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European Journal of Medicinal Chemistry 85 (2014) 95e106

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European Journal of Medicinal Chemistry

journal homepage: http: / /www.elsevier .com/locate/ejmech

Original article

Synthesis and biological evaluation of andrographolide analogues asanti-cancer agents*

Ranjan Preet c, 1, Biswajit Chakraborty a, 1, Sumit Siddharth c, Purusottam Mohapatra c,Dipon Das c, Shakti Ranjan Satapathy c, Supriya Das b, Nakul C. Maiti b, Prakas R. Maulik a,Chanakya Nath Kundu c, *, Chinmay Chowdhury a, *

a Chemistry Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, Indiab Structural Biology and Bioinformatics Division, CSIR-Indian Institute of Chemical Biology, 4 Raja S. C. Mullick Road, Kolkata 700032, Indiac Cancer Biology Division, KIIT School of Biotechnology, KIIT University, Campus-11, Patia, Bhubaneswar, Odisha 751024, India

a r t i c l e i n f o

Article history:Received 21 October 2013Received in revised form23 July 2014Accepted 24 July 2014Available online 24 July 2014

Keywords:AndrographolideC14 ester analoguesEpoxy diastereomersAnti-cancerHEK-293MCF-7Normal cellsApoptosis

Abbreviations: AG, andrographolide; MTT, 3-(4diphenyltetrazolium bromide; DAPI, 4,6-diamidino-2ADP-ribose)polymerase.* This paper is dedicated to Dr. Pradeep Kumar Dutt

of Chemistry Division, Indian Institute of Chemical Bi* Corresponding authors.

E-mail addresses: cnkundu@gmail.com (C.N. K(C. Chowdhury).

1 Contributed equally.

http://dx.doi.org/10.1016/j.ejmech.2014.07.0880223-5234/© 2014 Elsevier Masson SAS. All rights re

a b s t r a c t

A new family of andrographolide analogues were synthesized and screened in vitro against kidney (HEK-293) and breast (MCF-7) cancer cells. The anti-cancer effects of the active analogues (2b, 2c and 4c) weredetermined by multiple cell based assays such as MTT, immunostaining, FACS, western blotting andtranscriptional inhibition of NF-kB activity. Importantly, these compounds were found to possess higheranti-cancer potency than andrographolide and low toxicity to normal (VERO and MCF-10A) cells.Increased level of Bax/Bcl-xL ratio, caspase 3, and sub G1 population, higher expression level of tumorsuppressor protein p53 and lower expression level of NF-kB suggested potent apoptotic property of theactive analogues. Data revealed that the andrographolide derivative-mediated cell death in cancer cellswas p53 dependent.

© 2014 Elsevier Masson SAS. All rights reserved.

1. Introduction

Andrographis paniculata Nees (Acanthaceae) is considered asone of the most important medicinal plants in India, China andother Asian countries due to its popular use in traditional systemsof medicines [1]. Andrographolide 1 (Fig. 1), a major phytocon-stituent of the plant, has been recognized as an important phar-macophore because of its key role as inducer of apoptosis againstdifferent types of cancers [2] in addition to other pharmacologicaleffects [3] (e.g., anti-viral [3a], anti-inflammatory [3b], antimalarial[3c], anti-hyperglycemic [3d], immunostimulatory [3e] etc.).

,5-dimethylthiazol-2-yl)-2,5--phenylindole; PARP, poly(-

a, a former scientist and headology, Kolkata 700032, India.

undu), chinmay@iicb.res.in

served.

However, despite its impressive biological activities, the majordrawback of andrographolide is poor oral bioavailability [4] makingit difficult to prepare formulations for clinical use. Thus, only well-designed derivatives of andrographolide might have the potentialto be developed as anti-cancer chemotherapeutic agents. Indeed, agrowing interest has been observed in recent times for designing,synthesizing and subsequently screening different analogues ofandrographolide in order to discover lead(s) having better phar-macological profile than the parent compound. Towards thisendeavor, few promising compounds having ester functionality atC14 of 1 have recently been identified by Stanslas [5a] (14-acetylandrographolide), Nanduri [5b] (14-cinnamoyl-8,17-epoxy-andrographolide), Rajagopal [5c] (DRF 3188), and us [5d] (14-succinylandrographolide). In our previous report [5d], we alsoestablished the important role of the a-alkylidene-g-butyrolactonemoiety of andrographolide to account for its cytotoxicity in humanleukemic cell lines (U937, K562 and THP1). In continuation of ourwork [5d,6] in lead identification from bioactive natural products,we therefore became interested to check the anti-cancer potentialof the ester analogs of 2 and their epoxy derivatives 3e4 as well

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Fig. 1. Andrographolide and its designed analogues.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e10696

(Fig. 1). The rationale for choosing the aforesaid compounds wasprimarily the following: (a) an ester moiety at C14 of androgra-pholide has been shown to be important for cytotoxicity [5]; (b) theester moiety (or the halogen in the side chain of the estermoiety) ofcompounds (2e4) may serve as good leaving group to facilitate thebinding with intracellular glutathione (GSH) [7], thereby triggeringsuicide of the cell leading to apoptosis; (c) esters might act like pro-drugs, being hydrolyzed by esterases or under physiological pH torelease andrographolide 1 (or its epoxy derivatives) in vivo [8].Besides, compounds 3e4 differing in their epoxide configurationscould be important for structureeactivity relationship (SAR)studies [9].

Despite several reports on the structural modifications ofandrographolide [10], chemo-selective functionalizations at C14hydroxyl are limited in number [5,11]. In this report, we describethe synthesis of novel C14-ester analogues 2e4 and their cytotoxiceffects (in vitro) on kidney (HEK-293) and breast (MCF-7) cancercells. We have also demonstrated the apoptotic properties of themost active analogues and compared their cytotoxicity with that innormal cells.

2. Results and discussion

2.1. Chemistry

Andrographolide (~100 g) was isolated from the leaves of A.paniculata and used as starting material for derivatization. Thesynthetic pathways used in the present work are outlined inScheme 1. We chose to carry out chemoselective esterification ofandrographolide at C14 hydroxy, which is allylic in nature. Towardsthis objective, the other hydroxyls were converted (Scheme 1) into3,19-isopropylidene derivative 5 by treatment with 2,2-dimethoxypropane and p-toluenesulfonic acid (cat.). Compound 5was initially reacted with chloroacetyl chloride in dry THF in thepresence of pyridine (1.5 eqv.) and 4-dimethylaminopyridine (cat.)to afford the intermediate ester 6a with 66% yield. The corre-sponding bromo-ester 6b was obtained (63% yield) by reactingbromoacetyl bromide with 5, while the iodo derivative 6c wasprepared (65% yield) by treating 6a with sodium iodide in acetoneat rt for 3 h. The isopropylidene moiety of products 6aec was thenremoved by exposing the products to aqueous acetic acid (3:7),affording the targeted compounds 2aec which were then purified(>95%) by HPLC separations. Thereafter, we directed our efforts forchemoselective epoxidation of the exocyclic double bond (D8(17)) ofintermediates 6aec. Towards this end, compound 6a was treatedwithm-chloroperbenzoic acid (m-CPBA) in dry dichloromethane tofurnish a diastereomeric mixture of epoxides 7a and 8a with 51%yield. Pleasingly, this mixture was separated through silica gel(100e200 mesh) column chromatography (7a:8a ¼ 2:3). This

reaction protocol was subsequently applied on compounds 6b and6c, which afforded the corresponding diastereomeric epoxides 7b/8b (54%, 7b:8b ¼ 1:2) and 7c/8c (52%, 7c:8c ¼ 2:5), respectively(Scheme 1). Thereafter, the isopropylidene moiety of the epoxides(7aec and 8aec) was removed using aqueous acetic acid (3:7)leading to the formation of the targeted products 3aec and 4aec asshown in Scheme 1. These compounds were finally purified (>95%)using HPLC separations.

As epoxidation of intermediate 6 resulted in a diastereomericmixture, we tried to make this reaction stereoselective by changingthe strategy. Pleasingly, replacing intermediate 6 with 5 for epox-idation usingm-CPBA ensured a totally stereoselective formation ofepoxide 9with 76% yield (Scheme 2). The absolute stereochemistryat C8 of product 9was found to be S by single crystal X-ray analysis(see Fig. S1 of the Supplementary material). Notably, a previousstudy [12] on this reaction failed to get the expected epoxide 9;instead, a deprotected product was isolated. The facial selectivityoperating in b-epoxidation of compound 5 is possibly influenced bythe hydroxyl group which takes up b-orientation by rotationaround the C11eC12 single bond (for energy minimized conforma-tions see Fig. S2 of the Supplementary Material) to become proxi-mate to the reactive double bond and directs (through hydrogenbonding [13]) the approach of m-chloroperbenzoic acid towards it.However, attempted esterification at C14 hydroxy of 9 employingchloroacetyl chloride, pyridine and DMAP (cat.), as shown inScheme 2, did not furnish ester 8a; instead, an intractable solidresulted. After having this disappointing result, we performed thisreaction using bromoacetyl bromide; this resulted in the formationof the expected product 8b, but with a discouraging yield of 35%only (Scheme 2). Due to the poor yield and lack of the consistencyof the reaction for esterification of epoxide 9, the former strategy(Scheme 1) seemed to be a better option for the generation of suchanalogues. However, the structure determination of epoxide 8bfrom the latter study (Scheme 2) helped us to identify the stereo-chemical outcomes of the conversion of intermediate 6 described inScheme 1 unambiguously. In 1H NMR, the olefinic proton (C12eH)signal appeared as triplet (t) at around d 6.99 ppm for di-astereomers 7, but d 7.15 ppm in other diastereomers 8. The ste-reochemical assignments were further supported by NOESYanalysis.

2.2. Biology

2.2.1. Anticancer properties of the synthesized compoundsTo check the anticancer effects of the synthesized compounds,

we initially performed an MTT assay in human embryonic kidneycancer cells (HEK-293) and compared its effect with the normalmonkey kidney cells (VERO). VERO cells are non cancerous in na-ture and are derived from the normal kidney epithelial cells. Cells

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Scheme 1. Synthesis of andrographolide analogues 2e4. Reagents and conditions: (a) 2,2-dimethoxypropane, p-TsOH (cat.), acetone, reflux, 2 h, 70%; (b) chloroacetyl chloride/bromoacetyl bromide, pyridine, DMAP(cat.), dry THF, 0 �C, 30 min, 66% for 6a, 63% for 6b; (c) NaI, acetone, rt, 3 h, 65%; (d) acetic acid/water (7:3), rt, 30 min, 67e75%; (e) m-CPBA,0 �C, DCM, 3 h, 51e54%.

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Scheme 2. Approach for stereoselective epoxidation and subsequent esterifications.Reagents and conditions: (a) m-CPBA, dry DCM, 0 �C to rt, 3 h, 76%; (b) chloroacetylchloride, pyridine, DMAP (cat.), dry THF, 0 �C to rt, 2 h, 0%; (c) bromoacetyl bromide,pyridine, DMAP (cat.), dry THF, 0 �C to rt, 2 h, 35%.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106 97

were treated with increasing concentrations of the compounds for48 h and cell viability was measured. Among the derivativesscreened, only three compounds (2b, 2c, 4c) were found to besignificantly active (LC50 ~ 8e12 mM), while others caused 50% cell(HEK-293) death at concentrations only above 20 mM. The LC50 ofthe remaining compounds has been provided in supplementarymaterial (Table S2). Compounds 2b, 2c and 4c displayed LC50 of12, 8, and 12 mM, respectively, in HEK-293 cells, but were notsignificantly toxic in normal (VERO) cells (Fig. 2A) as 50% cell deathwas not reached upto tested concentrations (40 mM). Androgra-pholide (AG) kills 50% of HEK-293 cells at ~25 mM but is non-cytotoxic to VERO cells (Fig. 2A) at this range. The relative anti-cancer potency of the investigational compounds (2b, 2c and 4c)in HEK-293 and VERO cells with some of the already known anti-cancer agents like andrographolide, etoposide and 5-fluorouracil isprovided in Table S3 of the Supplementary material.

Additionally, we checked the cell viability of these compounds(2b, 2c and 4c) in MCF-7 (breast cancer) andMCF-10A cells (normalbreast epithelial cells) as well. The tested compounds displayedLC50 of 8.4, 6, and 7 mM, respectively against MCF-7 cells, while innormal cells (MCF-10A) 50% cell death was not observed up to40 mM (Fig. 2B). Andrographolide (AG) caused 50% cell death inMCF-7 cells at 16 mM but not in MCF-10A even up to 40 mM.

Though 14-acetylandrographolide was reported [5a] to be anactive compound against MCF-7 cell lines (GI50 ¼ 5.49 ± 1.5 mM),there is no report about its activity against kidney cancer cells(HEK-293). In the present study, 14-(2-bromo/iodoacetyl)androg-rapholide (2b/2c) displayed comparable results in MCF-7 cells andsignificant cytotoxicity in HEK-293 cells. On the other hand, 14-acetyl-8,17-b-epoxyandrographolide was previously found [5b] tobe inactive in most of cancer cells except few (ovarian/renal); wehowever observed that 14-(2-iodoacetyl)-8,17-b-epoxyan-drographolide (4c) was active against kidney and breast cancer aswell. Surprisingly, compound 3c, the other diastereomer, did not

appear as an active compound. Detailed study about the role ofepoxide configuration in the activity profile is currently in progress.

2.2.2. Investigational compounds cause apoptosis in kidney cancercells without affecting the phases of cell cycle

To analyze the effect of 2b, 2c and 4c on the cell cycle regulationand apoptosis, HEK-293 cells were treated with these compoundsin a dose dependent manner for 48 h, followed by analysis of thecells by flow cytometry after propidium iodide staining (Table 1)(see also Fig. S3 in the Supplementary material). It was noted thatexposure of increasing concentrations (0e20 mM) of the investi-gational compounds led to an increase in the Sub G1 phase popu-lation, representing apoptosis without arresting the cells in anyphase of cell cycle. A greater than four-fold increase in apoptosis incomparison to the untreated cells was observed at 20 mMconcentration.

2.2.3. Immunocytochemistry experiment of caspase 3For further studies on apoptosis caused by the test com-

pounds, we performed an immunocytochemistry experiment of

Fig. 2. A. MTT cell viability assay. Compound 2b/2c/4c and andrographolide (AG) cause a decrease in cell (HEK-293) survival. The sign , , and represent HEK-293/investigational compound, VERO/investigational compound, HEK-293/AG, and VERO/AG, respectively. Data represented here is the mean of three independent experiments and thevalues are mean ± SD. Statistical significance was determined by paired t-test. *p < 0.05. B. MTT cell viability assay. Compounds 2b/2c/4c trigger decrease in cell (MCF-7) survival.The signs , and represent MCF-7/2b, MCF-7/2c, MCF-7/4c, respectively, while the signs , , and represent MCF-10A/2b, MCF-10A/2c, and MCF-10A/4c,respectively. However, MCF-7/AG and MCF-10A/AG are represented by and , respectively. Data represented are the mean of three independent experiments and the valuesare mean ± SD. Statistical significance was determined by paired t-test. *p < 0.05.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e10698

caspase 3 in HEK-293 cells. An increased expression of caspase 3was observed when HEK-293 cells were treated with the fixedconcentration (respective LC50 value) of 2b/2c/4c for 48 h(Fig. 3). Higher accumulation of caspase 3 was observed intreated cells in comparison to untreated and andrographolide(AG) treated cells.

2.2.4. Transcriptional inhibition of NF-kB activity by theinvestigational compounds

The overexpression of NF-kB transcription factor activity isconsidered as the hallmark in initiation, promotion and growth ofcancer, and thus inhibition of this activity of NF-kB is an importantapproach in the discovery of chemotherapeutic agents. To

Table 1Quantitative analysis of cell cycle profile.a

Apoptosis GI S G2/M

2bControl 24.26 ± 0.8 53.21 ± 0.8 19.9 ± 0.5 2.63 ± 0.75 mM 60.44 ± 0.7 25.16 ± 0.8 10.44 ± 0.5 3.96 ± 0.710 mM 67.33 ± 0.6 19.57 ± 0.5 10.28 ± 0.7 2.82 ± 0.915 mM 76.62 ± 0.9 14.98 ± 0.6 6.69 ± 0.6* 1.71 ± 0.820 mM 88.84 ± 1.1 9.01 ± 1.2 1.57 ± 1.1* 0.58 ± 1.32c5 mM 43.09 ± 0.5* 24.77 ± 0.5* 9.21 ± 0.6* 2.14 ± 1.110 mM 74.03 ± 0.7* 15.60 ± 0.8* 7.57 ± 0.6 2.74 ± 0.815 mM 78.50 ± 0.6 14.80 ± 0.6 5.67 ± 0.9 1.28 ± 0.720 mM 89.69 ± 0.8 7.96 ± 0.7 1.66 ± 0.7 0.69 ± 0.94c5 mM 62.05 ± 0.7 25.38 ± 0.7* 9.87 ± 0.6 2.70 ± 0.810 mM 77.99 ± 0.8* 15.61 ± 0.9* 4.98 ± 0.8* 1.41 ± 0.915 mM 79.55 ± 0.6 14.98 ± 0.7 4.63 ± 0.9 1.31 ± 0.820 mM 92.93 ± 1.1* 5.37 ± 1.1 1.18 ± 1.2 0.52 ± 1.3

Data represent the mean ± SD of experiments in triplicate. Statistical significancewas determined by paired t-test. *p < 0.05.

a Quantitative analysis of cell cycle profile was done using histogram data (pro-vided in Fig. S3 in the Supplementary material) analyzed by Cell Quest Pro software(Becton Dickinson, CA).

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106 99

determine the role of the test compounds on transcriptional ac-tivity of NF-kB gene in HEK-293 cells, a luciferase based assay wasused following our earlier protocol [14e18]. We observed that therelative luciferase activity of the NF-kB gene decreased in a dosedependent manner after treatment of the cells with the investi-gational compounds (Fig. 4). Approximately five-fold reduction ofluciferase activity was observed at 5 mM concentration of 2c, but at10 mM concentration for both 2b and 4c.

Additionally, in order to determine the effect of the investiga-tional compounds on apoptosis in HEK-293 cells, a DAPI (40,6-diamidino-2-phenylindole) nuclear staining method and determi-nation of anti-cell migratory activity were also used (see Fig. S4 inthe Supplementary material for details).

2.2.5. Mechanism of actionTo further confirm the apoptotic effect of investigational com-

pounds and study the mechanism of anti-cancer action, we havemeasured the expression of different pro-apoptotic and anti-apoptotic protein markers in HEK-293 (expressed wt p53) andHEK-293 p53 �/� cells after exposure to these agents (Fig. 5A). Theleft panel of Fig. 5A demonstrates the increase in expression of Baxprotein, while the level of Bcl-xL was reduced significantly withsimultaneous increase in the PARP cleaved product (86 kDa) intreated HEK-293 cells. An increase in the tumor suppressor proteinp53 with a significant decrease in the level of NF-kB was also notedwhen HEK-293 cells were treated with the investigational com-pounds. Interestingly, no significant alteration was noted in theexpression level of the above mentioned protein biomarkers in thetreated HEK-293 p53 �/� cells (right panel of Fig. 5A).

Thus it appeared that investigational compound mediatedapoptosis in HEK-293 cells is p53 dependent. Based on above result,we have drawn a scheme representing the mechanism of action ofthe compounds on kidney cancer cells (Fig. 5B). We propose thatcompounds (2b, 2c and 4c) promote the activation of p53, leadingto a downregulation of NF-kB with simultaneous upregulation ofBax/Bcl-xL ratio that triggers cytochrome C release followed byupregulation of caspase 3 and thereby causing apoptosis.

3. Biophysical studies

3.1. Binding efficacy and mode of binding of compound 2c

Among the active analogues (2b, 2c and 4c), compound 2cappeared to be the best; we, therefore, decided to study the bindingefficacy and mode of binding on this compound. DNA shows anintense absorption band at 260 nm and the intensity of this ab-sorption band was reduced significantly in the presence of 2c withincreasing concentration (Fig. 6A). These indicated that this com-pound interacts with DNA effectively and further investigationswere carried out focusing on the binding efficacy, mode of bindingand the interaction pattern. The binding isotherm (Fig. 6B) for DNA- 2c interaction was obtained by plotting fraction (q) of DNA boundto 2c versus total compound concentration [19]. The fraction (q)was calculated as q ¼ (A0 e Ai)/(A0 e Aa), where, A0 is the absor-bance of DNA in the absence of compound at 260 nm (or slightlyblue or red shifted), Ai is the absorbance of DNA at any compoundconcentration, and Aa is the absorbance of DNA at compoundconcentration for which maximum binding took place. Kb wasobtained from the reciprocal of the ligand (2c) concentration cor-responding to the half-saturation value of the binding isotherm.The measured binding constant was 2.5 � 105 M�1.

In addition, circular dichroism (CD) spectral analysis was car-ried out to measure the effect of drug molecule binding toconformational changes in DNA duplex structure (Fig. 7). The CDspectra of the DNA solution (500 mg/mL) in the absence and

presence of 2c (100 mM) were recorded after 2 h incubation at37 �C. The solution was half diluted with the same buffer for CDmeasurement. It was observed that the CD band at 275 nm (due tobase stacking) and the absorbance at 245 nm (due to the B-conformation of DNA) did not change significantly in the presenceof 2c, while EtBr (a well known DNA intercalator) which is oftenused as positive control [20] showed changes in the absorptionspectra (shape and absorbance). Earlier report [21] suggested thatsimple electrostatic interaction or groove binding of small mole-cules to DNA did not severely affect the absorbance at 275 nmband of the DNA. Similarly, changes in absorption of the band at245 nm implied the change of B-DNA conformation or the for-mation of a different conformation of DNA. Our results showedthat the CD spectra of the DNA in the presence and absence of thecompound were very similar; indicating that compound 2c causedno detectable conformational change of DNA and was not inter-calated like EtBr.

We further investigated the mode of binding of 2c to DNA usingDAPI (40,6-diamidino-2-phenylindole)-DNA complex. DAPI has ahigh affinity for the minor groove of A/T-rich binding sites [22] andbinds to double stranded DNA. It forms a complex that gives highfluorescence with a band maximum at 460 nm. However, DAPIalone is weakly fluorescent in water. When compound 2c wasadded to DAPI-DNA complex, the fluorescence of the solution wasreduced (Fig. S6 in Supplementary material) due to release of DAPIand incorporation of compound 2c to DNA. The result indicatedthat the compound was capable of binding to the minor groove ofDNA where DAPI also preferred to bind.

Finally, a molecular docking analysis was performed to furthercharacterize the binding interactions of compound 2c to DNA (seeTable S5 and Fig. S7 in Supplementary material). Out of the 100docking runs performed, favorable binding poses were obtainedwith those presenting the most negative binding free energies.Molecular docking results showed that compound 2c binds favor-ably to DNA minor groove and the calculated binding energywas �8.47 kcal/mol. The docking of netropsin, a well known minorgrove binder with DNA [23], showed binding energy �7.02 kcal/mol. Theminor groove bound state of 2c resulted in a close fit of themolecule along the wall of the minor groove (Fig. S7 inSupplementary material). The results of docking analysis withother compounds (2b, 4c) are also shown in Supplementarymaterial (Table S5 in Supplementary material). The docking

Fig. 3. Caspase 3 staining: HEK-293 cells treated with andrographolide (AG) and 2b/2c/4c were stained with anti-caspase-3 antibody and DAPI. Photographs were taken at 40�magnification. The fields of Caspase 3 staining were merged with respective DAPI stained nucleus with the help of IMAGE J software. Photographs presented here is the best of threeindependent experiments.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106100

results thus indicated that 2c can act as a minor groove binder likenetropsin.

Thus all the experiments and computation analysis favored thenotion that compound 2c effectively binds to the minor grove of

Fig. 4. Relative luciferase gene reporter assay of p5XIP10 B plasmid containing NF-kB construcompounds andrographolide (AG), 2b, 2c and 4c, respectively. Data represent the mean ± SDtest. *p < 0.05.

DNA with significant affinity. We therefore became interested tocheck the potentiality of 2c for DNA adduct formation. For thispurpose, we performed a model reaction between guanosine and2c in glacial acetic acid at 75 �C following the literature procedure

ct after transfection into HEK-293 cells and treatment with indicated concentrations ofof three independent experiments. Statistical significance was determined by paired t-

Fig. 5. A: Western blot of HEK-293 and HEK-293 p53 �/� cells. Western blot analysis of the investigational compounds (2b/2c/4c) and andrographolide (AG) for apoptotic markersin HEK-293 and HEK-293 p53 �/� cells after treatment for 48 h. The lower panel shows GAPDH as the loading control. B: Analogs (2b, 2c and 4c) of andrographolide cause apoptosisin kidney cancer cells by upregulating p53. Upward arrow indicates the upregulation and downward arrow indicates the downregulation.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106 101

[24]. However, even after heating the reaction mixture for 12 h, nosuch adduct formation was observed by TLC and this was furtherconfirmed from LCMS analysis.

3.2. Hydrolysis of C14 ester moiety of compound 2c anddetermination of the half life period (t1/2)

We also planned to study the hydrolysis of the C14 ester moietyof 2c at physiological pH (7.4) using buffer and employing suitableenzyme (esterase type). Before performing this experiment, weattempted to construct the requisite calibration curve using HPLCemploying different concentrations of 2c in PBS buffer (pH ¼ 7.4)without adding any enzyme. Surprisingly, it was observed that 2cwas slowly hydrolyzed to andrographolide in PBS buffer only. It isworthwhile to mention here that the prodrug isotaxel, an analogueof taxol, was converted to taxol at physiological pH and this facili-tated the delivery (in vivo) of taxol [25]. This gave us the impetus tocalculate the half life period of 2c in PBS buffer to check the pos-sibility of using this compound as a prodrug. Accordingly, the hy-drolysis experiment of compound 2c was carried out in PBS buffer

(at pH 7.4) and reaction kinetics was followed by HPLC throughmonitoring the amount of unreacted 2c at different time intervals.We observed an exponential decrease of the amount of 2c withtime and the reaction kinetics was found to be of first order. Thus,fitting to a first order rate equation themeasured rate constant of 2cwas 0.02 min�1 and corresponding half-life (t1/2) was 35 min (seeFig. S8 in Supplementary material). This experiment indicates that2c could serve as a prodrug under physiological pH. Besides, there isthe other possibility that esterases may also cause this hydrolysis(in vivo).

3.3. GSH binding of compound 2c

Next, we investigated the binding efficacy of 2c with GSH. Asester moiety of 2c is susceptible to hydrolysis in buffer of pH 7.4, wedecided to conduct this study in high dilution of the buffercompared to the substrate. Accordingly, ethanolic solutions of both2c and GSH were added to the PBS buffer where final concentra-tions of each substratewere adjusted to 10mM and that of buffer to2 mM. After heating this reaction mixture at 50 �C for 8 h under

Fig. 6. (A) Ultravioletevisible (UVevis) absorption spectra of DNA (100 mg/mL) after incubation with 2c in different concentrations in buffer (50 mM TriseHCl/NaCl, pH 7.5) at 37 �Cfor 2 h. Spectra were taken after six dilutions with the same buffer in which the final concentrations of 2c were calculated as 0 (a), 3.33 (b), 6.66 (c), 9.99 (d), 13.33 (e), 16.66 (f), and20.00 (g) mM. The absorption spectra were normalized at 230 nm to avoid contribution from turbidity due to the formation of complex. (B) shows the fraction (q) of DNA binding siteinvolved in complexation with the ligand. Details about the calculation of fraction of DNA occupied by the ligand are provided in the result section. The data were fitted to non-linear Boltzman curve fit using Origin software with the equation, y ¼ A2 þ (A1 � A2)/(1 þ exp((x � x0)/dx)), where, x0 ¼ half saturation point where the concentration of ligandequals Kd, i.e., ~4 mM, the reciprocal of which provides the binding constant Kb ¼ 2.5 � 105 M�1.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106102

argon atmosphere, two major products were found to be formed;these were separated by reverse phase HPLC and characterizedusing ESI mass and NMR spectroscopy (see Supplementarymaterialfor details). Both products were identified to be 14-deoxy-12-(glutathione-S-yl)-andrographolide (Fig. 8) but must differ in C12stereochemistry (diastereomeric at C12). The crucial evidence infavor of linkage through sulfur atom of glutathione (GSH) camefrom clear HMBC correlations observed between the signals forCH2S- protons and C12 carbon in both the isomers. Incidentally,earlier workers [7] reported formations of two products from thereaction of GSH with andrographolide or a different C14 ester and

220 240 260 280 300 320

-8

-4

0

4

8

mde

g

Wavelength (nm)

DNA DNA + 2C DNA + EtBr

Fig. 7. CD spectra of DNA in the presence of different compounds in aqueous buffer:black, red and blue line represent the spectra of only DNA (500 mg/mL), DNA in thepresence of compound 2c (50 mM) and DNA in the presence of EtBr (150 mM),respectively. (For interpretation of the references to colour in this figure legend, thereader is referred to the web version of this article.)

identified them as 14-deoxy-12-(glutathione-S-yl)andrographolideand 14-deoxy-12-(glutathione-amino)andrographolide. But in ourcase, there was no formation of any 14-deoxy-12-(glutathione-amino)andrographolide. However, formations of our products canbe explained through a possible mechanism where sulfhydryl (SH)group of GSH undergo Michael addition to the C12eC13 doublebond of 2c followed by elimination of its ester group at C14. Thisstudy demonstrates that 2c makes covalent binding with GSH andis capable of reducing the level of the intracellular GSH augmentingits cytotoxic activity.

4. Conclusion

A new family of C14-ester analogs of andrographolide and theira and b diastereomeric epoxy derivatives were synthesized [26].The anti-cancer activity of these compounds was tested in kidney(HEK-293) and breast cancer cells (MCF-7) and compared with theresults in corresponding normal (VERO and MCF-10A) cells. MTTcell viability assay revealed that analogs 2b, 2c and 4c exertedsignificant cytotoxicity (8e12 mM in HEK-293 cells and 6e8.4 mM inMCF-7 cells) in cancer cells and low toxicity towards normal cells.Importantly, these analogues showed more potency than androg-rapholide. Increase in apoptotic nuclei after DAPI staining, sub G1population after FACS analysis, and Bax/Bcl-xL ratio, cleavage ofpoly (ADP) polymerase, caspase 3, and p53, and decreased level ofNF-kB promoter activity pointed to the significant apoptotic prop-erty of these compounds. Compound 2c turned out to be the mostactive in both kidney and breast cancer cells suggesting its potentialto be utilized in drug discovery through further optimization. Ascheme representing the mechanism of action of the investiga-tional compounds (2b, 2c and 4c) on kidney cancer cells is shown(Fig. 5B). We propose that the compounds activate p53, a tumorsuppressor protein, leading to the downregulation of NF-kB withsimultaneous upregulation of Bax/Bcl-xL ratio, thereby causingapoptosis. These compounds were shown to act as minor groovebinder of DNA. The study will aid in the development of novel anti-

1

35

9

12

141'

2'

4'

9'

17

19

HO

HO

S

O

ONH

O

OH

NH2

O

HNO

OHO

Fig. 8. 14-Deoxy-12-(glutathione-S-yl)-andrographolide.

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106 103

cancer agents based on andrographolide and provide insight intoanti-cancer mechanism.

5. Experimental

5.1. Chemistry

5.1.1. General methodsAndrographolide 1was isolated (~100 g, 0.54% yield) from dried

and powdered leaves of A. paniculata following the reported pro-cedure [27]. All solvents were distilled prior to use. All the reactionswere performed under argon atmosphere and anhydrous condi-tions unless otherwise noted. Reactions were monitored by thin-layer chromatography (TLC) on TLC aluminium sheets 20 � 20 cmsilica gel 60F254 and silica gel 60 RP-18 F254s. Visualization of thedeveloped chromatogramwas performed by UV (254 nm, 365 nm)light or iodine or Liebermann solution. The analytical and semipreparative HPLC was performed using X-Bridge columns (C18,5 mm, 4.6 � 250 mm and C18, 5 mm,10 � 250 mm). 1H and 13C NMRspectra were recorded in 300 or 600 MHz spectrometer. Chemicalshifts (d) were reported in parts per million (ppm) downfield fromtetramethylsilane (d ¼ 0.00) with the residual protons of deuter-ated solvents used [CDCl3: 1H NMR, d ¼ 7.26 ppm (s), 13C NMR,d ¼ 77.0 ppm (t); CD3OD : 1H NMR, d ¼ 4.86 ppm (s), 3.32 ppm (s),13C NMR, d ¼ 49.0 ppm (multiplet)]. Coupling constants (J) wereexpressed in hertz (Hz) and spin multiplicities are given as ‘s’(singlet), ‘d’ (doublet), ‘dd’ (double doublet), ‘t’ (triplet), ‘dt’(doublet of triplet), ‘m’ (multiplet) ‘br’ (broad) and brs (broadsinglet).

5.1.2. Typical procedure for the synthesis of the intermediatechloroester 6a

Initially, the hydroxyl groups at C3 and C19 of andrographolide 1were protected according to our earlier protocol [5d] resulting inthe synthesis of 3,19-isopropylideneandrographolide 5.

To awell stirred solution of compound 5 (250mg, 0.64 mmol) indry THF (5 mL) maintained at 0 �C, chloroacetyl chloride (0.06 mL,0.67 mmol), pyridine (0.07 mL, 0.96 mmol), and a catalytic amountof DMAP (4-dimethylaminopyridine) were added sequentiallykeeping the pH of the reaction mixture neutral or just basic. Thereaction mixture was then stirred for 30 min under argon atmo-sphere. After completion of the reaction (monitored by TLC), thesolvent was evaporated under reduced pressure at 0 �C; theresulting residue was then mixed with water (10 mL) and ether(10 mL). The mixture was neutralized using 1(N) acetic acid andwashed with saturated Na2CO3 solution (10 mL). The organic ex-tracts were dried over anhydrous Na2SO4, filtered and concentrated

in vacuo. The residue was purified by silica gel (100e200 mesh)column chromatography using 20% ethyl acetate in petroleumether (v/v) as eluent to afford the product 6a (66%).

The same procedurewas adopted for the synthesis of product 6busing bromoacetyl bromide.

5.1.3. Procedure for the synthesis of 6cCompound 6a (200 mg, 0.42 mmol) was dissolved in dry

acetone and the mixture was stirred with NaI (128 mg, 0.85 mmol)for 4 h at rt under argon atmosphere. After completion of the re-action (TLC), the solvent was evaporated under reduced pressureand the residue was extracted with EtOAc (3 � 10 mL). The organicextracts were dried over anhydrous Na2SO4 and concentrated invacuo; the residue was purified by silica gel (100e200 mesh) col-umn chromatography using 20% ethyl acetate in petroleum ether(v/v) as eluent to furnish the product 6c (65%).

5.1.4. Typical procedure for the synthesis of the epoxides 7a and 8aTo an ice cooled solution of 6a (300 mg, 0.64 mmol) in dry

dichloromethane under argon atmosphere, m-chloroperbenzoicacid (143 mg, 0.83 mmol) was added portion wise over a period of30 min; the reaction mixture was then allowed to come to rt over aperiod of 40 min and stirred for another two and half hr at rt. Aftercompletion of the reaction (TLC), it was extracted with EtOAc(2 � 20 mL), and washed with saturated Na2CO3 solution (15 mL),and brine (10 mL), respectively. The organic extracts were driedover anhydrous Na2SO4, filtered and evaporated in vacuo. The res-idue obtained was purified over silica gel (100e200 mesh) using22% ethyl acetate in petroleum ether (v/v) resulting in the separa-tion of the diastereomeric epoxides 7a and 8a (2:3) in 51% yield.

The same procedure was adopted for the preparations of otherepoxides 7b/8b and 7c/8c using substrates 6b and 6c, respectivelyand these diastereomeric mixtures were also separated success-fully using silica gel (100e200 mesh) chromatography leading toisolation of 7b/8b (54%, 1:2) and 7c/8c (52%, 2:5).

The spectral data of 6aec, 7a/8a, 7b/8b and 7c/8c have beenprovided under “Supplementary material”.

5.1.5. General procedure for the deprotection of isopropylidenemoiety of intermediate 6

To a well stirred solution of compound 6 (0.05 mmol) in 1,4-dioxan (3 mL), 1.5 mL acetic acid-water (7:3, v/v) was added. Thereaction mixture was allowed to stir at room temperature for30 min. After completion of the reaction (TLC), the mixture wasevaporated under reduced pressure. The residue was mixed withwater (10 mL) and extracted with ethyl acetate (3 � 20 mL). Thecombined organic extracts were dried over anhydrous Na2SO4,filtered and concentrated under reduced pressure. The residueobtained was purified through semi preparative HPLC using amixture of water, methanol, acetic acid as mobile phase to get thedesired product 2 (69e74%).

The same procedure was adopted for the deprotection of otherintermediates 7aec, 8aec resulting in the formations of 3aec and4aec, respectively.

The spectral data of compounds 2a, 3aec, 4aeb have beenprovided under “Supplementary material”.

5.1.6. Andrographolide-14a-O-bromoacetate (2b)Yellow gum, yield 70%, IR (KBr) nmax : 3291, 2927, 1759, 1268,

1194, 1081, 1029 cm�1; 1H NMR (CDCl3, 600 MHz): d 7.07 (1H, td,J¼ 6,1.2 Hz), 5.97 (1H, d, J¼ 6 Hz), 4.89 (1H, s), 4.57 (1H, dd, J¼ 11.4,6 Hz), 4.49 (1H, s), 4.26 (1H, dd, J ¼ 11.4, 1.8 Hz), 4.17 (1H, d,J ¼ 10.8 Hz), 3.86 (1H, d, J ¼ 12 Hz), 3.82 (1H, d, J ¼ 12 Hz),3.48e3.46 (1H, m), 3.32 (1H, d, J¼ 10.8 Hz), 2.85 (1H, brd), 2.65 (1H,brd), 2.47e2.41 (3H, m),1.97 (1H, td, J¼ 12.6, 4.2 Hz),1.84e1.80 (4H,

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106104

m), 1.74e1.72 (1H, m), 1.63 (1H, brs), 1.24 (3H, s), 1.31e1.17 (2H, m),0.66 (3H, s); 13C NMR (CDCl3, 75 MHz): d 168.6, 167.1, 151.7, 146.5,123.1, 109.1, 80.4, 71.1, 69.5, 64.1, 55.7, 55.1, 42.8, 38.8, 37.6, 36.9,28.1, 25.4, 24.7, 23.6, 22.7, 15.1; MS (ESI) 495.21 [MþNa]þ. HRMS(ESI) m/z calculated for C22H32BrO6 [MþH]þ 471.1382, found471.1380.

5.1.7. Andrographolide-14a-O-iodoacetate (2c)Yellow solid, yield 69%, m.p. 148e150 �C. IR (KBr) nmax: 3275,

2923, 1753, 1250, 1073, 1028 cm�1; 1H NMR (CDCl3, 300 MHz):d 7.08 (1H, t, J ¼ 6.15 Hz), 5.94 (1H, d, J ¼ 5.4 Hz), 4.91 (1H, s),4.59e4.53 (2H, m), 4.25 (1H, d, J ¼ 10.5 Hz), 4.17 (1H, d, J ¼ 11.1 Hz),3.76 (1H, d, J ¼ 9.6 Hz), 3.69 (1H, d, J ¼ 9.6 Hz), 3.49e3.47 (1H, m),3.33e3.29 (1H, m), 2.83e2.70 (1H, m), 2.50e2.41 (4H, m),2.04e1.92 (2H, m), 1.83e1.58 (6H, m), 1.25 (3H, s), 0.68 (3H, s); 13CNMR (CDCl3, 75 MHz): d 168.8, 168.7, 151.6, 146.4, 123.2, 109.1, 80.3,70.9, 69.2, 64.05, 55.7, 56.0, 42.8, 38.8, 37.6, 36.9, 29.0, 25.5, 23.6,22.6, 15.2, �7.0; MS (ESI) 541.49 [MþNa]þ. HRMS (ESI) m/z calcu-lated for C22H31INaO6 [MþNa]þ 541.1063, found 541.1065.

5.1.8. Andrographolide-8,17-b-epoxide-14a-O-iodoacetate (4c)Colorless gum (yield 67%) IR (Neat) nmax : 3435, 2939,1755,1032,

736 cm�1; 1H NMR (CDCl3, 600 MHz): d 7.13 (1H, t, J ¼ 6.9 Hz), 5.92(1H, d, J ¼ 4.8 Hz), 4.55 (1H, dd, J ¼ 11.1, 6.3 Hz), 4.23 (1H, d,J ¼ 11.4 Hz), 4.18 (1H, d, J ¼ 10.8 Hz), 3.76 (1H, d, J ¼ 10.2 Hz), 3.72(1H, d, J¼ 10.2 Hz), 3.49e3.47 (1H, m), 3.35 (1H, d, J¼ 10.8 Hz), 2.65(1H, d, J ¼ 1.8 Hz), 2.54 (1H, brs), 2.09e2.04 (2H, m), 1.99e1.80 (3H,m), 1.78e1.67 (2H, m), 1.63e1.57 (2H, m), 1.48e1.43 (2H, m), 1.27(3H, s), 1.22e1.17 (3H, m), 0.83 (3H, s); 13C NMR (CDCl3, 150 MHz):d 168.7, 152.1, 122.5, 80.1, 70.9, 69.0, 63.9, 58.1, 54.6, 53.9, 50.1, 42.6,39.4, 37.3, 35.7, 27.4, 23.4, 22.7, 21.2, 15.3, �6.2; (ESI) 557.18[MþNa]þ. HRMS (ESI) m/z calculated for C22H31NaIO7 [MþNa]þ

557.1012, found 557.1015.

5.2. Biology

5.2.1. Maintenance of cell linesThe HEK-293 (human kidney cancer cells), HEK-293 p53 �/�,

VERO (Kidney epithelial cells of African Green monkey) and MCF-7 cells (breast cancer cells) were maintained in DMEM supple-mented with 10% FBS, 1.5 mM L-Glutamine and 1% antibiotic (100 Uof Penicillin and 10 mg of streptomycin per ml in 0.9% normal sa-line) under 5% CO2 at 37 �C in a humidified CO2 incubator. MCF-10Acells (normal breast epithelial cells) was grown in DMEM/F-12(50:50, v/v) medium supplemented with 10% (v/v) FBS, 100 U/mLof penicillin, 100 mg/mL of streptomycin, 0.5 mg/mL of hydrocorti-sone, 100 ng/mL of cholera toxin, 10 ng/mL of epidermal growthfactor and 1% (w/v) L-Glutamine in 5% CO2 at 37 �C in a humidifiedCO2 incubator. Cell culture reagents and other growth supplementswere procured from HiMedia, India; all the antibodies were pur-chased from Cell Signaling Technology, USA. SiRNA targeting p53(catalog no. # sc-37459) was purchased from Santa Cruz Biotech-nology, Inc., Santa Cruz, CA, USA.

5.2.2. Cell viability assayThe anchorage dependent viability of the cells after treatment

with the synthesized andrographolide analogues was measuredusing MTT [3-(4,5-dimethylthiazol-2yl-)-2,5-diphenyl tetrazoliumbromide] cell proliferation assay according to our earlier protocol[14e18]. Cytotoxicity of the investigational compounds wasmeasured by MTT assay. Cells were plated in 96-well plates at adensity of 10,000 cells per well/200 mL of the medium. Cultureswere treated with different concentrations of the investigationalcompounds for 48 h. The cells were washed with 1� PBS and thenMTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium

bromide) (2.5 mg/mL) was added to each well. It was then incu-bated at 37 �C for 6 h. Detergent solution (10% NP-40 with 4 mMHCL) was added to each well of culture plate and the color intensitywas measured at 570 nm using microplate reader (Berthold,Germany).

5.2.3. NF-kB luciferase assayLuciferase was used as a reporter gene to measure the activity of

promoters and to study the transfection efficiency. The Luciferaseassay was carried out according to the protocol mentioned earlier[14]. HEK-293 cells were seeded in six well plates. After reaching70% confluency, the cells were transfected with two plasmids, NF-kB (2.0 mg) and b-Gal (1.0 mg) by Calcium Phosphate method andincubated for 6e8 h. After incubation, the transfection media werereplacedwith fresh serum containingmedia and incubated for 12 h.Then the cells were treated with the analogues (2b/2c/4c) andandrographolide (25 mM) for 48 h. Finally, the cells were harvestedand washed twice with PBS, then lysed with lysis buffer and theefficiency of transfection was normalized to b-galactosidase activ-ity. Luciferase activity was measured by microplate reader (Multi-mode ELISA Reader, Tristar, Berthold technologies, Germany).

5.2.4. Analysis of cell cycle by flow cytometryA FACS analysis was carried out to confirm the apoptosis of cells

after 2b/2c/4c treatment for 48 h. After the end of the treatment,cells were harvested, washed with PBS containing RNase-A, andfixed with 70% ethanol. Later they were stained with 0.1 ml of PI(50 mg/ml) and finally analyzed by Flow Cytometry (Becton andDickinson, CA) using Cell Quest Pro Software (Becton and Dick-inson, CA).

5.2.5. Western blot analysisFor western blot analysis, approximately 5 � 105 cells/plate of

HEK-293 and HEK-293 p53�/� were seeded on 60 mm tissueculture discs, treated with the LC50 concentration of 2b/2c/4c andandrographolide and incubated for 48 h. Total cellular lysates wereprepared using modified RIPA lysis buffer (50 mM tris, 150 mMNaCl, 0.5 mM deoxycholate, 1% NP 40, 0.1% SDS, 1 mM Na3VO4,5 mM EDTA, 1 mM PMSE, 2 mM DTT, 10 mM b-glycerophosphate,50 mM NAF, 0.5% triton X-100, protease inhibitor cocktail), 80 mg ofprotein was loaded, and separated on an SDS-PAGE gel electro-phoresis apparatus. Proteins were transferred on to PVDF mem-branes and western blotting was performed by using anti-p53,anti-Bax, anti-Bcl-xL, anti-PARP, anti-NF-kB and anti-GAPDHantibodies.

5.2.6. Knockdown of p53 in HEK-293 cellsThe wild type expression level of p53 was knocked down in

HEK-293 cells according to the protocol referred earlier [18]. Inbrief, HEK-293 cells were grown on 60mm tissue culture dishes. Onattaining 70% confluency, the cells were transfected with 4 mg ofp53 SiRNA (catalog no. # sc-37459, Santa Cruz Biotechnology, Inc.,Santa Cruz, CA, USA) using Lipofectamine 2000® as the transfectionreagent in serum and antibiotic free media. The cells were thenincubated for 8 h, following which the transfection media wasreplaced with normal serum containing media. The cells were thentreated with the LC50 concentrations of the investigational com-pounds (2b, 2c and 4c) along with andrographolide and incubatedfor 48 h. Total cellular lysates were prepared using modified RIPAlysis buffer and western blot analysis was performed as mentionedabove.

5.2.7. Immunocytochemistry analysis of caspase-3 expressionHEK-293 cells were seeded in a 96 well plate and incubated at

37 �C in 5% CO2 incubator for 24 h. After attaining 70% confluence,

R. Preet et al. / European Journal of Medicinal Chemistry 85 (2014) 95e106 105

the cells were treated with the compounds at LC50 value of the 2b/2c/4c and incubated for 48 h. After incubation, the media wasremoved and washed with 1� PBS. The cells were fixed with ace-tone:methanol in 1:1 ratio and the plate was kept for 20 minat �20 �C followed by blocking with 2% BSA and triton X-100 in 1�PBS. Anti-caspase 3 antibody (cat #9662 from Cell SignallingTechnology) was added, and incubated for 2 h at 37 �C, then sec-ondary antibody conjugated to FITC was added and incubated for1 h at 37 �C. DAPI was used as a nuclear stain and fluorescent mi-croscope (Nikon, Japan) photographs were taken at 40�magnification.

5.2.7.1. Statistical analysis. A two-tailed Student's t-test wasemployed and P < 0.05 was considered to be statistically significant.

5.3. Biophysical studies

5.3.1. UVevis absorption spectroscopic studyThe UVevis absorption spectrophotometric studies were carried

out with 1 cm path length quartz cuvette using UVevis spectro-photometer; (UV-2401PC, Shimadzu). The spectra were scanned inthe wavelength range 200e400 nm. Stock solution of each of thecompounds having different concentration was prepared in 60%ethanol in 50 mM TriseHCl/NaCl buffer (pH 7.5) and then diluted tovaried concentration with the buffer. Then the absorption spectrafor different concentrations were taken to obtain the molarextinction coefficient which was used to determine the actualconcentration of the compound throughout the experiment. CT-DNA concentration was kept fixed at 50 mg/mL and the concen-tration of the compound was varied from 0 to a higher concen-tration as required for individual compounds. The DNA-compoundcomplex was incubated at 37 �C for 2 h and the absorption spectrawere taken with proper dilution in the range 200e400 nm.

5.3.2. Circular dichroism (CD) spectroscopic studyCD spectroscopic measurements were performed at 25 �C with

cuvette having path length 1 mm using CD spectrometer (Jasco J-815) with the sensitivity of 100 millidegree and in continuousscanning modewith a speed of 100 nm/min; band-width was 1 nmand the number of accumulations was three. Scans were taken from190 to 250 nm. CT-DNA (1000 mg/mL) was used for recordingspectra in the presence and absence of compound 2c (100 mM). EtBr(300 mM) was used as positive control. Spectra were recorded after2 h of incubation at 37 �C.

Acknowledgments

This work is financially supported by Council for Scientific andIndustrial Research network project (CSC0108). Partial financialsupport from Department of Science and Technology, Ministry ofScience and Technology, NewDelhi (project no: SR/S1/OC-42/2009)is gratefully acknowledged. PRM thanks CSIR (New Delhi) for anEMS grant.

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2014.07.088.

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[26] Though epoxides are considered as structural alert in drug discovery, wechose epoxidation in the derivatizations of the andrographolide; the idea isthat once the lead compound having epoxide moiety is identified, the epoxygroup could be used as synthetic handle for further lead optimization.

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