In-vitro cytotoxicity and cell cycle analysis of two novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC(50) of 3-5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G(1) phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

Alveolar macrophage cytotoxic activity is inhibited by 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), a carcinogenic component of cigarette smoke

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a carcinogenic compound of cigarette smoke that generates electrophilic intermediates capable of damaging DNA. Recently, we have shown that NNK can modulate mediator production by alveolar macrophages (AM) and bronchial and alveolar epithelial cells, suggesting that cigarette smoke can alter lung immune response. Thus, we investigated the effect of NNK and cigarette smoke extract (CSE) on AM capacity to eliminate tumoral cells. Rat AM cell line, NR8383, was treated with NNK (500 μM) or CSE (3%) and stimulated with lipopolysaccharide (10 ng/ml). The release of cytotoxic mediators, tumor necrosis factor (TNF) and reactive oxygen species (ROS), was measured in cell-free supernatants using ELISA and superoxide anion production. TNF- and ROS-dependent cytotoxicity were studied using a ⁵¹Chromium-release assay and WEHI-164 and P-815 cell lines. Treatment of AM with NNK and CSE for 18 h significantly inhibited AM TNF release. CSE exposure resulted in a significant increase of ROS production, whereas NNK did not. TNF-dependent cytotoxic activity of NR8383 and freshly isolated rat AM was significantly inhibited after treatment with NNK and CSE. Interestingly, although ROS production was stimulated by CSE and not affected by NNK, CSE inhibited AM ROS-dependent cytotoxicity. These results suggest that NNK may be one of the cigarette smoke components responsible for the reduction of pulmonary cytotoxicity. Thus, NNK may have a double pro-carcinogenic effect by contributing to DNA adduct formation and inhibiting AM cytotoxicity against tumoral cells.     

In-Vitro Cytotoxicity and Cell Cycle Analysis of Two Novel bis-1,2, 4-triazole derivatives: 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl)-1,2,4-triazol-3-yl]-butane (MNP-16)

In the present study, we have tested the cytotoxic and DNA damage activity of two novel bis-1,2,4 triazole derivatives, namely 1,4-bis[5-(5-mercapto-1,3,4-oxadiazol-2-yl-methyl)-thio-4-(p-tolyl)-1,2,4-triazol-3-yl]-butane (MNP-14) and 1,4-bis[5-(carbethoxy-methyl)-thio-4-(p-ethoxy phenyl) -1,2,4-triazol-3-yl]-butane (MNP-16). The effect of these molecules on cellular apoptosis was also determined. The in-vitro cytotoxicity was evaluated by a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay as well as Trypan blue dye exclusion methods against human acute lymphoblastic leukemia (MOLT4) and lung cancer cells (A549). Our results showed that MNP-16 induced significant cytotoxicity (IC₅₀ of 3–5 μM) compared with MNP-14. The cytotoxicity induced by MNP-16 was time and concentration dependent. The cell cycle analysis by flow cytometry (fluorescence-activated cell sorting [FACS]) revealed that though there was a significant increase in the apoptotic population (sub-G₁ phase) with an increased concentration of MNP-14 and 16, there was no cell cycle arrest. Further, the comet assay results indicated considerable DNA strand breaks upon exposure to these compounds, thereby suggesting the possible mechanism of cytotoxicity induced by MNP-16. Hence, we have identified a novel molecule (MNP-16) which could be of great clinical relevance in cancer therapeutics.     

3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones: a novel group of cytotoxic agents

A series of novel 3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones 3 have been synthesized which displayed potent cytotoxicity towards human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. In contrast, the related N-arylmaleamic acids 4 possessed little or no cytotoxicity in these four screens. Molecular modeling revealed certain interplanar and bond angles and interatomic distances which were perceived to contribute to the observed bioactivity as well as providing suggestions for future structural modifications of the piperidones 3. Evaluation of representative compounds in series 3 and 4 on the activity of human N-myristoyltransferase revealed that, at the maximum concentration utilized, namely 250 microM, only weak inhibiting properties were displayed by some of the compounds in series 4. Various members of series 3 and 4 were well tolerated in mice.     

Synthesis, cytotoxic effect and antiviral activity of 1-(beta-D-arabinofuranosyl)-5-bromo-N4-substituted cytosine and 1-(beta-D-arabinofuranosyl)-5-bromo-4-methoxypyrimidin-2(1H)-one derivatives

A convenient and mild synthesis of 5-bromo-N4-substituted-1-(beta-D-arabinofuranosyl)cytosine and 5-bromo-O4-methyl-1-(beta-D-arabinofuranosyl)pyrimidin-2(1H)-one derivatives by selective oxyfunctionalization of the corresponding 4-thionucleosides with 3,3-dimethyldioxirane is reported. The cytotoxicity and the antiviral activity against parainfluenza 1 (Sendai virus) of all new synthesized products are also reported.     

3,5-Bis(Phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones: A Novel Group of Cytotoxic Agents

A series of novel 3,5-bis(phenylmethylene)-1-(N-arylmaleamoyl)-4-piperidones 3 have been synthesized which displayed potent cytotoxicity towards human Molt 4/C8 and CEM T-lymphocytes as well as murine P388 and L1210 leukemic cells. In contrast, the related N-arylmaleamic acids 4 possessed little or no cytotoxicity in these four screens. Molecular modeling revealed certain interplanar and bond angles and interatomic distances which were perceived to contribute to the observed bioactivity as well as providing suggestions for future structural modifications of the piperidones 3. Evaluation of representative compounds in series 3 and 4 on the activity of human N-myristoyltransferase revealed that, at the maximum concentration utilized, namely 250?μM, only weak inhibiting properties were displayed by some of the compounds in series 4. Various members of series 3 and 4 were well tolerated in mice.     

Pt(II) complexes with N-(3-pyridyl)-2-(4-(trifluoromethyl)phenyl)diazenecarboxamide and their reactions with glutathione

The reaction between [PtCl(dmso)(en)]Cl (dmso=dimethyl sulfoxide, en=ethylenediamine) and N-(3-pyridyl)-2-(4-(trifluoromethyl)phenyl)diazenecarboxamide (L) was studied using multinuclear NMR spectroscopy. The water-soluble complexes [PtCl(en)(L-N1)](+) (1) and [Pt(en)(L-N1)(2)](2+) (2) were isolated and their reactions with glutathione (GSH) were investigated to assess the oxidation properties of coordinated L. Both species 1 and 2 oxidized GSH to GSSG, while the reduced form of L (semicarbazide, SL) remained coordinated to Pt(2+). In complex 1 the labile chloride ion was substituted by the thiol moiety of GSH, which gave rise to the release of en in excess GSH over a period of 7 days. Complexes [PtCl(dmso)(en)]Cl, 1, 2 and ligand L were tested against T24 bladder carcinoma cells. Ligand L and complexes 1 and 2 showed higher cytotoxicity than [PtCl(dmso)(en)]Cl.     

Reversal of multidrug resistance by 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide through the reversible inhibition of P-glycoprotein

Overexpression of P-glycoprotein (P-gp) is one of the major obstacles to successful cancer chemotherapy. In this study, we examined the ability of 4-chloro-N-(3-((E)-3-(4-hydroxy-3-methoxyphenyl)acryloyl)phenyl)benzamide (C-4) to reverse multidrug resistance (MDR) in P-gp expressing KBV20C cells. Treatment of KBV20C cells with C-4 led to a dramatic increase in paclitaxel- or vincristine-induced cytotoxicity without any cytotoxicity by itself. In parallel, C-4 treatment caused an increase in apoptotic cell death by paclitaxel or vincristine. Furthermore, C-4 treatment significantly increases in intracellular accumulation of fluorescent P-gp substrate rhodamine 123, indicating that C-4 treatment leads to reversal of the MDR phenotype resulting from an increased accumulation of anticancer drugs by inhibiting drug efflux function of P-gp. This notion is further supported by the observation that C-4 treatment potentiates paclitaxel-induced G(2)/M arrest of the cell cycle. In addition, the drug efflux function of P-gp was reversibly inhibited by C-4 treatment, while the expression level of P-gp was not affected. Collectively, our results describe the potential of C-4 to reverse the P-gp-mediated MDR phenotype through reversible inhibition of P-gp function, which may make it an attractive new agent for the chemosensitization of cancer cells.     

N-alkylated cyclen cobalt(III) complexes of 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol DNA alkylating agent as hypoxia-activated prodrugs

A series of cobalt complexes of the potent DNA minor groove alkylator 1-(chloromethyl)-3-(5,6,7-trimethoxyindol-2-ylcarbonyl)-2,3-dihydro-1H-pyrrolo[3,2-f]quinolin-5-ol (seco-6-azaCBI-TMI) were prepared from a series of N-substituted cyclen ligands. The final N-substituted complexes carried formal overall charges ranging from +2 to -2 and showed limited improvements in solubility. They showed similar stabilities to that of the complex with the unsubstituted cyclen ligand, and large but variable attenuation of the cytotoxicity of the free alkylator (2-30-fold), compared to 150-fold for the unsubstituted ligand. However, they had oxic/hypoxic ratios (2-22-fold) comparable to that of the unsubstituted cyclen complex (5).     

A 2-nitroimidazole carbamate prodrug of 5-amimo-1-(chloromethyl)-3-[(5,6,7-trimethoxyindol-2-yl)carbony l]-1,2-dihydro-3H--benz[E]indole (amino-seco-CBI-TMI) for use with ADEPT and GDEPT.

The synthesis of a 2-nitroimidazol-5-ylmethyl carbamate prodrug 10 of the potent minor groove alkylating agent amino-seco-CBI-TMI 3 is described. Chemical, radiolytic, and enzymic reductions of a model 2-nitroimidazol-5-yl carbamate 8 show release of the amine effector upon reduction. Prodrug 10 gives a ten fold increase in cytotoxicity against human ovarian carcinoma SKOV3 cells in the presence of E. coli B nitroreductase (NTR) and a 21-fold increase in cytotoxicity against a SKOV3 cell line (SC3.2) transfected with the gene for NTR. The cytotoxicity of 10 increased 15- to 40-fold under hypoxia. Prodrug 10 has significant potential as a prodrug for use in ADEPT and GDEPT applications, and as a hypoxia-selective cytotoxin.     

6-Bicycloaryl substituted (S)- and (R)-5,6-dihydro-2H-pyran-2-ones: Asymmetric synthesis,and anti-proliferative properties

(R)-Goniothalamin, is a member of styryl lactones, possesses selective cytotoxicity against cancer cell lines. In this work, replacement of styryl substituent with 2-naphthyl and 3-quinoyl gave new analogues which may have less conformational changes compared to the lead compound. Anti-proliferative tests indicated that 2-naphthyl substituted (R)-5,6-dihydro-2H-pyran-2-one has slightly better cytotoxicity than (R)-goniothalamin. To clarify the effect of 2-naphthyl substituent additional aryl substituted (R)-5,6-dihydro-2H-pyran-2-ones have been synthesized enantioselectively and tested against PC-3 and MCF-7 cell lines.     

Non-classical antifolates, 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines and 2,4-Diamino-6(5H)-oxopyrimidines,synthesis and antitumor studies

A series of non-classical antifolates, namely 5-(N-phenylpyrrolidin-3-yl)-2,4,6-triaminopyrimidines (25a-i) and 2,4-diamino-(N-phenylpyrrolidin-3-yl)-6(5H)-oxopyrimidines (26a,b,c,f,h,i) was synthesized and evaluated for their in vitro cytotoxicity. Reacting aniline derivatives with 1,4-dibromo-2-butanol gave 1-phenyl-3-pyrrolidinols (19a--i), which were oxidized to pyrrolidin-3-ones (20a-i). The Knoevenagel reaction of 20a-i with malononitrile or ethyl cyanoacetate gave 3-(dicyanomethylene)- (21a-i) and 3-[cyano(ethoxycarbonyl)methylene]-pyrrolidines (22a,b,c,f,h,i), respectively, which were subsequently reduced to the corresponding 3-(dicyano)methyl- or 3-[cyano(ethoxycarbonyl)methyl)]pyrrolidines (23a-i and 24a,b,c,f,h,i, respectively). Condensation of either 23a-i or 24a,b,c,f,h,i with guanidine afforded the target compounds. The cytotoxicity of these compounds was evaluated based on their ability to inhibit various human tumors (human colon adenocarcinoma COLO 205, lung carcinoma H23 and its adriamycin resistant cell line H23/0.3, T-cell leukemia MOLT-4, promyelocytic leukemia HL-60, and T-cell acute lymphocytic leukemia CCRF-CEM) cell growth in culture. These studies revealed that the 2,4,6-triaminopyrimidine derivatives were more cytotoxic than the 2,4-diamino-6(5H)-oxopyrimidine counter parts, in which the latter was inactive in all testing systems. The 2,4,6-triaminopyrimidine derivatives bearing halogen substituent on the phenyl ring (25f,h,i) were cytotoxic in all cultured leukemia cell growth. Among these compounds, 5-(4-fluoro and 4-chlorophenyl)-2,4,6-triaminopyrimidines (25e and 25h, respectively) were more potent than methotrexate (MTX) in inhibiting of H23/0.3 cell growth. These compounds inhibit the folate metabolic pathways as indicated by tritium release from [5-3H]deoxyuridine in MTX sensitive human fibrosarcoma HT-1080 cells. Dihydrofolate reductase is the major target for 25f,h,i, as shown by leucovorin (LV) rescue of MTX cytotoxicity.     

Cytotoxicity of 6,16-disubstituted analogues of (-)-vincadifformine

Eight analogues of (-)-16-chloro-1-dehydro-6S-bromovincadifformine 1 were synthesized and evaluated for cytotoxicity in L1210 cell culture. None of the new compounds was more active than 1 but the modulation at C6, C16 and on the aromatic ring at C10 informs about structure-activity relationships within this series.     

Synthesis and cytotoxic evaluation of certain 4-(phenylamino)furo[2,3-b]quinoline and 2-(furan-2-yl)-4-(phenylamino)quinoline derivatives

Certain 4-(phenylamino)furo[2,3-b]quinoline and 2-(furan-2-yl)-4-(phenylamino)quinoline derivatives were synthesized and evaluated in vitro against the full panel of NCIs 60 cancer cell lines. The preliminary results indicated these tricyclic 4-(phenylamino)furo[2,3-b]quinolines were more cytotoxic than their corresponding 2-(furan-2-yl)-4-(phenylamino)quinoline isomers. For the 4-(phenylamino)furo[2,3-b]quinolines, compounds 2a and 3d are two of the most potent with a mean GI50 value of 0.025 microM in each case. Inactivity of 2b and 2c (positional isomers of 2a) indicated that both electronic environment, and the distance between intercalating pharmacophore and H-bond-donating MeO group are important. For the 2-(furan-2-yl)-4-(phenylamino)quinoline isomers, compound 12 (a mean GI50 of 4.36 microM), which bears a para-COMe substituent, is more active than its meta-substituted counterpart 13 (10.5 microM). However, the electron-donating MeO substituent is preferred at the meta-position, and the cytotoxicity for the meta-substituted derivatives decreased in the order: MeO derivative 14b (3.05 microM) > oxime 16 (6.85 microM) > ketone 13 (10.5 microM) > methyl oxime 18 (20.6 microM).     

Novel side chain modified Delta8(14)-15-ketosterols

Synthesis of five novel Delta8(14)-15-ketosterols comprising modified side chains starting from ergosterol is described. Ergosteryl acetate was converted into (22E)-3beta-acetoxy-5alpha-ergosta-8(14),22-dien-15-one through three stages in 32% overall yield; further transformations of the product obtained led to (22E)-3beta-hydroxy-5alpha-ergosta-8(14),22-dien-15-one, (22S,23S)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one, (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol and (22R,23R)-3beta-hydroxy-22,23-isopropylidenedioxy-5alpha-ergost-8(14)-en-15-one. New Delta8(14)-15-ketosterols were evaluated for their cytotoxicity and effects on sterol biosynthesis in human hepatoma Hep G2 cells in comparison with known 3beta-hydroxy-5alpha-cholest-8(14)-en-15-one. Among the compounds tested, (22R,23R)-3beta-hydroxy-22,23-oxido-5alpha-ergost-8(14)-en-15-one was found to be the most potent inhibitor of sterol biosynthesis (IC(50)=0.6+/-0.2microM), whereas (22R,23R)-5alpha-ergost-8(14)-en-15-on-3beta,22,23-triol exhibited the highest cytotoxicity (TC(50)=12+/-3microM at a 24h incubation).     

Synthesis and biological evaluation of 1-(2,4,5-trisubstituted phenyl)-3-(5-cyanopyrazin-2-yl)ureas as potent Chk1 kinase inhibitors

Based on the X-ray crystallography of our lead compound 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-cyanopyrazin-2-yl)urea in the checkpoint kinase 1 (Chk1) enzyme, we modified R4, and to a lesser extent, R2, and R5 of the phenyl ring, and made a variety of N-aryl-N'-pyrazinylurea Chk1 inhibitors. Enzymatic activity less than 20 nM was observed in 15 of 41 compounds. Compound 8i provided the best overall results in the cellular assays as it abrogated doxorubicin-induced cell cycle arrest (IC50=1.7 microM) and enhanced doxorubicin cytotoxicity (IC50=0.44 microM) while displaying no single agent activity.     

Toxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to cultured cancer cells

Toxicity of eight 22,23-dihydroxystigmastane derivatives (four pairs of (22R,23R)- and (22S,23S)-isomers differing in steroid backbone structure) to human breast carcinoma MCF-7 cells was compared. For every pair of structurally related compounds, (22R,23R) isomer was found to be significantly more toxic than (22S,23S) isomer. Computational analysis showed that side chain of (22R,23R)-22,23-dihydroxystigmastane derivatives is rigid, whereas that of (22S,23S)-isomers is rather flexible. Structure of steroid backbone significantly affects cytotoxicity of (22R,23R)-22,23-dihydroxystigmastane derivatives to human breast carcinoma MCF-7 cells, human ovary carcinoma CaOv cells, and human prostate carcinoma LnCaP cells. (22R,23R)-3β,22,23-trihydroxystigmast-5-ene and (22R,23R)-3β,22,23-trihydroxystigmast-5-en-7-one, both comprising equatorial 3β-hydroxyl group, exhibited the highest cytotoxicity, while the most polar 28-homobrassinolide and 28-homocastasterone, both comprising 2α,3α-dihydroxy groups, exhibited the lowest toxicity. Binding of (22R,23R)-22,23-dihydroxystigmastane derivatives to plasmatic membrane was suggested to be important for cytotoxicity.     

Esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinones with melphalan as multifunctional anticancer agents

Eight esters of 2-(1-hydroxyalkyl)-1,4-dihydroxy-9,10-anthraquinone with melphalan were prepared and tested for their antitumor activity (S-180) and cytotoxicity. 2-[1-[4-(p-Bis(2-chloroethyl)-aminophenyl)-butanoyloxy]methyl]-1,4-dihydroxy-9,10-anthraquinone and 2-[1-[4-(p-bis(2-chloroethyl)-aminophenyl)-butanoyloxy]ethyl]-1,4-dihydroxy-9,10-anthraquinone showed remarkable antitumor activity (T/C, 265 and 272%).     

Evaluation of 5-[1-(2-halo(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines as inhibitors of herpes simplex virus

A series of 5-[1-(2-haloethyl(or nitro)ethoxy-2-iodoethyl)]-2'-deoxyuridines (3-7) and related uracil analogs (9-10) were prepared using 5-vinyl-2-deoxyuridine (2) and 5-vinyl uracil (8) as starting materials. The regiospecific reaction of 2 and 8 with iodine monochloride and an alcohol provided the target compounds 3-10. These analogs were evaluated in vitro for inhibitory activity against thymidine-kinase (TK) positive and negative strains of herpes simplex virus type-1. The compounds 3-10 were either weak or non-inhibitory to HSV-1 replication. All compounds investigated exhibited low host cell cytotoxicity.     

Influence of polymer architecture and molecular weight of poly(2-(dimethylamino)ethyl methacrylate) polycations on transfection efficiency and cell viability in gene delivery

Nonviral gene delivery with the help of polycations has raised considerable interest in the scientific community over the past decades. Herein, we present a systematic study on the influence of the molecular weight and architecture of poly(2-(dimethylamino)ethyl methacrylate) (PDMAEMA) on the transfection efficiency and the cytotoxicity in CHO-K1 cells. A library of well-defined homopolymers with a linear and star-shaped topology (3- and 5-arm stars) was synthesized via atom transfer radical polymerization (ATRP). The molecular weights of the polycations ranged from 16 to 158 kDa. We found that the cytotoxicity at a given molecular weight decreased with increasing number of arms. For a successful transfection a minimum molecular weight was necessary, since the polymers with a number-average molecular weight, M(n), below 20 kDa showed negligible transfection efficiency at any of the tested polyelectrolyte complex compositions. From the combined analysis of cytotoxicity and transfection data, we propose that polymers with a branched architecture and an intermediate molecular weight are the most promising candidates for efficient gene delivery, since they combine low cytotoxicity with acceptable transfection results.