Evaluation of Antimycobacterial, Antiplasmodial and Cytotoxic Activities of Preussomerins Isolated from the Lichenicolous Fungus Microsphaeropsis sp. BCC 3050

• Abstract
• Introduction
• Materials and Methods
• Fungal material
• Isolation
• Isolates
• Biological assays
• Results and Discussion
• Acknowledgements
• References

Abstract

A new preussomerin isomer, 3′-O-demethylpreussomerin I, five known preussomerins E - I, and two known deoxypreussomerins, deoxypreussomerin A and bipendensin (palmarumycin C₁₁), were isolated from a lichenicolous fungus Microsphaeropsis sp. BCC 3050. These structures were elucidated by spectroscopic methods, especially 1D- and 2D-NMR. The preussomerins were evaluated for their antimycobacterial and antiplasmodial activities as well as cytotoxicity against KB, BC-1 and vero cell lines.

Introduction

Preussomerins belong to a class of fungal metabolites previously isolated from dung-colonizing fungi Preussia isomera [1], [2], Sporormiella vexans [3], an unidentified coelomycetes (MF 5916) [4], and from an endophytic fungus Harmonema dematioides [5]. All together 10 isomers, preussomerins A-I and 3′-O-demethyl-1-epipreussomerin C, have been reported. These compounds are known to exhibit antifungal activity [2], and their activity as inhibitors of ras farnesyl-protein transferase has also been reported [4]. Understandably, their unique skeleton has been attracting the interests of synthetic chemists and syntheses of this class of compounds have recently been appeared in the literature [6], [7].

In our continued search for bioactive natural products from microbial resources in Thailand [8], [9], we came across preussomerins and deoxypreussomerins, as bioactive chemical constituents of a lichen fungus Microsphaeropsis sp. BCC 3050. A crude extract of the strain BCC 3050 showed antimycobacterial and antiplasmodial activity. Bioassay guided fractionation led to the isolation of five known preussomerins E (1), F (2), G (3), H (4) and I (5) together with a new derivative, 3’-O-demethylpreussomerin I (6). Two deoxypreussomerins, deoxypreussomerin A (7) and bipendensin (palmarumycin C₁₁) (8), were also isolated from the same culture broth. Here we report the isolation, structural elucidation and biological evaluation of the identified preussomerins.[]

Materials and Methods

Fungal material

The producing fungus was isolated from a lichen sample, Dirinaria applanata collected from Phu Teen-Suan-Sai Forest, Loei province, Northeastern Thailand, by Mr. Ek Sangvichien, Ramkhamhaeng University, and deposited at the BIOTEC Culture Collection as BCC 3050.

Isolation

A culture maintained on PDA (22 °C, 5 days) was inoculated into 12 × 1 L Erlenmeyer flasks each containing 250 mL of bacto-malt extract broth (MEB), and incubated at 22 °C for 21 days on rotary shakers (250 rpm). The flask cultures were filtered and the residual wet mycelia were digested in MeOH (1 L), left to stand for 2 days at room temperature, and filtered. To the MeOH filtrate was added H₂O (100 mL) and the mixture washed with hexane (500 mL). The aqueous MeOH layer was partially concentrated under reduced pressure. The residue (ca. 50 mL) was dissolved in EtOAc (300 mL), and washed with H₂O (100 ml). Concentration under reduced pressure gave a brown solid (1.6 g). The crude mixture was passed through a Sephadex LH-20 column (3.5 × 26 cm) using MeOH as eluent. Fractions of 150 - 325 mL were combined and subjected to silica gel column chromatography (8 × 15 cm; stepwise gradient elution with each 100 mL of CH₂Cl₂/hexane in the order of 20 : 80, 30 : 70, 40 : 60, 50 : 50, 80 : 20). The following four fractions were collected in the order of elution : Fr-A (containing compound 7), Fr-B (compound 3), Fr-C [a mixture of 3, 4 and 5 (major)] and Fr-D [a mixture of 1, 2, 6 (major) and 8]. Fr-A was further purified by preparative HPLC using a reversed phase column (Prep Nova-Pak HR C₁₈, 6 μm, 40 × 100 mm) with MeCN/H₂O = 60 : 40 as eluent at a flow rate of 20 mL/min to obtain compound 7 : 8 mg, t_R 18 min, yellow powder, [α]²⁵ _D: -269° (c 0.21, CH₂Cl₂). Fr-B was subjected to silica gel column chromatography (5 × 17 cm; step gradient elution with CH₂Cl₂/hexane) to obtain 3 : 180 mg, R_f 0.17 (CH₂Cl₂/hexane = 50 : 50), light yellow powder, m. p. 223 - 225 °C, [α]²⁵ _D: -673° (c 0.67, CH₂Cl₂). Fr-C was subjected to repeated silica gel column chromatography (CH₂Cl₂/hexane, gradient elution) to obtain pure compounds 3 (10 mg), 4 (16 mg, R_f 0.15 for CH₂Cl₂/hexane = 50 : 50) and 5 (236 mg, R_f 0.10 for CH₂Cl₂/hexane = 50 : 50) as light yellow powders: 4, m. p. 238 - 240 °C, [α]²⁶ _D: -541° (c 0.075, CH₂Cl₂); 5, m. p. 124 - 126 °C, [α]²⁵ _D: -487° (c 0.78, MeOH). Fr-D was fractionated by silica gel column chromatography (5 × 15 cm, step gradient elution with each 250 mL of CH₂Cl₂/hexane, 30 : 70, 40 : 60, 50 : 50, 60 : 40 and 70 : 30). The CH₂Cl₂/hexane = 50 : 50 elute (33 mg, containing a mixture of 1 and 2) was further separated by preparative. HPLC (MeOH/H₂O = 55 : 45) to yield 1 (8 mg, t_R 38 min) and 2 (14 mg, t_R 47 min) as yellow powders: 1, [α]²⁶ _D: -374° (c 0.024, MeOH); 2, [α]²⁶ _D: -364° (c 0.045, MeOH). The CH₂Cl₂/hexane = 70 : 30 elute (76 mg) was subjected to preparative HPLC (MeCN/H₂O = 35 : 65 for 25 min, then with 50 : 50) to obtain 6 (24 mg, t_R 17 min) and 8 (7 mg, t_R 34 min): compound 8, yellow powder, [α]²⁶ _D: -140° (c 0.16, MeOH).

Isolates

3′-O-demethylpreussomerin I (6): yellow powder; m. p. 238 - 240 °C; IR (KBr): ν_max = 3421, 1690, 1659, 1594, 1473, 1288, 1270, 1221, 1096, 1028, 987, 947, 833, 798 cm^-1; UV (CHCl₃): λ_max (log ε) = 256 (4.13), 311 (3.52), 370 (3.62) nm; HRMS (ESI-TOF, negative): m/z = 379.0447 [M-H]^- (calc. for C₂₀H₁₁O₈, 379.0454); ¹H NMR (CDCl₃): δ = 10.17 (1H, s, 9-OH), 7.68 (1H, d, J = 7.5 Hz, H-9′), 7.42 (1H, dd, J = 8.1, 7.9 Hz, H-8′), 7.08 (1H, d, J = 8.1 Hz, H-7′), 7.07 (1H, d, J = 9.2 Hz, H-7), 6.97 (1H, d, J = 9.1 Hz, H-8), 4.80 (1H, dd, J = 3.1, 2.9 Hz, H-3′), 4.27 (1H, d, J = 3.8 Hz, H-3), 3.86 (1H, d, J = 3.9 Hz, H-2), 3.37 (1H, dd, J = 18.4, 3.2 Hz, H-2′a), 3.05 (1H, dd, J = 18.3, 2.5 Hz, H-2′b), 2.35 (1H, br s, 3′-OH); ¹³C NMR data (Table [1]).

Biological assays

Growth inhibitory activity against M. tuberculosis H₃₇Ra was determined using the Microplate Alamar Blue Assay (MABA) [10]. The assay for activity against P. falciparum K1 was performed using the protocol previously reported [11] which follows the microculture radioisotope technique described by Desjardins [12]. Cytotoxicity of the purified compounds against human epidermoid carcinoma (KB cells), human breast cancer (BC-1 cells) and African green monkey kidney fibroblast (vero cells) were tested using the colorimetric method described by Skehan et. al [13].

Results and Discussion

The structures of five preussomerins 1 - 5 were elucidated by NMR analyses (¹H, ¹³C, DEPT, ¹H-¹H COSY, HMQC and HMBC) and identified as preussomerins E - I, respectively. Spectral data (¹H-NMR, IR, UV) of these compounds were identical to those reported in the literature [2], [4]. In the present work, we successfully assigned all carbons in compounds 4 and 5, which have never been reported, by analyses of 2D NMR spectral data (Table [1]). As for compound 2 (preussomerin F), the ¹³C-NMR chemical shift of a methine carbon (C-9′) and several assignments of carbons were different from the published data [2]. Our assignments, based on the analyses of DEPTs, HMQC and HMBC spectra, are listed (Table [1]).

The new derivative, 6, showed similar ¹H- and ¹³C-NMR spectral data to those of 5, except for the absence of the signal due to the methoxy group on C-3′ and the appearance of a hydroxy proton at δ_H = 2.35 ppm. The IR and UV spectra of 6 were also very similar to those of 5. On the basis of these spectral data, compound 6 is therefore a 3′-O-demethyl analogue of 5. The molecular formula of C₂₀H₁₂O₈, elucidated by HRMS and ¹³C-NMR, was in accord with this proposal. Detailed analysis of the NMR spectra (¹H, ¹³C, DEPTs, COSY, HMQC and HMBC) led to the full assignment of protons and carbons (Table [1], Fig [1]), and the structure depicted as 6 was confirmed. The small coupling constants of 2.9 and 3.1 Hz between H-3′ and the two methylene protons (H-2′) in the ¹H-NMR spectrum of 6, are close to those of 5 (2.7 and 2.9 Hz) and the values indicate that H-3′ must be placed in the pseudo-equatorial position in the rigid, pseudochair conformation. Thus, compound 6 should have same sense of configuration at C-3′ with compound 5 whose stereochemistry at this moiety has earlier been elucidated [4].

The absolute stereochemistry of preussomerin A had previously been established [2], and all the preussomerins, except for 5 (not recorded), are reported to show large negative rotation values in MeOH or CH₂Cl₂. From the optical rotation of 6, [α]²⁵ _D: -533° (c 0.51, MeOH), it is apparent that this compound also possesses same sense of absolute configuration. Compounds 1 - 5 from our isolation also showed large negative rotation values (see Materials and Methods section).

The structural features of compounds 5 and 6 indicated that they may be artifacts due to the addition of MeOH or H₂O across the electrophilic C-2′/C-3′ double bond of compound 3. To prove this hypothesis, following experiments were conducted. The crude MeOH extract from mycelia was subjected to direct analysis by HPLC-UV using a reversed-phase column (Nova-Pak 8NV C₁₈; 4 μm, 4 × 100 mm) with MeCN/H₂O = 40 : 60 as an eluent at a flow rate of 1 mL/min. Peaks due to compounds 3, 5 and 6 were detected at retention times of 7.5, 17.5 and 24 min, respectively (UV observed at 254 nm). The peak assignments were established by independent injections of pure, authentic compounds 3, 5 and 6, and co-injections of these samples with the extract, and by comparison of the UV spectra (200 - 400 nm) of each individual compound to those of authentic substances. A small portion of the mycelia was taken and extracted with absolute EtOH, and the extract was also analyzed by HPLC-UV from which the peak due to compound 5 was completely absent, while compounds 3 and 6 and other minor isomers were detected. These results clearly indicate that, as for the cultures of the strain BCC 3050, preussomerin I (5) is an isolation artifact due to the reaction of preussomerin G (3) with MeOH during the extraction of mycelia. It is not certain whether or not compound 6 is also an artifact due to the Michael addition of H₂O to 3 in the MeOH extraction of the wet mycelia or it is a real constituent of the fungus BCC 3050.

The structures of two deoxypreussomerins, 7 and 8, were elucidated by NMR analyses, and their spectral data were consistent with those reported for deoxypreussomerin A [4] and bipendensin [14] (palmarumycin C₁₁) [15], respectively.

Compounds 1 - 8 were tested for antimycobacterial and antiplasmodial activity. All the preussomerins, 1 - 6, and deoxypreussomerin A (7) showed moderate activities (Table [2]). Tuberculosis and malaria are by far the most serious of the world’s deadly diseases, and the search for new drug leads is an urgent need due to the emergence of drug-resistant strains of both mycobacteria and parasites. This is the first report on the in vitro activity of preussomerins against M. tuberculosis and P. falciparum. Preussomerins 1 - 6 also showed significant cytotoxicity against two cancer cell lines, KB and BC-1, and vero cells. Compounds 3, 4, 5 and 7 are reported to be inhibitors of ras farnesyl-protein transferase (bovine brain) with an IC₅₀ range of 1.2 - 17 μM [4]. Thus, our results of cytostatic activity to mammalian cells may possibly be related with this mechanism. It is interesting to note that deoxypreussomerin A (7) showed rather weak cytotoxicity to vero cells while retaining its antimycobacterial and antiplasmodial activities.

The fungal genus Microsphaeropsis (Coelomycetes) has been known to be a rich source of bioactive secondary metabolites; recent examples include macrosphelides A and B (inhibitors of cell adhesion) from Microsphaeropsis sp. FO-5050 [16], [17], L-755,807 (a bradykinin binding inhibitor) from Microsphaeropsis sp. [18], TAN-1496 A, C and E (diketopiperazine antibiotics) from Microsphaeropsis sp. FL-16 144 [19], anthraquinones and betanone derivatives from sponge-associated Microsphaeropsis sp. [20], microsphaeropsisin (a new antifungal substance) from marine-derived Microsphaeropsis sp. [21], and an unusual fatty acid and its glyceride from M. olivacea [22]. This is the first report on the occurrence of preussomerins from this genus. All previous isolations of this unusual class of compounds were from coprophilous (dung-colonized) fungi of the family Sporormiaceae except for a report on the isolation of preussomerin D from an endophytic fungus Hormonema dematioides [5].

Acknowledgements

Financial support from the Biodiversity Research and Training Program (BRT) and the Thailand-Tropical Diseases Research Programme (T-2) is gratefully acknowledged. We are also grateful to Professor D. L. Hawksworth and Mr. E. Sangvichien for the identification of the fungus used in this study. One of us (Y. T.) thanks NSTDA for the Senior Research Fellowship Award.

References

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• 19 Funabashi Y, Horiguchi T, Iinuma S, Tanida S, Harada S. TAN-1496 A, C and E, diketopiperazine antibiotics with inhibitory activity against mammalian DNA topoisomerase I.  Journal of Antibiotics. 1994;  47 1202-18
  PubMedSearch in Google Scholar
• 20 Brauers G, Edrada R A, Ebel R, Proksch P, Wray V, Berg A. et al . Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis species: novel inhibitors of protein kinases.  Journal of Natural Products. 2000;  63 739-45
  CrossrefPubMedSearch in Google Scholar
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  CrossrefPubMedSearch in Google Scholar
• 22 Yu C -M, Curtis J M, Wright J LC, Ayer S W, Fathi-Afshar Z R. An unusual fatty acid and its glyceride from the marine fungus Microsphaeropsis olivacea .  Canadian Journal of Chemistry. 1996;  74 730-5
  PubMedSearch in Google Scholar

Dr. Masahiko Isaka

National Center for Genetic Engineering and Biotechnology (BIOTEC)

National Science and Technology Development Agency (NSTDA)

Rama 6 Road

Bangkok 10400

Thailand

Email: isaka@biotec.or.th

References

• 1 Weber H A, Baenziger N C, Gloer J B. Structures of preussomerin A: an unusual new antifungal metabolite from the coprophilous fungus Preussia isomera .  Journal of the American Chemical Society. 1990;  112 6718-9
  CrossrefPubMedSearch in Google Scholar
• 2 Weber H A, Gloer J B. The Preussomerins: Novel antifungal metabolites from the coprophilous fungus Preussia isomera Cain.  Journal of Organic Chemistry. 1991;  56 4355-60
  CrossrefPubMedSearch in Google Scholar
• 3 Soman A G, Gloer J B. Sporovexins A-C and a new preussomerin analog: antibacterial and antifungal metabolites from the coprophilous fungus Sporormiella vexans .  Journal of Natural Products. 1999;  62 659-61
  CrossrefPubMedSearch in Google Scholar
• 4 Singh S B, Zink D L, Liesch J M, Ball R G, Goetz M A, Bolessa E A. et al . Preussomerins and deoxypreussomerins: Novel inhibitors of ras farnesyl-protein transferase.  Journal of Organic Chemistry. 1994;  59 6296-302
  CrossrefPubMedSearch in Google Scholar
• 5 Polishook J D, Dombrowski A W, Tsou N N, Salituro G M, Curotto J E. Preussomerin D from the endophyte Hormonema dematioides .  Mycologia. 1993;  85 62-4
  PubMedSearch in Google Scholar
• 6 Shannon C, Heathcock C H. Total syntheses of (±)-preussomerins G and I.  Organic Letters. 1999;  1 3-5
  CrossrefPubMedSearch in Google Scholar
• 7 Ragot J P, Steeneck C, Alcaraz M -L, Taylor R JK. The synthesis of 1,8-dihydroxynaphthalene-derived natural products: palmarumycin CP₁, palmarumycin CP₂, palmarumycin C₁₁, CJ-12,371, deoxypreussomerin A and novel analogues.  Journal of Chemical Society, Perkin Transactions. 1999;  1 1073-82
  PubMedSearch in Google Scholar
• 8 Isaka M, Jaturapat A, Kladwang W, Punya J, Lertwerawat Y, Tanticharoen M. et al . Antiplasmodial compounds from the wood-decayed fungus Xylaria sp. BCC 1067.  Planta Medica. 2000;  66 473-5
  Thieme ConnectPubMedSearch in Google Scholar
• 9 Nilanonta C, Isaka M, Kittakoop P, Palittapongarnpim P, Kamchonwongpaisan S, Pittayakhajonwut D. et al . Antimycobacterial and antiplasmodial cyclodepsipeptides from the insect pathogenic fungus Paecilomyces tenuipes BCC 1614.  Planta Medica. 2000;  66 756-8
  Thieme ConnectPubMedSearch in Google Scholar
• 10 Collins L, Franzblau S G. Microplate Alamar Blue Assay versus BACTEC 460 System for high-throughput screening of compounds against Mycobacterium tuberculosis and Mycobacterium avium .  Antimicrobial Agents and Chemotherapy. 1997;  41 1004-9
  PubMedSearch in Google Scholar
• 11 Isaka M, Punya J, Lertwerawat Y, Tanticharoen M, Thebtaranonth Y. Antimalarial activity of macrocyclic trichothecenes isolated from the fungus Myrothecium verrucaria .  Journal of Natural Products. 1999;  62 329-31
  CrossrefPubMedSearch in Google Scholar
• 12 Desjardins R E, Canfield C J, Chulay J D. Quantitative assessment of antimalarial activity in vitro by semiautomated microdilution technique.  Antimicrobial Agents and Chemotherapy. 1979;  16 710-8
  PubMedSearch in Google Scholar
• 13 Skehan P, Storeng R, Scudiero D, Monks A, McMahon J D, Vistica D. et al . New colorimetric cytotoxicity assay for anticancer-drug screening.  Journal of National Cancer Institute. 1990;  82 1107-12
  CrossrefPubMedSearch in Google Scholar
• 14 Kouam , Mpondo T N, Lavaud C, Massiot G, Nuzillard J -M, Connolly J D,. et al . Bipendensin, an unusual phenolic acetal from Afzelia bipendensis .  Natural Product Letters. 1993;  3 299-303
  PubMedSearch in Google Scholar
• 15 Krohn K, Michel A, Flörke U, Aust H -J, Draeger S, Schulz B. Palmarumycins C₁-C₁₆ from Coniothyrium sp.: isolation, structure elucidation, and biological activity. Justus Liebigs Annalen der Chemie 1994: 1099-108
  Search in Google Scholar
• 16 Hayashi M, Kim Y P, Hiraoka H, Natori M, Takamatsu S, Kawakubo T. et al . Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. I. Taxonomy, fermentation, isolation and biological activities.  Journal of Antibiotics. 1995;  48 1435-9
  PubMedSearch in Google Scholar
• 17 Takamatsu S, Kim Y P, Hayashi M, Hiraoka H, Natori Mkomiyama K. et al . Macrosphelide, a novel inhibitor of cell-cell adhesion molecule. II. Physicochemical properties and structural elucidation.  Journal of Antibiotics. 1996;  49 95-8
  PubMedSearch in Google Scholar
• 18 Tony-Lam Y K, Hensens O D, Ransom R, Giacobbe R A, Polishook J, Zink D. L-755,807, a new non-peptide bradykinin inhibitor from an endophytic Microsphaeropsis sp.  Tetrahedron. 1996;  52 1481-6
  CrossrefPubMedSearch in Google Scholar
• 19 Funabashi Y, Horiguchi T, Iinuma S, Tanida S, Harada S. TAN-1496 A, C and E, diketopiperazine antibiotics with inhibitory activity against mammalian DNA topoisomerase I.  Journal of Antibiotics. 1994;  47 1202-18
  PubMedSearch in Google Scholar
• 20 Brauers G, Edrada R A, Ebel R, Proksch P, Wray V, Berg A. et al . Anthraquinones and betaenone derivatives from the sponge-associated fungus Microsphaeropsis species: novel inhibitors of protein kinases.  Journal of Natural Products. 2000;  63 739-45
  CrossrefPubMedSearch in Google Scholar
• 21 Höller U, König G M, Wright A D. Three new metabolites from marine-derived fungi of the genera Coniothyrium and Microsphaeropsis.  Journal of Natural Products. 1999;  62 114-8
  CrossrefPubMedSearch in Google Scholar
• 22 Yu C -M, Curtis J M, Wright J LC, Ayer S W, Fathi-Afshar Z R. An unusual fatty acid and its glyceride from the marine fungus Microsphaeropsis olivacea .  Canadian Journal of Chemistry. 1996;  74 730-5
  PubMedSearch in Google Scholar

Dr. Masahiko Isaka

National Center for Genetic Engineering and Biotechnology (BIOTEC)

National Science and Technology Development Agency (NSTDA)

Rama 6 Road

Bangkok 10400

Thailand

Email: isaka@biotec.or.th