In search of cyclooxygenase inhibitors, anti-Mycobacterium tuberculosis and anti-malarial drugs from Thai flora and microbes.

Malaria continues to be a major infectious disease of the developing world and the problem is compounded not only by the emergence of drug resistant strains but also from a lack of a vaccine. The situation for tuberculosis (TB) infection is equally problematic. Once considered a "treatable" disease for which eradication was predicted, TB has re-emerged as highly lethal, multi-drug resistant strains after the outbreak of AIDS. Worldwide, the disease causes millions of deaths annually. Similarly, treatments for chronic inflammatory diseases such as arthritis have been impeded due to the potentially lethal side effects of the new and widely prescribed non-steroidal anti-inflammatory compounds. Thais have utilized bioresources from plants and some microorganisms for medicine for thousands of years. Because of the need for new drugs to fight malaria and TB, with radically different chemical structures and mode of actions other than existing drugs, efforts have been directed towards searching for new drugs from bioresources. This is also true for anti-inflammatories. Although Thailand is considered species-rich, only a small number of potential bioresources has been investigated. This article briefly describes the pathogenesis of 2 infectious diseases, malaria and TB, and modern medicines employed in chemotherapy. Diversities of Thai flora and fungi and their chemical constituents with antagonistic properties against these 2 diseases are described in detail. Similarly, anti-inflammatory compounds, mostly cyclooxygenase (COX) inhibitors, are also described herein to demonstrate the potential of Thai bioresources to provide a wide array of compounds for treatment of diseases of a different nature.

Immobilization of malarial (Plasmodium falciparum) dihydrofolate reductase for the selection of tight-binding inhibitors from combinatorial library.

A simple procedure for selection of tight-binding inhibitors of mutant dihydrofolate reductases from Plasmodium falciparum (PfDHFRs) based on preferential binding to the enzyme immobilized on a Sepharose column has been described. PfDHFRs with a cysteine residue at the C-terminal have been prepared in order to immobilize to a thiopropyl-Sepharose gel via S-S linkage. The amount of immobilized DHFRs was estimated to be 4-5 mg/g of dried gel, and the activities of bound DHFRs were comparable to that of free enzymes. The prepared immobilized enzyme has been used for the selection of tight-binding inhibitors from combinatorial libraries, based on the affinities of each ligand with the enzyme. Free ligands were then identified and analyzed quantitatively by high-performance liquid chromatography-mass spectrometry, and the components with high binding affinity of the library could thus be realized. Results could be confirmed by quantitative analysis of the bound ligands released from the enzyme by guanidine hydrochloride treatment.

Antiplasmodial, antimycobacterial, and cytotoxic principles from Camchaya calcarea.

Chemical exploration of Camchaya calcarea (family Compositae) has led to the isolation of nine known sesquiterpene lactones 1 - 9, together with caffeic acid methyl ester 10. Sesquiterpenes 1, 2, 3, 4, 5, 7, and 8 exhibited moderate antiplasmodial activity, but showed potent antimycobacterial activity. Interestingly, the cytotoxicity of sesquiterpene lactones 1, 2, and 4 towards small-cell lung cancer cell line (NCI-H187) is stronger (two orders of magnitude) than towards the Vero cell line. Caffeic acid methyl ester (10) was cytotoxic against NCI-H187 and BC cell lines, however the ester 10 showed only mild antimycobacterial activity.

Bioactive compounds from the seed fungus Menisporopsis theobromae BCC 3975.

Eight new compounds (2-9), together with a known dithiodiketopiperazine (1), were isolated from the seed fungus Menisporopsis theobromae BCC 3975. The structures of these substances were elucidated by analyses of spectroscopic data. Compounds 1 and 4 exhibited moderate cytotoxicity against BC-1 cell lines with IC50 values of 29.2 and 57.4 microM, respectively. Cytotoxicity of 1, 2, 4, and 9 against the NCI-H187 cell line showed respective IC50 values of 22.9, 20.3, 1.8, and 56.6 microM. Compounds 2 and 4 exhibited antimalarial activity with IC50 values of 2.95 and 28.8 microM, respectively. Substances 1, 4, 7, 8, and 9 possessed weak antimycobacterial activity (MIC 154.8-952.3 microM), while compounds 2 and 3 showed potent antimycobacterial activity with respective MIC values of 1.24 and 7.14 microM.

Transformation of an irregularly bridged epidithiodiketopiperazine to trichodermamide A.

[reaction: see text] An unusually bridged epidithiodiketopiperazine, pretrichodermamide A (3), was isolated from the fungus Trichoderma sp. BCC 5926. During the extensive effort to crystallize 3 for X-ray crystallographic analysis, conversion of this compound to trichodermamide A with coproduction of S(8) occurred.

Bioactive substances from insect pathogenic fungi.

Insect pathogenic fungi have opened up a relatively untapped area of natural product research which, unfortunately, has not received much attention to date. Found in wild abundance in wet tropical Thailand, the insect fungi are shown to contribute not only as controllers of insect populations but also as rich sources of structurally novel biologically active substances.

Combretastatins D-3 and D-4, new macrocyclic lactones from Getonia floribunda.

Chemical investigation of biologically active compounds of Getonia floribunda led to the isolation of two new macrocyclic lactones, combretastatins D-3 (1) and D-4 (2). The structures of these compounds were confirmed by spectroscopic analyses. Combretastatin D-3 (1) exhibited cytotoxicity towards the small-cell lung cancer cell line (NCI-H187, IC50=13.0 +/- 0.2 microg/mL) but was inactive against KB, BC-1, and Vero cell lines. Combretastatin D-3 (1) showed weak antitubercular activity with a minimum inhibitory concentration (MIC) of 100.0 microg/mL, and was inactive towards the malarial parasite. Combretastatin D-4 (2) was inactive in all antitubercular, antiplasmodial, and cytotoxic assays.

Antiviral and antiplasmodial spirodihydrobenzofuran terpenes from the fungus Stachybotrys nephrospora.

Two known spirodihydrobenzofuran terpenes (1 and 2) were isolated from a mycelium extract of the fungus Stachybotrys nephrospora BCC 3900. Compound 1 (Mer-NF5003F or stachybotrydial) exhibited potent antiviral activity (the IC50 value of 4.32 microg/mL) comparable to the standard drug, acyclovir, while compound 2 was inactive against the HSV-1 virus. Both 1 and 2 possessed antiplasmodial activity (IC50 values of 0.85 and 0.15 microg/mL for 1 and 2, respectively), and were not toxic towards the Vero cell line. A regiospecific conversion of the dialdehyde 1 to the lactone 2 proceeded simply under acidic conditions.

Azaphilone pigments from a yellow mutant of the fungus Monascus kaoliang.

Azaphilone pigments, monascusones A (1) and B (2), together with two known azaphilones, monascin (3) and FK17-P2b2 (4), were isolated from the CH2Cl2 extract of a yellow mutant of the fungus M. kaoliang grown on rice. Structures of the isolated compounds were elucidated by analyses of spectroscopic data. Monascusone A (1), the major metabolite of M. kaoliang, showed no antimalarial (against Plasmodium falciparum), antitubercular (against Mycobacterium tuberculosis H37Ra), and antifungal (toward Candida albicans) activities. Compound 1 exhibited no cytotoxicity against BC (breast cancer) and KB (human epidermoid carcinoma of cavity) cell lines.

Isolation and structure elucidation of a novel antimalarial macrocyclic polylactone, menisporopsin A, from the fungus Menisporopsis theobromae.

An antimalarial macrocyclic polylactone, menisporopsin A (1), was isolated from a cell extract of the seed fungus Menisporopsis theobromae. The structure of 1 was elucidated on the basis of spectroscopic analysis and chemical transformations, with the absolute configuration established by application of the modified Mosher method and by using chiral HPLC. Menisporopsin A (1) possesses an unprecedented residue, 2,4-dihydroxy-6-(2,4-dihydroxy-n-pentyl)benzoic acid. This compound exhibited antimalarial activity, with an IC(50) value of 4.0 microg mL(-1), and antimycobacterial activity (MIC value of 50 microg mL(-1)).

A novel ascochlorin glycoside from the insect pathogenic fungus Verticillium hemipterigenum BCC 2370.

Vertihemipterin A, the ascochlorin glycoside, and its aglycone, 4',5'-dihydro-4'-hydroxyascochlorin, were isolated from the insect pathogenic fungus Verticillium hemipterigenum BCC 2370. A new analog, 8'-hydroxyascochlorin, and five known compounds, ascochlorin, LL-Z1272delta, 8',9'-dehydroascochlorin, ascofuranone and ascofuranol, were also isolated from the same fermentation broth. Structures of these compounds were elucidated by spectroscopic methods. Stereochemistry of the known compound, 4',5'-dihydro-4'-hydroxyascochlorin, was addressed by NMR spectral analyses and the modified Mosher's method. Antiviral (HSV-1) and cytotoxic activities of these ascochlorin analogs were evaluated.

Ketene acetal and spiroacetal constituents of the marine fungus Aigialus parvus BCC 5311.

Aigialone (1) and aigialospirol (2), two structurally unique compounds, were isolated from the mangrove fungus Aigialus parvus BCC 5311. The structure of the new ketene acetal 1 was elucidated by spectral analysis, and its relative stereochemistry was determined by X-ray crystallography. The stereochemistry of aigialospirol (2), elucidated by NMR spectral analysis, suggested that this compound is possibly derived from hypothemycin (3), a metabolite previously isolated from this same fungus.

Lakoochins A and B, new antimycobacterial stilbene derivatives from Artocarpus lakoocha.

Two new stilbene derivatives, lakoochins A (1) and B (2), were isolated from the roots of Artocarpus lakoocha. The structures of 1 and 2 were elucidated by analysis of their spectral data. Lakoochins A (1) and B (2) exhibited antimycobacterial activity with the respective MIC values of 12.5 and 50 microg/mL. While 1 was cytotoxic against the BC (breast cancer) cell line (IC(50) 6.1 microg/mL) but inactive (at 20 microg/mL) toward KB (nasopharyngeal carcinoma) cells, compound 2 possessed cytotoxicity against the BC and KB cell lines with IC(50) values of 3.1 and 6.1 microg/mL, respectively.

Target guided synthesis of 5-benzyl-2,4-diamonopyrimidines: their antimalarial activities and binding affinities to wild type and mutant dihydrofolate reductases from Plasmodium falciparum.

The resistance to pyrimethamine (PYR) of Plasmodium falciparum arising from mutation at position 108 of dihydrofolate reductase (pfDHFR) from serine to asparagine (S108N) is due to steric interaction between the bulky side chain of N108 and Cl atom of the 5-p-Cl aryl group of PYR, which consequently resulted in the reduction in binding affinity between the enzyme and inhibitor. Molecular modeling suggested that the flexible antifolate, such as trimethoprim (TMP) derivatives, could avoid this steric constraint and should be considered as new, potentially effective compounds. The hydrophobic interaction between the side chain of inhibitor and the active site of the enzyme around position 108 was enhanced by the introduction of a longer and more hydrophobic side chain on TMP's 5-benzyl moiety. The prepared compounds, especially those bearing aromatic substituents, exhibited better binding affinities to both wild type and mutant enzymes than the parent compound. Binding affinities of these compounds correlated well with their antimalarial activities against both wild type and resistant parasites. Molecular modeling of the binding of such compounds with pfDHFR also supported the experimental data and clearly showed that aromatic substituents play an important role in enhancing binding affinity. In addition, some compounds with 6-alkyl substituents showed relatively less decrease in binding constants with the mutant enzymes and relatively good antimalarial activities against the parasites bearing the mutant enzymes.

Novel biologically active bibenzyls from Bauhinia saccocalyx Pierre.

Four new bibenzyls, bauhinols A-D (1-4), together with the two known bibenzyls 5 and 6, were isolated from the roots of Bauhinia saccocalyx, and their structures were elucidated by analyses of spectroscopic data. Bauhinol A (1) exhibits significant cytotoxicity towards NCI-H187 (small-cell lung cancer), BC (breast cancer), and KB (oral-cavity cancer) cell lines, with IC50 values of 2.7-4.5 microg/ml. Bauhinol B (2) is cytotoxic against NCI-H187 (IC50 = 1.1 microg/ml) and BC (IC50 = 9.7 microg/ml) cell lines, but inactive toward the KB cell line (at 20 microg/ml). Compound 2 also is mildly antifungal towards Candia albicans (IC50 = 28.9 microg/ml). Bibenzyl 6 is active against NCI-H187 (IC50 = 14.1 microg/ml) and BC (IC50 = 4.0 microg/ml) cells, but inactive (at 20 microg/ml) toward the KB cell line. Compounds 1, 2, and 6 show mild antimycobacterial activities, with MIC values of 25-50 microg/ml, but are inactive at 20 microg/ml against the K1 malarial parasite strain (Plasmodium falciparum). While bauhinol A (1) is inactive against cyclooxygenase 1 (COX-1) and cyclooxygenase 2 (COX-2), compounds 2 and 6 inhibit both COX-1 and COX-2, with IC50 values comparable to those of the standard drug, aspirin (Table 3).

Micromonosporin A, a novel 24-membered polyene lactam macrolide from Micromonospora sp. isolated from peat swamp forest.

A novel 24-membered polyene lactam macrolide, micromonosporin A (=(3E,5E,7Z,15E,17E,19E,21E)-9,11,13-trihydroxy-14,19,24-trimethyl-1-azacyclotetracosa-3,5,7,15,17,19,21-heptaen-2-one; 1) was isolated from the actinomycete, Micromonospora sp. strain TT1-11, which was isolated from a very acidic peat swamp forest.

Enniatin production by the entomopathogenic fungus Verticillium hemipterigenum BCC 1449.

Optimal fermentation conditions for enniatin production using the entomopathogenic fungus Verticillium hemipterigenum BCC 1449 have been investigated. Among various liquid media tested, highest efficiency of enniatin production was achieved by fermentation in yeast extract sucrose. Application of this condition to large-scale fermentation resulted in the isolation of three new analogs, O1, O2 and O3, which are closely related isomers that were characterized as an inseparable mixture, along with seven known enniatins.

New diketopiperazines from the entomopathogenic fungus Verticillium hemipterigenum BCC 1449.

Two new diketopiperazines, bisdethiodi(methylthio)-1-demethylhyalodendrin and 1-demethylhyalodendrin tetrasulfide, together with two known cyclodepsipeptides, enniatins B and B4, and two known pyrones, pyrenocines A and B, were isolated from a culture broth of the entomopathogenic fungus Verticillium hemipterigenum BCC 1449. These structures were elucidated using spectroscopic methods and X-ray crystallography. Antimalarial and cytotoxic activities of these compounds were evaluated.

New antimycobacterial and antimalarial 8,9-secokaurane diterpenes from Croton kongensis.

Two new 8,9-secokaurane diterpenes, ent-8,9-seco-7alpha,11beta-diacetoxykaura-8(14),16-dien-9,15-dione (1) and ent-8,9-seco-8,14-epoxy-7alpha-hydroxy-11beta-acetoxy-16-kauren-9,15-dione (2), together with two known compounds, ent-8,9-seco-7alpha-hydroxy-11beta-acetoxykaura-8(14),16-dien-9,15-dione (3) and ent-7beta-hydroxy-15-oxokaur-16-en-18-yl acetate, were isolated from Croton kongensis. This is the first report on the presence of 8,9-secokauranes in the plant genus Croton. Diterpenes 1-3 exhibited antimycobacterial activity with minimum inhibitory concentrations (MICs) of 25.0, 6.25, and 6.25 microg/mL, respectively, and possessed antimalarial activity with IC(50) ranges of 1.0-2.8 microg/mL. They also demonstrated comparable cytotoxicity toward the Vero (IC(50) ranged from 0.9 to 3.2 microg/mL), KB (IC(50) from 1.2 to 13.8 microg/mL), and BC cell lines (IC(50) from 1.1 to 2.2 microg/mL, except for compound 1, which was inactive against BC cells).

Synthesis of ethyl 5-phenyl-6-oxa-1-azabicyclo[3.1.0]hexane-2-carboxylate derivatives and evaluation of their antimalarial activities.

Derivatives of ethyl 5-phenyl-6-oxa-1-azabicyclo[3.1.0]hexane-2-carboxylate (14-20), with side chains varying from three to five carbon atoms and bearing various substituents, have been prepared from ethyl 2-phenyl-1-pyrroline-5-carboxylate (12). Their in vitro activity against P. falciparum (K1 strain) and antimycobacterium and also their cytotoxic activity against Vero cell have been evaluated.