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In the present paper, a semisynthetic preparation of mallotus B is reported via base-mediated intramolecular rearrangement of rottlerin (1), which is ...
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Semisynthesis of Mallotus B from Rottlerin: Evaluation of Cytotoxicity and Apoptosis-Inducing Activity Shreyans K. Jain,†,‡ Anup S. Pathania,‡,§ Samdarshi Meena,†,‡ Rajni Sharma,†,‡ Ashok Sharma,† Baljinder Singh,†,‡ Bishan D. Gupta,† Shashi Bhushan,‡,§ Sandip B. Bharate,*,‡,⊥ and Ram A. Vishwakarma*,†,‡,⊥ †

Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India Academy and Scientific & Industrial Research (AcSIR), Anusandhan Bhawan, 2 Rafi Marg, New Delhi-110001, India § Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India ⊥ Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India ‡

S Supporting Information *

ABSTRACT: Mallotus B (2d) is a prenylated dimeric phloroglucinol compound isolated from Mallotus philippensis. There have been no reports on the synthesis or biological activity of this compound. In the present paper, a semisynthetic preparation of mallotus B is reported via basemediated intramolecular rearrangement of rottlerin (1), which is one of the major constituents of M. philippensis. The homodimer “rottlerone” was also formed as one of the products of this base-mediated intramolecular reaction. Rottlerin (1), along with rottlerone (2c) and mallotus B (2d), was evaluated for cytotoxicity against a panel of cancer cell lines including HEPG2, Colo205, MIAPaCa-2, PC-3, and HL-60 cells. Mallotus B (2d) displayed cytotoxicity for MIAPaCa-2 and HL-60 cells with IC50 values of 9 and 16 μM, respectively. Microscopic studies in HL-60 cells indicated that mallotus B (2d) induces cell cycle arrest at the G1 phase and causes defective cell division. It also induces apoptosis, as evidenced by distinct changes in cell morphology. delta (PKC-δ),16 although the inhibition of PKC-δ by rottlerin has been considered controversial.17 Literature evidence18−28 indicates that rottlerin (1) does not modulate a single welldefined molecular target of cancer but interacts with numerous biochemical pathways; its full mechanism of action is not yet understood.29 Mallotus B is a prenylated dimeric phloroglucinol isolated from M. philippensis,30,31 but no reports exist on its synthesis, semisynthesis, or pharmacological evaluation at present. Herein, we report on the chemical transformation of rottlerin (1) to mallotus B (2d) and the evaluation of the cytotoxicity and apoptosis-inducing activity of the latter compound. Rottlerin (1) is a dimeric phloroglucinol compound that comprises two substituted phloroglucinol units linked to each other via a methylene group. This kind of dimeric phloroglucinol compound can be obtained synthetically via coupling of two phloroglucinol partners in the presence of formaldehyde under acidic conditions.32,33 It has been reported that 1 is unstable under alkaline and acidic conditions.34−39 During our efforts on semisynthetic modifications of rottlerin,

Mallotus philippensis (Lam.) Muell. Arg. var. philippensis (Euphorbiaceae) is distributed widely in the rainforests of Southeast Asia and is commonly known as the Kamala tree. This species is a rich source of biologically active compounds and has also been used as a dye-yielding plant. It is a common plant used in the Indian system of medicine, and thus its medicinal properties have been well documented in Ayurveda.1 More than 170 chemical constituents have been isolated from the genus Mallotus, with the majority belonging to the phloroglucinol class of compounds.2 Furthermore, a diverse range of biological activities have been reported for this plant including antiallergic,3 anthelmintic,4 antiatherosclerotic,5 antibacterial,6 antifertility,7 antiherpes,8 antioxidant,9,10 antiplasmodial,6 antitubercular,11 aphrodisiac,7 and cytotoxic effects.12 Among various dimeric phloroglucinol compounds reported from M. philippensis, only rottlerin (1) (a major constituent) has been documented for its biological potential. Rottlerin was initially isolated as a Kamala dye in 1893.13,14 Later, it was discovered that rottlerin is an inhibitor of several kinases such as p38-regulated/activated protein kinase (PRAK), mitogenactivated protein kinase-activated protein kinase 2 (MAPKAPK-2), protein kinase B (PKB, Akt), Ca2+/calmodulindependent protein kinase (CaMK),15 and protein kinase C© XXXX American Chemical Society and American Society of Pharmacognosy

Received: June 1, 2013

A

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Figure 1. Intramolecular transformation of rottlerin (1) to rottlerone (2c), mallotus B (2d), and the benzylated products, 2a, and 2b.

Table 1. 1H and 13C NMR Data for Compounds 2a−2c in CDCl3 (δ in ppm, J in Hz in parentheses)a 2a position 2 2a, 2b 3 4 4a 5 5-OH 6 7 7-OH 8 8a 9 10 11 12 13, 17 14, 16 15 1′ 2′ 3′, 7′ 4′, 6′ 5′ 1″ 2″ 3″, 7″ 4″, 6″ 5″

δC 78.0, 28.0, 124.6, 116.9, 103.4, 160.3,

C CH3 CH CH C C

2b δH 1.56, s 5.46, d (12) 6.66, d (12)

δC 77.7, 27.9, 125.6, 117.7, 107.0, 159.6,

2c δH

C CH3 CH CH C C

1.56, s 5.50, d (12) 6.51, d (12)

δC 78.1, 28.0, 126.8, 117.2, 106.5, 159.2,

C CH3 CH CH C C

δH 1.56, s 5.49, d (12) 6.68, d (12)

3.49, s 93.7, CH 167.3, C

6.14, s

115.0, C 164.3, C

103.8, C 162.7, C

14.17, s 106.5, 155.8, 192.8, 142.3, 127.4, 135.6, 127.4, 128.6, 128.2, 70.3, 136.0, 128.2, 128.9, 130.1,

C C C CH CH C CH CH CH CH2 C CH CH CH

7.78, d (16) 8.13, d (16) 7.62−7.38, mb

5.11, s 7.62−7.38, mc

13.94, s 108.9, 154.8, 193.7, 142.5, 127.8, 135.5, 127.6, 128.4, 128.1, 76.3, 136.5, 128.1, 128.5, 130.2, 27.9, 141.2, 128.3, 128.9, 125.9,

C C C CH CH C CH CH CH CH2 C CH CH CH CH2 C CH CH CH

7.79, d (16) 8.09 d (16) 7.81−7.13 mb

105.0, 155.4, 192.8, 143.3, 126.8, 135.4, 129.0, 128.4, 126.2,

C C C CH CH C CH CH CH

7.84, d (16) 8.20, d (16)

4.77, s 7.81−7.13 mc

3.98, s

29.7, CH2

3.82, s

7.81−7.13 md

H NMR spectra were recorded at 500 MHz and the 13C NMR spectra at 125 MHz. bMultiplet for five aromatic protons of C-13 through C-17. Multiplet for five aromatic protons of C-3′ through C-7′. dMultiplet for five aromatic protons of C-3′ through C-7′.

a1 c

products, 2c and 2d are known in the literature as rottlerone40 and mallotus B,30,31 but 2a and 2b are new structures. Rottlerin (1) has been reported as a potent cytotoxic agent;21,24 however, the anticancer potential of rottlerone (2c) and mallotus B (2d)

when this compound (1) was reacted with benzyl bromide in the presence of potassium carbonate, the formation of four major products was observed, as depicted in Figure 1. All these products were isolated and characterized. Among these four B

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Figure 2. Major HMBC correlations (blue arrows) for compounds 2a−2c.

and 2b were identical except for the presence of another Cbenzyl functionality at C-6 in 2b. The structure of 2b was confirmed on the basis of HMBC correlations and was identified as 6-benzyl-7-hydroxy-5-benzyloxy-8-(phenylprop-2en-1-oyl)-2,2-dimethylchromene. Compounds 2c [(orange solid; Rf 0.40 in ethyl acetate− hexane (2:8)] and 2d [(colorless solid; Rf 0.25 in ethyl acetate− hexane (1:1)] were identified as rottlerone40 and mallotus B,30,31 respectively. The UV maxima (λmax 358 and 290 nm) and mp (220−225 °C) of 2c were identical to those reported for rottlerone. Furthermore, the 1H NMR spectrum of 2c clearly indicated the loss of the acetyl phloroglucinol portion of the rottlerin (1) structure. The HRESIMS of 2c showed a pseudomolecular ion peak at m/z 657.2486 corresponding to a molecular formula of C41H37O8. On the basis of 13C NMR, DEPT135, and HMBC analysis, compound 2c was identified as rottlerone, which is a symmetrical dimer of the chromene part of rottlerin (1). The major HMBC correlations of 2c are shown in Figure 2. In 1937, McGookin et al.40 obtained rottlerone (2c) by refluxing rottlerin (1) with aqueous saturated barium hydroxide solution.34,40 Although rottlerone (2c) is a known compound, its NMR data have not been reported previously. Product 2d was identified as mallotus B by comparison of its spectroscopic data with literature values.30,31 Compound 2d was formed via intramolecular rearrangements in the chromene portion of the rottlerin structure. The conversion of rottlerin (1) to mallotus B (2d) involves two key intramolecular reactions: (a) opening of the chromene ring to form a free ArOH at C-5 and a prenyl functionality at position C-6 and (b) cyclization of the phenylprop-2-en-1-oyl functionality with the Ar-OH at C-7, leading to the formation of a dihydro-γ-pyrone system. The assignments of the 1H and 13C NMR spectroscopic data for compounds 2a−2c are shown in Table 1. Since products 2c and 2d do not contain a benzyl functionality, the reaction of rottlerin (1) with potassium carbonate without the addition of benzyl bromide was carried out. In this reaction, as expected, only the formation of products 2c and 2d was observed. Although the primary objective of this work was not to develop a synthetic route for 2d, an attempt was made to improve the yield for compound 2d (which was 20 >20 >20 0.2

>20 >20 >20 0.05

8 >20 9 0.19

>20 >20 >20 1.2

9 15 16 0.06

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EXPERIMENTAL SECTION

General Experimental Procedures. All chemicals were obtained from Sigma-Aldrich Company and used as received. 1H, 13C, and DEPT NMR spectra were recorded on Bruker-Avance DPX FT-NMR 500 and 400 MHz instruments. Chemical data for protons are reported in parts per million (ppm) downfield from tetramethylsilane and are referenced to the residual proton in the NMR solvent (CDCl3, 7.26 ppm; CD3OD, 3.31 ppm). ESIMS were recorded on an Agilent 1100 LC-QTOF mass spectrometer. Plant Material. The authentic plant material, Mallotus philippensis, was collected by the Biodiversity and Applied Botany Division of Indian Institute of Integrative Medicine (CSIR), Jammu, in May 2007 from the Himalayan range of the Jammu region of India. The plant material was identified by Dr. S. N. Sharma. A specimen sample (accession number, 21722) was preserved in Janaki Ammal Herbarium at the IIIM (CSIR), Jammu, India. Extraction and Isolation. The dried and powdered (2 kg) plant material was extracted by cold percolation with ethanol to afford 248 g of extract (12.4%). This extract was suspended in water and sequentially fractionated with hexane (14.8 g), chloroform (20.4 g, 1.02% of plant material), and n-butanol (12.8 g). The chloroform fraction was purified using silica gel column chromatography eluted with increasing proportions of ethyl acetate in petroleum ether to produce 808 mg of rottlerin (1, 0.04% of plant material, 0.32% of crude extract). This compound was identified by comparison of its 1H NMR spectrum and melting point with literature values.31 Transformation of Rottlerin (1) to Mallotus B (2d) and Other Products. Rottlerin (1, 250 mg, 1 equiv) and potassium carbonate (200 mg, 3 equiv) were added to dry acetone. To this reaction mixture was added dropwise benzyl bromide (3.5 equiv) at 0 °C. The reaction mixture was stirred at 40 °C for 10 h and was then partitioned between water and ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, with the solvent evaporated in vacuo and the crude product purified using preparative TLC to furnish four products, 2a− 2d. The same reaction was then performed in the absence of benzyl bromide, where only two major products, 2c (65%) and 2d (5%), were formed. 7-Hydroxy-5-benzyloxy-8-(phenylprop-2-en-1-oyl)-2,2-dimethylchromene (2a): orange-red solid; isolated amount 22 mg; yield 11%; mp 138−140 °C; IR (CHCl3) νmax 3400, 2921, 1632, 1599, 1543, 1343, 1223, 1158, 1117 cm−1; for 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 413 [M + H]+; HRESIMS m/z 413.1754 [M + H]+ (calcd for C27H25O4+, 413.1747).

a

Cells were grown in 96-well culture plates and treated with various concentrations of each test compound for 48 h. Thereafter, cells were incubated with MTT solution for 2 h, and the optical density of formazan crystals was measured as described in the Experimental Section. HEPG2, hepatocellular carcinoma cells; Colo205, colon cancer cells; MIAPaCa-2, pancreatic cancer cells; PC-3, prostate cancer cells; HL-60, human promyelocytic leukemia cells. Compounds 2b and 2c were inactive against all cell lines used (IC50 > 20 μM).

Next, the effects of compounds 1, 2a, and 2d were investigated on cell cycle phase distribution in human leukemia HL-60 cells. Rottlerin (1) arrested the cells at the G1 phase at a low concentration (10 μM), which was preceded by DNA damage as analyzed by cell cycle phase distribution using flow cytometry (Figure 4). Untreated HL-60 cells showed a 45% G1 population, while rottlerin-treated HL-60 cells at 10 μM exhibited a 67% G1 population and a 20% sub-G0 DNA fraction, indicative of apoptosis population, as analyzed by Modfit software. The apoptosis induced by 1 was increased in a concentration-dependent manner, with 64%, 80%, and 84% of the cells showing apoptotic populations at 20, 30, and 40 μM, respectively (Figure 4). Compound 2a was found to be less effective at 10 and 20 μM, with 3% and 9% apoptotic cells, which increased to 39% and 83% at 30 and 40 μM, respectively. Mallotus B (2d) demonstrated robust G1 arrest at 10, 20, and 30 μM, where more than 75% cells showed G1 arrest in HL-60 cells at 10 μM as compared to 45% in the untreated control (Figure 4). The effect of compounds 1, 2a, and 2d on mitochondrial membrane potential loss in human leukemia HL-60 cells was also investigated. The mitochondrial membrane potential (MMP) loss in treated and untreated cells was measured using the rhodamine-123 dye (Rh-123), which is reduced by healthy mitochondria in a fluorescent probe, for which the fluorescence was measured using a flow cytometer in the FL-1 channel. The untreated control cells showed about 5% MMP loss, while HL-60 cells treated with 1 exhibited 61% and 89% MMP loss at 30 and 40 μM, respectively (Figure 5). D

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Figure 4. Cell cycle analysis of compounds 1, 2a, and 2b using HL-60 cells. HL-60 cells (0.44 × 106/mL/24 well plate) were treated with 1, 2a, and 2d for 24 h at 10, 20, 30, and 40 μM. Cells were stained with propidium iodide (PI, 10 μg/mL), to determine DNA fluorescence and cell cycle phase distribution, as described in the Experimental Section. Data were analyzed using Modfit software (Verity Software House Inc., Topsham, ME, USA) for the proportions of different cell cycle phases. The fraction of cells from apoptosis at the G1, S, and G2 phases analyzed from FL2-A vs cell counts are shown as percentages. Data are representative of three similar experiments.

Figure 5. Compounds 1, 2a, and 2d induced mitochondrial potential loss in HL-60 cells. Cells (0.44 × 106/mL/24-well plate) were treated with these compounds at 10, 20, 30, and 40 μM for 24 h. Cells were stained with rhodamine-123 (200 nM) for 30 min and analyzed in FL-1 vs count channels of a flow cytometer. Data are representative of three experiments at different time periods. Rottlerone (2c): orange-red solid; isolated amount 190 mg; yield 59%; mp 220−225 °C; IR (CHCl3) νmax 3400, 2922, 1595, 1344, 1287, 1124 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 657 [M + H]+; HRESIMS m/z 657.2486 [M + H]+ (calcd for C41H37O8+, 657.2483). Mallotus B (2d): colorless solid; isolated amount 6 mg; yield 2.4%; mp 218−220 °C (lit. 230 °C);30,31 IR (CHCl3) νmax 3369, 2922, 1627,

6-Benzyl-7-hydroxy-5-benzyloxy-8-(phenylprop-2-en-1-oyl)-2,2dimethylchromene (2b): orange-red solid; isolated amount 25 mg; yield 10.2%; mp 80−82 °C; IR (CHCl3) νmax 3028, 2923, 1632, 1588, 1336, 1285, 1146 cm−1; 1H NMR and 13C NMR data, see Table 1; ESIMS m/z 503 [M + H]+; HRESIMS m/z 503.2233 [M + H]+ (calcd for C34H31O4+, 503.2217). E

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Figure 6. Effect of compounds 1, 2a, and 2d on cellular and nuclear morphology of HL-60 cells. Cells were treated with the indicated concentrations of compounds 1, 2a, and 2d for 24 h and visualized for cellular morphology.

Figure 7. Effect of compounds 1, 2a, and 2d on cellular and nuclear morphology of HL-60 cells. Cells were treated with the indicated concentrations of compounds 1, 2a, and 2d for 24 h. Cells were stained simultaneously with the DNA-binding Hoechst 33258 dye as described in the Experimental Section. Nuclear morphology and the formation of apoptotic bodies were visualized on a fluorescent microscope. Condensed nuclei and the apoptotic bodies are indicated by white arrows, while the nuclei of untreated control were round in shape. Data are representative of three experiments, and the magnification of the pictures is 30× on an Olympus 1X 70 inverted microscope. 1447, 1319, 1110 cm−1; the 1H NMR and 13C NMR data matched reported values;30,31 ESIMS m/z 519 [M + H]+; HRESIMS m/z 519.2014 [M + H]+ (calcd for C30H31O8+, 519.2013). Cell Culture, Growth Conditions, and Treatment. HL-60 human promyelocytic leukemia, PC-3 prostate cancer, HEPG2 hepatocellular carcinoma, MIAPaCa-2 pancreatic cancer, and Colo205 colon cancer cells were obtained from the National Cancer Institute (NCI), Bethesda, MD, USA. The cells were grown in RPMI1640 or MEM medium supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), L-glutamine (0.3 mg/mL), pyruvic acid (0.11 mg/mL), and 0.37% NaHCO3. Cells were grown in a CO2 incubator (Thermocon Electron Corporation, MA, USA) at 37 °C in an atmosphere of 95% air and 5% CO2 with 98% humidity. Camptothecin was used as a positive control in this study. Cell Proliferation Assay. Cells were plated in 96-well plates at a density of 6000 cells/well in 100 μL of medium per well overnight. The next day, cultures were incubated with 1, 10, 30, 50, and 100 μM concentrations of test compound and incubated for 48 h. MTT [3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] dye, at a concentration of 2.5 mg/mL, was added for 3 h at 37 °C. The supernatant was aspirated, and MTT-formazan crystals were dissolved in 150 μL of DMSO; the OD was measured at λ540 (reference wavelength, λ620) on an ELISA reader (BioTek Instruments Inc., VT, USA). Cell growth was calculated by comparing the absorbance of treated versus untreated cells.41 Cell Cycle Analysis. Cell cycle phase distribution progress for HL60 cells treated with 1, 2a, and 2d was investigated by propidium

iodide (PI) staining using a flow cytometer (FACSCalibur; BectonDickinson, CA, USA). Cells were incubated with compounds at 10, 20, 30, and 40 μM for 24 h and were collected at 400g, washed with icecold PBS, and fixed with ice-cold 70% ethanol at 4 °C overnight. On the next day, the cells were centrifuged again, washed with PBS, and incubated with RNase (0.2 mg/mL) at 37 °C for 1 h followed by staining with PI (10 μg/mL) for 30 min in the dark. Cells were analyzed using flow cytometry in the list mode on 10 000 events for FL2-A vs FL2-W. The sub-G0 (apoptosis), G1, G2, and S phase cell fractions were analyzed using Modfit software.42 Effect of Compounds 1, 2a, and 2d on Mitochondrial Membrane Potential. Changes in mitochondrial transmembrane potential (Ψmt) as a result of mitochondrial perturbation were measured after staining with rhodamine-123. HL-60 cells were seeded in 12-well plates and incubated with compounds 1, 2a, and 2d at the indicated concentrations for 24 h. Rhodamine-123 (200 nM) was added 30 min before the termination of the experiments. Cells were collected at 400g and washed once with PBS, and mitochondrial membrane potentials were measured in FL-1 channel vs counts using a Becton-Dickinson FACS Calibur flow cytometer.43 Hoechst Staining. Human leukemia HL-60 cells were incubated with compounds 1, 2a, and 2d for 24 h at 10, 20, 30, and 40 μM. Cells were collected at 400g, washed with PBS twice, and fixed in 1 mL of fixing solution containing cold acetic acid−methanol (1:3) for 3 h on ice. Cells were collected at 400g and suspended in 50 μL of fixing solution. Cells were spread on a glass slide, dried overnight at room temperature, and then stained with Hoechst 33258 (5 μg/mL in 0.45 M disodium phosphate and 0.01 M citric acid containing 0.05% Tween F

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20) for 30 min at room temperature. Slides were washed with PBS, and 40 μL of mounting fluid containing PBS−glycerol (1:1) was poured over the slide, which was covered with a glass coverslip and sealed with nail polish. Cells were observed under a microscope at 30× (Olympus IX 70) for any nuclear morphological changes. For phasecontrast microscopy, cells were photographed using the microscope after treatment.

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ASSOCIATED CONTENT

* Supporting Information S

Spectroscopic data scans of compounds 1 and 2a−2d. This material is available free of charge via the Internet at http:// pubs.acs.org

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AUTHOR INFORMATION

Corresponding Authors

*(S.B.B.) Tel: +91-191-2569000 (ext. 345). Fax: +91-1912569333. E-mail: [email protected]. *(R.A.V.). Tel: +91-191-2569111. Fax: +91-191-2569333. Email: [email protected]. Notes

The authors declare no competing financial interest. This is IIIM communication number IIIM/1589/2013.

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ACKNOWLEDGMENTS The authors gratefully acknowledge D. Singh and S. Aravinda for analytical support. S.K.J. and A.S.P. are Senior Research Fellows receiving financial support from CSIR, New Delhi. S.M. is a Junior Research Fellow receiving financial support from UGC, New Delhi, India. The authors would like to express special thanks to Dr. S. N. Sharma, Biodiversity and Applied Botany Division, IIIM Jammu, India, for providing authentic plant material. This research was supported in part by a grant from the CSIR 12th FYP project (BSC-0108).

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