Synthesis and in vitro antitumoral activity of new hydrazinopyrimidine-5-carbonitrile derivatives

A new series of 2-amino-6-(2-alkyl or arylidenehydrazinyl)-4-(dialkylamino)pyrimidine-5-carbonitriles, 5-24, were synthesized in satisfactory overall yield, using 2-amino-4-(dialkylamino)-6-hydrazino-5-pyrimidinecarbonitriles 3, 4, as key intermediates, by applying classical synthetic methods to construct the hydrazone moiety at C-6 of the pyrimidine ring. Hydrazinopyrimidine derivatives 5-24 were evaluated for their in vitro anticancer activity toward cell lines of nine different types of human cancers. Some of the newly prepared compounds demonstrated inhibitory effects on the growth of a wide range of cancer cell lines generally at 10(-5)M level and in some cases at 10(-7)M concentrations.     

Synthesis and pharmacological assessment of derivatives of isoxazolo[4,5-d]pyrimidine

A series of new 5-alkyl and 5-arylisoxazolo[4,5-d]pyrimidinones (5a-g, 6-8) were prepared from 4-amino-3-oxo-isoxazolidine-5-carboxylic acid amide. Some of the aryl derivatives of isoxazolo[4,5-d]pyrimidine were tested pharmacologically in comparison with Diazepam. Compounds 5b-d and 7 demonstrated interesting anxiolytic activity.     

Herpes Simplex Virus Thymidine Kinase Gene-Transfected Tumor Cells; Sensitivity to Antiherpetic Drugs

Abstract

A series of antiherpetic 5-substituted 2′-deoxyuridine derivatives (i. e. BVDU) and guanine derivatives (i. e. ganciclovir) have been evaluated for their cytostatic activity against murine mammary carcinoma FM3A cell lines that are deficient in cytosol thymidine kinase, but transfected by the herpes simplex virus type 1 (HSV-1)- or type 2 (HSV-2)-specified thymidine kinase gene. Most compounds were endowed with a markedly higher cytostatic activity against the HSV TK gene-transfected tumor cells than against wild-type tumor cells. The principal target for cytostatic activity of the BVDU derivatives proved thymidylate synthase, whereas the guanine derivatives inhibited HSV TK gene-transfected tumor cell proliferation by competing with cellular DNA polymerase(s) and subsequent incorporation into the cellular genome.

     

Design, synthesis and activity against human cytomegalovirus of non-phosphorylatable analogs of toyocamycin, sangivamycin and thiosangivamycin

A number of 7-alkyl 4-aminopyrrolo[2,3- pyrimidine derivatives related to toyocamycin, sangivamycin and thiosangivamycin have been prepared and tested for their activity against human cytomegalovirus (HCMV). Only the thioamide substituted derivatives demonstrated biological activity.     

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.     

Biological activity of bis-benzimidazole derivatives on DNA topoisomerase I and HeLa, MCF7 and A431 cells

Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H(2) receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.     

Synthesis of new iso-C-nucleoside analogues from 2-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)ethanal

Treatment of 2-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)ethanal with malononitrile, cyanoacetamide and 2-cyano-N-(4-methoxyphenyl)acetamide, respectively, in the presence of aluminium oxide yielded 2-cyano-4-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)crotonic acid derivatives. Cyclization with sulfur and triethylamine was performed to synthesize the 2-amino-5-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)thiophene-3-carbonic acid derivatives, which were treated with triethyl orthoformate/ammonia and triethyl orthoformate, respectively, to furnish 6-(methyl 2-O-benzyl-4,6-O-benzylidene-3-deoxy-alpha-D-altropyranosid-3-yl)thieno[2.3-d]pyrimidine derivatives. Deprotection in two steps afforded 2-amino-5-(1,6-anhydro-3-deoxy-beta-D-altropyranos-3-yl)thiophene-3-carbonitrile and 6-(1,6-anhydro-3-deoxy-beta-D-altropyranos-3-yl)thieno[2.3-d]pyrimidine derivatives, respectively.     

Synthesis and in vitro cytotoxic and antiviral activities of 1-(2,5,6-trideoxy-6-halogenohept-5-enofuranurononitrile)thymine and derivatives

All 1-(2,5,6-trideoxy-6-halogenohept-5-enofuranurononitrile)thymine and their 3'-O-TBDMS derivatives have been prepared and their configuration established. Some of these compounds are endowed with a cytotoxic or cytostatic activity in cell culture. The single most important factor affecting the cytotoxicity of these compounds is the presence on the molecule of a soft (electrofugal) halogen atom.     

5-Heteroatom-substituted pyrazoles as canine COX-2 inhibitors: Part 2. Structure-activity relationship studies of 5-alkylethers and 5-thioethers

Structure-activity relationship (SAR) studies of novel 2-[3-trifluoromethyl-5-alkyl(thio)ether pyrazo-1-yl]-5-methanesulfonyl pyridine derivatives for canine COX enzymes are described. The 4-cyano-5-alkyl ethers were found to have excellent potency and selectivity, whereas the 5-thioethers were potent but less selective than the ether analogs in a canine whole blood (CWB) COX-2 assay.     

Synthesis, 18F-radiolabelling and biological evaluations of C-6 alkylated pyrimidine nucleoside analogues

Synthesis of pyrimidine derivatives with a side-chain attached to the C-6 of pyrimidine ring (6-14) is reported. Target compounds 8 and 12 were subjected to in vitro phosphorylation tests, determination of their binding affinities to herpes simplex virus (HSV-1) thymidine kinase (TK) and catalytic turnover constants. Fluorinated pyrimidine derivative 12 (40 microM) exhibited better binding affinity for HSV-1 TK than acyclovir (ACV, 170 microM) and ganciclovir (GCV, 48 microM). Catalytic turnover constant (k(cat)) of 12 (0.08 s(-1)) was close to the k(cat) values of ACV (0.10 s(-1)) and GCV (0.10 s(-1)). Furthermore, compounds 8 and 12 showed no cytotoxic effects in HSV-1 TK-transduced and non-transduced cell lines. Besides, compounds 8 and 12 did not exhibit antiviral or cytostatic activities against several viruses and malignant tumor cell lines that were evaluated. The new fluorinated pyrimidine derivative 16 that is phosphorylated by HSV-1 TK could be developed as non-toxic PET-tracer molecule. Thus, 18F labelling of the precursor 14 was performed by nucleophilic substitution using [18F] tetrabutylammonium fluoride as the fluorinating reagent.     

Regulation of deoxyribonucleotide biosynthesis during in vivo bacteriophage T4 DNA replication. Intrinsic control of synthesis of thymine and 5-hydroxymethylcytosine deoxyribonucleotides at precise ratio found in DNA.

The kinetics of the de novo formation of pyrimidine deoxyribonucleotides is the same after infection by wild type bacteriophage T4, which generate very low steady state levels of deoxytibonucleotides, and by T4 DNA synthesis-negative mutatants (Dna-), which accumulate high levels, suggesting that the control is not by a feedback mechanism. In this study, the ratio of the de novo synthesis of dTMP to HmdCMP derivatives was measured by determining the total thymine and 5-hydroxylxytosine (HmCyt) deoxyribonucleotides synthesized by the reductive pathways from [6-3H]uracil including those in DNA and any degradation products excreted into the medium. The ratio of the de novo synthesis of Thy/HmCyt derivatives remained constant at 2.1 +/- 0.1 for at least 45 min after infection by wild type phage, i.e. precisely at the Thy/HmCyt ratio in T4 DNA. On infection by phage mutated in the Dna-genes 32, 41, 44, or 45, the ratio still remained close to 2 to 1 for at least 25 min. Only after the pyrimidine deoxyribonucleotide concentrations reached levels about 100-fold greater than the initial values did the ratio begin to increase. However, a mutant of the structural gene for T4 DNA polymerase showed some increase in ratio by 15 min. Mutants of gene 1 (HmdCMP kinase) were distinct in that the Thy/HmCyt ratio dropped to about 1.0 by 25 min, and then remained quite constant. Uniquely, in these mutants a significant quantity of 5-hydroxymethyluracil or a derivative was found, about 40% being in the medium. The product was shown to be derived by deamination of a 5-HmCyt derivative. All Dna- mutants tested excreted 35 to 50% of their thymine derivatives, mostly as thymine, into the medium. Neither thymine nor 5-hydroxymethyluracil derivates were excreted after wild type phage infection. We propose that pyrimidine deoxyribonucleotide synthesis is regulated at a Thy:HmCyt ratio of 2:1 as an intrinsic property of a complex of enzymes synthesizing and channeling deoxyribonucleotides for T4 DNA replication and not exclusively by effector-sensitive mechanisms.     

Synthesis and biological evaluation of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidines as dual EGFR/ErbB-2 kinase inhibitors

A series of 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-substituted-phenoxy)pyrimidine derivatives were elaborately designed based on the skeleton of Lapatinib, and evaluated for their potential to inhibit epidermal growth factor receptor (EGFR) and ErbB-2 tyrosine kinase activities and antiproliferative activities against A431 and SKOV-3 cell lines. Among these synthesized pyrimidine derivatives, 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-acrylamidophenoxy)pyrimidine (6), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-cyanoacetamidophenoxy)pyrimidine (9), 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-{3-[6-(4-amino)pyrimidinyl]amino) phenoxy}pyrimidine (11) and 4-[3-chloro-4-(3-fluorobenzyloxy)anilino]-6-(3-phenoxyacetamidophenoxy)pyrimidine (14) could significantly inhibit dual EGFR/ErbB-2 kinase activities (IC(50)=37/29 nM, 48/38 nM, 61/42 nM, 65/79 nM, respectively). And compounds 6 and 11 also showed the most potent antiproliferative activities in vitro, with the IC(50) value of 6 being 3.25 μM for A431 and 0.89 μM for SKOV-3, as for 11, 4.24 μM for A431 and 0.71 μM for SKOV-3, respectively. Docking study was also performed to determine the possible binding model.     

Biological activity of bis-benzimidazole derivatives on DNA topoisomerase I and HeLa,MCF7 and A431 cells

Benzimidazoles of both natural and synthetic sources are the key components of many bio-active compounds. Several reports have shown antifungal, antiviral, H₂ receptor blocker and antitumor activities for benzimidazoles and their derivatives. In this study, we synthesized twelve bis-benzimidazole derivatives by selecting di(1H-benzo[d]imidazol-2-yl)methane as the main compound. The numbers of carbons at 2 positions of bis-benzimidazole derivatives were changed from 1 to 4, and derivatives were synthesized with methyl substitutions at 5- and/or 6- positions. The compounds were screened via in vitro plasmid superciol relaxation assays using mammalian DNA topoisomerase I and cytostatic assays were carried out against HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma) and A431 (skin epidermoid carcinoma) cells for selected derivatives. Our results suggest that the malonic acid derivatives of bis-benzimidazoles, namely, bis(5-methyl-1H-benzo[d]imidazol-2-yl)methane and bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)methane, were remarkably active compounds in interfering with DNA topoisomerase I and the former compound was also found to be cytotoxic against MCF7 and A431 cells. The inhibitory effects obtained with these derivatives are significant as these compounds can be potential sources of anticancer agents.     

Synthesis and biological evaluation of new 2,4,6-trisubstituted pyrimidines and their N-alkyl derivatives

A series of new 2,4,6-trisubstituted pyrimidines and their N-alkyl bromide derivatives were prepared based upon methoxy substituted azachalcones as the starting materials. All newly synthesized compounds were screened for their anti-proliferative, cytotoxic, antibacterial activities and DNA/protein binding affinity. In vitro cell proliferation inhibitory and cell cytotoxic effects of 2,4,6-trisubstituted pyrimidines (1–9) and their N-alkyl bromide derivatives (2a-c, 3a-c, 5a-c, 6a-c, 8a-c, 9a-c) were obtained with the help of the 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) cell proliferation, LDH cytotoxicity detection, and microdilution assays. The antimicrobial activity for these compounds was also evaluated following the European Pharmacopoeia 8.0 protocol. The interactions of these compounds with DNA or bovine serum albumin were investigated by the spectrophotometric titration method. When the cytotoxic analysis and anticancer properties of the compounds were examined, most of the compounds significantly exhibited an anti-proliferative potency on cancer cells (IC₅₀ ∼ 2–10 µg/mL) and caused a cytotoxic effect as low as control drugs, 5-fluorouracil, and cisplatin (∼7–15%). Because the compound-DNA adducts are hyperchromic or hypochromic, they caused variations in their spectra. This situation shows they can be linked to DNA by the groove binding mode at a binding constant range of 2.0 × 104 and 2.4 × 105 M⁻¹. The antimicrobial screening results revealed that our new compounds for some human Gram(+) and Gram(−) pathogen bacteria showed remarkable activity with MIC values between <7.81 and 125 µg/mL. Overall, incorporation of alkyl chain to pyrimidines in the generation of N-alkyl bromides has resulted in showing differences in DNA/protein binding affinity, along with anti-proliferative and cytotoxic activity in favor of new compounds.     

Identification of guanine and adenine adducts in DNA alkylated by dibromodulcitol in vitro and in vivo

DNA reacted with dibromodulcitol in neutral solution yielded 3- and 7-alkyl substituted purines after hydrolysis at neutral pH-value at 37°C. The alkylated products were identified by mass spectrometry and by comparison of their UV absorption spectra and chromatographic properties on thin-layer chromatography (TLC) and various columns with those of the corresponding galactitylpurine derivatives obtained by synthetic route from alkylation of the appropriate nucleic bases or nucleosides. The labelled alkylpurines occurring in DNA of Yoshida tumour cells treated with [³H]dibromodulcitol in vivo were also indentified by co-chromatography of labelled DNA hydrolysate with synthetic 3- and 7-alkyl substituted purines. On the basis of the same chromatographic behaviour 3-(1-deoxy-3,6-anhydrogalactit-1-yl)adenine, 7-(1-deoxygalactit-1-yl)guanine, 7-(1-deoxy-3,6-anhydrogalactit-1-yl)guanine and 1,6-di(guanin-7-yl)-1,6-dideoxygalactitol were identified as main alkylated products in tumor cell DNA after in vivo treatment with dibromodulcitol.     

Nucleosides 7(9): synthesis, structure, and biological activity of new 6-arylidenamino-2-thio- and 2-benzylthiopyrimidine N-nucleosides

The condensation of 6-amino-2-thioxo-2,3-dihydro-1H-pyrimidine-4-one [compound (1)] with aromatic aldehydes (2) afforded azomethine derivatives (3). The formed azomethines underwent glycosidation with α-acetobromoglucose (4) to form the corresponding pyrimidine N-glycosides (6) and not S-glycosides (5). The interaction of (3) with 1-O-acetyl-2, 3, 5-tri-O-benzoyl-β-D-ribofuranose (8) afforded the corresponding pyrimidine N-riboside (10) and not S-riboside (9). Deacetylation and debenzoylation of each of (6) and (10) by using methanolic sodium methoxide afforded the corresponding free N-nucleosides (7) and (11), respectively. Next, the reaction of 2-benzylthio-6-benzylidenaminouracil (13) with (4) and (8) did not yield the corresponding protected N-nucleosides (14) and (17), whereas it afforded (15) and (18), respectively. The latter compounds (15) and (18) were stirred in methanolic sodium methoxide to yield the corresponding free N-nucleosides (16) and (19), respectively. The structures of products have been elucidated and reported and also some of the products were screened for their antimicrobial activity. Graphical Abstract:     

Synthesis, cytostatic and anti-HIV evaluations of the new unsaturated acyclic C-5 pyrimidine nucleoside analogues

A series of the novel C-5 alkynyl pyrimidine nucleoside analogues (1-14) in which the sugar moiety was replaced by the conformationally restricted Z- and E-2-butenyl spacer between the phthalimido and pyrimidine ring were synthesized by using Sonogashira cross-coupling reaction. Cytostatic activity evaluation of the novel compounds showed that E-isomers exhibited, in general, better cytostatic activities than the corresponding Z-isomers. E-isomer 14 exhibited the best cytostatic effect against all evaluated malignant cell lines, particularly against hepatocellular carcinoma (Hep G2, IC(50)=4.3microM). However, this compound was also cytotoxic to human normal fibroblasts (WI 38). Its Z-isomer 7 showed highly specific antiproliferative activity against Hep G2 (IC(50)=18microM) and no cytotoxicity to WI 38. Moreover, compounds 3, 4 and 14 expressed some marginal inhibitory activity against HIV-1 and HIV-2.     

Single-strand DNA triple-helix formation

Chemical modification studies provide evidence that single-stranded oligodeoxyribonucleotides can form stable intrastrand triple helices. Two oligonucleotides of opposite polarity were synthesized, each composed of a homopurine-homopyrimidine hairpin stem linked to a pyrimidine sequence which is capable of folding back on the hairpin stem and forming specific Hoogsteen hydrogen bonds. Using potassium permanganate as a chemical modification reagent, we have found that two oligodeoxyribonucleotides of sequence composition type 5'-(purine)8(N)4(pyrimidine)8(N)6(pyrimidine)8-3' and 5'-(pyrimidine)8N6(pyrimidine)8N4(purine)8-3' undergo dramatic structural changes consistent with intrastrand DNA triple-helix formation induced by lowering the pH or raising the Mg2+ concentration. The intrastrand DNA triple helix is sensitive to base mismatches.     

Quantitative assessment of Tet-induced oxidation products of 5-methylcytosine in cellular and tissue DNA

Recent studies showed that Ten-eleven translocation (Tet) family dioxygenases can oxidize 5-methyl-2’-deoxycytidine (5-mdC) in DNA to yield the 5-hydroxymethyl, 5-formyl and 5-carboxyl derivatives of 2’-deoxycytidine (5-HmdC, 5-FodC and 5-CadC). 5-HmdC in DNA may be enzymatically deaminated to yield 5-hydroxymethyl-2’-deoxyuridine (5-HmdU). After their formation at CpG dinucleotide sites, these oxidized pyrimidine nucleosides, particularly 5-FodC, 5-CadC, and 5-HmdU, may be cleaved from DNA by thymine DNA glycosylase, and subsequent action of base-excision repair machinery restores unmethylated cytosine. These processes are proposed to be important in active DNA cytosine demethylation in mammals. Here we used a reversed-phase HPLC coupled with tandem mass spectrometry (LC-MS/MS/MS) method, along with the use of stable isotope-labeled standards, for accurate measurements of 5-HmdC, 5-FodC, 5-CadC and 5-HmdU in genomic DNA of cultured human cells and multiple mammalian tissues. We found that overexpression of the catalytic domain of human Tet1 led to marked increases in the levels of 5-HmdC, 5-FodC and 5-CadC, but only a modest increase in 5-HmdU, in genomic DNA of HEK293T cells. Moreover, 5-HmdC is present at a level that is approximately 2–3 and 3–4 orders of magnitude greater than 5-FodC and 5-CadC, respectively, and 35–400 times greater than 5-HmdU in the mouse brain and skin, and human brain. The robust analytical method built a solid foundation for dissecting the molecular mechanisms of active cytosine demethylation, for measuring these 5-mdC derivatives and assessing their involvement in epigenetic regulation in other organisms and for examining whether these 5-mdC derivatives can be used as biomarkers for human diseases.