Antimycobacterial agents. Novel diarylpyrrole derivatives of BM212 endowed with high activity toward Mycobacterium tuberculosis and low cytotoxicity

On the basis of suggestions derived either from a pharmacophoric model for antitubercular agents or from a structure-activity relationship analysis of many pyrroles previously described by us, we report here the design and synthesis of new analogues of 1,5-(4-chlorophenyl)-2-methyl-3-(4-methylpiperazin-1-yl)methyl-1H-pyrrole (BM212). Various substituents with different substitution patterns were added to both positions 1 and 5 of the pyrrole nucleus to evaluate their influence on the activity toward Mycobacterium tuberculosis (MTB) and atypical mycobacteria. Biological data showed that, although some nontuberculosis mycobacterial strains were found to be sensitive, MIC values were higher than those found toward MTB. The best compound (1-(4-fluorophenyl)-2-methyl-3-(thiomorpholin-4-yl)methyl-5-(4-methylphenyl)-1H-pyrrole, 5) possessed a MIC of 0.4 microg/mL (better than BM212 and streptomycin) and a very high protection index (160), better than BM212, isoniazid, and streptomycin (6, 128, and 128, respectively). Finally, molecular modeling studies were performed to rationalize the activity of the new compounds in terms of both superposition onto a pharmacophoric model for antitubercular compounds and their hydrophobic character.     

Novel combretastatin analogues endowed with antitumor activity

We studied the anticancer activity of a series of new combretastatin derivatives with B-ring modifications. The structure-activity relationship (SAR) information confirmed the importance of cis-stereochemistry and of a phenolic moiety in B-ring. We selected the benzo[b]thiophene and benzofuran combretastatin analogues 11 (ST2151) and 13 (ST2179) and their phosphate prodrugs (29 and 30) for their high antitumor activity in in vitro and in vivo models. Cell exposure to IC50 of 11, 13, and CA-4 led to the arrest of various cell types in the G2/M phase of the cell cycle and induction of apoptosis. Mainly, 11 and 13 induced the formation of multinucleated cells with abnormal chromatin distribution, with only a minimal effect on the microtubule organization, with respect to CA-4. Interestingly, both the pharmacokinetic profile of 29 and its in vivo antitumor effect and those of 30, active even after oral administration, suggest additional pharmacological differences between these compounds and CA-4P.     

Novel nitrofurazone derivatives endowed with antimicrobial activity

The N-alkylated derivatives from nitrofurazone were synthesised and evaluated in vitro for their efficacy as antimicrobial agents against representative strains, including methicillin-resistant Staphylococcus aureus (MRSA). The derivative 2a demonstrated greater activity than the prototype and was comparable to currently used antimicrobial drugs.     

Quinazoline derivatives with antitubercular activity

4-Quinazolinol was prepared by the reaction of anthranilic acid and formamide. The hydroxy group was converted into the thiol function by treatment with phosphorus(V)sulfide, and the subsequent alkylation of the thiol group was carried out with alkylhalides under the conditions of phase-transfer catalysis. The structure of the substances was confirmed by 1H, 13C NMR, IR, and MS. Most of the synthesized compounds exhibited antimycobacterial activity against the strains of Mycobacterium tuberculosis, Mycobacerium avium, Mycobacterium fortuitum, Mycobacterium kansasii and Mycobacterium intracellulare. 4-(S-Butylthio)quinazoline (3c) was even more active than isoniazide against atypical strains of mycobacteria.     

Undersulfated and glycol-split heparins endowed with antiangiogenic activity

Tumor neovascularization (angiogenesis) is regarded as a promising target for anticancer drugs. Heparin binds to fibroblast growth factor-2 (FGF2) and promotes the formation of ternary complexes with endothelial cell surface receptors, inducing an angiogenic response. As a novel strategy to generate antiangiogenic substances exploiting binding to FGF2 while preventing FGF receptor (FGFR) activation, sulfation gaps were generated along the heparin chains by controlled alkali-catalyzed removal of sulfate groups of iduronic acid 2-O-sulfate residues, giving rise to the corresponding epoxide derivatives. A new class of heparin derivatives was then obtained by opening the epoxide rings followed by oxidative glycol-splitting of the newly formed (and the preexisting) nonsulfated uronic acid residues. In vitro these heparin derivatives prevent the formation of FGFR/FGF2/heparan sulfate proteoglycan ternary complexes and inhibit FGF2-stimulated endothelial cell proliferation. They exert an antiangiogenic activity in the chick embryo chorioallantoic membrane assay, where the parent heparin is inactive. Low and very low molecular weight derivatives of a prototype compound, as well as its glycine and taurine derivatives obtained by reductive amination of glycol-split residues, retained the angiostatic activity. A significant relationship was found between the extent of glycol-splitting and the FGF2-antagonist/angiostatic activities of these heparin derivatives. Molecular dynamics calculations support the assumption that glycol-split residues act as flexible joints that, while favoring 1:1 binding to FGF2, disrupt the linearity of heparin chains necessary for formation of active complexes with FGFRs.     

Novel analgesic/anti-inflammatory agents: diarylpyrrole acetic esters endowed with nitric oxide releasing properties

The design of compounds that are able to inhibit cyclooxygenase (COX) and to release nitric oxide (NO) should give rise to drugs endowed with an overall safer profile for the gastrointestinal and cardiovascular systems. Herein we report a new class of pyrrole-derived nitrooxy esters (11a-j), cyclooxygenase-2 (COX-2) selective inhibitors endowed with NO releasing properties, with the goal of generating new molecules able to both strongly inhibit this isoform and reduce the related adverse side effects. Taking into account the metabolic conversion of nitrooxy esters into corresponding alcohols, we also studied derivatives 12a-j. All compounds proved to be very potent and selective COX-2 inhibitors; nitrooxy derivatives displayed interesting ex vivo NO-dependent vasorelaxing properties. Compounds 11c, 11d, 12c, and 12d were selected for further in vivo studies that highlited good anti-inflammatory and antinociceptive activities. Finally, two selected compounds (11c and 12c) tested in human whole blood (HWB) assay proved to be preferential inhibitors of COX-2.     

联合氯法齐明方案治疗耐多药肺结核的临床研究

目的：探讨氯法齐明联合其他抗结核药物治疗耐多药肺结核的临床疗效。方法：72例耐多药肺结核患者,随机分为试验组32例和对照组40例,观察治疗6、18个月末痰结核菌阴转率、胸部X线病灶和空洞变化情况等。结果：治疗6个月末试验组的涂阳阴转率、培阳阴转率、病灶显吸率分别为81．3％、84．4％、37．5％,均高于对照组的57．5％、62．5％、15．00．4,差异有统计学意义（P〈0．05）,其他结果2组无明显差异（P〉0．05）。结论：联合氯法齐明方案治疗耐多药肺结核可以提高疗效、缩短疗程,特别是在强化期具有明显的优势。     

含氯法齐明联合方案治疗耐多药结核疗效分析

目的评价含氯法齐明联合方案治疗耐多药结核病的效果。方法对28例耐多药复治肺结核病病人使用含氯法齐明联合方案进行抗结核治疗,每月监测痰涂片找抗酸杆菌及结核菌培养。治疗后12个月细菌学检结果均为阴性为治愈。观察其治疗效果,并分析治疗失败与纤维病灶面积/总病灶面积比值的相关性。结果自身对照显示含氯法齐明联合方案对耐多药肺结核治愈率78.6%,优于一线抗结核药物联合方案差异有统计学意义（P〈0.05）。纤维病灶面积/总病灶面积〈50%组治疗成功率高于≥50%组,差异有统计学意义（P〈0.01）。结论含氯法齐明联合方案对耐多药结核病有较好疗效。     

Novel A-ring and B-ring modified combretastatin A-4 (CA-4) analogues endowed with interesting cytotoxic activity

A novel class of combretastatins, modified at A-ring or both A- and B-rings, mainly by replacement with benzofuran or benzo[b]thiophene, were synthesized. The new heterocombretastatins showed good cytotoxic activity on BMEC and H-460 cell lines. The aminocombretastatin 9f potently inhibits cell growth of BMEC and combretastatin-resistant HT-29 cell lines, with potential interest to treat colon carcinoma. Heterocombretastatins 9a,b inhibit tubulin polymerization similarly to CA-4 by having a binding to colchicine site five times stronger.     

A novel atypical retinoid endowed with proapoptotic and antitumor activity

The novel atypical retinoid E-3-(4'-hydroxy-3'-adamantylbiphenyl-4-yl)acrylic acid (ST1926, 4) exhibited a potent antiproliferative activity on a large panel of human tumor cells. Despite almost complete loss of ability to activate RARs, the compound was an effective apoptosis inducer and surprisingly produced DNA damage, that likely contributes to the proapoptotic activity. Following oral administration, 4 was well tolerated and caused tumor growth inhibition in the ovarian carcinoma, A2780/DX, and in the human melanoma, MeWo, growing in nude mice, thus supporting the therapeutic interest of the novel agent.     

A novel bitriazolyl acyclonucleoside endowed with dual antiproliferative and immunomodulatory activity

A novel bitriazolyl acyclonucleoside was discovered to exhibit powerful antiproliferative effects on different cancer cell lines through caspase-dependent apoptosis and at the same time stimulate the immune response in dendritic cells via Toll-like receptor 7 (TLR7) signaling. This promising compound with dual anticancer and immunomodulatory activity may represent a new generation of highly efficacious drug candidates for use in cancer therapy.     

Novel alpha-melanocyte stimulating hormone peptide analogues with high candidacidal activity

alpha-Melanocyte stimulating hormone (alpha-MSH) is an endogenous linear tridecapeptide with potent antiinflammatory effects. We recently demonstrated that alpha-MSH and its C-terminal sequence Lys-Pro-Val (alpha-MSH (11-13)) have antimicrobial effects against two major and representative pathogens: Staphylococcus aureus and Candida albicans. In an attempt to improve the candidacidal activity of alpha-MSH and to better understand the peptide structure-antifungal activity relations, we designed and synthesized novel peptide analogues. Because previous data suggested that antimicrobial effects of alpha-MSH were receptor-mediated, we chose to focus on the sequence alpha-MSH (6-13), which contains the invariant core sequence His-Phe-Arg-Trp (6-9) that is important for binding to the known melanocortin receptors and also contains the sequence Lys-Pro-Val (11-13) that is known to be important for antimicrobial activity. In this structure-activity study, we discovered several compounds that have greater candidacidal activity than alpha-MSH. The peptide [d-Nal-7,Phe-12]-alpha-MSH (6-13) was the most potent of the analogues tested. The present results are very encouraging because they show the great potential of these peptides as a truly novel class of candidacidal compounds.     

Inhibitors for proteins endowed with catalytic and non-catalytic activity which recognize pTyr

Reversible phosphorylation of Tyr residues in proteins plays a central role in the transduction of signals. For both SH2 domains and for protein tyrosine phosphatases (PTPs) the phosphate group of phosphotyrosine (pTyr) of peptides provides a key affinity element, but its highly charged nature and its hydrolytic lability render it unsuitable in inhibitor design. The research in the recent years has been addressed to find pTyr bioisosters devoid of the phenylphosphate moiety and more potent inhibitors with less peptidic character. Several derivatives were prepared as pTyr bioisosters, and their activity appears to depend on the nature of the substrate, peptidic or low-molecular weight compounds, in which they are placed. In the field of PTPs, the research was mainly focused on new and selective PTP1B inhibitors, possibly useful in the treatment of Type 2 diabetes. The discovery of non-peptidic low molecular weight compounds able to inhibit PTP1B, by means of docking procedures and HTS screening, and the presence of secondary binding sites on PTP1B afforded new potent and selective inhibitors; several leads devoid of negative charges were also found. To date, however, few compounds have been tested In vivo and found to show a significant activity in diabetic mouse models. Other neutral compounds, mainly quinones, were found to inhibit CD45 and Cdc25. Several papers have appeared in recent years on the discovery of new Grb2, Src, Syk, and Lck SH2 domains binding antagonists. In this field very good inhibitors derived from high affinity peptides were found, with less peptidic character and with a reduced number of negative charges; however the presence of some negative charges, especially the one present on the pTyr bioisoster moiety, seems to be indispensable. As regards Grb2, Src and Lck SH2 domains, rigidification of the starting high affinity binding peptides afforded derivatives with improved affinity; cellular activity was achieved by modification of the side chains of these inhibitors.     

Androstane derivative devoid of anabolic-virilizing effects and endowed with an antiglucocorticoid activity

2-Carbodecyloxy-17 alpha-methylandrosta-1,4-dien-11 beta,17 beta-dihydroxy-3-one (decylroxibolone, BR 917) is a new androstane derivative, esterified with decyl alcohol, carrying a methyl group in the 17 position, a hydroxyl group in the 11 beta position and a carboxyl group in position 2. Unlike norandrostenolone decanoate, decylroxibolone did not cause any weight increase of the levator ani muscle and of the seminal vesicles in castrated rats, nevertheless exerting a marked antiglucocorticoid activity. This new steroid agent can consequently act positively on the nitrogen metabolism, being concurrently devoid of undesired virilizing anabolic effect.     

Novel modular DNA aptamer for human thrombin with high anticoagulant activity

Aptamers based on nucleic acids are a promising alternative to antibodies in therapy and diagnostics. Several DNA aptamers against human thrombin have been developed by selection from random libraries: a 15-mer and its derivatives, a 29-mer, and a 31-mer. Some of them are patented and already under clinical trial. The 15-mer structure was determined by X-ray and NMR and turned out to be a monomolecular antiparallel G-quadruplex. The other aptamers mentioned above have higher inhibitory activity than the initial 15-mer, but there are not yet structural data explaining this phenomenon. Here, the initial 15-mer, 31-mer, and novel RA-36 aptamers are compared to establish the structure-function correlation, providing a solid ground for further rational aptameric drug design. For the molecular dynamic simulation of aptamers, the force field parmbsc0 was ported onto GROMACS, and the main stabilizing parameters were revealed, leading to the novel DNA aptamer RA-36. The functional properties of the DNA aptamers were studied by conventional coagulation tests, which do not directly elucidate the mechanism of thrombin inhibition by aptamers. Improved turbidimetric measurements provided data to develop detailed kinetics showing that the 31-mer and RA-36, in contrast to the 15-mer, are competitive inhibitors. These data revealed RA-36 to be an efficient thrombin inhibitor with a dose-dependent effect. Animal tests of the studied DNA aptamers suggested an unexpected species-specificity of the novel RA-36.     

Novel terphenyls and 3,5-diaryl isoxazole derivatives endowed with growth supporting and antiapoptotic properties

A new study on terphenyl and diaryl-isoxazole and -isoxazoline derivatives, maintaining a common 3-adamantyl-4-hydroxyphenyl moiety, has been conducted to find compounds with growth supporting and antiapoptotic properties. Unexpectedly, diphenyisoxazole derivatives bearing a nitro group replacing the carboxylic function have been found with the highest cell protective activity within the series, in complete and in serum-free conditions. Inhibition of apoptosis induced by daunorubicin has also been observed for the most active compound.