Bioorganic & Medicinal Chemistry Letters 24 (2014) 3865–3868

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Bioorganic & Medicinal Chemistry Letters

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

Synthesis and evaluation of triazole linked glycosylated18b-glycyrrhetinic acid derivatives as anticancer agents

http://dx.doi.org/10.1016/j.bmcl.2014.06.0540960-894X/� 2014 Elsevier Ltd. All rights reserved.

⇑ Corresponding authors. Tel.: +91 33 2569 3240; fax: +91 33 2355 3886.E-mail addresses: kuladip.jana@gmail.com (K. Jana), kaushik@jcbose.ac.in

(K. Biswas), akmisra69@gmail.com (A.K. Misra).1 Both the authors contributed equally.

Pravat Kumar Parida a,1, Abhijit Sau a,1, Tamashree Ghosh a, Kuladip Jana a,⇑, Kaushik Biswas a,⇑,Sanghamitra Raha b, Anup Kumar Misra a,⇑a Bose Institute, Division of Molecular Medicine, P 1/12, C.I.T. Scheme VII M, Kolkata 700054, Indiab Visva Bharati University, Centre of Biotechnology, Santiniketan, West Bengal, India

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

Article history:Received 15 April 2014Revised 18 June 2014Accepted 19 June 2014Available online 27 June 2014

Keywords:18b-Glycyrrhetinic acidTriazoleCarbohydrateClick chemistryAnticancer agent

A series of glycosyl triazol linked 18b-glycyrrhetinic acid (GA) derivatives have been synthesized using1,3-dipolar cycloaddition reaction of per-O-acetylated glycosyl azide derivatives (4a–h) with propargylester of 18b-glycyrrhetinic acid (GA) (2 and 3) following the concept of ‘Click chemistry’. The synthesizedtriazole derivatives were de-O-acetylated to furnish compounds (7a–h and 8a–c) with free hydroxylgroups in the carbohydrate moieties, which were evaluated for their anticancer potential against humancervical cancer cells (HeLa) and normal kidney epithelial (NKE) cells. GA (1), compound 7d, compound 7gand compound 8c showed promising anticancer activities.

� 2014 Elsevier Ltd. All rights reserved.

For a long time, Licorice (glycyrrhiza glabra) root has been usedas traditional medicine1 in various parts of the world because of itswide range of biological activities.2 It has also been used in the foodand beverage industries as herbal sweetener3 and flavouringagents.4 Licorice root contains a large number of bioactive com-pounds such as, flavonoids, saponins and polyphenols. Glycyrrhizin(GN) and its triterpenoid metabolite, 18b-glycerrhetinic acid (GA;1) are abundantly present in the Licorice roots and responsiblefor its various therapeutic properties.5 Natural products serve asimportant feedstock in the medicinal chemistry for the develop-ment of novel therapeutics.6,7 From the earlier studies, it wasobserved that the therapeutic properties of 18b-glycerrhetinic acid(GA; 1) and its precursor glycyrrhizin (GN) emerge from the triter-pene skeleton present in them.8 Although a diverse range of GAderivatives have been prepared and evaluated for their therapeuticpotential,8 a lot of scopes remain to utilize GA derivatives in thedevelopment of novel therapeutics.

Cervical cancer is a frequently found cancer amongst womenthroughout the world and most of the preventive strategies arequite expensive.9 Many of the chemotherapeutic agents used forthe treatment of cancer have been found to be associated with poor

prognosis, largely due to their cytotoxic effects on normal cells aswell.10,11 The current thrust in the medicinal chemistry is todevelop novel therapeutics with improved efficacy and low toxic-ity by the chemical modifications of natural sources. Till date, alarge number of chemotherapeutic agents used for the treatmentof cancer are derived from the natural products. Based on the ther-apeutic potentials of GA reported earlier,1,2,8 it was envisaged thatdevelopment of glycosylated triazolyl GA derivatives could lead tonovel molecules of improved therapeutic potential. Since the car-boxylic functional group of GA remained unaffected in most ofthe earlier attempts of its derivatizations, it was decided to deriv-atize GA using the carboxylic group present in it. In the recent past,‘Click chemistry’ has been applied for the coupling of alkyne andazide derivatives to make triazole derivatives of various naturalproducts with improved medicinal properties.12 Synthesis of anovel series of glycosylated triazolyl 18b-glycerrhetinic acid deriv-atives by the reaction of glycosyl azide derivatives with propargylester of 18b-glycerrhetinic acid under copper catalyzed clickreaction conditions and their evaluation as anticancer agents ispresented in this communication.

Reaction of 18b-glycyrrhetinic acid (GA; 1) with propargylbromide in the presence of sodium hydride in DMF furnished prop-argyl ester of 18b-glycyrrhetinic acid (2) as the major product(69%) together with a minor quantity (15%) of dipropargylated18b-glycyrrhetinate derivative (3). Treatment of compound 2 with2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl azide13 (4a; 2 equiv) in

3866 P. K. Parida et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3865–3868

the presence of copper(II) sulfate pentahydrate and D-glucose inDMSO–water (1:1, v/v) at 80 �C furnished 2,3,4,6-tetra-O-acetyl-b-D-glucopyranosyl-1,2,3-triazolyl-18b-glycyrrhetinate (5a). Since,D-glucose has been used as a reducing agent in aqueous reactionconditions14,15 earlier, it was decided to test the reducing potentialof D-glucose in the copper mediated aqueous 1,3-cycloadditionreaction. After a set of experimentation, it was observed that1,4-substituted 1,2,3-triazole derivative (5a) was formed in 90%yield by the reaction of compound 2 (1 mmol) with compound4a (1.1 mmol) in the presence of CuSO4�5H2O (2.5 mL, 0.1 M aqsoln) and D-glucose (2.5 mL, 2 M aq soln) in DMSO–H2O (1:2) at70 �C in 30 min. A series of glycosyl 1,2,3-triazolyl GA derivatives(5a–h) were synthesized in excellent yields by the reaction of com-pound 2 with a series of glycosyl azides (4a–h)13 following theoptimized reaction conditions. Similarly, di-propargylated GAderivative (3) was also reacted with 4a, 4b and 4d to give bis-triaz-olyl derivatives (6a–c) in excellent yields. Exclusive formation of1,4-substituted 1,2,3-triazole derivatives between glycosyl azidesand GA-propargyl ester (2 and 3) was observed under the reactionconditions, which was confirmed from the NMR spectral analysis.Appearance of C4 and C5 of the triazole moieties in the range of d147.1 and 120.1–120.8, respectively, in the 13C NMR spectra unam-biguously confirmed the formation of 1,4-disubstituted 1,2,3-tria-zole derivative.16 Finally, de-O-acetylation of compounds 5a–hand 6a–c using sodium methoxide furnished compounds 7a–hand 8a–c with free hydroxyl groups in the sugar moieties in quan-titative yield (Scheme 1). Synthesized compounds have been eval-uated for their anticancer potential using various biological assays.

In order to address the comparative efficacy as anti-canceragents, 18b-glycerrhetinic acid (GA; 1) and its natural precursor,glycyrrhizin (GN) as well as synthesized compounds (7a–h and

18β-Glycyrrhetinic acid (

HO

O

H H

OO

H

O

O

H

2 (69%)

+

HO

O

H H

OO

H N NN R

5a-h

R =

a

b

per-O-acetylated-β-D-glucosyl (a) (90%)per-O-acetylated-β-D-galactosyl (b) (92%)per-O-acetylated-α-D-mannosyl (c) (90%)per-O-acetylated-α-L-rhamnosyl (d) (86%)per-O-acetylated-β-L-fucosyl (e) (84%)per-O-acetylated-β-D-lactosyl (f) (85%)per-O-acetylated-β-D-cellobiosyl (g) (82%)per-O-acetylated-β-D-maltosyl (h) (85%)

HO

O

H H

OO

H NN

N R

7a-h

R

c Quantitative

RN3 (4a-h)

Scheme 1. Reagents and conditions: (a) propargyl chloride, NaH (70% oil coated), DMF, rocompounds 5a–e; 60 min for compounds 5f–h and 6a–c; (c) 0.1 M CH3ONa, CH3OH, roo

8a–c) were assessed for their ability to block proliferation ofhuman cervical cell line (HeLa) in comparison to the normal kidneyepithelial cell line (NKE). Resveratrol, which is well known to blockcancer cell proliferation17 was used as a positive control in thisexperiment. Both HeLa as well as NKE cells were treated with var-ied concentrations of the compounds and cell proliferation wasmeasured by MTT assay.18,19 The antiproliferative activity of thetest compounds in terms of IC50 is presented in Table 1. Compara-tive efficacy of the compounds which significantly inhibit the pro-liferation in HeLa cells are shown in Figure 1a. It was observed thatboth compound GA (1) and 7d showed significantly higher efficacy(IC50 12.22 and 13.76 lM, respectively) in blocking proliferation ofHeLa cells in comparison to Resveratrol (IC50 20.35 lM) as well asfound ineffective in blocking the NKE proliferation (IC50 values ofGA (1), 7d and Resveratrol were 47.38, 61.30 and 46.57 lM,respectively) (Fig. 1b, Table 1). Since compound 7g and glycyrrhi-zin (GN) displayed comparable efficacy in blocking proliferationof both HeLa and NKE cells [(IC50 = 22.49 and 22.41 lM, respec-tively) and (IC50 = 37.77 and 29.32 lM, respectively)], they werenot considered for further biological evaluation. In comparison toGA (1) and compound 7d, compound 8c showed only moderateIC50 values for HeLa (21.50 lM) and NKE cells (50.56 lM) andwas not considered for further evaluation. GA (1), compound 7dand Resveratrol were used for further biological activities toestablish their anticancer potential.

Since, the inhibition of proliferation is often associated with theincrease in apoptosis, compounds 1, 7d and Resveratrol (positivecontrol) were selected for testing their ability to induce apoptosisof the HeLa cells.20 The induction of apoptosis was measured byflow cytometric estimation of annexinV/PI staining,21,22 which ispresented in Figure 2. An increased induction of apoptosis was

1)

H

OO

H

3 (15%)

O

O

H H

OO

H

6a-c

NNNR

N NN R

R =

b

per-O-acetylated-β-D-glucosyl (a) (85%)per-O-acetylated-β-D-galactosyl (b) (85%)per-O-acetylated-α-L-rhamnosyl (c) (80%)

O

O

H H

OO

H

8a-c

NNN

NN

N R

c Quantitative

om temperature, 4 h; (b) CuSO4�5H2O, D-glucose, DMSO–H2O (1:2), 70 �C, 30 min form temperature, 3 h.

Table 1The IC50 values of the compounds for the inhibition of proliferation of HeLa and NKEcells

Compounds IC50 (lM)

HeLa cells NKE cells

GA (1) 12.22 ± 5.63 47.38 ± 7.85GN 37.77 ± 4.12 29.32 ± 6.037a >40 >407b >40 >407c >40 31.77 ± 5.877d 13.76 ± 5.30 61.30 ± 8.737e >40 27.897f >40 >407g 22.49 ± 5.58 22.41 ± 8.827h >40 >408a >40 34.77 ± 8.888b >40 >408c 21.50 ± 2.24 50.56 ± 5.70Resveratrol 20.35 ± 3.59 46.57 ± 7.08

Figure 1. (a) Comparative efficacy of GA (1), 7d, 7g, 8c and Resveratrol in inhibitingproliferation of HeLa cells and (b) comparative efficacy of GA (1), 7d, 7g, 8c andResveratrol in inhibiting proliferation of NKE cells measured by MTT proliferationassay. Data represented are mean ± SEM of three separate experiments.

Figure 2. (a) Apoptosis inducing ability of the compounds GA (1) and 7d by flowcytometric analysis of annexinV/PI staining; (b) quantitative presentation ofapoptosis inducing ability of the GA (1) and 7d by density plot; (c) apoptosisinduction in HeLa cells in response to the compounds GA (1) and 7d using ELISAbased apoptosis detection assay; (d) microscopically visual confirmation ofapoptosis by TUNEL assay where the apoptotic cells stain fluorescent green(indicated by the arrows).

P. K. Parida et al. / Bioorg. Med. Chem. Lett. 24 (2014) 3865–3868 3867

observed in the HeLa cells treated with compound 7d compared toGA (1) treated cells (Fig. 2a and b). Resveratrol displayed compara-ble induction in apoptosis as in the case of compound 7d. Apopto-tic abilities of compounds GA (1) and 7d in HeLa cells were alsoquantitatively measured using an ELISA based apoptosis detectionkit. It works by the sensitivity of the apoptotic DNA to the formam-ide denaturation leading to the single stranded DNA and detectedby using an antibody.23 Although, significant induction of apopto-sis in both GA (1) and compound 7d was observed, compound 7d

displayed more apoptotic ability on HeLa cells compared to GA(1) (Fig. 2c). Visual confirmation of apoptotic ability of compounds

Figure 3. Mitochondrial damage in HeLa and NKE cell lines by measuring thechanges in mitochondrial permeability using flow cytometric analysis of JC1 stainedcells. Data are representative of three independent experiments and bar graphshows mean ± SEM (⁄⁄p <0.01, ⁄⁄⁄p <0.001).

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1 and 7d was also achieved by using a Terminal Deoxynucleotidyltransferase dUTP Nick End Labeling (TUNEL) assay using a TUNELkit.24 Significantly higher number of TUNEL positive cells (FITC+ve) were observed in the cells treated with compound 7d thanGA (1) treated cells (Fig. 2d).

Since, the mitochondria plays a vital role in apoptotic events,20

it was investigated whether the observed apoptosis induction inresponse to the compounds was preceded by the mitochondrialdamage. The mitochondrial damage in the HeLa cells and NKE cellswas assessed by induction of mitochondrial permeability. A drasticalteration of the redox status of the mitochondria in the cells inresponse to the compounds GA (1), 7d and Resveratrol wereestimated by flow cytometric evaluation of JC-1 staining, whichis presented in Figure 3. The left panel in Figure 3 shows clearchange in the mitochondrial redox status in HeLa cells, which isevident from the shift of red to green fluorescence or a decreasein the red/green ratio, indicating the increase in the mitochondrialpermeability in response to the compounds (⁄⁄⁄p <0.001 vs DMSO).Compound 7d induced the most mitochondrial damage, as evidentfrom the lowest red/green ratio (Fig. 3, left panel). Although, com-pound 7d induced significant mitochondrial damage in HeLa cells,it did not cause any significant alterations in the mitochondrialpermeability of NKE cells as evident from the minimal shift inred/green fluorescence ratio (Fig. 3; right panel). However, GA (1)induced significant mitochondrial damage in the NKE cells as well(⁄⁄p <0.01) (Fig. 3, right panel). These results indicate that com-pound 7d is effective in causing mitochondrial damage specificallyto the cancer cells without affecting the normal cells.

In summary, a series of novel glycosylated 1,2,3-triazol linked18b-glycyrrhetinic acid derivatives were synthesized in excellent

yields. The reaction condition is operationally simple, mild, repro-ducible and high yielding. D-Glucose has been used as a reducingagent in the cycloaddition reaction for the first time. Synthesizedcompounds were evaluated for their anticancer potential againstHeLa cells as well as normal cells (NKE). Among the compoundstested, GA (1) and 7d were found to have promising therapeuticpotential against cervical cancer to be considered for their furtherbiological studies in details, which are under progress and will bedisclosed in due course.

Acknowledgments

P.P., A.S. and T.G. thank CSIR, New Delhi for providing researchfellowships. This work is supported by CSIR, New Delhi [Project No.02(0038)/11/EMR-II (AKM)] and Bose Institute.

Supplementary data

Supplementary data ((i) experimental reaction conditions forthe synthesis of compounds, reaction protocols for the biologicalassays and copies of NMR spectra of all synthesized acetylatedderivatives (1, 2, 3, 5a–h and 6a–c).) associated with this articlecan be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.06.054.

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