Aminopterin Analogues Research Articles

464 POSTER Pharmacokinetics of talotrexin (PT-523), a novel aminopterin analogue, in patients with non-small cell lung cancer

Background. Talotrexin, Nα-(4-Amino-4-deoxypteroyl)-N5-hemiphthaloyl)-L-ornithine (PT-523) is a nonpolyglutamatable antifolate which has demonstrated improved antitumor activity in a broad spectrum of cancer models by targeting DHFR to inhibit tumor growth. Talotrexin binds more tightly (15-fold, Ki 0.35 pM) to DHFR than methotrexate (MTX)1. In lung cancer cell lines, talotrexin inhibits tumor cell proliferation at subto low nanomolar concentrations and is more potent than MTX in all cell lines tested (Data on file, Hana Biosciences, Inc.). We conducted a dose escalation study of talotrexin administered intravenously (IV) over a 5 minute infusion on Days 1, 8, on a 21-day cycle in patients with non-small cell lung cancer (NSCLC). The objectives of this study were to determine the maximum tolerated dose (MTD), dose limiting toxicities (DLT) pharmacokinetic (PK) profile, and the preliminary response data. This report describes the PK behavior of talotrexin in NSCLC patients.

Pharmacokinetics of PT523, a novel aminopterin analogue, in patients with solid tumors

2052 Background: Nα-(4-Amino-4-deoxypteroyl)-N5-hemiphthaloyl)-L-ornithine (PT523) is a nonpolyglutamatable aminopterin analogue selected for clinical evaluation based upon properties that confer potential therapeutic advantages over classic and nonclassic antifolates. This report describes the pharmacokinetic (PK) behavior of PT523 in cancer patients as determined in the first phase I trial of the drug. Methods: Adult patients with refractory solid tumors received PT523 as a 5 min iv infusion every 7 days for 3 weeks. Plasma samples were obtained at -5, 4, 10, 15, 20, 30, 45 min; 1, 2, 3, 4, 6, 8, 24, and 48 h after starting the first weekly infusion. Urine was collected and pooled from 0–8, 8–24, and 24–48 h. An LC/MS assay was used to measure PT523 in plasma and urine. Interday accuracy and precision were both <15% at the lowest concentrations measured in plasma (0.50 ng/mL) and urine (50 ng/mL). PK parameters were estimated by standard noncompartmental methods. Results: The PK of PT523 was characterized in 24 patients with normal renal and hepatic function, and a median age of 52 years (range, 28 - 77 years). Data was obtained from groups of at least three patients receiving doses of 5, 6.7, 9, 12, and 16 mg/m2. The PT523 concentration in plasma decreased in a polyexponential manner and the terminal log-linear phase was achieved 4–6 h after dosing. In the 7 patients receiving doses of 16 mg/m2, the mean peak drug concentration in plasma (Cmax) was 5,650 ± 300 ng/mL and the median plasma concentration 48 h after dosing was 1.7 ng/mL (range, 1.0 - 23.4 ng/mL). The apparent biological half-life (t1/2,z), total body clearance (CL) and apparent volume of distribution at steady-state (Vss) were all independent of the dose. Mean ± SD values of PK parameters for the entire cohort of 24 patients were: CL, 1.16 ± 0.31 L/h/m2; t1/2,z, 5.3 ± 1.2 h; Vss, 7.7 ± 1.5 L/m2. The mean amount of the dose excreted as unchanged drug in urine over 48 h was 40 ± 15%. Conclusions: PT523 exhibits linear PK with moderate interpatient variability when administered as a 5 min iv infusion at doses of 5 - 16 mg/m2. Renal clearance is a major route of elimination. Association between the CL of PT523 and creatinine clearance should be evaluated in a future study to assess whether dose modification is warranted for patients with diminished renal function. No significant financial relationships to disclose.

Synthesis and in Vitro Antifolate Activity of Rotationally Restricted Aminopterin and Methotrexate Analogues

Heretofore unknown analogues of aminopterin (AMT) and methotrexate (MTX) in which free rotation of the amide bond between the phenyl ring and amino acid side chain is prevented by a CH(2) bridge were synthesized and tested for in vitro antifolate activity. The K(i) of the AMT analogue (9) against human dihydrofolate reductase (DHFR) was 34 pM, whereas that of the MTX analogue (10) was 2100 pM. Both compounds were less potent than the parent drugs. However, although the difference between AMT and MTX was <2-fold, the difference between 9 and 10 was 62-fold, suggesting that the effect of N(10)-methyl substitution is amplified in the bridged compounds. The K(i) values of 9 and 10 as inhibitors of [(3)H]MTX influx into CCRF-CEM human leukemia cells via the reduced folate carrier (RFC) were 0.28 and 1.1 muM, respectively. The corresponding K(i) and K(t) values determined earlier for AMT and MTX were 5.4 and 4.7 muM, respectively. Thus, in contrast to its unfavorable effect on DHFR binding, the CH(2) bridge increased RFC binding. In a 72 h growth assay with CCRF-CEM cells, the IC(50) values of 9 and 10 were 5.1 and 140 nM, respectively, a 27-fold difference that was qualitatively consistent with the observed combination of weaker DHFR binding and stronger RFC binding. Although rotationally restricted inhibitors of other enzymes of folate pathway enzymes have been described previously, 9 and 10 are the first reported examples of DHFR inhibitors of this type.

ChemInform Abstract: Synthesis and Antifolate Evaluation of the Aminopterin Analogue with a Bicyclo[2.2.2]octane Ring in Place of the Benzene Ring.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis and antifolate evaluation of the aminopterin analogue with a bicyclo[2.2.2]octane ring in place of the benzene ring

N-[4-[[2,4-diamino-6-pteridinyl)methyl]amino]bicyclo[2.2.2]octane-1-carbonyl]- l-glutamic acid ( 1) was synthesized and tested for antifolate activity. N-(4-Aminobicyclo[2.2.2]octane-1-carbonyl- l-glutamic acid dimethyl ester ( 6), the side chain precursor to subject compound 1, was synthesized readily via reported bicyclo[2.2.2]octane-1,4-dicarboxylic acid monoethyl ester ( 2). The side chain precursor 6 was alkylated by 6-(bromomethyl)-2,4-pteridinediamine ( 7). Subsequent ester hydrolysis then afforded 1. Antifolate and antitumor evaluation of 1 verses L1210 dihydrofolate reductase (DHFR) and three tumor cell lines (L1210, S180, and HL60) showed it to be ineffective. Although compound 1 was very similar to aminopterin structurally, the bicyclo[2.2.2]octane ring system in place of the phenyl ring in the p-aminobenzoate moiety effectively negates the stoichiometric binding displayed by many classical DHFR inhibitors bearing appropriate aromatic ring systems in the side chain.

PT523 and Other Aminopterin Analogs with a Hemiphthaloyi-L­ ornithine Side Chain: Exceptionally Tight-binding Inhibitors of Dihydrofolate Reductase which are Transported by the Reduced Folate Carrier but cannot form Polyglutamates

Abstract: Nonpolyglutamatable antifolates are potentially of therapeutic interest for the treatment of tumors that are inherently refractory or have become resistant, to classical antifolates as a result of decreased expression of the enzyme folylpolyglutamate synthetase. An interesting class of water-soluble nonpolyglutamatable analogs of aminopterin (AMT) have been developed, which are much more cytotoxic because they bind more tightly to dihydrofolate reductase (DHFR) and also utilize the reduced folate carrier (RFC) pathway more efficiently for influx into the cell. This review summarizes the in vitro and in vivo preclinical data on the initial lead compound, Na-(4-amino-4-deoxypteroyi)-N &gt;-hemiphthaloyi-L-ornithine (PT523}. In addition, the synthesis and in vitro biochemical and biological properties of several types of second-generation analogs are discussed. Analogs modified in the B-ring of the pteridine moiety have been found to be of particular interest because their affinity for DHFR and their influx rate into cells via the RFC pathway are even greater than those of PT523. The hemiphthaloylornithine moiety, which is larger and more hydrophobic than the glutamate side chain of classical antifolates, appears to be chiefly responsible for the exceptionally high biological potency of PT523 and its B-ring analogs.

Analogues of methotrexate in rheumatoid arthritis. 2. Effects of 5-deazaaminopterin, 5,10-dideazaaminopterin, and analogues on type II collagen-induced arthritis in mice.

Twenty-six compounds derived from the 5-deaza- and 5,10-dideazaaminopterin series of aminopterin analogues were evaluated for antiarthritic activity in the mouse type II collagen model. New compounds in the 5-deaza series were prepared by alkylation of an appropriate N-substituted (4-aminobenzoyl)-L-glutamic acid dialkyl ester or N-(5-amino-2-thenoyl)-L-glutamate diester with a 2,4-diamino-5-alkyl-6-(bromomethyl)-5-deazapteridine. The resultant 5-deazaaminopterin diesters were saponified to provide the target 5-deaza analogues. 5,10-Dideazaaminopterins were synthesized by similar alkylation of the carbanions of appropriate 4-carboxyphenylacetic, (5-carboxy-2-thienyl)acetic, or (5-carboxy-2-pyridyl)acetic acid dimethyl esters. The diesters of the 2,4-diamino-4-deoxy-10-carboxy-5,10-dideazapteroic acid types so obtained were saponified and then readily decarboxylated by heating in Me2SO solution to provide the 2,4-diamino-5,10-dideazapteroic acid-type intermediates. Peptide coupling with diethyl L-glutamate followed by ester hydrolysis at room temperature afforded the new 5,10-dideazaaminopterin analogues. 5-Deazaaminopterins bearing an alkyl substituent at the 5-position were generally quite effective as antiinflammatory agents. Thus 5-propyl-5-deazaaminopterin, 5-methyl-10-propargyl-5-deazaaminopterin, 5-methyl-10-allyl-5-deazaaminopterin, 5-ethyl-5-deazamethotrexate, and 2,5-disubstituted thiophene analogue of 5-methyl-5-deazaaminopterin showed potencies greater than methotrexate by intraperitoneal or oral administration and were active over a considerably broader dose range. Useful activity in the 5,10-dideaza series was only observed for 5,10-dideazaaminopterin and its 10-methyl analogue. Alkyl substitution at C-5 or C-10 was generally detrimental to antiinflammatory activity in this series.

Analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523) modified in the side chain: synthesis and biological evaluation.

Four heretofore undescribed side chain analogues of N alpha-(4-amino-4-deoxypteroyl)-N delta-hemiphthaloyl-L-ornithine (PT523, 4) were synthesized via straightforward methods of antifolate chemistry, and their properties were compared with those of PT523 and two related compounds with the aim of defining the contribution of the hemiphthaloyl-L-ornithine moiety to the exceptional in vitro antitumor activity of this novel non-polyglutamatable aminopterin analogue. The IC50 values of N alpha-(4-amino-4-deoxypteroyl)-N beta-hemiphthaloyl-L-2,3-diaminopropanoic acid (10) and N alpha-(4-amino-4-deoxypteroyl)-N gamma-hemiphthaloyl-L-2,4- diaminobutanoic acid (9) against A549 human non-small-cell lung carcinoma cells in culture were 23 and 22 nM, whereas those of PT523 and N alpha-(4-amino-4-deoxypteroyl)-N epsilon-hemiphthaloyl-L-lysine (8) were 1.3 and 5.2 nM. A decrease in the in vitro activities of 8 and 9 relative to PT523 was also observed against the panel of cell lines used by the National Cancer Institute to screen new drugs. However the potency of 8 and 9 remained several times greater than that of the historical control methotrexate against many of the cell lines in the screening panel. In an in vivo tumor model, SCC-VII murine squamous cell carcinoma, 9 and methotrexate were well tolerated as 5-day continuous infusions at doses of 0.52 and 0.75 mg/kg/day, whereas the highest tolerated dose of PT523 on this schedule was 0.19 mg/kg/day, in agreement with its lower IC50 in culture. To assess the importance of the hemiphthaloyl group in PT523, N alpha-(4-amino-4-deoxypteroyl)-N delta-isophthaloyl-L-ornithine (11), N alpha-(4-amino-4-deoxypteroyl)-N delta-terephthaloyl-L-ornithine (12), and N alpha-(4-amino-4-deoxypteroyl)-N delta-(4,5-dichlorohemiphthaloyl)-L-ornithine (13) were also synthesized. The IC50 values of 11 and 12 against A549 cells were 45 and 3300 nM, as compared with 1.3 nM for PT523 and 23 nM for methotrexate. In a clonogenic assay against SCC25 human squamous cell carcinoma cells, the IC50 values of 11 and 12 were 2.9 and 72 nM, as compared with 0.3 nM for PT523 and 27 nM for methotrexate. Thus, activity was decreased by moving the aromatic carboxyl group in PT523 to the meta position and was further diminished by moving it to the para position. The IC50 of the halogenated analogue 13 against SCC25 human head and neck squamous carcinoma cells was 18 nM, suggesting lack of tolerance for this 4,5-disubstitution in the phthaloyl moiety. Our results suggest that the combination of a hemiphthaloyl group and three CH2 groups in the side chain are critical determinants of the potent in vitro activity of PT523.

Synthesis and biological activities of tetrahydroquinazoline analogs of aminopterin and methotrexate

Abstract(6R,6S)‐5,8‐Dideaza‐5,6,7,8‐tetrahydroaminopterin (1) and (6R,6S)‐5,8‐dideaza‐5,6,7,8‐tetrahydromethotrexate (2) were synthesized as potential inhibitors of dihydrofolate reductase (DHFR) and as antitumor agents. Cyclohexanone‐4‐carboxaldehyde dimethyl acetal, a key intermediate [10] was synthesized from cyclohexane‐1,4‐dione monoethylene ketal, which was converted via a Wittig reaction to its exocyclic 4‐methylene derivative which in turn, was converted to the 4‐aldehyde via a hydroboration‐oxidation sequence. Selective protection of the 4‐aldehyde as the dimethylacetal and cyclization with dicyandiamide afforded the 6‐dimethylacetal of 2,4‐diamino‐5,6,7,8‐tetrahydroquinazoline. Protection of the 2,4‐diamino moieties and selective deprotection of the 6‐aldehyde followed by reductive amination with p‐aminobenzoyl‐L‐glutamate afforded 2,4‐bisacetamido‐5,8‐dideaza‐5,6,7,8‐tetrahydroaminopterin (11). Deprotection of 11 afforded 1. Compound 2 was obtained from 11 via N10‐methylation and deprotection. The N10‐methyl analogue 2 was 2–10 fold more potent than 1 as an inhibitor of various DHFRs. In the in vitro preclinical screening program of the National Cancer Institute, compound 2 inhibited the growth of eighteen of the twenty nine tumor cell lines in culture at a GI50 &gt; 1.0 × 10−8 M.

Studies on analogues of classical antifolates bearing the naphthoyl group in place of benzoyl in the side chain.

Analogues of classical antifolates with the 4-aminobenzoyl group replaced by 4-amino-1-naphthoyl were synthesized for study after molecular modeling indicated ample spatial accommodation for the naphthalene ring and even larger groups in models based on reported X-ray crystallographic data describing the binding of methotrexate to human dihydrofolate reductase (DHFR). The side-chain precursors, N-(4-amino- and 4-(methylamino)-1-naphthoyl)-L-glutamic acid diethyl esters, were synthesized, and the 2,4-diamino-substituted heterocyclic groups were attached using several methods. Target compounds included naphthoyl analogues of aminopterin (AMT), methotrexate (MTX), 5-deazaAMT, 5-deazaMTX, 5-methyl-5-deazaAMT, 5-methyl-5-deazaMTX, and 5,8-dideazaAMT. A 5,6,7,8-tetrahydronaphthoyl analogue of 5-deazaAMT was also prepared. None of the naphthoyl analogues showed loss in binding to DHFR compared with the corresponding antifolate bearing the benzoyl group, thus confirming the anticipated bulk tolerance. Only the 5,6,7,8-tetrahydronaphthoyl analogue displayed reduced antifolate effects. Substrate activity toward folylpolyglutamate synthetase was, however, severely compromised. The naphthoyl compounds were transported into L1210 cells 3-6 times more readily than MTX, and despite apparently low levels of intracellular polyglutamylation, each compound was found to be significantly more potent than MTX in inhibiting tumor cell growth in vitro in three lines (L1210, HL60, and S180). The MTX, 5-methyl-5-deazaAMT, and 5-methyl-5-deazaMTX analogues were evaluated in vivo alongside MTX against E0771 mammary adenocarcinoma in mice. All three proved more effective than MTX in retarding the tumor growth. The naphthoyl analogue of 5-deazaAMT strongly inhibited DHFR from Pneumocystis carinii, Toxoplasma gondii, and rat liver giving IC50 (pM) values of 0.53, 2.1, and 1.6 respectively, but this compound did not inhibit in vitro growth of T. gondii, thus indicating lack of transport.

Phase-selective AC adsorptive stripping voltammetric assay for aminopterin and 10-Edam in human serum

Aminopterin was studied as a model compound for its analogues which maintain the pteridine ring in their structure. Its adsorptive behaviour on mercury was studied and the DC adsorptive stripping and phase-selective AC adsorptive stripping conditions were optimized. 10-Edam, an aminopterin analogue, was studied and shown to behave similarly to aminopterin. Phase-selective AC voltammetry provided the best signal and gave a detection limit of 4 × 10 −12 M aminopterin in aqueous solution employing an accumulation time of 10 min. The optimized method was applied to the analysis of both aminopterin and 10-Edam respectively in human serum. After extraction with a C 18 reversed-phase cartridge the detection limit of the method was 1 × 10 −8 M aminopterin and the overall assay percentage recovery was 73.5% ( n = 5) at a concentration of 5 × 10 −7 M aminopterin in serum. The analysis of 10-Edam at the same concentration in serum yielded the higher percentage recovery of 94.46% ( n = 5) following the same procedure.

Synthesis and biological activity of the 2-desamino and 2-desamino-2-methyl analogs of aminopterin and methotrexate

The previously undescribed 2-desamino and 2-desamino-2-methyl analogues of aminopterin (AMT) and methotrexate (MTX) were synthesized from 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile. The AMT analogues were obtained via a three-step sequence consisting of condensation with di-tert-butyl N-(4-aminobenzoyl)-L-glutamate, heating with formamidine or acetamidine acetate, and mild acidolysis with trifluoroacetic acid. The MTX analogues were prepared similarly, except that 2-amino-5-(chloromethyl)pyrazine-3-carbonitrile was condensed with 4-(N-methylamino)benzoic acid and the resulting product was annulated with formamidine or acetamidine acetate to obtain the 2-desamino and 2-desamino-2-methyl analogues, respectively, of 4-amino-4-deoxy-N10-methylpteroic acid. Condensation with di-tert-butyl L-glutamate in the presence of diethyl phosphorocyanidate followed by ester cleavage with trifluoroacetic acid was then carried out. Retention of the L configuration in the glutamate moiety during this synthesis was demonstrated by rapid and essentially complete hydrolysis with carboxypeptidase G1 under conditions that likewise cleaved the L enantiomer of MTX but left the D enantiomer unaffected. The 2-desamino and 2-desamino-2-methyl analogues of AMT and MTX inhibited the growth of tumor cells, but were very poor inhibitors of dihydrofolate reductase (DHFR). These unexpected results suggested that activity in intact cells was due to metabolism of the 2-desamino compounds to polyglutamates.

Synthesis and In Vitro Biological Evaluation of β,γ-Methano Analogues of Methotrexate and Aminopterin

Summaryβ,γ-Methano derivatives of methotrexate (MTX) and aminopterin (AMT) were synthesized with the aim of assessing the effect of this side-chain modification on dihydrofolate reductase (OHFR) inhibition, folylpolyglutamate synthetase (FPGS) inhibition, and tumor cell growth inhibition. Mixed carboxylic-carbonic anhydride (MCA) coupling of 4-amino-4-deoxy-N10-methylpteroic acid (mAPA) and dimethyl trans-α-(2-carboxycyclopropyl) glycinate, followed by alkaline hydrolysis, afforded N-( 4-amino-4-deoxy-N10-methylpteroyl)-α-( trans-2-carboxycyclopropyl)glycine (β,γ-methanoMTX) as a mixture of the four possible diastereomers with trans substitution on the cyclopropane ring. N-(4-Amino-4-deoxypteroyl)-trans-α.-(2-carboxycyclopropyl) glycine (β,γ-methanoAMT) , also as a diastereomer mixture, was obtained from 4-amino-4-deoxy-N10 - formylpteroic acid (fAPA) and dimethyl trans-α-(2-carboxycyclopropyl)-glycinate by MCA coupling and alkaline hydrolysis of the ester and N10-formyl groups. β,γ-MethanoMTX and β,γ-methanoAMT may be viewed as MTX and AMT analogues with a conformationally restricted side chain. In vitro biological activity data for these novel compounds support the view that the active site of DHFR, already known for its ability to tolerate modification of the γ-carboxyl group of MTX and AMT, can likewise accommodate substitution on the β- and γ-carbons.

Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogs of methotrexate and tetrahydrofolic acid

Synthesis of the 10-methyl and 10-ethyl analogues of 5,10-dideazatetrahydrofolic acid (DDTHF), a potent inhibitor of glycinamide ribotide (GAR) formyltransferase, is reported. Key intermediates in the process were 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acid. Condensation of the piperidine enamines of branched 4-(p-carbomethoxyphenyl)butyraldehydes with (acetoxymethylene)malononitrile afforded 1,1-dicyano-4-piperidinobutadiene 5a,b. Subsequent reaction with alcoholic ammonium hydroxide yielded the appropriately substituted 2-amino-3-cyanopyridines 6a,b. Ring closure with guanidine gave 10-methyl- and 10-ethyl-4-amino-4-deoxy-5,10-dideazapteroic acids (7a,b). Coupling with diethyl glutamate followed by ester hydrolysis afforded 10-alkyl-5,10-dideazaminopterin analogues 9a,b. Hydrolysis of the 4-amino group of 7a,b yielded the 10-alkylpteroic acids, which were coupled with diethyl glutamate, hydrogenated over PtO2, and saponified to afford 10-alkyl-5,10-dideazatetrahydrofolic acids 13a,b. Aminopterin analogues 9a,b were effective inhibitors of DHFR derived from L1210, but were less potent than methotrexate for inhibition of growth of L1210 in culture. The 10-ethyl (13b) analogue of 5,10-DDTHF was about twice as potent an inhibitor of L1210 cell growth as 5,10-DDTHF, but was only 1/7 as potent for inhibition of GAR formyltransferase. 10-Methyl analogue 13a was similar in potency to 5,10-DDTHF. All of the compounds showed moderately improved transport into L1210 cells relative to methotrexate.

ChemInform Abstract: Synthesis and Antifolate Activity of 5‐Methyl‐5,10‐dideaza Analogues of Aminopterin and Folic Acid and an Alternative Synthesis of 5,10‐Dideazatetrahydrofolic Acid, a Potent Inhibitor of Glycinamide Ribonucleotide Formyltransferase.

AbstractOlefination of the carboxaldehydes (II), obtained by reduction of the nitriles (I), with the Wittig reagent (III) gives the key intermediates (IV).

The 2-desamino and 2-desamino-2-methyl analogues of aminopterin do not inhibit dihydrofolate reductase but are potently toxic to tumor cells in culture.

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTThe 2-desamino and 2-desamino-2-methyl analogs of aminopterin do not inhibit dihydrofolate reductase but are potently toxic to tumor cells in cultureAndre Rosowsky, Ronald A. Forsch, James H. Freisheim, and Richard G. MoranCite this: J. Med. Chem. 1989, 32, 3, 517–520Publication Date (Print):March 1, 1989Publication History Published online1 May 2002Published inissue 1 March 1989https://doi.org/10.1021/jm00123a001RIGHTS & PERMISSIONSArticle Views106Altmetric-Citations6LEARN 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 (510 KB) Get e-Alerts Get e-Alerts

Synthesis and antifolate activity of 5-methyl-5,10-dideaza analogues of aminopterin and folic acid and an alternative synthesis of 5,10-dideazatetrahydrofolic acid, a potent inhibitor of glycinamide ribonucleotide formyltransferase.

The title compounds were prepared in extensions of a general synthetic approach used earlier to prepare 5-alkyl-5-deaza analogues of classical antifolates. Wittig condensation of 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (2a) and its 5-methyl analogue 2b with [4-(methoxycarbonyl)benzylidene] triphenylphosphorane gave 9,10-ethenyl precursors 3a and 3b. Hydrogenation (DMF, ambient, 5% Pd/C) of the 9,10-ethenyl group of 3b followed by ester hydrolysis led to 4-[2-(2,4-diamino-5-methylpyrido[2,3-d]pyrimidin-6-yl)ethyl]ben zoi c acid (5), which was converted to 5-methyl-5,10-dideazaaminopterin (6) via coupling with dimethyl L-glutamate (mixed-anhydride method using i-BuOCOCl) followed by ester hydrolysis. Standard hydrolytic deamination of 6 gave 5-methyl-5,10-dideazafolic acid (7). Intermediates 3a and 3b were converted through concomitant deamination and ester hydrolysis to 8a and 8b. Peptide coupling of 8a,b (using (EtO)2POCN) with diesters of L-glutamic acid gave intermediate esters 9a and 9b. Hydrogenation of both the 9,10 double bond and the pyrido ring of 9a and 9b (MeOH-0.1 N HCl, 3.5 atm, Pt) was followed by ester hydrolysis to give 5,10-dideaza-5,6,7,8-tetrahydrofolic acid (11a) and the 5-methyl analogue 11b. Biological evaluation of 6, 7, 11a, and 11b for inhibition of dihydrofolate reductase (DHFR) isolated from L1210 cells and for growth inhibition and transport characteristics toward L1210 cells revealed 6 to be less potent than methotrexate in the inhibition of DHFR and cell growth. Compounds 6, 11a, and 11b were transported into cells more efficiently than methotrexate. Growth inhibition IC50 values for 11a and 11b were 57 and 490 nM, respectively; the value for 11a is in good agreement with that previously reported (20-50 nM). In tests against other folate-utilizing enzymes, 11a and 11b were found to be inhibitors of glycinamide ribonucleotide formyltransferase (GAR formyltransferase) from one bacterial (Lactobacillus casei) and two mammalian (Manca and L1210) sources with 11a being decidedly more inhibitory than 11b. Neither 11a nor 11b inhibited aminoimidazolecarboxamide ribonucleotide formyltransferase. These results support reported evidence that 11a owes its observed antitumor activity to interference with the purine de novo pathway with the site of action being GAR formyltransferase.

Methotrexate analogues. 32. Chain extension, alpha-carboxyl deletion, and gamma-carboxyl replacement by sulfonate and phosphonate: effect on enzyme binding and cell-growth inhibition.

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.

Methotrexate analogues—27: Dual inhibition of dihydrofolate reductase and folylpolyglutamate synthetase by methotrexate and aminopterin analogues with a γ-phosphonate group in the side chain

γ-Phosphonate analogues of methotrexate (MTX) and aminopterin (AMT) were synthesized from 4-amino-4-deoxy- N 10-methylpteroic acid and 4-amino-4-deoxy- N 10-formylpteroic acid, respectively, by reaction with methyl d,1-2-amino-4-phosphonobutyrate followed by gentle alkaline hydrolysis. The products were compared with the corresponding d,l-homocysteic acid derivatives as inhibitors of dihydrofolate reductase and folylpolyglutamate synthetase, and as inhibitors of cell growth in culture. The γ-phosphonates were somewhat less active than either the γ-sulfonates or the parent drugs as inhibitors of murine dihydrofolate reductase. The MTX γ-sulfonate and γ-phosphonate analogues were equally inhibitory toward mouse liver folylpolyglutamate synthetase ( K i = 190 μM), but in the AMT series the γ-phosphonate ( K i = 8.4 μM) was more potent than the γ-sulfonate ( K i = 45 μM). The AMT analogues were consistently more inhibitory than the MTX analogues against cultured L1210 murine leukemia cells, but neither the γ-phosphonates nor the γ-sulfonates were as potent as their respective parent drugs. The γ-phosphonate analogue of MTX was three times more potent than MTX against the MTX-resistant mutant line L1210/R81, but the AMT γ-phosphonate was less potent than AMT; however, these differences were small in comparison with the level of resistance to all these compounds in the L1210/R81 line. The results suggest than n 10-methyl and n 10-unsubstituted compounds altered at the γ-position do not necessarily follow identical structure-activity patterns in every test system.

ChemInform Abstract: Synthesis and Biological Activities of 5‐Deaza Analogues of Aminopterin and Folic Acid.

AbstractDie Synthese von 5‐Deazaaminopterin (IIIa) und seines 5‐Methyl‐Analogons (IIIb) sowie der 5‐Deaza‐5‐methylfolsäure (IIIc) aus den geschützten 5‐Cyanouracilen (I) wird beschrieben.