Bioorganic & Medicinal Chemistry Letters 24 (2014) 4865–4870

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Bioorganic & Medicinal Chemistry Letters

journal homepage: www.elsevier .com/ locate/bmcl

Synthesis and anti-proliferative activities of new derivativesof embelin q

http://dx.doi.org/10.1016/j.bmcl.2014.08.0520960-894X/� 2014 Elsevier Ltd. All rights reserved.

q IIIM publication no.: IIIM/1668/2014.⇑ Corresponding authors. Tel.: +91 191 2569111; fax: +91 191 2569333.

E-mail addresses: sbharate@iiim.ac.in (S.B. Bharate), ram@iiim.ac.in(R.A. Vishwakarma).

Baljinder Singh a,b, Santosh K. Guru c, Rashmi Sharma b,d, Sonali S. Bharate e, Inshad Ali Khan b,d,Shashi Bhushan c, Sandip B. Bharate b,f,⇑, Ram A. Vishwakarma a,b,f,⇑a Natural Products Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, Indiab Academy of Scientific & Innovative Research (AcSIR), Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, Indiac Cancer Pharmacology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, Indiad Clinical Microbiology Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, Indiae Preformulation Laboratory, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, Indiaf Medicinal Chemistry Division, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu 180001, India

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

Article history:Received 29 May 2014Revised 8 August 2014Accepted 22 August 2014Available online 1 September 2014

Keywords:EmbelinMannich reactionAnticancerAntimicrobialWater soluble

Embelin (1), a benzoquinone isolated from Embelia ribes is known to possess variety of biological activ-ities. Despite of several promising biological activities, preclinical efforts on embelin were hamperedbecause of its poor aqueous solubility. In order to address the solubility issue, herein, we have synthe-sized a series of Mannich products of embelin by treating it with various secondary amines. The synthe-sized compounds were screened for antiproliferative and antimicrobial activities. In cytotoxicityscreening, the benzyl-piperidine linked derivative 8m was found to possess better antiproliferative activ-ity 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 lM, respectively. The mechanistic study ofcompound 8m revealed that it exhibits cytotoxicity via induction of apoptosis and mitochondrial mem-brane potential loss. Further, the compounds were tested for antimicrobial activity where dimethyl-amino- 8a and piperidine linked derivative 8b displayed antibacterial activity against Staphylococcusaureus with MIC values of 8 and 16 lg/mL, respectively. Mannich derivatives did now show improvedaqueous solubility, however their hydrochloride salts 8a�HCl, 8b�HCl and 8m�HCl showed significantlyimproved aqueous solubility without affecting biological activities of parent Mannich derivatives.

� 2014 Elsevier Ltd. All rights reserved.

Embelin (1), natural hydroxy benzoquinone with alkyl substitu-tion is one of the main constituent of plant Embelia ribes (family:Myrsinaceae). Embelin is reported to possess various biologicalactivities 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 activityvia inhibition of X-linked inhibitor of apoptosis protein (XIAP).9,10

Embelin also displayed activity against multiple sclerosis and otherautoimmune inflammatory diseases via suppression of dendriticcell functions and limits autoimmune encephalomyelitis throughthe TGF-b/b-catenin and STAT3 signaling pathways.11 Embelininhibits inducible and constitutive STAT3 activation through theinduction of tyrosine phosphatase such as PTEN, which makes itpotentially effective suppressor of tumor cell survival, proliferation,

and angiogenesis.12 Embelin also inhibits the production ofpro-inflammatory cytokines (TNF-a and IL-1b) in acute andchronic model of skin inflammation in mice.13 Ahn et al. proposedthe mechanism of embelin in suppression of tumor cell survival,proliferation, invasion, angiogenesis, and inflammation via inhibi-tion of NF-jB signaling.14 Toxicity studies revealed that embelinis safe to use and doses of 10 mg to 3 g/kg given orally to ratsand mice did not show any toxic effects.15

Several researchers have synthesized embelin derivatives andtested for different biological activities. Pena et al. synthesizedantibacterial dihydropyran and dihydropyridine derivatives 2, 3of embelin.16 Chen et al. have reported embelin derivative 4 withXIAP inhibiting activity.17 Viault et al. have designed and synthe-sized chemical library based upon embelin scaffold.18 Novel pyr-ano-embelin derivatives were synthesized through dominoKnoevenagel hetero Diels–Alder reactions of embelin with parafor-maldehyde and electron-rich alkenes.19 Mahendran et al. havesynthesized condensation products (5) of embelin with various

O

OH3C(H2C)10 OH

HO

O

OH3C(H2C)10 N

H

HOO

O

OH3C(H2C)10 O

HOO

O

OOH

HOHN

OClH . H2N

R

n

n=1-3

O

O(CH2)10CH3HO

OHNN

2

1

3

6

7

O

OH3C(H2C)10 N

H

HOR

5

F

MIC (µM)S. aureus = 1.8MRSA = 0.8

O

MIC (µM)S. aureus = 3.9MRSA = 3.8

Antioxidant (ABTS) IC50 = 0.18 µg/mL

O

OOH

HO

4XIAP inhibitionKi (µM) = 0.18

NN O

S. aureus = 18 mm zone inhibition at 100 µg

Inactive against cancer

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

4866 B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4865–4870

substituted aromatic primary amines having antioxidant activity.4

Sushama and Alaudeen have synthesized azo derivatives (6) ofembelin possessing antibacterial activity.20 Despite of havingpromising biological activities, embelin failed to advance to pre-clinical development stage due to its poor solubility.21 Recently,Lamblin et al. synthesized series of embelin derivatives (7) withimproved solubility, however compounds were devoid of antipro-liferative activity.22 The structures of representative embelinderivatives are summarized in Figure 1.

O

OOH

HO

N

N

N N

O

N

NN

N

N

N

NN

O

O

NN

HO

NN

HCHO, 2o amineMethanol

R =1

8a 8b 8c 8d 8e 8f

8g 8h 8i 8j

8k 8l 8m

10

O

OOH

HO

8a-m

10

R

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

O

OOH

HO

n

n=10

O

OOH

HO

n

n=10

N

8a, 8b or 8m

CHCl3:MeOHHCl gas

8a, 8b or 8m hydrochloride salt

R1

R2.HClN

R1

R2

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

In order to address the issue of poor solubility, we decided tointroduce nitrogen containing heterocycles into the embelin scaf-fold; and to prepare salts. All the synthesized compounds 8a–mwere tested for antiproliferative activity as well as antimicrobialactivity. Hydrochloride salts of selected active compounds werescreened for these biological activities.

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

The formation of Mannich adduct was confirmed by the appear-ance of singlet at d 4.0 ppm and absence of aromatic proton (C6-H)at d 6.0 ppm. Further, the confirmation was done by 2D NMR anal-ysis of compound 8a. The 13C NMR spectrum of embelin and itsderivatives did not show all the ring carbon signals, but showedonly two peaks at 117.41 (C-3) and 103.87 (C-6).24 According to

Table 1Antiproliferative activity of embelin (1) and its derivatives 8a–m

Entry Antiproliferative activitya (IC50 in lM)

HCT-116 MCF-7 MIAPaCa-2 PC-3

1 65 28 56 608a >100 82 >100 908b 86 94 74 788c 82 >100 92 948d >100 >100 >100 1008e 22 70 62 688f 92 100 84 1008g 78 >100 86 828h 100 >100 >100 868i >100 66 >100 1008j 40 >100 50 588k >100 >100 >100 1008l 96 >100 100 >1008m 30 41 34 368m salt 29 nd nd nd

a HCT-116, human colon carcinoma cell line; MCF-7, human breast cancer cellline; MIAPaCa-2, human pancreatic cancer cell line; PC-3, human prostate cancercell line.

IC50

, µM

Time, h

IC50, 6 h= 630 µMIC50, 12h = 500 µMIC50, 24h = 42 µMIC50, 48h = 30 µM

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 differentconcentrations of compound 8m for indicated time intervals. Cells were incubatedwith MTT solution and optical density of formazon crystals was measured asdescribed in experimental section. Data are Mean ± SD (n = 8 wells), and represen-tative of three similar experiments.

B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4865–4870 4867

the reported literature, the 13C NMR of 2,5-dihydroxy-3-alkyl-1,4-benzoquinones do not show ring carbon peaks particularly thoseattached to oxygen atoms due to fluxional effect caused by intra-molecular hydrogen bonding.4 The synthesis of embelin deriva-tives 8a–m is depicted in Scheme 1.

Hydrochloride salts of selected compounds were prepared bypassing HCl gas to the solution of compounds viz. 8a, 8b and 8m(Scheme 2). The 1H NMR showed slight shift in the chemical shiftvalues due to the formation of quaternary nitrogen salt. In com-pound 8a, two methyl groups appeared at d 2.89 ppm and twoCH2 protons appeared at d 4.0 ppm whereas in 8a�HCl it shiftedto d 3.19 and 4.28 ppm, respectively. The spot of hydrochloride saltremained at the bottom of TLC whereas parent compound showedRf value of 3–4 in mobile phase CHCl3/MeOH (9.5:0.5).

The in vitro antiproliferative activity of synthesized compounds8a–m along with embelin (1) was determined by MTT assay in fourdifferent cell lines including HCT-116 (colon), MCF-7 (breast), MIA-PaCa-2 (pancreatic), and PC-3 (prostate). The results are summa-rized in Table 1. Compound 8m showed better activity in all thetested cell lines with IC50 values of 30, 41, 34 and 36 lM against

Figure 3. Cell cycle analysis of compound 8m treated HCT-116 cells. HCT-116 cells wereto determine DNA fluorescence and cell cycle phase distribution. Data are representative obetween control and treated groups or the entire intra group using one way ANOVA withp-Values ⁄<0.001 were considered significant.

HCT-116, MCF-7, MIAPaCa-2 and PC-3 cells, respectively. The con-centration and time-dependent inhibition of HCT-116 cell prolifer-ation was studied for compound 8m. It showed varyingcytotoxicity potential (IC50) at different time points as depictedin Figure 2. Compounds 8e and 8j have shown inhibition againstHCT-116 cells with IC50 values of 22 and 40 lM, respectively. Theparent compound embelin (1) exhibits IC50 values of 65, 28, 56and 60 lM against HCT-116, MCF-7, MIAPaCa-2 and PC-3 cells,respectively. The reported IC50 value for embelin (1) in PC-3 cellline is 3.7 lM10 whereas in our experiment we have obtained thisvalue as 60 lM. The difference between two values may be due tothe fact that reported IC50 was obtained when PC-3 cells wereexposed to embelin (1) for 5 days,10 whereas we have performedthe assay for 48 h.

Apoptosis is often termed as programmed cell death, a processthat generally occurs in multicellular organisms. It leads to mor-phological changes in cell including blebbing, cell shrinkage,nuclear fragmentation, chromatin condensation, and chromosomalDNA fragmentation and finally causing cell death. It has beenobserved that many cytotoxic drugs show anticancer activity byinducing apoptosis.25 Thus, in order to address the cell deathcaused by compounds 8m, the extent of apoptotic death in HCT-116 cell lines was assessed using flow cytometry through determi-nation of sub-G1 cell population by propidium iodide (PI) staining.Due to DNA degradation by the apoptosis-associated endonucle-ases, the DNA content measurement by staining with DNA specificfluorochrome, propidium iodide discriminates apoptotic cells.26 Asdepicted in Figure 3, the percentage of apoptotic cells exposed tocompound 8m increased in a concentration-dependent mannerafter 24 h of incubation. The sub-G1 (G0) apoptotic populationwas found to be 3, 34 and 68% following 20, 30 and 40 lM of 8mtreatment compared to control (untreated cells—2%). As depictedin Figure 3, the compound 8m also affected the G2 phase of the cellcycle in HCT-116 cells.

To confirm the apoptosis induction results obtained from cellcycle analysis, nuclear morphological changes of cells by fluores-cence microscopy was studied. After the treatment at 20, 30, and40 lM of compound 8m, characteristic changes of apoptosis suchas nuclear condensation, membrane blebbing and formation ofapoptotic bodies were observed in the morphology of treated cellsin a concentration-dependent manner, whereas the nuclei of

treated with different concentrations (20, 30 and 40 lM) of compound 8m for 24 hf one of three similar experiments at different time period. Comparisons were madepost Bonferroni test through GraphPad Prism 5.00.288 statistical analysis software.

Control 8m, 20 µM 8m, 30 µM 8m, 40 µM

Figure 4. Effect of compound 8m on cellular and nuclear morphology of HCT-116 cells. Cells were treated with indicated concentrations of compound 8m for 24 h timeperiod. Cells were visualized for cellular morphology. Second row shows nuclear morphology of cells after staining with Hoechst 33258 dye.

Figure 5. Compound 8m induced mitochondrial membrane potential loss in human leukaemia HCT-116 cells. Cells (0.44 � 106/ml/24 well plate) were treated with thesecompounds at 20, 30, and 40 lM concentration for 24 h time period. Cells were stained with rhodamine-123 (200 nM) for 30 min and analyzed in FL-1 vs. counts channels offlow cytometer. Data are representative of one of three similar experiments at different time period. Comparisons were made between control and treated groups or theentire intra group using one way ANOVA with post Bonferroni test through GraphPad Prism 5.00.288 statistical analysis software. p-Values ⁄<0.001 were consideredsignificant.

4868 B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4865–4870

untreated cells was found to be of normal intact morphology. Theresults suggested that compound 8m was able to induce apoptoticcell morphology in HCT-116 cells (Fig. 4).

The mitochondrial membrane potential (MMP) provides muchof the cells energy needs and as such is very tightly regulated.Mitochondrial permeability transition, during which the electro-chemical gradient across the mitochondrial membrane is lost, isa key step in the induction of apoptosis. Loss of MMP leads to depo-larization of mitochondrial membrane resulting in mitochondrialdysfunction and ultimately a cell death.27,28 The loss of MMP alsoresults in release of several pro-apoptotic factors that activatecaspases, which induces apoptosis. Thus, the effect of compound8m on MMP loss in HCT-116 cells was investigated. The MMP lossin treated and untreated cells is measured by rhodamine-123 dye(Rh-123) which is reduced by healthy mitochondria into fluorescent

probe whose fluorescence is measured by flow cytometer in FL-1channel. Compound 8m induced loss of MMP in HCT-116 cells ina dose-dependent manner (Fig. 5). The untreated control cellsshowed 2% MMP loss, whereas the compound 8m-treatedHCT-116 cells showed 11%, 53% and 72% MMP loss at 20, 30 and40 lM concentrations, respectively (Fig. 5).

Since, the synthesized compounds showed cytotoxicity againstselected panel of cell lines, we decided to screen these compoundsfor their effect on the growth of different bacterial and fungalstrains. Thus, compounds 8a–m along with embelin (1) wereevaluated for antibacterial and antifungal activities againstStaphylococcus aureus ATCC 29213, Escherichia coli ATCC 25922,Candida albicans ATCC 90028 and Aspergillus fumigatus MTCC1811 (Table 2). S. aureus is known to be associated with severaldisease conditions including cellulitis, keratitis, osteomyelitis,

Table 3Aqueous solubility of embelin, its selected derivatives and their hydrochloride salts

Entry Aqueous solubilitya (lg/mL)

1 <58a <58b <58m <58a�HCl 8008b�HCl 4008m�HCl >1500

a Average of three determinations.

Table 2Antimicrobial activity of embelin (1) and its derivatives 8a–m

Entry Antimicrobial activity MIC in lg/mL

S. aureusATCC29213

E. coli ATCC25922

C. albicansATCC 90028

A. fumigatesMTCC 1811

1 8 >256 >256 >2568a 8 >256 >256 >2568b 16 >256 >256 >2568c 32 >256 >256 >2568d 32 >256 >256 >2568e >256 >256 >256 >2568f >256 >256 >256 >2568g 128 >256 >256 >2568h 32 >256 >256 >2568i 256 >256 >256 >2568j 32 >256 >256 >2568k >256 >256 >256 >2568l 32 >256 >256 >2568m 128 >256 >256 >2568a salt 8 nd nd nd8b salt 16 nd nd ndCiprofloxacin 0.125 0.007 — —Amphotericin B — — 0.5 0.5

B. Singh et al. / Bioorg. Med. Chem. Lett. 24 (2014) 4865–4870 4869

septic arthritis and mastitis. It belongs to the major class of humanpathogens and is the most common cause of increasing hospitaldeath rate by 35%.29 The compounds 8a, 8b, 8c, 8d, 8h, 8j and 8lwere found to be potent against S. aureus with MIC values in therange of 8–32 lg/mL. The compound 8a and 8b were most potenthaving MIC values of 8 and 16 lg/mL. The tested compounds didnot show any effect on the growth of Gram-negative bacteria anddifferent fungal strains.

The thermodynamic equilibrium aqueous solubility of embelin(1) along with selected derivatives and their salts were determinedusing 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 <5 lg/ml. However, salts of 8a, 8band 8m showed significantly improved aqueous solubility(Table 3). The hydrochloride salts 8a�HCl and 8b�HCl displayedsimilar antibacterial activity against S. aureus compared to theirparent compounds 8a and 8b (results shown in Table 2). Similarly,the salt 8m�HCl also showed similar antiproliferative activity inHCT-116 cells (IC50 29 lM) compared to its parent Mannichderivative 8m (results shown in Table 1).

In summary, new derivatives of embelin were prepared withthe aim to improve solubility, cytotoxicity and antimicrobialactivity. These efforts led to identification of water-solubleembelin Mannich adduct 8m�HCl as better antiproliferativeagent than embelin (1) with improved solubility. The mechanis-tic studies involving cell cycle analysis and fluorescence micros-copy indicated that 8m induces apoptotic cell death and alsocauses the loss of mitochondrial membrane potential in HCT-116 cells. Further, compounds 8a�HCl and 8b�HCl were identified

as potent antibacterial agents against S. aureus with improvedsolubility.

Acknowledgments

The authors are thankful to Deepika Singh and S. Aravinda foranalytical support. This research was supported in part by a grantfrom the CSIR 12th FYP project (BSC-205). S.S.B. thanks ICMR, NewDelhi for the award of research associateship.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.bmcl.2014.08.052.

Experimental section, scanned copies of NMR spectra including1H NMR, 13C NMR and HPLC. This material is available free ofcharge via the internet at www.sciencedirect.com.

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(1, 0.25 mmol) in MeOH (3 mL) was added secondary amines (1.0 mmol) and37% formaldehyde solution (1.0 mmol). The mixture was stirred at roomtemperature for 12–24 h and then concentrated under reduced pressure. Thereaction mixture was then dried under vacuum and the final products 8a–mwere washed with hexane and diethyl ether mixture giving 75–90% of pureproducts.

24. 2-((4-Benzylpiperidin-1-yl)methyl)-3,6-dihydroxy-5-undecylcyclohexa-2,5-diene-1,4-dione (8m): Yield, 80%; red colored solid; mp 169–172 �C; 1H NMR(400 MHz, DMSO-d6, ppm): d 7.30–7.16 (m, 5H), 3.82 (s, 2H), 2.81–2.75 (m,3H), 2.23 (t, 2H, J = 8.0 Hz), 1.84–1.64 (m, 6H), 1.52–1.42 (m, 2H), 1.36–1.16 (m,18H), 0.86 (t, 3H, J = 8.0 Hz); 13C NMR (100 MHz, DMSO-d6, ppm): d 139.45 (C),128.95 (CH), 128.95 (CH), 128.19 (CH), 128.19 (CH), 125.94 (CH), 114.48 (C),98.85 (C), 51.46 (CH2), 51.16 (CH2), 43.25 (CH2), 43.21 (CH2), 41.67 (CH2), 35.01(CH2), 34.45 (CH), 31.26–22.05 (s, 10 � CH2 of chain C10–100), 13.90 (CH3);HRMS: m/z calcd 482.3278 for C30H43NO4+H+ (482.3265).

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