Synthesis, cytotoxicity, and antiviral activity of some acyclic analogues of the pyrrolo[2,3-d]pyrimidine nucleoside antibiotics tubercidin, toyocamycin, and sangivamycin

A number of 7-[(1,3-dihydroxy-2-propoxy)methyl]pyrrolo[2,3d-d]pyrimidine derivatives that are structurally related to toyocamycin and sangivamycin and the seco nucleosides of tubercidin, toyocamycin, and sangivamycin were prepared and tested for their biological activity. Treatment of the sodium salt of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]-pyrimidine with 1,3-bis(benzyloxy)-2-propoxymethyl chloride afforded compound 3, which without isolation was debrominated to obtain 4-amino-5-cyano-7-[[1,3-bis(benzyloxy)-2- propoxy]methyl]pyrrolo[2,3-d]pyrimidine. Although catalytic hydrogenolysis failed, the benzyl ether functionalities of 4 were successfully cleaved by boron trichloride to afford 4-amino-5-cyano-7-[(1,3-dihydroxy-2- propoxy)methyl]pyrrolo[2,3-d]pyrimidine. Conventional functional group transformation of the cyano group of 6 provided a number of novel 5-substituted derivatives. Tubercidin (8a), toyocamycin (8b), and sangivamycin (8c) were treated separately with sodium metaperiodate and then with sodium borohydride to afford the 2',3'-seco derivatives 9a-c, respectively. The acyclic nucleoside 4-chloro-2-(methylthio)-7-[[1,3-bis(benzyloxy)-2- propoxy]methyl]pyrrolo[2,3-d]pyrimidine was aminated, desulfurized with Raney Ni, and then debenzylated to provide the tubercidin analogue 11. Cytotoxicity evaluation against L1210 murine leukemic cells in vitro showed that although the parent compounds tubercidin (8a), toyocamycin (8b), and sangivamycin (8c) were very potent growth inhibitors, the acyclic derivatives 6, 7a-c, and 9a-c had only slight growth-inhibitory activity. Evaluation of compounds 6, 7a, 7b, 7c, 9a, 9b, 9c, 11 for cytoxicity and activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) revealed that only the carboxamide (7a) and the thioamide (7c) were active. Compound 7c was the more potent of the two, inhibiting HCMV but not HSV-1 at concentrations producing little cytotoxicity.     

Synthesis and antiviral activity of certain 4-substituted and 2,4-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines

Treatment of the sodium salt of 4-chloro-2-(methylthio)pyrrolo[2,3-d]pyrimidine (2) with (2-acetoxyethoxy)methyl bromide (3) has provided 4-chloro-2-(methylthio)-7[(2-acetoxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (4). Ammonolysis of 4 at room temperature gave 4-chloro-2-(methylthio)-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (5). However, ammonolysis of 5 at 130 degrees C furnished 4-amino-2-(methylthio)-7-[(2-hydroxyethoxy)methyl]-pyrrolo[2,3- d]pyrimidine (6), which on desulfurization with Raney Ni yielded 4-amino-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidine (7) (acyclic analogue of tubercidin). The oxidation of 6 with m-chloroperbenzoic acid provided the sulfone derivative 8. A nucleophilic displacement of the 2-methylsulfonyl group from 8 with methoxide anion provided 4-amino-2-methoxy-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (9). Demethylation of 9 with iodotrimethylsilane gave 4-amino-2-hydroxy-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (10). Treatment of 2,4-dichloropyrrolo[2,3-d]pyrimidine (11) with 3 gave the protected acyclic compound 12, which on deacetylation and ammonolysis under controlled reaction conditions gave 2,4-dichloro-7-[(2-hydroxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidine (13) and 4-amino-2-chloro-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3- d]pyrimidine (14), respectively. The condensation of 2-acetamido-4-chloropyrrolo[2,3-d]pyrimidine (15) with 3 gave the protected acyclic compound 16, which on concomitant deacetylation and ammonolysis with methanolic ammonia at an elevated temperature yielded 2,4-diamino-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (17) in moderate yield. In tests involving human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1), only slight activity and cytotoxicity were observed. The most active compounds (12 and 13) were slightly more active against HCMV than acyclovir, but both compounds were inactive against HSV-1. The activity against HCMV, however, was not well separated from cytotoxicity leading to the conclusion that these compounds did not merit further study.     

Synthesis, cytotoxicity, and antiviral activity of certain 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine nucleosides related to toyocamycin and sangivamycin

A number of 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine derivatives related to the nucleoside antibiotics toyocamycin and sangivamycin were prepared and tested for their biological activity. Treatment of the sodium salt of 4-amino-6-bromo-5-cyanopyrrolo[2,3-d]pyrimidine (1) with (2-acetoxyethoxy)methyl bromide (2) afforded a mixture of 4-amino-6-bromo-5-cyano-7-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d] pyrimidine (3) and the corresponding N1 isomer. Debromination of this mixture gave the corresponding 4-amino-5-cyano-7-[(2-acetoxyethoxy)-methyl]pyrrolo[2,3-d]pyrimidi ne (4) and 4-amino-5-cyano-1-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d]pyrimidin e (5). Deacetylation of 4 and 5 furnished 4-amino-5-cyano-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (6) and the corresponding N1 isomer (7), respectively. The sites of attachment for the acyclic moiety for 6 and 7 were assigned on the basis of UV spectral studies as well as 13C NMR spectroscopy. Conventional functional group transformation of 6 provided a number of novel 5-substituted derivatives (8-10), including the sangivamycin derivative 8. The methyl formimidate derivative 10 was converted to the thioamide derivative 11 and the carbohydrazide derivative 12. Compounds 6 and 8-12 were tested for cytotoxicity to L1210 murine leukemic cells in vitro. None of these compounds caused significant inhibition of cell growth. Evaluation of compounds 4 and 6-12 for activity against human cytomegalovirus (HCMV) and herpes simplex virus type 1 (HSV-1) revealed that only the thioamide (11) was active. It inhibited HCMV but not HSV-1 at concentrations producing only slight cytotoxicity in human foreskin fibroblasts (HFF cells) and KB cells.     

Synthesis and antiviral activity of certain 4- and 4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines

In vitro evaluation of a series of previously prepared tubercidin analogues revealed that certain 5-halogen-substituted analogues were active against human cytomegalovirus (HCMV) at concentrations lower than those that produced comparable cytotoxicity in uninfected cells. In contrast, tubercidin was cytotoxic at all antiviral concentrations. Even though the antiviral selectivity of the 5-substituted compounds was slight, this observation led us to prepare a series of acyclic analogues. Treatment of the sodium salt of 4-chloropyrrolo[2,3-d]pyrimidine (2) with (2-acetoxyethoxy)methyl bromide (2a) provided the acyclic nucleoside 4-chloro-7-[(2-acetoxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine (3). A nucleophilic displacement of the 4-chloro group with methoxide, methylamine, and dimethylamine yielded the corresponding 4-substituted compounds 4, 5, and 6, respectively, in good yield. Electrophilic substitution (chlorination, bromination, and iodination) was effected at the C-5 position of compound 3 with N-chlorosuccinimide, N-bromosuccinimide, and iodine monochloride, respectively, in methylene chloride. Removal of the acetyl group from these intermediates (7a-9a) with methanolic ammonia at room temperature afforded the 5-chloro (7b), 5-bromo (8b), and 5-iodo (9b) derivatives of 4-chloro-7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidine. Treatment of compounds 7b-9b with methanolic ammonia at an elevated temperature produced the corresponding 5-halotubercidin analogues 10, 11, and 12, respectively. An alternate procedure for the preparation of these 4,5-disubstituted 7-[(2-hydroxyethoxy)methyl]pyrrolo[2,3-d]pyrimidines involved an electrophilic substitution prior to the condensation of the heterocycle with 2a. Treatment of 2 with N-chlorosuccinimide and N-bromosuccinimide gave compounds 13a and 13b, respectively. The condensation of 13a and 13b with 2a and subsequent treatment with methylamine and ethylamine furnished the corresponding 5-halo-4-substituted-pyrrolo[2,3-d]pyrimidines 14a, 14b, 14c, and 14d, respectively. Evaluation of the target compounds (4-6, 7b-9b, 10-12, and 14a-14d) for cytotoxicity and activity against HCMV and herpes simplex virus type 1 (HSV-1) revealed that all compounds except the 5-halogen-substituted compounds 10, 11, and 12 were inactive. Compounds 10, 11, and 12 were active against both viruses at noncytotoxic concentrations. The activity of compound 11 was particularly noteworthy, being at least 10-fold more potent than acyclovir.     

Synthesis and biological activity of certain 6-substituted and 2,6-disubstituted 2'-deoxytubercidins prepared via the stereospecific sodium salt glycosylation procedure

A number of 6-substituted and 2,6-disubstituted pyrrolo[2,3-d]pyrimidine 2'-deoxyribonucleosides were prepared by the direct stereospecific sodium salt glycosylation procedure. Reaction of the sodium salt of 4-chloro-6-methyl-2-(methylthio)pyrrolo[2,3-d]pyrimidine (6a) or 4,6-dichloro-2-(methylthio)pyrrolo[2,3-d]pyrimidine (6b) with 1-chloro-2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranose (9) provided the corresponding N7 2'-deoxy-beta-D-ribofuranosyl blocked derivatives (8a and 8c) which, on ammonolysis, gave 4-amino-6-methyl-2-(methylthio)-7-(2-deoxy-beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11a) and 4-amino-6-chloro-2-(methylthio)-7-(2-deoxy-beta-D-erythro-pentofuranosyl )pyrrolo[2,3-d]pyrimidine (11b), respectively. Dethiation of 11a and 11b afforded 6-methyl-2'-deoxytubercidin (10a) and 6-chloro-2'-deoxytubercidin (10b), respectively. Dehalogenation of 10b provided an alternate route to the reported 2'-deoxytubercidin (3a). Application of this glycosylation procedure to 4,6-dichloro and 4,6-dichloro-2-methyl derivatives of pyrrolo[2,3-d]pyrimidine (15a and 15b) gave the corresponding blocked 2'-deoxyribonucleosides (18a and 18b), which on ammonolysis furnished 10b and 4-amino-6-chloro-2-methyl-7-(2-deoxy-beta-D-erythro- pentofuranosyl)pyrrolo[2,3-d]pyrimidine (17), respectively. This stereospecific attachment of the 2-deoxy-beta-D-ribofuranosyl moiety appears to be due to a Walden inversion at the C1 carbon by the anionic heterocyclic nitrogen. Controlled deacylation of 4-chloro-7-(2-deoxy-3,5-di-O-p-toluoyl-beta-D-erythro-pentofuranosyl) pyrrolo[2,3-d]pyrimidine (20a) gave 4-chloro-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d] pyrimidine (20b). Dehalogenation of 20b gave the 2'-deoxynebularin analogue 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine (19), and reaction of 20b with thiourea gave 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (21). All of these compounds were tested in vitro against certain viruses and tumor cells. Only compounds 12a, 20b, and 21 showed significant activity against measles in vitro, and the activity is comparable to that of ribavirin. Although compounds 3a and 12b are slightly more active than ribavirin against HSV-2 in vitro, they are relatively more toxic to Vero cells. Compounds 3a and 20b exhibited moderate cytostatic activity against L1210 and P388 leukemia in vitro but are considerably less active than 2-chloro-2'-deoxyadenosine (1).     

Effect of acyclic pyrimidines related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine on herpesviruses

A series of pyrimidines related to the potent antiherpetic agent 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (1, BW B759U), all containing the same acyclic chain, have been synthesized. Some of the compounds were derivatives of the naturally occurring bases, cytosine, uracil, and thymine; others included compounds in which the 5-position of the cytosine and uracil moieties were substituted by bromo, iodo, fluoro, methyl, and amino groups. Other variations of the cytosine derivatives were the 5-aza, 2-mercapto, 4-methylamino, 4-dimethylamino, and isocytosine congeners. A 4-aminopyrimidine adduct was also made. Antiviral testing showed that 1-[(1,3-dihydroxy-2-propoxy)methyl]cytosine (18, BW A1117U) was equivalent to the guanine analogue in potency against human cytomegalovirus and Epstein Barr virus. Other compounds in the series were largely inactive in antiviral screening against the herpesviruses.     

Synthesis of classical and a nonclassical 2-amino-4-oxo-6-methyl-5-substituted pyrrolo[2,3-d]pyrimidine antifolate inhibitors of thymidylate synthase.

Compounds 2-5 were designed as potential antifolate nonpolyglutamatable inhibitors of thymidylate synthase (TS). These analogues are structurally related to 2-amino-4-oxo-5-substituted quinazolines and 2-amino-4-oxo-5-substituted pyrrolo[2, 3-d]pyrimidines which have shown excellent inhibition of TS and, for the quinazoline, significant promise as clinically useful antitumor agents. Compounds 2-4 were synthesized by appropriate amine exchange reactions on pivaloyl-protected 5-dimethylaminomethyl-substituted 6-methyl pyrrolo[2,3-d]pyrimidine 7 which in turn was obtained from the Mannich reaction of pivaloylated-6-methyl pyrrolo[2, 3-d]pyrimidine 6. In instances where the amine exchange reaction was sluggish, the Mannich base was quaternized with methyl iodide which afforded much faster exchange reaction with improved yields. For compound 5, 4-mercaptopyridine was used as the nucleophile and reacted with 7. The analogues 2-4 inhibited Lactobacillus casei (lc) TS and recombinant human (h) TS with IC50 in the 10(-4) to 10(-5) M range. Compound 5 inhibited lcTS and hTS 20% at 26 and 25 microM, respectively. In addition, compound 5 inhibited the growth of Pneumocystis carinii and Toxoplasma gondii cells in culture by 76% at 32 x 10(-6) M and 50% at 831 x 10(-6) M, respectively.     

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.     

Synthesis and antiviral activity of some 7-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine analogues of sangivamycin and toyocamycin

The sodium salt of 4-amino-3-cyanopyrazolo[3,4-d]pyrimidine (1) was condensed with (2-acetoxyethoxy)methyl bromide (2) to provide the corresponding protected acyclic nucleoside, 4-amino-3-cyano-1-[(2-acetoxyethoxy)methyl]-pyrazolo[3,4-d]pyrimid ine (3). Treatment of 3 with sodium methoxide in methanol provided a good yield of methyl 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- formimidate (4). Treatment of the imidate (4) with sodium hydrogen sulfide gave the thiocarboxamide derivative 5. Aqueous base transformed 4 into 4-amino-1-[(2-hydroxyethoxy)methyl]pyrazolo[3,4-d]pyrimidine-3- carboxamide (6) in good yield. Treatment of 5 with mercuric chloride furnished the toyocamycin analogue 7. Evaluation of compounds 1, 3-7 revealed that only the heterocycle (1) and the thiocarboxamide acyclic nucleoside (5) were active. Compound 5 was the more potent with activity against human cytomegalovirus and herpes simplex virus type 1.     

Synthesis and antitumor evaluation in mice of certain 7-deazapurine (pyrrolo[2,3-d]pyrimidine) and 3-deazapurine (imidazo[4,5-c]pyridine) nucleosides structurally related to sulfenosine, sulfinosine, and sulfonosine

7-Deaza (pyrrolo[2,3-d]pyrimidine) and 3-deaza (imidazo[4,5-c]pyridine) congeners of sulfenosine (5a and 9), sulfinosine (6a and 10), and sulfonosine (7a) have been prepared and evaluated for their antileukemic activity in mice. Amination of 2-amino-7-beta-D-ribofuranosylpyrrolo[2,3-d]pyrimidine-4(3H)-th ion e (4a) and its 2'-deoxy analogue (4c) with a chloramine solution gave the corresponding 4-sulfenamides (5a and 5c, respectively), which on selective oxidation with m-chloroperoxybenzoic acid (MCPBA) gave the respective diastereomeric 2-amino-7-beta-D-ribofuranosyl-pyrrolo[2,3-d]pyrimidine-4-sulfinamide (7-deazasulfinosine, 6a) and its 2'-deoxy derivative (6c). A similar amination of 7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine-4(3H)- thione (4b) gave the corresponding 4-sulfenamide derivative (5b). Oxidation of 5b with 1 molar equiv of MCPBA furnished (R,S)-7-(2-deoxy-beta-D-erythro-pentofuranosyl)pyrrolo[2,3-d]pyrimidine- 4- sulfinamide (6b), whereas use of excess of MCPBA afforded the corresponding sulfonamide derivative (7b). Treatment of 3-deaza-6-thioguanosine (8) with a chloramine solution gave 3-deazasulfenosine (6-amino-1-beta-D- ribofuranosylimidazo[4,5-c]pyridine-4-sulfenamide, 9). Controlled oxidation of 9 with MCPBA afforded 3-deazasulfinosine (10). As gauged by increases in the mean postinoculation life spans of L1210 inoculated mice, none of these nucleosides exhibited biologically significant activity (T/C greater than or equal to 125). Even so, antileukemic activity appeared to be influenced, albeit not uniformly, by structural modifications in the base and carbohydrate moieties of sulfenosine and sulfinosine. Thus, while several of the compounds were lacking in cytotoxic activity, eight others (4c, 5a, 5c, 6a, 6b, 7b, 9, and 10) were estimated to have reduced body burdens of viable L1210 cells by 16-77%.     

Synthese von Pyrrolo[2,3-d]pyrimidinen

Synthesis of Pyrrolo[2,3-d]pyrimidines A simple synthesis of pyrrolo[2,3-d]pyrimidines is described. Basic materials are 2-amino-3-cyano-pyrroles. The yields are good or excellent.     

Synthesis and activity of novel 5-substituted pyrrolo[2,3-d]pyrimidine analogues as pp60(c-Src) tyrosine kinase inhibitors

Therapy with receptor tyrosine kinase inhibitors provides an improved treatment option in a number of diseases such as cancer, myocardial infection, osteoporosis, stroke, and neurodegeneration. We have designed, synthesized, and evaluated a series of novel 2-amino-5-[(benzyl)imino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one 7a and 2-amino-5-[(substituted-benzyl)imino]methyl-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidine-4-one 7b-e derivatives as potential tyrosine kinase inhibitors. These compounds were synthesized by condensation reaction using 2-tritylamino-4-oxo-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidine-5-carbaldehyde 5 and appropriate benzylamines followed by detritylation. Compounds were evaluated for their inhibitory activity toward tyrosine phosphorylation for the pp60c-Src tyrosine kinase. Compounds 7a, 7d, and 7e demonstrated potent inhibitory activities against pp60c-Src tyrosine kinase with IC50 values of 13.9, 34.5, and 78.4 microM, respectively. Dihalogenated compounds 7d and 7e have 3 to 7-times lower IC50 values than that of the parent compound 7a.     

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

Bridge homologation of the previously reported classical two-carbon-bridged antifolates, a 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine (1) [which is a 6-regioisomer of LY231514 (Alimta)] and a 6-subsituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine, afforded the three-carbon-bridged antifolates analogues 4 and 5, with enhanced inhibitory activity against tumor cells in culture (EC(50) values in the 10(-8)-10(-7) M range or less). These two analogues were synthesized via a 10-step synthetic sequence starting from methyl 4-bromobenzoate (14), which was elaborated to the alpha-chloromethyl ketone (8) followed by condensation with 2,6-diamino-pyrimidin-4-one (7) to afford the substituted furo[2,3-d]pyrimidine 9 and the pyrrolo[2,3-d]pyrimidine 10. Subsequent coupling of each regioisomer with diethyl-l-glutamate followed by saponification afforded 4 and 5. The biological results indicate that elongation of the C8-C9 bridge of the classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidine and 6-substituted 2-amino-4-oxopyrrolo[2,3-d]pyrimidine are highly conducive to antitumor activity in vitro, despite a lack of increase in inhibitory activity against the target enzymes. This supports our original hypothesis that truncation of the B-ring of a highly potent 6-6 ring system to a 6-5 ring system can be compensated by bridge homologation to restore the overall length of the molecule.     

Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase and as potential antitumor agents

Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.     

Synthesis and antiallergy activity of 10-oxo-10H-pyrido[1,2-a]thieno[3,2-d]pyrimidines and 10-oxo-10H-pyrido[1,2-a]thieno[3,4-d]pyrimidines

Synthesis and antiallergy activity of 10-oxo-10H-pyrido[1,2-a]thieno[3,2-d]pyrimidines (2 and 3) and 10-oxo-10H-pyrido[1,2-a]thieno[3,4-d]pyrimidines (4 and 5) are described. The activity, shown by these compounds in the rat passive cutaneous anaphylaxis (PCA) test, is compared to the PCA data previously reported for a series of 4-oxo-4H-pyrido[1,2-a]thieno[2,3-d]pyrimidines. 10-Oxo-N-1H-tetrazol-5-yl-10H-pyrido[1,2-a]thieno[3,4-d]pyri midine (2b), 10-oxo-7-(1H-tetrazol-5-yl)-10H-pyrido[1,2-a]thieno[3,4-d]py rimidine (4e), and 3,10-dihydro-10-oxo-7-(1H-tetrazol-5-yl)-1H-pyrido[1,2-a]thieno[3, 4-d] pyrimidine (7e) gave a 100% inhibition in the rat PCA test at a dose of 5 mg/kg. The activity displayed by these compounds is comparable to that of the most active compounds in the 4-oxo-4H-pyrido[1,2-a]thieno[2,3-d]pyrimidine series.     

Synthesis and cytotoxicity of 4-amino-5-oxopyrido[2,3-d]pyrimidine nucleosides

A number of nucleoside analogues have been either used clinically as anticancer drugs or evaluated in clinical studies, while new nucleoside analogues continue to show promise. In this article, we report synthesis and cytotoxicity of a series of new pyrido[2, 3-d]pyrimidine nucleosides. 2-Amino-3-cyano-4-methoxypyridine was converted, in two steps, to 4-amino-5-oxopyrido[2,3-d]pyrimidine. A variety of 1-O-acetylated pentose sugar derivatives were condensed with silylated 4-amino-5-oxopyrido[2,3-d]pyrimidine, followed by protection, to afford a series of 4-amino-5-oxopyrido[2, 3-d]pyrimidine nucleosides. Further derivatizations provided an additional group of pyrido[2,3-d]pyrimidine nucleosides. These nucleosides were evaluated for in vitro cytotoxicity to human prostate cancer (HTB-81) and mouse melanoma (B16) cells as well as normal human fibroblasts (NHF). A number of compounds (1a,b, 2a-c,f, 3f+4d) showed significant cytotoxicity to cancer cells, with 4-amino-5-oxo-8-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine (1b) being the most potent proliferation inhibitor (EC(50): 0.06-0.08 microM) to all types of cells tested. However, a selective inhibition to the cancer cells was observed for 4-amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (2b), which is a potent inhibitor of HTB-81 (EC(50): 0.73 microM) and has a favorable in vitro selectivity index (28).     

Antitumor and antiviral activity of synthetic alpha- and beta-ribonucleosides of certain substituted pyrimido[5,4-d]pyrimidines: a new synthetic strategy for exocyclic aminonucleosides

A novel and direct synthesis of the antiviral and antitumor agent 4-amino-8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidine (ARPP, 8) and its alpha-anomer (11) has been developed. Treatment of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (1) with 2,3-O-isopropylidene-D-ribofuranosylamine gave an anomeric mixture of 2,4,6-trichloro-8-(2,3-O-isopropylidene-beta- and -alpha-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidines (3 and 4) in a ratio of 1.0:0.7. A nucleophilic displacement of the 4-chloro group of 3 and 4 with NH3 furnished 4-amino-2,6-dichloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino ] pyrimido[5,4-d]pyrimidine (6) and its alpha-anomer (9), respectively. Catalytic hydrogenation of 6 and 9, followed by deisopropylidenation gave ARPP (8) and the alpha-anomer 11, respectively. Similarly, 3 and 4 have been transformed to 4-methoxy-8-(beta-D-ribofuranosylamino)pyrimido-[5,4-d]pyrimidine (MRPP, 14) and its alpha-anomer (17). Application of this procedure to 3 with NH2Me or NHMe2 resulted in the synthesis of 4-(methylamino)- and 4-(dimethylamino)-8-(beta-D-ribofuranosylamino)pyrimido [5,4-d]pyrimidine (24 and 27, respectively). A synthesis of 8-(beta-D-ribofuranosylamino)pyrimido[5,4-d]pyrimidin-4(3H)-one (21) has also been accomplished from 3 in three steps. Selective hydrogenation of 6 furnished 4-amino-6-chloro-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino] pyrimido[5,4-d]pyrimidine (36), the structure of which was established by single-crystal X-ray diffraction analysis. Deisopropylidenation of 36 gave 6-chloro-ARPP (37). Extended treatment of 36 with NH3 furnished 4,6-diamino-8-[(2,3-O-isopropylidene-beta-D-ribofuranosyl)amino]pyrimido [5,4-d]pyrimidine (34), which on deisopropylidenation gave 6-amino-ARPP (35). An unambiguous synthesis of 34 and 36 has also been accomplished by the reaction of 4,6,8-trichloropyrimido[5,4-d]pyrimidine (28) with 2, followed by the treatment with NH3. Nucleophilic displacement studies with 1, 6, and 28 indicated the reactivity of the halogens in these compounds is in the order of 8 greater than 4 greater than 6 greater than 2. The structures of 3 and 9 have been assigned on the basis of 1H NMR data and further confirmed by single-crystal X-ray diffraction analysis. The exocyclic aminonucleosides synthesized during this study were tested for their activity against several RNA and DNA viruses in vitro and against L1210, WI-L2, and LoVo/L in cell culture. The effect of these compounds on the de novo nucleic acid biosynthesis has been studied. Compound 14 (MRPP) exhibited enhanced activity against L1210 in vivo, when compared to ARPP (8).     

Isomeric N-methyl-7-deazaguanines: synthesis, structural assignment, and inhibitory activity on xanthine oxidase

The N-methyl isomers of 2-amino-3,7-dihydro-4H-pyrrolo[2,3-d]pyrimidin-4-one (2a) have been synthesized regiospecifically and their structures assigned. The 3-methyl compound 3 was obtained by alkylation of the parent chromophore 2a with dimethyl sulfate, and the 1-methyl isomer 5b was obtained by condensation of ethyl 2-cyano-4,4-diethoxybutyrate with N-methylguanidine and subsequent cyclization. Methylation of 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (7b), however, with methyl iodide in the presence of 50% NaOH, by phase-transfer techniques, followed by the replacement of halide by hydroxyl, yielded the 7-methyl compound 2b. The N-methyl isomers of 2a were all found to be inhibitors of xanthine oxidase from cow's milk. While the 3-methyl isomer 3 exhibits a Ki of 40 microM, the 7- and 1-isomers show Ki values of 4.5 and 3 microM, respectively.     

Synthese von 4-Phenyl-pyrido[2,3-d]pyrimidinen

Synthesis of 4-Phenyl-Pyrido[2,3-d]pyrimidines 4-Phenylpyrido[2,3-d]pyrimidines and -2(1H)ones can be synthesized from 2-amino-3-benzoylpyridines and formamide or urea. Title compounds bearing a basic substituent at position 2 are prepared from 2-chloropyrido [2,3-d]pyrimidines and primary or secondary amines.     

Synthesis of 7 beta-(4-oxo-3,4-dihydrothieno[2,3-d] pyrimidyl-2-thioacetamido)cephalosporanic acid derivatives

Synthesis of new cephalosporins possessing thieno [2,3-d]pyrimidine is described in the present paper. Potassium salts of 2-thio-4-oxo-3,4-dihydrothieno [2,3-d]pyrimidines, which gave then 2-carboxymethylmercapto-4-oxo-3,4-dihydrothieno [2,3-d]pyrimidines, were prepared from 2-amino-3-carbethoxythiophene derivatives. These compounds were allowed to react with 7-aminocephalosporamic acid (ACA) benzhydryl esters or with 7 beta-chloroacetamidocephalosporanic acid derivatives to obtain new cephalosporins. Antibacterial activities of these new cephalosporins are summarized in the present paper as well.