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Current Topics in Medicinal Chemistry, 2015, 15, 973-979 973

Crystallographic Study and Biological Evaluation of 1,4-dimethyl-N-alkyl-carbazoles†

Carmela Saturnino1,#, Anna Caruso2,#, Pasquale Longo3, Anna Capasso1, Attilio Pingitore2, Maria Cristina Caroleo2, Erika Cione2, Mariarita Perri2, Francesco Nicolò4, Viviana Mollica Nardo4, Luigi Monsù Scolaro4,5, Maria Stefania Sinicropi2,*, Maria Rosaria Plutino5,* and Hussein El-Kashef6

1Dip. di Farmacia, Università di Salerno, Via Giovanni Paolo II 132, 84084-Salerno, Italy; 2Dip. di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Via Pietro Bucci, 87036-Arcavacata di Rende (CS), Italy; 3Dip. di Chimica e Biologia, Università di Salerno, Via Giovanni Paolo II 132, 84084-Salerno, Italy; 4Dipartimento di Scienze Chimiche, Università degli Studi di Messina, and Consorzio Interuniversitario di Ricerca in Chimica dei Metalli nei Sistemi Biologici (CIRCMSB), Viale F. Stagno d’Alcontres 31, Vill. S. Agata, 98166 Messina, Italy; 5Istituto per lo Studio dei Materiali Nanostrutturati, ISMN - CNR, UO di Palermo, c/o Dipartimento di Scienze Chimiche, Università degli Studi di Messina, Viale F. Stagno d’Alcontres 31, Vill. S. Agata, 98166 Messina, Italy; 6Department of Chemistry, Assiut University, 71516-Assiut, Egypt

Abstract: The 9-(bromoalkyl)-1,4-dimethyl-9H-carbazole (2a-d) derivatives, characterized by the presence of five or seven methylenic spacer groups bonded to the carbazole nitrogen, have been synthesized from the corresponding 1,4-dimethyl-9H-carbazole and appropriate dibromoalkane following a general synthetic method. All the prepared species have been fully characterized by means of IR, and 1H and 13C NMR spectroscopy, GC-MS and Elemental analysis. Good crystals of the 2c have been obtained and the crystal structure has been solved by means of X-ray diffractometry. In order to study the cytotoxic effect of 2a, 2b, 2c, 2d carbazole derivatives on A2780 ovarian cancer cells, we performed MTT assay after exposure of this cell population to those compounds in a concentration range from 1 to 10µM. Finally, we want to verify whether the cytotoxic effect of the 2c carbazole is mediated by apoptotic mechanisms, by performing chromatin condensation assay on the A2780 cell cultures upon the carbazole treatment at concentration of 10 µM for 72h. All to-gether our data demonstrate that carbazole derivatives exert inhibitory effects on ovarian cancer cell growth, highlighting a stronger and a dose-dependent anti proliferative activity displayed by 2c carbazole, designating this compound, as a bet-ter candidate in the treatment of human ovarian cancer.

Keywords: A2780 ovarian cancer cells, Apoptosis, Carbazole derivatives, X-Ray structure, Heterocycles, MTT.

INTRODUCTION

In recent years, many studies have been focused on the design and synthesis of new ligands able to recognize specif-ic DNA sequences. Some DNA ligands have several biologi-cal activities and some of these have interesting anticancer properties [1-2].

With the availability of modern spectral techniques used to determine the selectivity of binding at DNA, such as NMR, the X-ray diffractometry, and the foot printing, it has been possible to determine the nature of the chemical processes involved in the interaction of these substances with DNA. *Address correspondence to these authors at the Dip. di Farmacia e Scienze della Salute e della Nutrizione, Università della Calabria, Via Pietro Bucci, 87036-Arcavacata di Rende (CS), Italy; Tel: +39-0984-493200; Fax: +39-0984-493107; E-mail: s.sinicropi@unical.it and Istituto per lo Studio dei Materiali Nanostrutturati, ISMN - CNR, UO di Palermo, c/o Dipartimento di Scienze Chimiche, Università degli Studi di Messina, Viale F. Stagno d’Alcontres 31, Vill. S. Agata, 98166 Messina, Italy; Tel: +39-090-6765734; Fax: +39-090-3974108; E-mail: rosaria.plutino@pa.ismn.cnr.it #These authors equally contributed to the manuscript. †The paper is dedicated to the memory of Mr. G. Irrera, who recently passed the way.

In the last fifteen years with the identification of oncogenes, onco-suppressor genes and genes that codify for proteins involved in DNA repair, regulation of cell cycle, differentia-tion and cell death, there has been an amplification of knowledge concerning biology of cancer and mechanism of action of many anticancer drugs [3].

Several studies showed that the first step in the mecha-nism of action of many anticancer drugs is the reversible or irreversible binding to the DNA. The molecules can interca-late into the DNA, between the base pairs of the double he-lix, bind a major or minor groove of DNA, or alkylate one or more nitrogen bases [1, 4-6].

Consequently, this allows the synthesis of the selected “target” in experimental models both in vitro and in vivo with good anticancer properties.

Then considerable advances in cancer therapy have been achieved as a result of the discovery of alkylating and inter-calanting agents. The alkylating agents [3] are compounds that react through covalent bond formation with nucleophilic centers (amino groups, thiol, hydroxyl, phosphate, etc.) of biological compounds (proteins, nucleic acids, etc.) which are thereby damaged.

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974 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 11 Saturnino et al.

Among these compounds, important agent is the water soluble cis-platinum particularly active in testicular and ovarian cancers, for which nephro and mielotoxicity repre-sent serious side effects [7].

The intercalating agents are instead compounds, with dif-ferent chemical structures, able to bind tightly but reversibly the DNA, damaging it and preventing the replication of can-cer cells.

Some of these drugs are also capable to produce free rad-icals as ethidium bromide which, after hydrogen interactions with DNA bases, leads to mutations such as deletions and insertions [2].

In nature there are several alkaloids with carbazolic core (Fig. 1), extracted from different plants of the genus Murray Glycosmis [8], and Clausen in the family Rutaceae [9-10], able to intercalation between the bases of DNA, to induce apoptosis (in different ways) and oxidative stress resulting in the formation of radicals, and to activate transcription of the p53 gene [11]. For many natural and synthetic carbazoles [12-13], cytotoxicity can be related to DNA-dependent en-zyme inhibition such topoisomerase I/II [2] and telomerase. But also other target such as cyclin dependent kinase and estrogen receptors have emerged thus rendering these com-pounds suitable for estrogen-dependent tumor such as breast or ovarian cancer [14-15].

Fig. (1). Carbazole structure.

To date ovarian tumors are classified as chemosensitive but not chemocurable tumors. Indeed, therapeutic approach involves surgery with maximal cytoreduction, subsequent chemotherapy and exceptionally radiotherapy [16]. The de-velopment of resistence to the traditional chemotherapeutic agents has challenged the scientists to design new therapeutic tools. In the present study we have prepared carbazole deriva-tives (2a-d) and evaluated the cytotoxic activity of these us-ing A2780 human cell line as in vitro model system for ovari-an cancer treatment. Since the cis-PtCl2(NH3)2 (cis-platin) is the first platinum(II) derivative and it is still one of the most active and clinically effective anti-cancer agent used, in the last years a number of platinum (II) species catch great atten-tion thanks to their ability to bind covalently or through inter-calation of the aromatic ligands to DNA, or even acting as drug-delivery systems [17]. In this regard, the promising re-sults obtained with the present study, encourage us to use these compounds as precursors of some transition metal com-plexes, in particular platinum (II) derivatives, endowed with citotoxic ability and potential anti-cancer activity.

RESULTS

The 9-(bromoalkyl)-1,4-dimethyl-9H-carbazole (2a-d) derivatives have been prepared following a general synthetic method [18 a-b]. In particular, a mixture of 1,4-dimethyl-9H-carbazole (1) and dry DMF was stirred at rt until clear solu-

tion was obtained. NaH 60% oil dispersion was added at 0°C and after appropriate dibromoalkyl was added and the mix-ture was stirred for 5 h at room temperature. Water was then added and the resulting mixture was extracted with EtOAc. The residue obtained was purified by silica gel column chromatography (Et2O/hexane, 2/3 as eluent) to give pure compound. The carbazoles 1a-b were prepared as described in the literature [18].

Scheme 1. Preparation of carbazole derivatives 2a-d. X-Ray Crystallography

The geometry of compound 2c, at the solid state, has been determined by means of crystallographic study starting from single crystal diffraction data (see Table 1). A prospec-tive view of the molecule and the content of asymmetric unit are illustrated in Fig. (2). Selected bond distances, angles and some relevant torsion angles are reported in Table 2. The UA is made up by neutral 6-bromo-9-(5-bromopentyl)-1,4-dimethyl-9H-carbazole (2c).

In the title molecule 6-bromo-9-(5-bromopentyl)-1,4-dimethyl-9H-carbazole (2c) the bond lengths and angles show normal values comparable with those observed in the related compounds [19-21], the tricyclic carbazole system is essentially planar with the two outer rings forming a dihedral angle of 1.42° (9). From the mean plane of phenyl ring C(2)-

C(16) the Br(1) atom, bonded to C(13) at 1.902(4) Å devi-ates of -0.1255(5) Å; while the two methyl groups bonded to the second phenyl group are essentially planar to it.

Pairs of molecules made in relations by crystallographic inversion centres result in weak interactions involving bro-mine atoms, Br(2) C(3) 3.958(4).

The molecular packing, shown in Fig. (3) well as strong hydrogen bonds and significant interactions of stacking are determined by the usual Van der Waals interactions (see Figs. S1 and S2 in Supporting Information).

The amine nitrogen, sp2 hybridized, and the consequent marked shortening of the distance N(1)-C(3) and N(1)-C(2) contribute significantly to the enrichment in electric charge, the aromatic system, delocalising its electronic doublet [18 c,d].

Carbazoles Derivatives Affects Cell Viability in Human Ovarian Cancer Cells

In order to examine the cytotoxic effect of 2a-d carbazole derivatives on A2780 ovarian cancer cells, we performed MTT assay after exposure of this cell population to those compounds in a concentration range from 1 to 10 µM. Time

N

CH3

CH3

R

R'NH

CH3

CH3

R

1 2

NaH,Br(CH2)nBr

2a R= OCH3 R'= (CH2)5Br2b R= OCH3 R'= (CH2)7Br2c R= Br R'= (CH2)5Br2d R= Br R'= (CH2)7Br

1a R= OCH3 1b R= Br

DMF

NH

Crystallographic Study and Biological Evaluation Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 11 975

course preliminary experiments demonstrated that the effec-tiveness of carbazole derivatives is maximal after 72 h of treatment (data not shown), so that we chose this time for our experimental design. As illustrated in Fig. (4), panel a, treatment of A2780 cell population with 10 µM of 2a-d car-bazole derivatives yielded to a significant reduction of cell viability which was less than 35% after exposure of the ovar-ian human cell line to the carbazole 2c. Since this compound have shown a greater efficacy than the other carbazoles we performed a dose-response experiments. To this end A2780 cells were exposed to 2c concentrations ranging from 0.1 nM to 10 µM and 72h after treatment cultures were assayed through MTT test. The results have shown that the cytotoxic effect of 2c carbazole was dose-dependent (Fig. 4b) with an EC50 value of 1.36 x 10-8 and a goodness of fit, R2 of 0.9434 (Fig. 4c). Table 1. Crystal data and structure refinement for 2c.

Compound 2c

Empirical formula C19H21Br2N

Molecular mass 423.19

Temperature (K) 296(2) K

Crystal system Monoclinic

Space group P21/c

Z 4

a (Å) 4.7453(10)

b (Å) 17.7002(3)

c (Å) 20.8745(3)

α (°) 90

β (°) 95.7480(10)

γ (°) 90

V(A3) 1744.49(5)

Dcalcd (g cm3) 1.611

Measured reflections 73770

F(000) 848

R[int] 0.0407

GOF on F2 1.035

R1 a [I > 2σ (I)] 0.0473

wR2b 0.0885

Carbazoles Derivative 2c Induces Apoptosis in A2780 Human Ovarian Cancer Cells

To verify whether the cytotoxic effect of the 2c carbazole is mediated by apoptotic mechanisms, we performed DAPI staining on the A2780 cell cultures upon the carbazole treatment at concentration of 10 µM for 72h. As shown in

Fig. (5, panel b), DAPI assay revealed in the exposed cul-tures the presence of dramatic morphologic changes of cell nuclei with gross abnormality of nuclear size and shape and DNA fragmentation indicative of apoptotic cell death. No morphologic changes of cell nuclei were observed in control cultures exposed to vehicle alone (Fig. 5, panel a) Analysis of chromatine condensation by DAPI technique. A2780 ovarian cancer cells were treated with DMSO as a vehicle (a). A2780 ovarian cancer cells were treated with 2c for 72h. The white arrows show the gross abnormality in nuclear size and shape as apoptotic marker. Figure is representative of three independent experiments. Original magnification 40x.

Fig. (2). ORTEP view of 2c with thermal ellipsoids drawn at the 35% probability level. Table 2. Selected Bond lengths (Å) angle(°) and torsion an-

gle(°) of compound 2c.

Compound 2c

Br(1)-C(9) 1.907(3)

Br(2)-C(1) 1.917(5)

C(6)-N(1) 1.383(4)

N(1)-C(5) 1.452(4)

N(1)-C(6)-C(7) 129.1(3)

N(1)-C(6)-C(11) 109.4(3)

C(10)-C(9)-Br(1) 119.2(3)

C(5)-N(1)-(C4)-C(3) -173.54(11)

C(19)-C(1)-C(2)-C(3) -1.66(13)

EXPERIMENTAL SECTION

General Methods and Materials

Commercial reagents were purchased from Aldrich, Acros Organics and Alfa Aesar and used without additional purification. Melting points were determined on a Kofler melting point apparatus. IR spectra were recorded on a Jasco FT-IR-4200 spectrophotometer. Mass spectra were obtained by using a JEOL JMS GCMate spectrometer at ionising

976 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 11 Saturnino et al.

Fig. (3). Mercury view illustrating the Br-H interactions.

Fig. (4). Effect of carbazoles on A2780 cell viability. a) A2870 cells treated with different concentrations of 2a-d for 72 hours as described in Materials and Methods b, c) Dose-Response of increasing concentrations of the most active compound (2c); EC50 value:1.36 x 10-8 R2 value (goodness of fit): 0.9434 (Tukey-Kramer post hoc test following significant ANOVA).

Fig. (5). DAPI Chromatin Condensation Staining in A2780 ovarian cancer cells.

Crystallographic Study and Biological Evaluation Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 11 977

potential of 70 eV (EI). 1H NMR and 13C NMR spectra were recorded on a Brüker 300 MHz spectrometer. Chemical shifts are expressed in parts per million downfield from tet-ramethylsilane as an internal standard. Thin layer chroma-tography (TLC) was performed on silica gel 60F-264 (Merck). Elemental analyses were performed on a Perkin-Elmer 240C elemental analyzer.

Synthetic Procedures

A mixture of 1,4-dimethyl-9H-carbazole (1a-b) (3.00 mmol) and dry DMF (15 mL) was stirred at rt until clear solution was obtained. NaH 60% oil dispersion (4.00 mmol) was added at 0°C. After 15 min stirring, appropriate dibro-moalkyl (9.00 mmol) was added and the mixture was stirred for 5 h at rt. Water (100 mL) was then added and the result-ing mitxure was extracted with EtOAc (2 x 100 mL). The residue obtained was purified by silica gel column chroma-tography (Et2O/hexane, 2/3 as eluent) to give pure compound (2a-d).

9-(5-Bromopentyl)-6-methoxy-1,4-dimethyl-9H-carbazole (2a). Brown solid, (60% yield). Mp 88°C. IR (KBr) (cm-1): 2929, 1421, 1296, 1212, 1038, 807, 720, 641. 1H NMR (CDCl3): δ 1.30-2.00 (m, 6H, CH2), 2.76 (s, 3H, CH3), 2.82 (s, 3H, CH3), 3.29-3.36 (m, 2H, CH2Br), 3.86 (s, 3H, OCH3), 4.39-4.44 (m, 2H, NCH2), 6.79 (d, J = 6.90 Hz, 1H, Ar), 6.86-7.05 (m, 2H, Ar), 7.18-7.25 (m, 1H, Ar), 7.64 (s, 1H, Ar). 13C NMR (CDCl3): 153.65, 129.08, 120.63, 113.54, 109.24, 106.94, 56.50, 44.93, 33.70, 32.64, 30.03, 24.72, 21.16, 20.49. GC-MS [m/z (% int.rel.)]: 374 (40) [M+.]; 238 (100); 224 (10); 195 (15); 180 (14); 152 (5). Anal. Calcd for C20H24BrNO: C, 64.18; H, 6.46; N, 3.74. Found: C, 64.20; H, 6.49; N, 3.71.

9-(7-Bromoheptyl)-6-methoxy-1,4-dimethyl-9H-carbazole (2b). Yellow solid, (43% yield). Mp 116°C. IR (KBr) (cm-1): 2931, 1419, 1298, 1215, 1040, 803, 715, 643.1H NMR (CDCl3): δ 1.30-1.50 (m, 6H, CH2), 1.70-1.90 (m, 4H, CH2), 2.70 (s, 3H, CH3), 2.80 (s, 3H, CH3), 3.30-3.49 (m, 2H, CH2Br), 3.90 (s, 3H, OCH3), 4.40-4.60 (m, 2H, NCH2), 6.86 (d, J = 6.90 Hz, 1H, Ar), 7.03-7.14 (m, 2H, Ar), 7.27-7.32 (m, 1H, Ar), 7.70-7.74 (m, 1H, Ar). 13C NMR (CDCl3): 153.47, 139.58, 136.32, 131.40, 128.88, 124.26, 122.08, 120.39, 117.50, 113.39, 109.16, 106.69, 56.39, 44.97, 33.98, 32.80, 30.69, 28.72, 28.18, 26.94, 21.02, 20.34. GC-MS [m/z (% int.rel.)]: 402 (45) [M+.]; 238 (100); 224 (10); 210 (5); 195 (12); 180 (8); 152 (3). Anal. Calcd for C22H28NOBr: C, 65.67; H, 7.01; N, 3.48. Found: C, 65.70; H, 7.03; N, 3.45.

6-Bromo-9-(5-bromopentyl)-1,4-dimethyl-9H-carbazole (2c). White solid, (35% yield). Mp 88°C. IR (KBr) (cm-1): 2919, 1711, 1464, 1228, 1012, 796, 651. 1H NMR (CDCl3): δ 1.30-2.10 (m, 6H, CH2), 2.70 (s, 3H, CH3), 2.80 (s, 3H, CH3), 3.35-3.45 (m, 2H, CH2Br), 4.30-4.50 (m, 2H, NCH2), 6.84 (d, J = 6.60 Hz, 1H, Ar), 7.04 (d, J = 6.60 Hz, 1H, Ar), 7.15-7.30 (m, 1H, Ar), 7.45 (d, J = 8.40 Hz, 1H, Ar), 8.20 (s, 1H, Ar). 13C NMR (CDCl3): 144.71, 139.69, 139.23, 131.71, 129.68, 127.69, 125.60, 125.27, 121.32, 117.60, 111.87, 110.05, 44.82, 33.44, 32.44, 29.85, 25.58, 20.99, 20.28. GC-MS [m/z (% int.rel.)]: 423 (67) [M+.]; 286 (100); 207 (14); 291 (18); 165 (8). Anal. Calcd for

C19H21Br2N: C, 53.93; H, 5.00; N, 3.31. Found: C, 53.96; H, 5.02; N, 3.28.

6-Bromo-9-(7-bromoheptyl)-1,4-dimethyl-9H-carbazole (2d). White solid, (50% yield). Mp 105°C. IR (KBr) (cm-1): 2921, 1709, 1472, 1234, 1016, 798, 649. 1H NMR (CDCl3): δ 1.30-1.60 (m, 6H, CH2), 1.70-2.10 (m, 4H, CH2), 2.79 (s, 3H, CH3), 2.83 (s, 3H, CH3), 3.30-3.60 (m, 2H, CH2Br), 4.40-4.60 (m, 2H, NCH2), 6.93 (d, J = 7.20 Hz, 1H, Ar), 7.12 (d, J = 6.90 Hz, 1H, Ar), 7.27 (dd, J1 = 7.50 Hz, J2 = 3.30 Hz, 1H, Ar), 7.27 (dd, J1 = 8.40 Hz, J2 = 1.80 Hz, 1H, Ar), 8.28-8.30 (m, 1H, Ar). 13C NMR (CDCl3): 139.68, 139.22, 131.61, 129.62, 127.58, 125.17, 121.31, 121.20, 117.64, 111.74, 110.10, 44.91, 34.03, 32.79, 30.63, 29.28, 28.70, 28.18, 26.92, 21.02, 20.28. GC-MS [m/z (% int.rel.)]: 451 (62) [M+.]; 286 (100); 207 (15); 191 (20); 165 (8); 55 (9). Anal. Calcd for C21H25 Br2N: C, 55.90; H, 5.58; N, 3.10. Found: C, 55.93; H, 5.55; N, 3.12.

Cell Culture

Carbazole cytotoxicy were studied on human ovarian cancer cell line (A2780). A2780 were purchased from Amer-ican Type Cell Culture Collection. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin, (hereafter referred as complete medium; all reagents were from Invitrogen, Gibco, Milan, Italy) in an atmosphere of 5% CO2 at 37 °C. Expo-nentially growing cells were used in all experiments. Cell viability was assessed with trypan blue dye exclusion using an automated cell counter (Cell countess, Life Tecnologies).

Carbazole derivatives were dissolved in DMSO with a stock concentration of 100 mM/L at -20 °C and diluted at desired concentration in complete medium before adding to the cell cultures. The final concentration of DMSO in medi-um was less than 0.1% (v/v). DMSO treated cells were used as vehicle control. Before each experiment cells were switched to medium without serum for 24 hours.

In vitro Cytotoxicity Assay

MTT Assay. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay was performed according to the protocol described for the first time by Mossman with some modification [22a-b]. Briefly, A2780 cell line at con-centration of 8x104 cells were seeded in 12-well plates over-night in complete medium. Cells were then washed twice in PBS and switched to medium without serum. After 24 hours cell cultures were washed twice in PBS and then treated with of each carbazole compounds at concentration of 10 µM in complete medium for 72 hours. Control cultures were ex-posed to complete medium containing 0.1% DMSO (v/v). Both in control and in treated cultures the medium contain-ing DMSO or carbazole derivatives was refreshed every 24 h. At the end of the incubation period, 10 µL of 1mg/mL MTT (Sigma-Aldrich, Milan, Italy) was added to each well and plates were incubated for 2 h at 37 °C. Then the medium was changed and 1 mL of Isopropanol/HCl 0.04 N was add-ed to each well to dissolve the formazan crystals. Dye ab-sorbance in each well was measured by ELISA reader at 595 nm. The cytotoxicity, C (%), was calculated as follows: C(%)= [1-A(ctw)/A(cw)x100] where A(ntw) and A(vw) were the absorbance of carbazole-treated cells and control

978 Current Topics in Medicinal Chemistry, 2015, Vol. 15, No. 11 Saturnino et al.

well, respectively. Each sample was run in triplicate. Values are expressed as mean of three different experiments ± SEM. Statistical analysis was performed using one-way ANOVA followed by Tukey Kramer as post-hoc test.

DAPI Chromatin Condensation Assay

A2780 were cultured and treated with carbazole derivates as described. After incubation with nucleases, cells were then fixed with 10% formaldehyde for 1 h on ice, centri-fuged and suspended in 10 µM 4,6-diamidino-2-phenylindole (DAPI) dissolved in the reaction mixture buff-er. Samples were visualized by epi-fluorescence microscopy, and images were acquired with a color camera.

Crystallographic Structure Determination

Crystals of 6-Bromo-9-(5-bromopentyl)-1,4-dimethyl-9H-carbazole (2c), suitable for X-ray diffraction study, were grown by slow evaporation of concentrated methanol solu-tions. Single-crystal X-ray diffraction data for 2c were col-lected on a Bruker APEX II equipped with a CCD area detec-tor and utilizing Mo-Kα radiation (λ=0.71073 Å) at room temperature. Data were collected and reduced by SMART and SAINT softwares in the Bruker packages [23]. The struc-tures were solved by direct methods SIR04 [24] and then de-veloped by least squares refinement on F2 [SHELXTL.97] [25]. The complete conditions of data collection and structure are given in Table 1. All non-H atoms were placed in calcu-lated positions and refined as anisotropic”. All hydrogen at-oms were refined in isotropic approximation in riding model with the Uiso(H) parameters equal to 1.2 Ueq(Ci), for methyl groups equal to 1.5 Ueq(Cii), where U(Ci) and U(Cii) are respectively the equivalent thermal parameters of the carbon atoms to which corresponding H atoms are bound. Refine-ment of F2 was against all reflections. The weighted R-factor wR and goodness of fit S are based on F2, conventional R-factors R are based on F, with F set to zero for negative F2. The threshold expression of F2 > 2σ(F2) is used only for cal-culating R-factors(gt) etc., and is not relevant to the choice of reflections for refinement. Graphical elaborations was per-formed with WinGX (package software) [26]. Whole struc-tural parameters are reported as supplementary material in Tables S1-S3.

CONCLUSION

Administration of a mixture of platinum derivative and paclitaxel was the first line standard for patients with ad-vanced epithelial ovarian cancer [27]. However, the initial positive response to therapy is limited by the development of resistance to these chemotherapeutic agents thus rendering ovarian cancer refractory to other anticancer drugs and to radiation. Currently there is much interest in the develop-ment of agents which could be useful in therapy manage-ment of this tumor. In order to provide a mechanistic feed-back into the efficiency and usefulness of these carbazoles as potential anti-cancer agents, we investigated their antiprolif-erative effect using a human ovarian cancer cell line, A2780 as in vitro model system for ovarian cancer treatment. To highlight the antiproliferative effects of the novel carbazole derivatives we employed the MTT assay that detected the activity of mitochondrial succinate dehydrogenase [22]. Our

results demonstrated that all the compounds inhibited cell proliferation (Fig. 4 panel a). It is worth mentioning that among the carbazoles showing moderate to high anti-tumor activity, only the derivative 2c displayed a dose-dependent stronger antiproliferative activity against A2780 cells (Fig. 4 panel b), as confirmed by the EC50 value of 1.36 x 10-8 (Fig. 4 panel c). We then further investigated the mechanism involved in the cellular growth inhibitory action of the compound 2c. Our results demonstrated that this effect is mediated through the occurrence of programmed cell death as evidenced by DAPI condensation chromatin stain-ing (Fig. 5 panel b).

All together our data demonstrate that carbazole deriva-tives exert inhibitory effects on ovarian cancer cell growth, highlighting a stronger and a dose-dependent anti prolifera-tive activity displayed by the derivative 2c, designating this compound, whose crystal structure have been solved, as a better candidate in the treatment of human ovarian cancer.

CONFLICT OF INTEREST

The authors confirm that this article content has no con-flict of interest.

ACKNOWLEDGEMENTS

This work was financially supported by Ministero dell'Istruzione, Università e Ricerca (MIUR, PRIN 2010-2011, prot. 010C4R8M8_003) and Consiglio Nazionale delle Ricerche (CNR), Italy. M.R. Plutino thanks Mr. G. Irrera (ISMN-CNR) for technical assistance. Ely Lilli Italia s.p.a. (CD) supporting grant to E. Cione.

SUPPLEMENTARY MATERIAL

Figs. (S1 and S2) showing the unit cell of compound 2c, and Tables S1-S3 giving complete crystallographic data, bond distances, bond angles, anisotropic thermal parameters, and hydrogen atom coordinates.

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Received: June 26, 2013 Accepted: October 28, 2014