Indian Journal of Chemistry Vol. 4 1B, July 2002, pp. 1 472- 1476

Biotransformations, antibacterial evaluations of the sesquiterpenoids and diterpenoids +

M Rama Rao", U Venkatesham", K V Sridevi", P Srinivasa Reddyh, Kaiser Jamilh & Y Venkateswarlu"'*

"Natural Products Laboratory, Organic Chemistry Division-I bBiology and Biotechnology Division, Indian Institute of Chemical Technology, Hyderabad 500 007, India

Tel.: 9-40-7 1 93 1 67. Fax: +9 1 -40-7 1705 12; e-mail : luchem@iicLap.nic.in

Received 12 March 2001; accepted (revised) 26 November 2001

Incubation of 6�,7�-epoxy-4�-hydroxyguaian- 1O-ene 1 with fungus Aspergillus niger affords 4�, 1 3-dihydroxyguaian-6, l O-diene 4 and 4�, 1 2-dihydroxy-guaian-6, lO-diene 5, whereas with a plant pathogenic fungus Glomerella cingulata yields exclusively compound 4. In separate experiments incubation of (EEE)-7�,8�-epoxy- I -isopropyl-4,8, 1 2-trimethylcyclo­tetradeca- I ,3, I I -triene 2 and deoxosarcophine 3 with Aspergillus niger afford exclusively (EEE)-7�,8a-dihydroxy- l ­isopropyl-4,8, 1 2-trimethylcyclotetradeca- I ,3, I l -triene 6 and 9�-hydroxydeoxosarcophine 7, respectively. The structures of compounds 4, 5, 6 and 7 have been confirmed by spectral data. Antibacterial evaluations of these compounds have been conducted.

We have been investigating the biotransformations of terpenoids using a plant pathogenic fungus G. cingu­latal .5 and with A. niger6•7• Biotransforrnations are useful methods for producing medicinal compounds and also important for introducing chemical functions into inaccessible sites of molecules to enhance the biological activity. In particular we were interested in the oxidation of the isopropyl group of the tricyclic sesquiterpenoid by using A. niger and G. cingulata5• 6B,7B-Epoxy-4B-hydroxyguaian- 10-ene 1 is a ses­quiterpenoid isolated from the soft coral Nephthea chabrolii (Nephthedae)8, (EEE)-7B,8B-epoxy- l ­isopropyl-4,8, 12-trimethy1cyclotetradeca- l ,3, l l-triene 2 and deoxosarcophine 3 cembrane diterpenoids were previously isolated from the soft coral Sarcophyton Sp.9. IO, in which the compound 3 has been shown to facilitate neuromuscular transmission in a rat dia­phramlO• The present paper describes the biotransfor­mations of compounds 1 , 2 and 3 by using plant pathogenic fungus G. cingulata and A. niger and we have carried out the antibacterial activities of com­pounds 1 to 7.

Compound 1 ( l 00 mg) was incubated with G. cingulata for eight days. The culture medium was then extracted with EtOAc followed by column

+ nCT Communication No. 4450

chromatography, to afford compound 4 ( lOmg, 20%) and unconverted starting material (40mg, ca 80%) was recovered from the ethyl acetate extract.

Similarly compound 1 ( 100 mg) was incubated with A. niger for eight days. Two main products 4 (5mg, ca 5%) and 5 (4mg, ca 4%) were detected by TLC analysis of the ethyl acetate extract of the culture medium after biotransformation. Further, these products were not detected by TLC analysis of the culture of G. cingulata and A. niger, to which substrate was not fed. From above results it was evident that G. cingulata and A. niger transformed 1 into 4, and 4 and 5, respectively.

Similarly, in separate experiments compounds 2 ( 100 mg) and 3 ( l 00 mg) were incubated with A. niger for eight days. Then each culture medium was extracted with EtOAc followed by column chromatography, to afford compound 6 ( 10 mg, ca 1 0%) and 7 (8 mg, ca 8%), respectively, along with unconverted compounds 2 (40mg, ca 40%) and 3 (45mg, ca 45%). However, compounds 2 and 3 were not transformed when incubated with G. cingulata.

Compound 4 obtained as a viscous liquid, [a]D +2.4 (c 0.5, CHCI3), showed no absorptions in UV light and had a molecular ion at mlz 236 (M+), analyzed for C I5H2402 by elemental analysis which required four degrees of unsaturation. The IR

RAO et al. : BIOTRANSFORMATIONS OF SESQUITERPENOIDS AND DITERPENOIDS 1473

1 1

3 HO 1 3

14 1

G. cingulata •

A. niger •

HO

HO OH

+

�

HO

HO OH

A. niger HO •

2 6

3

A. niger •

absorption at 3400 cm-I indicated the presence of a hydroxyl group. The I H NMR spectrum of compound 4 displayed signals for the presence of a quaternary methyl bearing hydroxyl at 8 1 .25 (3H, s), a secondary methyl at 8 1 .0 (3H, d, J = 7.5Hz). Further, the downfield spectrum displayed signals due to presence of trisubstituted double bond at 8 5.65 ( l H, br s), an exocyclic methylene at 8 4.78 ( lH, br s) and 4.70 ( lH, br s) and a methylene bearing hydroxyl group at 8 3.42 (2H, d, J = 7.5Hz). These observations were further supported by its I3C NMR spectral signals at 8 153 . 18 (s), 1 44.38 (s), 1 26.64 (d), 1 07.07 (t), 80.5 (s) and 65. 1 5 (t). From the literature survey compound 4 was reminiscent to guaian-6,1O-dien-4f3-01 1 1 , except having a methylene bearing hydroxyl at 8 3.42 (2H, d, J = 7.5 Hz) and absence of methyl signal at 8 0.92 (3H, d, J = 6.5 Hz), indicating that one of the isopropyl methyl groups was hydroxylated. Thus, from the above findings the structure of compound 4

7

reflux 600C 30 min

2

was established as 4f3, 1 3-dihydroxyguaian-6, 1 O­diene. The above biotransformation of compound 1 to compound 4 could be a useful tool for synthesis of xeniolone 8 related diterpenoidsl2•

Compound 5 obtained as a viscous liquid [a]o -54.2 (c 0.5, CHCh), showed no absorptions in UV light and had a molecular ion at mJz 2 1 8 (M+-H20), analyzed for C1sH220 by elemental analysis which required four degrees of unsaturation. The IR absorption at 3380 cm- I indicated the presence of a hydroxyl group. The IH NMR spectrum of compound 5 showed signals for the presence of three quaternary methyls bearing hydroxyl group at 8 1 .25 (3H, s) and 1 .38 (6H, s). Further, the downfield spectrum showed signals due to the presence of a trisubstituted double bond at 8 5.9 1 ( l H, br s), and an exocyclic methylene protons at 8 4.75 ( l H, br s), and 4.70 ( l H, br s). The down field shift of trisubstituted double bond proton indicated the presence of hydroxyl group at allylic

1474 INDIAN J. CHEM., SEC B, JULY 2002

carbon connected with two methyl groups. From the above spectral data the structure of compound 5 was established as 4�, 1 2-dihydroxyguaian-6, 1 0-diene.

Compound 6 obtained as a colourless prisms, mp 82°C, [a]o -65.20 (c 0.5, CHCh) and had molecular ion at mlz 306 (M+), analyzed for C2oH3402 by elemental analysis which required four degrees of unsaturation. The IR absorptions at 1 6 10, 1 650 cm-I and UV absorption at 245 nm (E 1 4000) indicated the presence of a conjugated diene system.

The I H NMR spectrum of compound 6 displayed signals for the presence of an isopropyl group at 8 1 .05 (6H, d, J = 7 Hz), a quaternary methyl bearing hydroxyl at 8 1 .25 (3H, s), two vinylic methyls at 8 1 .45 (3H, s) and 1 .74 (3H, s). Further, the down field spectrum displayed signals for a conjugated trisubsti­tuted double bond protons at 8 6.05 ( tH, d, J = 1 1 .5 Hz) and 5 .96 ( tH, d, J = 1 1 .5 Hz), a tri­substituted double bond proton at 8 5 . 1 0 (m, IH), and a hydroxy-methine proton at 8 3.58 ( tH, d, J = 1 0.5 Hz). These observations were further supported by its I3C NMR spectral signals at 8 1 47.43 (s), 1 37.07 (s), 1 34.69 (s), 1 25.89 (d), 1 2 1 .86 (d), 1 1 8 .90 (d), 75.55 (s) and 7 1 .2 1 (d). The IH NMR and I3C NMR chemical shifts of compound 6 were virtually the same as those of 2, except for the epoxy functionality [ IH NMR; 8 2.86 ( tH, t, J = 6.5 Hz), I 3C NMR; 8 6 1 .9 (d), 60.4 (s)], which had been replaced by two hydroxyl groups [ IH NMR, 8 3.58 ( l H, d, J = 10.5 Hz), I3C NMR, 8 75.55 (s) and 7 1 .2 1 (d)] in compound 6. Thus from the above findings the structure of compound 6 was established as (EEE)-7�, 8a-dihydroxy- l -isopropyl-4,8, 1 2-trimethylcyclotetra­deca- l ,3 , 1 1 -triene 6. In order to confirm the structure of 6, compound 2 was treated with 2 % H2S04 in acetone to yield diol 61 3. The physical properties (mp, [a]o) and spectral data eH NMR, I3C NMR) of compound 6 obtained from 2% H2S04 in acetone were found to be similar to compound obtained by biotransformation method.

Compound 7 obtained as viscous liquid, [a]o -40.0 (c, 1, CHCh), showed molecular ion at mlz 3 1 8 (M+) and analyzed for C2oH3003 by elemental analysis which required six degrees of unsaturation. Its IH NMR spectrum showed the presence of two trisubstituted olefins at 8 5 .27 ( tH, d, J = 1 0 Hz) and 5.23 ( lH, m), a methine and a methylene of the dihydrofuran at 8 5 .44 ( tH, m) and 4.52 (2H, d, J = 4.3 Hz), a hydroxy bearing methine proton at 8 3.66

( l H, dd, J = 4.3 Hz), an epoxy methine proton at 8 2.52 ( tH, t, J = 6.5 Hz). Further the IH NMR spectrum showed three olefinic methyls at 8 1 .89 (3H, s), 1 .76 (3H, s) and 1 .66 (3H, s) and an oxygenated methyl at 8 1 .25 (3H, s). The foregoing spectral data was reminiscent to deoxosarcophine 3 except having hydroxy methine at 8 3.66 ( tH, dd, J = 6.0, 4.3 Hz), which was located at C-9 by the IH_ IH COSY spectrum. In the IH_IH COSY spectrum of compound 7, the hydroxy methine proton at 8 3 .66 ( tH, dd, J = 4.3 Hz), correlates with the methylene protons at 8 2. 1 8 (2H, m, H- lO) which in tum is correlated with olefinic proton at 8 5.23 ( tH, m, H- l l ). The relative stereochemistry of C-9 hydroxy in compound 7 was derived from the chemical shift values of the corresponding 9a and 9�-hydroxysarcophines, which are biotransformation products of sarcophine with A. niger7• The C-9 hydroxymethine proton in compound 7 resonated at 8 3.66 ( tH, dd, J = 6.0, 4.3 Hz) close to the value of 9�-hydroxysarcophine which resonated at 8 3.92 ( tH, dd, J = 6.0, 4.3 Hz), in contrast to the 9a­hydroxysarcophine which resonated at 8 2.92 ( tH, dd, J =6.0, 4.3 Hz). Hence the structure of compound 7

was established as 9� - hydroxydeoxosarcophine 7.

Antibacterial activity The antibacterial activity was assayed by disk

susceptibility tests according to the NCCLSI4. Excess moisture was allowed to absorb for 1 0 min before applying dried disks containing the compound. The compound was dissolved in DMSO, which did not influence the growth of bacteria. Ampicillin was also performed as a positive control according to the standard method. Inocula were adjusted to 0.5 Mc Farland turbidity. The test plates were incubated at 37°C and zones of inhibition were recorded after 24 hr. All the compounds were tested (Table I) against Gram-positive bacteria Bacillus sphaericus (ATCC # 14577), Bacillus subtilis (ATCC # 605 1 ), Staphylococcus aureus (ATCC # 9144) and Gram­negative bacteria Escherichia coli (A TCC # 25922), Pseudomonas aeruginosa (ATCC # 25619), Salmonella typhi (ATCC # 23564). Compounds 2, 4, 5, 6 and 7 showed moderate activity against Gram­positive and Gram-negative bacteria when compared to positive control Ampicillin.

Experimental Section General. The IH NMR (200MHz) and I3C NMR

(50MHz) spectra were recorded on a Varian Gemini

RAO et al. : BIOTRANSFORMATIONS OF SESQUITERPENOIDS AND DITERPENOIDS 1475

Table I - Antibacterial activity of compounds 1-7 ( lOO Ilgldisk)

Compd Gram-positive bacteria Gram-negative bacteria B. sphaeriicus B. subtilis S. aureus E. coli P. aeruginosa S. tyhimurium

1 8 9 2 1 1 3 10 9 4 8 1 0 5 9 9 6 9 1 0 7 1 2 12

Ampicillin 1 9 22

Values for zone of inhibition are shown in mmldia *Positive control: Ampicillin (50 Ilgldisk)

200 MHz spectrometer using TMS as internal standard (chemical shifts in &, ppm and J values in Hz); UV and IR spectra on a Shimadzu 240C instrument and mass spectra on a Finnigan-MAT 1020 instrument. Optical rotations were measured on a JASCO DIP-70 polarimeter. Elemental analysis was carried out on Elementar vario EL.

Preculture of G. cingulata. Spores of G. cingulata, which had been preserved at low temperature, were inoculated into sterilized culture medium ( 1 .5% sucrose, 1 .5% glucose, 0.5% peptone, 0.05% MgS04. 7H20, 0.05% KCI, 0. 1 % K2HP04 and 0.001 % FeS04. 7H20 in distilled water) in a flask which was shaken for 3 days at room temperature.

Preculture of A. niger (ATCC#9642). Spores of A. niger, which had been preserved at low temperature, inoculated into sterilized culture medium [sucrose (30 gIL + potassium chloride (0.5 gIL) + ferrous sulfate (0.01 gIL) + PH 7.3 + 0.2] in a flask which was shaken at 37°C for 3 days.

Biotransformation of compound 1. Precultured G. cingulata was transferred into four 250mL Erlenmeyer flasks containing loomL of medium and stirred for 3 days. After the growth of the fungus G. cingulata, compound 1 (25 mg) was added separately to the each flask containing medium and the organism was cultivated for 8 days.

Isolation of metabolite 4. After eight days of fermentation, the culture medium and mycelium were separated by filtration. The medium was extracted with EtOAc (3x50 mL). The mycelium was also extracted with EtOAc. The combined EtOAc extracts were dried over anhydrous Na2S04 and the solvent was evaporated under reduced pressure to yield a crude extract (200 mg). It was subjected to silica gel chromatography to afford compound 4 ( 10 mg) and unconverted compound 1 (80 mg).

8 8 9 12 9

8 9 8 1 0 9

1 0 10 8 1 0 9 8 9 9 1 1 10 21 18 22 1 0

Biotransformation of compound 1 . Precultured A. niger was transferred into four 250 mL Erlenmeyer flasks containing l oomL of medium and stirred for 3 days. After the growth of A. niger, compound 1 (25 mg) was added separately to the each flask containing medium and the organism was further cultivated for 8 days.

Isolation of metabolites 4 and 5. After eight days of fermentation, the culture medium and mycelium were separated by filtration. The medium was extracted with EtOAc (3x75 mL). The combined EtOAc extract was dried over anhydrous Na2S04 and the solvent was evaporated to yield a crude extract (220 mg) which was subjected to silica gel chromatography to afford compounds 4 (5 mg, ca 5%), 5 (4 mg, ca 4%) and unreacted compound 1 (30mg, ca 30%).

Biotransformation of compound 2. Precultured A. niger was transferred into four 250mL Erlenmeyer flasks containing l oomL of medium and stirred for 3 days. After the growth of A. niger, compound 2 (25 mg) was added separately to the each flask containing medium and the organism was further cultivated for 8 days.

Isolation of metabolite 6. After 8 days of fermentation, the culture medium and mycelium were separated by filtration. The medium was extracted with EtOAc (3x75 mL). The combined EtOAc extract was dried over anhydrous Na2S04 and the solvent was evaporated to yield a crude extract ( 1 50 mg) which was subjected to silica gel chromatography to give compound 6 ( 10 mg, ca 10%), and unreacted compound 2 (40mg, ca 40%).

Biotransformation of compound 3. Precultured A. niger was transferred into four 250mL Erlenmeyer flasks containing l oomL of medium and stirred for 3 days. After the growth of A. niger, compound 3 (25 mg) was added separately to the each flask

1476 INDIAN J. CHEM., SEC B, JULY 2002

containing medium and the organism was further cultivated for 8 days.

Isolation of metabolite 7. After 8 days of fermentation, the culture medium and mycelium were separated by filtration. The medium was extracted with EtOAc (3x75 mL). The combined EtOAc extract was dried over anhydrous Na2S04 and the solvent was evaporated to yield a crude extract (250 mg) which was subjected to silica gel chromatography to give compound 7 (8 mg, ca 8%), and unreacted compound 3 (45mg, ca 45%).

Antibacterial assay of compounds 1-7: Antibacterial activity was assayed by disk susceptibility tests according to the NCCLS. Inocula were adjusted to a density of 0. 10 at 625nm in nutrient broth and spread on nutrient agar plates (Hi­Media, India). Disks (5mm) were moistened with different compounds in l OOllg concentration placed at the centre of petri plates. Compounds 1-7 were tested against Gram-positive bacteria, B. sphaeriicus, B. subtilis, S.aureus and Gram-negative bacteria E. coli, P. aeruginosa and S. typimurim.

4�,13-Dihydroxyguaian-6,10-diene 4: Colourless viscous liquid, [a]o +2.40 (c 0.5, CHCh); IR (neat) : 3400 (OH), 3060, 1 640, 1 140, 955, 870 cm-I ; IH NMR (CDCI3, 200 MHz): 5 5.65 ( lH, br s), 4.78 ( l H, br s), 4.70 ( lH, br s), 3.42 (2H, d, J = 7.5 Hz), 2.58-2.32 (4H, m), 2. 18- 1 .70 (7H, m), 1 .25 (3H, s), 1 .0 (3H, d, J = 7.5 Hz); 1

3C NMR (CDCl3, 100 MHz): 5 153. l 8 (s) 144.38

(s), 126.64 (d), 107.07 (t), 80.5 (s), 65. 1 5 (t), 54.79 (d), 47. l 1 (d), 45.87 (t), 40. 13 (t), 36.8 (d), 29.7 (t), 28.85 (t), 24.82 (q), 15.32 (q); ElMS: mJz 236 [M+] (25). Anal. Ca1cd for CIsH2402: C, 76.2266; H, 10.2359. Found: C 76.2258, H, 10.2352%.

4�,12-Dihydroxyguaian-6,10-diene 5: Colourless viscous liquid, [a]o -54.20 (c 0.5, CHCh); IR (neat) : 3380 (OH), 3055, 1640, 1 140, 960, 880 cm- I ; IH NMR (CDCh, 200 MHz): 8 5.9 1 ( l H, br s), 4.75 ( 1H, br s), 4.70 ( 1H, br s), 2.56-2.32 (4H, m), 2. 15- l .70 (6H, m), l .38 (6H, s), 1 .25 (3H, s); ElMS: mJz 2 18 [M+-H20] (26); Anal. Ca1cd for CIsH220: C , 82. l609; H, 10. 1565. Found: C, 82. 1614; H, 10. 1560%.

(EEE)-7�,8a-Dihydroxy-1-isopropyl-4,8,12-tri­methylcyclotetradeca-1 ,3,1l-triene 6: Colourless prisms; mp 82°C; [a]o -65.20 (c 0.5, CHCh); UV (MeOH) (E): 245 ( 14000), 250 nm ( 1 5 000); IR (KBr) : 3450 (OH), 1650, 16 10, 1460, 1 370, 1 165, 750 cm- I ; IH NMR (CDCh, 200 MHz): 8 6.05 ( 1H, d, J = 1 1 .5 Hz), 5 .96 ( l H, d, J = 10.5 Hz), 5 . 10 ( 1H, m), 3.58 ( lH, d, J=1O.5 Hz), 1 .74 (3H, s), 1 .45 (3H, s),

1 .25 (3H, s), 1 .05 (6H, d, J = 7Hz); I3C NMR (CDCh, 50 MHz): 5 147.43 (s), 137.07 (s), 1 34.69 (s), 125.89 (d), 1 2 1 .6 (d), 1 18 .90 (d), 75.55 (s), 7 1 .21 (d), 40. 10 (t), 36.36 (t), 35 .30 (d), 3 1 . 1 5 (t), 27.66 (t), 25.95 (q), 24.07 (t), 23 .08 (t), 22.44 (q), 2 1 . 1 5 (q), 16.32 (q) and 1 5.41 (q); ElMS: mJz 306 [M+] (30). Anal. Ca1cd for C2oH3402: C, 78.3780; H, 1 1 . 18 14. Found: C, 78.3774; H, 1 1 . 1 8 1 9%.

9�-Hydroxydeoxosarcophine 7: Viscous liquid, [a]D -40.0 (c 1 , CHCh); IR (CHCh) : 3460 (OH), 1680, 1460, 1385, 1250, 1050, 960, 765 cm-I ; IH NMR (CDCh, 200 MHz): 8 5.44 ( tH, m), 5.27 ( lH, d, J = 10 Hz), 5 .23 ( tH, m), 4.52 (2H, d, J = 4.3 Hz), 3.66 ( lH, dd, J = 6.0, 4.3 Hz), 2.52 ( IH, t, J = 6.5 Hz), 1 .89 (3H, s), 1 .76 (3H, s), 1 .66 (3H, s), 1 .25 (3H, s); ElMS: rnJz 3 18 [M+] . Anal. Ca1cd. for C2oH3003: C, 75.4326; H, 9.4952. Found: C, 75.4321 ; H 9.4958%.

Acknowledgement We are thankful to the Department of Ocean

Development for financial assistance, the Director nCT and Dr J S Yadav for their constant encouragement, and UGC and CSIR, New Delhi for providing fellowships to M R R, U V and K S, respectively.

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