J . Med. Chem. 1986,29,655-660
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Methotrexate Analogues. 26. Inhibition of Dihydrofolate Reductase and Folylpolyglutamate Synthetase Activity and in Vitro Tumor Cell Growth by Methotrexate and Aminopterin Analogues Containing a Basic Amino Acid Side Chain Andre Rosowsky,*+James H. Freisheim,t Richard G. Moran,s Vishnu C. Solan,+Henry Bader,t Joel E. Wright,+ and Mary Radike-Smith* Dana-Farber Cancer Institute a n d Department of Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, Department of Biochemistry, Medical College of Ohio, Toledo, Ohio 43699, and Division of HematologylOncology, Children’s Hospital of Los Angeles, Los Angeles, California 90027. Received July 22, 1985 Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Om), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with O m in place of Glu was also synthesized. The MTX analogues were obtained (i) by reaction of 4-amino-4-deoxy-~0-methylpteroic acid (mAPA) and N”-Boc-cY,w-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or (ii) by reaction of p-nitrophenyl-mAPA and Nu-Boc-cu,w-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Om) was with silylated N6-Boc-L-ornithine in synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate DMF at 55 “C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Om was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (ICm = 0.072 FM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 & 0.06 wM). The MTX analogue (mAPA-Om) was likewise active against both enzymes, with an ICboof 0.160 fiM for DHFR and a Ki of 20.4 f 7.7 WMfor FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Om but lacked activity as FPGS inhibitors. The positively charged amino group appears to be detrimental to cellular uptake, as evidenced by the low cytotoxicity of these compounds (IC, = 0.40-2.4 fiM) in comparison with MTX and AMT (IC5o = 0.002 1M) against wild-type L1210 cells. On the other hand, mAPA-Orn and APA-Orn were both more potent than the corresponding Glu derivatives MTX and AMT against L1210/R81 cells, suggesting that in these MTX-resistant cells there may occur a “self-potentiation” process involving enhanced antifolate activity via interference with the polyglutamylation of reduced folates. APA-Orn is the most potent dual inhibitor of DHFR and FPGS discovered to date, but its effectiveness as a therapeutic agent may require some form of prodrug modification to neutralize the terminal amino group of the side chain.
As part of a larger research program on analogues of methotrexate (MTX, 1) as antifolates,’ we synthesized N”-(4-amino-4-deoxy-~o-methylpteroyl)-~-lysine (2) and converted it to W-iodoacety12 and Ne-(4’-fluoresceinyl)thiocarbamoyP derivatives. The iodoacetamide was found to be a potent active-site-directed irreversible inhibitor of dihydrofolate reductase (DHFR),2whereas the fluorescein derivative proved useful in detecting DHFR overproduction associated with gene amplification in MTX-resistant cells3g4and in identifying the presence of a defect in MTX tran~port.~ Use of 2 and of the corresponding L-ornithine analogue 3 has been made to prepare fluorescent Nd-and W -[ [5-(N,N-dimethylamino)-l-naphthyl]sulfonyl] derivatives with strong DHFR affinity.6-8
I, R=Me; n = 2 ; X = C O O H (mAPA-L-Glu. M T X ) 2, R=Me: n = 4 : X=NH, ( m A P A - L - L y s ) 3, R=Me: n = 3 : X=NH, (mAPA-L-Orn) 4. R=Me: n.2: X = N H 2 (mAPA-L-Dab) 5. R = M e ; n = l : X=NHz (mAPA-L-Dap) 6, R=H; n.3; X = N H 2 ( A P A - L - O r n )
Analogues of 2 with fewer CH2 groups in the side chain or with hydrogen in place of methyl at N’O were required as synthetic intermediates in our program and were themselves of interest as potential inhibitors of DHFR and folylpolyglutamate synthetase (FPGS), another folate enzyme we have been studying as a potential target for ~hemotherapy.~-’~ Recently, Shane15reported that hog Dana-Farber Cancer Institute. Medical College of Ohio. 5 Children’s Hospital of Los Angeles. 0022-2623/86/1829-0655$01.50/0
liver FPGS is inhibited by pteroyl-L-ornithine and 5,6,7,8-tetrahydropteroyl-~-ornithine. The present paper describes the preparation of the L-ornithine, ~-2,4-diaminobutyric acid, and ~-2,3-diaminopropionicacid anaFor a recent account of progress in this field, the following review may be consulted: Montgomery, J. A,; Piper, J. R. In ”Folate Antagonists as Therapeutic Agents”; Sirotnak, F. M., Burchall, J. J., Ensminger, W. D., Montgomery, J. A,, Eds.; Academic Press, Orlando, FL, 1984; pp 219-260. Rosowsky, A.; Wright, J. E.; Ginty, C.; Uren, J. J . Med. Chem. 1982, 25; 960. Rosowskv. A,: Wrieht. J. E.: Shaairo. H.: Beardslev. G. P.: Lazarus,H. i.Biol Chem. 1982, i57, ’14 162. Freisheim, J. H.; Susten, S. S.: Delcamp, T. J.; Rosowsky, A.; Wright, J. E.; Kempton, R. J.; Blankenship, D. T.; Smith, P. L.; Kumar, A. A. In “Pteridines and Folic Acid Derivatives”; Blair, J. A., Ed.; Walter De Gruyter: Berlin, 1983; pp 223-227. Rosowsky, A.; Wright, J. E.; Cucchi, C. A.; Boeheim, K.; Frei, E., I11 Biochem. Pharrnacol. 1985, 35, 356. Kumar, A. A.; Freisheim, J. H.; Kempton, R. J.; Anstead, G. M.; Black, A. M.; Judge, L. J. Med. Chern. 1983,26, 111. Kumar, A. A.; Kempton, R. J.; Anstead, G. M.; Freisheim, J. H. Biochemistry 1983,22, 390. Susten, S. S.; Kempton, R. J.; Black, A. M.; Freisheim, J. H. Biochem. Pharmacol. 1984, 33, 1957. Moran, R. G. Adu. Exptl. Med. Biol. 1983, 163, 327. Rosowsky, A,; Moran, R. G.; Forsch, R.; Colman, P.; Wick, M. Biochem. Pharmacol. 1984, 33, 155. Rosowsky, A.; Forsch, R. A.; Freisheim, J. H.; Moran, R. G.; Wick, M. J . Med. Chem. 1984, 27, 600. Moran, R. G.; Colman, P. D.; Rosowsky, A.; Forsch, R. A.; Chan, K. K. Mol. Pharrnacol. 1985,27, 156. Moran, R. G.; Rosowsky, A.; Colman, P.; Forsch, R. A.; Solan, V. C.; Bader, H.; Harvison, P.; Kalman, T. I. In “Second Workshop on Folyl and Antifolyl Polyglutamates”; Goldman, I. D., Ed.; Praeger Publishers: Philadelphia, 1985; pp 51-64. Forsch, R. A.; Freisheim, J. H.; Moran, R. G.; Rosowsky, A. Proc. Am. Assoc. Cancer Res. 1985, 26, 22. Cichowicz, D.; Cook, J.; George, S.; Shane, B. In “Second Workshop on Folyl and Antifolyl Polyglutamates”; Goldman, I. D., Ed.; Praeger Publishers: Philadelphia, 1985; pp 7-13. “
0 1986 American Chemical Society
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656 Journal of Medicinal C h e m i s t r y , 1986, Vol. 29, No. 5
Rosowshy et al.
logues 3-5 or MTX, as well as the synthesis of the Lcarbonyl)oxy]imino]-2-phenylacetonitrilez4instead of ornithine analogue 6 of aminopterin (AMT). Compounds tert-butyloxycarbonyl azide. Compounds 9-1 1 gave a 3-5 were prepared from 4-amin0-4-deoxy-N~~-methyl- positive ninhydrin test and showed the expected NMR pteroic acid (7)2J6while 6 was obtained from 4-amino-4features including a 6 1.4 singlet which is characteristic of deoxy-WO-formylpteroicacid (8).11916J7Compounds 3-6 the Boc group. The Nw-Bocderivatives were of sufficient were tested as inhibitors of DHFR from L E 1 0 murine purity to be used directly in subsequent coupling reactions. leukemia cells and of FPGS from normal mouse liver. In H,NCHCOOH addition, the growth-inhibitory activity of 3-6 was evalu(CH,),,NHBoc ated in culture against L1210 and L1210/R81 cells, the latter of which are highly resistant to MTX. Although they 9. n = 3 show slightly lower activity than MTX or AMT as DHFR 10, n = 2 11, n =1 inhibitors, the L-ornithine derivatives 3 and 6 display striking activity against FPGS and may therefore be Condensation of the amino acid 9-11 with 7 was acviewed as a lead for further analogue synthesis. complished with the aid of the peptide-bond-forming reagent diethyl phosphorocyanidate (DEPC),28which we have used extensively in the past to prepare MTX anal o g ~ e s . The ~ ~ reaction ~ ~ ~ was ~ ~conducted ~ ~ ~ ~ at room ~ - ~ ~ temperature in DMF, with 2 molar equiv each of DEPC and N,iV-diisopropylethylamine. The carboxy group of the amino acid was left unprotected. Compounds 12-14 were 7. R'=Me; R 2 = H (mAPA) obtained in yields that varied according to the number of 8 . R'=CHO: R2 = H 15. R'=CHO; R ~ = ~ - N O , C , H , methylene groups in the side chain (12,87%; 13,72%; 14, 16, R ' = M ~ ;R ~ = . ~ - N O , C , H , %YO), but there was a severe decrease in the yield for 14 in comparison with 12 and 13. This may reflect an inChemistry. Because of our extensive prior experience herent instability of the amino acid 11 under the conditions with 7 as a precursor of MTX analogues with modified side of the DEPC coupling reaction. One possibility is that 11 chains,2J0J1Jg22we elected to prepare 3-5 from this commay suffer facile ring closure to an imidazolidin-2-one. pound and suitable Nu-protected a,w-diaminoalkanoic acids. However, instead of using a benzyloxycarbonyl (Cbz) group to block the terminal NH2 group of the amino N~rcH2~--@owcHcooH I acid, as we had done in the preparation of 2,2,3we used the tert-butyloxycarbonyl (Boc) group, which Kumar and co(CH,)"NHR~ workers showed can be cleaved readily with trifluoroacetic H2N acid.6 12. n.3; R'=Me; R2=Boc N*-Boc-L-ornithine (9) was prepared from bis(L13. n.2; R'=Me: R2=Boc ornithinat~)copper(II)~~ in 60% overall yield by reaction 14. n = l ;R'=Me; R2=Boc 17. n =3; R'=CHO: R2=Boc with 2- [ [ (tert-butyloxycarbonyl)oxy]imino]-2-phenyl18, n = 3 : R ' = H ; Rz=Boc acetonitrile in aqueous dioxane followed by copper seA modification of the usual workup procedure for DEPC questration with EDTA.24 The same procedure was used reactions that is worth noting is that, in the isolation of to prepare Ny-Boc-~-2,4-diaminobutyric acid (10) but could 12-14, solid NaHCO, was added prior to the evaporation not be applied to N6-Boc-~-2,3-diaminopropionic acid (11) of the DMF and tertiary amine. A pH decrease is normally since the reaction of Cu(I1) with ~-2,3-diaminopropionic observed in this step, presumably because of the diethyl acid is known to produce a coordination complex involving phosphate that is left behind. We reasoned that this could the two amino groups, rather than the a-amino group and produce cleavage of the acid-labile Boc group and in fact carboxy group, as ligands.25 Compound 11 was therefore observed such cleavage by TLC. When NaHCO, was inprepared from N"-tosyl-L-asparagine,26using the literature cluded in the workup, loss of Boc was avoided and the routez7with the exception of the introduction of the N6-Boc product was isolated in pure state and higher overall yield. group, which was carried out with 2-1[(tert-butyloxyRemoval of the Nw-Bocgroup in 12-14 was accomplished easily with trifluoroacetic acid at room temperature, and Pioer. J. R.: Montpomerv. J. A. J . Ore. Chem. 1977. 42. 208: the products (3-5) were purified by cation-exchange see also earlier referencescited in this paper for other methods of synthesis of 7. chromatography on sulfoxyethylcellulose or anion-exFor references to methods of synthesis of 4-amino-4-deoxychange chromatography on DEAE-cellulose, with 0.1 M pteroic acid, the immediate precursor of 8, see: Nair, M. G.; NH4HCO3 as the eluent in each system.29 Adapa, S. R.; Bridges, T. W. J . Org. Chem. 1981, 46, 3152. An inherent drawback of DEPC coupling in the prepaRosowsky, A.;Forsch, R.; Uren, J.; Wick, M.; Kumar, A. A.; ration of MTX analogues from 7 is that the amino acid Freisheim, J. H. J . Med. Chem. 1983, 26, 1719. component has to be used in excess. This is undesirable Rosowsky, A.;Forsch, R. A.; Yu, C.-S.; Lazarus, H.; Beardsley, G. P. J . Med. Chern. 1984, 27, 605. when the amino acid (e.g., 11) is in limited supply. In a Rosowsky, A.; Forsch, R. A.; Freisheim, J. H., Galivan, J.; recent paperz2we reported that p-nitrophenyl4-amino-4Wick, M. J . Med. Chern. 1984,27, 888. deoxy-NlO-formylpteroate (15) affords a 70% yield of
A
Moran, R. G., Colman, P. D.; Forsch, R. A.; Rosowsky, A. J . Med. Chem. 1984, 27, 1263. Rosowsky, A.; Freisheim, J. H.; Bader, H.; Forsch, R. A.; Susten, S. s.;Cucchi, C. A.; Frei, E., 111J. Med. Chem. 1985, 28,
660 (paper 25 in this series). Taurins, A. Can. J . Res., Sect. E 1950, 28E, 762. Rosowsky, A,; Wright, J. E. J . Org. Chern. 1983, 48, 1539. Albert, A. Biochem. J . 1952, 50, 690. Zaoral, M.;Rudinger, J. Coll. Czech. Chern. Cornnun. 1959,24, 1993. Rudinger, J.; Poduska, K.; Zaoral, M. Coll. Czech. Chern.
rOmmun. 1960,25,2022.
(28) Shioiri, T.; Hamada, Y. J . Org. Chem. 1978, 43, 3631. (29) Upon completion of this synthetic work, we learned that compounds 4 and 5 were synthesized independently at Southern Research Institute via a different route involving reaction of 2,4-diamino-6-bromomethylpteridine with N*-aroyl-N"-Boca,w-diaminoalkanoic acids. We are grateful to Dr. James Piper for bringing this to our attention while the present paper was being written; see Piper, J. R.; McCaleb, G. S.; Montgomery, J. A., Schmid, F. A.; Sirotnak, F. M. J . Med. Chern. 1985,28, 1016.
Journal of Medicinal Chemistry, 1986, Vol. 29, No. 5
M e t h o t r e x a t e Analogues
Table I. Inhibition of Enzyme Activity and Cell Growth by Compounds 2-6 enzymes DHFR:* FPGS:c cells:" ICw, pM L121O/R81 Ki,pM L1210 compd ICM,pM 0.065 0.40 220 2 d 0.160 20.4e 1.30 3 86 0.120 d 2.42 290 4 5 0.180 d 0.44 405 1.30 32 0.072 0.19 6 MTX 220 g 0.002 0.035 0.002 84 AMT .e 0.035 See ref 38 for a Forty-eight hours of continuous drug exposure. assay method; ICw values are for DHFR concentrations of 0.07 pM in the assay mixture. CSeeref 10 for assay method. d
