Three New Constituents from the Roots of Erythrina variegata and TheirAntibacterial Activity against Methicillin-Resistant Staphylococcus aureus

by Hitoshi Tanaka*a), Ikunori Atsumia), Osamu Shirotab), Setsuko Sekitab), Eiji Sakaic), MasaruSatod), Jin Muratae), Hiroko Murata f) , Dedy Darnaedig), and Ih-Sheng Chenh)

a) Faculty of Pharmacy, Meijo University, Yagoto, Tempaku-ku, Nagoya 468-8503, Japan(phone: þ81-52-832-1781; fax: þ81-52-834-8090; e-mail: hitoshi@ccmfs.meijo-u.ac.jp)

b) Kagawa School of Pharmaceutical Sciences, Tokushima Bunri University, Shido, Sanuki,Kagawa 760-2193, Japan

c) Gifu Pharmaceutical University, Mitahora-higashi, Gifu 502-8585, Japand) Department of Oral Biology, Asahi University School of Dentistry, 1851-Hozumi, Mizuho,

Gifu 501-0296, Japane) Botanical Gardens, Graduate School of Science, The University of Tokyo, Hakusan, Bunkyo-ku,

Tokyo 112-0001, Japanf) Faculty of Pharmaceutical Sciences, Setsunan University, Nagaotoge-cho, Hirakata, Osaka 573-0101,

Japang) Herbarium Bogoriense, Research Center for Biology, Indonesian Institute of Sciences, Jalan Raya

Jakarta-Bogor Km 46 Cibinong 16911, Indonesiah) School of Pharmacy, Kaohsiung Medical University, Kaohsiung, Taiwan, R.O.C.

Two new isoflavonoids, eryvarins V and W (1 and 2, resp.), and a new chromen-4-one derivative,eryvarin X (3), along with three known isoflavonoids, 4–6, were isolated from the roots of Erythrinavariegata. Their structures were established by spectroscopic analyses. Compound 1 is a rare naturallyoccurring isoflavanone which possesses a OH group at C(3). Among the new compounds 1–3, 2exhibited a potent antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA)strains.

Introduction. – Erythrina variegata L. (Leguminosae) is distributed in severaltropical and subtropical regions around the world and has been used for the treatmentof microbial infection in China [1]. The plant has been found to be a rich source ofisoflavonoids [2 –6]. Our previous investigations have demonstrated that someisoflavonoids, such as bidwillon B [7], erycristagallin [8], eryvarin Q [5], eryvarin U[6], and orientanol B [8], isolated from E. variegata, possess potent antibacterialactivities against methicillin-resistant Staphylococcus aureus (MRSA) strains. In ourcontinuing screening of antibacterial compounds against MRSA, three new com-pounds, eryvarins V, W, and X (1, 2, and 3, resp.), together with three knownisoflavonoids 4 – 6, were isolated from the roots of E. variegata (Fig. 1). We describehere the isolation and structure elucidation of the three new compounds, 1 –3, and theirantibacterial potency against MRSA strains.

Results and Discussion. – Chromatographic purification of the CH2Cl2-solubleportions, obtained from the acetone extracts of the E. variegata roots, afforded three

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� 2011 Verlag Helvetica Chimica Acta AG, Z�rich

new compounds, 1 – 3, along with three known compounds 4– 6. The isolated knowncompounds were identified as auriculatin (4) [9], erystagallin A (5) [10], andphaseollin (6) [11] by comparison of their spectroscopic data with those of authenticsamples or reported values.

Eryvarin V (1) was obtained in the racemic form, and its molecular formula wasdetermined as C25H26O6 from the HR-EI-MS, exhibiting the Mþ ion peak at m/z422.1733. This compound was confirmed to be a 3-hydroxyisoflavanone [12], based onits characteristic spectral data, i.e., the conjugated C¼O group (1660 cm�1) in the IRspectrum and a set of CH2 H-atom signals (d(H) 4.38 (d, J¼11.7, 1 H�C(2)), 4.95 (d,J¼11.7, 1 H�C(2))) in the 1H-NMR spectrum (Table 1). The 1H-NMR spectrumdisplayed a singlet for an aromatic H-atom (d(H) 7.44 (s, H�C(5))), and signals for a2,2-dimethylpyran group (d(H) 1.44, 1.45 (s, each 3 H, Me(5’’), Me(6’’)), 5.74 (d, J¼9.9,H�C(3’’)), and 6.43 (d, J¼9.9, H�C(4’’))), and a 3,3-dimethylallyl (prenyl) group(d(H) 1.66 (s, Me(5’’’)), 1.79 (s, Me(4’’’)), 3.30 (d, J¼7.3, CH2(1’’’)), 5.19 (t, J¼7.3,H�C(2’’’))), as well as a 2,4-dihydroxyphenyl moiety (d(H) 6.31 (dd, J¼8.4, 2.2,H�C(5’)), 6.32 (d, J¼2.2, H�C(3’)), 7.31 (d, J¼8.4, H�C(6’))). The location of thesinglet aromatic H-atom at C(5) was determined from the HMBC spectrum, whichdisplayed correlations from H�C(5) to C(4), C((7), and C(9) (Fig. 2). The presence ofthe dimethylpyran and 2,4-dihydroxyphenyl groups was determined from both the1H,1H-COSY and HMBC spectra. The assignment of the dimethylpyran group fused tothe C(6)¼C(7) side was deduced from the HMBC spectrum, revealing correlationsH�C(5)/C(7), H�C(5)/C(4’’), H�C(3’’)/C(6), H�C(4’’)/C(6), and H�C(4’’)/C(7).Furthermore, the position of the dimethylpyran moiety was confirmed by the NOESYspectrum, which exhibited an NOE interaction between H�C(5) and H�C(4’’). Theattachment of the 2,4-dihydroxyphenyl group at C(3) was determined from both theNOESY (NOE interaction: CH2(2)/H�C(6’)) and the HMBC spectra (correlation:CH2(2)/C(1’)). The location of the prenyl group at C(8) was established by the HMBCexperiment, indicating correlations from CH2(1’’’) to C(7), C(8), and C(9). Thus,eryvarin V was characterized as 7-(2,4-dihydroxyphenyl)-7,8-dihydro-7-hydroxy-2,2-dimethyl-10-(3-methylbut-2-en-1-yl)-2H,6H-benzo[1,2-b : 5,4-b’]dipyran-6-one (1).

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1) Arbitrary atom numbering. For systematic names, see Exper. Part.

Fig. 1. Structures of compounds 1–3

Eryvarin W (2) was assigned the molecular formula of C25H26O4 from the HR-EI-MS (m/z 390.1823 (Mþ )). The UV spectrum and the characteristic singlet of the CH2

H-atom at C(6) (d(H) 5.56) in the 1H-NMR spectrum indicated that 2 has a

Fig. 2. Selected 1H,1H-COSY (——) , NOESY (H$H) , and HMBC (H!C) correlations of compound 1

Table 1. 1H-NMR Data of Compounds 1–31). d in ppm, J in Hz. C-Atom numbering as indicated inFig. 1.

1a) 2a) 3b)

H�C(1) 7.19 (d, J¼8.1)CH2(2) or H�C(2) 4.38 (d, J¼11.7), 6.56 (d, J¼8.1) 7.96 (s)

4.95 (d, J¼11.7)H�C(5) 7.44 (s) 7.88 (s)CH2(6) 5.56 (s)H�C(7) 7.13 (d, J¼8.1)H�C(8) 6.85 (d, J¼8.1)CH2(1’) 3.37 (d, J¼7.3) 3.43 (d, J¼7.3)H�C(2’) 5.28 (t, J¼7.3) 5.33 (t, J¼7.3)H�C(3’) 6.32 (d, J¼2.2)Me(4’) 1.79 (s) 1.78 (s)H�C(5’) 6.31 (dd, J¼8.4, 2.2)Me(5’) 1.65 (s) 1.79 (s)H�C(6’) 7.31 (d, J¼8.4)CH2(1’’) 3.62 (d, J¼7.3) 3.59 (d, J¼7.3)H�C(2’’) 5.40 (t, J¼7.3) 5.24 (t, J¼7.3)H�C(3’’) 5.74 (d, J¼9.9)H�C(4’’) 6.43 (d, J¼9.9)Me(4’’) 1.88 (s) 1.85 (s)Me(5’’) 1.44 (s)c) 1.66 (s) 1.76 (s)Me(6’’) 1.45 (s)c)CH2(1’’’) 3.30 (d, J¼7.3)H�C(2’’’) 5.19 (t, J¼7.3)Me(4’’’) 1.79 (s)Me(5’’’) 1.66 (s)HO�C(3) 8.57 (s)HO�C(9) 8.38 (s)HO 3.83 (s) 2.64 (br. s)HO 8.41 (br. s) 6.25 (br. s)

a) In (D6)acetone. b) In CDCl3. c) Chemical shifts may be interchanged.

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pterocarpene skeleton [13]. The 1H-NMR spectrum displayed signals of two sets ofortho-coupled aromatic H-atoms (d(H) 6.56 (d, J¼8.1, H�C(2)), 7.19 (d, J¼8.1,H�C(1)); and 6.85 (d, J¼8.1, H�C(8)), 7.13 (d, J¼8.1, H�C(7))) and of two prenylgroups (d(H) 1.65 (s, Me(5’)), 1.79 (s, Me(4’)), 3.37 (d, J¼7.3, CH2(1’)), 5.28 (t, J¼7.3,H�C(2’)); and d(H) 1.66 (s, Me(5’’)), 1.88 (s, Me(4’’)), 3.62 (d, J¼7.3, CH2(1’’)), 5.40 (t,J¼7.3, H�C(2’’))). The position of the ortho-coupled aromatic H-atoms at C(1) andC(2) were determined from both the NOESY (NOE interaction: H�C(2)/HO�C(3))and the HMBC spectra (correlations: H�C(1)/C(3), H�C(1)/C(4a), H�C(1)/C(11a),H�C(2)/C(4), and H�C(2)/C(11b)) (Fig. 3). The assignment of the other ortho-coupled aromatic H-atoms at C(7) and C(8) was obtained from both the NOESY(NOE interactions: CH2(6)/H�C(7) and H�C(8)/HO�C(9)) and the HMBC spectra(correlations: H�C(7)/C(9), H�C(7)/C(10a), H�C(8)/C(6b), and H�C(8)/C(10)).The location of the prenyl group at C(4) was confirmed by the HMBC experiment,indicating cross-peaks from CH2(1’) to C(3), C(4), and C(4a). The other prenyl groupat C(10) was also determined from the HMBC spectrum, which exhibited correlationsof CH2(1’’) with C(9), C(10), and C(10a). Thus, eryvarin W was characterized as 4,10-bis(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c] [1]benzopyran-3,9-diol (2).

Eryvarin X (3) has the molecular formula C19H22O4, which was established by theHR-EI-MS (m/z 314.1513 (Mþ )). This compound was determined as 3-hydroxy-4H-chromen-4-one on the basis of its peculiar spectral data; i.e., the conjugated C¼O group(1620 cm�1) in the IR spectrum, a singlet for an olefinic H-atom (d(H) 7.96) at C(2) inthe 1H-NMR spectrum, and a signal for an O-atom-carrying quaternary C-atom (d(C)141.1 (C(3))) in the 13C-NMR spectrum (Table 2). The assignment of C(3) wasobtained from the HMBC experiment, which indicated a correlation of H�C(2) withC(3) (Fig. 4). The 1H-NMR spectrum displayed a singlet for an aromatic H-atom(d(H) 7.88 (s, H�C(5)) and signals for two prenyl groups (d(H) 1.78 (s, Me(4’)), 1.79 (s,Me(5’)), 3.43 (d, J¼7.3, CH2(1’)), 5.33 (t, J¼7.3, H�C(2’)); and 1.76 (s, Me(5’’)), 1.85 (s,Me(4’’)), 3.59 (d, J¼7.3, CH2(1’’)), 5.24 (t, J¼7.3, H�C(2’’))). The assignment of asinglet aromatic H-atom at C(5) was deduced from the HMBC experiment, revealingcorrelations from H�C(5) to C(4), C(7), and C(9). The location of the prenyl group atC(6) was confirmed by both the NOESY (NOE interaction: CH2(1’)/H�C(5)) and the

Fig. 3. Selected 1H,1H-COSY (——) , NOESY (H$H) , and HMBC (H!C) correlations of compound 2

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HMBC spectra (correlations: CH2(1’)/C(5), CH2(1’)/C(6), and CH2(1’)/C(7)). Theposition of the other prenyl group at C(8) was determined on the basis of the HMBCspectrum, which exhibited correlations from CH2(1’’) to C(7), C(8), and C(9). Thus,eryvarin X was characterized as 3,7-dihydroxy-6,8-bis(3-methylbut-2-en-1-yl)-4H-1-benzopyran-4-one (3).

The anti-MRSA activities of the new compounds 1 –3 against 13 MRSA strainswere evaluated by determining the minimum inhibitory concentration (MIC) andminimum bactericidal concentration (MBC) according to the broth dilution methoddescribed in [5]. The results are compiled in Table 3. Among the three compounds,eryvarin W (2), which possesses a pterocarpene skeleton with both prenyl groups at theC(4) and C(10) positions, exhibited the highest anti-MRSA activity. The MIC andMBC values were comparable to those of the representative anti-MRSA antibiotic,vancomycin. Eryvarin V (1) also showed a moderate antibacterial activity, whileeryvarin X (3) failed to inhibit the growth of the MRSA strains tested at 50 mg/ml.

Fig. 4. Selected NOESY (H$H) and HMBC (H!C) correlations of compound 3

Table 2. 13C-NMR Data of Compounds 1–3. d in ppm. C-Atom numbering as indicated in Fig. 1.

1a) 2a) 3b) 1a) 2a) 3b)

C(1) 118.6 C(2’) 160.8 123.7 120.8C(2) 74.6 108.9 137.6 C(3’) 104.4 131.3 135.8C(3) 74.7 157.2 141.1 C(4’) 157.7 17.9 18.0C(4) 191.2 117.5 173.1 C(5’) 107.4 25.9 25.9C(4a) 153.3 C(6’) 128.6C(5) 123.7 123.3 C(1’’) 23.4 22.3C(6) 116.6 65.9 126.5 C(2’’) 78.5 123.0 120.6C(6a) 106.8 C(3’’) 130.3 132.0 135.4C(6b) 119.3 C(4’’) 122.2 17.9 18.0C(7) 157.9 116.6 157.8 C(5’’) 28.6 25.9 25.8C(8) 117.4 112.9 114.7 C(6’’) 28.6C(9) 160.9 155.8 154.4 C(1’’’) 22.3C(10) 114.0 112.8 115.6 C(2’’’) 122.8C(10a) 153.2 C(3’’’) 131.8C(11a) 148.1 C(4’’’) 18.0C(11b) 110.0 C(5’’’) 25.9C(1’) 116.9 23.1 29.6

a) In (D6)acetone. b) In CDCl3.

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Eryvarin W (2 : 4,10-diprenylpterocarpene derivative) could be a leading compound todevelop new therapeutics for MRSA infections.

Experimental Part

General. Column chromatography (CC): silica gel (SiO2; 230–400 mesh; Merck). TLC: precoatedSiO2 60 F254 plates (Merck); detection by UV light or I2 vapor. Optical rotations: JASCO DIP-370 digitalpolarimeter and JASCO J-725 spectropolarimeter. UV Spectra: Beckman DU-530 spectrophotometer inMeOH; lmax (log e) in nm. IR Spectra: JASCO IR-810 spectrophotometer; n in cm�1. 1H- and 13C-NMR,1H,1H-COSY, NOESY, HSQC, and HMBC spectra: JEOL ALPHA-600 spectrometer at 600 (1H) and150.8 MHz (13C); d in ppm rel. to Me4Si as internal standard, J in Hz. EI- and HR-EI-MS: JEOL JMS-SX102A spectrometer; in m/z (rel. %). The MIC and MBC values of the isolated new compounds 1–3against the 13 MRSA strains were determined as reported in [5].

Plant Materials. The roots of E. variegata were collected in January 2001 from Bogor BotanicalGarden, Indonesia [3], and in November 2001 from Kaohsiung, Taiwan, R.O.C [6].

Extraction and Isolation. The dried roots of E. variegata (1.53 kg), collected from Bogor BotanicalGarden, Indonesia, were extracted (2�3 d) with acetone (18 l) at 238. Upon evaporation, the acetoneextract produced a brownish residue (209.2 g), which was successively extracted with hexane, CH2Cl2,and AcOEt. The CH2Cl2 extract (65.4 g) was fractionated by CC (SiO2; CHCl3/acetone; then CHCl3/MeOH of increasing polarity) to give the main fractions, Frs. A1–A33. This procedure was described inour previous article [3]. Fr. A25 was subjected to CC (SiO2; CHCl3/acetone 3 : 1!1 :1) to afford twentyfractions, Frs. A25-1–A25-20. Fr. A25-6 was submitted to repeated CC (SiO2; CHCl3/acetone 10 : 1; thenbenzene/AcOEt 3 : 1) to yield eryvarin V (1; 13.6 mg).

The finely powdered roots of E. variegata (3.19 kg), collected from Kaohsiung, Taiwan, wereextracted (2�2 d) with acetone (73 l) at 238, and the solvent was removed to yield a residue that wasdivided into hexane-, CH2Cl2-, and AcOEt-soluble fractions. The CH2Cl2-soluble fraction (228.4 g) wasapplied to CC (SiO2; CHCl3/acetone of increasing polarity) to afford seven main fractions, Frs. B1–B7.This procedure was described in [6]. Fr. B3 was subjected to CC (SiO2; CHCl3/acetone 40 : 1!20 : 1) togive 24 fractions, Frs. B3-1–B3-24. Fr. B3-5 was separated by CC (SiO2; hexane/acetone 7 : 1!3 : 1; thenCHCl3/acetone 20 : 1!5 : 1) to yield eryvarin X (3 ; 15.5 mg) and phaseollin (6 ; 11.0 mg). Fr. B4 wasapplied to CC (SiO2; CHCl3/acetone 10 : 1.5!1 : 1) to give twenty fractions, Frs. B4-1 –B4-20. Fr. B4-6and B4-7 were separated by CC (SiO2; benzene/AcOEt 5 :1!3 : 1; then hexane/acetone 3 :1!1 : 1) tofurnish eryvarin W (2 ; 4.7 mg), auriculatin (4 ; 34 mg), and erystagallin A (5 ; 41 mg).

Eryvarin V (¼ 7-(2,4-Dihydroxyphenyl)-7-hydroxy-2,2-dimethyl-10-(3-methylbut-2-en-1-yl)-7,8-di-hydro-2H,6H-pyrano[3,2-g]chromen-6-one¼7-(2,4-Dihydroxyphenyl)-7,8-dihydro-7-hydroxy-2,2-di-methyl-10-(3-methylbut-2-en-1-yl)-2H,6H-benzo[1,2-b :5,4-b’]dipyran-6-one; 1). Colorless oil. [a]D¼ �0.

Table 3. Antibacterial Activity of Compounds 1–3 against Methicillin-Resistant Staphylococcus aureus(MRSA) Strains

Compound MIC [mg/ml] MBC [mg/ml]

MIC Valuesa) MIC50b) MIC90

c) MBC Valuesa) MBC50b) MBC90

c)

Eryvarin V (1) 12.5–25 12.5 12.5 12.5–50 25 25Eryvarin W (2) 1.56–3.13 3.13 3.13 3.13–25 25 25Eryvarin X (3) >50 –d) – – – –Vancomycine) 0.78–1.56 1.56 3.13 3.13–25 6.25 25

a) MIC or MBC values against 13 MRSA strains tested. b) Minimum concentration needed to inhibit thegrowth or recovery of 50% of MRSA strains. c) Minimum concentration needed to inhibit the growth orrecovery of 90% of MRSA strains. d) Not tested. e) Positive control.

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CD (c¼2.37 · 10�5, MeOH): no ellipticity. UV: 205 (4.47), 225 (sh, 4.14), 257 (4.30), 344 (3.54). IR(KBr): 3400, 1660, 1600, 1520, 1470, 1380, 1280, 1170, 1120, 1040. 1H- and 13C-NMR: Tables 1 and 2, resp.EI-MS: 422 (36, Mþ ), 407 (12), 389 (19), 312 (21), 297 (100), 271 (96), 255 (26), 242 (21), 227 (21), 215(43). HR-EI-MS: 422.1733 (Mþ , C25H26Oþ

6 ; calc. 422.1729).Eryvarin W (¼4,10-Bis(3-methylbut-2-en-1-yl)-6H-[1]benzofuro[3,2-c]chromene-3,9-diol¼4,10-

Bis(3-methylbut-2-en-1-yl)-6H-benzofuro[3,2-c] [1]benzopyran-3,9-diol ; 2). Colorless oil. UV: 200(5.05), 255 (sh, 4.14), 322 (sh, 4.21), 334 (4.32), 351 (4.21). IR (KBr): 3600, 1615, 1420, 1380, 1290,1160, 1030. 1H- and 13C-NMR: Tables 1 and 2, resp. EI-MS: 390 (100, Mþ ), 346 (14), 334 (45), 319 (11),290 (15), 278 (36). HR-EI-MS: 390.1823 (Mþ , C25H26Oþ

4 ; calc. 390.1831).Eryvarin X (¼ 3,7-Dihydroxy-6,8-bis(3-methylbut-2-en-1-yl)-4H-chromen-4-one¼3,7-Dihydroxy-

6,8-bis(3-methylbut-2-en-1-yl)-4H-1-benzopyran-4-one; 3). Colorless oil. UV: 202 (4.28), 236 (4.31),309 (3.99). IR (KBr): 3400, 1620, 1600, 1450, 1380, 1340, 1280, 1190, 1090. 1H- and 13C-NMR: Tables 1and 2, resp. EI-MS: 314 (75, Mþ ), 299 (9), 297 (9), 271 (9), 259 (28), 243 (100), 229 (10), 215 (27), 203(48), 149 (20). HR-EI-MS: 314.1513 (Mþ , C19H22Oþ

4 ; calc. 314.1518).Antibacterial Assay. The antibacterial activity against 13 MRSA strains was evaluated by

determining MIC and MBC values. The assay procedure was carried out as described in [5].

REFERENCES

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[3] H. Tanaka, M. Hirata, H. Etoh, H. Shimizu, M. Sako, J. Murata, H. Murata, D. Darnaedi, T. Fukai,Phytochemistry 2003, 62, 1243.

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[6] H. Tanaka, M. Sudo, M. Hirata, M. Sako, M. Sato, I.-S. Chen, T. Fukai, Heterocycles 2005, 65, 871.[7] M. Sato, H. Tanaka, R. Yamaguchi, K. Kato, H. Etoh, Int. J. Antimicrob. Agents 2004, 24, 241.[8] H. Tanaka, M. Sato, S. Fujiwara, M. Hirata, H. Etoh, H. Takeuchi, Lett. Appl. Microbiol. 2002, 35,

494.[9] K. V. Subba Raju, G. Srimannarayana, B. Ternai, R. Stanley, K. R. Markham, Tetrahedron 1981, 37,

957.[10] H. Tanaka, T. Tanaka, H. Etoh, Phytochemistry 1997, 45, 835.[11] D. R. Perrin, C. P. Whittle, T. J. Batterham, Tetrahedron Lett. 1972, 13, 1673.[12] C. M. Kim, Y. Ebizuka, U. Sankawa, Chem. Pharm. Bull. 1989, 37, 2879.[13] A. V. K. Prasad, R. S. Kapil, S. P. Popli, Indian J. Chem., Sect. B 1985, 24, 236.

Received March 5, 2010

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