Inhibition of hog liver folylpolyglutamate synthetase by 5-substituted 5,8-dideaza analogues of folic acid bearing a terminal L-ornithine residue.

Five new N alpha-(5,8-dideazapteroyl)-L-ornithines have been prepared using multistep synthetic sequences. These include N alpha-[5-(trifluoromethyl)-5,8-dideazapteroyl]-L-ornithine, 3, as well as N alpha-[5-(trifluoromethyl)-5,8-dideazaisopteroyl]-L-ornithine, 4, and its 5-fluoro and 5-chloro analogues. Both of the compounds containing a 5-(trifluoromethyl) group (3 and 4) were found to be excellent inhibitors of homogeneous hog liver folylpolyglutamate synthetase, having Ki values in the same range as N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, 2, (approximately 10 nM). However, the bridge-reversed isomer of 2 was 60-fold less inhibitory than 2.     

Inhibition of murine thymidylate synthase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin

A series of 5,8-dideaza analogues of folic acid, isofolic acid, aminopterin, and isoaminopterin were evaluated for inhibition of thymidylate synthase, TS, from mouse L1210 leukemia cells with 10-propargyl-5,8-dideazafolic acid, CB3717, 4a, as the reference inhibitor. These compounds were also tested as inhibitors of human dihydrofolate reductase, DHFR, obtained from WIL2 cells. None of the analogues studied were as potent as 4a toward TS; however, 9-methyl-5,8-dideazaisoaminopterin, 6d, was only 2.5-fold less effective. Compound 4a was prepared by direct alkylation of the di-tert-butyl ester of 5,8-dideazafolic acid followed by hydrolysis of the resulting diethyl ester, which resulted from concomitant transesterification. It was found to be identical with a sample of 4a prepared by earlier methodology by using a variety of spectroscopic techniques. Its isomer, 9-propargyl-5,8-dideazaisofolic acid, 4b, which was synthesized by an analogous approach, was found to be dramatically less inhibitory toward TS than 4a. Each of the 2,4-diamino derivatives, including those possessing an allyl or propargyl group at N9, was an excellent inhibitor of DHFR, having a level of potency similar to that of methotrexate, MTX. However, many of these 5,8-dideazaaminopterin analogues were far more inhibitory toward TS than MTX.     

Synthesis of 5-chloro-5,8-dideaza analogues of folic acid and aminopterin targeted for colon adenocarcinoma

Several classical quinazoline analogues of folic acid bearing chloro or methyl substituents at position 5 were evaluated as inhibitors of the growth of four human gastrointestinal adenocarcinoma cell lines in vitro. The preparation of two of these, 5-chloro-5,8-dideazaisofolic acid, 1e, and 5-chloro-5,8-dideazaisoaminopterin, 2a, is reported for the first time. In addition, a new synthetic route to 5-chloro-5,8-dideazaaminopterin, 2b, is described. For compounds having a 2,4-diamino configuration, the presence of chlorine at position 5 afforded superior growth inhibitory potency. However, compound 1e was substantially less effective than its 5-methyl counterpart.     

Inhibition of mammalian folylpolyglutamate synthetase and human dihydrofolate reductase by 5,8-dideaza analogues of folic acid and aminopterin bearing a terminal L-ornithine

Six new 5,8-dideaza analogues of folic acid and aminopterin containing a terminal L-ornithine residue were prepared by using multistep synthetic sequences. Each was evaluated as an inhibitor of hog liver folylpolyglutamate synthetase and human dihydrofolate reductase. Structural modifications at positions 2, 4, 5, and 10 were included to help define structure-activity relationships for compounds of this type. The compound N alpha-(4-amino-4-deoxy-5-chloro-5,8-dideazapteroyl)-L-ornithine (3f) was identified as the most potent inhibitor of mammalian folylpolyglutamate synthetase reported thus far (Ki congruent to 2 nM). Its 4-oxy counterpart, N alpha-(5-chloro-5,8-dideazapteroyl)-L-ornithine, was only 5-fold less inhibitory than 3f toward folylpolyglutamate synthetase but was found to be a much weaker inhibitor of dihydrofolate reductase than 3f.     

Inhibition of adenylosuccinate lyase by L-alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR)

L-Alanosyl-5-aminoimidazole-4-carboxylic acid ribonucleotide (alanosyl-AICOR) has been synthesized enzymatically using 4-(N-succino)-5-aminoimidazole-4-carboxamide ribonucleotide (SAICAR) synthetase in conjunction with 5-aminoimidazole-4-carboxylic acid ribonucleotide and L-2-amino-3-(N-hydroxy-N-nitrosoamino)propionic acid (alanosine). The product was characterized by chromatography, ultraviolet spectrum and NMR spectrum at 300 MHz. Alanosyl-AICOR was not a substrate of adenylosuccinate lyase from rat skeletal muscle, but it was an apparent competitive inhibitor in both of the reactions catalyzed by the enzyme. The KI values for alanosyl-AICOR were approximately 1.5 and 1.3 microM in the SAICAR and adenylosuccinate cleavage reactions respectively. These KI values were essentially the same as the Km values for the two substrates of adenylosuccinate lyase. They compare with an accumulation of 70 microM alanosyl-AICOR in leukemic nodules of mice treated with alanosine [A. K. Tyagi and D. Cooney, Cancer Res. 40, 4390 (1980)]. Thus, inhibition of adenylosuccinate lyase may account for much of the inhibitory effect exerted by alanosyl-AICOR in vivo. We confirmed the previous observation that alanosyl-AICOR is an inhibitor of adenylosuccinate synthetase.     

Virtual screening of human 5-aminoimidazole-4-carboxamide ribonucleotide transformylase against the NCI diversity set by use of AutoDock to identify novel nonfolate inhibitors

AICAR transformylase (5-aminoimidazole-4-carboxamide ribonucleotide transformylase) is a folate-dependent activity of the bifunctional protein ATIC (AICAR transformylase and IMP cyclohydrolase) and is responsible for catalyzing the penultimate step of the de novo purine biosynthetic pathway. As such, AICAR transformylase has been proposed as a potential target for antineoplastic drug design. Virtual screening of the human AICAR transformylase active site by use of AutoDock against the NCI diversity set, a library of compounds with nonredundant pharmacophore profiles, has revealed 44 potential inhibitor candidates. In vitro inhibition assay of 16 soluble compounds from this list revealed that eight compounds with novel scaffolds, relative to the general folate template, had micromolar inhibition. Subsequent extension of docking trials on compounds with similar scaffolds from the entire NCI-3D database has unveiled 11 additional inhibitors that were confirmed by the in vitro inhibition assay. In particular, one compound, NSC30171, had nanomolar inhibition (K(i) = 154 nM, IC(50) = 600 nM) against AICAR transformylase. These 19 inhibitors serve as novel templates/scaffolds for development of more potent and specific non-folate-based AICAR transformylase inhibitors.     

5-氨基咪唑-4-甲酰胺盐酸盐的电位滴定法测定

5-氨基咪唑-4-甲酰胺盐酸盐(C4H6N4O·HCI,5-aminoimidazole-4-carboxamide hydrochloride,1),为治疗肝炎类药物阿卡明(aicamin)的组分,其含量测定多采用重氮化反应,以KI淀粉溶液为指示剂,该法不够灵敏.本文采用电位滴定法,准确度略高,能满足药物质控的要求.     

N10-substituted 5,8-dideazafolate inhibitors of glycinamide ribonucleotide transformylase

A series of 5,8-dideazafolates bearing ethyl, isopropyl, cyclopropylmethyl, propargyl, 3-cyanopropyl, carboxymethyl, 2-carboxyethyl, phenacyl, 3-fluorobenzyl, and 5-uracilylmethyl substituents at N10 were tested as inhibitors of purified L5178Y glycinamide ribonucleotide transformylase (GAR TFase), which requires 10-formyltetrahydrofolate as cofactor. All of these cofactor analogues exhibited competitive inhibition against N10-formyl-5,8-dideazafolate, with Ki's ranging from 2 to 32 microM.     

Synthesis of 10-acetyl-5,8-dideazafolic acid: a potent inhibitor of glycinamide ribonucleotide transformylase

10-Acetyl-5,8-dideazafolic acid has been synthesized in good yield from the parent compound, 5,8-dideazafolic acid. This quinazoline folate analogue showed no activity as a substrate for the folate-requiring de novo purine biosynthetic enzyme glycinamide ribonucleotide transformylase isolated from the murine lymphoma cell line L5178Y, but proved to be a potent competitive inhibitor, Ki = 1.3 microM, of the purified enzyme.     

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 and 5-aminoimidazole-4-carboxamide riboside exert synergistic anticancer action against human breast cancer and hepatocellular carcinoma

Aim:

To investigate the influences of methotrexate (MTX) on the anticancer actions and pharmacokinetics of 5-aminoimidazole-4-carboxamide riboside (AICA riboside) in human breast cancer and hepatocellular carcinoma.

Methods:

Human breast cancer cell line MCF-7 and human hepatocellular carcinoma cell line HepG2 were examined. The cell proliferation was assessed using a sulforhodamine B assay. Western blotting and radioactivity assays were used to analyze the phosphorylation of AMPK. The DNA synthesis was analyzed with BrdU incorporation. Nude mice bearing MCF-7 cell xenografts were used to for in vivo study. MTX (50 mg/kg, ip, per week) and AICA riboside (200 mg/kg, ip, every other day) were administered the animals for 2 weeks. The concentrations of AICA riboside and its active metabolite AICA ribotide in the plasma and tumors were measured with HPLC.

Results:

Synergistic cytotoxicity in vitro was observed with MTX (0.1, 0.5, and 1 μmol/L) combined with AICA riboside (0.25–1 mmol/L) in MCF-7 cells, and with MTX (0.5 and 1 μmol/L) combined with AICA riboside (0.5 and 1 mmol/L) in HepG2 cells. MTX (1 μmol/L) significantly enhanced the AICA riboside-induced AMPK activation and BrdU incorporation in both MCF-7 and HepG2 cells. Co-treatment with MTX and AICA riboside exerted more potent inhibition on the tumor growth in nude mice than either drug alone. After injection of AICA riboside (200 mg/kg, iv) in nude mice bearing MCF-7 xenografts, MTX (50 mg/kg, iv) significantly increased the concentrations of AICA riboside and its active metabolite AICA ribotide in tumors.

Conclusion:

MTX and AICA riboside exert synergistic anticancer action against MCF-7 and HepG2 cells in vitro and in vivo. MTX increases the concentration of AICA riboside and its active metabolite AICA ribotide in tumors in vivo.     

Effect of 5-aminoimidazole-4-carboxamide riboside (AICA-r) on isolated thoracic aorta responses in streptozotocin-diabetic rats.

Diabetes mellitus alters the vascular responsiveness to several vasoconstrictors and vasodilators. 5-amino-4-imidazole-carboxamide riboside (AICA-r), a nucleoside corresponding to AICA-ribotide and an intermediate of the de novo pathway of purine biosynthesis, was recently proposed as a new insulinotropic tool in non-insulin-dependent diabetes mellitus. The aim of the present study was to define whether AICA-r affects altered vascular responsiveness to vasoconstrictors and vasodilators in the thoracic aorta of neonatal streptozotocin (STZ)-diabetic rats. The results of this study indicate that a 1-month treatment with AICA-r significantly increases the body weight in diabetic rats; significantly decreases the blood glucose level of diabetic rats (from 302+/-47 to 135+/-11 mg/dL, p<0.001); does not significantly affect the fast, slow, and total components of responses to noradrenaline in all the experimental groups; reverses the increased Emax values of noradrenaline in diabetic rats to near-control values; reverses the completely abolished responses of acetylcholine (pD2 and percent relaxation) in diabetic rats to control values; and reverses the decreased pD2 values of sodium nitroprussiate in diabetic rats to control values. In conclusion, AICA-r treatment in neonatal STZ-diabetic rats improved increased blood glucose levels, accelerated weight gain, reversed endothelial dysfunction, and normalized vascular responses.     

Synthesis and biological activities of 5-deaza analogues of aminopterin and folic acid

N-[p-[[(2,4-Diaminopyrido[2,3-d]pyrimidin-6-yl)methyl] amino]benzoyl]-L-glutamic acid (1a, 5-deazaaminopterin) and the 5-methyl analogue (1b) were synthesized in 14 steps from 5-cyanouracil (4a) and 5-cyano-6-methyluracil (4b), respectively, by exploitation of the novel pyrimidine to pyrido[2,3-d]pyrimidine ring transformation reaction. The 5-cyanouracils 4 were treated with chloromethyl methyl ether to the 1,3-bis(methoxymethyl)uracils (5, which were treated with malononitrile in NaOEt/EtOH to give the pyrido[2,3-d]pyrimidines 6. Diazotization of 6 in concentrated HCl afforded the 7-chloro derivatives 8 in high yield. After reduction of 8, the 7-unsubstituted products 9 were reduced in the presence of Ac2O and the products, 6-(acetamidomethyl)pyridopyrimidines 10, were converted into the 6-acetoxymethyl derivatives 12 via nitrosation. After removal of the N-methoxymethyl groups from 12, the 6-(acetoxymethyl)pyrido[2,3-d]pyrimidine-2,4(1H,3H)-diones 14 were converted into 2,4-diamino-6-(hydroxymethyl)pyrido[2,3-d]pyrimidine (15a) and its 5-methyl analogue 15b by the silylation-amination procedure. Compounds 15 were brominated to the 6-bromomethyl derivatives 16, which were treated with diethyl (p-aminobenzoyl)-L-glutamate, and the products 17 were saponified to afford 5-deazaaminopterin (1a) and its 5-methyl analogue 1b. Compound 1b was also prepared by an alternative procedure in 10 steps from cyanothioacetamide and ethyl beta-(ethoxymethylene)acetoacetate via 2,4-diamino-6-(hydroxymethyl)-5-methylpyrido[2,3-d]pyrimidine (15b). 5-Deaza-5-methylfolic acid (2) was also prepared in four steps from 15b. The aminopterine analogues 1 showed significant anticancer activity in vitro and in vivo, whereas the folic acid analogue 2 did not exhibit any significant toxicity.     

Augmentation of reduced folate carrier-mediated folate/antifolate transport through an antiport mechanism with 5-aminoimidazole-4-carboxamide riboside monophosphate

5-Aminoimidazole-4-carboxamide riboside (AICAR), an agent with diverse pharmacological properties, augments transport of folates and antifolates. This report further characterizes this phenomenon and defines the mechanism by which it occurs. Exposure of HeLa cells to AICAR resulted in augmentation of methotrexate, 5-formyltetrahydrofolate, and 5-methyltetrahydrofolate initial rates and net uptake in cells that express the reduced folate carrier (RFC). This did not occur in cells that express only the proton-coupled folate transporter and accumulated folates by this mechanism. Transport stimulation correlated with the accumulation of 5-aminoimidazole-4-carboxamide ribotide monophosphate (ZMP), the monophosphate derivative of AICAR, within cells as established by liquid chromatography. When ZMP formation was blocked with 5-iodotubercidin, an inhibitor of adenosine kinase, folate transport stimulation by AICAR was absent. When cells first accumulated ZMP and were then exposed to 5-iodotubercidin or AICAR-free buffer, the ZMP level markedly decreased and folate transport stimulation was abolished. Extracellular ZMP inhibited RFC-mediated folate influx, and the presence of intracellular ZMP correlated with inhibition of folate efflux. The data indicate that intracellular ZMP trans-stimulates folate influx and inhibits folate efflux, which, together, produce a marked augmentation in the net cellular folate level. This interaction among ZMP, folates, and RFC, a folate/organic phosphate antiporter, is consistent with a classic exchange reaction. The transmembrane gradient for one transport substrate (ZMP) drives the uphill transport of another (folate) via a carrier used by both substrates, a phenomenon intrinsic to the energetics of RFC-mediated folate transport.     

Synthesis and antifolate properties of 10-alkyl-5,10-dideaza analogues 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.     

Inhibition of papilloma formation by analogues of 7,8-dihydroretinoic acid

The design of analogues of 7,8-dihydroretinoic acid (7,8-dihydro-RA) was based on reported biological activities of this retinoid and its dihydro-TMMP(1) analogue and on structural hypotheses. 7-Oxa-7,8-dihydroretinoids (5, 6) were prepared by O-alkylation of phenoxides by methyl 8-bromo-3,7-dimethyl-2,4,6-octatrienoate. In some cases, C-alkylation also occurred. 7-Aza-8-oxo-7,8-dihydroretinoids (12, 13) were synthesized from benzeneamines and the acyl cyano or bromo derivative of the monomethyl ester of 3,7-dimethyl-2,4,6-octatriene-1,8-dioic acid. These monomethyl ester precursors were synthesized from the known analogous aldehyde via an O-trimethylsilyl cyanohydrin. 7-(2,3,5-Trimethylphenoxy)-3,5-dimethyl-2,4,6-octatrienoic acid (6b) was the most active of the 7-oxa-7,8-dihydro-RAs in inhibiting DMBA-initiated and TPA-promoted mouse-skin papillomas. The ED(50) was about 4-fold that of etretinate. Two additional 7-oxa-7,8-dihydro-RAs exhibited modest activity in the papilloma assay. Some of the 7-oxa-7,8-dihydro-RAs bind to CRABP and RARalpha.