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Antifungal dimeric chalcone derivativekamalachalcone E from MallotusphilippinensisRoshan R. Kulkarnia, Santosh G. Tupeb, Suwarna P. Gamplec,Macchindra G. Chandgudea, Dhiman Sarkarc, Mukund V.Deshpandeb & Swati P. Joshiaa Division of Organic Chemistry, CSIR-National ChemicalLaboratory, Pune, 411008, Indiab Biochemical Sciences Division, CSIR-National ChemicalLaboratory, Pune, 411008, Indiac Combi Chem-Bio Resource Centre, CSIR-National ChemicalLaboratory, Pune, 411008, IndiaPublished online: 07 Oct 2013.

To cite this article: Roshan R. Kulkarni, Santosh G. Tupe, Suwarna P. Gample, Macchindra G.Chandgude, Dhiman Sarkar, Mukund V. Deshpande & Swati P. Joshi , Natural Product Research(2013): Antifungal dimeric chalcone derivative kamalachalcone E from Mallotus philippinensis,Natural Product Research: Formerly Natural Product Letters, DOI: 10.1080/14786419.2013.843178

To link to this article: http://dx.doi.org/10.1080/14786419.2013.843178

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Natural Product Research, 2013 http://dx.doi.org/10.1080/14786419.2013.843178

Antifungal dimeric chalcone derivative kamalachalcone E from Mallotus philippinensis

Roshan R. Kulkarnia, Santosh G. Tupeb, Suwarna P. Gamplec, Macchindra G. Chandgudea,

Dhiman Sarkarc, Mukund V. Deshpandeb and Swati P. Joshia*

aDivision of Organic Chemistry, CSIR-National Chemical Laboratory, Pune 411008, India; bBiochemical Sciences Division, CSIR-National Chemical Laboratory, Pune 411008, India; cCombi Chem-Bio Resource Centre, CSIR-National Chemical Laboratory, Pune 411008, India

(Received 3 April 2013; final version received 7 August 2013)

From the red coloured extract (Kamala) prepared through acetone extraction of the fresh whole uncrushed fruits of Mallotus philippinensis, one new dimeric chalcone (1) along with three known compounds 1-(5,7-dihydroxy-2,2,6-trimethyl-2H-1-benzo­pyran-8-yl)-3-phenyl-2-propen-1-one (2), rottlerin (3) and 40-hydroxyrottlerin (4) were isolated. The structure of compound 1 was elucidated by 1D and 2D NMR analyses that included HSQC, HMBC, COSY and ROESY experiments along with the literature comparison. Compounds 1– 4 were evaluated for antifungal activity against different human pathogenic yeasts and filamentous fungi. The antiproliferative activity of the compounds was evaluated against Thp-1 cell lines. Compounds 1 and 2 both exhibited IC50 of 8, 4 and 16 mg/mL against Cryptococcus neoformans PRL518, C. neoformans ATCC32045 and Aspergillus fumigatus, respectively. Compound 4, at 100 mg/mL, showed 54% growth inhibition of Thp-1 cell lines.

Keywords: Mallotus philippinensis; dimeric chalcone; kamalachalcone E; rottlerin; 40-hydroxyrottlerin; antifungal

1. Introduction

Mallotus (Family: Euphorbiaceae) is a large genus of trees and shrubs distributed chiefly in the

tropical and subtropical regions of the Old World with around 20 species in India (Widen and

Puri 1980). The genus is represented in Maharashtra by five species of which Mallotus philippinensis, or Kamala tree, is a branched tree distributed throughout the state (Singh et al.

2001). Red dye, Kamala, secreted on the surface of the fruits, is used medicinally and also as a

dye. It has purgative properties and is also used in external applications for parasitic infections of

the skin and also as a lithontriptic and styptic (Wealth of India 1998). Earlier work on the

chemical components of different parts of M. philippinensis has revealed the presence of several triterpenes (Bandopadhyay et al. 1972; Nair & Madhusudana Rao 1993), flavonoids (Crombie

et al. 1968; Lounasmaa et al. 1975; Widen & Puri 1980; Ahluwalia et al. 1988; Nguyen et al.

2010a), lignans (Nguyen et al. 2010b), chalcones and dimeric chalcone derivatives (Tanaka et al.

1998; Furasawa et al. 2005).

In our programme on the isolation of bioactive molecules from higher plants, Kamala,

obtained as acetone washings from the fresh whole uncrushed fruits, was examined. This led to

the isolation of one new dimeric chalcone (1) along with three other known compounds 1-(5,7­

dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one (2), rottlerin (3)

*Corresponding author. Email: sp.joshi@ncl.res.in

q 2013 Taylor & Francis

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Figure 1. The structures of compounds isolated from M. philippinensis.

and 40-hydroxyrottlerin (or 17-hydroxyrottlerin 4) (Figure 1). The structure of 1 was elucidated by 1D and 2D NMR analyses that included heteronuclear single quantum coherence (HSQC),

heteronuclear multiple bond connectivity (HMBC), COrrelation SpectroscopY (COSY) and

Rotating frame Overhauser Effect SpectroscopY (ROESY) experiments and comparison of the

spectroscopic data with previous reports.

We report here the isolation and identification of compounds 1– 4 along with the biological evaluation for antifungal activity and antiproliferative activity against leukaemia cell line.

2. Results and discussion

Compound 1 was isolated as an orange amorphous powder. The molecular formula was

determined as C60H56O18 from HR-ESI-MS which showed a pseudomolecular peak at

1087.3338 [M þ Na]þ indicating 33 indices of hydrogen deficiency.

The 1H NMR spectra displayed eight methyl singlets at dH 1.29, 1.45, 1.50, 1.58, 1.77, 1.88, 2.20 and 2.41. The aromatic region displayed 2 four-proton broad doublets at dH 6.86 and 7.56.

These along with 2 one-proton doublet at dH 7.89 (J ¼ 15.4Hz) and 7.83 (J ¼ 15.4 Hz) and two-proton doublets at dH 7.72 (J ¼ 15.4 Hz) indicated two para hydroxy-substituted trans­cinnamoyl moieties. The 13C and DEPT spectra revealed 60 resonances consisting of 8 methyls,

3 methylenes, 15 methines and 34 quaternary carbons that included two cinnamoyl carbonyl

carbons at dC 191.0 and 192.9 along with two acetyl carbonyl carbons at dC 203.3 and 203.5. Methyls at dH 2.20 (dC 32.3) and dH 2.41 (dC 32.2) showed HMBC correlations with the

acetyl carbonyl carbons at dC 203.3 and 203.5, respectively, corresponding to two acetyl groups. Presence of 28 quaternary carbons and 12 methine carbons in the aromatic region

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3 Natural Product Research

indicated towards a total of six benzene rings including those of two cinnamoyl groups and two

olefinic groups indicating 1 being similar to the previously isolated kamalachalcones A, B

(Tanaka et al. 1998) and D (Furasawa et al. 2005), formed presumably by the dimmerisation

of 4-hydroxyrottlerin (2) and containing the unique A –B –C ring system. The presence of the

A–B –C ring system was confirmed by detailed analysis of the HMBC and COSY spectra as

follows. Methyls at dH 1.45 (dC 29.9, C-19) and 1.50 (dC 26.6, C-20) showed two-bond HMBC

correlations with the quaternary carbon at dC 78.9 (C-18) and three-bond correlations with the methylene at dC 40.5 (C-17). Similarly, methyls at dH 1.29 (dC 20.2, C-19

0) and 1.58 (dC 27.9, C­200) showed two-bond HMBC correlations with the quaternary carbon at dC 79.1 (C-18

0) and three-bond correlations with the methine at dC 45.6 (C-17

0). The proton at dH 2.05 (dC 40.5, C­17) showed two-bond correlations with the methine at dC 24.2 (C-16), while the methine proton

at dH 4.84 (H-160) displayed three-bond correlations with the quaternary carbon at dC 79.1 (C­

180). The COSY correlations observed between the protons at dH 4.84 and 2.23 confirmed the C­

160 –C-170 connectivity. These correlations confirmed the A –B –C ring system, while HMBC

correlation of the methine proton at dH 4.84 with the aromatic methine carbons at dC 155.4 (C-4) and 100.3 (C-30) confirmed its position at the site of dimerisation. These values were very similar

to those reported for kamalachalcones A, B (Tanaka et al. 1998) and D (Furasawa et al. 2005). In

kamalachalcones A and B (Tanaka et al. 1998), position C-60 is assigned for the quaternary carbons at dC 166.4 and 166.5, respectively. These values provided assignment of dC 164.3 to position C-60 in 1 allowing rest of the assignments (Figure S12). Strong four-bond HMBC

correlations observed between the methylene protons at dH 3.73 (H-210) and dC 106.6 allowed

assignment of this carbon to position 10 and by analogy, a quaternary carbon at dC 106.1 to position 1. Four-bond HMBC correlations of the methylene protons at dH 3.87 (H-21) with a quaternary carbon at dC 104.9 allowed its assignment to C-300. The methyl dH 2.20 showed three-bond HMBC correlations with the carbon at dC 104.9 (C-3

00) which was thus identified as H-800 . Other HMBC correlations are shown in Figure S12. The HSQC and HMBC correlations are

given in Table S1. Relative stereochemistry was analysed with the help of ROESY spectrum.

ROESY peaks were observed between the protons at dH 4.84 (H-160) and 2.58 (H-16). ROESY

correlation was also observed between the proton at dH 2.58 (H-16) and the methyl at dH 1.45 (H-19). These two sets of correlations confirmed their orientation on the same side. The ROESY

correlation of H-160 and the methyl at dH 1.29 confirmed their location at position 190. These correlations indicated trans-fused B and C rings with H-160 as b. The methine at dH 2.58 (H-16) showed ROESY correlations with the methyl groups at dH 1.29, 1.45 and 1.58. Rest of the ROESY correlations given in Figure S13 allowed assignment of the cinnamoyl groups and hence

their position. Thus, 1 was identified as a new natural product to which name kamalachalcone E

is assigned.

Compound 2 was identified as 1-(5,7-dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl)-3­

phenyl-2-propen-1-one (Lounasmaa et al. 1975). Compounds 3 and 4 were identified as rottlerin and 40-hydroxyrottlerin, respectively, by comparison of their NMR spectra (Furasawa et al.

2005) and mass spectroscopy.

All the compounds were evaluated for antifungal susceptibility testing against human

pathogens Candida albicans ATCC 10231, C. albicans ATCC 24433, Candida glabrata NCYC 388, Candida tropicalis ATCC 750, Cryptococcus neoformans ATCC 32045, C. neoformans PRL 518, C. neoformans ATCC 34664, Aspergillus niger ATCC 10578, A. fumigatus NCIM 902

and Aspergillus flavus NCIM 519. Compounds 1 and 2 exhibited IC50 of 8, 4 and 16 mg/mL

against C. neoformans PRL 518, C. neoformans ATCC 32045 and A. fumigatus NCIM 902,

respectively. Compounds 1, 3 and 4 were also tested for their antiproliferative activity against Thp-1 cell lines. Compound 4, at 100 mg/mL, exhibited 54% growth inhibition of Thp-1 cells.

Standard drug paclitaxel exhibited 83% inhibition at the same concentration.

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3. Experimental

3.1. General experimental procedures

The 1H and 13C NMR spectra were recorded on a Bruker Avance III Ultra Shield NMR

instrument (Bruker AG, Fallanden, Switzerland; 1H: 500 MHz, 200 MHz and 13C: 125 MHz,

50 MHz) at 258C. HMBC spectrum was recorded with twofold low-pass J-filter optimised from

135 to 160 Hz and long-range correlations optimised for 7 Hz. ESI-MS was recorded with a

Waters Acquity LC –MS instrument (Waters Corporation, Milford, MA, USA) and HR-ESI-MS

with a Thermo-scientific Q-Exactive spectrometer (Thermo Scientific, Orlando, FL, USA). All

the solvents used were distilled prior to use. Column chromatography (CC) was performed with

silica gel purchased from Thomas Baker (Chemicals) Pvt. Ltd. (Mumbai, India), and preparative

TLC was carried out using TLC plates supplied by Merck Ltd. (Mumbai, India).

3.2. Plant material

Fruits of M. philippinensis were collected from Bhimashankar Forest, Pune, in March 2012.

A herbarium was deposited in Botanical Survey of India, Pune (No. PCOPAVMA2).

3.3. Extraction and isolation

Whole uncrushed fruits (1.3 kg) were extracted with acetone (6 L £ 3 £ 14 h) at room

temperature. The acetone solubles were filtered and concentrated under reduced pressure to

provide reddish dye extract (54.6 g, 2.5% based on fresh fruit weight). The acetone extract,

53.0 g, was separated by CC using 15% acetone: petroleum ether followed by 1% methanol:

chloroform as the mobile phases to collect six fractions (MP1 –MP6). Fraction MP1 (1.9 g) was

subjected to CC in 15% acetone:petroleum ether followed by gradient of methanol in chloroform

from 0.1% to 2.0% to collect 13 fractions (MP1a – MP1m). Compound 2 (20 mg) was isolated

from fraction MP1c by preparative TLC in 10% acetone:petroleum ether. Compound 3 was themajor compound in fraction MP2. Fraction MP2 (3 g) was subjected to CC in 10% acetone:

petroleum ether followed by gradient of methanol in chloroform from 1% to 2% to collect seven

fractions (MP2a – MP2 g). Compound 3 (1.5 g) was isolated by repeated crystallisations inacetone. Fraction MP4 (5 g) was subjected to CC in methanol:chloroform gradient from 1% to

5% to collect 10 fractions (MP4a–MP4j). From fractions MP4 g and MP4 h, compound 1(30 mg) was obtained as an orange precipitate. Fractions MP4c and MP4d were subjected to

preparative TLC in 5% methanol in chloroform to isolate compound 4 (20 mg).

3.3.1. Kamalachalcone E (1)

Orange amorphous solid, UV (DMSO) lmax (log 1) 210 (4.57), 300 (4.65), 400 (4.58) nm; HR­

ESI-MS m/z: 1087.3338 (calculated for C60H59O18Na, 1087.3359); 1H NMR (500MHz, DMSO­

d6) d: 1.29 (3H, s, H-190), 1.45 (3H, s, H-19), 1.50 (3H, s, H-20), 1.58 (3H, s, H-200), 1.77 (3H, s,

H-900/H-900 0), 1.90 (1H, bs, H-17), 1.88 (3H, s, H-900/H-900 0), 2.05 (1H, d, 9.6 Hz, H-17), 2.20 (3H,s, H-800), 2.23 (1H, bs, H-170), 2.41 (3H, s, H-800 0), 2.58 (1H, d, J ¼ 10.3 Hz, H-16), 3.73 (2H, m,

H-210), 3.87 (2H, m, H-21), 4.84 (1H, s, H-160), 6.86 (4H, bt, J ¼ 8.1 Hz, H-12 and H-14 or H­120 and H-140), 7.56 (4H, bd, J ¼ 3.5 Hz, H-11 and H-15 or H-110 and H-150), 7.72 (2H, d,J ¼ 15.4 Hz, H-9 and H-90), 7.83 (1H, d, J ¼ 15.4Hz, H-8), 7.89 (1H, d, J ¼ 15.4 Hz, H-80). 13CNMR (125 MHz, DMSO-d6) d: 7.9 (C-9

00/C-900 0), 8.4 (C-900/C-900 0), 15.9 (C-21), 16.2 (C-210),20.2 (C-190), 24.2 (C-16), 26.6 (C-20), 27.9 (C-200), 29.9 (C-19), 32.2 (C-800 0), 32.3 (C800), 40.5(C-17), 45.6 (C-1700), 70.5 (C-160), 78.9 (C-18), 79.1 (C-180), 100.3 (C-30), 102.7 (C-500/C-500 0),103.1 (C-500/5000), 103.4 (C-3/C-100), 103.6 (C-3/C-100), 104.8 (C-1000), 104.9 (C-300), 105.7 (C-300 0),106.1 (C-1), 107.0 (C-10), 107.7 (C-50), 108.1 (C-5), 116.2 (C-12/C-120/C-14/C-140), 116.2

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(C-12/C-120/C-14/C-140), 116.3 (C-12/C-120/C-14/C-140), 116.3 (C-12/C-120/C-14/C-140), 123.1 (C-80), 123.2 (C-8), 126.0 (C-10), 126.1 (C-100), 130.6 (C-11/C-110/C-15/C-150), 130.6 (C-11/C­110/C-15/C-150), 130.7 (C-11/C-110/C-15/C-150), 130.7 (C-11/C-110/C-15/C-150), 143.4 (C-90), 144.1 (C-9), 155.1 (C-2), 155.1 (C-20), 155.4 (C-4), 157.2 (C-200), 158.4 (C-2000/C-600 0/C-40), 159.5(C-200 0/C-6000/C-40), 159.8 (C-400/C-400 0), 160.20 (C-6/C-600 0/C-13/C-130), 160.22 (C-6/C-600),160.22 (C-13/C-130), 160.3 (C-200 0/C-6000/C-40), 160.5 (C-13/C-130), 159.6 (C-400/400 0), 164.3(C-60), 191.0 (C-70), 192.9 (C-7), 203.3 (C-700) and 203.5 (C7000).

3.3.2. 1-(5,7-Dihydroxy-2,2,6-trimethyl-2H-1-benzopyran-8-yl)-3-phenyl-2-propen-1-one (2)

Orange amorphous solid; UV (acetone) lmax (log 1) 220 (5.63), 240 (6.54), 340 (6.58) nm. 1H

NMR (200 MHz, CDCl3) d: 1.56 (6H, s, H-21 and H-22), 2.10 (3H, s, H-20), 5.52 (1H, d,J ¼ 9.9 Hz, H-3), 6.62 (1H, d, J ¼ 9.9 Hz, H-4), 7.40 – 7.48 (3H, m, H-15, H-17 and H-19),

7.60 – 7.64 (2H, m, H-16 and H-18), 7.78 (1H, d, J ¼ 15.5 Hz, H-12), 8.15 (1H, d, J ¼ 15.5 Hz,H-13), 14.40 (1H, s, OH). 13C NMR (50 MHz, CDCl3) d: 7.1 (C-20), 27.8 (C-21 and 22), 77.0(C-2), 101.8 (C-210), 102.4 (C-200), 106.3 (C-16), 116.6 (C-4), 125.2 (C-3), 127.7 (C-13), 128.2(C-15 and C-19), 128.9 (C-16 and C-18), 130.0 (C-17), 135.6 (C-14), 142.1 (C-13), 154.4 (C-9),

156.0 (C-5), 164.2 (C-7), 193.1 (C-11).

3.3.3. 4 0-Hydroxyrottlerin (4)

Dark red powder; 1H NMR (500MHz, acetone-d6) d: 1.48 (3H, s, H-21/H-22), 1.52 (3H, s, H-21/H-22), 1.92 (3H, s, H-90), 2.63 (3H, br. s., H-80), 3.59 (1H, d, J ¼ 15.0Hz, H-20), 3.71 (1H, d,J ¼ 15.0Hz, H-20), 5.29 (1H, d, J ¼ 9.8 Hz, H-3), 6.77 (d, J ¼ 9.8 Hz, H-4), 6.92 (2H, J ¼ 8.5Hz,H-16, H-18), 7.52 (2H, J ¼ 8.5 Hz, H-15, H-19), 7.66 (1H, d, J ¼ 15.6 Hz, H-13), 8.23 (1H, d,J ¼ 15.6Hz, H-12). 13CNMR (125MHz, acetone-d6) d: 8.4 (C-9

0), 19.1 (C-20), 28.1 (C-21/C-22),28.2 (C-21/C-22), 33.1 (C-80), 77.6 (C-2), 101.9 (C-30/C-50), 101.9 (C-8), 104.4 (C-30/C-50), 106.8(C-10), 108.9 (C-6/C-10), 110.4 (C-6/C-10), 116.9 (C-16, C-18), 120.8 (C-4), 122.2 (C-3), 125.3(C-12), 128.8 (C-14), 130.6 (C-15, C-19), 140.9 (C-13), 156.2 (C-5), 156.2 (C-9/C-200), 156.2 (C-9/C-200), 160.4 (C-17), 162.3 (C-40/60), 163.0 (C-7), 163.3(C-40/60), 187.9 (C-11), 204.2 (C-70).

3.4. Antifungal activity

Evaluation for the antifungal susceptibility was carried out using microbroth dilution method

according to the recommendations of the Clinical Laboratory Standards Institute (CLSI)

(NCCLS 1997, 1998) (Table 1).

Table 1. Antifungal activities of compounds 1 and 2.

Concentration in mg/mL causing 50% growth inhibition (IC50)

1 2

C. albicans ATCC 10231 C. albicans ATCC 24433 C. glabrata NCYC 388 C. tropicalis ATCC 750 C. neoformans PRL 518 C. neoformans ATCC 32045 C. neoformans ATCC 34664 A. niger ATCC 10578 A. fumigatus NCIM 902 A. flavus NCIM 519

.256

.256

.256

.256 8 4

.256 99 16

.256

.256

.256

.256

.256 8 4

.256 216 16

.256

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3.5. Antiproliferative activity on human Thp-1 cell line (3-(4,5-dimethylthiazol-2-yl-)-2,5­diphenyltetrazolium bromide cell proliferation assay)

The activity was carried out according to the published protocol (Sreekanth et al. 2009).

4. Conclusion

This work led to the identification of new dimeric chalcone kamalachalcone E (1) along with

three known chalcone derivatives (2– 4). Compounds 1 and 2 showed significant inhibition of human pathogenic fungi. The results support the traditional use of Kamala.

Supplementary material

Supplementary material relating to this article is available online, alongside Figures S1 –S13 and

Table S1.

Acknowledgements The authors thank the Council of Scientific and Industrial Research, India, for fellowship.

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