Synthesis and Cytotoxicity Studies of Novel Triazolo-Benzoxazepine as New Anticancer Agents

Synthesis and Cytotoxicity Studies of Novel Triazolo-Benzoxazepine as New Anticancer Agents

Biswadip Banerji1,*, Sumit Kumar Pramanik1,Priyankar Sanphui2, Sameer Nikhar1 and SubhasC. Biswas2

1Department of Chemistry, CSIR-Indian Institute ofChemical Biology, 4 Raja S.C. Mullick Road, Kolkata,700032, India2Department of Cell Biology & Physiology, CSIR-IndianInstitute of Chemical Biology, 4 Raja S.C. Mullick Road,Kolkata, 700032, India,*Corresponding author: Biswadip Banerji,[email protected]

Cancer continues to be one of the biggest threats tothe human civilization because there is no cure of it.Small heterocyclic molecule with low molecular weightand novel structural feature is therapeutically highlydemanding. These molecules have the capability todisrupt signaling pathways leading to anticancer activi-ties. Therefore, the search for new anticancer agentscontinues to draw attention to the research commu-nity. In this study, a small triazolo-benzoxazepine scaf-folds was synthesized using a one-pot four-stepsynthetic methodology involving click reaction. Smalllibraries of 12 compounds were successfully synthe-sized and screened them against different cancer celllines. Low micromolar anticancer activity was recordedusing MTT assay, and further confirmation of cell deathwas obtained by phase contrast, fluorescent, and con-focal images.

Key words: biological screening, chemical biology, drugdesign

Received 15 December 2012, revised 25 April 2013 andaccepted for publication 3 May 2013

The continuous discovery of novel heterocyclic scaffoldsprovides new tools to modulate or modify various diseasestates (1–3). The advantage of a new scaffold is that itmay disrupt a signal pathway or block an active site of anenzyme (4–6). Thus, synthesis of new hetereocycles bynewer and easier techniques draws attention to the syn-thetic organic chemists. Medium-sized ring-fused hetero-cycle exhibits lots of important biological properties (7–10).Moreover, attaching suitable substituent as well as install-ing another fused five- or six-member rings on the scaffoldhas enhanced the activity many times. In recent times,

chandrasekhar’s group had shown the synthesis of triazol-o-benzoxazepine and briefly studied their antifungal andantibacterial properties (11). In our laboratory, in an ongo-ing program on the search for new anticancer agents, wewere interested to synthesize new ring-fused heterocyclesand test their efficacies against different cancer cell lines.Benzoxazepine is one of the privileged heterocycle scaf-folds, which have not yet been extensively studied as anti-cancer agents (12–14). Moreover, we believe that triazolering–fused benzoxazepine may act as new scaffolds. Thus,we were interested to synthesize a small library of triazolering–fused benzoxazepine scaffolds with different substitu-ents installed on the smaller ring (15,16). Here, in thisstudy, we report a one-pot efficient synthesis of theabove-mentioned scaffold by a four-step organic reactionsand disclose their hitherto unknown anticancer properties.For the synthesis of the said scaffold, we have used silvercocatalyzed Sonogashira coupling and click reaction asthe first and the last step, respectively. In all 12 examplesof triazolo-benzoxazepines were synthesized by this proto-col and screened against Neura 2a (neuroblastoma cell),Hek 293 (kidney cancer), and Hep G2 (liver cancer) celllines. The anticancer property of these molecules was fur-ther investigated by taking images from inverted phasecontrast microscope, fluorescent microscope, and confo-cal studies.

Methods and Materials

ChemistryAll the reagents and solvents were purchased from AcrosOrganics (Geel, Belgium). Solvents were freshly distilled bystandard procedures prior to use. Flash chromatographywas performed on silica gel (Merck, 100–200 mesh) as thestationery phase. All 1H and 13C-NMR spectra wererecorded on a Bruker 300 and 600 MHz spectrometer.For 1H NMR, tetramethylsilane (TMS) served as internalstandard (d = 0), and data are reported as follows: chemi-cal shift, integration, multiplicity (s = singlet, d = doublet,t = triplet, q = quartet, m = multiplet), and coupling con-stant(s) in Hz. For 13C NMR, TMS (d = 0) or CDCl3(d = 77.26) was used as internal standard, and spectrawere obtained with complete proton decoupling. Massspectra were obtained on a Jeol MS station 700. IR Spec-tra were recorded on a JASCO FT/IR–4200 spectrometer.Melting points were recorded on a Yanaco MP-500

ª 2013 John Wiley & Sons A/S. doi: 10.1111/cbdd.12164 1

Chem Biol Drug Des 2013

Research Article

micro-melting point apparatus, and the crystal structuresof these compounds were recorded on Bruker Smart-APEX II CCD Diffractometer.

One-pot sequential reaction protocol to synthesizetriazolo-benzoxazepinesAlkyne substrate (1) (1.61 g, 10 mmol) was dissolved inDMF (10 mL) with AgCl (143 mg, 1 mmol), Pd(PPh3)2Cl2(70.1 mg, 0.1 mmol), TEA (30 mmol), and different aryliodides (1.2 equiv.) at room temperature. After 1-h stirring,the reaction mixture was cooled to 0 °C, and aq. NaNO2

(3 equiv.)/HCl [1 (M) 2 mL] was added, and the mixturewas stirred for 0.5 h. After this period, aq. NaN3 solutionwas added drop wise into it and stirred vigorously for20 min. Finally, AgCl (0.1 equiv.) was added to it andheated to 80 °C for 8 h. In all the steps, reaction wasmonitored by checking TLC, and finally, the triazole prod-uct was formed (Rf = 0.6 at ethyl acetate: hexane = 7:1).It was extracted with ethyl acetate, washed with brine(3 9 10 mL) solution. After that the organic layer wasseparated, dried over sodium sulfate, filtered, and thenevaporated under reduced pressure. The residuewas finally purified by column chromatography (silica gel100–200, ethyl acetate-hexane) to get the correspondingtriazolo-benzoxazepine as pale yellow solid in 40–45%yield.

3-Naphthalen-1-yl-4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4a)Light yellow solid, m.p. = 191.5–192.6 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 4.42 (2 H, s), 4.63 (2 H, s),7.23–7.11 (3 H, m), 7.38–7.32 (3 H, m), 7.69–7.51 (3 H,m), 7.97–7.93 (1 H, m), 8.10 (1 H, d, J = 8.4); 13C NMR(150 MHz, DMSO): d (in ppm) 56.49, 70.33, 122.24,123.84, 125.84, 126.64, 127.74, 127.84, 127.94, 128.60,128.66, 128.70, 129.98, 132.07, 137.17, 137.30; HRMS(EI+): m/z Calcd for C20H15N3O (M)+ 313.1215, Found: m/z 313.1206; FTIR (c/cm, KBr): 3382, 3065, 2925, 2857,2430, 2122, 1723, 1587, 1488, 1456, 1286, 1124, 1043,754.

4-(4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine)-benzoic acid methyl ester (4b)Light yellow solid, m.p. = 216.3–217.2 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 3.88 (3 H, s), 4.56 (2 H, s),4.77 (2 H, s), 7.46–7.45 (2 H, m), 7.60–7.56 (1 H, m),7.81 (2 H, d, J = 8.4), 8.01 (1 H, d, J = 7.8), 8.10 (2 H, d,J = 8.4); 13C NMR (75 MHz, CDCl3): d (in ppm) 52.23,58.66, 68.26, 122.42, 127.29, 129.05, 129.56, 130.01,130.27, 130.54, 130.66, 130.86, 134.65, 136.69, 144.42,166.67; HRMS (FAB+): m/z Calcd for C18H16N3O3 (M + H)+

322.1192, Found: (m + H)/z 322.1190; FTIR (c/cm, KBr):3423, 2918, 2857, 1657, 1436, 1313, 1027, 953, 754.

3-(4-Nitro-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4c)Light yellow solid, m.p. = 172.6–173.4 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 4.35 (2 H, s), 4.68 (2 H,s), 6.74–6.67 (2 H, m), 7.16–7.11 (4 H, m), 7.34 (2 H,d, J = 7.8); 13C NMR (150 MHz, DMSO): d (in ppm)56.70, 67.21, 121.59, 122.29, 123.83, 125.88, 126.67,127.20, 127.76, 127.92, 128.73, 130.04, 137.35,154.53; HRMS (EI+): m/z Calcd for C16H12N4O3 (M)+

308.0909, Found: m/z 308.09O1; FTIR (c/cm, KBr):3486, 2918, 2850, 1689, 1627, 1460, 1293, 1206,1095.

3-p-Tolyl-4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4d)Light yellow solid, m.p. = 222.1-223.4 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 2.35 (3 H, s), 4.56 (2 H, s),4.70 (2 H, s), 7.10–6.98 (2 H, m), 7.39–7.23 (1 H, s),7.44–7.37 (2 H, m), 7.62–7.53 (2 H, m), 8.01 (1 H, d,J = 7.8); 13C NMR (75 MHz, CDCl3): d (in ppm) 21.47,58.67, 68.17, 118.07, 122.35, 124.75, 127.46, 129.04,129.68, 130.11, 130.47, 131.68, 136.94, 138.52, 145.62;HRMS (FAB+): (m + H)/z Calcd for C17H16N3O (M)+

278.1293, Found: (m + H)/z 278.1294; FTIR (c/cm, KBr):3380, 3030, 2922, 2857, 2432, 2238, 2122, 1910, 1731,1587, 1492, 1459, 1354, 1289, 1109, 1077, 896, 818,760.

3-(2-Bromo-phenyl)-4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4e)Light yellow solid, m.p. = 194.5–195.8 °C, 1H NMR(300 MHz, DMSO): d (in ppm) 4.64 (2 H,s), 4.68 (2 H, s),7.31 (1 H, t, J = 7.8), 7.58–7.41 (4 H, m), 7.73–7.60 (2 H,m), 8.11 (1 H, d, J = 7.8); 13C NMR (75 MHz, CDCl3): d(in ppm) 58.90, 68.26, 123.64, 127.59, 128.30, 128,80,129.27, 130.33, 130.51, 130.87, 131.87, 132.36, 133.18,136.69, 144.86; HRMS (FAB+): (m + H)/z Calcdfor C16H13BrN3O (M + H)+ 342.0242, Found: m/z342.0228; FTIR (c/cm, KBr): 3061, 2962, 2929, 2859,1724, 1607, 1486, 1447, 1376, 1279, 1202, 1120, 1081,983, 902, 761.

3-(3-Bromo-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4f)Light yellow solid, m.p. = 189.5–190.1 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 4.43 (2 H, s), 4.67 (2 H,s), 7.20–7.03 (2 H, m) 7.35–7.28 (1 H, s), 7.93–7.40(3 H, m), 7.95 (2 H, d, J = 8.4); 13C NMR (75 MHz,CDCl3): d (in ppm) 60.91, 68.22, 117.97, 124.73,128.18, 128.65, 129.08, 129.44, 129.56, 129.86,130.32, 133.20, 136.08, 138.01; HRMS (EI+): m/zCalcd for C16H12BrN3O (M)+ 341.0164, Found: m/z341.0151.

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Banerji et al.

3-(5-Methyl-2-nitro-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4g)Yellow solid, m.p. = 179.7–180.2 °C, 1H NMR (300 MHz,DMSO): d (in ppm) 2.42 (3 H, s), 4.62 (2 H, s), 4.81 (2H, s), 7.31 (2 H, d, J = 7.8), 7.51 (2 H, d, J = 3.8), 7.93–7.51 (2 H, m), 8.07 (1 H, d, J = 8.1); 13C NMR(150 MHz, CDCl3): d (in ppm) 22.82, 58.09, 69.04,121.36, 122.43, 129.34, 130.13, 130.47, 130.69, 131.31,131.96, 132.07, 132.90, 136.67, 137.01, 138.32, 144.01;HRMS (EI+): m/z Calcd for C17H14N4O3 (M)+ 322.1066,Found: m/z 322.1071; FTIR (c/cm, KBr): 3356, 2926,2430, 2123, 1728, 1584, 1487, 1453, 1290, 1095, 1040,753.

3-(4-Trifluoromethyl-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4h)Light yellow solid, m.p. = 201.8–202.0 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 4.26 (2 H, s), 4.60 (2 H,s), 6.67–6.12 (2 H, m), 7.10–7.03 (2 H, m); 7.25–7.18 (3H, m), 7.38 (1 H, d, J = 8.7); 13C NMR (150 MHz,CDCl3): d (in ppm) 58.74, 68.06, 123.49, 126.31,126.37, 126.56, 126.29, 128.06, 128.63, 128.74,131.35, 132.89, 134.98, 136.96, 146.34; HRMS (FAB+):(m + H)/z Calcd for C17H13F3N3O (M + H)+ 332.1011,Found: (m + H)/z 332.1005; FTIR (c/cm, KBr): 2920,2864, 1727, 1610, 1460, 1376, 1324, 1166, 1115,1069, 895, 844, 759.

3-Thiophen-2-yl-4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4i)Light yellow solid, m.p. = 219.8–220.0 °C, 1H NMR(600 MHz, CDCl3): d (in ppm) 4.64 (2 H, s), 4.94 (2 H, s),7.16 (1 H, t, J = 5.4) 7.42 (1 H, d, J = 5.4), 7.45 (1 H, d,J = 8.4), 7.51–7.47 (2 H, m), 7.62 (1 H, t, J = 6.6), 8.11 (1H, d, J = 7.8); 13C NMR (150 MHz, CDCl3): d (in ppm)59.54, 68.64, 122.41, 125.37, 126.08, 127.78, 129.28,130.38, 131.99, 133.60, 136.68, 140.27, 144.35; HRMS(EI+): m/z Calcd for C14H11N3OS (M)+ 269.0623, Found: m/z 269.0622.

3-(2-Bromo-5-methyl-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4j)Light yellow solid, m.p. = 198.6–199.4 °C, 1H NMR(600 MHz, CDCl3): d (in ppm) 2.36 (3 H, s), 4.64 (2 H,s), 4.69 (2 H, s), 7.13 (1 H, d, J = 8.4), 7.38 (1 H, s),7.52–7.48 (2 H, m), 7.58 (1 H, d, J = 7.8), 7.64–7.62(1 H, m), 8.11 (1 H, d, J = 7.8); 13C NMR (150 MHz,CDCl3): d (in ppm) 20.80, 58.98, 68.24, 120.16, 122.40,129.32, 130.34, 130.47, 130.69, 131.41, 131.81,132.87, 133.01, 136.73, 137.66, 139.32, 145.01;HRMS (EI+): m/z Calcd for C17H14BrN3O (M)+ 355.0320,Found: m/z 355.0301; FTIR (c/cm, KBr): 3367, 2955,2919, 2854, 1732, 1461, 1375, 1214, 1022, 892, 813,758.

3-(4-Bromo-phenyl)- 4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine (4k)Light yellow solid, m.p. = 207.1–209.0 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 4.62 (2 H, s), 4.79 (2 H, s),7.65–7.26 (7 H, m), 8.07 (1 H, d, J = 7. 8); 13C NMR(75 MHz, CDCl3): d (in ppm) 58.55, 68.22, 122.40,122.81, 129.04, 129.24, 129.52, 130.36, 130.55, 130.68,132.20, 136.78, 144.49;HRMS (FAB+): (m + H)/z Calcdfor C16H13BrN3O (M + H)+ 342.0242, Found: (m + H)/z342.0215; FTIR (c/cm, KBr): 3583, 3058, 2923, 2857,2116, 1951, 1723, 1958, 1563, 1485, 1407, 1348, 1284,1204, 1114, 1078, 988, 937, 900, 757.

2-(4H,6H-[1,2,3]triazolo[1,5-a][4,1]-benzoxazepine)-benzoic acid methyl ester (4l)Light yellow solid, m.p. = 190.7–191.0 °C, 1H NMR(300 MHz, CDCl3): d (in ppm) 3.81 (3 H, s), 4.60 (2 H, s),4.61 (2 H, s),7.52–7.49 (2 H, m) 7.57–7.54 (1 H, m), 7.66–7.60 (3 H, m), 7.97 (1 H, d, J = 7.8),8.10 (1 H, d, J = 8.1);13C NMR (150 MHz, CDCl3): d (in ppm) 52.32, 58.12,67.98, 122.42, 128.89, 129.27, 129.31, 130.19, 130.40,130.58, 131.11, 131.41, 131.57, 131.85, 136.89, 144.84,167.65; HRMS (EI+): m/z Calcd for C18H15N3O3 (M)+

321.1113, Found: m/z 321.1121.

Biology

Cell cultureNeuroblastoma cells, HEK 293, and human hepatocellularcarcinoma cell line Hep G2 were procured from theNational Centre for Cell Sciences (NCCS, Pune, India) andwere grown in Dulbecco’s modified Eagle’s medium(DMEM; Invitrogen Life Technologies, Grand Island, NY,USA), supplemented with 10% fetal bovine serum (FBS)and antibiotics (penicillin/streptomycin and gentamicin).Cells were cultured at 37 °C in 95% air and 5% CO2

humidified incubators. Hep G2 cells were seeded at adensity of 105/well plated in 96-well plates. Cells were typ-ically grown to 60–70% confluence, rinsed in phosphate-buffered saline (PBS), and placed into serum-free mediumovernight prior to treatments. After overnight incubation,the Hep G2, HEK 293 cells, and neuroblastoma cellswere treated with these compounds separately at theconcentration of 10, 50, and 100 lM. After 48 h, themedium was removed, and 50 lL of fresh medium wasadded along with 10 lL of MTT (5 mg/mL). MTT solu-tion was slowly removed after 4 h, and the purple crys-tals were solubilized in 1.4 mL of DMSO. Theabsorbance was measured at test wavelength of550 nm in Elisa Plate Reader. Cell viability was quanti-fied by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltet-razolium bromide), a yellow tetrazole assay where theviable cells were determined by the reduction of the yel-low MTT into purple formazan product by mitochondrialdehydrogenase present in metabolically active cells.

Chem Biol Drug Des 2013 3

Novel Triazolo-Benzoxazepine

JC-1 staining of HEK 293 cellsHere, the cells were treated with these compounds forovernight, then stained with JC-1 dye for checking mito-chondrial health. The cells were then studied and imagedunder a fluorescence microscope. Cells with proper mito-chondrial health shows punctuated red staining along withgreen scattered staining, whereas the cells with mitochon-drial dysfunction shows only scattered green staining with-out any red puncta.

Active caspase 3 staining of HEK 293 cellsHere, the HEK 293 cells were treated with these com-pounds for overnight. The cells were fixed with 4% para-formaldehyde (PFA) and immunostained using primaryantibody against active caspase 3 and alexa fluor 546 assecondary antibody. The cells were imaged under a confo-cal microscope.

Results and Discussion

We wish to report a one-pot efficient synthesis of the triaz-olo-benzoxazepines scaffold by a four-step organic reac-tions and disclose their hitherto unknown anticancerproperties. For the synthesis of the said scaffold, we haveused silver cocatalyzed Sonogashira coupling and clickreaction as the first and the last step, respectively (17,18).In all 12 examples of triazolo-benzoxazepines were synthe-sized by this protocol and screened against Neura 2a(glioblastoma cell), Hek 293 (kidney cancer), and Hep G2(liver cancer) cell lines. The anticancer property of thesemolecules was further investigated by taking images frominverted phase contrast microscope, fluorescent micro-scope, and confocal studies. The images indicate that thebest molecule (4c) results cell death through mitochondrialmembrane dysfunction, whereas rest of the moleculesmay follow a different pathway.

We initiated the synthesis by preparing a common startingmaterial, substituted propargyl ether, 1, Scheme 1. Typi-cally, 2-amino benzyl alcohol was treated with conc.H2SO4 in propargyl alcohol at 0 °C to produce the corre-sponding propargyl ether, 1 in 95% yields. In order tofunctionalize 1, the triple bond was substituted with differ-ent aromatic functionalities. Well-known Sonogashira cou-pling was employed for this purpose. It is worthy tomention here that, we have used silver salts instead of CuIin the reaction and successfully got the desired product inmoderate to good yields. Among the various silver salts(AgCl, AgBr, Ag2CO3, AgI, Ag2O, AgNO3, and AgSO4)screened, AgCl gave the best yield (75–80%) of the Sono-gashira product. The reactions were monitored closely byperiodically checking TLC, and usual aqueous workupwas performed on the Sonogashira products, 2. The total12 different aromatic substituted alkynes were successfullysynthesized under this new condition using different aro-matic iodides. With the Sonogashira product, 2 in hand,our next target were to synthesize the triazole-fused het-erocyclic scaffold. Accordingly, the product was subjectedto diazotization reaction (NaNO2, 1 M HCl, 0 °C), followedby nucleophilic displacement of the in situ generated diazogroup by sodium azide, which resulted into the corre-sponding azides, 3. The azides, 3 thus synthesized weredirectly used for the final [3 + 2] cycloaddition reactionwithout any further purification. Accordingly, it was sub-jected to typical [3 + 2] cycloaddition condition; however,here also, we had successfully used AgCl instead of CuIand got the desired triazolo-benzoxazepines, 4 in 65–70%yield (Figure 1A). It is noteworthy to mention here that thisreaction can also proceed in simple heating without thepresence of any catalyst (Ag/Cu-salts) but is extremelysluggish (50% conversion after 12 h) and did not go to thecompletion at all. In the above four-step methodology, So-nogashira, diazotization, and azidation reaction do notrequire any column purification, it can be directly used forthe final [3 + 2] cycloaddition step. With the different triaz-

A B

C

D

Scheme 1: Reagent and conditions: (a) Conc. H2SO4, 0 °C to rt, 2 h; (b) Pd(PPh3)2Cl2, AgCl, TEA, DMF, rt, 4 h; (c) NaNO2, 1 M HCl,NaN3, 0 °C; (d) AgCl, TEA, THF, 60 °C, 6–8 h.


Banerji et al.

oles (4a–l) in hand, we further explored the possibility ofsynthesizing the same product by one-pot sequential pro-cess of the four steps. We started our sequence of reac-tions from silver cocatalyzed Sonogashira reaction,followed by diazotization and nucleophilic displacement ofazide, and finally intramolecular [3 + 2] cycloaddition reac-tion were performed in one-pot manner. Gratifyingly, in allthe cases, the corresponding triazolo-benzoxazepineswere produced in 40–45% yields as off-white to pale yel-low solids.

Single X-ray crystal structure of triazolo-benzoxazepines4c and 4k are depicted in the above ORTEP diagram inthe Figure 1B. The crystal structures unambiguously provethe fused 6-7-5 nature of the new heterocycles.

To determine the biological efficacy of these newly synthe-sized compounds, in vitro cell culture system has beenused. The viability of three different cancer cells, namelyNeura 2a (a neuroblastoma cell line), Hek 293 (a kidneycancer cell line), and Hep G2 (a liver cancer cell line) inpresence and absence of these compounds had beenchecked (19,20). The cells were maintained in DMEM sup-plemented with 10% heat-inactivated FBS and 0.5% peni-cillin–streptomycin at 37 °C in humidified (5% CO2, 95%air) atmosphere.

Cell viability was quantified by MTT [3-(4,4-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide], a yellow tetrazoleassay where the viable cells were determined by thereduction of the yellow MTT into purple formazan productby mitochondrial dehydrogenase present in metabolicallyactive cells. For screening the activity, the cultured cellswere exposed to these compounds at three different con-centrations (10, 50, and 100 lM) and incubated for 48 h.Viability was assessed by MTT assay as described. Intotal, seven compounds (4c, 4e, 4f, 4g, 4i, 4j, and 4l)showed significant reduction in the amount of viable cellsin all the three cell lines screened. A known cytotoxic com-pound, 5-fluorouracil was used as control compound tothe cellular response of these compounds. Some of themhave shown efficacy in killing cancer cells, and compound4c have comparable activity with 5-fluorouracil. The resultsare shown graphically below, Figure 2A–C, respectively. Ofseven compounds, 4c causes maximum cell death,whereas 4e, 4j, and 4l also cause significant reduction ofviable cells in this screening assay. This result was furtherconfirmed by microscopic studies using phase contrast,fluoroscent, and confocal microscopes. Thus, cells wereexamined under an inverted phase contrast microscope(21). For example, Hek 293 cells were treated with twobest compounds 4c and 4e for 48 h, and images weretaken. As shown in Figure 3, there was massive cell death

A

B

Figure 1: (A) Lists of triazolo-benzoxazepines synthesized under thereaction condition. (B) X-ray crystalstructure of compounds 4c and 4k

(ORTEP diagram).



A

B

C

Figure 2: Cytotoxicity studies againstHek 293 (A), Neura 2a (B), Hep G2 (C)cell lines, respectively. Asterisks denotestatistical significance differencesbetween control cells and treated cells:*p < 0.001.


Banerji et al.

in response to these two compounds as compared withcontrol.

One of the key features of apoptosis in most of the casesis the disruption of active mitochondria. JC-1 dye is widelyused to study mitochondrial health (i.e., indicator of mito-chondrial membrane potential). This membrane permeabledye can exist as either green fluorescent monomer atdepolarized membrane potential or as a reddish orangefluorescent J-aggregate at hyperpolarized membranepotential. Cells with proper mitochondrial health showpunctuated red staining along with green scattered stain-ing, whereas the cells with mitochondrial dysfunction show

only scattered green staining without any red puncta. Tocheck the mitochondrial health of the cells, Hek 293 cellswere treated with the said compounds (22). Hek 293 cellswere incubated with these compounds at 50 lM for 20 h.Cells were then stained with JC-1 dye (10 lg/mL) for15 min at 37 °C, and live cells were imaged under a fluo-rescence microscope. As shown in Figure 4, cells treatedwith compound 4c result in loss of mitochondrial mem-brane potential as punctuated red staining has diminishedcompared with control. This result suggests that com-pound 4c may cause cell death at least by parts throughmitochondrial dysfunction. Regarding the other compoundtested, there is partial loss of red puncta after 20 h,

Figure 3: Phase contrast images showing cell death.

Figure 4: JC-1 staining of HEK 293cells. Cells were treated with 4c and 4e

for overnight, then stained with JC-1dye for checking mitochondrial health.The cells were then studied and imagedunder a fluorescence microscope. First,second, and third row, respectively,show control cells and cells treated with4c and 4e. First column representsgreen fluorescent monomer of JC-1,second column represents reddishorange fluorescence of J-aggregate,and third column shows mergedimages. Scale bar represents 25 lm.



indicating a partial effect on mitochondrial function. Mito-chondria may be affected at a later stage or cell deathmay result through other pathways by this compound.Currently, we are pursuing experiments to identify celldeath pathways in other compounds.

Caspase 3 is cleaved in response to mitochondrial disruptionand activated, hence a well-known marker for apoptosis (23).We wanted to check whether compound 4c activates cas-pase 3. To achieve this goal, we treated Hek 293 cells with50 lM of this compound for 20 h. The cells were then fixedwith 4% PFA, and fixed cells were immunostained using pri-mary antibody specific for cleaved caspase 3 & Alexa fluor546 as secondary antibody. The cells were imaged underconfocal microscope. Result shows an elevated active cas-pase 3 levels in treated cells (Figure 5). This result clearly indi-cates that compound 4c induces cell death by apoptosis.

Conclusion and Future Directions

In conclusion, in this study, we have demonstrated one-potsynthesis of silver cocatalyzed 12 novel triazolo-benzoxaze-pines. Some of these molecules showed anticancer activi-ties at low micromolar range when subjected to differentcancer cell lines. One of the molecules (4c) may cause celldeath through mitochondrial pathway of apoptosis as itdisrupts mitochondrial membrane potentials and activatescaspase 3. We will modify further these compounds toachieve more potent anticancer agents. Moreover, detailedmechanistic studies will be carried out to know the causeof cell death by these molecules.

Acknowledgments

S.K.P and P.S. thank CSIR for fellowship. Authors wish tothank to the reviewers for the positive criticism and thesuggestions. Financial assistance from ‘BenD’ project(CSIR) is gratefully acknowledged.

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Supporting Information

Additional Supporting Information may be found in theonline version of this article:

Appendix S1. Representative 1H, 13C NMR data of someselected benzoazulenes (CCDC 863898 and CCDC863899 contain the supplementary crystallographic datafor this paper for compound 4c and 4k respectively. Thesedata can be obtained free of charge from The CambridgeCrystallographic Data Centre via www.ccdc.cam.ac.uk/data_request/cif).



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Synthesis and Cytotoxicity Studies of Novel Triazolo-Benzoxazepine as New Anticancer Agents