Aki Nishihara-Tsukashima

Spoxazomicins A–C, novel antitrypanosomal alkaloids produced by an endophytic actinomycete, Streptosporangium oxazolinicum K07-0460 T

Three novel antitrypanosomal alkaloids, named spoxazomicins A–C, were isolated by silica gel column chromatography and HPLC from the culture broth of a new endophytic actinomycete species, Streptosporangium oxazolinicum K07-0460T. The structures of the spoxazomicins were elucidated by NMR and X-ray crystal analyses and shown to be new types of pyochelin family antibiotic. Spoxazomicin A showed potent and selective antitrypanosomal activity with an IC50 value of 0.11 μg ml−1 in vitro without cytotoxicity against MRC-5 cells (IC50=27.8 μg ml−1).

In vitro and in vivo antimalarial activity of puberulic acid and its new analogs, viticolins A–C, produced by Penicillium sp. FKI-4410

In the course of screening for antimalarial agents, five tropolone compounds were isolated from the culture broth of Penicillium sp. FKI-4410. Two were known compounds, puberulic acid and stipitatic acid. Three were new analogs of puberulic acid, designated viticolins A–C. Among them, puberulic acid exhibited potent antimalarial inhibition, with IC50 values of 0.01 μg ml−1 against chloroquine-sensitive and -resistant Plasmodium falciparum strains in vitro. Furthermore, puberulic acid showed weak cytotoxicity against human MRC-5 cells, with an IC50 value of 57.2 μg ml−1. The compound also demonstrated a therapeutic effect in vivo, which compared well against the currently used antimalarial drugs, and thus shows promise as a leading candidate for development into a new antimalarial compound.

Borrelidin, a potent antimalarial: stage-specific inhibition profile of synchronized cultures of Plasmodium falciparum

Borrelidin, a potent antimalarial: stage-specific inhibition profile of synchronized cultures of Plasmodium falciparum

Antitrypanosomal peptaibiotics, trichosporins B-VIIa and B-VIIb, produced by Trichoderma polysporum FKI-4452

6.0 before sterilization), in a 500-ml Erlenmeyer flask. The inoculated flask was incubated in a rotary shaker (210 r.p.m.) at 271 Cf or 3d ays. For production of 1, 2, and the other known trichosporins (3–7), a 1-ml portion of the seed culture was transferred to each of nine 500-ml Erlenmeyer flasks containing 100 ml of the production medium, consisting of 3.0% soluble starch, 2.0% soybean meal, 1.0% glycerol, 0.3% dry yeast, 0.3% KCl, 0.2% CaCO3 ,0 .05% MgSO4� 7H2O, and 0.05% KH2PO4 (adjusted to pH 6.5 before sterilization), fermentation taking place on a rotary shaker (210 r.p.m.) at 271 Cf or 6d ays. To the whole culture broth (1.0 l) was added 1.0 l of ethanol, followed by filtration. The filtrate was concentrated under reduced pressure to remove ethanol and then extracted with 1.0 l of ethyl acetate (pH 2). The ethyl acetate layer was concentrated under reduced pressure to afford a crude extract (745 mg). The ethyl acetate extract (426 mg) was applied to an ODS column (Pegasil Prep ODS-751512A, 20f� 120 mm, Senshu Scientific Co., Tokyo, Japan) pre-equilibrated with 20% methanol. The column was eluted with 20, 40 and 60% methanol stepwise (120 ml each) and the active principles were eluted with 80% methanol (120 ml), followed by concentration in vacuo to yield a brown material (91.0 mg). The material was purified by HPLC using a Pegasil ODS column (20f� 250 mm, Senshu Scientific Co.) with 70% CH3CN at 5 ml min � 1 detected at UV 210 nm. The retention times of the active fractions 1, 2, 3, and 4 were 16, 18, 20, and 24 min, respectively. The active fractions 1 and 2 were concentrated in vacuo to dryness to afford trichosporins B-V (3, 9.9 mg) and B-VIb (4, 10.0 mg), respectively, as white powders. The active fraction 3 (8.4 mg) was purified by HPLC using an XBridge C8 column (10f� 250 mm, Waters Co., Milford, MA, USA) with 50%

Jogyamycin, a new antiprotozoal aminocyclopentitol antibiotic, produced by Streptomyces sp. a-WM-JG-16.2

Jogyamycin, a new antiprotozoal aminocyclopentitol antibiotic, produced by Streptomyces sp. a-WM-JG-16.2

Pyrenocine I, a new pyrenocine analog produced by Paecilomyces sp. FKI-3573

Human African trypanosomiasis, also known as sleeping sickness, is caused by two subspecies of the parasitic protozoan, Trypanosoma brucei. Unique to sub-Saharan Africa, human African trypanosomiasis has, in past epidemics, caused significant and widespread mortality and morbidity. The World Health Organization estimated that human African trypanosomiasis caused 52 000 deaths in 2004, projecting a continuing fall to a total of 36 000 in 2015 (http://www.dndi.org/).1 There are significant problems with the four parenteral drugs currently used for treatment (suramin, pentamidine, eflornithine and melarsoprol) as they are highly toxic, expensive, difficult to administer, and parasite resistance to them is increasing. Although a new combination therapy using drugs was introduced in 2009, only one molecule is currently in clinical development for treatment of human African trypanosomiasis (http://www.dndi.org/).2 Therefore, there is an urgent need for new antitrypanosomal drugs that are more effective, safer, affordable easier to use and which, ideally, have a novel mode of action. Our research group has focused on the screening of antitrypanosomal agents from microbial metabolites.3–5 Our ongoing studies have led to the discovery of a novel pyrenocine, pyrenocine I (1), which was isolated from the culture broth of Paecilomyces sp. FKI-3573, along with the known pyrenocines A (2)6 and B (3)6 and citreoviridin (4) (Figure 1).7 All of the compounds exhibit in vitro antitrypanosomal activity. In this article, we report their fermentation, isolation, structure elucidation and antitrypanosomal activity. Fungal strain FKI-3573 was isolated from a soil sample collected in Hilo, HI, USA. The ITS rDNA sequence of FKI-3573 was a 95.6% match to Paecilomyces lilacinus ATCC 10114 (GenBank accession number AY213665). From this information, combined with morphological characteristics, FKI-3573 was identified to be a member of the Paecilomyces genus.8 A stock culture of strain FKI-3573 was inoculated into 100 ml of seed medium, consisting of glucose (2.0%), Polypepton (0.5%; Nihon Pharmaceutical, Tokyo, Japan), yeast extract (0.2%), KH2PO4 (0.1%), MgSO4 7H2O (0.05%) and agar (0.1%), adjusted to pH 5.7 before sterilization. The mixture was subsequently incubated on a rotary shaker at 27 1C for 3 days. Seed culture (1 ml) was transferred into each of 10 Erlenmeyer flasks (500 ml) together with 100 ml of production medium containing potato dextrose broth (2.4%), malt extract (0.5%), Mg3(PO4)2 8H2O (0.5%) and agar (0.1%). The mixture was adjusted to pH 6.0 before sterilization. Fermentation was carried out on a rotary shaker at 27 1C for 3 days, followed by stationary culture for 10 days. The culture broth (1.0 l) was added 1.0 l of EtOH. It was concentrated under reduced pressure after removing the mycelia by centrifugation and filtration. The resulting aqueous solution was extracted with EtOAc and the organic layer was concentrated to dryness in vacuo to afford a yellow solid (488 mg). It was applied to ODS column chromatography (eluted with 20, 40 and 60% aqueous CH3CN). The concentrated 20% CH3CN eluate (42.8 mg) was purified by HPLC (column, Pegasil ODS (20f 250 mm, Senshu Scientific, Tokyo, Japan); mobile phase, 30% aqueous CH3CN; flow rate, 5.0 ml min 1; detection, UV at 210 nm). Pyrenocine B (3, 16.2 mg) and 1 (3.5 mg) were obtained at 16 and 18 min, respectively. The 40% CH3CN eluate (65 mg) was purified by HPLC (column, Pegasil ODS (20f 250 mm); mobile phase, 40% aqueous CH3CN containing 0.1% TFA; flow rate, 5.0 ml min 1; detection, UV at 210 nm) to yield 15 mg of pyrenocine A (2). The 60% CH3CN eluate (70 mg) was also purified by HPLC (column, XBridge Prep C8 (10f 250 mm, Waters, Milford, MA, USA); mobile phase, 32% aqueous CH3CN; flow rate, 3.0 ml min 1; detection, UV at 210 nm) to produce 9.9 mg of citreoviridin (4), which was identified by comparison of spectral data with published values.9 Compound 1 was obtained as a light yellow amorphous solid ([a]D1⁄4 1.0; c1⁄40.1 in MeOH; UV (MeOH) lmax (e): 229 (16 000), 289 nm (4700)). The molecular formula was elucidated to be C11H14O4 by HR-FAB-MS (found, 209.0805 [M-H] ; calculated, 209.0819 for C11H13O4). The IR spectrum showed characteristic absorptions at 3431, 1697, 1633 and 1558 cm 1, suggesting the

Sinefungin VA and dehydrosinefungin V, new antitrypanosomal antibiotics produced by Streptomyces sp. K05-0178.

Two new nucleotide antibiotics, named sinefungin VA and dehydrosinefungin V, were separated by cation exchange column chromatography and purified by HPLC from the culture broth of Streptomyces sp. K05-0178, together with the known antibiotics, sinefungin, dehydrosinefungin and KSA-9342. The structures of the two novel sinefungin analogs were elucidated by spectroscopic studies, including various NMR and advanced peptide chemical methods. Sinefungin VA consists of adenosine and ornithylvalylalanine, whereas dehydrosinefungin V consists of 4′,5′-dehydroadenosine and ornithylvaline. Sinefungin VA showed potent antitrypanosomal activity with an IC50 value of 0.0026 μg ml−1 in vitro without cytotoxicity against MRC-5 cells. Dehydrosinefungin V showed moderate antitrypanosomal activity (IC50=0.15 μg ml−1).

In vitro and in vivo antiprotozoal activities of bispolides and their derivatives

During the course of our screening program to discover new antiprotozoal (antimalarial and antitrypanosomal) chemicals, we have evaluated isolates from soil microorganisms, as well as compounds from the antibiotic libraries of the Kitasato Institute for Life Sciences and Nimura Genetic Solutions. We have previously reported various microbial metabolites exhibiting potent antimalarial1–4 and antitrypanosomal properties.5–7 We have recently found that the known 20-membered ring macrodiolide antibiotics, the bispolides,8 together with new derivatives, exhibit selective antitrypanosomal and potent antimalarial activities, both in vitro and in vivo. Here, we report the antitrypanosomal and antimalarial profiles of bispolides, their derivatives (Figure 1) and the related 16-membered ring macrodiolide antibiotic as elaiophylin (azalomycin B)9,10 (Figure 1) in comparison with those of clinically used antitrypanosomal drugs, such as suramin and eflornithine, and two clinically used antimalarial drugs, artemisinin and chloroquine. We also present some conclusions on structure–activity relationships. Bispolides A1, A3, B1 and B3 were purified from the culture broth of Microbispora sp. A34030.8 Derivatives of bispolide A1 (derivatives I and II) were prepared as follows. The 13,13¢-dimethoxy compound, bispolide A3, prepared from bispolide A1 by reaction with 0.04%-HCl in MeOH, was reduced by NaBH3CN in EtOH to give the 13,13¢dideoxy compound (derivative I (ESI-MS m/z 1123.69114; C62 H100 Na1 O16)). Partial hydrolysis of derivative I in the presence of p-toluenesulfonic acid in aqueous acetonitrile gave derivative II (FAB-MS m/z 841 (M+)). Elaiophylin was obtained from the antibiotic library of the Kitasato Institute for Life Sciences. In vitro antiprotozoal activities against Trypanosoma brucei brucei strain GUTat 3.1, Plasmodium falciparum strains K1 (drugresistant) and FCR3 (drug-sensitive), and cytotoxicity against human diploid embryonic cell line MRC-5 were measured as described previously.1,5 In vivo antitrypanosomal activity for T. b. brucei strain S427 was measured as described previously.6 Test compounds were solubilized in an aqueous mixture of 10% DMSO-Tween 80 and EtOH (7:3) and administered i.p. to mice on the next day (day 1) following infection with parasites (day 0). Subsequently, the compounds were successively administered (i.p.) to the infected mice once a day for 3 days (days 2–4). Efficacies of compounds were determined by the parasitemia levels and the mean of survival days (MSD), compared with that of the untreated control mice. Table 1 shows the in vitro antiprotozoal activities of bispolides, their derivatives, elaiophylin and some standard antiprotozoal drugs. Bispolides, derivative I (13,13¢-dideoxybispolide A1) and elaiophylin showed the more potent antimalarial activity against the drug-resistant K1 strain of P. falciparum, in the 260–620 ng ml 1 range. The half-maximal inhibitory concentration (IC50) values against the drug-sensitive FCR3 strain of P. falciparum were similar to those against the K1 strain of P. falciparum (data not shown). The antimalarial activity was similar to that of chloroquine, but 43–103-fold less than that of artemisinin. However, the antimalarial activity of derivative II was 14-fold less than that of bispolide A3. With respect to antitrypanosomal activity, bispolides and derivative I showed the most potency against the GUTat 3.1 strain of T. b. brucei, in the 57–150 ng ml 1 range. The antitrypanosomal activities were 10–40-fold more potent than that of the standard drugs, suramin and eflornithine. However, the antitrypanosomal activities of derivative II and elaiophylin were 3–6-fold less than that of bispolide A3. The in vitro cytotoxicities of bispolides, their derivatives, elaiophylin and some standard antiprotozoal drugs are presented in Table 1. Among them, the IC50 values of bispolide B1 and elaiophylin were 0.96 and 0.87mg ml 1, respectively. The IC50 values of bispolide A1, A3 and B3, and derivative I were in the range of 1.4–3.9mg ml 1, whereas that of derivative II was 15.6mg ml 1. To compare the antiprotozoal activities and cytotoxicities, we introduced selectivity indexes (SIs: cytotoxicity (IC50 for the MRC-5 cells)/antimalarial or antitrypanosomal activity (IC50 for the K1 strain or the GUTat 3.1 strain), as presented in Table 1. In the case of the MRC-5 cells/K1 strain, bispolide A1 showed a medium SI, with a ratio of 15. Other bispolides and elaiophylin showed a low SI, with ratios of 2–9. In the case of the MRC-5 cells/GUTat 3.1 strain, bispolides A1 and B3 showed a moderate SI, with ratios of 57–62. Other bispolides and elaiophylin showed a medium or low SI (ratios of 2–26). Among the bispolides, we were interested in bispolides A3 and B3, which had an SI of 26 and 62, respectively. Among the tested bispolides, B3 showed the more potent antitrypanosomal activity and highest SI, whereas bispolide A3 exhibited potent antitrypanosomal activity but with medium SI. The preliminary in vivo antitrypanosomal activities of bispolides A3 and B3 were measured in the T. b. brucei S-427 acute mouse model. At a dose of 25 mg kg 1, bispolide B3 did not achieve cure but did extend the MSD to 9.5 days, representing a 1.4-fold increase over control MSD (7.0 days). The same dose of bispolide A3 did not achieve cure but also extended the MSD. Under the same conditions, suramin showed a curative effect (MSD: 430 days) at a dose of 1 mg kg 1. The bispolide B3 data suggest that it is possibly a new candidate compound for discovering new antitrypanosomal drugs with more potent activity. These efficacy tests, including in vivo antimalarial activity, are being investigated further. The Journal of Antibiotics (2010) 63, 275–277 & 2010 Japan Antibiotics Research Association All rights reserved 0021-8820/10

In vitro antitrypanosomal activity of some phenolic compounds from propolis and lactones from Fijian Kawa ( Piper methysticum )

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Panowamycins A and B, new antitrypanosomal isochromans produced by Streptomyces sp. K07-0010

During our search to discover new antitrypanosomal compounds, eight known plant compounds (three phenolic compounds and five kawa lactones) were evaluated for in vitro activity against Trypanosoma brucei brucei. Among them, we found two phenolic compounds and three kawa lactones possessing an α-pyrone influenced antitrypanosomal property. In particular, β-phenethyl caffeate, farnesyl caffeate and dihydrokawain exhibited high or moderate selective and potent antitrypanosomal activity in vitro. We detail here the antitrypanosomal activity and cytotoxicities of the compounds, in comparison with two commonly used antitrypanosomal drugs (eflornithine and suramin). Our findings represent the first report of the promising trypanocidal activity of these compounds.