Thirteen 2,4-diamino-5-methyl-6-(monosubstituted anilino)methylpyrido2,3-dpyrimidines 5-17 were synthesized as potential Pneumocystis carinii (pc) and Toxoplasma gondii (tg) dihydrofolate reductase ...(DHFR) inhibitors and as antitumor agents. Compounds 5-17 were designed to investigate the structure-activity relationship of monomethoxy and monohalide substitution in the phenyl ring and N10-methylation of the C9-N10 bridge. The synthetic route to compounds 5-12 involved the reductive amination of a common intermediate, 2,4-diamino-5-methylpyrido2, 3-dpyrimidine-6-carbonitrile (18), with the appropriate anilines. N10-Methylation was achieved by reductive methylation. In contrast to previous reports of trimethoprim, the removal of methoxy and chloro groups from the phenyl ring in the 2, 4-diamino-5-methyl-6-(substituted anilino)methylpyrido2, 3-dpyrimidine series generally did not decrease DHFR inhibitory activity. The monosubstituted phenyl analogues 5-12 were as potent against pcDHFR and tgDHFR as the previously reported disubstituted phenyl analogues. N10-Methylation generally resulted in a marginal increase in potency against both pcDHFR and tgDHFR. Compounds 5, 7, and 9 were evaluated and shown to inhibit the growth of T. gondii cells in culture at nanomolar concentrations. Compounds 6-8, 9, 11, and 16 were selected by the National Cancer Institute for evaluation in an in vitro preclinical antitumor screening program. All six compounds showed GI50 values in the 10(-7)-10(-9) M range in more than 20 cell lines.
Classical and nonclassical isosteric C8−N9 bridged analogues of the multitargeted antifolate LY231514 were synthesized as inhibitors of thymidylate synthase (TS), dihydrofolate reductase (DHFR), and ...as antitumor and antiopportunistic infection agents. The syntheses of the analogues were accomplished by reductive amination of the appropriate anilines with 2-amino-4-oxo-5-cyanopyrrolo2,3-dpyrimidine (28) followed by saponification of the ethyl esters, for the classical analogue 6. The N9-methyl analogues were obtained from the N9−H precursors by reductive methylation. In general, the nonclassical compounds 7 − 17 were similar in potency to TMP against Toxoplasma gondii DHFR, with selectivity ratios greater than 38 and 21 for 11 and 16, respectively. These compounds were poor inhibitors of Pneumocystis carinii DHFR and rat liver DHFR. The nonclassical analogues were also inactive against TS. The classical analogue 6 was a marginal inhibitor of isolated human TS (IC50 = 46 μM) and of human DHFR (IC50 = 10 μM), however, it was a potent inhibitor of the growth of two human head and neck squamous cell carcinoma cell lines and of CCRF−CEM human lymphoblastic leukemia cells in culture and was similar to LY231514 against ZR-75-1 human breast carcinoma cell line. Evaluation of 6 against MTX-resistant sublines indicated that DHFR is not the major target of 6. Metabolite protection studies of the growth inhibitory activity of 6 suggest that TS is a major target of this drug and that polyglutamyl forms of 6 may serve as the intracellular TS inhibitors. These studies also suggest that 6 has a site of action in addition to sites in the folate pathway.
A series of lipophilic soft drugs structurally related to the nonclassical dihydrofolate reductase (DHFR) inhibitors trimetrexate and piritrexim have been designed, synthesized, and evaluated in DHFR ...assays, with special emphasis on the inhibition of P. carinii DHFR. The best inhibitors, encompassing an ester bond in the bridge connecting the two aromatic systems, were approximately 10 times less potent than trimetrexate and piritrexim. The metabolites were designed to be poor inhibitors. Furthermore, molecular dynamics simulations of three ligands in complex with DHFR from Pneumocystis carinii and from the human enzyme were conducted in order to better understand the factors determining the selectivity. A correct ranking of the relative inhibition of DHFR was achieved utilizing the linear interaction energy method. The soft drugs are intended for local administration. One representative ester was selected for a pharmacokinetic study in rats where it was found to undergo fast metabolic degradation to the predicted inactive metabolites.
A novel N-{2-amino-4-methyl(pyrrolo2,3-dpyrimidin-5-yl)ethylbenzoyl}-l-glutamic acid (3a) was designed and synthesized as a potent dual inhibitor of thymidylate synthase (TS) and dihydrofolate ...reductase (DHFR) and as an antitumor agent. Compound 3b, the N7-benzylated analogue of 3a, was also synthesized as an antitumor agent. The synthesis of 3a was accomplished via a 12-step sequence which involved the synthesis of 2-amino-4-methylpyrrolo2,3-dpyrimidine (10) in 5 steps from 2-acetylbutyrolactone. Protection of the 2-amino group of 10 and regioselective iodination at the 5-position followed by palladium-catalyzed coupling afforded intermediate 14 which was converted to 3a by reduction and saponification. Similar synthetic methodology was used for 3b. X-ray crystal structure of the ternary complex of 3a, DHFR, and NADPH showed that the pyrrolo2,3-dpyrimidine ring binds in a “2,4-diamino mode” in which the pyrrole nitrogen mimics the 4-amino moiety of 2,4-diaminopyrimidines. This is the first example of a classical pyrrolo2,3-dpyrimidine antifolate shown to have this alternate mode of binding to DHFR. Compounds 3a and 3b were more inhibitory than LY231514 against TS from Lactobacillus casei and Escherichia coli. Analogue 3a was also more inhibitory against DHFR from human, Toxoplasma gondii, and Pneumocystis carinii. Evaluation of 3a against methotrexate (MTX)-resistant cell lines with defined mechanisms indicated that cross-resistance of 3a was much lower than that of MTX. Metabolite protection studies and folylpoly-γ-glutamate synthetase studies suggest that the antitumor activity of 3a against the growth of tumor cells in culture is a result of dual inhibition of TS and DHFR. Compound 3a inhibited the growth of CCRF-CEM and FaDu cells in culture at ED50 values of 12.5 and 7.0 nM, respectively, and was more active against FaDu cells than MTX. In contrast, compound 3b was inactive against both cell lines. Compound 3a was evaluated in the National Cancer Institute in vitro preclinical antitumor screening program and afforded IG50 values in the nanomolar range against a number of tumor cell lines.
Osteoblast differentiation and function can be studied in situ in the metaphysis of growing long bones. Proliferation and apoptosis dominate in the primary spongiosa subjacent to the growth plate, ...and differentiation and function dominate in the proximal metaphysis. Apoptosis of osteocytes dominates at the termination of the trabeculae in diaphyseal marrow. As parathyroid hormone regulates all phases of osteoblast development, we studied the in vivo regulation by human parathyroid hormone (1-34) (PTH) of apoptosis in bone cells of the distal metaphysis of young male rats. Rats were given PTH at 80 μg/kg per day, once daily, for 1–28 days. Bone cells were defined for flow cytometry as PTH1-receptor-positive (PTH1R
+) and growth factor-receptor-positive (GFR
+) cells. Apoptotic cells stained positive for either TdT-mediated dUTP-X nick end labeling (TUNEL) or annexin V (annV
+) were detected by either flow cytometry or immunohistochemistry. Apoptosis was also assessed at the tissue level by RNAse protection and caspase enzyme activity assays. PTH increased apoptotic osteoblasts in the proliferating zone and apoptotic osteocytes in the terminal trabecular zone, by 40%–60% within 2–6 days of PTH treatment, but values became equivalent to controls after 21–28 days of treatment. This transient increase was confirmed in PTH1R
+, GFR
+ bone cells isolated by flow cytometry. There was no detectable change in the steady-state mRNA levels of selected apoptotic genes. Starting at 3 days, at the tissue level, PTH inhibited activity of caspases, which recognize the DEVD peptide substrate (caspases 2, 3, and/or 7), but not those caspases recognizing LEHD or YVAD peptide sequences. We speculate that the localized and tissue level effects of PTH on apoptosis can be explained on the basis of its anabolic effect on bone. The transient increase in apoptosis in the proliferating zone and terminal trabecular zone may be the result of the increased activation frequency and bone turnover seen with daily PTH treatment. As once-daily PTH increases the number of differentiated osteoblasts, and as these and hematopoietic marrow cells dominate metaphyseal tissue, inhibition of caspase activity may contribute to their prolonged survival, enabling extension of trabecular bone into the diaphyseal marrow to increase bone mass.
Fifteen novel nonclassical and two classical 2,4-diamino-6-(benzylamino)pyrido2,3-dpyrimidine antifolates were synthesized as potential inhibitors of Pneumocystis carinii, (pc) Toxoplasma gondii, ...(tg) rat liver (rl), and human (h) recombinant dihydrofolate reductases (DHFR). These analogues lack a 5-methyl substitution which has been shown to be important for increased hDHFR inhibitory activity. In addition, they contain a reversal of the C9−N10 bridge present in folates and most antifolates. The synthesis of the compounds involved the reaction of 2,4,6-triaminopyrimidine with the sodium salt of nitromalonaldehyde to afford the key intermediate 2,4-diamino-6-nitropyrido2,3-dpyrimidine (7), in a single step. Reduction of 7 to the 2,4,6-triaminopyrido2,3-dpyrimidine (8), followed by reductive amination with the appropriate benzaldehydes or phenylacetaldehydes afforded the target compounds. N9 methylation of these analogues was carried out using formaldehyde and sodium cyanoborohydride. The analogues demonstrated significant inhibition of pcDHFR and tgDHFR. N9 methylation significantly increased DHFR inhibitory potency. Compound 11, the 3‘,4‘,5‘-trimethoxy-substituted analogue with a selectivity ratio of 9.4 for tgDHFR (compared to rlDHFR) was the most selective analogue of the nonclassical series. Compound 22, the N9 methyl 2‘,5‘-dimethoxy-substituted analogue was the most potent analogue against tgDHFR (IC50 = 6.3 nM) and was the second most selective analogue for tgDHFR (compared to rlDHFR) in the nonclassical series. The naphthyl-substituted analogues 23−25 were generally more potent against rlDHFR than against pcDHFR and tgDHFR. Selected analogues were also evaluated against Streptococcus faecium (sf) DHFR, Escherichia coli (ec) DHFR, Lactobacillus casei (lc) DHFR and tgDHFR with hDHFR as the mammalian reference, under slightly different assay conditions than those employed for rlDHFR. Analogues 11 and 22 had selectivity ratios of greater than 100 for tgDHFR (compared to hDHFR). Analogue 22 in particular, was the most selective analogue of the nonclassical series against tgDHFR (selectivity ratio = 303.5) with excellent potency (28 nM). Analogue 11, also displayed significant selectivity for sfDHFR (selectivity ratio = 4902). Compound 22 was evaluated in vivo for the inhibition of the growth of T. gondii trophozoites in mice, where at 50 mg/kg orally, it demonstrated distinct prolongation of survival without toxicity. Compounds 11, 12, and 21−23 were evaluated as antitumor agents in the National Cancer Institutes preclinical in vitro screening program. Compounds 12, 22, and 23 showed GI50s for tumor growth inhibition in the 10-6−10-7 M range.
Widespread organ transplantation programs, aggressive chemotherapy of cancer, and the AIDS epidemic have created large numbers of immunosuppressed patients who are at increased risk from ...opportunistic infections, including those caused by fungi. Fungal infections in AIDS patients include candiasis, cryptococcosis, histoplasmosis, and others, but the most common is pneumonia caused by Pneumocystis carinii. Reports of P. carinii pneumonia in the pre-AIDS era were sporadic, with fewer than 100 cases reported yearly in the United States, but with the AIDS epidemic, the number of cases of P. carinii pneumonia in the United States has been estimated at 50 000 annually, and P. carinii pneumonia has been the leading cause of death in AIDS patients in this country. Even with widespread prophylaxis for P. carinii pneumonia, the disease is still common in AIDS patients. Because of the seriousness and prevalence of disease caused by P. carinii, this review will focus on that organism. P. carinii was originally discovered in 1909, but was misidentified as a form of Trypanosoma cruzi. Although it was later recognized as an independent organism, its exact taxonomic position remained unsettled for 80 years. When modern techniques were applied, P. carinii was found to be related more closely to fungi than to protozoans. The full life cycle of the organism is still unclear. Horizontal transmission by the airborne route occurs in animals and probably occurs in humans.
Two series of nonclassical antifolates (2,4-diamino-5-deaza compounds 2−5 and 5,10-dideaza compounds 6−13) were synthesized as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii ...(pc) and Toxoplasma gondii (tg) organisms that are responsible for fatal opportunistic infections in AIDS patients. Rat liver (rl) DHFR served as the mammalian reference enzyme to determine selectivity. Syntheses of the target 5-deaza compounds were achieved by initial construction of the pivaloyl-protected 2,4-diamino-6-bromopyrido2,3-dpyrimidine 17 via a cyclocondensation of 2,4,6-triaminopyrimidine with bromomalonaldehyde. Sequential Heck coupling of 17 with styrene followed by ozonolysis afforded the 6-formyl derivative 19. Reductive amination of 19 with 3,4,5-trimethoxyaniline afforded the N10-H analog. The N10-Me and N10-Et analogs were synthesized by nucleophilic displacement of the 6-bromomethyl derivative 22 (obtained from the 6-formyl derivative 19 by reduction and bromination) with the appropriate N-alkylaniline. The trans-5,10-dideaza analogs 6−8 were synthesized via a Heck coupling of the appropriate methoxystyrene with 17, and selective reduction of the resulting 9,10-double bond afforded target compounds 9−11. Further reduction to the tetrahydro derivatives afforded analogs 12 and 13. The 5-deaza N10-Me 3,4,5-trimethoxy analog 3 maintained the best balance of potency and selectivity against both tgDHFR and pcDHFR. Compared to trimethoprim, compound 3 was only slightly less selective but was 300-fold more potent against tgDHFR. The 5,10-dideaza analogs were generally less potent and selective than the 5-deaza compounds.
The classical antifolate N-{4-(2,4-diamino-5-ethyl-7H-pyrrolo2,3-dpyrimidin-6-yl)sulfanylbenzoyl}-l-glutamic acid (2) and 15 nonclassical analogues (3−17) were synthesized as potential dihydrofolate ...reductase (DHFR) inhibitors and as antitumor agents. 5-Ethyl-7H-pyrrolo2,3-dpyrimidine-2,4-diamine (20) served as the key intermediate to which various aryl thiols and a heteroaryl thiol were appended at the 6-position via an oxidative addition reaction. The classical analogue 2 was synthesized by coupling the benzoic acid derivative 18 with diethyl l-glutamate followed by saponification. The classical compound 2 was an excellent inhibitor of human DHFR (IC50 = 66 nM) as well as a two digit nanomolar (<100 nM) inhibitor of the growth of several tumor cells in culture. Some of the nonclassical analogues were potent and selective inhibitors of DHFR from two pathogens (Toxoplasma gondii and Mycobacterium avium) that cause opportunistic infections in patients with compromised immune systems.
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