5-deazafolate analogues with a rotationally restricted glutamate or ornithine side chain: synthesis and binding interaction with folylpolyglutamate synthetase.

Rotationally restricted analogues of 5-deazapteroyl-L-glutamate and (6R,6S)-5-deaza-5,6,7,8-tetrahydropteroyl-L-glutamate with a one-carbon bridge between the amide nitrogen and the 6'-position of the p-aminobenzoyl moiety were synthesized and tested as substrates for folylpolyglutamate synthetase (FPGS), a key enzyme in folate metabolism and an important determinant of the therapeutic potency and selectivity of classical antifolates. The corresponding bridged analogues of 5-deazapteroyl-L-ornithine and (6R,6S)-5-deaza-5,6,7, 8-tetrahydropteroyl-L-ornithine were also synthesized as potential inhibitors. Condensation of diethyl L-glutamate with methyl 2-bromomethyl-4-nitrobenzoate followed by catalytic reduction of the nitro group, reductive coupling with 2-acetamido-6-formylpyrido[2, 3-d]pyrimidin-4(3H)-one in the presence of dimethylaminoborane, and acidolysis with HBr/AcOH yielded 2-L-[5-[N-(2-acetamido-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]glutaric acid (1). When acidolysis was preceded by catalytic hydrogenation, the final product was the corresponding (6R,6S)-tetrahydro derivative 2. A similar sequence starting from methyl N(delta)-benzyloxycarbonyl-L-ornithine led to 2-L-[5-[N-(2-amino-4(3H)-oxopyrido[2, 3-d]pyrimidin-6-yl)methylamino]-2, 3-dihydro-1-oxo-2(1H)-isoindolyl]-5-aminopentanoic acid (3) and the (6R,6S)-tetrahydro derivative 4. Compounds 3 and 4 were powerful inhibitors of recombinant human FPGS, whereas 1 and 2 were exceptionally efficient FPGS substrates, with the reduced compound 2 giving a K(m) (0.018 microM) lower than that of any other substrate identified to date. (6R,6S)-5-Deazatetrahydrofolate, in which the side chain is free to rotate, was rapidly converted to long-chain polyglutamates. In contrast, the reaction of 1 and 2 was limited to the addition of a single molecule of glutamic acid. Hence rotational restriction of the side chain did not interfere with the initial FPGS-catalyzed reaction and indeed seemed to facilitate it, but the ensuing gamma-glutamyl adduct was no longer an efficient substrate for the enzyme.     

(6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydrofolate and 6(R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-ornithine as potential antifolates and antitumor agents

(6R,6S)-5,8,10-Trideaza-5,6,7,8-tetrahydropteroic acid was synthesized in several steps from 4,4-(ethylenedioxy)-cyclohexanone and [4-(tert-butyloxycarbonyl)benzyl]triphenylphosphonium bromide and was elaborated to (6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-glutamic acid and (6R,6S)-5,8,10-trideaza-5,6,7,8-tetrahydropteroyl-L-ornithin e. Compound 1 was found to be a good substrate for partially purified mouse liver folypolyglutamate synthetase (FPGS), with a Michaelis constant (Km = 15 microM) comparable to that reported for the reduced folate substrate (6S)-5,6,7,8-tetrahydropteroyl-L-glutamic acid and for (6R,6S)-5,10-dideaza-5,6,7,8-tetrahydropteroyl-L-glutamic acid (DDATHF). However, in striking contrast to DDATHF, which is potently cytotoxic, 1 failed to inhibit tumor cell growth in culture at concentrations of up to 100 microM. These results suggested that the NH at position 8 of DDATHF is important for cytotoxic activity but not for polyglutamylation. Just as 1 was a good substrate for FPGS, the ornithine analogue 2 proved to be among the more potent competitive inhibitors of this enzyme discovered to date, with a Ki,s of 10 microM. While the binding affinity of 2 was lower than that reported for 5,6,7,8-tetrahydropteroyl-L-ornithine (H4PteOrn), very substantial FPGS inhibition was observed even though N5,N8, and N10 in H4PteOrn were replaced by carbon. Binding to FPGS thus appears to be tolerant of bioisosteric replacements made simultaneously in ring B and the bridge region. Neither 1 nor 2 was active in preventing cell growth in culture at concentrations of up 100 microM. The N delta-hemiphthaloyl derivative of 2, synthesized as a potential prodrug, was also inactive.     

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

Analogues of the antitumor antifolate methotrexate (MTX) were synthesized in which the glutamate (Glu) moiety was replaced by ornithine (Orn), 2,4-diaminobutyric acid (Dab), or 2,3-diaminopropionic acid (Dap). An aminopterin (AMT) analogue with Orn in place of Glu was also synthesized. The MTX analogues were obtained by reaction of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and N omega-Boc-alpha,omega-diaminoalkanoic acids in the presence of diethyl phosphorocyanidate, followed by deprotection with trifluoroacetic acid (TFA) or by reaction of p-nitrophenyl-mAPA and N omega-Boc-alpha,omega-diaminoalkanoic acids and subsequent treatment with TFA. The AMT analogue (APA-Orn) was synthesized by reaction of p-nitrophenyl 4-amino-4-deoxy-N10-formylpteroate with silylated N delta-Boc-L-ornithine in DMF at 55 degrees C for 3 days (45% yield), saponification (83%), and TFA cleavage (89%). APA-Orn was a potent inhibitor of both dihydrofolate reductase (DHFR) from L1210 mouse leukemia (IC50 = 0.072 microM) and partly purified folylpolyglutamate synthetase (FPGS) from mouse liver (Ki = 0.15 +/- 0.06 microM). The MTX analogue (mAPA-Orn) was likewise active against both enzymes, with an IC50 of 0.160 microM for DHFR and a Ki of 20.4 +/- 7.7 microM for FPGS inhibition. The other MTX analogues and the previously reported lysine derivative (mAPA-Lys) showed DHFR affinity similar to that of mAPA-Orn 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 (IC50 = 0.40-2.4 microM) in comparison with MTX and AMT (IC50 = 0.002 microM) 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.     

Synthesis and biological evaluation of 5-deazaisofolic acid, 5-deaza-5,6,7,8-tetrahydroisofolic acid, and their N9-substituted analogues

Prompted by recent disclosures concerning the potent antitumor activities of 5-deaza-5,6,7,8-tetrahydrofolic acid and 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (DDATHF), we have prepared 5-deazaisofolic acid (3a) and 5-deaza-5,6,7,8-tetrahydroisofolic acid (4a). Reductive condensation of 2,6-diamino-3,4-dihydro-4- oxopyrido[2,3-d]pyrimidine with di-tert-butyl N-(4-formylbenzoyl)-L-glutamate and subsequent deprotection with trifluoroacetic acid yielded 5-deazaisofolic acid in good yield. Catalytic hydrogenation of this analogue then gave 4a. The 9-CH3 and 9-CHO modifications of 3a and the 9-CH3 derivative of 4a were also synthesized. Each of the new analogues was evaluated with a variety of folate-requiring enzymes as well as MCF-7 cells in culture. Compound 4a had an IC50 of ca. 1 microM against MCF-7 cells and was nearly 100-fold less potent than DDATHF in this regard. The three oxidized isofolate analogues were all poor inhibitors of tumor cell growth.     

Methotrexate analogues. 32. Chain extension, alpha-carboxyl deletion, and gamma-carboxyl replacement by sulfonate and phosphonate: effect on enzyme binding and cell-growth inhibition

Analogues of methotrexate (MTX) and aminopterin (AMT) with aminophosphonoalkanoic, aminoalkanesulfonic, and aminoalkanephosphonic acid side chains in place of glutamate were synthesized and tested as inhibitors of folylpolyglutamate synthetase (FPGS) from mouse liver. The aminophosphonoalkanoic acid analogues were also tested as inhibitors of dihydrofolate reductase (DHFR) from L1210 murine leukemia cells and as inhibitors of the growth of MTX-sensitive (L1210) and MTX-resistant (L1210/R81) cells in culture. The optimal number of CH2 groups in aminophosphonoalkanoic acid analogues of AMT was found to be two for both enzyme inhibition and cell growth inhibition but was especially critical for activity against FPGS. Deletion of the alpha-carboxyl also led to diminished anti-FPGS activity in comparison with previously studied homocysteic acid and 2-amino-4-phosphonobutyric acid analogues. In the aminoalkanesulfonic acid analogues of MTX without an alpha-carboxyl, anti-FPGS activity was low and showed minimal variation as the number of CH2 groups between the carboxamide and sulfonate moieties was changed from one to four. In similar aminoalkanephosphonic acid analogues of MTX, anti-FPGS activity was also low, was comparable for two and three CH2 groups between the carboxamide and phosphonate moieties, and was diminished by monoesterification of the phosphonate group. These effects demonstrate that the alpha-carboxyl group of folate analogues is involved in binding to the active site of FPGS, and that an alpha-carboxyl group should be retained as part of the structure of FPGS inhibitors.     

Methotrexate analogues. 31. Meta and ortho isomers of aminopterin, compounds with a double bond in the side chain, and a novel analogue modified at the alpha-carbon: chemical and in vitro biological studies

Five heretofore undescribed analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized and tested as dihydrofolate reductase (DHFR) inhibitors and tumor cell growth inhibitors. The meta isomer of AMT was obtained from 2,4-diamino-6-(bromomethyl)pteridine and m-(aminobenzoyl)-L-glutamic acid, while the ortho isomer was obtained via the same route by using alpha-methyl gamma-tert-butyl o-(aminobenzoyl)-L-glutamate instead of the free acid. Analogues of MTX and AMT containing a double bond in the side chain were prepared from dimethyl D,L-2-amino-4-hexenedioate and 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively. Finally, a positional isomer of MTX with the CH2CH2COOH moiety moved from the alpha-carbon to the adjacent carboxamide nitrogen was synthesized from 3-[N-(carboxymethyl)amino]propanoic acid diethyl ester and 4-amino-4-deoxy-N10-methylpteroic acid. The positional isomers of AMT were weak DHFR inhibitors and showed very little growth-inhibitory activity against L1210 murine leukemia cells or the MTX-resistant L1210/R81 mutant line in culture. The MTX and AMT analogues with the CH2CH2COOH moiety replaced by a CH2CH = CHCOOH side chain showed anti-DHFR activity similar to that of the previously described saturated compound N-(4-amino-4-deoxy-N10-methylpteroyl)-L-2-aminoadipic acid, but were less potent than the parent drugs. The MTX analogue with the CH2CH2COOH side chain displaced from C to N was weakly bound to DHFR, confirming the importance of an intact CONH moiety, and showed greatly diminished cell growth inhibitory potency relative to MTX. None of the compounds was a substrate for folylpolyglutamate synthetase (FPGS) from mouse liver. Furthermore, inhibition of folic acid polyglutamylation in vitro at equimolar 500 microM concentrations of drug and substrate was negligible. The structural changes embodied in these five novel compounds are therefore too great for binding to the FPGS active site.     

Benzoyl ring halogenated classical 2-amino-6-methyl-3,4-dihydro-4-oxo-5-substituted thiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine antifolates as inhibitors of thymidylate synthase and as antitumor agents

In an attempt to circumvent resistance to and toxicity of clinically used folate-based thymidylate synthase (TS) inhibitors that require folylpoly-gamma-glutamate synthetase (FPGS) for their antitumor activity, we designed and synthesized two classical 6-5 ring-fused analogues, N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-fluorobenzoyl]-l-glutamic acid (4) and N-[4-[(2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]-2'-chlorobenzoyl]-l-glutamic acid (5), as TS inhibitors and antitumor agents. The key intermediates in the synthesis of these classical analogues were the mercaptans 10 and 11, which were obtained from the corresponding nitro compounds 6 and 7 respectively, by reduction of the nitro groups followed by diazotization of the amines. The syntheses of analogues 4 and 5 were achieved via the oxidative addition of the sodium salt of ethyl 2-halo-substituted-4-mercaptobenzoate (16 or 17) to 2-amino-6-methyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine (18) in the presence of iodine. The esters obtained from the reaction were deprotected and coupled with diethyl-l-glutamate followed by saponification. Compounds 4 and 5 were both more potent inhibitors of human TS (IC(50) values of 54 and 51 nM, respectively) than were PDDF and the clinically used ZD1694 and LY231514. Compounds 4 and 5 were not substrates for human FPGS up to 250 muM. In addition, 4 and 5 were growth inhibitory against CCRF-CEM cells as well as a number of other tumor cell lines in culture, and protection studies established TS as the principal target of these analogues.     

Folate analogues. 30. Synthesis and biological evaluation of N10-propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolic acid and related compounds

The 5,6,7,8-tetrahydro derivative (1) of the powerful thymidylate synthase inhibitor N10-propargyl-5,8-dideazafolic acid (PDDF) has been synthesized and evaluated for its antifolate activity. A convenient method for the preparation of the key intermediate 2-amino-6-(bromomethyl)-4-hydroxy-5,6,7,8-tetrahydroquinazoline (18) is described. Two closely related analogues of 1 were also synthesized and evaluated for their antifolate activity and thymidylate synthase inhibition. N10-Propargyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (1) and N10-methyl and N10-hydrogen analogues 2 and 3 were weaker inhibitors of Lactobacillus casei thymidylate synthase compared to PDDF. N10-Methyl-5,8-dideaza-5,6,7,8-tetrahydrofolate (2) exhibited the most potent antifolate activity against L. casei (IC50 = 2.8 nM) and Streptococcus faecium (IC50 = 0.57 nM). In intact and permeabilized murine leukemia L1210 cells, the replacement of the quinazoline moiety with its tetrahydro derivative resulted in a marked decrease in potency and a loss of the contribution of the propargyl substituent to enzyme inhibition, indicating an altered binding mode to thymidylate synthase.     

Synthesis of classical and nonclassical,partially restricted,linear, tricyclic 5-deaza antifolates

Seven novel 2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridine derivatives 3-9 with different benzyl and a benzoyl substitution at the N7 position were designed and synthesized, as classical and nonclassical, partially restricted, linear tricyclic 5-deaza antifolates. The purpose was to investigate the effect of conformational restriction of the C6-C9 (tau(1)) and C9-N10 (tau(2)) bonds via an ethyl bridge from the N10 to the C7 position of 5-deaza methotrexate (MTX) on the inhibitory potency against dihydrofolate reductase (DHFR) from different sources and on antitumor activity. The synthetic methodology for most of the target compounds was a concise five-step total synthesis to construct the tricyclic nucleus, 2,4-diamino-5-deaza-7H-6,7,8,9-tetrahydropyrido[3,4-g]pteridine (23), followed by regioselective alkylation of the N7 nitrogen. Biological results indicated that this partial conformational modification for the classical analogue N-[4-[(2,4-diamino-5-deaza-6,7,8,9-tetrahydropyrido[3,4-g]pteridin-7-yl)methyl]benzoyl]-L-glutamic acid 3 was detrimental to DHFR inhibitory activity as well as to antitumor activity compared to MTX or 5-deaza MTX. However, the classical analogue 3 was a better substrate for folypolyglutamate synthetase (FPGS) than MTX. These results show that a classical 5-deaza folate partially restricted via a bridge between the N10 and C7 positions retains FPGS substrate activity and that the antitumor activity of classical tricyclic analogues such as 3 would be influenced by FPGS levels in tumor systems. Interestingly, the nonclassical analogues 4-9 showed moderate to good selectivity against DHFR from pathogenic microbes compared to recombinant human DHFR. These results support the idea that removal of the 5-methyl group of piritrexim along with restriction of tau(1) and tau(2) can translate into selectivity for DHFR from pathogens.     

Design,synthesis, and biological activities of classical N-[4-[2-(2-amino-4-ethylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid and its 6-methyl derivative as potential dual inhibitors of thymidylate synthase and dihydrofolate reductase and as potential antitumor agents

Two novel analogues, N-[2-amino-4-ethyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid (2) and N-[2-amino-4-ethyl-6-methyl[(pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-l-glutamic acid (4), were designed and synthesized as potent dual inhibitors of thymidylate synthase (TS) and dihydrofolate reductase (DHFR) and as antitumor agents. Compound 2 had inhibitory potency against human DHFR similar to N-[4-[2-(amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (LY231514) and 1, whereas 4 was inactive against human DHFR. Both 2 and 4 were more potent than LY231514 against E. coliTS. Against human TS, 2 was 7-fold less potent than LY231514 and 4 showed similar inhibitory activity as LY231514. In contrast to 2, which was an efficient substrate of human folypolyglutamate synthetase (FPGS), 4 was a poor substrate of FPGS. Compound 2 showed GI50 values in the nanomolar range against more than 18 human tumor cell lines in the standard NCI preclinical in vitro screen.     

Methotrexate analogues-27. Dual inhibition of dihydrofolate reductase and folylpolyglutamate synthetase by methotrexate and aminopterin analogues with a gamma-phosphonate group in the side chain

gamma-Phosphonate analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid and 4-amino-4-deoxy-N10-formylpteroic acid, respectively, by reaction with methyl D,L-2-amino-4-phosphonobutyrate followed by gentle alkaline hydrolysis. The products were compared with the corresponding D,L-homocysteic acid derivatives as inhibitors of dihydrofolate reductase and folylpolyglutamate synthetase, and as inhibitors of cell growth in culture. The gamma-phosphonates were somewhat less active than either the gamma-sulfonates or the parent drugs as inhibitors of murine dihydrofolate reductase. The MTX gamma-sulfonate and gamma-phosphonate analogues were equally inhibitory toward mouse liver folylpolyglutamate synthetase (Ki = 190 microM), but in the AMT series the gamma-phosphonate (Ki = 8.4 microM) was more potent than the gamma-sulfonate (Ki = 45 microM). The AMT analogues were consistently more inhibitory than the MTX analogues against cultured L1210 murine leukemia cells, but neither the gamma-phosphonates nor the gamma-sulfonates were as potent as their respective parent drugs. The gamma-phosphonate analogue of MTX was three times more potent than MTX against the MTX-resistant mutant line L1210/R81, but the AMT gamma-phosphonate was less potent than AMT; however, these differences were small in comparison with the level of resistance to all these compounds in the L1210/R81 line. The results suggest that N10-methyl and N10-unsubstituted compounds altered at the gamma-position do not necessarily follow identical structure-activity patterns in every test system.     

Effect of structural modifications in the C7-C11 region of the retinoid skeleton on biological activity in a series of aromatic retinoids

A series of conformationally restricted analogues of (E)-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)propenyl ] benzoic acid--(E)-4-[1-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2 - propenyl]benzoic acid, (E)-4-[3-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2-bu ten- 2-yl]benzoic acid, trans-4-[2-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl) cyclopropyl]benzoic acid, 4-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-anthracenyl)benzoic acid, 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid, 6-(5,6,7,8-tetrahydro-3,5,5,8,8-pentamethyl-2-naphthalenyl)-2- naphthalenecarboxylic acid and 6-(5,6,7,8-tetrahydro-5,5,8,8-tetramethyl-2-naphthalenyl)-5-methyl-2- naphthalenecarboxylic acid--were synthesized and screened for retinoid biological activity. Comparison of the conformers of these analogues generated by molecular mechanics calculations with the biological activity profiles of these compounds indicates that geometric constraints required for high biological activity are imposed on the bridge joining the two aromatic ring systems by the retinoid receptor.     

Synthesis and in vitro biological activity of new deaza analogues of folic acid, aminopterin, and methotrexate with an L-ornithine side chain

The 5-deaza and 5,8-dideaza analogues of N alpha-pteroyl-L-ornithine (Pter-Orn), the 5-deaza, 8-deaza, and 5,8-dideaza analogues of N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-Orn), and the N delta-carboxymethyl derivative of N alpha-(4-amino-4-deoxy-N10-methylpteroyl)-L-ornithine (mAPA-Orn) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) and as inhibitors of tumor cell growth in culture. Reductive amination of 2-acetamido-6-formylpyrido[2,3-d]pyrimidine-4(3H)-one with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate followed by removal of the blocking groups afforded the 5-deaza analogue of Pter-Orn, whereas N-alkylation of methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate with 2-amino-6-(bromomethyl)quinazolin-4(3H)-one and deprotection gave the corresponding 5,8-dideaza analogue. Reductive coupling of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carbonitrile and 4-aminobenzoic acid followed by reaction with 95-97% formic acid yielded 4-amino-4-deoxy-5-deaza-N10-formylpteroic acid, which on condensation with methyl N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection gave the 5-deaza analogue of APA-Orn. A similar sequence starting from 2,4-diamino-quinazoline-6-carbonitrile led to the corresponding 5,8-dideaza compound, whereas treatment of 2,4-diamino-pyrido[3,2-d]pyrimidine-6-methanol with phosphorus tribromide followed by condensation with methyl N alpha-(4-aminobenzoyl)-N delta-(benzyloxycarbonyl)-L-ornithinate and deprotection afforded the 8-deaza analogue. For the preparation of the N delta-carboxymethyl derivative of mAPA-Orn, N alpha-(benzyloxycarbonyl)-L-ornithine was subjected to N delta-monoalkylation with glyoxylic acid and sodium cyanoborohydride, followed by N delta-acylation with ethyl trifluoroacetate, N alpha-deprotection by hydrogenolysis, condensation with 4-amino-4-deoxy-N10-methylpteroic acid, and N delta-deprotection by gentle treatment with ammonia. The 2,4-diamino derivatives all inhibited the growth of tumor cells in culture, with IC50 values of 0.2-2 microM, and inhibited purified DHFR with IC50 values of 0.02-0.08 microM. Deletion of ring nitrogens and N delta-carboxymethylation both increased potency in the cell growth assay; however, the ornithine derivatives were less potent than aminopterin or methotrexate.     

Effect of C9-methyl substitution and C8-C9 conformational restriction on antifolate and antitumor activity of classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines

N-[4-[1-methyl-2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (5) and its C8-C9 conformationally restricted E- and Z-isomers (6 and 7) were designed and synthesized in order to investigate the effect of incorporating a methyl group at the C9 position and of conformational restriction at the C8-C9 bridge of N-[4-[2-(2,4-diaminofuro[2, 3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamic acid (1) with respect to dihydrofolate reductase (DHFR) inhibitory activity as well as antitumor activity. The compounds were synthesized by a Wittig reaction of 2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine with ethyl 4-acetylbenzoate followed by catalytic reduction, hydrolysis, and standard peptide coupling with diethyl L-glutamate. The biological results indicated that the addition of a 9-methyl group to the C8-C9 bridge, as in 5, increased recombinant human (rh) DHFR inhibitory potency (IC(50) = 0.42 microM) as well as the potency against the growth inhibition of tumor cells in culture (CCRF-CEM EC(50) = 29 nM, A253 EC(50) = 28.5 nM, and FaDu EC(50) = 17.5 nM) compared with the 9-desmethyl analogue 1. However, the conformationally restricted 4:1 Z/E mixture of 7 and 6 was less potent than 5 in both assays, and the pure E-isomer 6 was essentially inactive. These three classical analogues were also evaluated as inhibitors of Lactobacillus casei, Escherichia coli, and rat and rh thymidylate synthase (TS) and were found to be weak inhibitors. All three analogues 5-7 were good substrates for human folylpolyglutamate synthetase (FPGS). These data suggested that FPGS is relatively tolerant to different conformations in the bridge region. Further evaluation of the cytotoxicity of 5 and 7 in methotrexate (MTX)-resistant CCRF-CEM cell sublines suggested that polyglutamylation was crucial for their mechanism of action. Metabolite protection studies of 5 implicated DHFR as the primary intracellular target. Compound 5 showed GI(50) values in 10(-9)-10(-7) M range against more than 30 tumor cell lines in culture.     

Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates(1)

We designed and synthesized a classical analogue N-[4-[(2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)thio]benzoyl]-L-glutamic acid (4) and thirteen nonclassical analogues 5-17 as potential dual thymidylate synthase (TS) and dihydrofolate reductase (DHFR) inhibitors and as antitumor agents. The key intermediate in their synthesis was 2-amino-6-ethyl-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidine, 22, to which various aryl thiols were conveniently attached at the 5-position via an oxidative addition reaction using iodine. For the classical analogue 4, the ester obtained from the reaction was deprotected and coupled with diethyl L-glutamate followed by saponification. Compound 4 was a potent dual inhibitor of human TS (IC(50) = 90 nM) and human DHFR (IC(50) = 420 nM). Compound 4 was not a substrate for human FPGS. Metabolite protection studies established TS as its principal target. Most of the nonclassical analogues were only inhibitors of human TS with IC(50) values of 0.23-26 microM.     

Folate analogues. 35. Synthesis and biological evaluation of 1-deaza, 3-deaza, and bridge-elongated analogues of N10-propargyl-5,8-dideazafolic acid

Structural modifications at the pyrimidine ring and at the C9,N10-bridge region of the thymidylate synthase (TS) inhibitors N10-propargyl-5,8-dideazafolate (1; PDDF; CB 3717), 2-desamino-N10-propargyl-5,8-dideazafolate (2, DPDDF), and 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (3, DMPDDF) have been carried out. Methods for the synthesis of 2-desamino-N10-propargyl-1,5,8-trideazafolate (4), 2-desamino-2-methyl-N10-propargyl-3,5,8-trideazafolate (5a), and 2-desamino-2-methyl-N10-propargyl-5,8-dideaza-1,2-dihydrofolate (6) have been developed. The bridge-extended analogues isohomo-PDDF (7) and isohomo-DMPDDF (8) contain an additional methylene group interposed between N10 and the phenyl ring of 1 and 3, respectively. All new compounds were evaluated as inhibitors of TS and the growth of tumor cells in culture. Selected analogues were tested as substrates of folylpolyglutamate synthetase (FPGS) and striking differences in substrate activity were observed among these compounds, indicating that structural modifications at the pyrimidine ring of classical antifolates profoundly influence their polyglutamylation. Enzyme inhibition data established that both N1 and N3-H of the pyrimidine ring are essential for efficient binding of quinazoline-type antifolates to human TS.     

Methotrexate analogues. 33. N delta-acyl-N alpha-(4-amino-4-deoxypteroyl)-L-ornithine derivatives: synthesis and in vitro antitumor activity

N delta-Acyl derivatives of the potent folylpolyglutamate synthetase (FPGS) inhibitor N alpha-(4-amino-4-deoxypteroyl)-L-ornithine (APA-L-Orn) were synthesized from N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-L-ornithine by reaction with an N-(acyloxy)succinimide or acyl anhydride, followed by deformylation with base. The N delta-hemiphthaloyl derivative was also prepared from 4-amino-4-deoxy-N10-formylpteroic acid by reaction with persilylated N delta-phthaloyl-L-ornithine, followed by simultaneous deformylation and ring opening of the N delta-phthaloyl moiety with base. The products were potent inhibitors of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, with IC50's ranging from 0.027 and 0.052 microM as compared with 0.072 microM for APA-L-Orn. Several of the N delta-acyl-N10-formyl intermediates also proved to be good DHFR inhibitors. One of them, N alpha-(4-amino-4-deoxy-N10-formylpteroyl)-N delta-(4-chlorobenzoyl)-L- ornithine, had a 2-fold lower IC50 than its deformylated product, confirming that the N10-formyl group is well tolerated for DHFR binding. While N delta-acylation of APA-L-Orn did not significantly alter anti-DHFR activity, inhibition of FPGS was dramatically diminished, supporting the view that the basic NH2 on the end of the APA-L-Orn side chain is essential for the activity of this compound against FPGS. N delta-Acylation of APA-L-Orn markedly enhanced toxicity to cultured tumor cells. However, N delta-acyl derivatives also containing an N10-formyl substituent were less cytotoxic than the corresponding N10-unsubstituted analogues even though their anti-DHFR activity was the same, suggesting that N10-formylation may be unfavorable for transport. Two compounds, the N delta-benzoyl and N delta-hemiphthaloyl derivatives of APA-L-Orn, with IC50's against L1210 cells of 0.89 and 0.75 nM, respectively, were more potent than either methotrexate (MTX) or aminopterin (AMT) in this system. These compounds were also more potent than MTX against CEM human lymphoblasts and two human head and neck squamous cell carcinoma cell lines (SCC15, SCC25) in culture. Moreover, in assays against SCC15/R1 and SCC25/R1 sublines with 10-20-fold MTX resistance, the N delta-hemiphthaloyl derivative of APA-L-Orn showed potency exceeding that of MTX itself against the parental cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Synthesis of classical, four-carbon bridged 5-substituted furo[2,3-d]pyrimidine and 6-substituted pyrrolo[2,3-d]pyrimidine analogues as antifolates

We report, for the first time, the biological activities of four-carbon-atom bridged classical antifolates on dihydrofolate reductase (DHFR), thymidylate synthase (TS), and folylpolyglutamate synthetase (FPGS) as well as antitumor activity. Extension of the bridge homologation studies of classical two-carbon bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine (2), afforded two four-carbon bridged antifolates, analogues 5 and 6, with enhanced FPGS substrate activity and inhibitory activity against tumor cells in culture (EC(50) < or = 10(-7) M) compared with the two-carbon bridged analogues. These results support our original hypothesis that the distance and orientation of the side chain p-aminobenzoyl-L-glutamate moiety with respect to the pyrimidine ring are a crucial determinant of biological activity. In addition, this study demonstrates that, for classical antifolates that are substrates for FPGS, poor inhibitory activity against isolated target enzymes is not necessarily a predictor of a lack of antitumor activity.     

Methotrexate analogues. 19. Replacement of the glutamate side chain in classical antifolates by L-homocysteic acid and L-cysteic acid: effect on enzyme inhibition and antitumor activity

Methotrexate (MTX) and aminopterin (AMT) analogues containing L-homocysteic acid or L-cysteic acid in place of L-glutamic acid were synthesized and tested as inhibitors of dihydrofolate reductase from L1210 cells and folyl polyglutamate synthetase from mouse liver. The ID50 against dihydrofolate reductase was comparable for the MTX and AMT analogues (0.04-0.07 microM), whereas the ID50 against folyl polyglutamate synthetase was 3- to 4-fold lower for the AMT analogues (40-60 microM) than for the MTX analogues (100-200 microM). Thus, N10-substitution has a greater effect on binding to folyl polyglutamate synthetase than dihydrofolate reductase. The cytotoxicity of these compounds was assayed in vitro against L1210 cells, and the AMT analogues again proved more potent (ID50 = 0.03-0.05 microM) than the MTX analogues (ID50 = 0.1-0.4 microM). A similarly increased potency was observed for the AMT analogues against L1210 leukemia in vivo. Though differential cell uptake cannot be ruled out as the basis of increased potency, it is possible that part of the activity of the AMT analogues involves interference with the intracellular polyglutamation of reduced folate cofactors, i.e., that they are "self-potentiating antifolates". Of the four compounds reported, the most active was N-(4-amino-4- deoxypteroyl )-L-homocysteic acid, which produced a 138% increase in life span (ILS) in L1210 leukemic mice when given on a modified bid X 10 schedule at a dose of 2 mg/kg. A comparable ILS was obtained with AMT itself at 0.24 mg/kg. Thus, replacement of gamma-CO2H by gamma-SO3H in the side chain does not decrease therapeutic effect. However, a higher dose is required, presumably to offset pharmacological differences reflecting the inability of the sulfonate group to be polyglutamated .     

Synthesis and inhibitory activities against aminopeptidase B and enkephalin-degrading enzymes of ketomethylene dipeptide analogues of arphamenines.

Three ketomethylene pseudodideptide analogues [(S)Lys psi(COCH2)(R and S)Phe (14 or 15 and 15 or 14) and (S)Lys psi(COCH2)(xi Trp (19)] of natural arphamenine A [(S)Arg psi(COCH2(R,S)Phe (1)] were easily prepared by a route involving two successive main reactions: a malonic ester alkylation with Z-protected lysine iodomethyl ketone and the introduction of a benzyl or (indol-3-yl)methyl moiety in position 2 of the resulting 4-ketodiester. The isomer of 1 with reversed sequence, (S)Phe psi(COCH2)(R,S)Arg (22) was synthesized by guanidylation and subsequent deprotection of Z-(S)Phe psi(COCH2)(R,S)Orn. The inhibitory effects of compounds 14, 15, 19, and 22, and the related ketomethylene dipeptides (S)Ala psi(COCH2)(R,S)Phe (3), (S)Phe psi(COCH2)(R,S)X [X = Ala (4), Orn (5)] and (S)Trp psi(COCH2)(R,S)Y [Y = Orn (6), Lys (7), Arg (8)] on aminopeptidase B (AP-B), and enkephalin-degrading enzymes [aminopeptidase N (APN) and neutral endopeptidase (NEP)] were compared with that of the model compound 1.