Synthesis, cytostatic activity and ADME properties of C-5 substituted and N-acyclic pyrimidine derivatives

The synthesis of the novel 5-alkyl pyrimidine derivatives, 5,6-dihydrofuro[2,3-d]pyrimidines and 5-alkyl N-methoxymethyl pyrimidine derivatives and evaluation of their cytostatic activities are described. The mechanism of antiproliferative effect of 5-(2-chloroethyl)-substituted pyrimidine 3 that exerted the pronounced cytostatic activity was studied in further details on colon carcinoma (HCT116) cells. The cell cycle perturbation analysis demonstrated severe DNA damage (G2/M arrest) pointing to a potential DNA alkylating ability of 3. Preliminary ADME data of 3 and its 6-methylated structural congener (6-Me-3) showed their high permeability and good metabolic stability.     

Relationship between the Structures and Cytotoxic Activities of 1,3,4-Thiadiazolo[3,2-a]pyrimidines

1,3,4-Thiadiazole derivatives having a partial structure of 2-ethylsulfonyl-7-methyl-5H-1,3,4-thiadiazolo[3,2-a]pyrimidin-5-one (TPSO₂-2) were synthesized, and their chemical reactivities and biological activities were investigated. TPSO₂-2 readily reacted with SH compounds and showed a high inhibitory effect against “SH enzyme,” but 2-acetylamino-5-ethylsulfonyl-1,3,4-thiadiazole (AEST), a TPSO₂-2 analog without a pyrimidine structure, did not react with those compounds and showed a smaller inhibitory effect against the enzyme. Furthermore, TPSO₂-2 showed a strong anti-yeast activity, while AEST did not. It is presumed that not only the electron-withdrawing sulfonyl group at the 2-position but also the pseudopurine skeleton appear to be responsible for revealing the biological and chemical activities of TPSO₂-2.     

Developmental changes in the activity of phosphatidylethanolamine N-methyltransferases in rat brain.

A complete separation of myo-inositol 1,4,5-[4,5-(32)P]trisphosphate prepared from human erythrocytes, and myo-[2-3H]inositol 1,3,4-trisphosphate prepared from carbachol-stimulated rat parotid glands [Irvine, Letcher, Lander & Downes (1984) Biochem. J. 223, 237-243], was achieved by anion-exchange high-performance liquid chromatography. This separation technique was then used to study the metabolism of these two isomers of inositol trisphosphate in carbachol-stimulated rat parotid glands. Fragments of glands were pre-labelled with myo-[2-3H]inositol, washed, and then stimulated with carbachol. At 5s after stimulation a clear increase in inositol 1,4,5-trisphosphate was detected, with no significant increase in inositol 1,3,4-trisphosphate. After this initial lag however, inositol 1,3,4-phosphate rose rapidly; by 15s it predominated over inositol 1,4,5-trisphosphate, and continued to rise so that after 15 min it was at 10-20 times the radiolabelling level of the 1,4,5-isomer. In contrast, after the initial rapid rise (maximal within 15s), inositol 1,4,5-trisphosphate levels declined to near control levels after 1 min and then rose again very gradually over the next 15 min. When a muscarinic blocker (atropine) was added after 15 min of carbachol stimulation, inositol 1,4,5-trisphosphate levels dropped to control levels within 2-3 min, whereas inositol 1,3,4-trisphosphate levels took at least 15 min to fall, consistent with the kinetics observed earlier for total parotid inositol trisphosphates [Downes & Wusteman (1983) Biochem. J. 216, 633-640]. Phosphatidylinositol bisphosphate (PtdInsP2) from stimulated and control cells were degraded chemically to inositol trisphosphate to seek evidence for 3H-labelled PtdIns(3,4)P2. No evidence could be obtained that a significant proportion of PtdInsP2 was this isomer; in control tissues it must be less than 5% of the total PtdInsP2 radiolabelled by myo-[2-3H]inositol. These data indicate that, provided that inositol 1,4,5-trisphosphate is studied independently of inositol 1,3,4-trisphosphate, the former shows metabolic characteristics consistent with its proposed role as a second messenger for calcium mobilization. The metabolic profile of inositol 1,3,4-trisphosphate is entirely different, and its function and source remain unclear.     

Synthesis of variously coupled conjugates of D-glucose, 1,3,4-oxadiazole, and 1,2,3-triazole for inhibition of glycogen phosphorylase

5-(O-Perbenzoylated-β-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-β-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-β-D-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid-DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN(3). These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, β-D-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, β-D-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and β-D-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(β-D-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K(i)=854μM, 2-(β-D-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K(i)=745μM).     

High expression levels of pyrimidine metabolic rate–limiting enzymes are adverse prognostic factors in lung adenocarcinoma: a study based on The Cancer Genome Atlas and Gene Expression Omnibus datasets

Reprogramming of metabolism is described in many types of cancer and is associated with the clinical outcomes. However, the prognostic significance of pyrimidine metabolism signaling pathway in lung adenocarcinoma (LUAD) is unclear. Using the Gene Expression Omnibus (GEO) and The Cancer Genome Atlas (TCGA) datasets, we found that the pyrimidine metabolism signaling pathway was significantly enriched in LUAD. Compared with normal lung tissues, the pyrimidine metabolic rate–limiting enzymes were highly expressed in lung tumor tissues. The high expression levels of pyrimidine metabolic–rate limiting enzymes were associated with unfavorable prognosis. However, purinergic receptors P2RX1, P2RX7, P2RY12, P2RY13, and P2RY14 were relatively downregulated in lung cancer tissues and were associated with favorable prognosis. Moreover, we found that hypo-DNA methylation, DNA amplification, and TP53 mutation were contributing to the high expression levels of pyrimidine metabolic rate–limiting enzymes in lung cancer cells. Furthermore, combined pyrimidine metabolic rate–limiting enzymes had significant prognostic effects in LUAD. Comprehensively, the pyrimidine metabolic rate–limiting enzymes were highly expressed in bladder cancer, breast cancer, colon cancer, liver cancer, and stomach cancer. And the high expression levels of pyrimidine metabolic rate–limiting enzymes were associated with unfavorable prognosis in liver cancer. Overall, our results suggested the mRNA levels of pyrimidine metabolic rate–limiting enzymes CAD, DTYMK, RRM1, RRM2, TK1, TYMS, UCK2, NR5C2, and TK2 were predictive of lung cancer as well as other cancers.

     

Synthesis of variously coupled conjugates of d-glucose, 1,3,4-oxadiazole, and 1,2,3-triazole for inhibition of glycogen phosphorylase

5-(O-Perbenzoylated-β-d-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-β-d-glucopyranosyl cyanide by Bu₃SnN₃ or Me₃SiN₃–Bu₂SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-β-d-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid–DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN₃. These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, β-d-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, β-d-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and β-d-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(β-d-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K_i = 854 μM, 2-(β-d-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K_i = 745 μM).     

In vitro inhibition effects of some new sulfonamide inhibitors on human carbonic anhydrase I and II

A new series of aromatic and heterocyclic sulfonamides, including six new derivatives, 2-(3-cyclohexene-1-carbamido)-1,3,4-thiadiazole-5-sulfonamide (CCTS), 4-(3-cyclohexene-1-carbamido) methyl-benzenesulfonamide (CCBS), 2-(9-octadecenoylamido)-1,3,4-thiadiazole-5-sulfonamide (ODTS), 2-(4,7,10-trioxa-tetradecanoylamido)-1,3,4-thiadiazole-5-sulfonamide (TDTS), 2-(coumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (COTS) and 2-(8-methoxycoumarine-3-carbamido)-1,3,4-thiadiazole-5-sulfonamide (MCTS), has been investigated. These sulfonamides were assayed for inhibition of human carbonic anhydrase I (hCA-I) and human carbonic anhydrase II (hCA-II) which were purified by affinity chromatography.     

Synthesis, antimicrobial, and anti-HIV-1 activity of certain 5-(1-adamantyl)-2-substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3-substituted aminomethyl-1,3,4-oxadiazoline-2-thiones

The reaction of 5-(1-adamantyl)-1,3,4-oxadiazoline-2-thione 2 with iodoethane, 2-dimethylaminoethyl chloride hydrochloride or 2-piperidinoethyl chloride hydrochloride in ethanolic potassium hydroxide yielded the corresponding 5-(1-adamantyl)-2-ethyl or substituted ethylthio-1,3,4-oxadiazoles 3a-c. Interaction of 2 with formaldehyde solution and primary aromatic amines or 1-substituted piperazines, in ethanol at room temperature yielded the corresponding 5-(1-adamantyl)-3-arylaminomethyl-1,3,4-oxadiazoline-2-thiones 4a-m or 5-(1-adamantyl)-3-(4-substituted-1-piperazinylmethyl)-1,3,4-oxadiazoline-2-thiones 5a-h, respectively. All the synthesized compounds were tested for in vitro activities against certain strains of Gram-positive and Gram-negative bacteria and the yeast-like pathogenic fungus Candida albicans. Compounds 2, 5a, and 5e were found as the most active derivatives, particularly against the Gram-positive bacteria. In addition, the antiviral activity of compounds 2, 4a-m, and 5a-h against HIV-1 using the XTT assay was carried out. Compound 2 produced 100%, 43%, and 37% reduction of viral replication at 50, 10, and 2microg/mL concentrations, respectively.     

Regulation of inositol phosphate metabolism in chemoattractant-stimulated human polymorphonuclear leukocytes. Definition of distinct dephosphorylation pathways for IP3 isomers

The metabolism of the calcium mobilizing inositol-1,4,5-trisphosphate (IP3) isomer was studied in myo-[3H]inositol labeled, chemoattractant-stimulated human polymorphonuclear neutrophils (PMNs), and in PMN lysates. It was determined that 1,4,5-IP3 is metabolized in vitro by two distinct pathways: 1) by sequential dephosphorylation to 1,4-IP2, 4-IP1, and inositol or 2) by ATP dependent conversion to 1,3,4,5-IP4, followed by dephosphorylation to form 1,3,4-IP3, 3,4-IP2, 3-IP1, and inositol. In PMNs stimulated with 0.1 microM N-formyl-methionyl-leucyl-phenylalanine (fMet-Leu-Phe), 1,4-IP2, 1,4,5-IP3, and IP4, were elevated by 5 s; whereas production of 1,3,4-IP3, 3,4-IP2, and IP1 occurred only after an initial lag (approximately 15 s). The predominant IP1 isomer formed in fMet-Leu-Phe-stimulated cells was 4-IP1. Production of 1,3,4-IP3 and 3,4-IP2 was markedly reduced (17 and 35% of control, respectively) in fMet-Leu-Phe-stimulated cells pretreated to prevent a rise in intracellular calcium ([Ca2+]i). PMNs were also stimulated with leukotriene B4 (LTB4) since this agent is a poor activator of the respiratory burst compared to fMet-Leu-Phe. Peak levels (5 s) of 1,4,5-IP3 were equivalent after stimulation with 0.1 microM fMet-Leu-Phe versus 0.1 microM LTB4 (320 +/- 38% versus 378 +/- 38% of control values, respectively; n = 5); however, at 30 s, 1,4,5-IP3 remained elevated only in fMet-Leu-Phe-stimulated cells. Similarly, elevation of [Ca2+]i was more prolonged in response to 0.1 microM fMet-Leu-Phe (greater than 3 min) versus LTB4 (1 min). Thus, signal transduction in PMNs may be modulated by both the duration of the initial 1,4,5-IP3 signal and by the metabolic pathway(s) utilized to convert this IP3 isomer to other, potentially active inositol phosphate products.     

Profiles of pyrimidine biosynthesis,salvage and degradation in disks of potato (Solanum tuberosum L.) tubers

In order to obtain general metabolic profiles of pyrimidine ribo- and deoxyribonucleotides in potato (Solanum tuberosum L.) plants, the in situ metabolic fate of various (14)C-labelled precursors in disks from growing potato tubers was investigated. The activities of key enzymes in potato tuber extracts were also studied. The following results were obtained. Of the intermediates in de novo pyrimidine biosynthesis, [(14)C]carbamoylaspartate was converted to orotic acid and [2-(14)C]orotic acid was metabolized to nucleotides and RNA. UMP synthase, a bifunctional enzyme with activities of orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23), exhibited high activity. The rates of uptake of pyrimidine ribo- and deoxyribonucleosides by the disks were high, in the range 2.0-2.8 nmol (g FW)(-1) h(-1). The pyrimidine ribonucleosides, uridine and cytidine, were salvaged exclusively to nucleotides, by uridine/cytidine kinase (EC 2.7.1.48) and non-specific nucleoside phosphotransferase (EC 2.7.1.77). Cytidine was also salvaged after conversion to uridine by cytidine deaminase (EC 3.5.4.5) and the presence of this enzyme was demonstrated in cell-free tuber extracts. Deoxycytidine, a deoxyribonucleoside, was efficiently salvaged. Since deoxycytidine kinase (EC 2.7.1.74) activity was extremely low, non-specific nucleoside phosphotransferase (EC 2.7.1.77) probably participates in deoxycytidine salvage. Thymidine, which is another pyrimidine deoxyribonucleoside, was degraded and was not a good precursor for nucleotide synthesis. Virtually all the thymidine 5'-monophosphate synthesis from thymidine appeared to be catalyzed by phosphotransferase activity, since little thymidine kinase (EC 2.7.1.21) activity was detected. Of the pyrimidine bases, uracil, but not cytosine, was salvaged for nucleotide synthesis. Since uridine phosphorylase (EC 2.4.2.3) activity was not detected, uracil phosphoribosyltransferase (EC 2.4.2.9) seems to play the major role in uracil salvage. Uracil was degraded by the reductive pathway via beta-ureidopropionate, but cytosine was not degraded. The activities of the cytosine-metabolizing enzymes observed in other organisms, pyrimidine nucleoside phosphorylase (EC 2.4.2.2) and cytosine deaminase (EC 3.5.4.1), were not detected in potato tuber extracts. Operation of the de novo synthesis of deoxyribonucleotides via ribonucleotide reductase and of the salvage pathway of deoxycytidine was demonstrated via the incorporation of radioactivity from both [2-(14)C]cytidine and [2-(14)C]deoxycytidine into DNA. A novel pathway converting deoxycytidine to uracil nucleotides was found and deoxycytidine deaminase (EC 3.5.4.14), an enzyme that may participate in this pathway, was detected in the tuber extracts.     

Investigation of the effects of some sulfonamide derivatives on the activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase from human erythrocytes

In this study, the in vitro effects of some sulfonamide derivatives, which are carbonic anhydrase inhibitors, on the enzymes activities of glucose-6-phosphate dehydrogenase, 6-phospho gluconate dehydrogenase and glutathione reductase were investigated. For this purpose, these three enzymes were purified from human erythrocytes. Purification procedure composed of four steps; preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and gel filtration chromatography on Sephadex G-200. 5-(3alpha-Hydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3alpha,12alpha-Dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3alpha,7alpha,12alpha-Trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3), 5-(3alpha,Acetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (4), 5-(3alpha,7alpha,12alpha-Triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (5), 5-(3,7,12-Trioxo-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (6), acetazolamide, and dorzolamide were tested in this experiment. Compounds 3, 5, and dorzolamide showed inhibitory effects on the activity of 6-phosphogluconate dehydrogenase, and I(50) values and K(i) constants were calculated as 0.0601 mM, 0.00253 mM, and 1.41 mM and 0.0878 +/- 0.0274 mM, 0.0042 +/- 0.0009 mM, and 3.1446 +/- 0.2081 mM, respectively. Glutathione reductase was also inhibited by 1 and 2. I(50) values and K(i) constants were 0.0471 mM and 0.0723 +/- 0.0388 mM for 1 and 0.0045 mM and 0.0061 +/- 0.0014 mM, for 2. If these sulfonamide derivatives are proposed as drugs, some of which are being used in glaucoma treatment such as acetazolamide and dorzolamide, these results should be taken into consideration concerning via these enzymes.     

Pentacycle derivatives as cannabinoid CB1 receptor ligands

Cannabinoid CB-1 receptors have been the focus of extensive studies since the first clinical results of rimonabant (SR141716) for the treatment of obesity and obesity-related metabolic disorders were reported in 2001. To further evaluate the properties of CB receptors, we have designed and efficiently prepared a series of pentacycle derivatives. Five of the new compounds which displayed high in vitro rCB1 binding affinities were assayed for binding to hCB2 receptor. Noticeably, 2-(5-(4-bromophenyl)-1-(2,4-dichlorophenyl)-4-(5-methyl-1,3,4-thiadiazol-2-yl)-1H-pyrazol-3-yl)-5-(1-(trifluoromethyl)cyclopropyl)-1,3,4-oxadiazole (16l) demonstrated good binding affinity and decent selectivity for rCB1 receptor (IC₅₀ = 1.72 nM, hCB2/rCB1 = 142).     

The in vitro and in vivo inhibitory effects of some sulfonamide derivatives on rainbow trout (Oncorhynchus mykiss) erythrocyte carbonic anhydrase activity

The in vitro and in vivo inhibitory effects of 5-(3alpha, 12alpha-dihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (1), 5-(3alpha, 7alpha, 12alpha-trihydroxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (2), 5-(3alpha, 7alpha, 12alpha-triacetoxy-5-beta-cholanamido)-1,3,4-thiadiazole-2-sulfonamide (3) and acetazolamide on rainbow trout (Oncorhynchus mykiss) (RT) erythrocyte carbonic anhydrase (CA) were investigated. The RT erythrocyte CA was obtained by affinity chromatography with a yield of 20.9%, a specific activity of 422.5EU/mg protein and a purification of 222.4-fold. The purity of the enzyme was confirmed by SDS-PAGE. Inhibitory effects of the sulfonamides and acetazolamide on the RT erythrocyte CA were determined using the CO2-Hydratase method in vitro and in vivo studies. From in vitro studies, it was found that all the compounds inhibited CA. The obtained I50 value for the sulfonamides (1), (2) and (3) and acetazolamide were 0.83, 0.049, 0.82 and 0.052 microM, respectively. From in vivo studies, it was observed that CA was inhibited by the sulfonamides (1), (2) and (3) and acetazolamide.     

Relationship Between Conformation and Antiviral Activity -IV. 5-Ethyl-2′-Deoxyurldine and 5-Ethyl-2′-Deoxycytidine +

Abstract

5-Ethyl-2′-deoxyuridine(EtdUrd), though a potent inhibitor of herpes simplex virus (HSV) replication, is rapidly catabolized to produce an inactive pyrimidine base by thymidine and/or uridine phosphorylases. 5-Ethyl-2′-deoxycytidine (EtdCyd) was synthesized to confer metabolic stability and thus improve efficacy against systemic HSV infections. EtdCyd was inactive against HSV in the presence or absence of deaminase inhibitors in VERO cells up to 2 mM. The relationship between molecular conformation and antiherpes activity for EtdUrd and EtdCyd is discussed.

     

Carbonic anhydrase inhibitors. The X-ray crystal structure of human isoform II in adduct with an adamantyl analogue of acetazolamide resides in a less utilized binding pocket than most hydrophobic inhibitors

We investigated the inhibitory activity of several 1,3,4-thiadiazole-sulfonamides against all catalytically active CA (EC 4.2.1.1), CA I–XV. The tail derivatizing the 5-position in the 1,3,4-thiadiazole-2-sulfonamide scaffold was observed to be critical as an inhibitory determinant of these compounds. The high resolution X-ray crystal structure of hCA II in complex with 5-(1-adamantylcarboxamido)-1,3,4-thiadiazole-2-sulfonamide, showed the adamantyl moiety of the inhibitor residing in a less utilized binding pocket than that of most hydrophobic inhibitors, lined by the amino acid residues Ile91, Val121 and Phe131. This binding site may explain the diverse inhibition profiles of 5-carboxamide- and sufonamide-derivatized 1,3,4-thiadiazole-2-sulfonamides and offers a hot spot for designing isoform selective inhibitors, considering that residues 91 and 131 are highly variable among the 13 catalytically active isoforms.     

Synthesis and analgesic/anti-inflammatory evaluation of fused heterocyclic ring systems incorporating phenylsulfonyl moiety

A series of pyrazolo[1,5-a]pyrimidine, triazolo[1,5-a]pyrimidine, and pyrimido[1,2-a]benzimidazole ring systems incorporating phenylsulfonyl moiety were synthesized via the reaction of 3-(N,N-dimethylamino)-1-aryl-2-(phenylsulfonyl)prop-2-en-1-one derivatives 2a,b with appropriate nitrogen nucleophiles. The analgesic and anti-inflammatory activities of the newly synthesized compound were investigated in vivo. 3-Bromo-2-phenyl-6-(phenylsulfonyl)-7-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5e) was found to have an excellent analgesic activity in comparison with indomethacin as a reference drug, while the highest anti-inflammatory effect was observed in the case of 2-(4-bromophenyl)-6-(phenylsulfonyl)-5-(4-methylphenyl)pyrazolo[1,5-a]pyrimidine (5d). From the structure-activity relationship (SAR) point of view, the analgesic/anti-inflammatory activity of pyrazolo[1,5-a]pyrimidine derivatives was found to be much higher than triazolo[1,5-a]pyrimidine and pyrimido[1,2-a]benzimidazole derivatives.     

Metabolism of D-myo-inositol 1,3,4,5-tetrakisphosphate by rat liver, including the synthesis of a novel isomer of myo-inositol tetrakisphosphate.

1. We have studied the metabolism of Ins(1,3,4,5)P4 (inositol 1,3,4,5-tetrakisphosphate) by rat liver homogenates incubated in a medium resembling intracellular ionic strength and pH. 2. Ins(1,3,4,5)P4 was dephosphorylated to a single inositol trisphosphate product, Ins(1,3,4)P3 (inositol 1,3,4-trisphosphate), the identity of which was confirmed by periodate degradation, followed by reduction and dephosphorylation to yield altritol. 3. The major InsP2 (inositol bisphosphate) product was inositol 3,4-bisphosphate [Shears, Storey, Morris, Cubitt, Parry, Michell & Kirk (1987) Biochem. J. 242, 393-402]. Small quantities of a second InsP2 product was also detected in some experiments, but its isomeric configuration was not identified. 4. The Ins(1,3,4,5)P4 5-phosphatase activity was primarily associated with plasma membranes. 5. ATP (5 mM) decreased the membrane-associated Ins(1,4,5)P3 5-phosphatase and Ins(1,3,4,5)P4 5-phosphatase activities by 40-50%. This inhibition was imitated by AMP, adenosine 5'-[beta gamma-imido]triphosphate, adenosine 5'-[gamma-thio]triphosphate or PPi, but not by adenosine or Pi. A decrease in [ATP] from 7 to 3 mM halved the inhibition of Ins(1,3,4,5)P4 5-phosphatase activity, but the extent of inhibition was not further decreased unless [ATP] less than 0.1 mM. 6. Ins(1,3,4,5)P4 5-phosphatase was insensitive to 50 mM-Li+, but was inhibited by 5 mM-2,3-bisphosphoglycerate. 7. The Ins(1,3,4,5)P4 5-phosphatase activity was unchanged by cyclic AMP, GTP, guanosine 5'-[beta gamma-imido]triphosphate or guanosine 5'-[gamma-thio]triphosphate, or by increasing [Ca2+] from 0.1 to 1 microM. 8. Ins(1,3,4)P3 was phosphorylated in an ATP-dependent manner to an isomer of InsP4 that was partially separable on h.p.l.c. from Ins(1,3,4,5)P4. The novel InsP4 appears to be Ins(1,3,4,6)P4. Its metabolic fate and function are not known.     

Novel free fatty acid receptor 1 (GPR40) agonists based on 1,3,4-thiadiazole-2-carboxamide scaffold

Free fatty acid receptor 1 (FFA1), previously known as GPR40 is a G protein-coupled receptor and a new target for treatment of type 2 diabetes. Two series of FFA1 agonists utilizing a 1,3,4-thiadiazole-2-caboxamide scaffold were synthetized. Both series offered significant improvement of the potency compared to the previously described 1,3,4-thiadiazole-based FFA1 agonists and high selectivity for FFA1. Molecular docking predicts new aromatic interactions with the receptor that improve agonist potency. The most potent compounds from both series were profiled for in vitro ADME properties (plasma and metabolic stability, Log D, plasma protein binding, hERG binding and CYP inhibition). One series suffered very rapid degradation in plasma and in presence of mouse liver microsomes. However, the other series delivered a lead compound that displayed a reasonable ADME profile together with the improved FFA1 potency.     

Synthetic studies on mitotic kinesin Eg5 inhibitors: Synthesis and structure–activity relationships of novel 2,4,5-substituted-1,3,4-thiadiazoline derivatives

The 2,4,5-substituted-1,3,4-thiadiazoline derivative 1a has been identified as a new class of mitotic kinesin Eg5 inhibitor. With the aim of enhancement of the mitotic phase accumulation activity, structure optimization of side chains at the 2-, 4-, and 5-positions of the 1,3,4-thiadiazoline ring of 1a was performed. The introduction of sulfonylamino group at the side chain at the 5-position and bulky acyl group at the 2- and 4-position contributed to a significant increase in the mitotic phase accumulation activity and Eg5 inhibitory activity. As a result, a series of optically active compounds exhibited an increased antitumor activity in a human ovarian cancer xenograft mouse model that was induced by oral administration.     

Reverse mutation tests in Salmonella typhimurium and chromosomal aberration tests in mammalian cells in culture on fluorinated pyrimidine derivatives

Reverse mutation (Ames) tests with Salmonella typhimurium TA98, TA100 and TA1537, and chromosomal aberration tests in vitro with a Chinese hamster fibroblast cell line (CHL), were carried out on fluorinated pyrimidine derivatives, such as 5-fluorouracil (5-FU), 1-(2-tetrahydrofuryl)-5-fluorouracil (FT), 5-fluorodeoxyuridine (FUdR), 1,3-bis(2-tetrahydrofuryl)-5-fluorouracil (FD-1) and a mixture of uracil and FT in the molar ratio 4 : 1 (UFT) (Fujii et al., 1978). For comparison, similar tests were also carried out on 4 anti-metabolic agents, a metabolite of FD-1 and a component of UFT, such as cytosine-1-beta-D-arabinofuranoside (AraC), 6-mercaptopurine (6-MP), 6-thioguanine (6-TG), 8-azaguanine (8-AG), 3-(2-tetrahydrofuryl)-5-fluorouracil (3-FT) and uracil. The anti-bacterial action of 4 fluorinated pyrimidine derivatives such as 5-FU, FT, FD-1 and UFT to TA100 was tested under the condition that buffer, S9 mix, S9 and albumin were present. 6-MP was only positive in the Ames test with TA100 in the system without S9 mix, while all others failed to show mutagenic activity. On the other hand, all compounds tested, except uracil, induced chromosomal aberrations on CHL cells in the system without metabolic activation. FT was degraded by S9, but there was no significant difference in the killing activity of FT among with buffer, S9 mix and albumin. The killing activity of 5-FU was the strongest with buffer, and it was slightly binding to albumin. The killing activity of 5-FU was mostly decreased by S9 mix. FD-1 showed the strongest anti-bacterial action when S9 mix was present but it was degraded by S9. UFT showed no anti-bacterial action in any conditions.