Synthesis and evaluation of sesquiterpene lactone inhibitors of phospholipase A2 from Bothrops jararacussu

Several sesquiterpene lactone were synthesized and their inhibitive activities on phospholipase A₂ (PLA₂) from Bothrops jararacussu venom were evaluated. Compounds Lac01 and Lac02 were efficient against PLA₂ edema-inducing, enzymatic and myotoxic activities and it reduces around 85% of myotoxicity and around 70% of edema-inducing activity. Lac05-Lac08 presented lower efficiency in inhibiting the biological activities studied and reduce the myotoxic and edema-inducing activities around only 15%. The enzymatic activity was significantly reduced. The values of inhibition constants (K_I) for Lac01 and Lac02 were approximately 740 μM, and for compounds Lac05-Lac08 the inhibition constants were approximately 7.622-9.240 μM. The enzymatic kinetic studies show that the sesquiterpene lactones inhibit PLA₂ in a non-competitive manner. Some aspects of the structure-activity relationships (topologic, molecular and electronic parameters) were obtained using ab initio quantum calculations and analyzed by chemometric methods (HCA and PCA). The quantum chemistry calculations show that compounds with a higher capacity of inhibiting PLA₂ (Lac01-Lac04) present lower values of highest occupied molecular orbital (HOMO) energy and molecular volume (VOL) and bigger values of hydrophobicity (LogP). These results indicate some topologic aspects of the binding site of sesquiterpene lactone derivatives and PLA₂.     

Koninginins, phospholipase A2 inhibitors from endophytic fungus Trichoderma koningii.

Many isolated compounds from endophytic fungus have been useful to human beings, mainly those with medicinal applications and particularly those that can be used in inflammatory processes. Trichoderma fungi produce substances known as koninginins that have great structural similarity to compounds like flavonoids and vitamin E, which are able to inhibit the phospholipase A(2) (PLA(2)). In this work, koninginins A, E and F (KonA, KonE and KonF, respectivamente) isolated from Trichoderma koningii had their capabilities of inhibiting edema-inducing, myotoxic and enzymatic activities of the total venom of Bothrops jararacussu (jararacu?u) snake analyzed, as well as one of its homolog forms of phospholipases A(2) (bjPLA(2)-group IIB) and human secreted PLA(2) protein fusion (hsPLA(2)-group IIA). KonA was not efficient in inhibiting the three activities analyzed in all the tests performed. Nevertheless, KonE and KonF present great capability in inhibiting the effects provoked not only by the venom but also by both PLA(2). The activities inhibition shown by KonE and KonF over the enzymes is significantly higher than those obtained over the total venom. KonE and KonF were slightly more efficient in the inhibition of the group IIB (bjPLA(2)) PLA(2) effects than in the inhibition of the group IIA (hsPLA(2)) PLA(2) effects. KonE and KonF structures are similar to vitamin E and, possibly, the action mode of these molecules is similar to the one produced by the vitamin. These results, apparently, indicate that koninginins E and F, as well as vitamin E, present structural regions that might be used as start points in seeking for new and specific anti-inflammatory drugs against such enzymes.     

Photochemical Study of Hexahydroquinoline Derivatives - A New Group of Calcium Antagonists

The photochemical stability of 2, 6, 6-trimethyl-3-carbmethoxy-4-phenyl-5-oxo-1, 4, 5, 6, 7, 8-hexahydroquinoline (HHQ) derivatives with different substituents on the phenyl ring (-Cl, -NO(2), -CF(3), -CH(3), -OCH(3)) has been studied.The process of photodegradation was studied by UV spectrophotometry. The rate of photodegradation was found to depend on the type and position of the substituent in the phenyl ring. The compounds most susceptible to the damaging effect of light proved to be those containing the nitro group, in particular with the substituent in the ortho position of the aromatic ring. Derivatives with alkyl (-CH(3)) and halo-alkyl (-CF(3)) substituents showed the greatest photochemical stability. The compounds with substituents in the ortho position were found to be much less photostable than the meta isomers. The quantum yield values obtained ranged from 10(-4) to 10(-3), indicating the occurrence of secondary photochemical processes initiated by the primary products of decomposition.     

Three-component synthesis of pyrano[2,3-d]-pyrimidine dione derivatives facilitated by sulfonic acid nanoporous silica (SBA-Pr-SO3H) and their docking and urease inhibitory activity

Background

A straightforward and efficient method for the synthesis of pyrano[2,3-d]pyrimidine diones derivatives from the reaction of barbituric acid, malononitrile and various aromatic aldehydes using SBA-Pr-SO₃H as a nanocatalyst is reported.

Results

Reactions proceed with high efficiency under solvent free conditions. Urease inhibitory activity of pyrano[2,3-d]pyrimidine diones derivatives were tested against Jack bean urease using phenol red method. Three compounds of 4a, 4d and 4l were not active in urease inhibition test, but compound 4a displayed slight urease activation properties. Compounds 4b, 4k, 4f, 4e, 4j, 4g and 4c with hydrophobic substitutes on phenyl ring, showed good inhibitory activity (19.45-279.14 μM).

Discussion

The compounds with electron donating group and higher hydrophobic interaction with active site of enzyme prevents hydrolysis of substrate. Electron withdrawing groups such as nitro at different position and meta-methoxy reduced urease inhibitory activity. Substitution of both hydrogen of barbituric acid with methyl group will convert inhibitor to activator.     

Photochemical study of hexahydroquinoline derivatives--a new group of calcium antagonists

The photochemical stability of 2,6,6-trimethyl-3-carbmethoxy-4-phenyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline (HHQ) derivatives with different substituents on the phenyl ring (-Cl, -NO2, -CF3, -CH3, -OCH3) has been studied. The process of photodegradation was studied by UV spectrophotometry. The rate of photodegradation was found to depend on the type and position of the substituent in the phenyl ring. The compounds most susceptible to the damaging effect of light proved to be those containing the nitro group, in particular with the substituent in the ortho position of the aromatic ring. Derivatives with alkyl (-CH3) and halo-alkyl (-CF3) substituents showed the greatest photochemical stability. The compounds with substituents in the ortho position were found to be much less photostable than the meta isomers. The quantum yield values obtained ranged from 10(-4) to 10(-3), indicating the occurrence of secondary photochemical processes initiated by the primary products of decomposition.     

Synthesis and evaluation of tricyclic dipyrido diazepinone derivatives as inhibitors of secretory phospholipase A2 with anti-inflammatory activity

A series of tricyclic dipyrido diazepinone derivatives 6(a-f) bearing different substituents at the tenth position of diazepinone ring were designed and are characterized by 1H NMR, FTIR and X-Ray crystallography studies. The synthesised derivatives are tested in-vitro phospholipase A2 (PLA2) enzyme inhibitory activity and in-vivo anti-inflammatory activity against purified group I and group II PLA2 enzymes from the snake venom and human pleural fluid. Compounds bearing aromatic ring with different substituents at different positions shown varied specificity. The 6f derivative with strong electron withdrawing nitro (-NO2) and trifluoromethyl (-CF3) groups at ortho and para positions respectively shown greater inhibitory activity. Inhibitory effect of the compound appeared to be direct interaction with active site and likely competes with substrates as supported by substrate dependent and calcium independent assays. The IC50 value of potent PLA2 inhibitor 6f was 22.1 microM and showed similar potency in the neutralization of in vivo PLA2 induced mouse paw edema and hemolytic activity.     

Effects of morin on snake venom phospholipase A2 (PLA2).

Flavonoids are potent anti-inflammatory compounds isolated from several plant extracts, and have been used experimentally against inflammatory processes. In this work, a PLA2 isolated from the Crotalus durissus cascavella venom and rat paw oedema were used as a model to study the effect of flavonoids on PLA2. We observed that a treatment of PLA2 with morin induces several modifications in the aromatic amino acids, with accompanying changes in its amino acid composition. In addition, results from circular dichroism spectroscopy and UV scanning revealed important structural modifications. Concomitantly, a considerable decrease in the enzymatic and antibacterial activities was observed, even though anti-inflammatory and neurotoxic activities were not affected. These apparent controversial results may be an indication that PLA2 possess a second pharmacological site which does not affect or depend on the enzymatic activity.     

5-Arylidene-2(<Emphasis Type="Italic">5H</Emphasis>)-furanone derivatives: synthesis and structure-activity relationship for cytotoxicity

Thirty-eight 5-arylidene-2(5H)-furanone derivatives possessing halo-, methoxy-, oxo-, dioxo-, and thiophenyl groups as well as anthraquinone and naphthquinone moieties were synthesized, and their cytotoxicity was evaluated against various cancer cell lines. The introduction of halogen atoms or nitro group at aromatic ring of 5-arylidene-2(5H)-furanone was shown to increase the cytotoxicity with 5-(3-nitrobenzylidene)-2(5H)-furanone (21) being the most potent. Among anthracenyl or naphthalenyl derivatives, (E)-5-[2-(1,4-dimethoxy-9,10-dioxo) anthracenyl]-2(5H)-furanone (34) showed the most potent cytotoxic activity.     

Isolation of a snake venom phospholipase A2 (PLA2) inhibitor (AIPLAI) from leaves of Azadirachta indica (Neem): Mechanism of PLA2 inhibition by AIPLAI in vitro condition

A compound (AIPLAI (Azadirachta indica PLA(2) inhibitor)) purified from the methanolic leaf extract of A. indica (Neem) inhibits the cobra and Russell's viper venoms (RVVs) phospholipase A(2) enzymes in a dose-dependent manner. Inhibition of catalytic and tested pharmacological properties of cobra venom (Naja naja and Naja kaouthia) PLA(2) enzymes by AIPLAI is significantly higher (P<0.05) compared to the inhibition of PLA(2) enzymes of crude RVV (Daboia russelli) when tested under the same condition. Kinetic study reveals that in in vitro condition, AIPLAI inhibits the purified N. kaouthia PLA(2) enzymes in a non-competitive manner. The AIPLAI is quite stable at room temperature. The present study shows that AIPLAI holds good promise for the development of novel anti-snake venom drug in future.     

Structure activity analysis of the pro-apoptotic,antitumor effect of nitrostyrene adducts and related compounds

In the present study, we outlined the part of the molecule mediating the prominent pro-apoptotic effect of the Michael adduct of ascorbic acid with p-chloro-nitrostyrene, a new synthetic phosphatase inhibitor. The nitrostyrene (NS) moiety was identified as the structure essential for apoptosis induction. NS and its ascorbic acid adducts displayed LC(50) values of 10-25 microM with no significant reduction of potency in okadaic acid resistant cells overexpressing the MDR1 P-glycoprotein. Induction of apoptosis by NS derivatives and the protein phosphatase 2A inhibitor cantharidic acid was proven by the analysis of caspase-3 activation and subsequent fragmentation of DNA. Further structure activity analysis revealed the necessity of the nitro group at the beta-position of the side chain. The pro-apoptotic potential of adducts of NS with pyrimidine- or pyridine-derivatives varied between NS and a progressive reduction in potency up to a nearly complete loss of cytotoxicity. Substitutions at the benzene core of NS suggested a prominent enhancement of toxicity only by substitutions at the 2- or 3-position. Heterocyclic aromatics can substitute for the benzene ring of NS albeit with a 2-3-fold reduced potency. In conclusion, nitrostyrene was identified as the core structure mediating the pro-apoptotic effect of a new synthetic phosphatase inhibitor. Further studies defined a nitrovinyl side chain attached to an aromatic ring as the pharmacophore structure of a new group of pro-apoptotic agents. These observations present the basis for the development of a new group of anticancer drugs.     

Purification and renal effects of phospholipase A(2) isolated from Bothrops insularis venom.

Bothrops insularis venom contains a variety of substances presumably responsible for several pharmacological effects. We investigated the biochemical and biological effects of phospholipase A(2) protein isolated from B. insularis venom and the chromatographic profile showed 7 main fractions and the main phospholipase A(2) (PLA(2)) enzymatic activity was detected in fractions IV and V. Fraction IV was submitted to a new chromatographic procedure on ion exchange chromatography, which allowed the elution of 5 main fractions designated as IV-1 to IV-5, from which IV-4 constituted the main fraction. The molecular homogeneity of this fraction was characterized by high-performance liquid chromatography (HPLC) and demonstrated by mass spectrometry (MS), which showed a molecular mass of 13984.20 Da; its N-terminal sequence presented a high amino acid identity (up to 95%) with the PLA(2) of Bothrops jararaca and Bothrops asper. Phospholipase A(2) isolated from B. insularis (Bi PLA(2) ) venom (10 microg/mL) was also studied as to its effect on the renal function of isolated perfused kidneys of Wistar rats (n=6). Bi PLA(2) increased perfusion pressure (PP), renal vascular resistance (RVR), urinary flow (UF) and glomerular filtration rate (GFR). Sodium (%TNa(+)) and chloride tubular reabsorption (%TCl(-)) decreased at 120 min, without alteration in potassium transport. In conclusion, PLA(2) isolated from B. insularis venom promoted renal alterations in the isolated perfused rat kidney.     

Isolation and characterization of ellagic acid derivatives isolated from Casearia sylvestris SW aqueous extract with anti-PLA2 activity

The Casearia sylvestris SW (Flacourtiaceae) is utilized in folk medicine (Brazil and all Latin American) to treat several pathologic processes as inflammation, cancer, microbial infection and snake bites. Studies showed that C. sylvestris aqueous extract can inhibit many toxic effects caused by snake venoms (or caused by phospholipase A₂ isolated) from different species, mainly of Bothrops genus. Inhibition of enzymatic and myotoxic activities, decrease of edema formation and increase of the survival rate of rats injected with lethal doses of bothropic venoms are some toxic effects inhibited by C. sylvestris. In this study, four ellagic acid derivatives from aqueous extracts of C. sylvestris were isolated, characterized, and tested against effects from both total venom and PLA₂ (Asp 49 BthTX-II) from the venom of Bothrops jararacussu. The isolated compounds were as follows: ellagic acid (A), 3′-O-methyl ellagic acid (B), 3,3′-di-O-methyl ellagic acid (C), 3-O-methyl-3′,4′-methylenedioxy ellagic acid (D). The inhibition constant values (Ki) for enzymatic activity, as well the IC₅₀ values found in the edematogenic and myotoxic activities, indicate that the ellagic acid is the best inhibitor of these activities, while compounds C and D are the substances with lowest capacity on inhibiting these same effects. Our results show that the presence of hydroxyls at position 3 or 3′ (compounds A and B) increases the capacity of these derivatives on inhibiting these toxic effects. However, the presence of methoxyl groups at position 3 or 3′ reduced, but did not completely inhibit the capacity of compounds C and D on inhibiting all the toxic effects studied.     

Benzisothiazoles and β-adrenoceptors: Synthesis and pharmacological investigation of novel propanolamine and oxypro-panolamine derivatives in isolated rat tissues

In an attempt to examine the ability of benzisothiazole-based drugs to interact with beta-adrenoceptors, a series of 1,2-benzisothiazole derivatives, which were substituted with various propanolamine or oxypropanolamine side chains in the 2 or 3 position, were synthesised and tested. The pharmacological activity of these compounds at the beta-adrenoceptors was examined using isolated rat atria and small intestinal segments, which preferentially express the beta1- and beta3-adrenoceptor-mediated responses, respectively. None of these products showed any beta-adrenoceptor agonistic activity. In contrast, the 2- and 3-substituted isopropyl, tert-butyl, benzyl, and piperonyl derivatives 2a-d and 3a-d elicited surmountable inhibition of the isoprenaline-induced chronotropic effects in the atria, suggesting competitive antagonism at the beta1-recognition site. The pA2 values revealed tert-butyl 3b and the isopropyl substituted piperonyl derivatives 3a to be the most effective. Remarkably, many of the 2-substituted propanolamines were less active than the corresponding 3-substituted oxypropanolamines. With the exception of compound 3b, none of these drugs antagonised the muscle relaxant activity of isoprenaline in the intestine, suggesting no effect on the beta3-adrenoceptors. These results confirm the ability of the benzisothiazole ring to interact with the beta-adrenoceptors, and demonstrate that 2-substitution with propanolamine or 3-substitution with oxypropanolamine groups yields compounds with preferential antagonistic activity at the cardiac beta1-adrenoceptors. The degree of antagonism depends strongly on both the nature of the substituent and its position on the benzisothiazole ring.     

R,S-1-(取代苯基)-4-[3-(萘-1-氧基)-2-羟基丙基]哌嗪类化合物的设计、合成及血管舒张活性

设计合成具有血管舒张活性的苯基哌嗪类系列化合物。以naftopidil活性代谢产物为先导化合物，根据已报道的苯基哌嗪类α₁-受体拮抗剂构效关系研究结论对先导化合物进行结构优化，设计并合成一系列新的苯基哌嗪类化合物，确定其结构，并通过测定其对苯肾上腺素引起家兔胸主动脉条收缩的抑制作用评价其血管舒张活性。发现其中5个化合物显示出不同程度的活性，其中化合物16的血管舒张活性较强，其在0.01和1 μmol·L^-1条件下血管收缩抑制率分别达到7.03%和22.72%，化合物16拟采用自发性高血压大鼠降压试验等进行进一步抗高血压活性研究。以上研究提示已报道苯基哌嗪类化合物的构效关系研究结论可适合于naftopidil的结构修饰。     

Stability Studies of Oxazolidine-Based Compounds Using 1H NMR Spectroscopy

A series of oxazolidine-based compounds with a variety of substituents in positions 2 and 3 was synthesized and their stability studied. Ring opened intermediates formed on addition of limiting amounts of D2O to oxazolidine solutions, as observed by NMR. As the hydrolysis reactions proceeded, a series of novel dimeric β-amino alcohol compounds formed via an internal reaction between ephedrine and the ring opened intermediates. 2-Phenyl substituted oxazolidine compounds containing electron withdrawing nitro substituents were more rapidly hydrolyzed than the unsubstituted derivative and methoxy substituted compounds, with the nitro substituents appearing to stabilize the ring opened intermediates. Two oxazolidine derivatives, with a methyl and proton at position 2, were found to be more stable to oxazolidine hydrolysis than the 2-phenyl substituted compounds. Oxazolidines incorporating phenyl substituents at position 3 were synthesized and found to be less stable than those incorporating a methyl substituent at position 3. These fundamental structure-activity relationships may be useful when choosing oxazolidine derivatives as synthetic intermediates and as prodrugs for the delivery of compounds containing either β-amino alcohol or aldehyde components. © 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 99:3362–3371, 2010     

QSAR modeling on dopamine D2 receptor binding affinity of 6-methoxy benzamides

QSAR modeling was performed on 58 (S) N-[(1-ethyl-2-pyrrolidinyl) methyl]-6-methoxy benzamides as dopamine (DA) D2 receptor antagonists to identify the structural requirements for DA D2 receptor binding affinity. The study pointed out that the presence of hydrophobic substituents at R3 position and electron-donating groups at R5 position increased the biological activity. Substitutions at phenyl ring favored the binding affinity of these benzamides. Ethyl group and iodine at R3 position were advantageous to the activity whereas nitro group at phenyl ring hindered the antagonistic activity.     

Anticancer bisquaternary heterocyclic compounds: a ras-ional design

A new family of symmetrical bisquaternary compounds with semirigid linkers have shown to be highly specific for Choline Kinase (ChoK) inhibition and to exert antitumoural activity in cell lines and in mice. A three-parameter regression equation has been derived which satisfactorily describes the ex vivo inhibitory potency of ChoK of the title compounds. The electronic effect of the group at position 4 of the cationic head plays a critical role although the hydrophobic contribution, especially that of the linker, favors the ChoK inhibitory activity. The antiproliferative activity (in vitro assay) is correlated with the ChoK inhibition (ex vivo assay) through the electronic effect and a squared term of the overall lipophilicity of the molecules. We also provide in vivo evidence that ChoK is a novel target for the design of antitumoural drugs. All these results suggest that ChoK plays a crucial role in the onset of carcinogenesis.     

Functional expression and characterization of a recombinant phospholipase A2 from sea snake Lapemis hardwickii as a soluble protein in E. coli.

Three full-length phospholipase A(2) (PLA(2)) cDNAs from sea snake Lapemis hardwickii venom were cloned and sequenced in our previous study. In order to investigate their biological functions, we established a fusion expression system for PLA(2)-9 in E. coli. The open reading frame encoding mature peptide of PLA(2)-9 was subcloned into the vector pTRX. The Trx-PLA(2)-9 fusion protein was expressed as a soluble protein by IPTG induction at 23 degrees C. The fusion protein was purified with metal-chelate affinity chromatography and then cleaved by enterokinase. The mature recombinant PLA(2)-9 was further purified by ion-exchange chromatography and a final yield of approximately 2.5mg pure PLA(2)-9 from 1l of bacteria culture was obtained. The catalytic activity of recombinant PLA(2)-9 (rPLA(2)-9) was measured and found to be similar to native enzyme. As the Austrelaps superbus PLA(2), which shares 90% nucleotide sequence similarity to PLA(2)-9, the rPLA(2)-9 displayed the anti-platelet aggregation effect. Site-directed mutagenesis of the two conserved residues, His-48 and Asp-49, resulted in the loss of catalytic activity, however did not affect the inhibition effect of platelet aggregation suggesting that these two activities of sea snake PLA(2)-9 may be dissociated.