Antimycobacterial Labdane Diterpenes from Leucas stelligera

Oct 11, 2013 - Compounds 1–4 exhibited selective antimycobacterial activity against Mycobacterium tuberculosis with IC50 values in the range ... P.T...
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Antimycobacterial Labdane Diterpenes from Leucas stelligera Roshan R. Kulkarni,† Ketaki Shurpali,‡ Vedavati G. Puranik,§ Dhiman Sarkar,‡ and Swati P. Joshi*,† †

Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune 411008, India Combi-Chem Bioresource Center, CSIR-National Chemical Laboratory, Pune 411008, India § Center for Materials Characterization, CSIR-National Chemical Laboratory, Pune 411008, India ‡

S Supporting Information *

ABSTRACT: Phytochemical investigation of the acetone extract of the aerial parts of Leucas stelligera afforded four new compounds (1−4) belonging to the labdane diterpene series as well as two known flavones, velutin (5) and chrysoeriol (6). Structure elucidation of the new compounds was carried out using 1D and 2D NMR spectroscopic data and single-crystal X-ray crystallography of compound 1. Compounds 1−4 exhibited selective antimycobacterial activity against Mycobacterium tuberculosis with IC50 values in the range 5.02−9.80 μg/mL.

rium tuberculosis H37Ra. Phytochemical analysis of the extract resulted in the isolation of four new labdane diterpenes (1−4) (Figure 1) along with two known flavones, velutin (5) and

Tuberculosis (TB) is a major, and still neglected, cause of death and disability, with around 2 million deaths and 9 million infections reported worldwide in 2009. The emergence of drugresistant strains and confluence with an HIV epidemic have turned TB into a global public health crisis.1 Although available drug regimens can cure most patients,2 the emergence of drugresistant TB3,4 and an insufficient global drug pipeline5 justify continued efforts toward the development of new drugs with a new mode of action and novel structures. There is currently a reemerging interest in natural products as a source of lead compounds for drug discovery efforts, particularly in the area of antibacterials.6 Several reviews on naturally occurring antimycobacterial compounds display a variety of structures, for example, diterpenes belonging to the labdane, clerodane, pimarane, kaurane, diterpene amide, diterpene alkaloid, and phorbol ester classes.6−9 The Lamiaceae is a family of significant economic and therapeutic importance. The constituents from a few species10,11 show antimycobacterial activity, while phytochemical analyses of Leucas volkensii12 and Anisochilus harmandii13 have led to the isolation of diterpenes as active principles. The genus Leucas is represented in India with 43 species,14 of which 21 are found in the state of Maharashtra.15 Leucas stelligera, locally called Goma, is an herb found in open spaces, along roadsides, and in fields. It is used as a fish poison16 as well as for wound healing in ethnoveterinary practice.17 The plant is nontoxic to humans, and the leaves are edible. L. stelligera has thus far not been investigated phytochemically. In our program on the discovery of biologically active compounds from plants found in Western Ghats of Maharashtra, India, the acetone extract of the aerial parts of L. stelligera demonstrated inhibitory activity against Mycobacte© 2013 American Chemical Society and American Society of Pharmacognosy

Figure 1. Compounds from L. stelligera..

chrysoeriol (6). Herein we report the isolation, structure elucidation, and evaluation of biological activities of compounds 1−4. Compounds 1, 2, and 4 were evaluated against M. tuberculosis, M. smegmatis mc2 155, and Escherichia coli DH5α, as well as against Thp-1 (human monocytic leukemia), HepG-2 (liver hepatocellular carcinoma), and MCF-7 (human breast adenocarcinoma) cell lines. Compound 3 was tested against M. tuberculosis and Thp-1 cell lines. Received: January 7, 2013 Published: October 11, 2013 1836

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Table 1. NMR Spectroscopic Data (δ 400 and 100 MHz, CDCl3) for Compounds 1, 2, 3, and 4 1 position

δC, type

1 2ax 2eq 3ax

31.8, CH2 18.6, CH2 41.6, CH2

3eq 4 5 6ax 6eq 7ax 7eq 8 9 10 11 12 13 14 15

36.6, 76.8, 43.2, 32.2, 22.2, 135.1, 143.7, 70.1,

16

174.5, C

17 18 19 20

16.2, 33.7, 22.0, 16.3,

■

33.3, C 46.3, CH 21.5, CH2 31.2, CH2 CH C C CH2 CH2 CH C CH2

CH3 CH3 CH3 CH3

2 δH (J in Hz) 1.49 (m) 1.48 m 1.48 m 1.16 dt (3.5, 12.9) 1.34 m 1.42 1.54 1.29 1.49 1.29 1.79

m m m m m m

1.66 m, 1.85 m 2.39 dt (1.3, 8.4) 7.11 bt (1.6) 4.77 bq (2.0)

δC 33.0, CH2 18.7, CH2 41.8, CH2

d (6.7) s s s

δC

m m m dt (3.6, 13.0)

36.5, CH2 18.6, CH2 42.2, CH2

1.33 m 33.5, C 47.0, CH 21.7, CH2 32.1, CH2 35.8, 95.0, 42.5, 29.3, 33.4, 89.9, 45.5, 99.2,

CH C C CH2 CH2 C CH2 CH

77.3, CH2 0.92 0.88 0.84 0.94

1.36 1.55 1.48 1.17

3 δH (J in Hz)

17.7, 22.0, 33.3, 18.2,

CH3 CH3 CH3 CH3

1.37 1.55 1.27 1.48 1.22 1.76

4 δH (J in Hz)

1.10 1.68 1.43 1.15

m, 1.57 m m m dt (3.9, 13.6)

δC 31.9, CH2 18.6, CH2

1.48 m 1.48 m, 1.55 m

41.7, CH2

1.15 dt (3.5, 13.2) 1.35 m

1.37 m 32.9, C 46.3, CH 20.7, CH2

m m m m m m

37.8, CH2

1.77 m, 2.02 m 2.00 (m) 2.32 d (13.1), 1.98 m 5.43 bs 3.62 d (8.5); 4.37 d (8.5) 0.91 d (6.6) 0.81 s 0.86 s 0.91 s

74.2, 61.2, 38.9, 20.9, 45.0, 73.6, 146.2, 111.1,

C CH C CH2 CH2 C CH CH2

1.06 1.55 1.28 1.55 1.55

dd (12.6, 2.8) m m m m

1.11 1.50 m, 1.78 m 1.52−1.63 m

27.3, CH3

5.95 dd (17.3, 10.4) 5.03 d (10.4); 5.22 d (17.3) 1.29 s

32.0, 33.1, 21.4, 24.8,

1.47 0.87 0.79 1.07

CH3 CH3 CH3 CH3

s s s s

δH (J in Hz)

33.3, C 46.4, CH 21.6, CH2 31.3, CH2 36.9, 77.3, 43.2, 33.0, 31.9, 144.7, 126.0, 60.6,

CH C C CH2 CH2 C CH CH2

1.39 1.53 1.28 1.27 1.27 1.78

m m m m m m

1.57 m, 1.78 m 2.2 m 5.63 t (6.9) 4.19 bs

58.5, CH2

4.20 bs

16.5, 33.7, 22.0, 16.2,

0.89 0.88 0.84 0.94

CH3 CH3 CH3 CH3

d (7.0) s s s

position of H3-17 (Figure 2).19 The genus Leucas is known to produce only labdane-type diterpenes.20−23 Hence, 1 most

RESULTS AND DISCUSSION The acetone extract from the aerial parts of L. stelligera was fractionated on a silica gel column. The fractions were purified further by repeated column chromatography and preparative TLC to afford four new diterpenes (1−4) together with the known velutin (5) and chrysoeriol (6). Compound 1 was obtained as white needles. The molecular formula was determined as C20H32O3 by HREIMS, which showed a molecular ion peak at m/z 320.2365 [M]+ indicating five indices of hydrogen deficiency. The IR spectrum showed absorptions for hydroxy (3565 cm−1) and carbonyl (1749 cm−1) groups, the latter suggestive of an α,β-unsaturated lactone moiety. The 1H NMR spectrum (Table 1) displayed singlets for three tertiary methyl groups at δH 0.84, 0.88, and 0.94 and one secondary methyl group at δH 0.92 (d, J = 6.7 Hz). This indicated 1 to be a labdane-type diterpene. The 1H and 13C NMR data (Table 1) were nearly superimposable on those of 6-acetoxy-9-hydroxy-13(14)-labden-16,15-olide18 except for the absence of a C-6 acetyloxy group in 1, based on the chemical shift differences of H-6 (δH 5.39) and C-6 (δC 70.2) methine in the known labdane compared to δH 1.29 (1H, m) and 1.54 (1H, m), and δC 21.5, respectively in 1. The HMBC correlation of the equatorial H-6 (δH 1.29) with C-5 (δC 46.3) confirmed this assignment. Analysis of the NOESY data revealed cross-peaks between H3-20 and H3-19. The NOESY correlations observed between H3-20 and H2-11 placed the hydroxy group at C-9 in an axial position. Suitable X-ray quality crystals of 1 were obtained by crystallization from cyclohexane/ acetone (7:3). The study confirmed the structure of 1 to have a trans-fused A/B ring junction as well as the relative configuration, as assigned by NOESY, with an equatorial

Figure 2. ORTEP diagram of compound 1. Ellipsoids are drawn at 50% probability.

likely belongs to the labdane rather than ent-labdane series of diterpenes. Thus, 1 was identified as a new natural product, 9hydroxylabd-13-en-16,15-olide. Compound 2 was obtained as a colorless, viscous oil. The molecular formula was determined as C20H34O3 by HREIMS, which showed a molecular ion peak at m/z 322.2515 [M]+ indicating four indices of hydrogen deficiency. The IR spectrum showed a stretching frequency (3427 cm−1) for a hydroxy group. The 1H NMR spectrum (Table 1) displayed singlets for three tertiary methyl groups at δH 0.81, 0.86, and 0.91 and one secondary methyl group at δH 0.91 (d, J = 6.6 Hz). The 13C NMR spectrum (Table 1) displayed the presence of two 1837

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quaternary carbons at δC 95.0 and 89.9 and a methine carbon at δC 99.2. These indicated 2 to be a labdane-type diterpene with two spiro-tetrahydrofuran rings C and D, which was confirmed by comparison with the known 9,13;15,16-diepoxy-15,16dimethoxylabdane,18 leopersin C, 15-epi-leopersin C,24 and velutine A, 15-epi-velutine A. 25 The observed HMBC correlations of H2-14 (δH 1.98 and 2.32) with δC 89.9 (C-13) and δC 99.2 (C-15) confirmed the assignment. The HMBC correlation of H2-11 (δH 1.77 and 2.02) with C-9 at δC 95.0 confirmed the spiro fusion of ring C with ring B at C-9. NOESY cross-peaks were observed between H3-20 and H3-19, while a trans-fused ring junction was inferred from the absence of NOESY correlations between H3-20 and H-5 and the similarity of the chemical shifts of ring junction carbons and protons to 1. The presence of the NOESY correlation between H3-20 and H8 confirmed an equatorial position of H3-17. The NOESY correlations between H3-20 and H2-11, H3-17 and H2-14, H2-12 and H2-15, and H-16 and H2-1 led to the assignment of relative configuration. Thus compound 2 was identified as a new labdane diterpene, 9,13:15,16-diepoxylabdan-15-ol. Compound 3 was obtained as colorless, fine crystals. The molecular formula was determined as C20H36O2 by HRESIMS, which showed a pseudomolecular peak at m/z 331.2612 [M + Na]+ indicating three indices of hydrogen deficiency. The IR spectrum showed absorptions for hydroxy (3418 cm−1) and olefinic (1644 cm−1) groups. The 1H NMR spectrum (Table 1) displayed singlets for five tertiary methyl groups at δH 0.79, 0.87, 1.07, 1.29, and 1.47. This indicated 3 to be a diterpene with a labdane skeleton and an acyclic side chain. The 13C NMR spectrum (Table 1) displayed two quaternary carbons at δC 73.6 and 74.2, a methine at δC 61.2, and a terminal olefinic group (δC 146.2 and 111.1), indicating 3 to be similar to sclareol.22 The sclareol-type skeleton was confirmed by analyses of the HMBC and COSY spectra as follows: the methyl at δC 27.3 showed an HMBC correlation with the methine at δH 5.95 (H-14) and a four-bond HMBC correlation with the methylene protons at δH 5.22 and 5.03 (H2-15). Similarly, H-14 exhibited an HMBC correlation with the methylene at δC 45.0 (C-12), while the methylene protons at δH 1.52−1.63 (H2-12) exhibited an HMBC correlation with the methyl at δC 27.3, a methylene at δC 20.9 (C-11), and a methine at δC 61.2 (C-9). These correlations confirmed the side chain connectivity. The methine at δC 61.2 (C-9) showed correlations with the methyl groups at δH 1.07 (H3-20) and 1.47 (H3-17). The methyl group at δH 1.07 gave an HMBC correlation with the methine carbon at δC 46.3 (C-5) and a quaternary carbon at δC 38.9 (C-10) as well as with the methylene carbon at δC 36.5 (C-1). This led to placement of the C-20 methyl (δC 24.8) and C-5 methine (δC 46.3) groups. Two methyl groups at δH 0.79 (H3-19) and 0.87 (H3-18) showed three-bond HMBC correlations with the methine at δC 46.3 and the methylene at δC 42.2 (C-3) and a two-bond HMBC correlation with a quaternary carbon at δC 32.9 (C-4). These sets of correlations established 3−4−5−10− 20−1 connectivity. H3-17 (δH 1.47) showed an HMBC correlation with the methylene at δC 37.8 and was assigned to C-7. Correlation of an axial proton at C-3 (δH 1.15) with the methylene carbon at δC 18.6 identified the latter as C-2 and the remaining methylene at δC 20.7 as C-6, confirming a sclareoltype skeleton as the basic structure for 3. The sclareol-type labdane skeleton was also confirmed by analysis of the H2BC spectrum, which unambiguously established 1−2−3, 5−6−7, and 9−11−12 connectivities.

Comparison of the 13C NMR values of 3 with those reported for all the known stereoisomers of sclareol, viz., sclareol, 8-episclareol, and 13-epi-sclareol,26 along with those of 8,13-episclareol,27 8-epi-ent-sclareol,28 and pinnatol A,29 revealed deviations in chemical shifts of the carbons 1, 2, 5, 7, 11, and 20. An upfield shift of C-5 (∼9 ppm) and a downfield shift of C-20 (∼9 ppm) can be explained only by the cis-fused A/B ring junction. An upfield shift of C-5 (∼10 ppm) and a downfield shift of C-19 (∼10 ppm) are well-known for cis-fused steroids.30,31 In trans-fused A/B rings, C-20 is shielded due to 1,3-diaxial interactions with Hax-5, Hax-2, and C-19. These are relieved in cis-fused A/B rings, explaining the downfield shift of C-20 in 3. The fused rings assume a nonsteroidal conformation, as revealed by the strong NOESY correlation between H3-19 (δH 0.79) and H3-20 (δH 1.07). This NOESY correlation is not compatible with steroidal-type A/B fusion. A strong NOESY correlation between H3-20 (δH 1.07) and H3-17 (δH 1.47) revealed a β-orientation of H3-17 and a boat conformation for ring B. A downfield shift of H3-17 of ∼0.31 ppm from sclareol can be explained on the basis of relief from shielding 1,3-diaxial interactions with H-6. These data support an A/B ring chair− boat conformation. Thus, by detailed 2D NMR spectroscopy and comparison with reference structures, 3 was identified as a new natural product and named labd-14-ene-8,13-diol, or cis-sclareol. Compound 3 belongs to the rare class of bicyclic diterpenes that have cis-fused rings. Compound 4 was obtained as a colorless gum. The molecular formula was determined as C20H36O3 by HREIMS, which showed a molecular ion peak at m/z 324.2689 [M]+ indicating three indices of hydrogen deficiency. The IR spectrum showed a stretching frequency for a hydroxy (3372 cm−1) group. The 1H NMR spectrum (Table 1) displayed singlets for three tertiary methyl groups at δH 0.84, 0.88, and 0.94, as well as one secondary methyl group at δH 0.89 (d, J = 7.0 Hz). This identified 4 as a labdane-type diterpene. The 13C NMR spectrum (Table 1) displayed the presence of two methylenes at δC 58.5 and 60.6, a methine at δC 126.0, and a quaternary carbon at δC 144.7. This indicated a substituted but2-ene-1,4-diol pattern of the acyclic part of the labdane skeleton. This substitution pattern was confirmed by comparison with the known bincatriol.32 The observed threebond HMBC correlations of H3-17 (δH 0.89) with the quaternary carbon at δC 77.3 fixed the location of the latter to C-9. The HMBC correlation of H-8 (δH 1.78) with C-9 (δC 77.3), C-10 (δC 43.2), and C-17 (δC 16.5), along with the HMBC correlation of H2-11 (δH 1.57 and 1.78) with C-9 (δC 77.3) and C-8 (δC 36.9), confirmed the structure. NOESY cross-peaks were observed between H3-20 and H3-19, while a trans-fused ring junction was inferred from the absence of NOESY correlations between H3-20 and H-5 and the similarity of the ring junction carbon and proton NMR data to 1 and 2. The presence of an NOE correlation between H3-20 and H-8 confirmed the equatorial position of H3-17. The observed NOE between H3-20 and H2-11 placed the acyclic part at C-9 in an equatorial position. On the basis of the NOESY correlation between H2-12 and H-14, the double-bond configuration at C13 was determined as Z. Thus compound 4 was identified as a new labdane diterpene, labd-13Z-ene-9,15,16-triol. Velutin (5) and chrysoeriol (6) were identified by comparison of the 1H and 13C NMR data with published data.33,34 1838

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recorded with a Thermo-Scientific Q-Exactive spectrometer, and HREIMS were recorded using an MSI-Autoconcept mass spectrometer. The molecular structure of compound 1 was unequivocally established using a Bruker SMART APEX CCD diffractometer with Mo Kα radiation. All of the solvents used were of analytical grade (Thomas Baker Ltd.). Column chromatography was carried out using silica gel mesh 234−400 (Thomas Baker Ltd.), and preparative TLC using TLC plates supplied by Merck Ltd. Human acute monocytic leukemia (Thp-1), liver hepatocellular carcinoma (HepG2), and breast cancer (MCF-7) cell lines were purchased from the National Cell Repository, India. Isoniazid, MTT, and paclitaxel were purchased from Sigma-Aldrich, USA. M. smegmatis mc2 155 (ATCC 607) was obtained from Astra Zeneka Bangalore, India, E. coli DH5α from the National Collection of Industrial Microorganisms, National Chemical Laboratory, India, and M. tuberculosis H37Ra (ATCC 25177) from MTCC, Chandigarh, India. Plant Material. L. stelligera, whole flowering plants, were collected from paddy fields in the Mulshi area, District Pune, India, on January 3, 2008. A herbarium sample was deposited in the Botanical Survey of India, Western Circle, Pune (Voucher No. SPJ-5), and authenticated by Dr. P. G. Diwakar. Roots were separated, and aerial parts were cleaned of adhering dust and unwanted plant material, shade dried, cut, and pulverized. Extraction and Isolation. Pulverized aerial parts (1.8 kg) were extracted with acetone (6 L × 3 × 14 h) at room temperature. The acetone solubles were filtered and concentrated under reduced pressure to yield a greenish extract (57.0 g, 3.0% based on dry plant weight), 55.0 g of which was separated by column chromatography (CC) using a gradient of acetone from 10% to 100% in petroleum ether to give 11 fractions (LS1−LS11). Fractions LS4 (5.5 g) and LS5 (2.3 g) were subjected separately to CC using 6% MeCN in CHCl3 as the mobile phase to afford 10 (LS4a−j) and 13 (LS5a−m) fractions, respectively. The fractions LS4f, LS4h, LS4i, LS5j, and LS5k were combined and subjected further to CC with 15% acetone in petroleum ether to afford compound 1 (100 mg). Fraction LS7 (4.1 g) was subjected to CC with a gradient of MeCN from 1% to 3% in CHCl3 to give 18 fractions (LS7a−r). Fraction LS7i (287.2 mg) was subjected to CC with 15% acetone in petroleum ether to give compounds 2 (100 mg) and 3 (38 mg). Fraction LS11 (4.9 g) was subjected to CC using a gradient of MeOH from 5% to 20% in CHCl3 to give eight fractions (LS11a−h). Fraction LS11b (3 g) was subjected to CC using a gradient of MeOH from 1% to 3% in CHCl3 to give six (LS11bi−bvi) fractions. Fractions LS11biv (670 mg) and LS11bv (1.9 g) were subjected separately to CC using an elution system of a gradient of acetone from 5% to 50% in petroleum ether to give 25 (LS11biv1−25) and 20 (LS11bv1−20) fractions, respectively. Fractions LS11biv23, LS11bv18, and LS11bv19 were combined (372.5 mg) and subjected to CC using a gradient of EtOAc from 30% to 50% in petroleum ether to give eight fractions. From fraction 8, compound 5 (15 mg) was obtained as a pale yellow precipitate. From LS11bvi, compound 6 (10 mg) precipitated out. The precipitate was filtered, and the filtrate (300 mg) was subjected to CC using a gradient of acetone in petroleum ether from 5% to 50% to give 10 fractions (LS11bvi1−10). Fraction LS11bvi1 was purified by successive preparative TLC using two different developing systems (25% MeCN in CHCl3 and 35% EtOAc in CHCl3) to afford compound 4 (20 mg).

Compounds 1, 2, 3, and 4 showed moderate inhibition against M. tuberculosis H37Ra with IC50 values in the range 5.02−9.80 μg/mL and IC90 values ranging from 10.85 to 46.52 μg/mL compared to the positive control (isoniazid), which showed an IC90 value of 0.05 ± 0.00 μg/mL. Compound 5 was inactive, and compound 6 was not tested (Table 2). Table 2. In Vitro Antimycobacterial Activity of Compounds against M. tuberculosis H37Ra samplea 1 2 3 4 extract a

IC50 (μg/mL) 5.02 5.55 5.95 9.8 8.94

± ± ± ± ±

1.07 0.92 1.09 1.94 1.67

IC90 (μg/mL) 19.67 14.88 10.85 46.52 43.98

± ± ± ± ±

3.27 2.93 2.47 7.91 9.28

Isoniazid IC90 0.055 ± 0.003 μg/mL

Compounds 1, 2, and 4 were also tested at their IC90 concentration for the inhibition of Escherichia coli DH5α and Mycobacterium smegmatis mc2 155, with no significant effect, thus indicating the specificity of these compounds against pathogenic mycobacteria (Table 3). The compounds were also Table 3. Inhibition (%) of 1, 2, and 4 against M. smegmatis mc2 155 and E. coli DH5α sample a

1 2a 4a extracta rifampicinb a b

M. smegmatis 31.56 47.83 30.57 19.47 0.20

± ± ± ± ±

6.09 3.74 3.58 0.16 0.03

E. coli 0.29 23.15 11.47 3.48 0.50

± ± ± ± ±

3.56 2.45 1.65 2.56 0.04

Concentration of the compounds used: IC90 values from Table 2. IC90, μg/mL

tested for their in vitro cytotoxicity against MCF-7, Thp-1, and HepG-2 cell lines (Table 4). Compound 3 was tested only against the Thp-1 cell line. Compounds 1 and 2 showed 40.70 ± 2.78% and 42.40 ± 1.67% inhibition against the MCF-7 cell line, respectively, at a high concentration of 100 μg/mL. Compound 3 showed 96.36 ± 0.09% inhibition at 100 μg/mL. These results indicate a selective inhibition of 1, 2, and 4 toward M. tuberculosis.

■

EXPERIMENTAL SECTION

General Experimental Procedures. Optical rotations were measured with a JASCO P-1020 polarimeter. IR spectra were recorded in CHCl3 with a Perkin-Elmer FT-IR spectrometer. The 1H and 13C NMR spectra were recorded on a Bruker Avance Ultra Shield NMR instrument (1H, 400 MHz; 13C, 100 MHz). ESIMS spectra were recorded with an API-QSTAR-PULSAR spectrometer. HRESIMS was

Table 4. In Vitro Cytotoxicity Data of the Isolated Compounds MCF-7 a

1 2a 4a extracta paclitaxel a

Thp-1

HepG-2

at IC90

at 100 μg/mL

at IC90

at 100 μg/mL

at IC90

13.37 ± 2.76 28.98 ± 2.23 3.54 ± 1.76 7.01 ± 3.87 107 ± 2.09b

40.70 ± 2.78 42.40 ± 1.67 3.54 ± 3.45 inactive 86.70 ± 0.98c

10.11 ± 1.67 8.18 ± 2.65 9.07 ± 3.43 inactive 98.61 ± 3.22b

18.83 ± 2.34 21.74 ± 2.78 9.07 ± 4.65 inactive 82.54 ± 1.75c

inactive 6.92 ± 1.12 21.48 ± 2.13 4.16 ± 3.21 78.92 ± 2.41b

at 100 μg/mL 16.84 ± 14.43 ± 21.48 ± inactive 87.91 ±

3.25 3.09 2.65 1.86c

Concentration of the compounds used: IC90 values from Table 2. bIC90, nM. c% inhibition at 100 μg/mL. 1839

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Compound 1: colorless crystals; mp 80.5 °C; [α]25D +14.0 (c 1.1, acetone); IR (CHCl3) νmax 3536, 1749 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 1; HREIMS [M]+ m/z 320.2365 (calcd for C20H32O3, 320.2351). Compound 2: colorless, viscous oil; [α]25D +4.8 (c 1.3, acetone); IR (CHCl3) νmax 3427 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13 C NMR (CDCl3, 100 MHz), see Table 1; HREIMS [M]+ m/z 322.2515 (calcd for C20H34O3, 322.2507). Compound 3: colorless, fine crystals; mp 69.5 °C; [α]25D −4.0 (c 1.0, acetone); IR (CHCl3) νmax 3418, 1644 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13C NMR (CDCl3, 100 MHz), see Table 1; HRESIMS [M + Na]+ m/z 331.2612 (calcd for C20H36O2Na, 331.2612). Compound 4: colorless gum, [α]25D +12.5 (c 0.8 acetone); IR (CHCl3) νmax 3372 cm−1; 1H NMR (CDCl3, 400 MHz), see Table 1; 13 C NMR (CDCl3, 100 MHz), see Table 1; HREIMS [M]+ m/z 324.2689 (calcd for C20H36O3, 324.2664). Antimycobacterial Activity. Compounds 1−5 were tested for their in vitro effects against M. tuberculosis H37Ra.35 M. tuberculosis H37Ra (ATCC 25177) cells were grown to logarithmic phase (OD 0.595−1.0) in a defined medium (M. pheli medium) under aerobic conditions in a shaker incubator (Thermo Electron Corporation, model 481) maintained at 150 rpm and 37 °C. After growth, the culture was sonicated for 2 min using a water bath sonicator. Sonicated cells were used for the inoculation in microplate wells. The culture (250 μL (∼105 cells/mL)) was added in the wells. Test samples dissolved in DMSO were added to the wells to achieve a concentration of 100 μg/mL for the preliminary screening. A dose−response curve of the active compounds was carried out by creating serial dilutions of the test samples, while isoniazid was used as a positive control. The plate was incubated in a CO2 incubator at 37 °C. The plate was taken out on the eighth day of incubation to measure the viable cell counts. The optical density of the culture was measured at 470 nm before the addition of XTT [2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide], which served as a blank. XTT (200 μM) was added, and the mixture was shaken for 1 min and incubated for 20 min at 37 °C. Subsequently, menadione (60 μM) was added, and the mixture was shaken for 1 min and incubated at 37 °C for 40 min. Finally, the optical density of the suspension was measured at 470 nm using a microplate reader. The IC50 and IC90 values were calculated (Table 2). Compounds 1, 2, 3, and 4 showed moderate inhibition against M. tuberculosis H37Ra, while compound 5 was inactive. The specificity of the active compounds was also evaluated on M. smegmatis mc2 155 using the IC90 values found on M. tuberculosis. Antimycobacterial Activity against M. smegmatis mc2 155. This was carried out on 1, 2, and 4. On the third day of incubation, the microplate was taken out to measure the viable cells. Optical density was measured at 470 nm before the addition of XTT. XTT (200 μM) was added and, after 1 min shaking, incubated for 20 min at 37 °C. Subsequently, menadione (60 μM) was added, and the contents were mixed for 1 min and incubated at 37 °C for another 20 min. The optical density was measured at 470 nm by using a microplate reader (Table 3). Inhibitory Activity against E. coli DH5α. Compounds 1, 2, and 4 were evaluated for the inhibition of E. coli DH5α cultures at IC90 concentrations obtained against M. tuberculosis. The effect on growth was calculated by measuring the absorbance of the culture at 620 nm after an incubation time of 6 h. No significant inhibition of growth of the organism was observed, confirming their specific action against M. tuberculosis (Table 3). Antiproliferative Activity (MTT Cell Proliferation Assay) (ref 36). Thp-1, HepG2, and MCF-7 cells lines were used for cytotoxicity testing. Cells (∼105 cells/mL) were allowed to adhere for 24 h at 37 °C, treated with 1, 2, and 4 at 100 μg/mL and at their respective IC90 values obtained against M. tuberculosis, and then incubated for 72 h for Thp-1, 120 h for HepG-2, and 192 h for MCF-7. Compound 3 was tested at 100 μg/mL against Thp-1. For the control, culture medium consisting of the corresponding concentration of DMSO was used. After incubation, 10 μL of 5 mg/mL of MTT was added to the cells, which were incubated for an additional 1 h at 37 °C. The formazan

crystals were solubilized in 200 μL of 2-propanol and incubated for another 4 h. The optical density was read on a microplate reader at 490 nm filter against a blank prepared from cell-free wells. The absorbance given by the cells treated with the carrier DMSO alone was taken as 100% cell growth. All the experiments were performed in triplicate, and the quantitative value was expressed as the average ± standard deviation (Table 4).

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ASSOCIATED CONTENT

S Supporting Information *

1

H and 13C NMR data of 1, 2, 3, and 4, HREIMS data of 1, 2, and 4, HRESIMS data of 3, and crystallographic data of 1. This material is available free of charge via the Internet at http:// pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*Tel: +91 20 25902327. Fax: + 91 20 25902627. E-mail: sp. [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We wish to thank the University Grants Commission, New Delhi, India, for financial support. For HRMS analysis, we wish to acknowledge Dr. S. Biswas and Mr. B. Senthilkumar, Division of Organic Chemistry, CSIR-National Chemical Laboratory, Pune, India.

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REFERENCES

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Journal of Natural Products

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