OH h. R .CH,CH,F
CH~CHZCO~H
\2
R3 A
(-CH,CH,OAc in 51 and 101) (-CH,CH,CH,OAc in 5g and log)
Table 111. Preparation of Dimethoxyantifolate Diesters 10 (Method C) aniline no. of starting equiv of % compd N10-substit material 9 yield 10a CH2C=CH 5a" 1.20 29 10b H 5bb 1.10 23 1 0 ~ CH3 5cc 1.04 51 10d CH2CH3 5dd 1.04 32 10e CH2CH=CH2 5e4 1.04 86 10f (CH2)20COCH, 5f 1.03 47 log (CH2)80COCH, 5ge 1.11 41 10h (CH2)zF 5h' 1.04 31 aSee ref 1. *Aldrich Chemical Co. C F ~S-C.J.; , Reiner, M.; Loo, T. L. J . Org. Chem. 1965,30, 1277. dMontgomery, J. A.; Piper, J. R.; Elliot, R. D.; Temple, C.; Roberts, E. C.; Shealy, Y. F. J . Med. Chem. 1979,22, 862. OSee ref 10.
the resulting bromide 9 with a series of N-substituted diethyl (4-aminobenzoy1)glutamates" to afford the 2,4dimethoxy diethyl esters loa-h (Table 111). It is well established that 2,4-dialkoxyquinazolinescan be selectively hydrolyzed by alkali to give the 2-alkoxy-4-hydroxyderivative,'* and this property was utilized to effect complete deprotection of loa-h in one step (8 equiv of NaOH at 60 O C) to the desired 2-methoxyquinazoline antifolates (lla-h). The same approach was used in the synthesis of the other C2-ether analogues 15a-d via the quinazoline 2,4-diether intermediates 14a-d. In the case of 15d the 2-hydroxyethoxy substituent was introduced by treating 2,4-dichloro-6-methylquinazolinewith sodium 2-hydroxyethoxide in DMF. It was then necessary to protect the free hydroxy groups in 12d as the benzoate esters before completing the elaboration of the molecule to 14e. The benzoic acid generated upon alkaline hydrolysis of 14e could be readily removed from the product 15d by trituration with MeCN. (17) Mead Johnson & Co., British Patent 920019, 1963. (18) Lange, N. A.; Sheibley, F. E. J . Am. Chem. SOC.1933,55,1188.
-
Introduction of sulfur at C2 was accomplished by exposure of 6 to thiourea in the presence of formic acid. The resulting thiol diethyl ester 16a was hydrolyzed to the 2-mercaptoquinazolineantifolate diacid 17a. Methylation of 16a (MeI-NH,OH) followed by alkaline hydrolysis yielded the corresponding 2-(methyl thioether) 16b (Table 11). Finally, 6 itself could be hydrolyzed by aqueous NaOH under carefully controlled conditions to give the 2chloroquinazoline antifolate 18 (Table 11).
e 16
COpH NH cI H
HN 9 c H 2 y " c o C F
/
CH,CH,CO,H
CECH 17
a, R=-H, b, R=-CH,
~
c
H
,
,,I,
y
C\H2
/
~
C02H NH cCH I o CH~CH~COZH
ClCH
18
Biological Evaluation The diacids 2a-h, lla-h, 15a-d, 17a,b, and 18 were tested as inhibitors of TS partially purified from L1210 mouse leukemia cells that overproduce TS due to amplification of the TS gene.ls The partial purification and assay method used in this study was as previously described and used a (f)-5,10-methylenetetrahydrofolic acid concentration of 200 pM.19920 la was included in each assay as a positive control (INN 20 nM). The ratio of I N ' S (defined as an inverse relative potency, IRP) could then be compared. The compounds were also tested for their inhibition of the growth of L1210 cells in culture,'l and the results again were expressed as the concentration required to inhibit cell growth by 50% (IC,o). These results are (19) Jackman, A. L.; Alison, D. L.; Calvert, A. H.; Harrap, K. R. Cancer Res. 1986, 46, 2810. (20) Sikora, E.; Jackman, A. L.; Newell, D. R.; Calvert, A. H. Biochem. Pharmacol. 1988, 37, 4047.
Marsham et al.
572 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 3
Table IV. Preparation of Antifolate Diacids 11 and 15 (Method D) mass inhibn inhibn of L1210 spectra, m/z of TS cell growth in compd C2-substit N10-substit yield mp, "C formula" [M - HI(IRP) culture: ICw, pM lla OCH, CH2CeCH 88 155-165 C=HuN407*HzO 491 1.4 1.9 25 150-160 C22HnN40,*1.2H20 453 311.8 6.4 llb OCH, H 467 11.6 7.0 83 240-245 CZ3HuN407.2.5HzO CH3 llc OCH3 481 6.6 12.0 CHZCH, 64 140-146 CuHmN4O,*H20 lld OCH, CHZCH=CH2 75 130-134 C=HaN,O,*HzO 493 17.1 16.0 lle OCH, 17b 150-175 Cz4HaN408*1.25HzO* 497 13.8 7.3 (CHz)zOH llf OCH, 0.3MeCN 511 38.1 80.0 (CH&@H 63 145-155 C~H~N40s.1.5H20 llg OCH, 499 7.5 17.0 (CHM 80 141-145 CuHZFN407.1.25HzO llh OCH, 53 134-136 C%HBN40,*2H20c 467 7.3 >100 15a OCHzCH3 CHZCeCH CH~CICH 57 159-164 C&sN40,*3H2Od 15b OPh 553 35.2 >lo0 87 134-140 C,H,N4O**H20 1 5 ~ OCHZCHZOCHS C H 2 C 4 H 535 4.2 100.0 CHZCECH 21 145-149 CBHBNd08*2HzOe 15d OCH2CHZOH 521 5.5 >lo0 "Anal. C, H, N except where stated otherwise. bSolidification of the product was induced by trituration with MeCN. The NMR spectrum indicated the presence of 0.3 mol of MeCN. H: calcd, 5.5; found 4.9. H: calcd, 5.2; found, 4.4. e H: calcd, 5.3; found, 4.8. %
collected in Tables I1 and IV. Results and Discussion The values of the inverse relative potency for the inhibition of partially purified L1210 TS and the ICw values for growth inhibition of L1210 cells are shown in Tables I1 and IV. The replacement of the C2-amino group by a variety of secondary and tertiary amines has resulted in compounds of reduced potency against both parameters. Loss of activity is particularly marked in the tertiary amine 2g, the imidazole derivative 2h, and the amino acid 2f. Even in the simple secondary amines the activities rapidly dwindle as the size of the alkyl group increases from methyl to ethyl. The incorporation of a hydroxyl group into the secondary amine to give 2e has some benefit in terms of potency against both the enzyme and cells, but this is not maintained when the hydroxyl is replaced by the amino group in 2c. As can be seen from Table IV,the methoxy group (in lla) can replace the C2-amine without appreciable loss of enzyme activity. Indeed a slight improvement in activity against L1210 cells is seen. As was the case in the C2-amino series,1° a propargyl substituent on NO ' appears to be optimum in the C2-methoxy series. The methyl ( l l c ) ,ethyl (lld),and 2-hydroxyethyl ( l l f ) analogues are also quite cytotoxic despite their reduced potency against the isolated enzyme. That the enzyme has a low tolerance of a hydrogen substituent at NO ' is reinforced by the relatively poor activity of 1 Ib. The cytotoxic activity of l l b is unlikely to be due to its activity against dihydrofolate reductase (EC 1.5.1.4, DHFR,Ki N 0.6 but could possibly result from improved transport into the cells or from enhanced polyglutamation within the cells. Larger ether groups at C2, although still active enzyme inhibitors, are less well tolerated by the enzyme and are much poorer cytotoxic agents. The C2-methylthio derivative 17b also appears to fall into this series, being intermediate in both TS and L1210 activity between the methoxy and ethoxy analogues, reflecting the relative volumes of these three substituents. The reduced activity of the C2-thiol 17a presumably results from a predominance of the 2-thione tautomer in aqueous solution. A chlorine substituent at C2 (18) is also quite well tolerated by the enzyme despite its high lipophilicity. The overall conclusion therefore is that the C2-amino group is not absolutely essential for activity as a TS inhibitor. The enzyme can accommodate a wide range of substituents in this position, but cytotoxicity is severely limited by the size of this group. The solubility of the C2-methoxy analogue 1la in aqueous sodium phosphate has been determined
temperature unless otherwise stated. N,N-Dimethylformamide (DMF) and N,N-dimethylacetamide (DMA) were purified by azeotropic distillation at 10 "Hg. EtOH was dried by distillation from sodium metal. MeCN (FisonsHPLC Grade) and CCh (BDH Technical Grade) were used without further purification. Solutions in organic solvents were dried over anhydrous Na2S04. Preparative chromatography was performed on Merck Kieselgel 60 (ART 9385 or ART 15111) packed in Corning glass HPLC columns. A flow rate of 10-30 mL/min was achieved with a Gilson 302 pump, and the eluent was passed through a Gilson 111 ultraviolet monitor set to detect at 254 nm. TLC was performed on precoated silica gel plates (Merck ART 5715), and the resulting chromatograms were visualized under UV light at 254 nm. Analytical HPLC was performed on a Hichrom S50DS1 Spherisorb Column System set to run isocratically at 60% MeOH + 0.2% CF3C02Hin water for final antifolate diacids and 70% MeOH + 0.2g CF3C02Hfor intermediates. Melting pointa were determined on a Kofler block or with a Biichi melting point apparatus and are uncorrected. Fast atom bombardment (FAB) mass spectra were determined with a VG MS9 spectrometer and Finnigan Incos data system, using DMSO as the solvent and glycerol as the matrix. The NMR spectra were determined on a Bruker AM200 (200-MHz) spectrometer. Chemical shifts are expressed in units of d (ppm), and peak multiplicities are designated as follows: s, singlet; d, doublet; dd, doublet of doublets; t, triplet; br a, broad singlet; m, multiplet. D i e t h y l N -[4-[N - [ (2-Chlor0-3,4-dihydro-4-0~0-6quinazolinyl)methyl]-N-prop-2-ynylamino]benzoyl]-~glutamate (6). A mixture of powdered 2-chloro-3,4-dihydro-4oxo-6-methylquinazoline (3) (6.34 g, 32.6 mmol), N-bromosuccinimide (6.38 g, 35.8 mmol), and benzoyl peroxide (0.16 g, 0.65 mmol) in C C 4 (250 mL) was stirred vigorously under reflux for 16 h. The cooled reaction mixture was filtered. The filter
(21) Jackman, A. L. Unpublished results.
(22) Solubility values were determined by Dr. J. J. Morris.
Table V
compd la 1la
solvent 0.01 M aqueous 0.20 M aqueous NaH9POI NaHIPOl initially "at ~ I H7 initially "at ~ I H7 solubility, pH attained solubility, pH attained ma/mL at saturation m d m L at saturation 0.05 5.76 0.06 6.78 1.2 4.93 6.6 5.67
(Table V)22 and as expected is many times that of la. Thus from the results reported here, we conclude that lla has comparable in vitro activity to la and the enhanced aqueous solubility makes this an interesting compound for further study. Experimental Section General Procedures. All procedures were carried out at room
Quinazoline Antifolate Thymidylate Synthase Inhibitors cake was washed with H 2 0 and dried in vacuo to give the crude bromomethyl compound 4 (7.98 g, 91%), which was used without purification. A mixture of 4 (7.9 g, 28.9 mmol), diethyl N-[4-(prop-2-ynylamino)benzoyl]- glutamate (5a)' (7.34 g, 20.4 mmol), powdered CaC03 (2.45 g, 24.5 mmol), and DMF (30 mL) was stirred a t 60 "C for 16 h under argon. The cooled reaction mixture was partitioned between EtOAc and H20. The organic phase was separated, washed with H20, dried, and evaporated to dryness. The crude oil was purified by chromatography eluting with CH2C12 followed by CH2C12-EtOAc (7:3 v/v). The product 6 (6.08 g, 54%) sintered above 150 "C and melted a t 177 "C; NMR (Me2SO-d6) 6 1.04,1.07 (2 t, 6 H, OCH2CHd,2.05 (m, 2 H, CHCH2CH2C02Et), 2.4 (t, 2 H, CHCH2CH,CO,Et), 3.2 (t, 1 H, CECH), 4.05, 4.1 (2 q, 4 H, OCH,CH,), 4.35 (br s, 2 H, CH2C=C), 4.8 (br s, 2 H, ArCH2N
