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Analytical and Bioanalytical Chemistry Sep 2003Molecularly imprinted microspheres were synthesised using the antitumor drug piritrexim (PTX) as a template molecule by aqueous microsuspension polymerisation and were...
Molecularly imprinted microspheres were synthesised using the antitumor drug piritrexim (PTX) as a template molecule by aqueous microsuspension polymerisation and were used as a high-performance liquid chromatographic stationary phase. The molecularly imprinted column exhibited strong retention behaviour to the template molecule. The influences of pH of the buffer and the ratio of methanol to buffer on the retention behaviour were investigated in detail. Results indicated that the baseline separation of PTX, trimetrexate (TMX), trimethoprim (TMP) and sulfamethazine (SMZ) was achieved when the pH value of the acetate buffer was above pH 3.5 and the ratio of methanol to the buffer was 6:4 (v/v). A gradient elution programme was employed to enhance the separation, which led to an improvement in sensitivity and a reduction in determination errors. The method developed was used to analyse urine samples supplemented with PTX. The recoveries of 5 microg mL(-1) PTX in the urine sample were 99.1+/-3.0% and 93.3+/-2.8% at the beginning and 24 h later, respectively.
Topics: Animals; Antineoplastic Agents; Chromatography, High Pressure Liquid; Hydrogen-Ion Concentration; Microspheres; Polymers; Pyrimidines; Trimetrexate
PubMed: 12845402
DOI: 10.1007/s00216-003-2086-8 -
American Journal of Clinical Oncology Jun 2003Piritrexim is a new antifolate that has shown activity in methotrexate-resistant tumors. Gemcitabine is an antimetabolite similar in structure to cytosine arabinoside... (Clinical Trial)
Clinical Trial
Piritrexim is a new antifolate that has shown activity in methotrexate-resistant tumors. Gemcitabine is an antimetabolite similar in structure to cytosine arabinoside with early studies demonstrating activity in a variety of cancers. It also has apparent synergistic activity with antifolates from initial work in tumor models. Paclitaxel is an antimicrotubule agent that has a wide spectrum of activity against a variety of solid tumors. The combination of gemcitabine, paclitaxel, and piritrexim was assessed in this phase I trial. Thirty patients were enrolled. The starting doses were piritrexim 25 mg orally twice daily (days 1-4, 15-18), paclitaxel 75 mg/m2 (days 1, 15), and gemcitabine 750 mg/m2 (days 1, 15), which then was escalated in a stepwise fashion. Four patients achieved stable disease while on study, whereas one patient with a poorly differentiated neuroendocrine tumor achieved a partial response. The main toxicity was myelosuppression. The maximum tolerated dose was thought to be piritrexim 25 mg orally three times daily (days 1-4), paclitaxel 150 to 175 mg/m2 (days 1, 15), and gemcitabine 1,000 mg/m2 (days 1, 15). The combination of these new antifolates with paclitaxel and gemcitabine appears safe and should be considered for phase II trials in known responsive tumors such as transitional cell carcinomas.
Topics: Adult; Aged; Aged, 80 and over; Antimetabolites, Antineoplastic; Antineoplastic Combined Chemotherapy Protocols; Deoxycytidine; Female; Folic Acid Antagonists; Humans; Male; Maximum Tolerated Dose; Middle Aged; Paclitaxel; Pyrimidines; Gemcitabine
PubMed: 12796601
DOI: 10.1097/01.coc.0000081607.16287.6f -
Journal of Medicinal Chemistry Apr 2003As part of an ongoing effort to discover novel small-molecule antifolates combining the enzyme-binding species selectivity of trimethoprim (TMP) with the potency of...
Further studies on 2,4-diamino-5-(2',5'-disubstituted benzyl)pyrimidines as potent and selective inhibitors of dihydrofolate reductases from three major opportunistic pathogens of AIDS.
As part of an ongoing effort to discover novel small-molecule antifolates combining the enzyme-binding species selectivity of trimethoprim (TMP) with the potency of piritrexim (PTX), 10 previously unreported 2,4-diamino-5-(2'-methoxy-5'-substituted)benzylpyrimidines (2-11) containing a carboxyl group at the distal end of the 5'-substituent were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic pathogens frequently responsible for life-threatening illness in people with impaired immune systems as a result of HIV infection or immunosuppressive chemotherapy. The selectivity index of DHFR inhibition was evaluated by comparing the potency of each compound against the parasite enzymes with its potency against rat liver DHFR. 2,4-Diamino-5-[5'-(5-carboxy-1-pentynyl)-2'-methoxybenzyl]pyrimidine (3) inhibited Pc DHFR with a selectivity index of 79 and was 430 times more potent than TMP. 2,4-Diamino-5-[5'-(4-carboxy-1-butynyl)-2'-methoxybenzyl]pyrimidine (2), with one less carbon than 3 in the side chain, had a selectivity index of 910 against Ma DHFR and was 43 times more potent than TMP. 2,4-Diamino-5-[5'-(5-carboxypentyl)-2'-methoxybenzyl]pyrimidine (6) had a selectivity index of 490 against Tg DHFR and was 320 times more potent than TMP. 2,4-Diamino-5-[5'-(6-carboxy-1-hexynyl)-2'-methoxybenzyl]pyrimidine (4), with one more carbon than 3, was less potent against all three of the parasite enzymes than either 3 or 6 and also had a lower selectivity index than 3 against the Pc enzyme. However, 4 was the only member of the series with a selectivity index of >300 against both Tg and Ma DHFR. Given that PTX is at least 10 times more potent against rat DHFR than against P. carinii or T. gondii DHFR and that the selectivity index of several of the compounds matches or exceeds that of TMP as well as PTX, our results suggest that it may be possible to develop clinically useful nonclassical antifolates that are both potent and selective against the major opportunistic pathogens of AIDS.
Topics: AIDS-Related Opportunistic Infections; Animals; Benzyl Compounds; Folic Acid Antagonists; Liver; Magnetic Resonance Spectroscopy; Mycobacterium avium; Pneumocystis; Pyrimidines; Rats; Species Specificity; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase; Toxoplasma
PubMed: 12699390
DOI: 10.1021/jm020466n -
Bioorganic & Medicinal Chemistry Jan 2003A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving...
Synthesis of new 2,4-Diaminopyrido[2,3-d]pyrimidine and 2,4-Diaminopyrrolo[2,3-d]pyrimidine inhibitors of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductase.
A concise new route allowing easy access to five previously unreported 2,4-diamino-6-(substituted benzyl)pyrido[2,3-d]pyrimidines (2a-e) was developed, involving condensation of 2,4-dipivaloylamino-5-bromopyrido[2,3-d]pyrimidine (6) with an organozinc halide in the presence of a catalytic amount of [1,1'-bis(diphenylphosphino)ferrocene]dichloropalladium(II).CH(2)Cl(2), followed by removal of the pivaloyl groups with base. Also prepared via a scheme based on the Taylor ring expansion/ring annulation synthesis were three heretofore undescribed 2,4-diamino-5-(substituted benzyl)-7H-pyrrolo[2,3-d]pyrimidines (3b-c). Standard spectrophotometric assays were used to compare the ability of 2a-e and 3b-c to inhibit dihydrofolate reductase (DHFR) from Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium, three examples of opportunistic pathogens to which AIDS patients are highly vulnerable because of their immunocompromised state. For comparison, 13 previously untested 2,4-diamino-6-(substituted benzyl)quinazolines (17a-m) were also evaluated as inhibitors of these enzymes, as well as the enzyme from rat liver. None of the quinazolines or pyridopyrimidines tested was more potent against the P. carinii enzyme than the structurally related reference compound piritrexim (1), and none showed selectivity for the P. carinii enzyme over the rat enzyme. One of the pyridopyrimidines (2c) showed 10-fold selectivity for T. gondii versus rat DHFR, and two of them (2b, 2c) showed selectivity for the M. avium enzyme. However, this gain in species selectivity was achieved at the cost of decreased in potency, as has been noted with many other lipophilic DHFR inhibitors.
Topics: Animals; Folic Acid Antagonists; Humans; Inhibitory Concentration 50; Leukemia; Liver; Mycobacterium avium; Pneumocystis; Pyrimidines; Rats; Species Specificity; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase; Toxoplasma; Tumor Cells, Cultured
PubMed: 12467708
DOI: 10.1016/s0968-0896(02)00325-5 -
Investigational New Drugs Nov 2002This was a single-agent phase II clinical trial of the antifol piritrexim in patients with advanced transitional cell carcinoma of the bladder. (Clinical Trial)
Clinical Trial
UNLABELLED
This was a single-agent phase II clinical trial of the antifol piritrexim in patients with advanced transitional cell carcinoma of the bladder.
METHODS
Patients with previously-treated, advanced urothelial carcinoma were treated with oral piritrexim at a dose of 25 mg three times daily for 5 consecutive days each week for 3 consecutive weeks followed by a 1-week rest period. Courses were repeated every 28 days.
RESULTS
Thirty-five patients were enrolled in the study, with 28 patients evaluable for survival and toxicity and 27 evaluable for response.
TOXICITY
Myelosuppression was the major dose-limiting toxicity, with WHO grade 3/4 thrombocytopenia in 4 patients, granulocytopenia in 1 patient, and anemia in 3 patients. Grade 3 nonhematologic toxicity consisted of neuropathy in 5 patients, hepatotoxicity in 2, nausea in 2, and 1 each with pulmonary toxicity and rash.
EFFICACY
Of the 27 patients evaluable for response, 2 (7%) achieved an objective response, lasting 112 and 142 days, respectively.
CONCLUSION
Piritrexim has minimal activity in patients with previously treated transitional cell carcinoma of the bladder, regardless of prior exposure to methotrexate, and further evaluation of this compound in this clinical setting is not warranted.
Topics: Aged; Aged, 80 and over; Antineoplastic Protocols; Carcinoma, Transitional Cell; Female; Humans; Male; Middle Aged; Pyrimidines; United States; Urologic Neoplasms; Urothelium
PubMed: 12448661
DOI: 10.1023/a:1020675017737 -
Journal of Medicinal Chemistry Nov 2002Seven 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...
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.
Topics: Animals; Antineoplastic Agents; Cell Division; Cell Line; Drug Resistance, Neoplasm; Escherichia coli; Folic Acid Antagonists; Heterocyclic Compounds, 3-Ring; Humans; Methotrexate; Molecular Conformation; Peptide Synthases; Pneumocystis; Recombinant Proteins; Species Specificity; Structure-Activity Relationship; Substrate Specificity; Tetrahydrofolate Dehydrogenase; Toxoplasma; Tumor Cells, Cultured
PubMed: 12408727
DOI: 10.1021/jm0202369 -
Bioorganic & Medicinal Chemistry Sep 2002Quantitative structure-activity relationship (QSAR) studies for 2,6-substituted 2,4-diaminopyrido[3,2-d] pyrimidine analogues of piritrexim (PTX) as inhibitors of...
Quantitative structure-activity relationship (QSAR) studies for 2,6-substituted 2,4-diaminopyrido[3,2-d] pyrimidine analogues of piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) are now made using topological indices. The results have shown that best models are obtained by multiparametric analysis. The predictive potential of the model is discussed on the basis of a cross-validation method.
Topics: Alcohol Oxidoreductases; Antineoplastic Agents; Enzyme Inhibitors; Humans; Inhibitory Concentration 50; Opportunistic Infections; Pneumocystis; Pyrimidines; Quantitative Structure-Activity Relationship; Regression Analysis
PubMed: 12110313
DOI: 10.1016/s0968-0896(02)00159-1 -
Journal of Molecular Biology Jul 2002The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been...
The crystal structures of two human dihydrofolate reductase (hDHFR) ternary complexes, each with bound NADPH cofactor and a lipophilic antifolate inhibitor, have been determined at atomic resolution. The potent inhibitors 6-([5-quinolylamino]methyl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9439) and (Z)-6-(2-[2,5-dimethoxyphenyl]ethen-1-yl)-2,4-diamino-5-methylpyrido[2,3-d]pyrimidine (SRI-9662) were developed at Southern Research Institute against Toxoplasma gondii DHFR-thymidylate synthase. The 5-deazapteridine ring of each inhibitor adopts an unusual puckered conformation that enables the formation of identical contacts in the active site. Conversely, the quinoline and dimethoxybenzene moieties exhibit distinct binding characteristics that account for the differences in inhibitory activity. In both structures, a salt-bridge is formed between Arg70 in the active site and Glu44 from a symmetry-related molecule in the crystal lattice that mimics the binding of methotrexate to DHFR.
Topics: Amino Acid Sequence; Animals; Catalytic Domain; Crystallography, X-Ray; Folic Acid Antagonists; Humans; Hydrogen Bonding; In Vitro Techniques; Macromolecular Substances; Models, Molecular; Molecular Sequence Data; NADP; Protein Conformation; Pyrimidines; Recombinant Proteins; Sequence Homology, Amino Acid; Tetrahydrofolate Dehydrogenase; Toxoplasma
PubMed: 12096917
DOI: 10.1016/s0022-2836(02)00469-2 -
Acta Biochimica Polonica 2001Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key...
Dihydrofolate reductase (DHFR, EC 1.5.1.3) is one of the enzymes active in the folate cycle which plays an important role in DNA synthesis. Inhibition of DHFR is a key element in the treatment of many diseases, including cancer and AIDS related infections. A search for new selective inhibitors is motivated by the resistance to common drugs observed in the course of treatment. In this paper, results of a detailed computer analysis of human DHFR interactions with the lipophilic inhibitor piritrexim (PTX) are presented. It was found that the NADPH cofactor contributes 30% of the total PTX-enzyme interaction energy. Substitution of the highly conserved Glu30 with alanine does not lead to the release of the inhibitor from the hDHFR pocket. The important L22F point mutation does affect PTX orientation but does not changethe binding energy. Simulations of the dynamics of binary hDHFR-PTX complexes were performed with the use of Extensible Systematic Force Field (ESFF) and the results indicate structural changes in the enzyme induced by NADPH binding.
Topics: Antineoplastic Agents; Binding Sites; Humans; Models, Molecular; Mutation; NADP; Point Mutation; Protein Binding; Pyrimidines; Software; Tetrahydrofolate Dehydrogenase
PubMed: 11996001
DOI: No ID Found -
Journal of Medicinal Chemistry Jan 2002A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines... (Comparative Study)
Comparative Study
Inhibition of Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium dihydrofolate reductases by 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines: marked improvement in potency relative to trimethoprim and species selectivity relative to piritrexim.
A series of previously undescribed 2,4-diamino-5-[2-methoxy-5-alkoxybenzyl]pyrimidines (3a-e) and 2,4-diamino-5-[2-methoxy-5-(omega-carboxyalkyloxy)benzyl]pyrimidines (3f-k) with up to eight CH2 groups in the alkoxy or omega-carboxyalkyloxy side chain were synthesized and tested for the ability to inhibit partially purified dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat liver in comparison with two standard inhibitors, trimethoprim (1) and piritrexim (2). The latter drug is known to be extremely potent but shows a marked preference for binding to mammalian DHFR, whereas the former is very selective for the parasite enzymes but is a much weaker inhibitor. The underlying strategy for the synthesis of compounds 3a-k was that a hybrid structure embodying some features of both 1 and 2 might possess a more favorable combination of potency and selectivity than either parent drug. The choice of analogues 3f-k was based on the idea that the acidic omega-carboxyl group might interact preferentially with a basic center in the active site of DHFR from any of the parasite species relative to the active site of mammalian DHFR. In addition, the omega-carboxyl group was expected to improve water solubility relative to 1 or 2. In standardized spectrophotometric assays with dihydrofolate as the substrate and NADPH as the cofactor, 2,4-diamino-5-[(2-methoxy-4-carboxybutyloxy)benzyl]pyrimidine (3g) inhibited Pc DHFR with an IC(50) of 0.049 microM and rat DHFR with IC(50) of 3.9 microM. Its potency against Pc DHFR was 140-fold greater than that of 1 and close to that of 2, and its selectivity index, defined as the ratio IC(50)(rat liver)/IC(50)(P. carinii), was 8-fold higher than that of 1 and >10(4)-fold higher than that of 2. Although it was less potent and less selective against Tg than Pc DHFR, it was very potent as well as highly selective against Ma DHFR, with an IC(50) of 0.0058 microM and an IC(50)(rat liver)/IC(50)(M. avium) ratio of >600. Because of this favorable combination of potency and selectivity relative to 1 and 2, compound 3g may be viewed as a promising lead in the search for new antifolates with potential clinical activity against P. carinii and other opportunistic pathogens in patients with AIDS.
Topics: Animals; Folic Acid Antagonists; Liver; Mycobacterium avium; Pneumocystis; Pyrimidines; Rats; Species Specificity; Structure-Activity Relationship; Tetrahydrofolate Dehydrogenase; Toxoplasma; Trimethoprim
PubMed: 11754594
DOI: 10.1021/jm010407u