J. Med. Chem.
1989,32, 1559-1565
1559
Inhibition of Mammalian Folylpolyglutamate Synthetase and Human Dihydrofolate Reductase by 5,8-Dideaza Analogues of Folic Acid and Aminopterin Bearing a Terminal L-Ornithine Shirish A. Patil,tJ Barry Shane,o James H. Freisheim," Shyam K. Singh,t and John B. Hynes*Bt Department of Pharmaceutical Sciences, Medical University of South Carolina, Charleston, South Carolina 29425, Department of Nutritional Sciences, university of California, Berkeley, California 94720, and Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699. Received September 16, 1988 Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2 , 4 , 5 , and 10 were included to help define structure-activity relationships for compounds of this type. The compound N~-(4-amino-4-deoxy-5-chloro-5,8dideazapteroy1)-L-ornithine(30 was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase was only 5-fold reported thus far (Ki 2 nM). Its 4-oxy counterpart, Nu-(5-chloro-5,8-dideazapteroyl)-~-ornithine, less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.
The enzyme folylpolyglutamate synthetase (FPGS) is responsible for the intracellular conversion of naturally occurring folates to poly-y-L-glutamyl derivatives. Numerous studies have shown that the formation of these metabolites is essential for the cellular retention of folates in mammalian cells and results in the generation of more efficient substrates for many of the enzymes of one-carbon metaboli~m.'-~ The intracellular synthesis of polyglutamates of classical folate antagonists such as methotrexate (MTX) has been shown to be an important determinant of cytotoxicity, since longer chain polyglutamates of MTX are selectively retained by tumor cells in the absence of extracellular drug."' Recently, impaired glutamylation of MTX has been identified as a primary mechanism of resistance to MTX in a CCRF-CEM human leukemia subline in vitro.8 Earlier, several mammalian cell lines auxotrophic for the end products of folate metabolism (methionine, glycine, thymidine, and a purine) were shown to be devoid of FPGS activity.*" These results suggest that a potent and selective inhibitor of FPGS could become a useful new chemotherapeutic agent. This paper describes our initial efforts directed toward achieving this objective. Numerous analogues of folic acid, MTX, and aminopterin modified in the amino acid region have been prepared as potential inhibitors of FPGS. However, all of the potent inhibitors reported thus far having Ki values lower than the K , values for reduced folate substrates contain a terminal L-ornithine residue. Initially, pteroyl-Lornithine (1) was found to be an effective inhibitor of hog 0
/I
H I
-
0 /I
COOH I
1
liver FPGS (Ki = 5.9 pM), while reduction to its 5,6,7,8tetrahydro derivative caused a 30-fold reduction in the Ki ~ a l u e . ' ~ J Subsequent ~ studies showed that the MTX analogue, in which an L-ornithine replaced the L-glutamate residue, had a Ki value in the same range as 1 against FPGS from K562, CCRF-CEM cells, and rat liver.l4,l5 The aminopterin derivative containing an L-ornithine was Medical University of South Carolina. Department of Oncology, Montefiore Medical Center, Bronx, NY 10467. 5 University of California, Berkeley. Medical College of Ohio. t Present address:
found to be considerably more potent toward FPGS, having Ki values around 0.2 pM toward both the human and murine liver enzymes.15J6 Recently, we reported the substrate activities of a wide variety of 5,a-dideaza analogues of folic acid and aminopterin using homogeneous hog liver FPGS.17J8 Several of these compounds were highly effective substrates, having activities approaching those of the best reduced folates. The compound 5-chloro-5,8-dideazaaminopterin (1) McGuire, J. J.; Coward, J. K. In Folates and Pterins; Blakley,
R. L., Benkovic, S. J., Eds.; Wiley Interscience: New York, 1984;Vol. 1, pp 135-190. (2) Cook, J. D.; Cichowicz, D. J.; George, S.; Lawler, A.; Shane, B. Biochemistry 1987, 26, 530. (3) Shane, B.; Stokstad, E. L. R. Annu. Reu. Nutr. 1985, 5, 115. (4) Balinska, M.; Galivan, J.; Coward, J. K. Cancer Res. 1981,41, 2751. (5) Fry, D.W.; Yalowich, J. C.; Goldman, I. D. J. Biol. Chem. 1982, 257, 1890. (6) Samuels, L. L.;Moccio, D. M.; Sirotnak, F. M. Cancer Res. 1985, 45, 1488. (7) Curt, G. A.; Jolivet, J.; Carney, D. N.; Bailey, B. D.; Drake, J. C.; Clendeninn, N. J.; Chabner, B. A. J . Clin. Invest. 1985, 76, 1323. (8) Wright, J. E.; Rosowsky, A,; Waxman, D. J.; Trites, D.; Cucchi, C. A.; Flatow, J.; Frei, E., I11 Biochem. Pharmacol. 1987, 36, 2209. (9) McBurney, M. W.; Whitmore, G. F. Cell (Cambridge, Mass.) 1974, 2, 173. (10) Taylor, R. T.; Hanna, M. L. Arch. Biochem. Biophys. 1977, 181, 331. (11) Moran, R. G. In Folyl and Antifolyl Polyglutamates; Goldman, I. D., Chabner, B. A,, Bertino, J. R., Eds.; Plenum: New York, 1983;pp 327-339. (12) Cichowicz, D.J.; Cook, J. D.; George, S.; Shane, B. In Proceedings of the Second Workshop on Folyl and Antifolylpolyglutamates; Goldman, I. D., Ed.; Praeger: New York, 1985;pp 7-13. (13) George, S.;Cichowicz, D. J.; Shane, B. Biochemistry 1987,26, 522. (14) McGuire, J. J.;Hsieh, P.; Franco, C. T.; Piper, J. R. Biochem. Pharmacol. 1986, 35, 2607. (15) Rosowsky, A.;Freisheim, J. H.; Moran, R. G.; Solan, V. C.; Bader, H.; Wright, J. E.; Radike-Smith, M. J. Med. Chem. 1986, 29, 655. (16) Clarke, L.;Rosowsky, A.; Waxman, D. J. Mol. Pharmacol. 1987, 31, 122. (17) Hynes, J. B.; Cichowicz, D. J.; Shane, B. In Chemistry and Biology of Pteridines; Cooper, B. A., Whitehead, V. M., Eds.; deGruyter: Berlin, 1986;pp 997-1000. (18) Cichowicz, D.J.; Hynes, J. B.; Shane, B. Biochim. Biophys. Acta 1988,957, 363.
0022-2623/89/1832-1559$01.50/00 1989 American Chemical Society
1560 Journal of Medicinal Chemistry, 1989, Vol. 32, No. 7
(2f) was reported to be the most effective substrate iden0
R1 2 a H b NH2 c H d NH2 e NH2 NH2
1
R2
x
H H H H CI
S S O
COOH
Patil e t al.
Scheme I. Synthesis of Nu-( 2-Desamino-lO-thia-5,8-dideazapteroyl)-~-ornithine
5
4
0 NH
CI
1
6
IN~OH
2f
tified thus far. Analogues where S or 0 replaced N1", 2b and -d, were less efficient than their nitrogen counterparts but were superior to MTX.17J8 The 2-desamino modifications 2a and -c were recently prepared and found to be 3- to 5-fold more cytotoxic toward L1210 leukemia cells in vitro than their 2-NH2-containingcounterparts 2b and -d.19 The enhancement in cytotoxicity caused by the removal of the 2-NH2 group in structurally related compounds was attributed to enhanced cellular uptake.20 It was of interest, therefore, to prepare the L-ornithine counterparts of 2a-d (3a-d) for evaluation as inhibitors
0
II
7a COOH
I
I
0
II
Sa Ri 3 a H b NH2 C H d NH2 e NH2
NH2
I
R2 H H H H CI
IC
X S S O
3a
0 NH
0
CI
I
COOH
3f
of FPGS. Also synthesized were the 5-chloro folate and aminopterin modifications bearing a terminal L-ornithine, 3e and -f. In previous studies, the presence of a chlorine at position five of the quinazoline nucleus consistently produced the best substrates for FPGS within a given set of analogue~.'~J*During the course of these studies, 5,8dideazapteroyl-L-ornithine was reported to have a Ki toward human FPGS in the 0.15 MMrange. Chemistry. The preparation of 2-desamino-lo-thia5&dideazapteroyl-~-ornithine(3a) was facilitated by the recent description of the synthesis of 6-(bromomethyl)3,4-dihydro-4-oxo-3-[ (pivaloyloxy)methyl]quinazoline (4).20 As shown in Scheme I, treatment of 4 with the sodium salt of ethyl p-mercaptobenzoate (5) gave the fully protected intermediate 6, which in the presence of base yielded 2desamino-10-thiapteroicacid (7a). Peptide bond formation (8) using diethyl to N6-(tert-butyloxycarbony1)-L-ornithine (19) Hynes, J. B.; Patil, S. A.; Hagan, R. L.; Cole, A.; Kohler, W.; Freisheim, J. H. J . Med. Chem. 1989, 32, 852. (20) Jones, T. R.; Thornton, T. J.; Flinn, A.; Jackman, A. L.; Newell, D. R.; Calvert, A. H. J . Med. Chem. 1989, 32, 847. (21) McGuire, J. J.; Bolanowska, W. E.; Piper, J. R. Biochem. Pharmacol. 1988, 37, 3931.
phosphorocyanidate (DEPC) afforded 9a, which upon treatment with trifluoroacetic acid produced the target compound 3a in reasonable overall yield. The remaining three new L-ornithine derivatives, 3b-d, were prepared in an analogous fashion from their corresponding pteroic acid analogues, 7b-d. The methods employed in preparing these 5,B-dideazapteroates have been described in previous communi~ations.'~~~~~~~ The synthesis of the 5-chloro-~-ornithineanalogue 3e was conducted as outlined in Scheme 11. The key intermediate 6-[(p-carboxyanilino)methyl]-5-chloro-2,4-diaminoquinazoline (10) was prepared as described earlier2* and then subjected to acid-catalyzed hydrolysis to yield 5-chloro-5,8-dideazapteroicacid (1 I), which was converted to the 10-(trifluoroacetyl) derivative 12a by using anhydrous trifluoroacetic anhydride. The coupling reaction of the latter compound to 8 was conducted by using isobutyl chloroformate as the activating reagent to afford 13a. This protected intermediate was not purified by silica gel chromatography after it was found that the trifluoroacetyl group was labile under the basic conditions required for elution. Instead, it was treated with ammonium hydroxide to yield 14a, which was completely characterized. Removal of the tert-butyloxycarbonyl group from the N6-amino (22) Oatis, J. E.,Jr.; Hynes, J. B. J. Med. Chem. 1977, 20, 1393. (23) Hynes, J. B.; Patil, S. A.; TomaiiE, A.; Kumar, A.; Pathak, A.; Tan, K.; Xianqiang, L.; Ratnam, M.; Delcamp, T. J.; Freisheim, J. H. J . Med. Chem. 1988,31,449. (24) Hynes, J. B.;Kumar, A.; TomaiiE, A.; Washtien, W. L. J . Med. Chem. 1987,30, 1515. (25) Susten, S. S.;Hynes, J. B.; Kumar, A.; Freisheim, J. H. Biochem. Pharmacol. 1985, 34, 2163.
New Analogues of Folic Acid and Aminopterin
Journal of Medicinal Chemistry, 1989, Vol. 32, No. 7 1561
Scheme 11. Synthetic Route to Na-(5-Chloro-5,8-dideazapteroyl)-~-ornithine
Table I. Comparison of the Kinetic Constanta of 5,8-Dideaza Analogues of Folic Acid and Aminopterin with Their L-Ornithine Counterparts for Homogeneous Hog Liver Folylpolyglutamate Synthetase"
0
10
2a 3a 2b 3b 2C
3c 2d 3d 2e 3e 2f 3f PteGlu
11 U
0
II
50
42
15c
33'
17c
35
95
21
22c
25"
0.42
14
2.8
2.1
1.2
4.9
5.9
1.0
8.7c
0.3
68
1750
0.2
57
2210
0.0083d
710
0.0017d 3470 93e
61e
5.1°
1 5.90 1.0 (6S)-HaPteGlu 7.7O 100e 100e "Standard error of the mean, K , < *20%, V,, < *lo%. Relative to results for (6S)-H4PteGlu normalized to 100. CReported previously; cf. ref 18. dEvaluated by using 0.4 nM FPGS. eReported previously; cf. ref 13.
12a
~
0
13a
0
8.1
0 COOH
CI
14a
3e
0
II
(1) (CH312CHCH2OCCI
( 2 ) H2NCHCOO-t-Bu
I
i U
(CH212COO-I- BU
~~
Table 11. Inhibition of Human Dihydrofolate Reductase by 5,8-Dideaza Analogues of Folic Acid and Aminopterin Containing a Terminal L-Ornithine or L-Glutamate I w , PM" compd R1 Rz X L-glutamate L-ornithine 2a H H S 0.65b 3a H H S 3.8 2b NH2 H S 0.33' 3b NHp H S 1.5 H O 0.26b 2C H 3c H H O 3.8 2d NH2 H 0 0.17c 3d NHp H 0 0.53 C1 NH 0.10 2e NHp 3e NH2 C1 NH 0.43 C1 NH 0.0038' 2f NHp 3f NH2 C1 NH 0.0030 MTX 0.0043 Limits of variability
