Investigation of new 2-aryl substituted Benzothiopyrano[4,3-d]pyrimidines as kinase inhibitors targeting vascular endothelial growth factor receptor 2.

Vascular Endothelial Growth Factor (VEGF) pathway has emerged as one of the most important positive modulators of Angiogenesis, a central process implicated in tumour growth and metastatic dissemination. This led to the design and development of anti-VEGF monoclonal antibodies and small-molecule ATP-competitive VEGFR-inhibitors. In this study, we describe the synthesis and the biological evaluation of novel 2-aryl substituted benzothiopyrano-fused pyrimidines 1a-i, 2a-i and 3a-i. The ability of the compounds to target the VEGF pathway was determined in vitro exploiting the compounds' antiproliferative efficacy against HUVEC cells. The VEGFR-2 inhibition was confirmed by enzymatic assays on recombinant human kinase insert domain receptor (KDR), by cell-based phospho-VEGFR-2 inhibition assays, and by ex vivo rat aortic ring tests. The selectivity profile of the best performing derivatives belonging to series 2 was further explored combining modeling studies and additional assays in a panel of human cell lines and other kinases.

Pyrroloquinolinone-based dual topoisomerase I/II inhibitor.

A new series of pyrroloquinolinones bearing different alkylamino side chains were synthesized and evaluated as cytotoxic compounds against three different human tumor cell lines (HeLa, HL-60 and A431). Some compounds showed interesting antiproliferative activity, in particular against A431 cells. The compounds were tested for their ability to counteract topoisomerase II relaxation activity and the most interesting one (3c) was tested also against topoisomerase I, resulting a dual inhibitor. The molecular interactions between 3c and the intracellular targets were finally investigated through molecular modeling simulations.

Quinazolinecarboline alkaloid evodiamine as scaffold for targeting topoisomerase I and sirtuins.

This paper reports the synthesis of a series of evodiamine derivatives. We assayed the ability to inhibit cell growth on three human tumour cell lines (H460, MCF-7 and HepG2) and we evaluated the capacity to interfere with the catalytic activity of topoisomerase I both by the relaxation assay and the occurrence of the cleavable complex. Moreover, whose effect on sirtuins 1, 2 and 3 was investigated. Finally, molecular docking analyses were performed in an attempt to rationalize the biological results.

Synthesis and biological evaluation of novel tamoxifen analogues.

A collection of compounds, structurally related to the anticancer drug tamoxifen, used in breast cancer therapy, were designed and synthesized as potential anticancer agents. McMurry coupling reaction was used as the key synthetic step in the preparation of these analogues and the structural assignment of E, Z isomers was determined on the basis of 2D-NOESY experiments. The compounds were evaluated for their antiproliferative activity on breast cancer (MCF-7), cervix adenocarcinoma (HeLa) and biphasic mesothelioma (MSTO-211H) human tumor cell lines. The estrogen like properties of the novel compounds were compared with those of the untreated controls using an estrogen responsive element-based (ERE) luciferase reporter assay and compared to 17ß-estradiol (E2). Finally, with the aim to correlate the antiproliferative activity with an intracellular target(s), the effect on relaxation activity of DNA topoisomerases I and II was assayed.

Camptothecin-7-yl-methanthiole: semisynthesis and biological evaluation.

The introduction of a methylenthiol group at position 7 of camptothecin was carried out in four steps. This preparation also yielded the corresponding disulfide, which behaves as a prodrug due to its reactivity with glutathione. Assessment of their antiproliferative activities, investigations of their mechanism of action, and molecular modeling analysis indicated that the 7-modified camptothecin derivatives described herein maintain the biological activity and drug-target interactions of the parent compound.

A novel tetrahydrobenzoangelicin with dark and photo biological activity.

The synthesis of 8,9,10,11-tetrahydro-5-(3-dimethylaminopropoxy)-4-methylbenzofuro[2,3-h]coumarin (5) is described. The new compound showed the ability to inhibit cell growth both upon UVA irradiation and in the dark. The investigation on the mechanism of action highlighted the capacity of 5 to covalently photoadd to thymine, as demonstrated by the isolation and characterization of the 4',5'-monoadduct. Furthermore, in the ground state 5 interferes with the topoisomerase II relaxation activity, suggesting that this enzyme could constitute a molecular target responsible for the dark antiproliferative effect.

Synthesis and antiproliferative activity of some ellipticine-like 11H-pyrido[a]carbazole derivatives.

Some modified 11H-pyrido[a]carbazoles (11H-PyC) and their corresponding tetrahydro derivatives (11H-THPyC) were prepared. A common multistep pathway characterized by conventional reactions, including a Fischer-indole-type synthesis, yielded the tetracyclic compounds. To improve cytotoxicity, 11H-PyC and 11H-THPyC derivatives were endowed with a diethylaminoethyl side chain. The antiproliferative activity was assessed in three human tumor cell lines, and a number of derivatives showed a cytotoxic effect in agreement with their capacity to form a molecular intercalative complex with DNA and to interfere with the relaxation activity of DNA topoisomerase II. In contrast, three derivatives that exhibited significant antiproliferative efficacy, showed no inhibition of topoisomerase II, thus suggesting an unexpected and novel mode of action for these ellipticine-like compounds independent of topoisomerase II activity.

Synthesis and biological evaluation of new camptothecin derivatives obtained by modification of position 20.

The preparation and biological evaluation of a novel series of dimeric camptothecin derivatives are described. All the new compounds showed a significant ability to inhibit human tumor cell growth with IC(50) values ranging from 0.03 to 12.2 µM. The interference with the activity of the nuclear enzymes topoisomerases has been demonstrated, highlighting the poison effect of one of the obtained byproducts toward topoisomerase I. A moderate antiangiogenic activity has been demonstrated for one of the obtained compounds. Moreover, the effects of four new compounds on caspases activity and ROS generation have been studied on transgenic mouse cell.

Pyridazinopsoralens of wide chemotherapeutic interest.

The synthesis of new 6,10-dimethylpyridazino[4,5-h]psoralens, carrying no (4), one (5), or two (6-9) dialkylaminoalkylcarboxamide side chains on the pyridazine ring is reported. All compounds exert a significant photoantiproliferative activity. Moreover, the derivatives characterised by the protonable side chains show a notable cytotoxicity in the dark. The investigation on the mechanism of action demonstrated the capacity to intercalate into DNA base pairs and to inhibit the relaxation activity of topoisomerase II.

Benzothiopyranoindole-based antiproliferative agents: synthesis, cytotoxicity, nucleic acids interaction, and topoisomerases inhibition properties.

Novel benzo[3',2':5,6]thiopyrano[3,2-b]indol-10(11H)-ones 1a-v were synthesized and evaluated for their antiproliferative activity in an in vitro assay of human tumor cell lines (HL-60 and HeLa). Compounds 1e-v, substituted at the 11-position with a basic side chain, showed a significant ability to inhibit cell growth with IC(50) values in the low micromolar range. Linear dichroism measurements showed that all 11-dialkylaminoalkyl substituted derivatives 1e-v behave as DNA-intercalating agents. Fluorimetric titrations demonstrated their specificity in binding to A-T rich regions, and molecular modeling studies were performed on the most active derivatives (1e, 1i, 1p) to characterize in detail the complexation mechanism of these benzothiopyranoindoles to DNA. A relaxation assay evidenced a dose-dependent inhibition of topoisomerase II activity that appeared in accordance with the antiproliferative capacity. Finally, for the most cytotoxic derivative, 1e, a topoisomerase II poisoning effect was also demonstrated, along with a weak inhibition of topoisomerase I-mediated relaxation.

A new psoralen derivative with enlarged antiproliferative properties.

Following our results with benzopsoralens as potent photochemotherapeutic agents, we report the antiproliferative evaluation of nitrogenated isoster upon and without UVA irradiation. The evaluated pyridazinopsoralen showed a higher photochemotherapeutic activity with respect to the well-known drug, 8-MOP, and a significant cytotoxicity, also in the dark. This result enlarges the interest in this tetracyclic psoralen derivative skeleton in the search of new anticancer agents.

DNA-targeting pyrroloquinoline-linked butenone and chalcones: synthesis and biological evaluation.

A series of conjugates of alpha,beta-unsaturated ketone systems, phenyl-butenone and diaryl-propenones (chalcones), with the tricyclic planar pyrroloquinoline nucleus were synthesised and evaluated for their anticancer properties. The aim was to target DNA by butenone and chalcones, and determine the occurrence of interactions with the macromolecule or related functional enzymes. The ability to inhibit cell growth was assayed on three human tumor cell lines, and the capacity to form molecular complexes with DNA was studied by linear flow dichroism (LD). The effect on the activity of the nuclear enzyme DNA topoisomerase II was also investigated. A noticeable cytotoxic effect was observed for all pyrroloquinoline-conjugated compounds 5 and 7a-c, particularly against human melanoma cell line JR8 (IC(50) 1.2-3.3 microM); the unconjugated chalcones (8a-c) and butenone had a lower or no effect at the tested concentrations. LD experiments confirmed the pyrroloquinoline nucleus as an efficacious carrier for intercalative complexation with DNA. The ability of pyrroloquinoline derivatives to intercalate between base pairs appears to inhibit the relaxation of supercoiled DNA by topoisomerase II, while they induce no significant DNA cleavage. Since the concentrations inhibiting the enzyme appear relatively high with respect to cytotoxicity, the effective intercalation could affect the activity of more DNA processing enzymes and these overall nuclear effects may induce cell death.

Synthesis and biological evaluation of pyrroloiminoquinone derivatives.

Synthesis of 10 pyrroloiminoquinone derivatives is presented. The strategy is based around the elaboration of a common intermediate by reaction with primary amines. All the compounds obtained have been subjected to antiproliferative activity with three different cell lines (NCI-H460, HeLa, and HL-60). The capacity of 4 selected compounds to affect the enzymatic activity of the nuclear enzyme DNA topoisomerase II and to form the typical DNA fragmentation which occurs in the apoptotic process is discussed here.

3,5-bis(3'-indolyl)pyrazoles, analogues of marine alkaloid nortopsentin: synthesis and antitumor properties.

A series of 10 bis-indolylpyrazoles of type 9, 10 were obtained by cyclization of diketones 8 using hydrazine monohydrate or methylhydrazine in refluxing acetic acid/THF. Derivatives 9a,c,d were selected, by the National Cancer Institute (NCI, Bethesda, USA), to be evaluated against the full panel of about 60 human tumor cell lines derived from nine human cancer cell types and showed antiproliferative activity in the micromolar range. In particular, 9d, the most active compound was effective against all the tested cell lines with a GI(50) mean value of 3.23 microM; TGI and LC(50) values were 14.5 and 58.9 microM having positive response on 91% and 41% of the tested cell lines, respectively.

Synthesis and antitumor properties of 2,5-bis(3'-indolyl)thiophenes: analogues of marine alkaloid nortopsentin.

A series of 11 bis-indolylthiophenes of type 8-10 were obtained by cyclization of diketones 4 and 7 using Lawesson's reagent. Derivatives 8c, 9c, 9d, and 10c were selected to be evaluated in the full panel of about 60 human tumor cell lines derived from nine human cancer cell types and showed antiproliferative activity generally in the micromolar range. The most sensitive cell lines were: CCRF-CEM, MOLT-4, HL60 (TB), and RPMI-8226 of the leukemia subpanel, HT29 and HCC-2998 cell lines of the colon sub-panel, NCI-H522 of the non-small cell lung cancer sub-panel, LOX IMVI of the melanoma sub-panel, and UO-31 of the renal cancer sub-panel.

New furan side tetracyclic allopsoralen derivatives: synthesis and photobiological evaluation.

Novel tetracyclic allopsoralen derivatives characterized by the condensation of a fourth cyclohexenylic (5-7) or benzenic (8-10) ring at the furan side and a methoxy (5 and 8), a hydroxy (6 and 9), or a dimethylaminopropoxy (7 and 10) side chain in the 10 position of the chromophore were prepared. Compounds 7 and 10 showed a strong photoantiproliferative activity, up to 3 orders of magnitude higher than that of the photochemotherapeutic drug 8-methoxypsoralen (8-MOP). The investigation into the mechanism of action demonstrated for 10 the capacity to intercalate between DNA base pairs in the ground state, to give rise to a covalent photoaddition upon UVA irradiation, and to inhibit polymerase chain reaction (PCR) in a sequence-specific manner. Conversely, compound 7 showed a limited capacity to form an intercalative complex and the lack of ability to photoadd to the macromolecule, thus revealing a novel and unusual behavior for an allopsoralen derivative.

Photoaddition of thienocoumarin derivatives to DNA: stoichiometry and kinetics of binding.

Photoreaction of the 6,9-dimethyl-4-methoxymethyl-2H-thieno[3,2-g]-1-benzopyran-2-one (compound I) and 4-acetoxymethyl-6,9-dimethyl-2H-thieno[3,2-g]-1-benzopyran-2-one (compound II) to DNA was studied. The quantitative evaluation of the photobound molecules was performed by means of inductively coupled plasma atomic emission spectrometry (ICP-AES), exploiting the presence of the sulphur atom inside the tricyclic chromophore. The concurrent estimation of the phosphorus atom, present exclusively in the macromolecule, allowed possible intercalation sites to be identified and their involvement in the photoaddition reaction to be determined. The development of a kinetic model made it possible to discriminate and evaluate the single kinetic events that constitute the overall photoaddition process of I and II to DNA.

Design, synthesis and photobiological properties of 3,4-cyclopentenepsoralens.

The QSAR directed synthesis of tetracyclic psoralen derivatives (3-5) characterised by the condensation of a cyclopentane ring at the level of the 3,4 double bond of the tricyclic psoralen moiety is reported. The new compounds present a methoxy (3), a hydroxy (4) or a dimethylaminopropoxy (5) side chain inserted in position 8 of the lead chromophore. The evaluation of photoantiproliferative activity on human tumour cell lines reveals for 5 an ability to inhibit cell growth significantly higher with respect to that of the reference drug, 8-MOP. Interestingly, the enhancement in antiproliferative activity is accompanied by the disappearance of skin phototoxicity. On the other hand, no significant photobiological activity was scored for 3 and 4. The ability to photoreact with DNA, evaluated by isolating the 4',5' monoadduct and by estimating the ability to form interstrand cross-links, appeared to be significant for 5, practically negligible for 3 and 4. Furthermore, a back-projection of the more active compound identifies structural features suitable for further synthetic modifications.

Markovian chemicals "in silico" design (MARCH-INSIDE), a promising approach for computer-aided molecular design I: discovery of anticancer compounds.

A simple stochastic approach, designed to model the movement of electrons throughout chemical bonds, is introduced. This model makes use of a Markov matrix to codify useful structural information in QSAR. The self-return probabilities of this matrix throughout time ((SR)pi(k)) are then used as molecular descriptors. Firstly, a calculation of (SR)pi(k) is made for a large series of anticancer and non-anticancer chemicals. Then, k-Means Cluster Analysis allows us to split the data series into clusters and ensure a representative design of training and predicting series. Next, we develop a classification function through Linear Discriminant Analysis (LDA). This QSAR discriminates between anticancer compounds and non-active compounds with a correct global classification of 90.5% in the training series. The model also correctly classified 86.07% of the compounds in the predicting series. This classification function is then used to perform a virtual screening of a combinatorial library of coumarins. In this connection, the biological assay of some furocoumarins, selected by virtual screening using the present model, gives good results. In particular, a tetracyclic derivative of 5-methoxypsoralen (5-MOP) has an IC50 against HL-60 tumoral line around 6 to 10 times lower than those for 8-MOP and 5-MOP (reference drugs), respectively. Finally, application of Iso-contribution Zone Analysis (IZA) provides structural interpretation of the biological activity predicted with this QSAR.

Novel pyrone side tetracyclic psoralen derivatives: synthesis and photobiological evaluation.

This study reports the synthesis of tetrahydrobenzo- (4-6) and benzopsoralen (7-9) derivatives obtained by condensing the fourth ring to the pyrone side of the tricyclic psoralen moiety. The new compounds are characterized by having a methoxy, a hydroxy, or a dimethylaminopropoxy side chain inserted at position 8 of the psoralen chromophore. The evaluation of the photoantiproliferative activity on human tumor cell lines along with skin phototoxicity on guinea pigs revealed an interesting photobiological pattern for the dimethylaminopropoxy derivatives 6 and 9: they are in fact able to exert an antiproliferative effect up to 1 order of magnitude higher than that of the well-known drug 8-MOP, but they are devoid of skin phototoxicity. The ability of both 6 and 9 to photoadd to DNA is demonstrated by the isolation and characterization of the 4',5'-monoadducts. AM1 calculations were also performed to gain further insight into the molecular basis of their photobiological behavior.