Bioorganic & Medicinal Chemistry Letters 21 (2011) 6808–6810

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Bioorganic & Medicinal Chemistry Letters

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New bioactive macrocyclic diterpenoids from Jatropha multifida q

Boddu Shashi Kanth a, Avula Satya Kumar a, Digambar Balaji Shinde a, Kothapalli Hari Babu b,Tuniki Venugopal Raju c, Chityal Ganesh Kumar d, Pombala Sujitha d, Biswanath Das a,⇑a Organic Chemistry Division-I, Indian Institute of Chemical Technology, Hyderabad 500 607, Indiab HPLC Section, Organic Chemistry Division-I, Indian Institute of Chemical Technology, Hyderabad 500 607, Indiac NMR-Division, Indian Institute of Chemical Technology, Hyderabad 500 607, Indiad Chemical Biology Laboratory, Indian Institute of Chemical Technology, Hyderabad 500 607, India

a r t i c l e i n f o a b s t r a c t

Article history:Received 18 July 2011Revised 8 September 2011Accepted 12 September 2011Available online 22 September 2011

Keywords:MultifidanolMultifidenolJatropha multifidaDiterpenoidCytotoxicityAntimicrobial activity

0960-894X/$ - see front matter � 2011 Elsevier Ltd.doi:10.1016/j.bmcl.2011.09.032

q Part 76 in the series, ‘‘Studies on novel phytochem⇑ Corresponding author. Tel./fax: +91 40 7160512.

E-mail address: biswanathdas@yahoo.com (B. Das

Two new macrocyclic diterpenoids, multifidanol (1) and multifidenol (2) along with several known com-pounds have been isolated from the stem of Jatropha multifida. The structures of the new compoundswere established from the extensive studies of their 1D and 2D NMR spectra. The cytotoxic and antimi-crobial activities of these two constituents were examined.

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Jatropha multifida Linn (Euphorbiaceae) is a shrub grows in dif-ferent parts of India.1 The plant possesses various medicinal prop-erties including antibiotic activity.2 The earlier chemicalexamination on the latex of the plant yielded cyclic peptides, phen-olics and glucosides.3 We reported previously some diterpenoidsfrom the plant.4 Re-investigation on the chemical constituents ofthe stem of the species we isolated two new minor macrocyclicditerpenoids, multifidanol (1) and multifidenol (2). Here we reportthe structure elucidation and biological (cytotoxic and antimicro-bial) activities of these new compounds.

Multifidanol (1) was obtained as a white solid. Its molecular for-mula was suggested to be C20H32O4 from its HRESIMS(m/z 359.2183 [M+Na]+). The IR spectrum revealed the presenceof hydroxyl and carbonyl groups in the molecule. The structureof the compound was settled from the detailed analysis of its 1Hand 13C NMR spectral data (Table 1) which suggested it to be alathyrane diterpenoid.5 All the signals for the protons and carbonsin the 1H and 13C NMR spectra, respectively, were assigned from 2DNMR (1H–1H COSY, HSQC, HMBC and NOESY) and DEPT experi-ments. These spectral data suggested that the compound 1 isstructurally related to the known compound, 15-epi-4(E)-jatro-grossidentadione (3) (isolated from Jatropha grossidentata)6

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icals’’.

).

(Fig. 1). However, in the former the ring A is saturated and the ketogroup has been converted into hydroxyl. In compound 3 there aretwo keto and two hydroxyl groups while in 1 only one keto andthree hydroxyl groups are present. In the 1H NMR spectrum ofthe latter H2-1 appeared at d 1.90 (1H, dd, J = 14.0, 9.0 Hz) and1.63 (1H, dd, J = 14.0, 2.0 Hz) while H-2, H-3 and H3-16 at d 1.96(1H, m), 4.01 (1H, d, J = 8.0 Hz) and 1.20 (3H, d, J = 7.0 Hz), respec-tively. The 1H–1H COSY experiment showed that H-2 was related toH2-1, H-3 and H3-16. The HMBC experiment (Fig. 2) suggested thatH-1a (d 1.90) was correlated to C-3 (d 82.9) and C-4 (d 145.1) whileH3-16 to C-1 and C-3. Thus it was clear that C-1–C-2 bond in 1 issaturated and a hydroxyl group is at C-3. The 1H and 13C NMR spec-tral data (Table 1) indicated that the rings B and C of both 1 and 3are similar.5 The relative stereochemistry of 1 was established byNOESY experiment (Fig. 2) which showed that H-3 was related toMe-16, Me-17 (d 1.28, s) and H-13 (d 3.04, m) having b-configura-tion. The structure of multifidanol (1) was thus clearly established.

Multifidenol (2), another new diterpenoid, was also isolated as awhite solid. Its molecular formula was deduced to be C20H30O4

from its HRESIMS (m/z 357.2034 [M+Na]+). This molecular formulaindicated that the compound 2 may be a dehydro derivative of 1(Fig. 1). The IR spectra of 2 showed that it contained hydroxyland carbonyl groups. Its 1H and 13C NMR spectral data (Table 2)were very similar to those of 1. However, these data suggested thatthe ring A of 2 was unsaturated having a double bond at C-1–C-2.In the 1H NMR spectrum H-1 appeared at d 5.33 (1H, s) while in the

Table 1NMR spectral data of compounds 1 and 2.a,b

Position Compound 1 Compound 2

1H NMRc Multiplicity (J in Hz) 13C NMR 1H NMRc Multiplicity (J in Hz) 13C NMR

1 1.90(a) dd (J = 14.0, 9.0) 42 5.33 s 128.61.63(b) dd (J = 14.0, 2.0)

2 1.96 m 40.2 148.53 4.01 d (J = 8.0) 82.9 4.82 br s 804 145.1 145.25 5.78 s 134.5 6.1 s 137.56 74.5 73.67 1.80(a) m 41.8 1.79(a) m 42.1

1.60(b) m 1.69–1.53(b) m8 1.58(a) m 19.3 1.69–1.53(a) m 19.4

0.80(b) m 0.78(b) m9 0.44 m 27.1 0.41 m 27.310 17.5 17.211 0.65 t (J = 8.0) 19 0.64 m 19.212 1.71–1.65(a) m 28.1 1.69–1.53(a) m 28.5

1.44(b) dd (J = 14.0, 3.0) 1.52–1.41(b) m13 3.04 m 38.9 2.82 m 38.114 212.8 212.315 83 87.816 1.2 d (J = 7.0) 16.9 1.94 s 13.917 1.28 s 29.7 1.26 s 29.318 0.99 s 28.5 0.99 s 28.819 0.71 s 14.9 0.72 s 14.920 1.21 d (J = 7.0) 16.9 1.12 d (J = 7.0) 16.6OH 5.28 br s 5.52 br s

3.8 br s 4.12 br s1.71–1.65 m 1.69–1.53 m

a The spectra were run in CDCl3 at 500 MHz (1H NMR) and 100 MHz (13C NMR).b The signals were assigned with the help of 2D NMR (1H–1H COSY, HMBC and NOESY) and DEPT experiments.c (a) and (b) Indicate the d-values of two protons attached to a carbon.

HOH

HO

H

O

H

H

OH

HOH

HO

H

O

H

H

OH1 2

HO

H

O

H

H

OH

O

3

1

2

3 4

5 6 7

8

9

1011

12

1314

15

16

17

19

1820

Figure 1. Chemical structures of compounds 1–3.

HOH

HO

H

O

H

H

OH

HOH

HO

H

O

H

H

OH

1 2

Figure 2. Key HMBC ( ) and NOESY correlations ( ).

Table 2Cytotoxic activity of compounds 1 and 2

Test cell line IC50 (50% inhibitory concentration in lM)

Multifidanol (1) Multifidenol (2) Doxorubicin (Control)

A-549 6.27 12.5 1Neuro-2a 6.35 5.6 1.2HeLa 15.4 7.56 <1MDA-231 7.04 5.39 <1MCF-7 6.39 8.57 1

B. S. Kanth et al. / Bioorg. Med. Chem. Lett. 21 (2011) 6808–6810 6809

13C NMR spectrum C-1 and C-2 at d 128.6 and 148.5, respectively.The H-3 in 2 resonated somewhat more downfield (d 4.82, 1H, br s)compared to the position of the corresponding proton (d 4.01, 1H,d, J = 8.0 Hz) in 1. The HMBC experiment (Fig. 2) showed that H-1was related to C-3 (d 80.0) and C-4 (d 145.2) while Me-16 (d 1.94, s)to C-1 and C-3. The NMR spectral data indicated that the remainingprotons and carbons of both the compounds 1 and 2 were similar.

The NOESY experiment (Fig. 2) on 2 revealed that H-3 was re-lated to Me-16, Me-17 (d 1.26) and H-13 (d 2.82, m), thus havingthe similar b-configuration as that of H-3 of 1. The structure ofmultifidenol (2) was thus clearly deduced. The absolute stereo-chemistry of the compound 3 is not known.6 We tried to applymodified Mosher’s method7 to determine the absolute stereo-chemistry of the structurally related new compounds 1 and 2.However, when we attempted to prepare a MPTA ester of 1 wegot a complex mixture which could not be purified.

Along with the new compounds, multifidanol (1) and multifide-nol (2) several other known compounds such as tetradecyl-(E)-ferulate,8 fraxidin,9 jatrothrin,10 jatropholone A,11 jatrophenone12

and japodagrone13 were also isolated. The structures of theseknown compounds were established by comparison of theirphysical (mp, [a]D) and spectral (IR, 1H and 13C NMR and MS)properties with those reported in the literature.

Table 3Antimicrobial activity of compounds 1 and 2

Test pathogen Minimum inhibitory concentration (MIC lg/ml)

Multifidanol (1) Multifidenol (2) Neomycin (Control)

Bacillus subtilis MTCC 121 4.68 — 18.75Staphylococcus aureus MTCC 96 18.75 4.68 18.75Staphylococcus aureus MLS16 MTCC 2940 18.75 4.68 18.75Micrococcus luteus MTCC 2470 — — 18.75Escherichia coli MTCC 739 4.68 — 18.75Klebsiella planticola MTCC 530 9.37 — 18.75Pseudomonas aeruginosa MTCC 2453 9.37 — 18.75

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Multifidanol (1) and multifidenol (2) were tested for in vitrocytotoxicity against five different cancerous cell lines, A-549(human alveolar adenocarcinoma), Neuro-2a (mouse neuroblas-toma), HeLa (human cervical cancer), MDA-231 (human breastadenocarcinoma) and MCF-7 (human breast adenocarcinoma).Doxorubicin was considered as the positive control. The MTT assaywas followed according to the method of Mosmann.14 IC50 valuewith each cell line was determined after four individual observa-tions (Table 2). Both the compounds, multifidanol (1) and multi-fidenol (2) showed significant cytotoxic activity against all ofthese cell lines. The IC50 values indicate that the absence of doublebond at C-1–C-2 increases the cytotoxic activity of multifidanol (1)against A-549 and MCF-7 cell lines while the presence of a doublebond at this position enhance the cytotoxic activity of multifidenol(2) against Neuro-2a, HeLa and MDA-231 cell lines.

The antimicrobial activity of the new compounds 1 and 2 wasalso evaluated against seven bacterial organisms, Bacillus subtilis(MTCC 121), Staphylococcus aureus (MTCC 96), S. aureus (MTCC2940), Micrococcus luteus (MTCC 2470), Escherichia coli (MTCC739), Klebsiella planticola (MTCC 530) and Pseudomonas aeruginosa(MTCC 2453). Neomycin was used as the positive control andmicrotiter broth dilution method15 was employed to evaluate theactivity. The minimum inhibitory concentration (MIC) value witheach organism was determined after four individual observations(Table 3).

The result was found to be impressive. Multifidanol (1) showedprominent activity against B. subtilis and E. coli while multifidenol(2) exhibited selective activity against S. aureus. The presence of adouble bond at C-1–C-2 in 2 may be responsible for this selectiveantimicrobial activity. Multifidanol (1) having saturated C-1–C-2bond showed lower activity against S. aureus compared to multi-fidenol (2) but it showed promising activity against various otherpathogens.

In conclusion, we have isolated two new macrocyclic diterpe-noids, multifidanol and multifidenol and established their struc-tures through extensive spectroscopic studies. Both thecompounds showed significant cytotoxic and antimicrobialactivities.

Acknowledgment

The authors thank CSIR and UGC, New Delhi for financialassistance.

References and notes

1. Chopra, R. N.; Badhwar, R. L.; Ghosh, S. In Poisonous Plants of India; ICAR: NewDelhi, 1965; Vol. II, p 792.

2. Aiyellagbe, O. O. Fitoterapia 2001, 72, 544.3. (a) Kosasi, S.; Van der Sluis, W. G.; Labadie, R. P. Phytochemistry 1989, 28, 2439;

(b) Kosasi, S.; Van der Sluis, W. G.; Boelens, R.; Hart, L. A.; Labadie, R. P. FEBSLett. 1989, 256, 91; (c) Van den Berg, A. J. J.; Horsten, S. F. A. J.; Van den Bosch, J.J. K.; Kroes, B. H.; Labadie, R. P. Phytochemistry 1995, 40, 597.

4. (a) Das, B.; Ravikanth, B.; Reddy, K. R.; Thirupathi, P.; Raju, T. V.; Sridhar, B.Phytochemistry 2008, 69, 2639; (b) Das, B.; Ravikanth, B.; Laxminarayana, K.;Rao, B. R.; Raju, T. V. Chem. Pharm. Bull. 2009, 57, 318; (c) Das, B.;Laxminarayana, K.; Krishnaiah, M.; Srinivas, Y.; Raju, T. V. Tetrahedron Lett.2009, 50, 4885.

5. Isolation of the constituents: The stems of Jatropha multifida were collectedfrom the botanical garden, Osmania University Campus, Hyderabad in March2010 and botanically identified. A voucher specimen (No. JM-590211) ispreserved in IICT herbarium. The shade dried plant material (2 kg) waspowdered and extracted thrice (72 h in each case) at room temperature withCHCl3–MeOH (1:1, 2 L). The combined extract was concentrated under vacuumto furnish a thick brown mass (51.4 g). The residue (51.0 g) was subjected tocolumn chromatography over silica gel and the column was eluted with themixtures of hexane and EtOAc. The following compounds were obtainedaccording to the increasing order of polarity: tetradecyl-(E)-ferulate (452 mg),fraxidin (265 mg), jatrothrin (142 mg), jatropholone A (620 mg), jatrophenone(334 mg) and japodagrone (11 mg). Besides these compounds a fraction elutedwith hexane/EtOAc (1:1) was found (TLC) to be a mixture of two compoundswhose polarities were very similar. These two compounds were separated byreverse phase HPLC (mobile phase: 85% acetonitrile in water) to obtain 1(retention time: 10.394 min) (4.5 mg) and 2 (retention time 10.658 min)(5.5 mg).Multifidanol (1): white solid, mp 204–206.½a29

D � �67.1 (c 0.21, CHCl3); IR (KBr): 3353, 1706, 1457, 1377 cm�1; 1H and 13CNMR (CDCl3): Table 1; ESIMS: m/z 359 [M+Na]+; HRESIMS: m/z 359.2183[M+Na]+ (calcd. For C20H32O4Na: m/z 359.2192).Multifidenol (2): white solid, mp 201–203.½a25

D � �142.5 (c 0.07, CHCl3); IR (KBr): 3362, 1709, 1624, 1238 cm�1; 1H and 13CNMR (CDCl3): Table 1; ESIMS: m/z 357 [M+Na]+; HRESIMS: m/z 357.2034[M+Na]+ (calcd. For C20H30O4Na: m/z 357.2036).

6. Schmeda-Hirschmann, G.; Tsichritzis, F.; Jakupovic, J. Phytochemistry 1992, 31,1731.

7. Hoye, T. R.; Jeffrey, C. S.; Shao, F. Nat. Protocols 2007, 2, 2451.8. Mohamed, M.; Helmut, R.; Andrea, P.; Guenter, A. J. Praktische Chemi. 1995, 337,

43.9. Yusupov, M. I.; Sidyakin, G. P. Chem. Nat. Prod. 1976, 11, 94.

10. Ravindranath, N.; Reddy, M. R.; Ramesh, C.; Ramu, R.; Prabhakar, A.; Jagadeesh,B.; Das, B. Chem. Pharm. Bull. 2004, 52, 608.

11. Purushothaman, K. K.; Chandrasekaran, S.; Cameron, A. F.; Cannolly, J. D.;Labbe, C.; Maltz, A.; Rycroft, D. S. Tetrahedron Lett. 1979, 20, 979.

12. Ravindranath, N.; Venkataiah, B.; Ramesh, C.; Jayaprakash, P.; Das, B. Chem.Pharm. Bull. 2003, 51, 870.

13. Aiyelaagbe, O. O.; Adesogan, K.; Ekundayo, O.; Gloer, J. B. Phytochemistry 2007,68, 2420.

14. Mosmann, T. J. Immunol. Methods 1983, 65, 55.15. (a) Amsterdam, D. In Antibiotics in Laboratory Medicine; Loman, V., Ed., 4th ed.;

Williams and Wilkins: Baltimore, MD, 1996; p 52e111; (b) Das, B.; Shinde, D.B.; Kanth, B. S.; Kamle, A.; Kumar, C. G. Eur. J. Med. Chem. 2011, 46, 3124.