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Four novel compounds consisting of two new pimaranes, lecheronol A (1) and lecheronol B (2), an acylated cycloartane, 3-O-β-lauroyl-cycloart-(23E)-en-25-ol (10), and a highly oxygenated novel chalconoid, α,β,3,4,5,2‘,4‘,6‘-octahydroxydihydrochalcone (12), were isolated along with seven known triterpene derivatives and three flavonol glucosides from Mycobacterium tuberculosis growth-inhibiting fractions of the CH2Cl2/MeOH (1:1) extract of the aerial parts of Sapium haematospermum. Compounds 1, 3 (3α-hydroxyolean-12-ene), 8 [3α-hydroxylup-20(29)-en], and 9 (cycloartanol) were found most active, with MIC values of 4, 12.2, 13.4, and 8 μg/mL, respectively. Cytotoxicity tests in Vero cells for compounds 1, 3, 8, and 9 gave IC50 values of 104.8, 127.2, 127.2, and 102.4 μg/mL, respectively.

The emergence of resistant strains of Plasmodium falciparumand Mycobacterium tuberculosisunderscores the need for novel drugs that are effective against these microorganisms.

Pyridines and pyrazines substituted with 1,2,4-oxadiazole-5-ones, 1,2,4-oxadiazole-5-thiones, and 1,3,4-oxathiazoline-2-ones were synthesized and tested against Mycobacterium tuberculosis. The two former ring systems were documented in the literature to act as carboxylic acid isosteres. The latter series was synthesized as possible synthetic intermediates to 1,2,4-thiadiazole-3-ones and was included in this study due to their interesting activity. Pivaloyloxymethyl derivatives of the isosteres were also prepared in order to increase their lipophilicity and therefore improve their cellular permeability. The derivatized isosteres were expected to be biotransformed by esterases to the active species after penetration of the mycobacterial cell wall. Biological properties of the compounds were compared with the unmodified polar isosteres of pyrazinoic and nicotinic acids. The majority of the compounds exhibited activities ranging from 0.5 to 16 times the potency of pyrazinamide.

Dicationic 2,5-bis(4-guanidinophenyl)furans 5a−5f, 2,5-bis[4-(arylimino)aminophenyl]furans 6a−6b and 6e−6k, and 2,5-bis[4-(alkylimino)aminophenyl]furans 6c−6d have been synthesized starting from 2,5-bis[tri-n-butylstannyl]furan. Thermal melting studies with poly dA•dT and the duplex oligomer d(CGCGAATTCGCG)2 demonstrated high DNA binding affinities for a number of the compounds. The binding affinities are highly dependent on structure and are significantly affected by substituents both on the phenyl rings of the 2,5-diphenylfuran nucleus and on the cationic centers. Of the 17 novel dicationic compounds synthesized, six (6a, 6b, 5b, 6f, 6h, 6i) exhibited MICs of 2 μg/mL or less versus Mycobacterium tuberculosis. Of the compounds screened against Candida albicans, three gave MICs of 2 μg/mL or less (5b, 6h, 6i), and two (5b, 6i) were fungicidal, unlike a standard antifungal drug fluconazole, which was fungistatic. In addition, one of the tested compounds (6i) exhibited a MIC of <1 μg/mL against Aspergillus fumigatus, while also being a fungicidal against this organism. Finally, when evaluated against an expanded fungal panel, compound 6h showed good activity against Cryptococcus neoformans and Rhizopus arrhizus.

Assay-guided fractionation of the antitubercular MeOH−CH2Cl2 extract obtained from Lippia turbinata led to the isolation of four novel triterpenoids&sbd;3β,25-epoxy-3α,21α-dihydroxy-22β-(3-methylbut-2-en-1-oyloxy)olean-12-ene-28-oic acid (1); 3β,25-epoxy-3α,21α-dihydroxy-22β-angeloyloxyolean-12-ene-28-oic acid (2); 3β,25-epoxy-3α,21α-dihydroxy-22β-tigloyloxyolean-12-ene-28-oic acid (3); and 3β,25-epoxy-3α-hydroxy-22β-(2-methylbutan-1-oyloxy)olean-12-ene-28-oic acid (4)&sbd;together with the known triterpenoids lantanilic acid (5), camaric acid (6), lantanolic acid (7), and rehmannic acid (8). The MIC values of 1−8 for growth inhibition of Mycobacterium tuberculosis were determined in the radiorespirometric BACTEC system.

Three novel triterpenes, 3,4-seco-olean-12-ene-3,28-dioic acid (4), 3α-hydroxyolean-11-en-28,13β-olide (5), and 3α-hydroxyoleane-11:13(18)-dien-28-oic acid (6), were isolated from the aerial parts of the Argentinean shrub, Junellia tridens. Another five compounds&sbd;oleanolic (1), oleanonic (2), and epioleanolic acids (3), all biosynthetically related to the three new oleananes, and epibetulinic acid (7) and sitosterol (8)&sbd;were also isolated. Structures were elucidated primarily by 1D and 2D NMR and mass spectrometry, and all protons and carbons of the three novel compounds were fully assigned by NMR. We report the minimum inhibitory concentrations of these compounds against Mycobacterium tuberculosis and conclude that they are responsible for antitubercular activity originally observed in the crude plant extract. LC-MS data is provided on the occurrence of triterpenes 1−6 in six other species of Junellia.

The metabolic response of freshly harvested, nude-mouse-derived Mycobacterium leprae to biophysical parameters was studied to facilitate an understanding of axenic culture requirements. Quantitation of intracellular ATP and the rate of [U-14C]palmitic acid incorporation into phenolic glycolipid I (PGL-I) were used as metabolic indicators after axenic incubation in modified Dubos medium under various biophysical conditions. PGL-I synthesis was optimal at 33 degrees C, whereas ATP was optimally maintained at less than or equal to 33 degrees C. Both metabolic indices showed sharp reductions at 37 degrees C. After 5 days of incubation, PGL-I synthesis and ATP maintenance showed pH optima of 5.1 to 5.6, with the higher value appearing optimal for ATP maintenance after extended incubation. Metabolic activity was negatively affected by strong reducing agents, and ATP maintenance was optimal when the gaseous environment was maintained at 2.5 to 10% oxygen. The results may partially explain the failure to cultivate the leprosy bacillus in vitro.

Measurement of intracellular ATP of Mycobacterium leprae after direct in vitro exposure to antimicrobial agents was evaluated as a rapid means of identifying potentially useful therapeutic agents. Nude mouse-derived M. leprae was incubated in an axenic modified Dubos medium in the presence or absence of antimicrobial agents for up to 3 weeks. ATP was then assayed by using the firefly bioluminescence technique. Rifampin, clofazimine, and ethionamide each effected a significantly accelerated rate of ATP decay compared with controls. Dapsone appeared inactive, possibly reflecting a general insensitivity of this system to compounds acting at certain loci. The system appeared suitable for assessing comparative activity of new structural analogs of clofazimine. Other active compounds included erythromycin, minocycline, chloramphenicol, gramicidin, and, to a lesser extent, cycloserine, cephalothin, ciprofloxacin, tetracycline, and gramicidin S. The penicillins, bacitracin, isoniazid, nalidixic acid, trimethoprim, polymyxin B, and griseofulvin were all inactive. The system appears sensitive to agents with various modes of action and may prove useful as a primary screen for antileprosy drugs.

In an attempt to study the structural dependence of antimycobacterial activity of the guaianolide dehydrocostus lactone and its derivatives, m-chloroperoxybenzoic acid oxidations of dehydrocostus lactone (1a) were performed. Three new monoepoxides, one previously synthesized diepoxide, and two new diepoxides were obtained. Two of the monoepoxides are C-10 epimers (3a, 3b), while the 4(15)-monoepoxide (2) has the 4α-O-configuration. The known diepoxide (4a) contains a C-10 α-epoxide and a β-epoxide at C-4. The diepoxides 4b and 4c, each with a C-4 α-epoxy group, differ in the configuration of the epoxide ring at C-10. Allylic oxidation of dehydrocostus lactone (1a) with selenium dioxide/tert-butyl hydroperoxide afforded the known 3-epizaluzanin C (1b). The relative configurations of compounds 1b−4c were established by 1D and 2D NMR techniques (¹H, ¹³C, COSY, NOESY, HMQC, and HMBC) as well as comparison with literature data. The molecular structures of lactones 1b, 4a, and 4c were determined by single-crystal X-ray diffraction. In radiorespirometric bioassays against Mycobacterium tuberculosis and Mycobacterium avium, dehydrocostus lactone (1a) exhibited minimum inhibitory concentrations of 2 and 16 μg/mL, respectively. In contrast, its monoepoxides (2, 3a, and 3b) and diepoxides (4a−c), as well as its hydrogenated derivatives and other analogues (1b, 1c, 5, and 6), showed significantly lower activities against M. tuberculosis.

Bioactivity-guided fractionation of the petroleum ether extract of Azorella madreporica Clos has led to the isolation of the novel, antitubercular mulinane diterpenoid 1. The structure has been elucidated on the basis of its 1D and 2D NMR spectra and by comparison with mulinolic acid 2 and a dehydration product 3 obtained from 1. The MIC of 1 for growth inhibition of the H37Rv strain of Mycobacterium tuberculosis was determined as 20 μg/mL. LC-MS and NMR have suggested the presence of this new compound in four other species of Azorella.

Leprosy affects over 10 million people in the world. The disease is a model of graded cell-mediated immunity, in this case to the causative organism, Mycobacterium leprae. The clinical manifestations are due to (i) bacterial progression, (ii) immunologic responses of the host, (iii) peripheral nerve damage due to either or both bacterial progression and immunologic responses of the host, and (iv) preventable secondary deformities following nerve damage, which account for most of the stigma of the disease. Treatment modalities are now available to control or minimize the effects of bacterial progression, harmful immunologic responses of the host, peripheral nerve damage, and secondary deformities. Unique biochemical characteristics of M. leprae reside in the cell wall and associated macromolecules. Some of these molecules are potent immunogens in humans, while others constitute the structural integrity of the bacillus. Proteins of M. leprae are currently under intensive investigation as a result of deoxyribonucleic acid cloning of M. leprae genes. Structure-function and antigenic relationships of M. leprae proteins should become available by using recombinant deoxyribonucleic acid procedures coupled with T- and B-cell cloning to advance our understanding of the immunologic reactions encountered in Hansen's disease. Until recently, the study of the immunology of leprosy has been stymied by the lack of immunologically specific M. leprae antigens. The definition of specific antigens and production of recombinant and synthetic immunologic reagents have fostered state-of-the-art research efforts into new immunodiagnostic procedures and development of a leprosy vaccine. Also discussed is progress in understanding of the mechanism(s) underlying the M. leprae-specific immunodeficiency associated with lepromatous leprosy, including the role of suppressor T cells and defective macrophage function. Metabolic studies of M. leprae suggest intact catabolic pathways and energy generation with purine bases and catalase as possible growth factors. Special attention may also need to be given to biophysical parameters for eventual in vitro cultivation. Rapid in vitro systems, using quantitation of bacillary metabolic activity, may soon replace the lengthy mouse footpad test for determining the viability and drug susceptibility of the leprosy bacillus.

Mycobacterium leprae replicates within mononuclear phagocytes, reaching enormous numbers in the macrophage-rich granulomas of lepromatous leprosy. To examine the capability of macrophages to digest M. leprae, we studied the intracellular fate of M. leprae organisms in normal and activated mouse macrophages by using the electron-dense secondary lysosome tracer Thoria Sol. Intracellular M. leprae organisms, surrounded by a characteristic electron-transparent zone, were contained within phagosomal vacuoles of macrophages cultured in vitro for 1 to 6 days. In normal macrophages, a majority of phagosomes containing freshly isolated live M. leprae cells resisted fusion with Thoria Sol-labeled lysosomes. The extent of fusion was not significantly affected by pretreatment of M. leprae with human patient serum high in specific immunoglobulin G and M antibodies. In contrast, a majority of phagosomes containing gamma-irradiated M. leprae cells underwent lysosome fusion in normal macrophages. In addition, increased phagolysosome fusion was observed with live M. leprae-containing phagosomes in macrophages activated with gamma interferon. Increased fusion was associated with an increase in the number of fragmented and damaged bacilli, suggesting that increased digestion followed fusion. This study indicates that activated macrophages may have an increased capacity for clearance of normally resistant M. leprae.

We previously demonstrated the potent in vitro activity of erythromycin against Mycobacterium leprae as determined by its effect on ATP pools and rates of palmitate oxidation and phenolic glycolipid I synthesis. In the present study, the relative in vitro activities of a number of new macrolides with superior pharmacokinetic properties were assessed. In addition, for the most active compounds, concentrations in serum were determined by bioassay during continuous administration in the feed of mice, and in vivo activity against M. leprae was assessed by the kinetic mouse footpad technique. Both clarithromycin and roxithromycin were more potent than erythromycin in vitro, with the former showing the highest activity in accelerating rates of ATP decay and reducing rates of palmitate oxidation. In mice, concentrations of clarithromycin in serum were higher than those of roxithromycin and erythromycin, with the latter undetectable even when administered at 0.1% (wt/wt) in the diet. When administered at 0.01% (wt/wt) in the diet, erythromycin and roxithromycin were unable to inhibit growth of M. leprae in mouse footpads whereas clarithromycin demonstrated bactericidal-type activity. On the basis of these data and other properties of macrolides, a clinical trial of clarithromycin in leprosy is warranted.

The ability of Mycobacterium leprae to oxidize palmitic acid during incubation in an axenic medium was studied. By using a Buddemeyer-type detection system, partially purified nude-mouse-derived M. leprae was found to produce 14CO2 from 14C-labeled palmitic acid in a linear fashion for at least 1 week. Procedures known to remove residual host tissue did not diminish the rate of 14CO2 evolution, indicating that bacterial metabolism was being measured. Palmitate oxidation was temperature sensitive, with an apparent optimum of 33 degrees C, but pH insensitive. Bacilli exposed to a variety of antileprosy drugs for 1 or 2 weeks displayed significantly reduced rates of 14CO2 evolution upon subsequent addition of 14C-labeled palmitic acid. This activity could be readily detected with 10(6) bacilli, thus indicating its potential for use in clinical susceptibility testing.

In a bioassay-guided search for antimycobacterial compounds from higher plants of the southeastern United States, we have chemically investigated the sea daisy (Borrichia frutescens) from coastal marshes of Louisiana for their active constituents. Bioactive chromatographic fractions provided two new triterpenes, (24R)-24,25-epoxycycloartan-3-one (1) and (23R)-3-oxolanosta-8,24-dien-23-ol (4), and (3αH,24R)-24,25-epoxycycloartan-3-ol (3a). Compound 3a had been previously isolated as a mixture of C-24 epimers. The structures of 1, 3a, and 4 were established by spectroscopic methods and chemical transformations, and the molecular structures of 1 and 4 were determined by single-crystal X-ray diffraction. In a radiorespirometric bioassay against Mycobacterium tuberculosis, the epoxycycloartanes 1 and 3a exhibited minimum inhibitory concentrations of 8 μg/mL. In contrast, the lanostadiene-type triterpene 4 showed no significant inhibition at 128 μg/mL, as did the acetate 3b. Cytotoxicity for Vero cells gave IC50 values of 71.8, 39.8, and 103.6 μg/mL for triterpenes 1, 3a, and 4, respectively.

ABSTRACTAn optimal assay for high-throughput screening for new antituberculosis agents would combine the microplate format and low cost of firefly luciferase reporter assays and redox dyes with the ease of kinetic monitoring inherent in the BACTEC system. The green fluorescent protein (GFP) of the jellyfish Aequorea victoriais a useful reporter molecule which requires neither substrates nor cofactors due to the intrinsically fluorescent nature of the protein. The gene encoding a red-shifted, higher-intensity GFP variant was introduced by electroporation into Mycobacterium tuberculosisH37Ra and M. tuberculosisH37Rv on expression vector pFPV2. A microplate-based fluorescence assay (GFP microplate assay [GFPMA]) was developed and evaluated by determining the MICs of existing antimycobacterial agents. The MICs of isoniazid, rifampin, ethambutol, streptomycin, amikacin, ofloxacin, ethionamide, thiacetazone, and capreomycin, but not cycloserine, determined by GFPMA were within 1 log2dilution of those determined with the BACTEC 460 system and were available in 7 days. Equivalent MICs of antituberculosis agents in the BACTEC 460 system for both the reporter and parent strains suggested that introduction of pFPV2 did not influence drug susceptibility, in general. GFPMA provides a unique tool with which the dynamic response of M. tuberculosisto the existing and potential antituberculosis agents can easily, rapidly, and inexpensively be monitored.