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Journal of Medicinal Chemistry Oct 1993A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and...
A series of eight previously undescribed 2,4-diaminothieno[2,3-d]pyrimidine analogues of the potent dihydrofolate reductase (DHFR) inhibitors trimetrexate (TMQ) and piritrexim (PTX) were synthesized as potential drugs against Pneumocystis carinii and Toxoplasma gondii, which are major causes of severe opportunistic infections in AIDS patients. 2,4-Diamino-5-methyl-6-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 3,4,5-trimethoxy or 2,5-dimethoxy substitution in the aryl/aralkyl moiety and 2,4-diamino-5-(aryl/aralkyl)thieno[2,3-d]pyrimidines with 2,5-dimethoxy substitution in the aryl/aralkyl moiety were obtained by reaction of the corresponding 2-amino-3-cyanothiophenes with chloroformamidine hydrochloride. The aryl group in the 5,6-disubstituted analogues was either attached directly to the hetero ring or was separated from it by one or two carbons, whereas the aryl group in the 5-monosubstituted analogues was separated from the hetero ring by two or three carbons. 2-Amino-3-cyano-5-methyl-6-(aryl/alkyl)thiophene intermediates for the preparation of the 5,6-disubstituted analogues were prepared from omega-aryl-2-alkylidene-malononitriles and sulfur in the presence of a secondary amine, and 2-amino-3-cyano-4-(aryl/aralkyl)thiophene intermediates for the preparation of the 5-monosubstituted analogues were obtained from omega-aryl-1-chloro-2-alkylidenemalononitriles and sodium hydrosulfide. Synthetic routes to the heretofore unknown ylidenemalononitriles, and the ketone precursors thereof, were developed. The final products were tested in vitro as inhibitors of DHFR from Pneumocystis carinii, Toxoplasma gondii, rat liver, beef liver, and Lactobacillus casei. A selected number of previously known 2,4-diaminothieno[2,3-d]pyrimidines lacking the 3,4,5-trimethoxyphenyl and 2,5-dimethoxyphenyl substitution pattern of TMQ and PTX, respectively, were also tested for comparison. None of the compounds was as potent as TMQ or PTX, and while some of them showed some selectivity in their binding to Pneumocystis carinii and Toxoplasma gondii versus rat liver DHFR, this effect was not deemed large enough to warrant further preclinical evaluation.
Topics: Animals; Antineoplastic Agents; Folic Acid Antagonists; Humans; Liver; Pneumocystis; Pyrimidines; Rats; Structure-Activity Relationship; Toxoplasma; Trimetrexate
PubMed: 8230096
DOI: 10.1021/jm00073a009 -
British Journal of Cancer Sep 1993Piritrexim is a lipid-soluble antifolate which, like methotrexate, has a potent capacity to inhibit dihydrofolate reductase. We performed a multicentre phase II study... (Clinical Trial)
Clinical Trial
Piritrexim is a lipid-soluble antifolate which, like methotrexate, has a potent capacity to inhibit dihydrofolate reductase. We performed a multicentre phase II study with piritrexim in patients with locally advanced or metastatic breast cancer. Twenty-four patients of which sixteen had received prior chemotherapy, were initially treated with 25 mg piritrexim orally administered trice daily for four days, repeated weekly, with provision for dose escalation or reduction according to observed toxicity. Of twenty-one patients evaluable for tumour response, one patient achieved a partial response which lasted for 24 weeks. Three patients had stable disease during 12 weeks of treatment, seventeen had progressive disease. Pirtrexim was generally well tolerated, in eighteen patients the dose could be escalated. Myelotoxicity was the most frequent observed toxicity of this piritrexim regimen. Leucopenia and thrombocytopenia grade 3/4 occurred in 38% of the patients sometime during treatment. Pharmacokinetic analysis of piritrexim in three patients during the first treatment cycle, revealed peak levels 1 to 2 h after an oral dose, with a trend towards a higher peak plasma levels and AUCs on the fourth dosing day compared with the first dosing day. In conclusion, orally administered piritrexim appears to be a regimen with little activity in patients with locally advanced or metastatic breast carcinoma.
Topics: Administration, Oral; Adult; Aged; Breast Neoplasms; Drug Evaluation; Female; Humans; Leukopenia; Middle Aged; Neoplasm Metastasis; Pyrimidines; Thrombocytopenia
PubMed: 8353055
DOI: 10.1038/bjc.1993.400 -
Journal of the National Cancer Institute Aug 1993
Topics: Aged; Antineoplastic Agents; Carcinoma, Squamous Cell; Drug Resistance; Folic Acid Antagonists; Head and Neck Neoplasms; Humans; Male; Methotrexate; Neoplasm Recurrence, Local; Pyrimidines
PubMed: 8331686
DOI: 10.1093/jnci/85.15.1248 -
Development and characterization of a rapid screening assay for identifying antipneumocystis agents.Antimicrobial Agents and Chemotherapy Aug 1993We developed a rapid assay for screening of compounds with potential antipneumocystis activity on the basis of incorporation of [35S]methionine into proteins newly...
We developed a rapid assay for screening of compounds with potential antipneumocystis activity on the basis of incorporation of [35S]methionine into proteins newly synthesized by Pneumocystis carinii. Unambiguous evidence that P. carinii synthesizes proteins in vitro was provided by immunoprecipitation studies demonstrating the incorporation of [35S]methionine into the major surface glycoprotein. Treatment with two clinically active antipneumocystis agents, atovaquone (10(-4) M) or pentamidine (10(-4) M), prevented this incorporation. Total [35S]methionine incorporation paralleled incorporation into the major surface glycoprotein, permitting rapid assessment of anti-P. carinii activity by scintillation counting. Treatment with pentamidine (1 x 10(-4) M), atovaquone, trimethoprim (1 x 10(-4) M)-sulfamethoxazole (7.9 x 10(-4) M), piritrexim (1 x 10(-7) M), RO11-8958 (1 x 10(-4) M), and amphotericin B (1 microgram/ml) resulted in a greater than 67% inhibition (P < 0.05) of [35S]methionine incorporation. No decrease in [35S]methionine incorporation was seen with dapsone (10(-5) M), trimethoprim (10(-4) M), recombinant mouse tumor necrosis factor (500 ng/ml), or gamma interferon. This rapid in vitro assay should be a useful adjunct in the development of new antipneumocystis agents.
Topics: Amphotericin B; Anti-Infective Agents; Antifungal Agents; Atovaquone; Cell Membrane Permeability; Fungal Proteins; Membrane Glycoproteins; Methionine; Microbial Sensitivity Tests; Naphthoquinones; Pentamidine; Pneumocystis; Precipitin Tests; Sulfur Radioisotopes; Trimethoprim, Sulfamethoxazole Drug Combination; Tumor Necrosis Factor-alpha
PubMed: 8215282
DOI: 10.1128/AAC.37.8.1674 -
The Journal of Otolaryngology Jun 1993Methotrexate is often used for induction or palliative therapy of advanced head and neck squamous cell carcinoma (HNSCC). However, resistance to this drug is a common... (Comparative Study)
Comparative Study
Methotrexate is often used for induction or palliative therapy of advanced head and neck squamous cell carcinoma (HNSCC). However, resistance to this drug is a common clinical problem, which may be conferred by several mechanisms, including: i) decreased level of folate transport proteins, required for entry of methotrexate into cells, and ii) increase in amount of dihydrofolate reductase (DHFR), the target enzyme of this drug. Two established, clonal cell lines of HNSCC, having equal growth rates, differed 9-fold in sensitivity to methotrexate. Cellular accumulation of radiolabelled methotrexate was measured, and did not differ between the two lines when corrected for the different volume of the cells. This suggested that the difference in drug sensitivity was not due to differential uptake. This was confirmed by the finding of a 22-fold difference in sensitivity to piritrexin, a lipophilic antifolate which enters cells by simple diffusion, and, unlike methotrexate, is not polyglutamated. These results suggest that a quantitative difference in DHFR between the two cell lines probably accounts for the differential sensitivity to antifolates. Screening of patient tumors for DHFR content and drug uptake may provide a basis upon which to recommend whether methotrexate treatment is indicated.
Topics: Antineoplastic Agents; Carcinoma, Squamous Cell; Drug Resistance; Drug Screening Assays, Antitumor; Humans; Mandibular Neoplasms; Methotrexate; Pyrimidines; Tetrahydrofolate Dehydrogenase; Tongue Neoplasms; Tumor Cells, Cultured
PubMed: 8371322
DOI: No ID Found -
Anti-cancer Drugs Jun 1993Chinese hamster ovary (CHO) T19 cells express a stable P-glycoprotein (P-170)-dependent multidrug resistance (MDR) phenotype and display a 24- to 29-fold...
Chinese hamster ovary (CHO) T19 cells express a stable P-glycoprotein (P-170)-dependent multidrug resistance (MDR) phenotype and display a 24- to 29-fold cross-resistance to the lipophilic antifolates piritrexim (PTX) and trimetrexate (TMTX). We have examined the ability of various modulators of the MDR phenotype to sensitize T19 cells to TMTX and PTX in a clonogenic assay. An almost complete reversal of TMTX resistance in T19 cells was achieved with several modulators of the MDR phenotype whereas only a partial sensitization of T19 cells to PTX was obtained with the most potent modulator. In an attempt to explore the apparent P-170-independent locus of protection against PTX, resistant T19 sublines were isolated after stepwise selection with PTX and TMTX. Thus, T19 cells made resistant to PTX displayed a dramatic decrease in P-170 mRNA levels despite the maintenance of the parental T19 MDR gene amplification, whereas T19 cells selected for TMTX resistance exhibited a further increase in P-170 mRNA levels. Hence, the modulation experiments together with the established lipophilic antifolate-resistant T19 variants suggest that although T19 cells possess a P-170-dependent MDR phenotype and display a similar cross-resistance to TMTX and PTX, the protective pathway need not be necessarily via P-170. Rather, a pathway appears to exist that protects T19 MDR cells from the cytotoxicity of PTX without requiring a P-170 function.
Topics: ATP Binding Cassette Transporter, Subfamily B, Member 1; Animals; CHO Cells; Carrier Proteins; Cricetinae; Drug Interactions; Drug Resistance; Membrane Glycoproteins; Methotrexate; Phenotype; Pyrimethamine; Pyrimidines; Quinidine; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Trimetrexate
PubMed: 8102912
DOI: 10.1097/00001813-199306000-00018 -
International Journal For Parasitology May 1993A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into...
A variety of anti-folate compounds have been tested for their ability to inhibit the growth of Babesia bovis as measured by the incorporation of [3H]hypoxanthine into the parasite's nucleic acids. Inhibitors of folate synthesis (including 7-methylguanosine and several sulpha drugs) were without effect but several structural analogues of folate were toxic. The most potent folate analogues were the lipophilic compounds piritrexim and trimetrexate, each causing 50% inhibition of [3H]hypoxanthine incorporation (IC50) at a concentration of 2.9 nM; other classical anti-folates such as pyrimethamine, methotrexate and trimethoprim were at least 100-fold less effective with IC50 values of 1.2, 0.29 and 0.50 microM, respectively. From these results we conclude that B. bovis does not synthesize folate de novo under cell culture conditions. However, the toxic effects of piritrexim and trimetrexate suggest that dihydrofolate reductase (DHFR) activity is essential for the parasite, most probably because of the role of this enzyme in the synthesis of thymidine nucleotides via thymidylate synthase.
Topics: Animals; Babesia bovis; Folic Acid; Folic Acid Antagonists; Pyrimethamine; Pyrimidines; Quinazolines; Trimethoprim; Trimetrexate
PubMed: 8359989
DOI: 10.1016/0020-7519(93)90016-r -
American Journal of Clinical Oncology Apr 1993We describe a patient with transitional cell carcinoma of the renal pelvis who developed respiratory dysfunction and an abnormal chest x-ray with diffuse interstitial... (Clinical Trial)
Clinical Trial
We describe a patient with transitional cell carcinoma of the renal pelvis who developed respiratory dysfunction and an abnormal chest x-ray with diffuse interstitial opacities while on chemotherapy with piritrexim, a methotrexate analog. Drug discontinuation resulted in complete resolution of the clinical and radiographic picture. The pulmonary toxicity is probably induced by piritrexim.
Topics: Aged; Antineoplastic Agents; Carcinoma, Transitional Cell; Humans; Kidney Neoplasms; Lung Neoplasms; Male; Pulmonary Fibrosis; Pyrimidines
PubMed: 8452107
DOI: 10.1097/00000421-199304000-00013 -
The Journal of Biological Chemistry Feb 1993We describe the characterization of an antitumor drug resistance following multiple step selection of hamster cells to the 2,4-diaminopyrimidines (DAP) metoprine,...
We describe the characterization of an antitumor drug resistance following multiple step selection of hamster cells to the 2,4-diaminopyrimidines (DAP) metoprine, pyrimethamine (Pyr), and trimethoprim (Tmp). Pyr and Tmp are DAP lipophilic antifolates currently used as antiparasitic and antibacterial antibiotics, respectively. Dihydrofolate reductase (DHFR) from hamster cells bore a low or poor affinity to these DAP as compared to the hydrophilic folate antagonist methotrexate (MTX). Metoprine-resistant cells over-expressed DHFR enzyme and consequently displayed a high level of resistance to both hydrophilic and lipophilic antifolates including DAP but maintained wild type sensitivity to pleiotropic drugs involved in multi-drug resistance (MDR). In contrast, although Pyr- and Tmp-resistant cells expressed parental levels of wild type DHFR, they displayed a high degree of resistance to DAP and, surprisingly, to the lipophilic MTX analogs piritrexim (PTX) and trimetrexate (TMTX), while maintaining sensitivity to MTX. These drug-resistant cells maintained wild type mRNA levels of the MDR gene product P-glycoprotein and showed collateral hypersensitivity to pleiotropic drugs. To study the underlying mechanism of this apparently new resistance phenotype, we have employed fluorescein-methotrexate (F-MTX) labeling of cells and its displacement by different antifolates. Parental AA8 and Pyr-resistant cells showed a similar level of F-MTX labeling, however, while DAP, TMTX, and PTX showed an efficient competitive displacement of F-MTX from AA8 cells, Pyr-resistant cells displayed a persistent retention of F-MTX labeling in the presence of high concentrations of these lipophilic antifolates. Pyr-resistant cells showed a wild type displacement of F-MTX with MTX. This DAP resistance phenotype was unstable as it was rapidly lost upon growth under nonselective conditions. Furthermore, when the antifolate resistance levels of Pyr-resistant cells were plotted versus the ratios of the 50% F-MTX displacement values obtained with resistant and parental AA8 cells, a good correlation (r2 > 0.98) was obtained. We conclude that Pyr-resistant cells possess a novel phenotype that derives its resistance to lipophilic antifolates solely from a predominant decrease in the accumulation of DAP and lipid-soluble analogs of MTX.
Topics: Animals; CHO Cells; Cell Survival; Cricetinae; Drug Resistance; Flow Cytometry; Folic Acid Antagonists; Leucovorin; Pyrimethamine; RNA, Messenger; Tetrahydrofolate Dehydrogenase; Trimethoprim
PubMed: 8440739
DOI: No ID Found -
British Journal of Cancer Feb 1993Piritrexim is a lipid-soluble inhibitor of dihydrofolate reductase (DHFR) that enters tumour cells rapidly by passive diffusion, cannot be polyglutamated, and is as... (Clinical Trial)
Clinical Trial
Piritrexim is a lipid-soluble inhibitor of dihydrofolate reductase (DHFR) that enters tumour cells rapidly by passive diffusion, cannot be polyglutamated, and is as effective as methotrexate in inhibiting DHFR. Bioavailability after oral dosing is approximately 75%. We performed a phase II study with oral piritrexim in non-chemotherapy pretreated patients with metastatic urothelial cancer. Thirty-three patients were treated with 25 mg three times daily for 5 consecutive days, repeated weekly, with provision for dose escalation or reduction according to the toxicity observed. Of 29 evaluable patients, one patient achieved a complete response of 19+ weeks duration, and ten patients achieved a partial response with a median duration of 22 weeks (range 16-48), for a total response rate of 38%. Piritrexim was generally well tolerated, with myelosuppression as the major toxicity, that frequently required dose modification. We conclude that piritrexim appears to be an active agent in patients with metastatic urothelial cancer when administered as a 5-day, low-dose oral schedule. It would be attractive to investigate the combination of piritrexim and cisplatin.
Topics: Administration, Oral; Adult; Aged; Antineoplastic Agents; Antineoplastic Combined Chemotherapy Protocols; Carcinoma, Transitional Cell; Cisplatin; Female; Humans; Male; Methotrexate; Middle Aged; Pyrimidines; Urologic Neoplasms
PubMed: 8431372
DOI: 10.1038/bjc.1993.71