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Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent

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Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolide analogues as anti-cancer agent
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/47543975 Synthesis, cytotoxicity, and structure-activity relationship (SAR) studies of andrographolideanalogues as anti-cancer...  Article   in  Bioorganic & medicinal chemistry letters · October 2010 DOI: 10.1016/j.bmcl.2010.09.126 · Source: PubMed CITATIONS 32 READS 189 7 authors , including:Bimolendu DasIndian Institute of Chemical Biology 11   PUBLICATIONS   88   CITATIONS   SEE PROFILE Rupashree SenJohns Hopkins University 30   PUBLICATIONS   563   CITATIONS   SEE PROFILE Rajneeta RoyIndian Institute of Chemical Biology 3   PUBLICATIONS   46   CITATIONS   SEE PROFILE Mitali ChatterjeeInstitute of Post-Graduate Medical Education… 164   PUBLICATIONS   3,043   CITATIONS   SEE PROFILE All content following this page was uploaded by Mitali Chatterjee on 04 June 2014. The user has requested enhancement of the downloaded file.  Synthesis, cytotoxicity, and structure–activity relationship (SAR) studiesof andrographolide analogues as anti-cancer agent Bimolendu Das a , Chinmay Chowdhury a, ⇑ , Deepak Kumar a , Rupashree Sen b , Rajneeta Roy a , Padma Das a ,Mitali Chatterjee b a Indian Institute of Chemical Biology (CSIR), 4, Raja S.C. Mullick Road, Kolkata 700 032, India b Institute of Post Graduate Medical Education and Research, 244 B, A.J.C. Bose Road, Kolkata 700 020, India a r t i c l e i n f o  Article history: Received 17 June 2010Revised 15 September 2010Accepted 27 September 2010Available online 23 October 2010 Keywords: AndrographolideC14-esterAnalogueAnti-cancerApoptosis a b s t r a c t A series of analogues of andrographolide, prepared through chemo-selective functionalization at C14hydroxy, have been evaluated for in vitro cytotoxicities against human leukemic cell lines. Two of theanalogues ( 6a ,  9b ) exhibited significant potency. Preliminary studies on structure–activity relationship(SAR) revealed that the  a -alkylidene- c -butyrolactone moiety of andrographolide played a major role intheactivityprofile.Thestructuresoftheanalogueswereestablishedthroughspectroscopicandanalyticaldata   2010 Elsevier Ltd. All rights reserved. Mankind has benefited enormously through drugs discoveredfrom natural resources. Roughly around 50% of the currently usedanti-cancer drugs were discovered from studies on these second-ary metabolites. 1 Therefore, the development of novel therapeuticagents relies largely on the library of natural products and/or nat-ural product based molecules. Primarily this involves synthetictransformation of the lead molecule(s) allowing rapid access of complex molecular entities with diversified structures.The herb  Andrographis paniculata  Nees (Acanthaceae) is popularinIndia,ChinaandotherAsiancountriesduetoitsdifferentusesintraditional medicine. The metabolites isolated from this herb aremainly diterpenoids, flavonoids and sterols. The major constituentis andrographolide, a labdane diterpene. Extracts of this herb andits various constituents are reported to have a broad range of bio-logicalactivities,suchasantidiabetic, 2 antimalarial, 3 antibacterial, 4 antiinflammatory, 5 hepatoprotective, 6 and others. 7 Besides these,the aerial parts of   A. paniculata  have been traditionally used asmedicine to treat cancer. 8 In recent past, the potent anti-canceractivities of andrographolide have indeed been established. 9 How-ever, despite its impressive biological activities, the major draw-back of andrographolide is poor water solubility making itdifficult to prepare formulations for clinical use. So, various semi-synthetic analogues are being developed 10 and evaluated in orderto find out a better lead. Notably, Jada et al. recently reported 10c that 14-acetylandrographolide is more potent (in vitro) againstleukemia compared to andrographolide. In another report, 10d DRF3188, a novel derivative of andrographolide having  a , b -unsatu-ratedestersidechainatC14,wasshowntohavebetteranti-canceractivity than the parent molecule.During the course of studies on structural modifications of bio-active natural products for value addition, 11 we have chosenandrographolide for chemo-selective functionalization at C14 hy-droxy in order to develop the pharmacophore(s) possessing betterapoptoticindexthanandrographolide. Andrographolide 1  contains(a) two double bonds ( D 12 and D 8(17) ), (b) an a -alkylidene- c -buty-rolactone, and (c) three hydroxyl groups at C3, C14, and C19.However, we opted for selective functionalization at C14 hydroxylgroup which is allylic in nature. The idea was that transformationof andrographolide into an appropriate ester derivative (at C14)shouldincreasethe solubilityas well as activity. If esterasescleave 0960-894X/$ - see front matter    2010 Elsevier Ltd. All rights reserved.doi:10.1016/j.bmcl.2010.09.126 ⇑ Corresponding author. Tel.: +91 33 2499 5862; fax: +91 33 2473 0284. E-mail address:  chinmay@iicb.res.in (C. Chowdhury). OOOHOHHO 13 8 1417 RO possible cleavage by esterase esterase 19 Figure 1.  Ester analogue of andrographolide and possible site of cleavage.Bioorganic & Medicinal Chemistry Letters 20 (2010) 6947–6950 Contents lists available at ScienceDirect Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl  the ester bond under physiological conditions releasing androgra-pholide in vivo, the ester analogue may be considered as a pro-drug 12 (Fig. 1). In pursuance of this goal, we have synthesized anew class of andrographolide analogues having ester linkage atC14 connected through a spacer with a terminal acid moiety andevaluated their cytotoxicities (in vitro) in human leukemic celllines. The results obtained so far are reported herein.Andrographolide was isolated in high yield (0.54%) from theleaves of   A. paniculata  and used as starting material for derivatiza-tion.Thesyntheticpathwaysusedinthepresentstudyaredepictedin Scheme 1. Initially, the hydroxyl groups at C3 and C19 of andrographolide were protectedto furnish 3,19-isopropylidenean-dro- grapholide  2 , which served as the key intermediate in thepreparation of analogue library. Synthesis of the desired ester  5a wassmoothlyachievedbytreatmentofintermediate 2 withsuccinicanhydride  4a  in dry dichloromethane in the presence of catalyticamountof4-dimethylaminopyridine(DMAP)atroomtemperature.Encouraged by the result, the higher homologue  5b  was prepared(42%)usingglutaricanhydride( 4b ). Removalof theisopropylidenemoiety of the products  5a  and  5b  was carried out by exposure toaqueous acetic acid (3:7) affording the targeted analogues  6a  and 6b inyieldsof72%and86%,respectively.Inthenextphase,thisreac-tion protocol was applied for the installation of   a , b -unsaturatedester atC14of intermediate 2  usingmaleicandphthalicanhydride( 7a  and  7b ). The expectedproducts  8a  and  8b  were producedwithmoderateyields(41%ineachcase). Subsequentremovalof theiso-propylidene group from the intermediates  8a  and  8b  furnished(96%and88%)thetargetedproducts 9a  and 9b ,respectively.Inordertounderstandtheroleofthe a -alkylidenepartattachedto the c -butyrolactone ring of andrographolide for structure–activityrelationship (SAR) studies, we planned to prepare the correspondingsaturated derivatives. Accordingly, chemo-selective reduction of thedouble bond ( D 12 ) of intermediate  2  was accomplished (56% yield)using sodium borohydride in methanol instead of the costly reagentNiCl 2  as used earlier. 13 Next, we adopted our earlier strategy forattachment of ester side chain at C14 of the saturated intermediate 3  using succinic, glutaric, maleic and phthalic anhydrides ( 4a ,  4b , 7a , and 7 b ), which resulted in the formation of the intermediateproducts  10a ,  10b ,  12a , and  12b , respectively. Finally, synthesis of the targeted saturated analogues  11a – b  and  13a – b  was performedwith good yields (86–89%) by deprotection of the isopropylidenegroup as depicted in scheme 1. Purifications of the final analogueswere performed through usual silica gel chromatography followedby semipreparative HPLC. 14 The structures of the products 15 wereestablished based on spectroscopic (IR, NMR and mass spectra) andanalytical data. 16 In vitro screening andstructure–activity relationship (SAR) studies :In vitro screening was carried out in selected human leukemic celllines(U937,K562,andTHP1);paralleltothis,cytotoxicitiesagainstnormal cell lines (NIH3T3 and L132) were also checked. The OHOOHOHHOOOO( ) n OOOHO( ) n n=1,2OOOHOHHOO( ) n 6a  (n=1, 72%) 6b  (n=2, 86%)OR 2 OO 7a :R 1 =R 2 =H 7b :R 1 +R 2 =OOOHOOR 1 R 2 OOOHOHHOOR 1 R 2 OOOHOHHOO( ) n 12 (70%) 3 (56%) 5a  (n=1, 36%), 5b  (n=2, 42%) 9a : R 1 =R 2 =H (96%) 9b:  R 1 +R 2 = -CH=CH-CH=CH- (88%)138121417 4a (  n=1 ) 4b (  n=2) 4a (  n=1 ) 4b (  n=2)  Andrographolide  510a  (n=1, 50%) 10b  (n=2, 52%) 126911 iiiOOOHOOHOOOHOOHOOOOOOHO( ) n OOiiiiii iv ivOOO( ) n n=1,2OOOHOOR 1 R 2 O 478410 -CH=CH-CH=CH-OR 2 OO 7a : R 1 = R 2 = H 7b : R 1 + R 2  = 7 -CH=CH-CH=CH-OOOHOHHOOR 1 R 2 13 iiiiii 11a  (n=1, 86%) 11b  (n=2, 88%) 13a:  R 1 = R 2 = H (86%) 13b : R 1  + R 2  =R 1 R 1 8a :R 1 =R 2 =H (41%) 8b :R 1 +R 2 =-CH=CH-CH=CH- (41%) iviv 12a : R 1 = R 2  = H (41%) 12b : R 1  = R 2  =-CH=CH-CH=CH- (42%)HO 2 CHO 2 CHO 2 CHO 2 CHO 2 CHO 2 CHO 2 CHO 2 C-CH=CH-CH=CH- (89%) Scheme 1.  Synthesis of C14-ester derivatives of andrographolide. Reagents and conditions: (i) 2,2-dimethoxypropane,  p -TsOH (cat.), acetone, reflux, 2h ; (ii) sodiumborohydride, MeOH, 0  Ctort, 1h;(iii) succinic orglutaric ormaleicor phthalicanhydride( 4a  or  4b  or  7a  or  7b ), 4-dimethylaminopyridine(cat.), DCM, rt, 4–6h; (iv)aceticacid/water (7:3), rt, 30min.6948  B. Das et al./Bioorg. Med. Chem. Lett. 20 (2010) 6947–6950  standard MTS-PMS cell viability assay 17 was carried out 18 fordetermining the antiproliferative activities of the compounds syn-thesized. The results of the studies are summarized in Table 1.Interestingly, two of the synthesized analogues ( 6a  and  9b ) inhib-ited the proliferation of U937 and THP1 cells by 50% or more atconcentration less than 6.5 l M (Table 1, entries 3 and 8), while others displayed the cytotoxicity at higher concentrations. Amongthe analogues synthesized, the most active agent was  6a , while  9b was slightly weaker (Table 1, entry 3 vs 8). Thus, analogue  6a emerged as the best lead candidate.It is interesting to note that activities of the unsaturated ( D 12 )compounds ( 6a – b  and  9a – b ) are lost when the double bond be-tweenC12andC13( a -alkylidene)is reducedleadingtotheforma-tion of analogues  11a – b  and  13a – b  (Table 1, entries 9–12). This observation clearly indicates that the presence of the  a -alkylidenepart attached to the  c -butyrolactone ring is crucial for exhibitingcytotoxicity. Possibly, the activity of andrographolide and theirunsaturated analogues ( 6a – b  and  9a – b ) is associated with theirability to promote alkylation of biological nucleophiles (e.g., enzymes)through Michael addition 19 to the  a -alkylidene- c -butyrolactonemoiety. Furthermore, the results of SAR studies showthat increasein lipophilicity by protection of 3,19-hydroxyl groups of androgra-pholide has detrimental effects on the activity profile (Table 1, en-tries1vs 3, 2vs 4, 5vs 7, 6vs 8). Andrographolideitself was foundtobesignificantlyactiveagainstTHP1cellsbut onlymoderatelysoagainst U937 cells (Table 1, entry 13). The IC 50  values of analogues 6a and 9b appeartobelowercomparedtoandrographolideagainstbothcancer andnormal cell lines. Thus, thescreening studiesindi-cate that analogues  6a  and  9b  are more cytotoxic than androgra-pholide but have lower specificity (toward normal cell lines) thanandrographolide. Further optimization of these structures ( 6a  and 9b ) is being carried out to remove this lacuna.In addition, evaluation of the induction of apoptosis by ana-logue  6a  was carried out using flow cytometry 20 and confocalmicroscopy. 21 Cells (U937) treated with an IC 50  concentration(5.47 l M) of   6a  for 24h showed Annexin-V positivity that wasten fold higher than untreated cells (46.02% vs 4.6%, Fig. 2). Theconfocal images of cells similarly treated with  6a  and stained withHoechst 33258, showed formation of apoptotic bodies along withmembrane blebbing, whereas untreated cells had intact nuclei(Fig. 3).Inconclusion,inourendeavortodeveloppromisinganti-canceragent(s) based on andrographolide, we have synthesized a newfamily of andrographolide analogues and evaluated their in vitroactivities against different leukemic cell lines. The structure–activityrelationship (SAR) studies indicated that the major role is played bythe a -alkylidene- c -butyrolactonemoietypresentinandrographolide.Twooftheanalogues( 6a and 9b )werefoundtobepromisingforfur-ther structural modifications guided by the information as obtainedin the SAR studies. Studies in this direction are in progress.  Acknowledgments This work is supported by the CSIR net-work projects (NWP0009 and IAP 0001). Partial financial support from DST (projectno: SR/S1/OC-42/2009), India is also gratefully acknowledged.B.D. and D.K. thank CSIR, New Delhi, for the award of fellowships. Supplementary data Supplementarydataassociatedwiththisarticlecanbefound,inthe online version, at doi:10.1016/j.bmcl.2010.09.126. References and notes 1. (a) Bonazzi, S.; Eidam, O.; Guttinger, S.; Watch, J.-Y.; Zemp, I.; Kutay, U.;Gademann, K.  J. Am. Chem. Soc.  2010 ,  132 , 1432; (b)Newman, D. J.; Cragg, G. M.  J. Nat. Prod.  2007 ,  70 , 461; (c) Newman, D. J.; Cragg, G. M.; Snader, K. M.  J. Nat.Prod.  2003 ,  66  , 1022.2. Husen, R.; Pihie, A. H. L.; Nallappan, M.  J. Ethnopharmacol.  2004 ,  95 , 205.3. Misra, P.; Pal, N. L.; Guru, P. Y.; Katiyar, J. C.; Srivastava, V.; Tandon, J. S.  Int. J.Pharmacognosy  1992 ,  30 , 263.4. Gupta, S.; Choudhry, M. A.; Yadava, J. N. S.; Srivastava, V.; Tandon, J. S.  Pharm.Biol.  1990 ,  28 , 273.5. Madav, S.; Tanda, S. K.; Lal, J.; Tripathi, H. 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Oncol.  2003 ,  3 , 147; (b) Jiang, C.-G.; Li, J.-B.; Liu, F.-R.; Wu, T.; Yu, M.;  Table 1 Evaluation of in vitro cell growth inhibitory effects of andrographolide analogues invarious cell lines a,b,c Entry Compound Cell growth inhibition in terms of IC 50  ( l M)U937 THP1 K562 L132 NIH3T31  5a  19.53 10.01 31.39 21.72 36.692  5b  15.63 26.15 36.46 15.13 ND3  6a  5.47 5.84 25.30 8.50 10.204  6b  9.76 7.53 19.55 19.50 22.065  8a  ND 14.84 ND 31.11 ND6  8b  14.55 12.16 26.92 18.84 16.037  9a  9.72 7.77 41.95 12.78 11.338  9b  6.27 5.68 41.12 10.91 8.019  11a  NA ND ND ND ND10  11b  NA NA NA NA NA11  13a  NA NA NA NA NA12  13b  NA NA NA NA NA13 Andrographolide 12.87 6.69 41.85 53.8 44.5 a U937, THP1, and K562 are human leukemic cell lines; whereas L132 andNIH3T3 are normal cell lines. b NA means not active and IC 50  >50 l M. c ND means not detected. Figure 2.  Induction of apoptosis by analogue  6a : Untreated U937 cells (A)following treatment with  6a  (5.47 l M) for 24h (B) was co-stained with AnnexinV-FITC and propidium iodide, and analyzed by flow cytometry. Figure 3.  Morphological and nuclear changes of U937 cells upon treatment withanalogue  6a ; untreated U937 cells (C) following treatment with  6a  (5.47 l M) for24h (D) stained with Hoechst 33258 and observed under a Leica confocalmicroscope (100  ). B. Das et al./Bioorg. Med. Chem. Lett. 20 (2010) 6947–6950  6949  Xu,H.-M.  Anticancer Res.  2007 ,  27  ,2439;(c)Geethangili,M.;Rao,Y.K.;Fang,S.-H.;Tzeng,Y.-M. Phytother. Res.  2008 ,  22 ,1336;(d)Yang,L.;Wu,D.;Luo,K.;Wu,S.;Wu,P. Cancer Lett.  2009 ,  276  ,180;(e)Li,J.;Cheung,H.-Y.;Zhang,Z.;Chan,G.K. L.; Fong, W.-F.  Eur. J. Pharmacol.  2007 ,  568 , 31.10. (a)Nanduri,S.;Nyavanandi, V.K.;Thunuguntla,S.S.R.;Kasu,S.;Pallerla,M.K.;Ram, P. S.; Rajagopal, S.; Kumar, R. A.; Ramanujam, R.; Babu, J. M.; Vyas, K.;Devi, A. 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A.  ChemBiochem  2005 ,  6  , 133.12. (a) Deshmukh, M.; Chao, P.; Kutscher, H. L.; Gao, D.; Sinko, P. J.  J. Med. Chem. 2010 ,  53 , 1038; (b) Wu, Z.; Patel, A.; Dave, R.; Yuan, X.  Bioorg. Med. Chem. Lett. 2010 ,  20 , 3851; (c)Schmid, B.; Chung, D.-E.;Warnecke, A.; Fichtner, I.; Kratz, F. Bioconjugate Chem. 2007 , 18 ,702;(d)Dai,J.-R.;Hallock,Y.F.;Cardellina,J.H.,II;Boyd, M. R.  J. Nat. Prod.  1999 ,  62 , 1427.13. Dai, G.-F.; Xu, H.-W.; Wang, J.-F.; Liu, F.-W.; Liu, H.-M.  Bioorg. Med. Chem. Lett. 2006 ,  16  , 2710.14. HPLC was performed on RP C18 column (300mm  7.8mm, 7.0 l m) usingmethanol/acetonitrile/water/acetic acid (49:10:40:1 or 34:10:55:1) as mobilephase; flow rate=3ml/min;  t  R   =6.8, 8.9, 16.9, 17.2, 17.8 and 18.4min (forcompounds  6a ,  6b ,  9a ,  9b ,  13a  and  13b , respectively).15. All the synthesized final analogues were generally preserved at 0 to  4  C andremained stable for months. Even allowing them to stand at rt for severalweeks did not lead to any decomposition (HPLC analysis). Besides, few of thefinalanalogues( 6a , 9a , 11a – b , 13a )werefoundtobesolubleinchloroformbutall of them were fairly soluble in methanol and dimethyl sulfoxide (DMSO) aswell.16.  General procedure for the synthesis of acid  5  /  8  /  10  /  12  (step iii) : To a well stirredsolution of compound  2 / 3  (0.512mmol) in dry dichloromethane (8mL) wereadded acid anhydride  4 / 7  (0.615mmol) and a catalytic amount (4mg) of 4-dimethylamino-pyridine (DMAP). The whole mixture was stirred under argonatmosphere at roomtemperature for 4–6h. The solvent was evaporated underreduced pressure and the residue was purified through silica gel (100–200mesh) column chromatography (ethyl acetate–petroleum ether) to afford thedesired product  5 / 8 / 10 / 12 .  3,19-Isopropylideneandrographolide-14- a -O-succinate  5a : gum, 36% yield; IR (neat):3421,2936,1738,1676cm  1 ; 1 HNMR(CD 3 OD,300MHz): d 6.98(1H,t,  J   =6.3Hz), 6.04 (1H, d,  J   =5.1Hz), 4.93 (1H, s), 4.63–4.57 (2H, m), 4.30 (1H, d,  J   =11.1Hz), 4.04 (1H, d,  J   =11.7Hz), 3.54–3.51 (1H, m), 3.20 (1H, d,  J   =11.7Hz), 2.64 (4H, s), 2.57–2.52 (2H, m), 2.47–2.43 (1H, m), 2.07–1.97(3H, m), 1.83–1.78 (3H, m), 1.41 (3H, s), 1.36–1.28 (6H, m), 1.22 (3H, s), 0.98(3H, s);  13 C NMR (CD 3 OD, 75MHz):  d  175.5, 173.6, 171.4, 151.7, 149.0, 125.6,109.3, 100.3, 78.0, 73.1, 69.5, 64.9, 57.1, 53.4, 39.5, 38.9, 38.7, 35.5, 29.9, 29.6,27.5, 26.5, 26.2, 25.7, 24.3, 16.5; ESI-MS:  m /  z   513.01 [M+Na] + . Anal. Calcd forC 27 H 38 O 8 : C, 66.10; H, 7.81. Found: C, 66.04; H, 7.86. General procedure for the synthesis of acid  6   /  9  /  11  /  13  (step iv) : To a well stirredsolution of ester intermediate  5 / 8 / 10 / 12  (0.50mmol) in 1,4-dioxane (3mL)was added a solution (3mL) of acetic acid/water (7:3, v/v). The whole reactionmixture was allowed to stir at room temperature for 30min and the solventswere evaporated under reduced pressure. The residue was mixed with water(10mL) and extracted with ethyl acetate (3  20mL). The combined organicextracts were washed with brine (10mL), dried over anhydrous Na 2 SO 4 ,filtered, and concentrated under reduced pressure. The residue was purifiedthrough silica gel (100–200 mesh) column chromatography (ethyl acetate–petroleum ether) followed by HPLC (see Ref. 14 for details).  Andrographolide-14 a -O-succinate  6a : gum, 72% yield; IR (neat): 3425, 2938,1739cm  1 ;  1 H NMR (CDCl 3 , 300MHz):  d  7.06 (1H, t,  J   =6.4Hz), 5.94 (1H, d,  J   =5.1Hz), 4.89 (1H, s), 4.56 (1H, dd,  J   =11.5, 6.4Hz), 4.53 (1H, s), 4.25–4.17(2H, m), 3.93 (2H, br), 3.52–3.49 (1H, m), 3.33 (1H, d,  J   =10.8Hz), 2.74–2.64(4H, m), 2.50–2.39 (3H, m), 2.00–1.96 (1H, m), 1.82–1.73 (5H, m), 1.30–1.13(6H, m), 0.67 (3H, s);  13 C NMR (CDCl 3 , 75MHz):  d  175.7, 172.1, 169.2, 150.9,146.5, 123.6, 108.7, 80.2, 71.5, 68.0, 64.0, 55.7, 54.9, 42.4, 38.7, 37.5, 36.7, 28.7,28.6, 27.8, 25.1, 23.6, 22.6, 14.9; ESI-MS:  m /  z   473.14 [M+Na] + . Anal. Calcd forC 24 H 34 O 8 : C, 63.98; H, 7.61. Found: C, 64.02; H, 7.56.17. Ganguly, S.; Bandyopadhyay, S.; Sarkar, A.; Chatterjee, M.  J. Microbiol. Methods 2006 ,  66  , 79.18.  Method of MTS-PMS cell viability assay : The anti-proliferative activity of andrographolide and its analogues were evaluated in U937, THP1, K562,NIH3T3 and L132 cells using MTS-PMS assay (see Ref. 17 for details). Briefly,cells (2.5–5.0  10 4 /200 l L) were seeded in 96-well tissue culture plates andincubated with compounds (0–50 l M) for 48h at 37  C, 5% CO 2 . Followingtreatment, MTS [3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulphonyl)-2 H  -tetrazolium, inner salt] (2.0mg/ml) and PMS (phenazinemethosulphate) (0.92mg/ml) were added in a ratio of 10:1 (20 l L per well).After incubation for 3h at 37  C, the resulting absorbances at 490nm weremeasured in an ELISA reader. The specific absorbance that representedformazan production was calculated by subtraction of backgroundabsorbance from total absorbance. The mean % viability was calculated asfollows:  Mean specific absorbance of treated cells  100/Mean specific absorbanceof untreated cells The results were expressed as IC 50  values, that is, the concentration thatinhibited50%ofcellgrowth,enumeratedbygraphicextrapolationusingGraphpad prism software (version 5).19. (a) Kundu, N. G.; Dasgupta, S. K.  J. Chem. Soc., Perkin Trans 1  1993 , 2657; (b)Finer-Moore, J. S.; Montfort, W. R.; Stroud, R. M.  Biochemistry  1990 ,  29 , 6977;(c) Hardy, L. W.; Finer-Moore, J. S.; Montfort, W. R.; Jones, M. O.; Santi, D. V.;Stroud, R. M.  Science  1987 ,  235 , 448.20.  Flow cytometric analysis : Double staining for Annexin V-FITC and propidiumiodide (PI) was performed. Briefly, U937 cells were incubated without or with 6a  (IC 50 =5.47 l M) for 24h at 37  C, 5% CO 2 . Cells were then washed twice inphosphate bufferedsaline (PBS, 0.02M, pH7.2) andresuspendedinAnnexin-Vbinding buffer (10mM HEPES, 140mM NaCl, 2.5mM CaCl 2 , pH 7.4). AnnexinV-FITCandpropidiumiodidewerethenaddedaccordingtothemanufacturer’sinstructions and incubated for 15min in the dark at 25  C. Data was acquiredusing a FACS Calibur flow cytometer and analyzed with Cell Quest Prosoftware.21.  Confocal microscopy : Apoptotic cells were characterized by nuclearcondensation of chromatin and/or nuclear fragmentation. Briefly, U937 cellswere incubated with an IC 50  concentration (5.47 l M) of   6a  (for 24h), washedwithicecoldPBSandstainedwithHoechst33258(10 l g/ml,30min).Thecellswere mounted on poly  L  -lysine coated slides and analyzed in a laser scanningconfocal microscope (Leica TCS SP2 system, Leica microsystem, Heidelberg,Germany;100  ).Atleast20microscopicfieldswereobservedforeachsample.6950  B. Das et al./Bioorg. Med. Chem. Lett. 20 (2010) 6947–6950 View publication statsView publication stats
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