A. Rosowsky

Abstract We have attempted to evaluate the degree to which hepatic arterial infusion of 5-fluoro-2′-deoxyuridine (FdUrd) or 5-fluorouracil produces higher hepatic and lower systemic drug concentrations than are achieved with corresponding peripheral venous infusions. Hepatic arterial catheters were placed for therapy in 15 patients with primary or metastatic liver cancer. Temporary hepatic venous catheters allowed direct sampling of drug levels in the hepatic venous effluent as well as measurement of hepatic blood flow. FdUrd was measured primarily by radioimmunoassay and fluorouracil by a high-pressure liquid chromatographic system. Due to the limited sensitivities of these assays, short (40 to 60 min) infusions at dose rates 10 to 100 times those conventionally used were given in order to produce drug levels that could be measured reliably. Even at dose rates of 0.5 to 40 mg/kg of body weight/hr for FdUrd and 5.6 mg/kg of body weight/hr for fluorouracil, steady state drug levels were achieved in the bloodstream in 30 to 40 min and hepatic extraction could be quantified. The hepatic extraction of FdUrd is high with an extraction ratio (hepatic arterial level — hepatic venous level/hepatic arterial level) of 0.69 to 0.92 and a clearance of 0.81 to 2.3 liters/min. The hepatic extraction of fluorouracil, however, appears to be lower with an extraction ratio of 0.22 to 0.45 and a clearance of 0.24 to 0.45 liters/min. With hepatic arterial drug infusion, 94 to 99% of FdUrd and 19 to 51% of fluorouracil is extracted in one pass. Hepatic venous levels, which are one measure of intrahepatic drug concentration in the hepatic and tumor capillary bed, were 4-fold higher for FdUrd infusion and 1.5-fold higher for fluorouracil infusion when drug was given by the hepatic arterial route. Systemic FdUrd levels with hepatic arterial infusion were only about 25% of corresponding systemic levels with peripheral venous infusion. Systemic fluorouracil levels with hepatic arterial infusion were also lower and were about 60% of corresponding systemic levels with peripheral venous infusion. These results support hepatic arterial infusion as a means to improve the therapeutic index of FdUrd and fluorouracil in the treatment of cancer in the liver. Although molar equivalent dose rates of FdUrd and fluorouracil were used in this study, the differences in FdUrd and fluorouracil pharmacology as noted above may not be applicable to conventional hepatic arterial therapy where much lower dose rates are used. Nonetheless, this type of analytical approach should prove valuable in the evaluation of other agents for hepatic arterial chemotherapy.

BACKGROUND Cyclophosphamide, which forms the nucleus for virtually all preparative regimens for autologous bone marrow transplantation (ABMT), is an alkylating agent of which cytotoxicity is not directly caused by the parent compound but by its biologically active metabolites. Its nonmyelosuppressive toxicity in the ABMT setting is cardiomyopathy. We attempted to determine any correlation between plasma levels of total cyclophosphamide and the subsequent development of cardiac dysfunction. PATIENTS AND METHODS Analyses of plasma levels and the derivation of plasma concentration-time curves (area under the curve [AUC]) were performed in 19 women with metastatic breast carcinoma, who received a continuous 96-hour infusion of cyclophosphamide, thiotepa, and carboplatin (CTCb) with ABMT. The assay for total cyclophosphamide measures the inactive parent compound; reliable assays of the active metabolites of cyclophosphamide are not yet available. RESULTS Six of 19 women developed moderate, but transient, congestive heart failure (CHF) as assessed by clinical and radiologic criteria. These patients had a significantly lower AUC of total cyclophosphamide (median, 2,888 mumol/L/h) than patients who did not develop CHF (median, 6,121 mumol/L/h) (P less than .002). Median duration of tumor response in these patients was also more durable; at least 22 months in patients with lower AUCs versus a median of 5.25 months in those with higher AUCs (P = .008). CONCLUSION These pharmacokinetic data support the premise that enhancement of cyclophosphamide activation may lead to both greater tumor cytotoxicity and increased but reversible end-organ toxicity. Early analysis of pharmacokinetic data may allow modulation of cyclophosphamide administration in an attempt to enhance therapeutic efficacy.

We have developed a human head and neck squamous cell carcinoma cell line (SCC-25/CP) which is relatively stably resistant to cis-diamminedichloroplatinum(II) (CDDP) after repeated exposure to escalating doses of the drug. The studies reported elucidate the mechanism(s) by which the SCC-25/CP cell line is resistant to CDDP. The SCC-25/CP cell line is approximately 30-fold resistant to CDDP, approximately 10-fold resistant to carboplatin, and about 9-fold resistant to iproplatin. Using [195mPt]CDDP, we examined the levels of platinum in whole cells and cellular fractions of both the SCC-25 and SCC-25/CP cells after 1 h exposure to 100 microM drug. The SCC-25 cells took up 30 pmol of platinum/10(6) cells in 1 h; 64% of the drug was in the nucleus and 21% in the cytosol. The SCC-25/CP cells took up 7 pmol of platinum/10(6) cells; of this, 41% was in the nucleus and 33% in the cytosol. The SCC-25 cell nuclei contained 331 pmol of platinum/mg protein and the cytosol 21 pmol of platinum/mg protein, whereas the SCC-25/CP cell nuclei contained 47 pmol of platinum/mg protein and the cytosol 8.1 pmol/mg protein. The release of drug from both cell lines followed a very similar course and was most rapid over the first 6 h. There was no difference in the non-protein sulfhydryl content of the cell lines. The protein sulfhydryl content, as measured by Ellman's procedure, indicated that the SCC-25/CP cell line has approximately a 2-fold increase in protein sulfhydryl content compared to the SCC-25 cell line. The SCC-25/CP cell line is about 2-fold resistant to cadmium chloride at 50% cell kill and about 2.5-fold resistant at 1 log kill compared to the SCC-25 cell line. Glutathione transferase activity in crude cytoplasmic extracts was measured and found to be approximately 2- to 3-fold higher in the CDDP resistant cells. The isoelectric point of the glutathione transferase isozyme was 4.8 in both the sensitive and resistant cell lines, suggesting induction of the predominant isozyme present in the parent cell line. By alkaline elution there was greater cross-link formation by CDDP in the SCC-25 cell line than in the SCC-25/CP cell line at the same drug concentrations. In conclusion, the mechanism of resistance of the SCC-25/CP cell line to CDDP is multifactorial, involving plasma membrane changes, increased cytosolic binding, and decreased DNA cross-linking.

Development of in vitro resistance to HN2 (also called mustargen or mechlorethamine hydrochloride), N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), and cisplatin [cis-diamminedichloroplatinum(II)] was achieved in two human cell lines, the Raji/Burkitt lymphoma and a squamous cell carcinoma of the tongue. A 10- to 20-fold increase in resistance relative to the parental line was achieved in 3-4 months of continuous selection pressure. At this time, further increase in selection pressure resulted in cell death, while removal of drug led to rapid loss of resistance. However, by holding selection pressure constant over 8-12 months, semistable clones ranging in resistance up to 8- to 12-fold were obtained. The half-life for resistance loss upon removal of drug was 2-3 months. In the presence of intermittent low concentrations of the alkylating agent, resistance has been maintained in excess of 9 months. With one exception, the growth kinetics of the resistant clones were slightly slower than those of the parental lines. Cross-resistance studies were performed against HN2, BCNU, cisplatin, phenylalanine mustard, and hydroperoxycyclophosphamide. There was, in general, a lack of cross-resistance. We conclude that stable resistance to alkylating agents is produced with difficulty. We propose that these semistable cloned human tumor lines represent clinically relevant models for the study of alkylating agent resistance and that the cross-resistance patterns among these cells have important therapeutic and mechanistic implications.

Study Objective: To study the activity of continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin (PFL) as induction chemotherapy in patients with previously untreated, advanced squamous cell carcinoma of the head and neck. Design: Nonrandomized, prospective trial. Setting: A comprehensive cancer center. Patients: Thirty-five patients (4 patients [11%], stage III; 31 patients [89%], stage IV [MO]), all evaluable for response and toxicity. Interventions: Two to three cycles of PFL before definitive, local-regional therapy (surgery and radiation therapy or radiation therapy alone). Chemotherapy included continuous intravenous infusion of cisplatin (25 mg/m2 body surface area daily, days 1 through 5); 5-fluorouracil (800 mg/ m2 body surface area daily, days 2 through 6); and leucovorin (500 mg/m2 body surface area daily, days 1 through 6) administered once every 28 days. Pathologic response was evaluated by surgical resection or biopsy. Serum-reduced folates were measured before and 18 hours after the initiation of chemotherapy. Results: A clinical response to PFL was achieved in 28 of 35 (80%) patients: 23 (66%) patients had a complete response (90% CI, 50% to 79%) and 5 (14%) patients, a partial response. A complete response was confirmed pathologically in 14 of 19 (74%) patients. The most common toxicity was mucositis (grade 2 to 3; 94% of patients). Dose reduction for toxicity was necessary in 11 (31%) patients. There were no treatment-related deaths. Serum levels of leucovorin and (6S)5-methyltetrahydrofolate were measured in 7 patients. After 18 hours, the mean leucovorin level (± SD) was 34.3 ± 1.5 μmol/L, of which only 8.0 ± 0.5% was the active 6S isomer. The mean serum (6S)5-methyltetrahydrofolate was 9.2 ± 0.6 ¼mol/L. Conclusions: Continuous infusion cisplatin, 5-fluorouracil, and high-dose leucovorin is a new and highly active chemotherapy regimen that can achieve clinical and pathologically confirmed complete responses in a substantial proportion of patients with advanced, local-regional squamous cell carcinoma of the head and neck. Further studies are needed to confirm the activity of PFL and to determine its potential impact on local tumor control and disease-free and overall survival.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTStructural features of 5,10-dideaza-5,6,7,8-tetrahydrofolate that determine inhibition of mammalian glycinamide ribonucleotide formyltransferaseSamuel W. Baldwin, Archie Tse, Lynn S. Gossett, Edward C. Taylor, Andre Rosowsky, Chuan Shih, and Richard G. MoranCite this: Biochemistry 1991, 30, 7, 1997–2006Publication Date (Print):February 19, 1991Publication History Published online1 May 2002Published inissue 19 February 1991https://doi.org/10.1021/bi00221a037RIGHTS & PERMISSIONSArticle Views183Altmetric-Citations77LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

Three patients are presented, the first ones of American origin, with a newly identified constitutional lipidosis previously described by Wolman from Israel. The clinical picture as seen to date shows poor weight gain, vomiting and diarrhea, increasing hepatosplenomegaly with abdominal protuberance, and death in nutritional failure by 2-4 months of age. One of the patients in this report had a similarly involved sibling; present data suggest recessive genetic transmission. Foam cells are found in the bone marrow, and vacuolated lymphocytes in the peripheral blood, each entirely similar to those seen in Niemann-Pick disease. Serum lipids are normal, and signs of adrenocortical insufficiency are moderate. An apparently pathognomic radiologic sign is the presence of greatly enlarged adrenal glands, easily visible by virtue of diffuse punctate calcific deposits, with normal shape of the gland preserved. Pathologic studies show disseminated foam cells, laden with cholesterol and neutral fat, most notable in the liver, spleen, lymph nodes, marrow, thymus, and small intestinal mucosa. In addition, neutral fat deposits are found in connective tissue cells, vascular endothelium, bile ducts, hepatic parenchymal cells, and adrenal cortical cells. The adrenal enlargement is produced by increase in the adrenal cortex, with secondary, possibly ischemic, necrosis and calcification. Direct analysis of unfixed tissue has shown that the cholesterol level of the liver is increased 15-20-fold, and of the spleen 4-5 times normal, with apparent major increase in triglycerides as well. Nervous system changes are milder. The accumulated sterol has been demonstrated to be genuine cholesterol in the tissues studied so that the mechanism of the disease does not appear to rest with disturbed biosynthesis of cholesterol.

Abstract 2,4‐Diamino‐5,6,7,8‐tetrahydropyrido[4,3‐ d ]pyrimidines with di‐ and trimethoxyaralkyl substitution at the 6‐position were synthesized from the N 6 ‐unsubstituted compound and appropriate aralkyl bromides in N,N ‐dimethylformamide solution containing a catalytic amount of sodium iodide. An improved method of preparation of 2,4‐diamino‐5,6,7,8‐tetrahydropyrido[4,3‐ d ]pyrimidine from 2‐amino‐6‐benzyl‐5,6,7,8‐tetrahydropyrido[4,3‐ d ]pyrimidin‐4(3 H )‐one was also developed, in which N 2 was protected by reaction with pivalic anhydride and the resulting product was subjected consecutively to reaction with 4‐chlorophenylphosphorodichloridate and 1,2,4‐triazole, ammonolysis to replace the 4‐imidazolido group and remove the N 2 ‐pivaloyl group, and catalytic hydrogenolysis to remove the 6‐benzyl group. In assays of the ability of the products to inhibit dihydrofolate reductase from Pneumocystis carinii , and Toxoplasma gondii , and rat liver the most active of the compounds tested was 2,4‐diamino‐6‐(2′‐bromo‐3′,4′,5′‐trimethoxybenzyl)‐5,6,7,8‐tetrahydropyrido[4,3‐ d ]pyrimidine. The concentration of this compound needed to inhibit enzyme activity by 50% was 0.51 μ M against the P. carinii enzyme, 0.09 μ M against the T. gondii enzyme , and 0.35 μ M against the rat enzyme. Thus, there was selectivity of binding to T. gondii enzyme, but not P. carinii enzyme, relative to rat enzyme. 2′,5′‐Dimethoxybenzyl analogues were less active than the corresponding 3′,4′,5′‐trimethoxybenzyl analogues, and compounds with a CH 2 CH 2 or CH 2 CH 2 CH 2 bridge were less active than those with a CH 2 bridge. 2,4‐Diamino‐6‐(2′‐bromo‐3′,4′,5′‐trimethoxybenzyl)‐5,6,7,8‐tetrahydropyrido[4,3‐ d ]pyrimidine showed greater selectivity than trimetrexate or piritrexim for the P. carinii and T. gondii enzyme, but was less selective than trimethoprim or pyrimethamine. However its molar potency against both enzymes was greater than that of trimethoprim, the antifolate most commonly used, in combination with sulfamethoxazole, for initial treatment of opportunistic P. carinii and T. gondii infections in patients with AIDS and other disorders of the immune system.

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.

The transport properties and growth-inhibitory potential of 37 classic and novel antifolate compounds have been tested in vitro against human and murine cell lines expressing different levels of the reduced folate carrier (RFC), the membrane-associated folate binding protein (mFBP), or both. The intracellular targets of these drugs were dihydrofolate reductase (DHFR), glycinamide ribonucleotide transformylase (GARTF), folylpolyglutamate synthetase (FPGS), and thymidylate synthase (TS). Parameters that were investigated included the affinity of both folate-transport systems for the antifolate drugs, their growth-inhibitory potential as a function of cellular RFC/mFBP expression, and the protective effect of either FA or leucovorin against growth inhibition. Methotrexate, aminopterin, N10-propargyl-5,8-dideazafolic acid (CB3717), ZD1694, 5,8-dideazaisofolic acid (IAHQ), 5,10-dideazatetrahydrofolic acid (DDATHF), and 5-deazafolic acid (efficient substrate for FPGS) were used as the basic structures in the present study, from which modifications were introduced in the pteridine/quinazoline ring, the C9-N10 bridge, the benzoyl ring, and the glutamate side chain. It was observed that RFC exhibited an efficient substrate affinity for all analogues except CB3717, 2-NH2-ZD1694, and glutamate side-chain-modified FPGS inhibitors. Substitutions at the 2-position (e.g., 2-CH3) improved the RFC substrate affinity for methotrexate and aminopterin. Other good substrates included PT523 (N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine), 10-ethyl-10-deazaaminopterin, and DDATHF. With respect to mFBP, modifications at the N-3 and 4-oxo positions resulted in a substantial loss of binding affinity. Modifications at other sites of the molecule were well tolerated. Growth-inhibition studies identified a series of drugs that were preferentially transported via RFC (2,4-diamino structures) or mFBP (CB3717, 2-NH-ZD1694, or 5,8-dideazaisofolic acid), whereas other drugs were efficiently transported via both transport pathways (e.g., DDATHF, ZD1694, BW1843U89, or LY231514). Given the fact that for an increasing number of normal and neoplastic cells and tissue, different expression levels of RFC and mFBP are being recognized, this folate antagonist structure-activity relationship can be of value for predicting drug sensitivity and resistance of tumor cells or drug-related toxicity to normal cells and for the rational design and development of novel antifolates.

The alkylating agents represent one of the most important classes of antitumor agents and play a major role in combination with other agents in the curative chemotherapy of selected human cancers. By repeatedly exposing cells to escalating doses of an alkylating agent, we have developed four human tumor cell lines which are relatively stably resistant to the drug with which the culture was treated. The response of these cell lines to a variety of alkylating agents was compared to the response of the parent cell lines to the same drug. The Raji/HN2 line was 7-fold resistant to nitrogen mustard and about 3-fold resistant to 4-hydroxyperoxycyclophosphamide, but it was not resistant to N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU), melphalan (MEL), busulfan, trimethyleneiminethiophosphoramide, 4-hydroperoxyifosfamide, or cisplatin [cis-diamminedichloroplatinum(II)] (CDDP). The Raji/BCNU line was 5.3-fold resistant to BCNU and 4-fold resistant to both MEL and CDDP. The Raji/CP line was 7-fold resistant to CDDP and 3-fold resistant to both nitrogen mustard and BCNU, but it was not resistant to busulfan, trimethyleneiminethiophosphoramide, or 4-hydroperoxyifosfamide. The SCC-25/CP line, which was 12-fold resistant to CDDP, was 5-fold resistant to MEL and 3-fold resistant to 4-hydroxyperoxycyclophosphamide. The SCC-25/CP line was almost 24-fold resistant to methotrexate after 30-min treatment and about 7-fold resistant to methotrexate after continuous treatment. None of the other cell lines was resistant to methotrexate. The survival of SCC-25 and SCC-25/CP cells exposed to several antineoplastic agents was examined over several logs of survival. The SCC-25/CP cells are highly resistant to CDDP; the ratio of the slopes of the survival curves (SCC-25/CP to SCC-25) of the two lines was 43. At survivals of 1%, resistance to MEL and BCNU became evident in the SCC-25/CP line. At survivals of 0.1%, resistance to mitomycin C and, to a lesser degree, to Adriamycin and vincristine was evident. It is more difficult to produce resistance to alkylating agents, even with extended selection pressure, than to other antineoplastic drugs such as antimetabolites and natural products. We found no evidence of pleiotropic resistance in any alkylating agent-resistant cell line. Our results suggest that a judicious choice of alkylating agents given in sequential or concurrent combination may be a rational treatment strategy with potential applications in the clinic.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and biological activity of L-5-deazafolic acid and L-deazaaminopterin: synthetic strategies to 5-deazapteridinesEdward C. Taylor, David C. Palmer, Thomas J. George, Stephen R. Fletcher, Chi Ping Tseng, Peter J. Harrington, G. Peter Beardsley, Donald J. Dumas, Andre Rosowsky, and Michael WickCite this: J. Org. Chem. 1983, 48, 25, 4852–4860Publication Date (Print):December 1, 1983Publication History Published online1 May 2002Published inissue 1 December 1983https://doi.org/10.1021/jo00173a014RIGHTS & PERMISSIONSArticle Views390Altmetric-Citations64LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe Chemistry of Fumagillin1D. S. Tarbell, R. M. Carman, D. D. Chapman, S. E. Cremer, A. D. Cross, K. R. Huffman, M. Kunstmann, N. J. McCorkindale, J. G. McNally Jr., A. Rosowsky, F. H. L. Varino, and R. L. WestCite this: J. Am. Chem. Soc. 1961, 83, 14, 3096–3113Publication Date (Print):July 1, 1961Publication History Published online1 May 2002Published inissue 1 July 1961https://doi.org/10.1021/ja01475a029Request reuse permissionsArticle Views531Altmetric-Citations51LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alertsclose Get e-Alerts

Abstract A retrospective study of clinical resistance to methotrexate (MTX) was performed on 29 archival specimens of frozen lymphoblasts obtained from children with acute lymphoblastic leukemia (ALL), including 19 at initial presentation and 10 at first relapse. Blasts were assayed for dihydrofolate reductase and MTX transport by flow cytometry using the fluorescent methotrexate analog, PT430 (Rosowsky et al, J Biol Chem 257:14162, 1982). In contrast to tissue culture cells, patient blasts were often heterogeneous for dihydrofolate reductase content. Of the 19 specimens at initial diagnosis, 7 exhibited dual blast populations, characterized by threefold to 10-fold differences in relative dihydrofolate reductase; the dihydrofolate reductase-overproducing populations comprised 12% to 68% of the total blasts for these specimens. Remission duration intervals for patients exhibiting dual blast populations were notably shorter than for patients expressing a single blast population with lower dihydrofolate reductase ( < or = 9 months v > or = 15 months, respectively), a difference that was statistically significant (P = .045). There was no apparent correlation between expression of increased dihydrofolate reductase at diagnosis and known patient and disease prognostic features (immunophenotype, age, sex, and white blood count). For the relapsed patients, 4 of 10 exhibited dual lymphoblast populations with elevated dihydrofolate reductase. The majority of the patient lymphoblast specimens were entirely competent for MTX transport and, likewise, expressed immunoreactive reduced folate carriers by indirect immunofluorescence staining with specific antiserum to the transporter. Three patients (2 at relapse and 1 at diagnosis) exhibited heterogeneous expression of imparied MTX transport (14% to 73% of blasts). In only 1 of these patients did the majority of the lymphoblasts (73%) show impaired MTX transport and for this specimen, immunoreactive carrier proteins were virtually undetectable. These results suggest that heterogeneous expression of elevated dihydrofolate reductase and impaired MTX transport are important modes of resistance in childhood ALL patients undergoing chemotherapy with MTX and that these parameters may serve as predictive indices of clinical response to MTX.

Abstract Although the mechanisms of resistance to methotrexate (MTX) are known in experimental tumors made resistant to this drug, little information is available regarding acquired resistance to MTX in patients. A competitive displacement assay using the fluorescent lysine analogue of MTX, N-(4-amino-4-deoxy-N10-methylpteroyl)-N epsilon-(4′-fluorescein- thiocarbamyl)-L-lysine (PT430), was developed as a sensitive method of detection of transport resistance to MTX in cell lines, as well as in blast cells from patients with leukemia. Rapid uptake of PT430 at high concentrations (20 mumol/L) in leukemic blasts resulted in achievement of steady-state levels within 2 hours. Subsequent incubation with the folate antagonists, MTX and trimetrexate (TMTX), which differ in the mode of carrier transport, produced characteristic patterns of PT430 displacement. Flow cytometric analysis of the mean fluorescence intensity in the human CCRF-CEM T-cell lymphoblastic leukemia cell line and its MTX-resistant subline clearly identified the presence of transport deficiency in the resistant subline. Analysis of blasts from 17 patients with leukemia, nine with no prior chemotherapy and eight previously treated with chemotherapy, found evidence of MTX transport resistance in two of the four patients who were treated with MTX and considered to be clinically resistant to the drug. The finding that blast cells of some patients with leukemia considered clinically resistant to MTX is due to decreased MTX transport has important implications for clinical use of this drug and for new drug development.

Four methotrexate (MTX)-resistant sublines of a human squamous cell carcinoma (SCC15) were established in culture by progressive dose escalation. The biochemical basis of resistance was studied. The line with the lowest resistance (R1) had a normal dihydrofolate reductase (DHFR) content but showed decreased MTX transport and polyglutamation. Lines of intermediate resistance (R2 and R3) showed an increased DHFR content and DHFR gene copy number and a defect in MTX transport. The line with the greatest resistance (R4) showed increased DHFR content and gene copy number but nearly normal MTX transport. These results demonstrate that multiple mechanisms of MTX resistance occur in human epithelial cells in culture. We also find evidence of alterations in DHFR gene expression. The MTX-resistant cells were either not cross-resistant or only partly cross-resistant to two lipophilic MTX ester derivatives. These compounds are of potential therapeutic interest for the treatment of MTX-resistant tumors.

The structure-activity relationship of human thymidylate synthetase (EC 2.1.1.45) was studied with two groups of folate analogs: (1) methotrexate (MTX) analogs modified at the glutamate residue and N10; and (2) tetrahydrofolate (H4PteGlu) analogs modified at N5 and N10. With respect to MTX analogs, it was found that: (1) substitution of the glutamate side chain by α-aminoadipic acid. α-aminopimelic acid or β-aminoglutaric acid slightly affects its Ki; (2) a free α-carboxyl group on the amino acid side chain of MTX, or any free carboxyl group in that vicinity plays an important role in the inhibitory potency of MTX analogs to the enzyme; (3)esterification or amidation of the α-carboxyl group of MTX decreases the inhibitory potency; and (4) free aspartyl or glutamyl conjugation through a peptide linkage to the γ-carboxyl group of the glutamate side chain decreases its Ki to the enzyme by 5- and 8-fold respectively. Tetrahydrofolate analogs formed by inserting an ethylene, iminyl or a carbonyl bridge between the nitrogen at N5 and N10 or by substitution at the N5 position were found to be poor inhibitors under our assay conditions.

The gamma-tert-butyl ester (1), gamma-hydrazide (2), gamma-n-butylamide (3), and gamma-benzylamide (4) derivatives of methotrexate (MTX) were synthesized from 4-amino-4-deoxy-N10-methylpteroic acid (APA) and the appropriate blocked L-glutamic acid precursors with the aid of the peptide bond forming reagent diethyl phosphorocyanidate. The affinity of these side chain modified products for dihydrofolate reductase (DHFR) from Lactobacillus casei and L1210 mouse leukemic cells was determined spectrophotometrically or by competitive radioligand binding assay, and their cytotoxicity was evaluated against L1210 leukemic cells in culture. The results provide continuing support for the view that the "gamma-terminal region" of the MTX side chain is an attractive site for molecular modification of this anticancer agent.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and antitumor activity of an acyclonucleoside derivative of 5-fluorouracilAndre Rosowsky, Sun-Hyuk Kim, and Michael WickCite this: J. Med. Chem. 1981, 24, 10, 1177–1181Publication Date (Print):October 1, 1981Publication History Published online1 May 2002Published inissue 1 October 1981https://doi.org/10.1021/jm00142a011Request reuse permissionsArticle Views350Altmetric-Citations51LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (723 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT2,4-Diaminothieno[2,3-d]pyrimidines as antifolates and antimalarials. 1. Synthesis of 2,4-diamino-5,6,4,8-tetrahydrothianaphtheno[2,3-d]pyrimidines and related compoundsA. Rosowsky, M. Chaykovsky, K. K. N. Chen, M. Lin, and E. J. ModestCite this: J. Med. Chem. 1973, 16, 3, 185–188Publication Date (Print):March 1, 1973Publication History Published online1 May 2002Published inissue 1 March 1973https://doi.org/10.1021/jm00261a002RIGHTS & PERMISSIONSArticle Views259Altmetric-Citations45LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (503 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTCysteine scavengers. 2. Synthetic .alpha.-methylenebutyrolactones as potential tumor inhibitorsAndre Rosowsky, Nickolas Papathanasopoulos, Herbert Lazarus, George E. Foley, and Edward J. ModestCite this: J. Med. Chem. 1974, 17, 7, 672–676Publication Date (Print):July 1, 1974Publication History Published online1 May 2002Published inissue 1 July 1974https://doi.org/10.1021/jm00253a003Request reuse permissionsArticle Views138Altmetric-Citations49LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (653 KB) Get e-Alertsclose Get e-Alerts

The result of a search at the LHC for heavy stable charged particles produced in pp collisions at $ \sqrt {s} = 7\;{\text{TeV}} $ is described. The data sample was collected with the CMS detector and corresponds to an integrated luminosity of 3.1 pb−1. Momentum and ionization-energy-loss measurements in the inner tracker detector are used to identify tracks compatible with heavy slow-moving particles. Additionally, tracks passing muon identification requirements are also analyzed for the same signature. In each case, no candidate passes the selection, with an expected background of less than 0.1 events. A lower limit at the 95% confidence level on the mass of a stable gluino is set at 398GeV/c 2, using a conventional model of nuclear interactions that allows charged hadrons containing this particle to reach the muon detectors. A lower limit of 311 GeV/c 2 is also set for a stable gluino in a conservative scenario of complete charge suppression, where any hadron containing this particle becomes neutral before reaching the muon detectors.

Hadronic event shapes have been measured in proton-proton collisions at sqrt(s)=7 TeV, with a data sample collected with the CMS detector at the LHC. The sample corresponds to an integrated luminosity of 3.2 inverse picobarns. Event-shape distributions, corrected for detector response, are compared with five models of QCD multijet production.

A measurement of the top-antitop production cross section in proton-proton collisions at a centre-of-mass energy of 7 TeV has been performed at the LHC with the CMS detector. The analysis uses a data sample corresponding to an integrated luminosity of 36 inverse picobarns and is based on the reconstruction of the final state with one isolated, high transverse-momentum electron or muon and three or more hadronic jets. The kinematic properties of the events are used to separate the top-antitop signal from W+jets and QCD multijet background events. The measured cross section is 173 + 39 - 32 (stat. + syst.) pb, consistent with standard model expectations.

The charge asymmetry in the production of top quark and antiquark pairs is measured in proton-proton collisions at a center-of-mass energy of 8 TeV. The data, corresponding to an integrated luminosity of 19.6 inverse femtobarns, were collected by the CMS experiment at the LHC. Events with a single isolated electron or muon, and four or more jets, at least one of which is likely to have originated from hadronization of a bottom quark, are selected. A template technique is used to measure the asymmetry in the distribution of differences in the top quark and antiquark absolute rapidities. The measured asymmetry is A[c,y] = [0.33 +/- 0.26 (stat) +/- 0.33 (syst)]%, which is the most precise result to date. The results are compared to calculations based on the standard model and on several beyond-the-standard-model scenarios.

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.

2,4-Diamino-5-[3′,4′-dimethoxy-5′-(5-carboxy-1-pentynyl)]benzylpyrimidine (6) and 2,4-diamino-5-[3′,4′-dimethoxy-5′-(4-carboxyphenylethynyl)benzylpyrimidine (7) were synthesized from 2,4-diamino-5-(5′-iodo-3′,4′-dimethoxybenzyl)pyrimidine (9) via a Sonogashira reaction with appropriate acetylenic esters followed by saponification, and were tested as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), Mycobacterium avium (Ma), and rat in comparison with the widely used antibacterial agent 2,4-diamino-5-(3′,4′,5′-trimethoxybenzyl)pyrimidine (trimethoprim, TMP). The selectivity index (SI) for each compound was calculated by dividing its 50% inhibitory concentration (IC50) against rat DHFR by its IC50 against Pc, Tg, or Ma DHFR. The IC50 of 6 against Pc DHFR was 1.0 nM, with an SI of 5000. Compound 7 had an IC50 of 8.2 nM against Ma DHFR, with an SI of 11000. By comparison, the IC50 of TMP was 12000 nM against Pc, 300 nM against Ma, and 180000 against rat DHFR. The potency and selectivity values of 6 and 7 were not as high against Tg as they were against Pc or Ma DHFR, but nonetheless exceeded those of TMP. Because of the outstanding selectivity of 6 against Pc and of 7 against Ma DHFR, these novel analogues may be viewed as promising leads for further structure–activity optimization.

As part of a search for dihydrofolate reductase (DHFR) inhibitors combining the high potency of piritrexim (PTX) with the high antiparasitic vs mammalian selectivity of trimethoprim (TMP), the heretofore undescribed 2,4-diamino-6-(2‘,5‘-disubstituted benzyl)pyrido[2,3-d]pyrimidines 6−14 with O-(ω-carboxyalkyl) or ω-carboxy-1-alkynyl groups on the benzyl moiety were synthesized and tested against Pneumocystis carinii, Toxoplasma gondii, and Mycobacterium avium DHFR vs rat DHFR. Three N-(2,4-diaminopteridin-6-yl)methyl)-2‘-(ω-carboxy-1-alkynyl)dibenz[b,f]azepines (19-21) were also synthesized and tested. The pyridopyrimidine with the best combination of potency and selectivity was 2,4-diamino-5-methyl-6-[2‘-(5-carboxy-1-butynyl)-5‘-methoxy]benzyl]pyrimidine (13), with an IC50 value of 0.65 nM against P. carinii DHFR, 0.57 nM against M. avium DHFR, and 55 nM against rat DHFR. The potency of 13 against P. carinii DHFR was 20-fold greater than that of PTX (IC50 = 13 nM), and its selectivity index (SI) relative to rat DHFR was 85, whereas PTX was nonselective. The activity of 13 against P. carinii DHFR was 20 000 times greater than that of TMP, with an SI of 96, whereas that of TMP was only 14. However 13 was no more potent than PTX against M. avium DHFR, and its SI was no better than that of TMP. Molecular modeling dynamics studies using compounds 10 and 13 indicated a slight binding preference for the latter, in qualitative agreement with the IC50 data. Among the pteridines, the most potent against P. carinii DHFR and M. avium DHFR was the 2‘-(5-carboxy-1-butynyl)dibenz[b,f]azepinyl derivative 20 (IC50 = 2.9 nM), whereas the most selective was the 2‘-(5-carboxy-1-pentynyl) analogue 21, with SI values of >100 against both P. carinii and M. avium DHFR relative to rat DHFR. The final compound, 2,4-diamino-5-[3‘-(4-carboxy-1-butynyl)-4‘-bromo-5‘-methoxybenzyl]pyrimidine (22), was both potent and selective against M. avium DHFR (IC50 = 0.47 nM, SI = 1300) but was not potent or selective against either P. carinii or T. gondii DHFR.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT2,4-Diaminothieno[2,3-d]pyrimidines as antifolates and antimalarials. 3. Synthesis of 5,6-disubstituted derivatives and related tetracyclic analogsA. Rosowsky, K. K. N. Chen, and M. LinCite this: J. Med. Chem. 1973, 16, 3, 191–194Publication Date (Print):March 1, 1973Publication History Published online1 May 2002Published inissue 1 March 1973https://doi.org/10.1021/jm00261a004Request reuse permissionsArticle Views304Altmetric-Citations37LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (509 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTQuinazolines. VI. Synthesis of 2,4-diaminoquinazolines from anthranilonitrilesAndre Rosowsky, James L. Marini, Marilyn E. Nadel, and Edward J. ModestCite this: J. Med. Chem. 1970, 13, 5, 882–886Publication Date (Print):September 1, 1970Publication History Published online1 May 2002Published inissue 1 September 1970https://doi.org/10.1021/jm00299a021RIGHTS & PERMISSIONSArticle Views248Altmetric-Citations29LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (665 KB) Get e-Alerts Get e-Alerts

We determined the mechanisms of resistance of human CCRF-CEM leukemia cells to methotrexate (MTX) vs. those to six novel antifolates: the polyglutamatable thymidylate synthase (TS) inhibitors ZD1694, multitargeted antifolate, pemetrexed, ALIMTA (MTA) and GW1843U89, the non-polyglutamatable inhibitors of TS, ZD9331, and dihydrofolate reductase, PT523, as well as DDATHF, a polyglutamatable glycinamide ribonucleotide transformylase inhibitor. CEM cells were made resistant to these drugs by clinically relevant intermittent 24 hr exposures to 5-10 microM of MTX, ZD1694, GW1843U89, MTA and DDATHF, by intermittent 72 hr exposures to 5 microM of ZD9331 and by continuous exposure to stepwise increasing concentrations of ZD9331, GW1843U89 and PT523. Development of resistance required only 3 cycles of intermittent drug exposure to ZD1694 and MTA, but 5 cycles for MTX, DDATHF and GW1843U89 and 8 cycles for ZD9331. The predominant mechanism of resistance to ZD1694, MTA, MTX and DDATHF was impaired polyglutamylation due to approximately 10-fold decreased folylpolyglutamate synthetase activity. Resistance to intermittent exposures to GW1843U89 and ZD9331 was associated with a 2-fold decreased transport via the reduced folate carrier (RFC). The CEM cell lines resistant to intermittent exposures to MTX, ZD1694, MTA, DDATHF, GW1843U89 and ZD9331 displayed a depletion (up to 4-fold) of total intracellular reduced folate pools. Resistance to continuous exposure to ZD9331 was caused by a 14-fold increase in TS activity. CEM/GW70, selected by continuous exposure to GW1843U89 was 50-fold resistant to GW1843U89, whereas continuous exposure to PT523 generated CEM/PT523 cells that were highly resistant (1550-fold) to PT523. Both CEM/GW70 and CEM/PT523 displayed cross-resistance to several antifolates that depend on the RFC for cellular uptake, including MTX (95- and 530-fold). CEM/GW70 cells were characterized by a 12-fold decreased transport of [3H]MTX. Interestingly, however, CEM/GW70 cells displayed an enhanced transport of folic acid, consistent with the expression of a structurally altered RFC resulting in a 2.6-fold increase of intracellular folate pools. CEM/PT523 cells displayed a markedly impaired (100-fold) transport of [3H]MTX along with 12-fold decreased total folate pools. In conclusion, multifunctional mechanisms of resistance in CEM cells have a differential impact on cellular folate homeostasis: decreased polyglutamylation and transport defects lead to folate depletion, whereas a structurally altered RFC protein can provoke expanded intracellular folate pools.

A structurally diverse library of 93 lipophilic di- and tricyclic diaminopyrimidine derivatives was tested for the ability to inhibit recombinant dihydrofolate reductase (DHFR) cloned from human and bovine isolates of Cryptosporidium parvum (J. R. Vásquez et al., Mol. Biochem. Parasitol. 79:153-165, 1996). In parallel, the library was also tested against human DHFR and, for comparison, the enzyme from Escherichia coli. Fifty percent inhibitory concentrations (IC(50)s) were determined by means of a standard spectrophotometric assay of DHFR activity with dihydrofolate and NADPH as the cosubstrates. Of the compounds tested, 25 had IC(50)s in the 1 to 10 microM range against one or both C. parvum enzymes and thus were not substantially different from trimethoprim (IC(50)s, ca. 4 microM). Another 25 compounds had IC(50)s of <1.0 microM, and 9 of these had IC(50)s of <0.1 microM and thus were at least 40 times more potent than trimethoprim. The remaining 42 compounds were weak inhibitors (IC(50)s, >10 microM) and thus were not considered to be of interest as drugs useful against this organism. A good correlation was generally obtained between the results of the spectrophotometric enzyme inhibition assays and those obtained recently in a yeast complementation assay (V. H. Brophy et al., Antimicrob. Agents Chemother. 44:1019-1028, 2000; H. Lau et al., Antimicrob. Agents Chemother. 45:187-195, 2001). Although many of the compounds in the library were more potent than trimethoprim, none had the degree of selectivity of trimethoprim for C. parvum versus human DHFR. Collectively, the results of these assays comprise the largest available database of lipophilic antifolates as potential anticryptosporidial agents. The compounds in the library were also tested as inhibitors of the proliferation of intracellular C. parvum oocysts in canine kidney epithelial cells cultured in folate-free medium containing thymidine (10 microM) and hypoxanthine (100 microM). After 72 h of drug exposure, the number of parasites inside the cells was quantitated by indirect immunofluorescence microscopy. Sixteen compounds had IC(50)s of <3 microM, and five of these had IC(50)s of <0.3 microM and thus were comparable in potency to trimetrexate. The finding that submicromolar concentrations of several of the compounds in the library could inhibit in vitro growth of C. parvum in host cells in the presence of thymidine (dThd) and hypoxanthine (Hx) suggests that lipophilic DHFR inhibitors, in combination with leucovorin, may find use in the treatment of intractable C. parvum infections.

Camptothecins are the only available antitumor agents which target the nuclear enzyme topoisomerase I. 9-Aminocamptothecin (9-AC) is a water-insoluble derivative of camptothecin which has demonstrated impressive antitumor activity in preclinical models. While two other water-soluble derivatives, CPT-11 and topotecan, have successfully completed Phase I and Phase II testing, biochemical and tissue culture studies suggest that camptothecin analogues differ in characteristics which may be important in determining antitumor activity. We performed a Phase I trial of 9-AC to determine the pharmacokinetics, dose-limiting toxicity, and maximum tolerated dose of this agent when administered as a 72-h continuous i.v. infusion. Thirty-one patients with resistant solid cancers received 5-60 microgram/m2/h 9-AC for 72 h, repeated at 3-week intervals. The drug was administered in a vehicle containing dimethylacetamide, polyethylene glycol, and phosphoric acid. Blood samples were collected and the lactone (closed ring) form of 9-AC was quantitated. The maximum tolerated dose of 9-AC was determined to be 45 microgram/m2/h. Dose-limiting toxicity consisted of neutropenia. Thrombocytopenia was also prominent. There were no significant nonhematological toxicities. Minimal responses were seen in patients with gastric, colon, and non-small cell lung cancer. Although significant interpatient variation in plasma 9-AC lactone levels was observed, pooled data were fit to a two-compartment model, with a terminal half-life of 36 h. Analyses of topoisomerase protein levels in peripheral blood cells indicated decreases in topoisomerase I accompanied by increases in topoisomerase II in two of three patients. 9-AC is an active antitumor agent and may be administered safely as a 72-h infusion in patients with cancer. Although Phase II trials with a 72-h infusion of 9-AC are warranted, alternate schedules should be evaluated given the dramatic preclinical activity seen with more prolonged administrations.

A systematic investigation of the properties of cerium fluoride monocrystals has been performed by the “Crystal Clear” collaboration in view of a p

Annals of the New York Academy of SciencesVolume 507, Issue 1 p. 252-271 Delivery of Lipophilic Drugs Using Lipoproteinsa J. MICHAEL SHAW, J. MICHAEL SHAW Alcon Laboratories, Inc. Fort Worth, Texas 76134-2099Search for more papers by this authorKALA V. SHAW, KALA V. SHAW Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorSAUL YANOVICH, SAUL YANOVICH Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorMICHAEL IWANIK, MICHAEL IWANIK Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorWILLIAM S. FUTCH, WILLIAM S. FUTCH Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorANDRÉ ROSOWSKY, ANDRÉ ROSOWSKY Dana-Farber Cancer Institute Boston, Massachusetts 02115Search for more papers by this authorLAWRENCE B. SCHOOK, LAWRENCE B. SCHOOK University of Illinois Urbana, Illinois 61801Search for more papers by this author J. MICHAEL SHAW, J. MICHAEL SHAW Alcon Laboratories, Inc. Fort Worth, Texas 76134-2099Search for more papers by this authorKALA V. SHAW, KALA V. SHAW Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorSAUL YANOVICH, SAUL YANOVICH Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorMICHAEL IWANIK, MICHAEL IWANIK Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorWILLIAM S. FUTCH, WILLIAM S. FUTCH Medical College of Virginia Richmond, Virginia 23298Search for more papers by this authorANDRÉ ROSOWSKY, ANDRÉ ROSOWSKY Dana-Farber Cancer Institute Boston, Massachusetts 02115Search for more papers by this authorLAWRENCE B. SCHOOK, LAWRENCE B. SCHOOK University of Illinois Urbana, Illinois 61801Search for more papers by this author First published: December 1987 https://doi.org/10.1111/j.1749-6632.1987.tb45806.xCitations: 41 a Supported by grants from the National Institutes of Health, National Cancer Institute, and the National Science Foundation. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL References 1 J. P. Segrest & J. J. Albers, Eds. 1986. Plasma lipoproteins, Part A, preparation, structure, and molecular biology. In Methods in Enzymology. Vol. 128. Academic Press. New York . 2 J. J. Albers & J. P. Segrest, Eds. 1986. Plasma lipoproteins, Part B, characterization, cell biology, and metabolism. In Methods in Enzymology. Vol. 129. Academic Press. New York . 3 Kreiger, M., L. Smith, R. Anderson, J. Goldstein, Y. Koa, H. Pownall, A. Gotto & M. Brown. 1979. Reconstituted low-density lipoprotein: A vehicle for the delivery of hydrophobic fluorescent probes in cells. J. Supramol. Struct. 10: 467– 478. 4 Remsen, J. & R. Shireman. 1981. Effect of low-density lipoprotein on the incorporation of benzo(a)pyrene by cultured cells. Cancer Res. 41: 3179– 3185. 5 Gal, D., M. OHashi, P. C. McDonald, H. J. Buchsbaum & E. R. Simpson. 1981. Low-density lipoprotein as a potential vehicle for chemotherapeutic agents and radionucleotides in the management of gynecologic neoplasms. Am J. Obstet. Gynecol. 139: 877– 885. 6 Mosley, S. T., J. L. Goldstein, M. S. Brown, J. R. Falck & R. G. Anderson. 1981. Targeted killing of cultured cells by receptor dependent photosensitization. Proc. Natl. Acad. Sci. 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Replacement of neutral lipids of low-density lipoprotein with esters of long-chain unsaturated fatty acids. J. Biol. Chem. 254: 3845– 3853. 17 Vitols, S. G., G. Gahrton & C. Peterson. 1984. Significance of low-density lipoprotein (LDL) receptor pathway for the in vitro accumulation of AD-32 incorporated into LDL in normal and leukemic white blood cells. Cancer Treat. Rep. 68: 515– 520. 18 Basu, S. K., J. L. Goldstein, R. G. Anderson & M. S. Brown. 1976. Degradation of cationized low-density lipoprotein and regulation of cholesterol metabolism in homozygous familial hypercholesterolemia fibroblasts. Proc. Natl. Acad. Sci. USA 73: 3178– 3182. 19 Goldstein, J. L. & M. S. Brown. 1977. The low-density lipoprotein pathway and its relation to atherosclerosis. Ann. Rev. Biochem. 46: 897– 930. 20 Mahley, R. W. & T. L. Innerarity. 1983. Lipoprotein receptors and cholesterol homeostasis. Biochim. Biophys. Acta 737: 197– 222. 21 Schneider, W. J., U. Beisiegel, J. L. Goldstein & M. S. Brown. 1982. Purification of the low-density lipoprotein receptor, an acidic glycoprotein of 164,000 molecular weight. J. Biol. Chem 257: 2664– 2673. 22 Goldstein, J. T., Y. K. Ho, S. K. Basu & M. S. Brown. 1979. Binding site on macrophages that mediates uptake and degradation of acetylated low-density lipoprotein producing massive cholesterol deposition. Proc. Natl. Acad. Sci. USA 76: 333– 337. 23 Brown, M. S. & J. L. Goldstein. 1983. Lipoprotein metabolism in the macrophage; implications for cholesterol deposition in atherosclerosis. Ann. Rev. Biochem. 52: 223– 261. 24 Nagelkerke, J. F., K. P. Barto & T. J. Van Berkel. 1983. In vivo and in vitro uptake and degradation of acetylated low-density lipoprotein by rat liver endothelial, Kupffer and Parenchymal cells. J. Biol. Chem. 258: 12,221– 12,227. 25 Via, D. P., H. A. Dresel, S. L. Cheng & A. M. Gotto. 1985. Murine macrophage tumors are a source of a 260,000-dalton acetyl-low-density lipoprotein receptor. J. Biol. Chem. 260: 7379– 7386. 26 Key, M., J. Talmadge, W. Fogler, C. Bucana & I. Fidler. 1982. Isolation of tumoricidal macrophages from lung melanoma metastases of mice treated systemically with liposomes containing a lipophilic derivative of muramyl dipeptide. J. Natl. Cancer Inst. 69: 1189– 1198. 27 Kleinerman, E., K. Erickson, A. Schroit, W. Fogler & I. Fidler. 1983. Activation of tumoricidal properties in human blood monocytes by liposomes containing lipophilic muramyl tripeptide. Cancer Res. 43: 2010– 2014. 28 Koff, W., S. Showalter, B. Hampar & I. Fidler. 1985. Protection of mice against fatal herpes simplex Type 2 infection by liposomes containing muramyl tripeptide. Science 228: 495– 497. 29 Spady, D. K., S. D. Turley & J. M. Dietschy. 1985. Receptor-independent low-density lipoprotein transport in the rat in vivo. J. Clin. Invest. 76: 1113– 1122. 30 Tabas, I. & A. R. Tall. 1984. Mechanism of the association of HDL, with endothelial cells, smooth muscle cells, and fibroblasts. J. Biol. Chem. 259: 13897– 13905. 31 Hoeg, J. M., J. C. Osborne & H. B. Brewer. 1982. Analysis of reversible lipoprotein-cell interactions. J. Biol. Chem. 257: 2125– 2128. 32 Chapman, M. J. 1986. Comparative analysis of mammalian plasma lipoproteins. In Methods of Enzymology. J. P. Segrest & J. J. Albers, Eds. 128: 70– 143. Academic Press. New York . 33 Heath, T., B. Macher & D. Papahadjopoulos. 1981. Covalent attachment of immunoglobulins to liposomes via glycosphingolipids. Biochim. Biophys. Acta 640: 66– 81. 34 Shaw, J. M., K. V. Shaw & L. B. Schook. 1987. Drug delivery particles and monoclonal antibodies. In Monoclonal Antibodies: Production, Techniques and Applications. L. B. Schook, Ed. 15: 285– 310. Marcel Dekker, Inc. New York . 35 Hynds, S. A., J. Welsh, J. M. Stewart, A. Jack, M. Soukop, C. S. McArdle, K. C. Calman, C. J. Packard & J. Sheperd. 1983. Low-density lipoprotein metabolism in mice with soft tissue tumors. Biochim. Biophys. Acta 795: 589– 595. 36 Adams, D. O. & T. A. Hamilton. 1984. The cell biology of macrophage activation. Ann. Rev. Immunol. 2: 283– 318. 37 Voyta, J. C., D. P. Via, C. E. Butterfield & B. R. Zetter. 1984. Identification and isolation of endothelial cells based on their increased uptake of acetylated-low-density lipoprotein. J. Cell Biol. 99: 2034– 2040. 38 Pitas, R. E., J. Boyles, R. W. Mahley & D. M. Bissell. 1985. Uptake of chemically modified low-density lipoproteins in vivo is mediated by specific endothelial cells. J. Cell Biol. 100: 103– 117. 39 Oldham, R. K. 1985. Biologicals and biological response modifiers: New approaches to cancer treatment. Cancer Invest. 3: 53– 70. 40 Parthosarathy, S., U. P. Steinbrecker, J. Barnett, J. L. Witztum & D. Steinberg. 1985. Essential role of phospholipase A2 activity in endothelial cell-induced modification of low-density lipoprotein. Proc. Natl. Acad. Sci. USA 82: 3000– 3004. 41 Plant, A. L., D. M. Benson & L. C. Smith. 1985. Cellular uptake and intracellular localization of Benzo(a)pyrene by digital fluorescence imaging microscopy. J. Cell Biol. 100: 1295– 1308. 42 Steinman, R., I. Mellman, W. Muller & Z. Cohn. 1983. Endocytosis and the recycling of plasma membrane. J. Cell Biol. 96: 1– 27. 43 Knott, T. J., S. C. Rall, T. L. Innerarity, S. F. Jacobson, M. S. Urdea, B. L. Wilson, L. M. Powell, R. J. Pease, R. Eddy, H. Nakai, M. Byers, L. M. Priestly, E. Robertson, L. B. Rall, C. Betsholtz, T. B. Shows, R. W. Mahley & J. Scott. 1985. Human apolipoprotein B: Structure of carboxyl-terminal domains, sites of gene expression, and chromosomal localization. Science 230: 37– 43. 44 Brown, M. S. & J. L. Goldstein. 1984. How LDL receptors influence cholesterol and atherosclerosis. Sci. Am. 251: 58– 66. 45 Shepherd, J. & C. J. Packard. 1986. Receptor-independent low-density lipoprotein catabolism. In Methods of Enzymology. J. J. Albers & J. P. Segrest, Eds. 129: 566– 590. Academic Press. New York . Citing Literature Volume507, Issue1Biological Approaches to the Controlled Delivery of DrugsDecember 1987Pages 252-271 ReferencesRelatedInformation

The serum half-life of 5-fluorouracil (5-FUra) in humans is best described as a biexponential decay function, with t1/2 alpha = 7.8 +/- 2.6 (S.E.) min and t1/2 beta = 36.8 +/- 13.5 min during initial courses of this drug alone. Pharmacokinetics of 5-FUra during courses of daily therapy (for 5 days) revealed prolongation of t1/2 in both components of the decay curve, which has not been previously reported. Despite the efficacy of thymidine (dThd) given as a continous i.v. infusion of 8 g/sq m/day in prevention of high-dose methotrexate toxicity, continuous infusion of dThd at this dose does not prevent the toxicity of 5-FUra orreverse inhibition of DNA and RNA synthesis by 5-fura. On the contrary, continuous infusion of dThd appears to increase the toxicity of 5-FUra during continuous dThd infusion revealed prolongation of the 5-FUra t1/2 which remained stable through the course of 5 days of 5-FUra with dThd. This protracted t1/2 is believed to account at least in part for the increased toxicity of 5-FUra with dThd. Dose-limiting mucositis, myelosuppression, and gastrointestinal toxicity were observed at 5-FUra doses ranging from one-half to two-thirds the customarily tolerated dose of 5-FUra alone in similar courses of daily bolus therapy (for 5 days).

Lipophilic 5'-(alkyl phosphate) esters of 1-beta-D-arabinofuranosylcytosine (ara-C) and several N4-acyl and 3'-O-acyl-2,2'-anhydro derivatives of ara-C were synthesized as potential prodrugs of ara-C 5'-monophosphate (ara-CMP). Alkylphosphorylation of ara-C, N4-palmitoyl-ara-C, and N4-stearoyl-ara-C was achieved in a single continuous operation by allowing the nucleoside to react with POCl3 in trimethyl or triethyl phosphate and adding the appropriate anhydrous alcohol directly to the intermediate phosphorodichloridate without isolation. Similar reaction of cytidine yielded cytidine 5'-(alkyl phosphate) esters, which on treatment with myristoyl or palmitoyl chloride in the presence of boron trifluoride gave 3'-O-acyl-2,2'-anhydro-ara-C 5'-(alkyl phosphate) esters. Ara-C 5'-(n-butyl phosphate) (1b), N4-palmitoyl-ara-C 5'-(n-butyl phosphate) (1h), and 2,2'-anhydro-3'-O-palmitoyl-ara-C 5'-(n-butyl phosphate) (2h) were tested against L1210/ara-C leukemia in mice in the hope that this kinase-deficient tumor would respond to treatment with these "prephosphorylated" derivatives, but no activity was observed. Of the simple 5'-(alkyl phosphate) esters tested in culture against l1210 leukemic cells, only ara-C 5'-(glyceryl phosphate) (1g) showed toxicity comparable to ara-CMP (ID50 = 0.35 and 0.65 microM, respectively), suggesting that beta-hydroxyalkyl phosphate esters may be worthwhile to examine further as prodrugs of ara-CMP.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of new chlorine-substituted derivatives of 2-tetraloneAndre Rosowsky, Josephine Battaglia, Katherine K. N. Chen, and Edward J. ModestCite this: J. Org. Chem. 1968, 33, 11, 4288–4290Publication Date (Print):November 1, 1968Publication History Published online1 May 2002Published inissue 1 November 1968https://doi.org/10.1021/jo01275a069Request reuse permissionsArticle Views289Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (477 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTQuinazolines. III. Synthesis of 1,3-Diaminobenzo[f]quinazoline and Related Compounds1-3Andre Rosowsky and Edward J. ModestCite this: J. Org. Chem. 1966, 31, 8, 2607–2613Publication Date (Print):August 1, 1966Publication History Published online1 May 2002Published inissue 1 August 1966https://doi.org/10.1021/jo01346a035Request reuse permissionsArticle Views251Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alertsclose Get e-Alerts

We present the results of beam tests with high-energy (8–375 GeV) electrons, pions, protons and muons of a sampling calorimeter based on the detection of Cherenkov light produced by shower particles. The detector, a prototype for the very forward calorimeters in the CMS experiment, consists of thin quartz fibers embedded in a copper matrix. Results are given on the light yield of this device, on its energy resolution for electron and hadron detection, and on the signal uniformity and linearity. The signal generation mechanism gives this type of detector unique properties, especially for the detection of hadron showers: narrow, shallow shower profiles and extremely fast signals. These specific properties were measured in detail. The implications for measurements in the high-rate, high-radiation Large Hadron Collider (LHC) environment are discussed.

This chapter discusses the structural changes that have been made in each of the above regions of methotrexate (MTX), the synthetic chemical methods used in preparing MTX analogs, and the fascinating structure–activity correlations made possible by the availability of these compounds. Because published biochemical and pharmacological data on MTX analogs are at this point extremely voluminous, the original literature should be consulted for additional details. Several attempts have been made to modify the pyrazine ring in MTX with the aim of gaining insight into the role that this region plays in the biological activity of the molecule. The effects on the biological activity of decreasing the size of the pyrazine ring in MTX and aminopterin (AMT) by one carbon, as in the purine analogs, are investigated.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTN.omega.-Alkoxycarbonylation of .alpha.,.omega.-diamino acids with 2-(trimethylsilyl)ethyl 4-nitrophenyl carbonateAndre Rosowsky and Joel E. WrightCite this: J. Org. Chem. 1983, 48, 9, 1539–1541Publication Date (Print):May 1, 1983Publication History Published online1 May 2002Published inissue 1 May 1983https://doi.org/10.1021/jo00157a034RIGHTS & PERMISSIONSArticle Views1031Altmetric-Citations32LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (476 KB) Get e-Alerts Get e-Alerts

n-Octyl, n-dodecyl, and n-hexadecyl alpha- and gamma-esters of methotrexate (MTX) were compared with the previously described alpha- and gamma-n-butyl esters and with MTX as inhibitors of dihydrofolate reductase (DHFR) and human leukemic lymphoblasts (CEM cells) in culture. The overall order of activity in both test systems was MTX greater than MTX gamma-esters greater than MTX alpha-esters. In the DHFR assay the activity of the alpha-esters followed the order C4 greater than C8 congruent to C12 greater than C16, whereas for the gamma-esters this order was C4 congruent to C8 greater than C12 greater than C16. On the other hand, the order of cytotoxic activity in culture in both series was C16 greater than C12 greater than C8 greater than C4. Increasing the alkyl chain length in the ester moiety therefore decreases DHFR affinity but increases cytotoxicity. The most potent member of the compounds tested was the gamma-n-hexadecyl ester, whose IC50 against CEM cells was 0.11 microM as compared with 0.025 microM for MTX. In a comparison of the effect of treatment with the gamma-n-hexadecyl ester (10(-5) M, 1 h) on DNA synthesis in CEM and CEM/MTX cells, the latter of which are 120-fold resistant to MTX by virtue of a transport defect, the ester produced only 4-fold less inhibition in the resistant line than in the parental line. These results suggest possible use of this compound or related derivatives in the treatment of MTX-resistant tumors with impaired transport.

Structural data are reported for two highly potent antifolates, 2,4-diamino-5-[3',4'-dimethoxy-5'-(5-carboxy-1-pentynyl)]benzylpyrimidine (PY1011), with 5000-fold selectivity for Pneumocystis carinii dihydrofolate reductase (pcDHFR), relative to rat liver DHFR, and 2,4-diamino-5-[2-methoxy-5-(4-carboxybutyloxy)benzyl]pyrimidine (PY957), that has 80-fold selectivity for pcDHFR. Crystal structures are reported for NADPH ternary complexes with PY957 and pcDHFR, refined to 2.2 A resolution; with PY1011 and pcDHFR, refined to 2.0 A resolution; and with PY1011 and mouse DHFR (mDHFR), refined to 2.2 A resolution. These results reveal that the carboxylate of the omega-carboxyalkyloxy side chain of these inhibitors form ionic interactions with the conserved Arg in the substrate binding pocket of DHFR. These data suggest that the enhanced inhibitory activity of PY1011 compared with PY957 is, in part, due to the favorable contacts with Phe69 of pcDHFR by the methylene carbons of the inhibitor side chain that are oriented by the triple bond of the 1-pentynyl side chain. These contacts are not present in the PY957 pcDHFR complex, or in the PY1011 mDHFR complex. In the structure of mDHFR the site of Phe69 in pcDHFR is occupied by Asn64. These data also revealed a preference for an unusual parallel ring stacking interaction between Tyr35 of the active site helix and Phe199 of the C-terminal beta sheet in pcDHFR and by Tyr33 and Phe179 in mDHFR that is independent of bound ligand. A unique His174-His187 parallel ring stacking interaction was also observed only in the structure of pcDHFR. These ring stacking interactions are rarely found in any other protein families and may serve to enhance protein stability.

Abstract We have recently explored the cytokinetic effects of thymidine given by continuous infusion in an animal model and have demonstrated an arrest of cell cycle traverse in rapidly proliferating tissues. Similar biologic effects have been observed in clinical trials upon infusing thymidine at a dose rate of 75 g/sq m/day. Fifteen courses of at least 5 days in duration have been administered to 11 patients with either leukemia or lymphoma. Steady-state serum thymidine levels were achieved in the range of 1-2 mM and the serum half-life of thymidine was approximately 90 min upon completion of the infusion. The associated toxicity included myelosuppression, headache, anorexia, nausea, vomiting, and diarrhea. The antitumor effect was dependent on the disease being treated and the percentage of blasts in the peripheral blood. Three patients with AML and three patients with T-cell leukemia responded to the thymidine infusions with an abrupt decrease in peripheral blast count. In contrast, no response was observed in two patients with poorly differentiated lymphocytic lymphoma of B-cell origin in a leukemic phase. One patient with T-cell leukemia had a marked clinical response with resolution of hepatosplenomegaly and another patient with the same diagnosis had partial clearing of the bone marrow. The cytokinetic effect on the bone marrow or peripheral blasts was monitored by microfluorimetry and labeling with precursors to monitor DNA synthesis. These data indicate an arrest of DNA synthesis during the period of thymidine infusion. The clinical effects observed with the high-dose thymidine infusions are correlated with measurements of thymidine kinase and phosphorylase levels detected in the blasts obtained from the leukemic patients. The relative ratios of thymidine kinase to phosphorylase are predictive of cellular sensitivity to the cytokinetic effects of thymidine.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 2. Facile method of preparation of lipophilic derivatives of methotrexate and 3',5'-dichloromethotrexate by direct esterificationA. RosowskyCite this: J. Med. Chem. 1973, 16, 10, 1190–1193Publication Date (Print):October 1, 1973Publication History Published online1 May 2002Published inissue 1 October 1973https://doi.org/10.1021/jm00268a029RIGHTS & PERMISSIONSArticle Views138Altmetric-Citations26LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (582 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPyrimido[4,5-c]isoquinolines. 2. Synthesis and biological evaluation of some 6-alkyl-, 6-aralkyl-, and 6-aryl-1,3-diamino-7,8,9,10-tetrahydropyrimido[4,5-c]isoquinolines as potential folate antagonistsAndre Rosowsky and Nickolas PapathanasopoulosCite this: J. Med. Chem. 1974, 17, 12, 1272–1276Publication Date (Print):December 1, 1974Publication History Published online1 May 2002Published inissue 1 December 1974https://doi.org/10.1021/jm00258a008Request reuse permissionsArticle Views334Altmetric-Citations27LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (588 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 3. Synthesis and biological properties of some side-chain altered analogsMichael Chaykovsky, Andre Rosowsky, Nickolas Papathanasopoulos, Katherine K. N. Chen, Edward J. Modest, Roy L. Kisliuk, and Yvette GaumontCite this: J. Med. Chem. 1974, 17, 11, 1212–1216Publication Date (Print):November 1, 1974Publication History Published online1 May 2002Published inissue 1 November 1974https://doi.org/10.1021/jm00257a015RIGHTS & PERMISSIONSArticle Views381Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (669 KB) Get e-Alerts Get e-Alerts

Calculations by the Hückel Molecular Orbital method have been made on ten non-classical aromatics, most of them non-alternant. Considerable resonance stabilization is predicted for all the systems studied. Except for charge distribution in the non-alternants, the calculated properties are mostly similar to those of normal benzenoid aromatics. The application to our systems of Craig's rules for predicting aromaticity is discussed.

As part of a larger search for potent as well as selective inhibitors of dihydrofolate reductase (DHFR) enzymes from opportunistic pathogens found in patients with AIDS and other immune disorders, N-[(2,4-diaminopteridin-6-yl)methyl]dibenz[b,f]azepine (4a) and the corresponding dihydrodibenz[b,f]azepine, dihydroacridine, phenoxazine, phenothiazine, carbazole, and diphenylamine analogues were synthesized from 2, 4-diamino-6-(bromomethyl)pteridine in 50-75% yield by reaction with the sodium salts of the amines in dry tetrahydrofuran at room temperature. The products were tested for the ability to inhibit DHFR from Pneumocystis carinii (pcDHFR), Toxoplasma gondii (tgDHFR), Mycobacterium avium (maDHFR), and rat liver (rlDHFR). The member of the series with the best combination of potency and species selectivity was 4a, with IC(50) values against the four enzymes of 0. 21, 0.043, 0.012, and 4.4 microM, respectively. The dihydroacridine, phenothiazine, and carbazole analogues were also potent, but nonselective. Of the compounds tested, 4a was the only one to successfully combine the potency of trimetrexate with the selectivity of trimethoprim. Molecular docking simulations using published 3D structural coordinates for the crystalline ternary complexes of pcDHFR and hDHFR suggested a possible structural interpretation for the binding selectivity of 4a and the lack of selectivity of the other compounds. According to this model, 4a is selective because of a unique propensity of the seven-membered ring in the dibenz[b,f]azepine moiety to adopt a puckered orientation that allows it to fit more comfortably into the active site of the P. carinii enzyme than into the active site of the human enzyme. Compound 4a was also evaluated for the ability to be taken up into, and retard the growth of, P. carinii and T. gondii in culture. The IC(50) of 4a against P. carinii trophozoites after 7 days of continuous drug treatment was 1.9 microM as compared with previously observed IC(50) values of >340 microM for trimethoprim and 0.27 microM for trimetrexate. In an assay involving [(3)H]uracil incorporation into the nuclear DNA of T. gondii tachyzoites as the surrogate endpoint for growth, the IC(50) of 4a after 5 h of drug exposure was 0.077 microM. The favorable combination of potency and enzyme selectivity shown by 4a suggests that this novel structure may be an interesting lead for structure-activity optimization.

ABSTRACT Competitive inhibitors of dihydrofolate reductase (DHFR) are used in chemotherapy or prophylaxis of many microbial pathogens, including the eukaryotic parasites Plasmodium falciparum and Toxoplasma gondii . Unfortunately, point mutations in the DHFR gene can confer resistance to inhibitors specific to these pathogens. We have developed a rapid system for testing inhibitors of DHFRs from a variety of parasites. We replaced the DHFR gene from the budding yeast Saccharomyces cerevisiae with the DHFR-coding region from humans, P. falciparum , T. gondii , Pneumocystis carinii , and bovine or human-derived Cryptosporidium parvum . We studied 84 dicyclic and tricyclic 2,4-diaminopyrimidine derivatives in this heterologous system and identified those most effective against the DHFR enzymes from each of the pathogens. Among these compounds, six tetrahydroquinazolines were effective inhibitors of every strain tested, but they also inhibited the human DHFR and were not selective for the parasites. However, two quinazolines and four tetrahydroquinazolines were both potent and selective inhibitors of the P. falciparum DHFR. These compounds show promise for development as antimalarial drugs.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 26. Inhibition of dihydrofolate reductase and folylpolyglutamate synthetase activity and in vitro tumor cell growth by methotrexate and aminopterin analogs containing a basic amino acid side chainAndre Rosowsky, James H. Freisheim, Richard G. Moran, Vishnu C. Solan, Henry Bader, Joel E. Wright, and Mary Radike-SmithCite this: J. Med. Chem. 1986, 29, 5, 655–660Publication Date (Print):May 1, 1986Publication History Published online1 May 2002Published inissue 1 May 1986https://doi.org/10.1021/jm00155a012RIGHTS & PERMISSIONSArticle Views459Altmetric-Citations35LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (921 KB) Get e-Alerts Get e-Alerts

gamma-tert-Butylaminopterin (gamma-tBAMT), the first example of an aminopterin (AMT) gamma-monoester, was synthesized, and new routes to the known N10-methyl analogue gamma-tert-butyl methotrexate (gamma-tBMTX) were developed. The inhibitory effects of gamma-tBAMT on the activity of purified dihydrofolate reductase (DHFR) from L1210 murine leukemia cells, the growth of L1210 cells and CEM human leukemic lymphoblasts in suspension culture, and the growth of several lines of human squamous cell carcinoma of the head and neck in monolayer culture were compared with the effects of gamma-tBMTX and the parent acids AMT and methotrexate (MTX). Patterns of cross-resistance to gamma-tBAMT, gamma-tBMTX, and AMT among several MTX-resistant cell lines were examined. In vivo antitumor activities of gamma-tBAMT and gamma-tBMTX were compared in mice with L1210 leukemia. While the activity of gamma-tBAMT was very close to that of gamma-tBMTX in the DHFR inhibition assay, the AMT ester was more potent than the MTX ester against cells in culture and against L1210 leukemia in vivo. Only partial cross-resistance was shown against gamma-tBMTX and gamma-tBAMT in cultured cells that were resistant to MTX by virtue of a transport defect or a combination of defective transport and elevated DHFR activity.

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.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 20. Replacement of glutamate by longer-chain amino diacids: Effects on dihydrofolate reductase inhibition, cytotoxicity, and in vivo antitumor activityAndre Rosowsky, Ronald Forsch, Jack Uren, Michael Wick, A. Ashok Kumar, and James H. FreisheimCite this: J. Med. Chem. 1983, 26, 12, 1719–1724Publication Date (Print):December 1, 1983Publication History Published online1 May 2002Published inissue 1 December 1983https://doi.org/10.1021/jm00366a012Request reuse permissionsArticle Views283Altmetric-Citations29LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (889 KB) Get e-Alertsclose Get e-Alerts

In a continuing effort to design small-molecule inhibitors of dihydrofolate reductase (DHFR) that combine the enzyme-binding selectivity of 2,4-diamino-5-(3',4',5'-trimethoxybenzyl)pyrimidine (trimethoprim, TMP) with the potency of 2,4-diamino-5-methyl-6-(2',5'-dimethoxybenzyl)pyrido[2,3-d]pyrimidine (piritrexim, PTX), seven previously undescribed 2,4-diamino-5-[2'-methoxy-5'-(substituted benzyl)]pyrimidines were synthesized in which the substituent at the 5'-position was a carboxyphenyl group linked to the benzyl moiety by a bridge of two or four atoms in length. The new analogues were all obtained from 2,4-diamino-5-(5'-iodo-2'-methoxybenzyl)pyrimidine via a Sonogashira reaction, followed, where appropriate, by catalytic hydrogenation. The new analogues were tested as inhibitors of DHFR from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three life-threatening pathogens often found in AIDS patients and individuals whose immune system is impaired as a result of treatment with immunosuppressive chemotherapy or radiation. The selectivity index (SI) of each compound was obtained by dividing its 50% inhibitory concentration (IC(50)) against Pc, Tg, or Ma DHFR by its IC(50) against rat DHFR. 2,4-Diamino-[2'-methoxy-5'-(3-carboxyphenyl)ethynylbenzyl]pyrimidine (28), with an IC(50) of 23 nM and an SI of 28 in the Pc DHFR assay, had about the same potency as PTX and was 520 times more potent than TMP. As an inhibitor of Tg DHFR, 28 had an IC(50) of 5.5 nM (510-fold lower than that of TMP and similar to that of PTX) and an SI value of 120 (2-fold better than TMP and vastly superior to PTX). Against Ma DHFR, 28 had IC(50) and SI values of 1.5 nM and 430, respectively, compared with 300 nM and 610 for TMP. Although it had 2.5-fold lower potency than 28 against Ma DHFR (IC(50) = 3.7 nM) and was substantially weaker against Pc and Tg DHFR, 2,4-diamino-[2'-methoxy-5'-(4-carboxyphenyl)ethynylbenzyl]pyrimidine (29), with the carboxy group at the para rather than the meta position, displayed 2200-fold selectivity against the Ma enzyme and was the most selective 2,4-diamino-5-(5'-substituted benzyl)pyrimidine inhibitor of this enzyme we have encountered to date. Additional bioassay data for these compounds are also reported.

We propose to use a 13 keV antiproton beam passing through a dense cloud of positronium (Ps) atoms to produce an H¯+ “beam”. These ions can be slowed down and captured by a trap. The process involves two reactions with large cross-sections under the same experimental conditions. These reactions are the interaction of p¯ with Ps to produce H¯ and the e+ capture by H¯ reacting on Ps to produce H¯+. Once decelerated with an electrostatic field and captured in a trap, the H¯+ ions could be cooled and the e+ removed with a laser to perform a measurement of the gravitational acceleration of neutral antimatter in the gravity field of the Earth.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 10. Direct coupling of methotrexate and diethyl L-glutamate in the presence of peptide bond-forming reagentsAndre Rosowsky and Cheng-Sein YuCite this: J. Med. Chem. 1978, 21, 2, 170–175Publication Date (Print):February 1, 1978Publication History Published online1 May 2002Published inissue 1 February 1978https://doi.org/10.1021/jm00200a005RIGHTS & PERMISSIONSArticle Views218Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (959 KB) Get e-Alerts Get e-Alerts

Nineteen previously undescribed 2,4-diamino-6-(arylmethyl)-5,6,7, 8-tetrahydroquinazolines (5a-m, 10-12) were synthesized as part of a larger effort to assess the therapeutic potential of lipophilic dihydrofolate reductase (DHFR) inhibitors against opportunistic infections of AIDS. Condensation of appropriately substituted (arylmethyl)triphenylphosphoranes with 4, 4-ethylenedioxycyclohexanone, followed by hydrogenation (H2/Pd-C) and acidolysis, yielded the corresponding 4-(arylmethyl)cyclohexanones, which were then condensed with cyanoguanidine to form the tetrahydroquinazolines. Three simple 2, 4-diamino-6-alkyl-5,6,7,8-tetrahydroquinazoline model compounds (9a-c) were also prepared in one step from commercially available 4-alkylcyclohexanones by this method. Enzyme inhibition assays against rat liver DHFR, Pneumocystis carinii DHFR, and the bifunctional DHFR-TS enzyme from Toxoplasma gondii were carried out, and the selectivity ratios IC50(rat)/IC50(P. carinii) and IC50(rat)/IC50(T. gondii) were compared. The three most potent inhibitors of P. carinii DHFR were the 2,5-dimethoxybenzyl (5j), 3, 4-dimethoxybenzyl (5k), and 3,4,5-trimethoxybenzyl (5l) analogues, with IC50 values of 0.057, 0.10, and 0.091 microM, respectively. The remaining compounds generally had IC50 values in the 0.1-1.0 microM range. However all the compounds were more potent against the rat liver enzyme than the P. carinii enzyme and thus were nonselective. The T. gondii enzyme was always more sensitive than the P. carinii enzyme, with most of the analogues giving IC50 values of 0.01-0.1 microM. Moderate 5-10-fold selectivity for T. gondii versus rat liver DHFR was observed with five compounds, the best combination of potency and selectivity being achieved with the 2-methoxybenzyl analogue 5d, which had an IC50 of 0.014 microM and a selectivity ratio of 8.6. One compound (5l) was tested for antiproliferative activity against P. carinii trophozoites in culture at a concentration of 10 microgram/mL and was found to completely suppress growth over 7 days. The suppressive effect of 5l was the same as that of trimethoprim (10 microgram/mL) + sulfamethoxazole (250 microgram/mL), a standard clinical combination for the treatment of P. carinii pneumonia in AIDS patients. Four compounds (5a,h,k,l) were tested against T. gondii tachyzoites in culture and were found to have a potency (IC50 = 0.1-0.5 microM) similar to that of pyrimethamine (IC50 = 0.69 microM), a standard clinical agent for the treatment of cerebral toxoplasmosis in AIDS patients. Compound 5h was also active against T. gondii infection in mice when given qdx8 by peritoneal injection at doses ranging from 62.5 (initial dose) to 25 mg/kg. Survival was prolonged to the same degree as with 25 mg/kg clindamycin, another widely used drug against toxoplasmosis. Three compounds (5j-l) were tested for antiproliferative activity against human tumor cells in culture. Among the 25 cell lines in the National Cancer Institute panel for which data were confirmed in two independent experiments, the IC50 for at least two of these compounds was <10 microM against 17 cell lines (68%) and in the 0. 1-1 microM range against 13 cell lines (52%). One compound (5j) had an IC50 of <0.01 microM against four of the cell lines. The activity profiles of 5k,l were generally similar to that of 5j except that there were no cells against which the IC50 was <0.01 microM.

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.

ABSTRACT There is a pressing need for drugs effective against the opportunistic protozoan pathogen Cryptosporidium parvum . Folate metabolic enzymes and enzymes of the thymidylate cycle, particularly dihydrofolate reductase (DHFR), have been widely exploited as chemotherapeutic targets. Although many DHFR inhibitors have been synthesized, only a few have been tested against C. parvum . To expedite and facilitate the discovery of effective anti- Cryptosporidium antifolates, we have developed a rapid and facile method to screen potential inhibitors of C. parvum DHFR using the model eukaryote, Saccharomyces cerevisiae . We expressed the DHFR genes of C. parvum , Plasmodium falciparum , Toxoplasma gondii , Pneumocystis carinii , and humans in the same DHFR-deficient yeast strain and observed that each heterologous enzyme complemented the yeast DHFR deficiency. In this work we describe our use of the complementation system to screen known DHFR inhibitors and our discovery of several compounds that inhibited the growth of yeast reliant on the C. parvum enzyme. These same compounds were also potent or selective inhibitors of the purified recombinant C. parvum DHFR enzyme. Six novel lipophilic DHFR inhibitors potently inhibited the growth of yeast expressing C. parvum DHFR. However, the inhibition was nonselective, as these compounds also strongly inhibited the growth of yeast dependent on the human enzyme. Conversely, the antibacterial DHFR inhibitor trimethoprim and two close structural analogs were highly selective, but weak, inhibitors of yeast complemented by the C. parvum enzyme. Future chemical refinement of the potent and selective lead compounds identified in this study may allow the design of an efficacious antifolate drug for the treatment of cryptosporidiosis.

We present the results of experimental studies of hadron showers in a copper/quartz-fiber calorimeter, based on the detection of Cherenkov light. These studies show that there are very significant differences between the signals from protons and pions at the same energies. In the energy range between 200 and 375 GeV, where these studies were performed, the calorimeter's response to protons was typically 10% smaller than the response to pions. On the other hand, the energy resolution was about 25% better for protons. In addition, the protons had a Gaussian line shape, whereas the pion response curve was asymmetric. These differences can be understood from the requirements of baryon number conservation in the shower development. They are expected to be present in any non-compensating calorimeter, to a degree determined by the e/h value.

Ten heretofore undescribed 2,4-diamino-5-chloroquinazoline analogues of trimetrexate (TMQ) and piritrexim (PTX) were synthesized and tested as inhibitors of dihydrofolate reductase (DHFR) from rat liver, Pneumocystis carinii, and Toxoplasma gondii. The most active quinazolines against both the P. carinii and the T. gondii enzyme were those with an ArCH2-NH or ArNHCH2 side chain. Among ArNH(CH2)n compounds with n = 1-3 and either 2',5'-dimethoxyphenyl or 3',4',5'-trimethoxyphenyl as the Ar moiety, activity decreased in the order n = 1 > n = 2 > n = 3. The best inhibitor of P. carinii DHFR, 2,4-diamino-5-chloro-6-[(N-methyl-3',4',5'-trimethoxyanilino)methy l] quinazoline (10) had an IC50 of 0.012 microM and was slightly more potent than TMQ and PTX. Compound 10 was also the best inhibitor of T. gondii DHFR, with an IC50 of 0.0064 microM corresponding again to a minor increase in activity over TMQ and PTX. However, as with these standard agents, 10 showed no appreciable selectivity for either the P. carinii or T. gondii enzyme relative to the rat liver enzyme. The highest selectivity achieved in this limited series was with 2,4-diamino-5-chloro-6-[N-(3',4',5'-trimethoxybenzyl)-N-methylamino] quinazoline (17) against T. gondii DHFR. While 17 (IC50 = 0.016 microM) was somewhat less potent than 10, its selectivity, as defined by the ratio IC50(rat liver)/IC50(T. gondii) was ca. 30-fold higher than that of TMQ or PTX. Two compounds, 2,4-diamino-5-chloro-6-[(3',4',5'-trimethoxyanilino)methyl] quinazoline (9) and 2,4-diamino-5-chloro-6-[N-(3',4',5'-trimethoxybenzyl) amino]quinazoline (15), were also tested against human DHFR and were found to have an IC50/[E] of 0.5, indicating that their binding was near-stoichiometric.

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.

We have examined the metabolism of the folate analog methotrexate (MTX) in the human parasite Leishmania major.These cells readily hydrolyzed MTX to N-lO-methyl-4-deoxy-4-aminopteroate (MAPA), such that following a 24-h incubation in 1 PM [3H]MTX approximately 30% of the cell-associated radioactivity was MAPA.MAPA also accumulated in the culture medium, exceeding the concentration of MTX after 24 h.Neither 7-hydroxy-methotrexate nor MTX polyglutamates were observed in cells or medium, even after a 72-h incubation with MTX.In contrast to MTX, folate is extensively polyglutamylated in L. major

Six previously undescribed N-(2,4-diaminopteridin-6-yl)methyldibenz[b,f]azepines with water-solubilizing O-carboxyalkyloxy or O-carboxybenzyloxy side chains at the 2'-position were synthesized and compared with trimethoprim (TMP) and piritrexim (PTX) as inhibitors of dihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc), Toxoplasma gondii (Tg), and Mycobacterium avium (Ma), three of the opportunistic organisms known to cause significant morbidity and mortality in patients with AIDS and other disorders of the immune system. The ability of the new analogues to inhibit reduction of dihydrofolate to tetrahydrofolate by Pc, Tg, Ma, and rat DHFR was determined, and the selectivity index (SI) was calculated from the ratio IC(50)(rat DHFR)/IC(50)(Pc, Tg, or Ma DHFR). The IC(50) values of the 2'-O-carboxypropyl analogue (10), with SI values in parentheses, were 1.1 nM (1300) against Pc DHFR, 9.9 nM (120) against Tg DHFR, and 2.0 nM (600) against Ma DHFR. The corresponding values for the 2'-O-(4-carboxybenzyloxy) analogue (12) were 1.0 nM (560), 22 nM (21), and 0.75 nM (630). By comparison, the IC(50) and SI values for TMP were Pc, 13 000 nM (14); Tg, 2800 nM (65); and Ma, 300 nM (610). For the prototypical potent but nonselective inhibitors PTX and TMX, respectively, these values were Pc, 13 nM (0.26) and 47 nM (0.17); Tg, 4.3 nM (0.76) and 16 nM (0.50); Ma, 0.61 nM (5.4) and 1.5 nM (5.3). Thus 10 and 12 met the criterion for DHFR inhibitors that combine the high selectivity of TMP with the high potency of PTX and TMX.

A new fluorescent methotrexate analogue (PT430) was synthesized as a reported ligand for dihydrofolate reductase. The analogue was prepared by attachment of lysine in place of the glutamate side chain of methotrexate and conjugation to fluorescein isothiocyanate via the epsilon-amino group of lysine. Spectrophotometric enzyme inhibition assays showed PT430 to be about one-tenth as potent as methotrexate against either Lactobacillus casei or L1210 mouse leukemia enzyme; competitive radioligand binding assays using tritiated methotrexate gave similar results. In assays of L1210 cell proliferation in culture, on the other hand, PT430 was 100-fold less toxic than methotrexate. In dilute solution, the fluorescence intensity of PT430 was 5-fold lower than that of equimolar fluorescein and diminished with decreasing pH. On complexation with dihydrofolate reductase, however, fluorescence intensity was enhanced 3- to 5-fold depending on the pH. Measurement of fluorescence increase with added ligand provided data for the determination of the stoichiometric ratio, dissociation constant, and extent of fluorescence enhancement. Specificity of PT430 for methotrexate binding sites was indicated by the observation of decreased fluorescence uptake in PT430-treated L1210 cells in the presence of methotrexate. Fluorescence uptake occurred faster, and to a greater extent, in methotrexate-resistant dihydrofolate reductase overproducing L1210/R6 cells than in the methotrexate-sensitive parent line. Therefore, PT430 may be used as a flow cytometry probe to detect methotrexate resistance based on dihydrofolate reductase overproduction.

Carbodiimide-mediated condensation of 4-amino-4-deoxy-N10-methylpteroic acid (APA) with several alkyl, aralkyl, and aryl amines, in the presence or absence of N-hydroxysuccinimide, was employed in order to prepare new lipid-soluble bis(amide) derivatives of methotrexate (MTX) as potential prodrugs. MTX dianilide was likewise prepared, in comparable yield, from APA and L-glutamic acid dianilide via the mixed carboxylic--carbonic anhydride method. Dihydrazide and bis(N-methylhydrazide) derivatives of MTX were formed readily from MTX diethyl ester. However, reaction with hydroxylamine led to MTX gamma-monohydroxamic acid as the sole isolated product. The bis adduct appears to form, but is unstable during workup. The identity of the product was confirmed by independent mixed anhydride synthesis from APA and the gamma-monohydroxamate of L-glutamic acid. Treatment of MTX dimethyl ester with N,N-dimethylhydrazine unexpectedly yielded MTX gamma-monomethyl ester. MTX dianilide was active against L1210 leukemia in mice, with a +155% increase in life span at a dose of 160 mg/kg given ip in 10% Tween 80 on a q3d X 3 schedule. The bis(p-chlorobenzylamide), bis(p-methoxybenzylamide), and dihydrazide were also active against L1210 leukemia in vivo, but to a lesser extent than the dianilide. The gamma-monohydroxamic acid derivative showed activity (+111% ILS at 40 mg/kg) similar to that of MTX and was found to bind to a partially purified dihydrofolate reductase preparation from L1210 cells with an ID50 of 0.005 microM as compared to 0.007 microM for MTX. In vivo experiments in mice indicated that the pharmacokinetic properties of this compound and of MTX are similar but failed to demonstrate any advantage over MTX in terms of selective uptake into tumor (sc implanted P388 leukemia) or improved penetration of the central nervous system. The activities of the dianilide, bis(benzylamide), and dihydrazide derivatives in vivo are of interest in view of their low toxicity relative to MTX against cells in culture, which suggests that these derivatives are probably acting as prodrugs in the intact animal.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTPteridines. I. .beta.-Ketosulfoxides and .alpha.-ketoaldehyde hemithioacetals as pteridine precursors. New selective synthesis of 6- and 7-substituted pteridinesAndre Rosowsky and Katherine K. N. ChenCite this: J. Org. Chem. 1973, 38, 11, 2073–2077Publication Date (Print):June 1, 1973Publication History Published online1 May 2002Published inissue 1 June 1973https://doi.org/10.1021/jo00951a022RIGHTS & PERMISSIONSArticle Views213Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (773 KB) Get e-Alerts Get e-Alerts

To understand the role of specific active site residues in conferring selective dihydrofolate reductase (DHFR) inhibition from pathogenic organisms such as Pneumocystis carinii (pc) or Pneumocystis jirovecii (pj), the causative agent in AIDS pneumonia, it is necessary to evaluate the role of these residues in the human enzyme. We report the first kinetic parameters for DHFR from pjDHFR and pcDHFR with methotrexate (MTX), trimethoprim (TMP), and its potent analogue, PY957. We also report the mutagenesis and kinetic analysis of active site mutant proteins at positions 35 and 64 of human (h) DHFR and the crystal structure determinations of hDHFR ternary complexes of NADPH and PY957 with the wild-type DHFR enzyme, the single mutant protein, Gln35Lys, and two double mutant proteins, Gln35Ser/Asn64Ser and Gln35Ser/Asn64Phe. These substitutions place into human DHFR amino acids found at those sites in the opportunistic pathogens pcDHFR (Q35K/N64F) and pjDHFR (Q35S/N64S). The Ki inhibition constant for PY957 showed greatest potency of the compound for the N64F single mutant protein (5.2 nM), followed by wild-type pcDHFR (Ki 22 nM) and then wild-type hDHFR enzyme (Ki 230 nM). Structural data reveal significant conformational changes in the binding interactions of PY957 in the hDHFR Q35S/N64F mutant protein complex compared to the other hDHFR mutant protein complexes and the pcDHFR ternary complex. The conformation of PY957 in the wild-type DHFR is similar to that observed for the single mutant protein. These data support the hypothesis that the enhanced selectivity of PY957 for pcDHFR is in part due to the contributions at positions 37 and 69 (pcDHFR numbering). This insight will help in the design of more selective inhibitors that target these opportunistic pathogens.

Journal of Heterocyclic ChemistryVolume 7, Issue 1 p. 197-200 Note [1] Benzopyrano[3,4-d] pyrimidines and [1] benzothiopyrano[3,4-d] pyrimidines. Two new heterocyclic ring systems † Andre Rosowsky, Andre Rosowsky The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this authorPing C. Huang, Ping C. Huang The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this authorEdward J. Modest, Edward J. Modest The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this author Andre Rosowsky, Andre Rosowsky The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this authorPing C. Huang, Ping C. Huang The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this authorEdward J. Modest, Edward J. Modest The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School, Boston, Massachusetts 02115Search for more papers by this author First published: February 1970 https://doi.org/10.1002/jhet.5570070131Citations: 17 † This is publication No. 701 from the Army Research Program on Malaria. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinked InRedditWechat Citing Literature Volume7, Issue1February 1970Pages 197-200 RelatedInformation

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and Properties of Bicyclic Oxetanes1A. ROSOWSKY and D. S. TARBELLCite this: J. Org. Chem. 1961, 26, 7, 2255–2260Publication Date (Print):July 1, 1961Publication History Published online1 May 2002Published inissue 1 July 1961https://doi.org/10.1021/jo01351a026RIGHTS & PERMISSIONSArticle Views204Altmetric-Citations15LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (755 KB) Get e-Alerts Get e-Alerts

Ten previously unreported 2,4-diaminothieno[2,3-d]pyrimidine lipophilic dihydrofolate reductase inhibitors were synthesized as potential inhibitors of Pneumocystis carinii and Toxoplasma gondii dihydrofolate reductase. Pivaloylation of 2,4-diamino-5-methylthieno[2,3-d]pyrimidine followed by dibromination with N-bromosuccinimide in the presence of benzoyl peroxide gave 2,4-bis(pivaloylamino)-6-bromo-5-(bromomethyl)thieno[2,3-d]pyrimidine, which after condensation with substituted anilines or N-methylanilines and deprotection with base yielded 2,4-diamino-6-bromo-5-[(substituted anilino)methyl]thieno[2,3-d]pyrimidines. Removal of the 6-bromo substituent was accomplished with sodium borohydride and palladium chloride. The reaction yields were generally good to excellent. The products were tested as inhibitors of dihydrofolate reductase (DHFR) from P. carinii, T. gondii, and rat liver. Although the IC50 could not be reached for the 6-unsubstituted compounds because of their extremely poor solubility, three of the five 6-bromo derivatives were soluble enough to allow the IC50 to be determined against all three enzymes. 2,4-Diamino-5-[3,5-dichloro-4-(1-pyrrolo)anilino]methyl]-6-bromothieno[2,3-d]pyrimidine was the most active of the 6-bromo derivatives, with an IC50 of 7.5 μM against P. carinii DHFR, but showed no selectivity for either P. carinii or T. gondii DHFR relative to the enzyme from rat liver.

Twenty-eight 2,4-diaminopteridines with alkyl and aralkyl groups at the 6- and 7-positions, five 1,3-diamino-7,8,9,10-tetrahydropyrimido [4,5-c]isoquinolines with an alkyl, alkylthio, or aryl group at the 6-position, and nine 4,6-diamino-1,2-dihydro-s-triazines with one or two alkyl groups at the 2-position and a substituted phenyl or naphthyl group at the 1-position were evaluated as inhibitors of dihydrofolate reductase enzymes from Pneumocystis carinii, Toxoplasma gondii, and rat liver. Halogen substitution at the 5- or 6-position of 2,4-diaminoquinazoline favored selective binding to the P. carinii enzyme but not the T. gondii enzyme. For example, the 50% inhibitory concentrations of 2,4-diamino-6-chloroquinazoline as an inhibitor of P. carinii, T. gondii, and rat liver dihydrofolate reductase were 3.6, 14 and 29 microM, respectively, corresponding to 12-fold selectivity for the P. carinii enzyme but only marginal selectivity for the T. gondii enzyme. Greater than fivefold selectivity for P. carinii but not T. gondii dihydrofolate reductase was also observed for the 2,4-diaminoquinazolines with 5-methyl, 5-fluoro, 5- and 6-bromo, 6-chloro, and 5-chloro-6-bromo substitution. In contrast, alkyl and aralkyl substitution at the 6- and 7-positions of 2,4-diaminopteridines was found to be a favorable feature for selective inhibition of the T. gondii enzyme and, in two cases, for both enzymes. Nine of the fifty-one compounds tested against P. carinii dihydrofolate reductase and four of the thirty compounds tested against T. gondii dihydrofolate reductase displayed fivefold or greater selectivity for the microbial enzyme versus the rat liver enzyme. The most selective against both enzymes was 2,4-diamino-6,7-bis(cyclohexylmethyl) pteridine, with a selectivity ratio 2 orders of magnitude greater than the value reported for trimetrexate and piritrexim. Since substitution at the 7-position is generally considered to be detrimental to the binding of 2,4-diaminop-teridines and related compounds to mammalian dihydrofolate reductase, the selectivity observed in this study with the 6,7-bis(cyclohexylmethyl) analog may represent a useful approach to enhancing selective inhibition of the enzyme from nonmammalian species.

ADVERTISEMENT RETURN TO ISSUEPREVNoteNEXTA Novel Method of Synthesis of 2,4-Diamino-6-arylmethylquinazolines Using Palladium(0)-Catalyzed Organozinc ChemistryAndre Rosowsky and Han ChenView Author Information Dana-Farber Cancer Institute and Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115 [email protected]Cite this: J. Org. Chem. 2001, 66, 22, 7522–7526Publication Date (Web):October 11, 2001Publication History Received25 May 2001Published online11 October 2001Published inissue 1 November 2001https://doi.org/10.1021/jo010536iCopyright © 2001 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views740Altmetric-Citations24LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (62 KB) Get e-AlertsSUBJECTS:Aromatic compounds,Cross coupling reaction,Halogens,Inhibitors,Reagents Get e-Alerts

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)

The activity of a series of folic acid analogues as substrates for partially purified mouse liver folylpolyglutamate synthetase was determined and the effects of substituents on the binding to, and catalytic processes of, this enzyme were inferred. A 4-amino group improved substrate activity primarily by decreasing the apparent Km while N10-methyl substitution substantially diminished utilization as a substrate, again, by effects on Km. Isosteric replacement of N-10 altered substrate activity. A free alpha-carboxyl group in the amino acid side chain was required for catalysis as was the presence of the side chain amide carbonyl group. Modification of the amino acid side chain length profoundly affected activity. Several observations were made that may be relevant to chemotherapy with folate antimetabolites: 1) 7-hydroxymethotrexate was a substrate for this enzyme; 2) substrate activity and substrate inhibition were observed with CB 3717, a potent inhibitor of thymidylate synthase; 3) potent classical dihydrofolate reductase inhibitors were identified that were either not substrates for mouse liver folylpolyglutamate synthetase (e.g., 4-amino-4-deoxy-N10-methylpteroyl-L-alpha-aminoadipate) or were much better substrates than methotrexate for this enzyme (e.g., aminopterin); and 4) leucovorin and methotrexate appeared to be substrates for the same synthetase, but leucovorin saturated the reaction at much lower concentrations. These results have implications for the design of folylpolyglutamate synthetase inhibitors and for the selection of dihydrofolate reductase inhibitors that are either not polyglutamated or are efficiently polyglutamated in vivo.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 19. Replacement of the glutamate side-chain in classical antifolates by L-homocysteic acid and L-cysteic acid: effect on enzyme inhibition and antitumor activityAndre Rosowsky, Ronald A. Forsch, James H. Freisheim, Richard G. Moran, and Michael WickCite this: J. Med. Chem. 1984, 27, 5, 600–604Publication Date (Print):May 1, 1984Publication History Published online1 May 2002Published inissue 1 May 1984https://doi.org/10.1021/jm00371a008Request reuse permissionsArticle Views244Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (726 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of the 2-chloro analogs of 3'-deoxyadenosine, 2',3'-dideoxyadenosine, and 2',3'-didehydro-2',3'-dideoxyadenosine as potential antiviral agentsAndre Rosowsky, Vishnu C. Solan, Joseph G. Sodroski, and Ruth M. RuprechtCite this: J. Med. Chem. 1989, 32, 5, 1135–1140Publication Date (Print):May 1, 1989Publication History Published online1 May 2002Published inissue 1 May 1989https://doi.org/10.1021/jm00125a031RIGHTS & PERMISSIONSArticle Views290Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (895 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTA new one-step synthesis of leucovorin from folic acid and of 5-formyl-5,6,7,8-tetrahydrohomofolic acid from homofolic acid using dimethylamine-borane in formic acidRonald A. Forsch and Andre RosowskyCite this: J. Org. Chem. 1985, 50, 14, 2582–2583Publication Date (Print):July 1, 1985Publication History Published online1 May 2002Published inissue 1 July 1985https://doi.org/10.1021/jo00214a037Request reuse permissionsArticle Views438Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (334 KB) Get e-Alertsclose Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis of mono- and bifunctional .alpha.-methylene lactone systems as potential tumor inhibitorsPaul A. Grieco, J. A. Noguez, Yukio Masaki, K. Hiroi, M. Nishizawa, Andre Rosowsky, Selma Oppenheim, and Herbert LazarusCite this: J. Med. Chem. 1977, 20, 1, 71–76Publication Date (Print):January 1, 1977Publication History Published online1 May 2002Published inissue 1 January 1977https://doi.org/10.1021/jm00211a014RIGHTS & PERMISSIONSArticle Views157Altmetric-Citations21LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (820 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSulfur dioxide extrusion from 2,5-diaryl-4-hydroxy-3-oxotetrahydrothiophene 1,1-dioxides. Novel synthesis of 1,4-diarylbutane-2,3-dionesMichael Chaykovsky, May H. Lin, and Andre RosowskyCite this: J. Org. Chem. 1972, 37, 12, 2018–2021Publication Date (Print):June 1, 1972Publication History Published online1 May 2002Published inissue 1 June 1972https://doi.org/10.1021/jo00977a034RIGHTS & PERMISSIONSArticle Views108Altmetric-Citations17LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (641 KB) Get e-Alerts Get e-Alerts

Journal of Heterocyclic ChemistryVolume 3, Issue 3 p. 387-388 Note Quinazolines. IV. a novel synthesis of 1,3-diamino-benzo[f]quinazolines from N1, N5-bis(2-naphthyl)biguanides†‡ Andre Rosowsky, Andre Rosowsky The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School at The Children's Hospital Medical Center, Boston, Mass. 02115Search for more papers by this authorEdward J. Modest, Edward J. Modest The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School at The Children's Hospital Medical Center, Boston, Mass. 02115Search for more papers by this author Andre Rosowsky, Andre Rosowsky The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School at The Children's Hospital Medical Center, Boston, Mass. 02115Search for more papers by this authorEdward J. Modest, Edward J. Modest The Children's Cancer Research Foundation and the Departments of Biological Chemistry and Pathology, Harvard Medical School at The Children's Hospital Medical Center, Boston, Mass. 02115Search for more papers by this author First published: September 1966 https://doi.org/10.1002/jhet.5570030340Citations: 16 † Paper III: A. Rosowsky and E. J. Modest, J. Org. Chem., 31, 2607 (1966). ‡ A. Rosowsky, N. Papathanasopoulos, M. E. Nadel, S. K. Sengupta, and E. J. Modest, Abstracts of Papers, 151st National Meeting, American Chemical Society, Pittsburgh, Pennsylvania, March 28, 1966, 1—1. AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL No abstract is available for this article. References (4) G. H. Hitchings and J. J. Burchall, in “ Advances in Enzymology,” Vol. 27, F. F. Nord, Ed., Interscience Publishers, Inc., New York, N. Y., 1965, pp. 417– 468, have given a comprehensive review of inhibitors of folate biosynthesis. (5) H. H. Hodgson and E. R. Ward, J. Chem. Soc., 327 (1947). (6) M. S. Newman and R. H. Galt, J. Org. Chem., 25, 214 (1960). (7) Satisfactory elemental analyses have been obtained for all new compounds reported here. (8) L. Neelakantan, J. Org. Chem., 22, 1587 (1957). (9) F. H. S. Curd and F. L. Rose, J. Chem. Soc., 729 (1946). (10) The use of diphenyl ether in a similar type of reaction was reported by Gompper and coworkers (ref. 13), who obtained 1-hydroxy-benzo[f]quinazoline in 89% yield on heating N1-carbethoxy-N3-(2-naphthyl)formamidine in this solvent for 2 minutes. (11) T. Bhattacharyya, P. K. Bose, and J. N. Ray, J. Indian Chem. Soc., 6, 279 (1929). (12) R. C. Shah and M. B. Ichaporia, J. Chem. Soc., 431 (1936). (13) R. Gompper, H. Noppel, and H. Schaefer, Angew. Chem., 75, 918 (1963). (14) K. Dziewonski, L. Sternbach, and A. Strauchen, Bull. Intern. Acad. Polon. Sci., Classe Sci. Math. Nat., 493 (1936). Chem. Abstr., 31, 3053 (1937). Citing Literature Volume3, Issue3September 1966Pages 387-388 ReferencesRelatedInformation

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTQuinazolines. V. Synthesis and proof of structure of 1,3-diamino-5,6-dihydrobenzo[f]quinazolineElizabeth P. Burrows, Andre Rosowsky, and Edward J. ModestCite this: J. Org. Chem. 1967, 32, 12, 4090–4092Publication Date (Print):December 1, 1967Publication History Published online1 May 2002Published inissue 1 December 1967https://doi.org/10.1021/jo01287a094RIGHTS & PERMISSIONSArticle Views85Altmetric-Citations13LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (454 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXT2,4-Diaminopyrido[3,2-d]pyrimidine Inhibitors of Dihydrofolate Reductase from Pneumocystis carinii and Toxoplasma gondiiAndre Rosowsky, Ronald A. Forsch, and Sherry F. QueenerCite this: J. Med. Chem. 1995, 38, 14, 2615–2620Publication Date (Print):July 1, 1995Publication History Published online1 May 2002Published inissue 1 July 1995https://doi.org/10.1021/jm00014a014Request reuse permissions Article Views397Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (903 KB) Get e-Alertsclose Get e-Alerts

Six new B-ring analogues of the nonpolyglutamatable antifolate Nalpha-(4-amino-4-deoxypteroyl)-Ndelta-hemiphthaloy l-L-ornithine (PT523, 3) were synthesized with a view to determining the effect of modifications at the 5- and/or 8-position on dihydrofolate reductase (DHFR) binding and tumor cell growth inhibition. The 5- and 8-deaza analogues were prepared from methyl 2-L-amino-5-phthalimidopentanoate and 4-amino-4-deoxy-N10-formyl-5-deaza- and -8-deazapteroic acid, respectively. The 5,8-dideaza analogues were prepared from methyl 2-L-[(4-aminobenzoyl)amino]-5-phthalimidopentanoate and 2, 4-diaminoquinazoline-6-carbonitriles. The Ki for inhibition of human DHFR by the 5-deaza and 5-methyl-5-deaza analogues was about the same as that of 3 (0.35 pM), 11-fold lower than that of aminopterin (AMT, 1), and 15-fold lower than that of methotrexate (MTX, 2). However the Ki of the 8-deaza analogue was 27-fold lower than that of 1, and that of the 5,8-dideaza, 5-methyl-5,8-dideaza, and 5-chloro-5,8-dideaza analogues was approximately 50-fold lower. This trend was consistent with the published literature on the corresponding DHFR inhibitors with a glutamate side chain. In colony formation assays against the human head and neck squamous carcinoma cell line SCC25 after 72 h of treatment, the 5- and 8-deaza analogues were approximately as potent as 3, whereas the 5,8-dideaza analogue was 3 times more potent. 5-Methyl and 5-chloro substitution was also favorable, with the 5-methyl-5-deaza analogue being 2. 5-fold more potent than the 5-deaza analogue. However the effect of 5-methyl substitution was less pronounced in the 5,8-dideaza analogues than in the 5-deaza analogues. The 5-chloro-5,8-dideaza analogue of 3 was the most active member of the series, with an IC50 = 0.33 nM versus 1.8 nM for 3 and 15 nM for MTX. The 5-methyl-5-deaza analogue of 3 was also tested at the National Cancer Institute against a panel of 50 human tumor cell lines in culture and was consistently more potent than 3, with IC50 values in the low-nanomolar to subnanomolar range against most of the tumors. Leukemia and colorectal carcinoma cell lines were generally most sensitive, though good activity was also observed against CNS tumors and carcinomas of the breast and prostate. The results of this study demonstrate that B-ring analogues of 3 inhibit DHFR activity and tumor cell colony formation as well as, or better than, the parent compound. In view of the fact that 3 and its B-ring analogues cannot form polyglutamates, their high cytotoxicity relative to the corresponding B-ring analogues of AMT is noteworthy.

Nα-(4-Amino-4-deoxypteroyl)-Nδ-hemiphthaloyl-l-ornithine (PT523) is an unusually tight-binding dihydrofolate reductase (DHFR) inhibitor and is efficiently taken up into cells via the reduced folate carrier (RFC). Unlike classical DHFR inhibitors with a glutamate side chain, such as methotrexate and aminopterin, PT523 cannot form polyglutamates. Thus, it resembles lipophilic antifolates such as trimetrexate in not requiring metabolic activation by folylpolyglutamate synthetase in order to produce its antifolate effect. However, in contrast to trimetrexate, PT523 retains growth inhibitory activity in cells with the multidrug resistance phenotype. As part of the preclinical development of this drug, we have performed systematic modification of several regions of the PT523 molecule, with the aim of defining the optimal structural features for DHFR binding, influx into cells via the RFC, and the ability to inhibit cell growth. The following structure-activity correlations have emerged from this ongoing investigation, and are discussed: (1) the hemiphthaloylornithine side chain has the optimal length; (2) the preferred location of the aromatic carboxyl group is the ortho position; and (3) replacement of the phenyl ring of the para-aminobenzoic acid moiety by naphthalene, of nitrogen at the 10-position of the bridge by carbon, and of nitrogen at the 5- and/or 8-position of the B-ring by carbon are all well tolerated. Several of the second generation analogs of PT523 are more potent DHFR inhibitors and better RFC substrates than PT523 itself, and are more potent inhibitors of tumor cell growth in culture.

N-[5-[N-(2-Amino-5-chloro-3,4-dihydro-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-l-glutamic acid (Figure 1, Scheme 2) and N-[5-[N-(5-chloro-3,4-dihydro-2-methyl-4-oxoquinazolin-6-yl)methylamino]-2-thenoyl]-l-glutamic acid (Figure 1, Scheme 3), the first reported thiophene analogues of 5-chloro-5,8-dideazafolic acid, were synthesized and tested as inhibitors of tumor cell growth in culture. 4-Chloro-5-methylisatin (10) was converted stepwise to methyl 2-amino-5-methyl-6-chlorobenzoate (22) and 2-amino-5-chloro-3,4-dihydro-6-methyl-4-oxoquinazoline (19). Pivaloylation of the 2-amino group, followed by NBS bromination, condensation with di-tert-butyl N-(5-amino-2-thenoyl)-l-glutamate (28), and stepwise cleavage of the protecting groups with ammonia and TFA yielded Figure 1, Scheme 2. Treatment of 9 with acetic anhydride afforded 2,6-dimethyl-5-chlorobenz[1,3-d]oxazin-4-one (31), which on reaction with ammonia, NaOH was converted to 2,6-dimethyl-5-chloro-3,4-dihydroquinazolin-4-one (33). Bromination of 33, followed by condensation with 28 and ester cleavage with TFA, yielded Figure 1, Scheme 3. The IC50 of Figure 1, Scheme 2 and Figure 1, Scheme 3 against CCRF-CEM human leukemic lymphoblasts was 1.8±0.1 and 2.1±0.8 μM, respectively.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTSynthesis and in vitro biological activity of new deaza analogs of folic acid, aminopterin, and methotrexate with an L-ornithine side chainAndre Rosowsky, Ronald A. Forsch, Henry Bader, and James H. FreisheimCite this: J. Med. Chem. 1991, 34, 4, 1447–1454Publication Date (Print):April 1, 1991Publication History Published online1 May 2002Published inissue 1 April 1991https://doi.org/10.1021/jm00108a032RIGHTS & PERMISSIONSArticle Views486Altmetric-Citations22LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

ABSTRACT The activities of 28 6-substituted 2,4-diaminoquinazolines, 2,4-diamino-5,6,7,8-tetrahydroquinazolines, and 2,4-diaminopteridines against Plasmodium falciparum were tested. The 50% inhibitory concentrations (IC 50 s) of six compounds were &lt;50 nM, and the most potent compound was 2,4-diamino-5-chloro-6-[ N -(2,5-dimethoxybenzyl)amino]quinazoline (compound 1), with an IC 50 of 9 nM. The activity of compound 1 was potentiated by the dihydropteroate synthase inhibitor dapsone, an indication that these compounds are inhibitors of dihydrofolate reductase. Further studies are warranted to assess the therapeutic potential of this combination in vivo.

Expansion of CH2THF pools in tissues of BALB/c mice bearing s.c.-implanted EMT6 mammary adenocarcinomas was measured after leucovorin administration. Twenty-four mice were treated with leucovorin at doses of 0, 45, 90, or 180 mg/kg/injection x 8 injections spaced over 48 h. Tumor and bone marrow cytosols were assayed for CH2THF by forming ternary complexes with thymidylate synthase and [3H]FdUMP. Tumor CH2THF pools were expanded significantly at the two higher doses. Marrow levels were not different from controls. Groups of tumor bearing mice were treated with saline, leucovorin, 5-fluorouracil or 5-fluourouracil plus leucovorin on an optimal dosage schedule. Measured plus leucovorin on an optimal dosage schedule. Measured from the last day of treatment, these tumors grew to 10 mm root-mean-square diameters in 3.5 +/- 1.4, 5.0 +/- 1.2, 6.5 +/- 1.5, and 9.3 +/- 1.2 days, respectively. Growth rates were significantly different from controls only in the latter two groups.

R67 dihydrofolate reductase (DHFR) is a novel bacterial protein that possesses 222 symmetry and a single active site pore. Although the 222 symmetry implies that four symmetry-related binding sites must exist for each substrate as well as for each cofactor, various studies indicate only two molecules bind. Three possible combinations include two dihydrofolate molecules, two NADPH molecules, or one substrate plus one cofactor. The latter is the productive ternary complex. To explore the role of various ligand substituents during binding, numerous analogues, inhibitors, and fragments of NADPH and/or folate were used in both isothermal titration calorimetry (ITC) and K(i) studies. Not surprisingly, as the length of the molecule is shortened, affinity is lost, indicating that ligand connectivity is important in binding. The observed enthalpy change in ITC measurements arises from all components involved in the binding process, including proton uptake. As a buffer dependence for binding of folate was observed, this likely correlates with perturbation of the bound N3 pK(a), such that a neutral pteridine ring is preferred for pairwise interaction with the protein. Of interest, there is no enthalpic signal for binding of folate fragments such as dihydrobiopterin where the p-aminobenzoylglutamate tail has been removed, pointing to the tail as providing most of the enthalpic signal. For binding of NADPH and its analogues, the nicotinamide carboxamide is quite important. Differences between binary (binding of two identical ligands) and ternary complex formation are observed, indicating interligand pairing preferences. For example, while aminopterin and methotrexate both form binary complexes, albeit weakly, neither readily forms ternary complexes with the cofactor. These observations suggest a role for the O4 atom of folate in a pairing preference with NADPH, which ultimately facilitates catalysis.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTMethotrexate analogs. 15. A methotrexate analogue designed for active-site-directed irreversible inactivation of dihydrofolate reductaseA. Rosowsky, J. E. Wright, C. Ginty, and J. UrenCite this: J. Med. Chem. 1982, 25, 8, 960–964Publication Date (Print):August 1, 1982Publication History Published online1 May 2002Published inissue 1 August 1982https://doi.org/10.1021/jm00350a015RIGHTS & PERMISSIONSArticle Views283Altmetric-Citations19LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (750 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTNucleosides. 1. 9-(3'-Alkyl-3'-deoxy-.beta.-D-ribofuranosyl)adenines as lipophilic analogs of cordycepin. Synthesis and preliminary biological studiesAndre Rosowsky, Herbert Lazarus, and Ayako YamashitaCite this: J. Med. Chem. 1976, 19, 11, 1265–1270Publication Date (Print):November 1, 1976Publication History Published online1 May 2002Published inissue 1 November 1976https://doi.org/10.1021/jm00233a001RIGHTS & PERMISSIONSArticle Views160Altmetric-Citations16LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (747 KB) Get e-Alerts Get e-Alerts

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTQuinazolines. I. Formation of a Guanidinoquinazoline during the Three-Component Synthesis of a 4,6-Diamino-1-aryl-1,2-dihydro-s-triazine1Andre Rosowsky, Heljo Kangur Protopapa, Paul J. Burke, and Edward J. ModestCite this: J. Org. Chem. 1964, 29, 10, 2881–2887Publication Date (Print):October 1, 1964Publication History Published online1 May 2002Published inissue 1 October 1964https://doi.org/10.1021/jo01033a018RIGHTS & PERMISSIONSArticle Views134Altmetric-Citations12LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (950 KB) Get e-Alertsclose Get e-Alerts