Accepted Manuscript Synthesis and anti-proliferative activities of new derivatives of embelin Baljinder Singh, Santosh K. Guru, Rashmi Sharma, Sonali S. Bharate, Inshad Ali Khan, Shashi Bhushan, Sandip B. Bharate, Ram A. Vishwakarma PII: DOI: Reference:

S0960-894X(14)00896-8 http://dx.doi.org/10.1016/j.bmcl.2014.08.052 BMCL 21950

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

29 May 2014 8 August 2014 22 August 2014

Please cite this article as: Singh, B., Guru, S.K., Sharma, R., Bharate, S.S., Khan, I.A., Bhushan, S., Bharate, S.B., Vishwakarma, R.A., Synthesis and anti-proliferative activities of new derivatives of embelin, Bioorganic & Medicinal Chemistry Letters (2014), doi: http://dx.doi.org/10.1016/j.bmcl.2014.08.052

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Synthesis and anti-proliferative activities of new derivatives of Leave this area blank for abstract info. embelin Baljinder Singh, Santosh K. Guru, Rashmi Sharma, Sonali S. Bharate, Inshad Ali Khan, Shashi Bhushan, Sandip B. Bharate,* Ram A. Vishwakarma* O HO

O

.HCl N

Antibacterial activity

OH O 8a.HCl S. aureus (MIC, µg/mL) = 8 Aq. solubility (µg/mL) = 800 10

HO 10

O Antiproliferative HO activity OH

O Embelin (1) HCT-116 (IC50, µM) = 65 S. aureus (MIC, µg/mL) = 8 Aq. solubility (µg/mL) = 1500

1

Bioorganic & Medicinal Chemistry Letters j o ur n al h om e p a g e : w w w . e l s e v i er . c o m

Synthesis and anti-proliferative activities of new derivatives of embelin Baljinder Singh,ab Santosh K. Guru,c Rashmi Sharma,bd Sonali S. Bharate,e Inshad Ali Khan,bd Shashi Bhushan,c Sandip B. Bharate,bf* Ram A. Vishwakarmaabf* a

Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India Academy of Scientific & Innovative Research (AcSIR), Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India c Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India d Clinical Microbiology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India e Preformulation Laboratory, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India f Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu-180001, India b

A R T IC LE IN F O

A B S TR A C T

Article history: Received Received in revised form Accepted Available online

Embelin (1), a benzoquinone isolated from Embelia ribes is known to possess variety of biological activities. Despite of several promising biological activities, preclinical efforts on embelin were hampered because of its poor aqueous solubility. In order to address the solubility issue, herein, we have synthesized a series of Mannich products of embelin by treating it with various secondary amines. The synthesized compounds were screened for antiproliferative and antimicrobial activities. In cytotoxicity screening, the benzyl-piperidine linked derivative 8m was found to possess better antiproliferative activity compared to parent natural product embelin against a panel of cell lines including HCT-116, MCF-7, MIAPaCa-2 and PC-3 with IC50 values of 30, 41, 34 and 36 µM, respectively. The mechanistic study of compound 8m revealed that it exhibits cytotoxicity via induction of apoptosis and mitochondrial membrane potential loss. Further, the compounds were tested for antimicrobial activity where dimethylamino- 8a and piperidine linked derivative 8b displayed antibacterial activity against Staphylococcus aureus with MIC values of 8 and 16 µg/mL, respectively. Mannich derivatives did now show improved aqueous solubility, however their hydrochloride salts 8a.HCl, 8b.HCl and 8m.HCl showed significantly improved aqueous solubility without affecting biological activities of parent Mannich derivatives.

Keywords: Embelin Mannich reaction Anticancer Antimicrobial Water soluble

2014 Elsevier Ltd. All rights reserved.

Embelin (1), natural hydroxy benzoquinone with alkyl substitution is one of the main constituent of plant Embelia ribes (family: Myrsinaceae). Embelin is reported to possess various biological activities including antimicrobial,1, 2 hepatoprotective,3 analgesic, anti-inflammatory,4 anticonvulsant,5 cytotoxicity,6 anxiolytic,7 antifertility,8 etc. Embelin is also known for anti-tumor activity via inhibition of X-linked inhibitor of apoptosis protein (XIAP).9, 10 Embelin also displayed activity against multiple sclerosis and other autoimmune inflammatory diseases via suppression of dendritic cell functions and limits autoimmune encephalomyelitis through the TGF-β/β-catenin and STAT3 signaling pathways.11 Embelin inhibits inducible and constitutive STAT3 activation through the induction of tyrosine phosphatase such as PTEN, which makes it potentially effective suppressor of tumor cell survival, proliferation, and angiogenesis.12 Embelin also inhibits the production of proinflammatory cytokines (TNF-α and IL-1β) in acute and chronic model of skin inflammation in mice.13 Ahn et al. proposed the mechanism of embelin in suppression of tumor cell survival, proliferation, invasion, angiogenesis, and inflammation via inhibition of NF-κB signaling.14 Toxicity studies revealed that embelin is safe to use and doses of 10 mg to 3 g/kg given orally to rats and mice did not show any toxic effects.15 -----*Corresponding author E-mail: [email protected] (RA Vishwakarma); [email protected] (SB Bharate) Fax: +91-191-2569333; Tel: +91-191-2569111 IIIM publication no.: IIIM/1668/2014

Several researchers have synthesized embelin derivatives and tested for different biological activities. Pena et al. synthesized antibacterial dihydropyran and dihydropyridine derivatives 2, 3 of embelin.16 Chen and co-workers have reported embelin derivative 4 with XIAP inhibiting activity.17 Viault et al. have designed and synthesized chemical library based upon embelin scaffold.18 Novel pyrano-embelin derivatives were synthesized through domino Knoevenagel hetero Diels–Alder reactions of embelin with paraformaldehyde and electron-rich alkenes.19 Mahendran et al. have synthesized condensation products (5) of embelin with various substituted aromatic primary amines having antioxidant activity.4 Sushama et al. have synthesized azo derivatives (6) of embelin possessing antibacterial activity.20 Despite of having promising biological activities, embelin failed to advance to preclinical development stage due to its poor solubility.21 Recently, Lamblin et al. synthesized series of embelin derivatives (7) with improved solubility, however compounds were devoid of antiproliferative activity.22 The structures of representative embelin derivatives are summarized in Figure 1. In order to address the issue of poor solubility, we decided to introduce nitrogen containing heterocycles into the embelin scaffold;

2

Bioorganic & Medicinal Chemistry Letters

and to prepare salts. All the synthesized compounds 8a-m were tested for antiproliferative activity as well as antimicrobial activity. Hydrochloride salts of selected active compounds were screened for these biological activities. O O

O

HO F

O H3C(H2C)10 O

O

HO

3 MIC (µM) S. aureus = 3.9 MRSA = 3.8

O

HO H3C(H2C)10

N H

OH O 4 XIAP inhibition Ki (µM) = 0.18

O O

2 MIC (µM) S. aureus = 1.8 MRSA = 0.8

O

H3C(H2C)10 R ClH . H2N

OH

O

O

O 1

HO R H3C(H2C)10

OH

n

O

O n=1-3 7 Inactive against cancer

O HO N N N

5 Antioxidant (ABTS) IC50 = 0.18 µg/mL

(CH2)10 CH3

N

N H

O 6 S. aureus = 18 mm zone inhibition at 100 µg

The preparation of semi-synthetic derivatives of embelin was directed on the substitution at C-6 position with various N-linked functionalities. To introduce N-linked functionalities in order to reduce the lipophilicity of embelin, Mannich reaction was employed. Treatment of embelin (1) with formaldehyde and different amines in methanol produced corresponding derivatives 8a-m.23 O

O HO

10

O

8a

8b

N

N

8c

O 8d

O

N N

N N 8k

N

N

N 8f

8e

N

N

8h

8i

N

O 8g

OH O

8a-m

R= N

N

R

Methanol

1

HO

HO

HCHO, 2o amine OH

Scheme 2 Synthesis of hydrochloride salts of 8a, 8b and 8m

OH O

Figure 1. Chemical structures of embelin (1) and its selected derivatives 2-7.

10

Hydrochloride salts of selected compounds were prepared by passing HCl gas to the solution of compounds viz. 8a, 8b and 8m (Scheme 2). The 1H NMR showed slight shift in the chemical shift values due to the formation of quaternary nitrogen salt. In compound 8a, two methyl groups appeared at δ 2.89 ppm and two CH2 protons appeared at δ 4.0 ppm whereas in 8a.HCl it shifted to δ 3.19 and 4.28 ppm respectively. The spot of hydrochloride salt remained at the bottom of TLC whereas parent compound showed Rf value of 3 to 4 in mobile phase CHCl3: MeOH (9.5:0.5).

HO

O HO H N

bonding.4 The synthesis of embelin derivatives 8a-m is depicted in Scheme 1.

8j N

N

8l

8m

Scheme 1 Synthetic scheme for the synthesis of embelin derivatives 8a-m

The formation of Mannich adduct was confirmed by the appearance of singlet at δ 4.0 ppm and absence of aromatic proton (C6-H) at δ 6.0 ppm. Further, the confirmation was done by 2D-NMR analysis of compound 8a. The 13CNMR spectrum of embelin and its derivatives did not show all the ring carbon signals, but showed only two peaks at 117.41 (C-3) and 103.87 (C-6).24 According to the reported literature, the 13C-NMR of 2,5-dihydroxy-3-alkyl-1,4-benzoquinones do not show ring carbon peaks particularly those attached to oxygen atoms due to fluxional effect caused by intramolecular hydrogen

The in vitro antiproliferative activity of synthesized compounds 8a-m along with embelin (1) was determined by MTT assay in four different cell lines including HCT-116 (colon), MCF-7 (breast), MIAPaCa-2 (pancreatic), and PC-3 (prostate). The results are summarized in Table 1. Compound 8m showed better activity in all the tested cell lines with IC50 values of 30, 41, 34 and 36 µM against HCT-116, MCF-7, MIAPaCa-2 and PC-3 cells respectively. The concentration and time-dependent inhibition of HCT-116 cell proliferation was studied for compound 8m. It showed varying cytotoxicity potential (IC 50) at different time points as depicted in Figure 2. Compounds 8e and 8j have shown inhibition against HCT116 cells with IC50 values of 22 and 40 µM respectively. The parent compound embelin (1) exhibits IC 50 values of 65, 28, 56 and 60 µ M against HCT-116, MCF-7, MIAPaCa-2 and PC-3 cells respectively. The reported IC 50 value for embelin (1) in PC-3 cell line is 3.7 µM10 whereas in our experiment we have obtained this value as 60 µM. The difference between two values may be due to the fact that reported IC50 was obtained when PC-3 cells were exposed to embelin (1) for 5 days,10 whereas we have performed the assay for 48 hrs. Table 1 Antiproliferative activity of embelin (1)and its derivatives 8a-m Entry Antiproliferative activity IC50 in µM

HCT-116 MCF-7 MIAPaCa-2 1 65 28 56 8a >100 82 >100 8b 86 94 74 8c 82 >100 92 8d >100 >100 >100 8e 22 70 62 8f 92 100 84 8g 78 >100 86 8h 100 >100 >100 8i >100 66 >100 8j 40 >100 50 8k >100 >100 >100 8l 96 >100 100 8m 30 41 34 8m salt 29 nd nd a HCT-116, human colon carcinoma cell line; MCF-7, human breast cell line; MIAPaCa-2, human pancreatic cancer cell line; PC-3, prostate cancer cell line.

PC-3 60 90 78 94 100 68 100 82 86 100 58 100 >100 36 nd cancer human

Apoptosis is often termed as programmed cell death, a process that generally occurs in multicellular organisms. It leads to morphological changes in cell including blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal

3 DNA fragmentation and finally causing cell death. It has been observed that many cytotoxic drugs show anticancer activity by inducing apoptosis.25 Thus, in order to address the cell death caused by compounds 8m, the extent of apoptotic death in HCT-116 cell lines was assessed using flow cytometry through determination of sub-G1 cell population by propidium iodide (PI) staining. Due to DNA degradation by the apoptosis-associated endonucleases, the DNA content measurement by staining with DNA specific fluorochrome, propidium iodide discriminates apoptotic cells.26 As depicted in Figure 3, the percentage of apoptotic cells exposed to compound 8m increased in a concentration-dependent manner after 24 h of incubation. The sub-G1 (G0) apoptotic population was found to be 3, 34 and 68% following 20, 30 and 40 µM of 8m treatment compared to control (untreated cells - 2%). As depicted in Figure 3, the compound 8m also affected the G2 phase of the cell cycle in HCT-116 cells. 700 600

IC50 , 6 h= 630 µM IC50 , 12h = 500 µM IC50 , 24h = 42 µM IC50 , 48h = 30 µM

IC 50 , µM

500 400

channel. Compound 8m induced loss of MMP in HCT-116 cells in a dose-dependent manner (Figure 5). The untreated control cells showed 2% MMP loss, whereas the compound 8m-treated HCT-116 cells showed 11%, 53% and 72% MMP loss at 20, 30 and 40 µ M concentrations, respectively (Figure 5). Since, the synthesized compounds showed cytotoxicity against selected panel of cell lines, we decided to screen these compounds for their effect on the growth of different bacterial and fungal strains. Thus, compounds 8a-m along with embelin (1) were evaluated for antibacterial and antifungal activities against Staphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922, Candida albicans ATCC 90028 and Aspergillus fumigatus MTCC 1811 (Table 2). S. aureus is known to be associated with several disease conditions including cellulitis, keratitis, osteomyelitis, septic arthritis and mastitis. It belongs to the major class of human pathogens and is the most common cause of increasing hospital death rate by 35%.29 The compounds 8a, 8b, 8c, 8d, 8h, 8j and 8l were found to be potent against S. aureus with MIC values in the range of 8-32 µg/mL. The compound 8a and 8b were most potent having MIC values of 8 and 16 µg/mL. The tested compounds did not show any effect on the growth of Gram-negative bacteria and different fungal strains.

300

Table 2 Antimicrobial activity of embelin (1), its derivatives 8a-m Antimicrobial activity Entry MIC in µg/mL

200 100 0 0

5

10

15

20

25

30

35

40

45

50

Time, h Figure 2. Cytotoxicity of compound 8m in HCT-116 cells at different time points. The cells were grown in 96-well culture plate and treated with different concentrations of compound 8m for indicated time intervals. Cells were incubated with MTT solution and optical density of formazon crystals was measured as described in experimental section. Data are Mean ± SD (n= 8 wells), and representative of three similar experiments.

To confirm the apoptosis induction results obtained from cell cycle analysis, nuclear morphological changes of cells by fluorescence microscopy was studied. After the treatment at 20, 30, and 40 µM of compound 8m, characteristic changes of apoptosis such as nuclear condensation, membrane blebbing and formation of apoptotic bodies were observed in the morphology of treated cells in a concentration-dependent manner, whereas the nuclei of untreated cells was found to be of normal intact morphology. The results suggested that compound 8m was able to induce apoptotic cell morphology in HCT-116 cells (Figure 4). The mitochondrial membrane potential (MMP) provides much of the cells energy needs and as such is very tightly regulated. Mitochondrial permeability transition, during which the electrochemical gradient across the mitochondrial membrane is lost, is a key step in the induction of apoptosis. Loss of MMP leads to depolarization of mitochondrial membrane resulting in mitochondrial dysfunction and ultimately a cell death.27, 28 The loss of MMP also results in release of several pro-apoptotic factors that activate caspases, which induces apoptosis. Thus, the effect of compound 8m on MMP loss in HCT-116 cells was investigated. The MMP loss in treated and untreated cells is measured by rhodamine-123 dye (Rh123) which is reduced by healthy mitochondria into fluorescent probe whose fluorescence is measured by flow cytometer in FL-1

1 8a 8b 8c 8d 8e 8f 8g 8h 8i 8j 8k 8l 8m 8a salt 8b salt Ciprofloxacin Amphotericin B

S. aureus ATCC 29213 8 8 16 32 32 >256 >256 128 32 256 32 >256 32 128 8 16 0.125 –

E.coli ATCC 25922 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 nd nd 0.007 –

C. albicans ATCC 90028 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 nd nd – 0.5

A. fumigates MTCC 1811

>256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 >256 nd nd – 0.5

The thermodynamic equilibrium aqueous solubility of embelin (1) along with selected derivatives and their salts were determined using 96-well plate based assay and results are shown in Table 3. Embelin (1) as well as its Mannich products were less water-soluble possessing solubility of

Synthesis and anti-proliferative activities of new derivatives of embelin.

Embelin (1), a benzoquinone isolated from Embelia ribes is known to possess variety of biological activities. Despite of several promising biological ...
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