Synthesis of 4β-carbamoyl epipodophyllotoxins as potential antitumour agents

A series of new 4β-carbamoyl epipodophyllotoxin analogues have been synthesized and evaluated for their anticancer activity against eleven cancer cell lines including Zr-75-1, MCF7, KB, Gurav, DWD, Colo 205, A-549, Hop62, PC3, SiHa and A-2780. Most of the compounds exhibited better growth-inhibition activities against tested cell lines than that of etoposide. Further, compounds 6g and 6i are also evaluated for their DNA topoisomerase-II (topo-II) inhibition activity and they exhibited significant inhibition of topo-II catalytic activity comparable to etoposide.     

Synthesis of 4β-N-polyaromatic substituted podophyllotoxins: DNA topoisomerase inhibition, anticancer and apoptosis-inducing activities

A new class of 4β-N-polyaromatic substituted podophyllotoxin congeners have been synthesized and evaluated for their DNA topoisomerase-II (topo-II) inhibition as well as anticancer potential in some human cancer cell lines. The ease of synthesis and interesting biological activities make the present series of polyaromatic-podophyllotoxin congeners as a promising new structure for the development of new anticancer agents based on podophyllotoxin scaffold.     

Synthesis and antioxidant activities of 3,5-dialkoxy-4-hydroxycinnamamides

A series of 3,5-dialkoxy-4-hydroxycinnamamides 6 and 7 was synthesized, and their antioxidant activity was assessed using the thiobarbituric acid reactive substance (TBARS) assay. Interestingly, cinnamamides with longer alkoxy groups on the C-3 and C-5 positions display enhanced inhibition, and most of the compounds in the series tested exhibit excellent lipid peroxidation inhibitory activities. Some cinamamides bearing hexyloxy or 2,6-di-tert-butyl-4-methyl phenol groups have submicromolar inhibitory activities.     

Synthesis and inhibition of Src kinase activity by 7-ethenyl and 7-ethynyl-4-anilino-3-quinolinecarbonitriles

A series of 7-ethynyl and 7-ethenyl-4-anilino-3-quinolinecarbonitriles were synthesized and tested for Src inhibition. Derivatives bearing a C-6 methoxy group and 2,4-dichloro-5-methoxyaniline at C-4 showed optimal inhibition of Src enzymatic and cellular activity. The ethenyl and ethynyl groups were incorporated at C-7 utilizing a Stille, Heck, or Sonogashira coupling reaction.     

Mechanism of biochemical action of substituted 4-methylcoumarins. Part 11: Comparison of the specificities of acetoxy derivatives of 4-methylcoumarin and 4-phenylcoumarin to acetoxycoumarins: protein transacetylase

Our earlier observations led to the identification of a microsomal enzyme termed as acetoxy drug: protein transacetylase (TAase) catalyzing the transfer of acetyl groups from acetylated polyphenols to the receptor proteins. TAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (1). The specificities of the acetoxy group on the benzenoid ring and position of the pyran carbonyl group of the coumarin with respect to oxygen heteroatom for the catalytic activity of TAase were also reported earlier. In this communication, we have demonstrated that the acetoxy coumarins and acetoxy dihydrocoumarins having a methyl group instead of a phenyl ring at the C-4, when used as the substrates, resulted in enhancement of TAase activity, while the saturation of double bond at C-3 and C-4 position had no effect on TAase activity. A comparison of the optimized structures of 1 and 7,8-diacetoxy-4-phenylcoumarin (2) suggested that the observed influence may be due to out of plane configuration of the phenyl ring at C-4. Further, the TAase-catalyzed activation of NADPH cytochrome c reductase and inhibition of aflatoxin B1 (AFB1)-DNA binding by acetoxy 4-phenylcoumarins and dihydrocoumarins were significantly lower as compared to those caused by acetoxy 4-methylcoumarins.     

Synthesis and antiviral evaluation of C-4-hydrazide derivatives of 2',3'-dideoxycytidine

Syntheses of three hitherto unknown derivatives of 2',3'-dideoxycytidine, namely C-4-(salicylic hydrazide)-ddC, C-4-(N-butyloxycarbonyl-isoleucine hydrazide)-ddC and its N-unprotected chlorhydrate salt have been carried out. These compounds do not induce inhibition of HIV-1 replication in cell culture experiments. Nevertheless, the modifications on the base moiety increased in all cases the lipophilicity of the parent molecule with an acceptable water solubility compared to ddC.     

The glycosaminoglycan chondroitin-4-sulfate alters progesterone secretion by porcine granulosa cells

Porcine granulosa cells from small (1-2 mm), medium (3-5 mm), and large (6-12 mm) antral follicles were cultured in monolayer for 2 to 3 days with 0 to 3 mg of chondroitin-4-sulfate (C-4-S)/ml in the presence or absence of 0.5 microgram follicle-stimulating hormone (NIH-FSH-S13)/ml. Testosterone (1.4 microgram/ml) was added to some cultures as substrate for estrogen synthesis. Progesterone and estrogen secreted into the media were measured by radioimmunoassay. Concentrations of C-4-S similar to concentrations of chondroitin sulfates (CS) reported for small antral or atretic follicles inhibited both basal and FSH-stimulated progesterone secretion. Progesterone secretion was not inhibited by C-4-S when pregnenolone was added to the media. Thus 3 beta-hydroxysteroid dehydrogenase activity was not inhibited by C-4-S. Estrogen secretion was also not inhibited by even the highest concentration of C-4-S tested. Testosterone did not influence C-4-S inhibition of progesterone secretion. Granulosa cells from medium-sized follicles were more sensitive to C-4-S than cells from small follicles. Granulosa cells from large follicles were completely resistant to C-4-S inhibition of progesterone secretion. These observations suggest that C-4-S may play a role in altering gonadotrophin-stimulated and basal progesterone secretion in follicles during differentiation of granulosa cells.     

Synthesis and in vitro cytotoxicity evaluation of β-carboline-combretastatin carboxamides as apoptosis inducing agents: DNA intercalation and topoisomerase-II inhibition

To explore a new set of cytotoxic agents, β-carboline-combretastatin carboxamide conjugates were designed, synthesized and evaluated for their in vitro cytotoxicity potential, DNA binding affinity and Topoisomerase-II (topo-II) inhibition activity. Among the designed hybrids, 10v and 10af have shown significant cytotoxic effect against A549 (lung cancer) cell line having IC₅₀ value 1.01 µM and 1.17 µM respectively. Further, it was speculated that treatment with compound 10v may induce apoptosis among A549 cells, which was supported by Hoechst staining, DCFDA, Annexin V-FITC and morphological assays. Flow cytometric analysis revealed that the hybrid 10v arrests A549 cells in G2/M phase of cell cycle in a dose dependent manner. Amongst the active hybrids, most potent hybrid 10v was tested for DNA topo-II inhibition activity. Moreover, to further support the biological activity and to infer the mode of interaction between ligands and DNA, spectroscopy and molecular docking studies were carried out. The docking and spectroscopy results showed that the ligands exhibited an intercalative mode of binding with DNA and could efficiently bind to DNA and form topo-II ternary complex. Based on these experiments, the hybrids 10v and 10af were identified as proficient new scaffolds which need to be developed as hit molecules for therapeutic interest.     

Synthesis and biological evaluation of substituted 6-alkynyl-4-anilinoquinazoline derivatives as potent EGFR inhibitors

A series of C-6 or C-3' alkynyl-substituted 4-anilinoquinazoline derivatives was prepared straightforwardly by a Sonogashira reaction of the corresponding bromo-substituted 4-anilinoquinazolines. Bioactive assay of these compounds for in vitro EGFR kinase inhibition demonstrated that the novel 6-hydroxypropynyl-4-anilinoquinazoline 5e was a very potent EGFR kinase inhibitor with an IC(50) of 14 nM.     

Synthesis and biological evaluation of 4β-sulphonamido and 4β-[(4'-sulphonamido)benzamide]podophyllotoxins as DNA topoisomerase-IIα and apoptosis inducing agents

A series of new 4β-sulphonamido and 4β-[(4'-sulphonamido)benzamide] conjugates of podophyllotoxin (11a-j and 15a-g) were synthesized and evaluated for anticancer activity against six human cancer cell lines and found to be more potent than etoposide. Some of the compounds 11b, 11d and 11e that showed significant antiproliferative activity in Colo-205 cells, were superior to etoposide. The flow cytometric analysis indicates that these compounds (11b, 11d and 11e) showed G2/M cell cycle arrest and among them 11e is the most effective. It is observed that this compound (11e) caused both single-strand DNA breaks as observed by comet assay as well as double-strand DNA breaks as indicated by γ-H2AX. Further 11e showed inhibition of topo-IIα as observed from Western blot analysis and related studies. Compounds caused activation of ATM as well as Chk1 protein indicating that the compound caused effective DNA damage. Moreover activation of caspase-3, p21, p16, NF-kB and down regulation of Bcl-2 protein suggests that this compound (11e) has apoptotic cell death inducing ability, apart from acting as a topo-IIα inhibitor.     

Synthesis and biological evaluation of 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives as GABA uptake inhibitors

A series of enantiomerically pure 4-hydroxy-4-(4-methoxyphenyl)-substituted proline and pyrrolidin-2-ylacetic acid derivatives have been synthesized starting from the respective N-protected 4-hydroxy derivatives via oxidation to the corresponding 4-oxo compounds, subsequent addition of organometallic reagents, final hydrolysis and deprotection. The major diastereoisomers obtained by the addition of the Grignard reagents were found to have opposite stereoconfigurations depending on whether cerium trichloride was present or absent as an additive. The final compounds were evaluated for their capability to inhibit the GABA transport proteins GAT1 and GAT3. 4-Hydroxyproline derivatives substituted with a tris(4-methoxyphenyl)methyloxyethyl residue at the nitrogen and a 4-methoxyphenyl group in 4-position showed, with the exception of the (2R,4R)-diastereomer, an improved inhibition at GAT3 compared to the derivatives missing the 4-methoxyphenyl group in 4-position. This may imply that an appropriate lipophilic group at the C-4 position of the proline moiety is beneficial for potent inhibition at GAT3.     

Synthesis and PKCtheta inhibitory activity of a series of 4-(indol-5-ylamino)thieno[2,3-b]pyridine-5-carbonitriles

The thieno[2,3-b]pyridine-5-carbonitrile with a 5-indolylamine at C-4 and a phenyl group at C-2 had a moderate activity against PKCθ. Optimization of the groups at C-4 and C-2 led to analog 29, which has an IC₅₀ value of 7.5 nM for the inhibition of PKCθ.     

Specificities of Calreticulin Transacetylase to acetoxy derivatives of 3-alkyl-4-methylcoumarins: Effect on the activation of nitric oxide synthase

Calreticulin Transacetylase (CRTAase) catalyzes the transfer of acetyl groups from polyphenolic acetates (PAs) to the receptor proteins and modulates their biological activities. CRTAase was conveniently assayed by the irreversible inhibition of cytosolic glutathione S-transferase (GST) by the model acetoxycoumarin, 7,8-diacetoxy-4-methylcoumarin (DAMC). We have studied earlier, the influence of acetoxy groups on the benzenoid ring, the effect of reduction of double bond at C-3 and C-4 position, the effect of methyl/phenyl group at C-4, and the influence of position of carbonyl group with respect to oxygen heteroatom in the benzopyran nucleus, for the catalytic activity of CRTAase. In this communication, we have extended our previous work; wherein we studied the influence of an alkyl group (ethyl, hexyl and decyl) at the C-3 position of the acetoxy coumarins on the CRTAase activity. The substitution at C-3 position of coumarin nucleus resulted in the reduction of CRTAase activity and related effects. Accordingly the formation of NO in platelets by C-3 alkyl substituted acetoxy coumarins was found to be much less compared to the unsubstituted analogs. In addition the alkyl substitution at C-3 position exhibited the tendency to form radicals other than NO.     

Further studies on ethenyl and ethynyl-4-phenylamino-3-quinolinecarbonitriles: identification of a subnanomolar Src kinase inhibitor

Several new ethynyl- and ethenyl-4-phenylamino-3-quinolinecarbonitriles were synthesized and tested for Src inhibition. Derivatives bearing an ethenyl or ethynyl substituent at C-6 showed decreased Src inhibitory activity. Incorporation of an ethenylpyridine N-oxide group at C-7 provided 20b, a 0.6 nM inhibitor of Src enzymatic activity with excellent cellular potency.     

Structural and functional bases of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of myometrium

Calix[4]arene C-97 (code is shown) is the macrocyclic compound which has lipophilic intramolecular higly-structured cavity formed by four aromatic cycles, one of which on the upper rim is modified by methylene bisphosphonic group. It was shown that calix[4]arene C-97 (100 microM) efficiently inhibits ATPase activity of myosin subfragment-1 from pig myometrium, the inhibition coefficient I(0.5) being 83 +/- 7 microM. At the same time, this compound at 100 microM concentration significantly increases the effective hydrodynamic diameter of myosin subfragment-1, that may be indicative of intermolecular complexation between the calix[4]arene and myosin head. Computer simulation methods (docking, molecular dynamics, involving the Grid) have been used to clarify structural basis of the intermolecular interaction of calix[4]arene C-97 with myosin subfragment-1 of the myometrium; participation of hydrophobic, electrostatic and pi-pi (stacking) interactions between calix[4]arene C-97 and amino acid residues of myosin subfragment-1, some of them being located near the active site of the ATPase has been found out.     

Investigation of the effect of varying the 4-anilino and 7-alkoxy groups of 3-quinolinecarbonitriles on the inhibition of Src kinase activity

Several 7-alkoxy-4-anilino-3-quinolinecarbonitriles were synthesized and evaluated for Src kinase inhibitory activity. Optimal inhibition of both Src enzymatic and cellular activity was seen with analogues having a 2,4-dichloro-5-methoxyaniline group at C-4. Compound 18, which has a 1-methylpiperidinemethoxy group at C-7, showed in vivo activity in a xenograft model.     

4-Alkyliden-beta-lactams conjugated to polyphenols: synthesis and inhibitory activity

A series of compounds combining the beta-lactam and polyphenol scaffold have been prepared and evaluated for inhibition of human leukocyte elastase and matrix metallo-proteases MMP-2 and MMP-9. The design of these compounds has been based on the 'overlapping-type' strategy where two pharmacophores are linked in a single molecule. The most powerful compound against elastase was an N-galloyl-4-alkyliden beta-lactam, [3-[1-(tert-butyl-dimethyl-silanyloxy)-ethyl]-4-oxo-1-(3,4,5-tris-benzyloxy-benzoyl)-azetidin-2-ylidene]-acetic acid ethylester, with an IC50 of 0.5 microM; while the most powerful against MMP-2 was a 4-alkyliden beta-lactam arylated on the C-3 hydroxy side chain (3,5-bis-benzyloxy-4-hydroxy-benzoic acid 1-(2-benzyloxycarbonylmethylene-4-oxo-azetidin-3-yl)-ethyl ester) with an IC50 of 4 microM. Of the total 35 compounds tested, high levels of inhibition of elastase and of MMPs were separately exerted by distinct molecules.     

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.     

Kinetic regularities and mechanisms of action of calix[4]arene C-99 on ATPase activity of myosin subfragment-1 of myometrium

It has been shown that calix[4]arene C-99 inhibited myosin subfragment-1 ATPase of myometrium. This inhibition is noncompetitive as to ATP and Mg2+. At the same time, this compound reduces the seeming enzymatic hydrolysis maximum rate of nucleoside triphosphate with respect to ATP and Mg2+. With the help of computer design the interaction of mentioned calix[4]arene with myosin subfragment-1 of myometrium has been investigated. Several mechanisms involved in the calix[4]arene C-99 inhibition of myosin head ATPase were supposed and participation of hydrogen, hydrophobic and electrostatic interactions in these mechanisms was discussed.     

Inhibition of sialidases from viral,bacterial and mammalian sources by analogues of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid modified at the C-4 position

The inhibition of sialidase activity from influenza viruses A and B, parainfluenza 2 virus,Vibrio cholerae, Arthrobacter ureafaciens, Clostridium perfringens, and sheep liver by a range of 2-deoxy-2,3-didehydro-N-acetylneuraminic acid analogues modified at the C-4 position has been studied. All substitutions tested resulted in a decrease in the degree of inhibition of the bacterial and mammalian sialidases. For sialidases from influenza viruses A and B, on the other hand, most of the substitutions tested either had no significant effect on binding or, in the case of the basic amino and guanidino substituents, resulted in significantly stronger inhibition. The results for parainfluenza 2 virus sialidase were mostly intermediate, in that inhibition was neither significantly increased nor decreased by most of the modifications. We conclude that only the influenza A and B sialidase active sites possess acid groups correctly positioned to participate in charge-charge interactions in the region of C-4 of bound substrate, and that the C-4 binding pockets of the bacterial and mammalian sialidases examined are considerably smaller than is observed for either the influenza virus or parainfluenza virus sialidases.This paper is dedicated to the memory of Professor Dr E. Zbiral.