Introduction

Breast cancer is a malignant cell growth in the breast. Breast cancer is the most common type of cancer diagnosed in women, excluding skin cancer. Almost one third (32%) of all cancers diagnosed in women are breast cancer. However, the incidence of breast cancer varies by race and ethnicity. American, Indian, and Native Alaskan women have the lowest incidence whereas Caucasian women have the highest incidence of breast cancer diagnoses. In recent years, there has been an explosion of life-saving treatment advances against breast cancer, bringing new hope and excitement. Instead of only one or two options, there are now many treatment choices that fight the complex mix of cells in each individual cancer. The decisions between surgery, then perhaps radiation, hormonal (anti-estrogen) therapy, and/or chemotherapy, can feel overwhelming.

Piperazines are currently the most important building blocks in drug discovery, with a high number of positive hits encountered in biological screens of this heterocycle and its congeners. The piperazine template forms the molecular backbone, possesses versatile binding properties with a frequently occurring binding motif, and provides potent and selective ligands for a range of different biological targets in medicinal chemistry. The piperazine scaffold and its analogues are important pharmacophores that can be found in biologically active compounds across a number of different therapeutic areas (Berkheij et al., 2005; Guo et al. 2004). These include anticancer (Gillet et al. 1998; Gabriel et al., 2000; Hulme et al., 1999), antifungal (Upadhayaya et al., 2004), antibacterial, antimalarial, antipsychotic agents (Choudhary et al., 2006), HIV protease inhibitors (Vacca et al., 1994; Askin et al., 1994; Rossel et al., 1995) and antidepressants (EGYT, 1975). MST-16[4,4-1,2-(ethanediyl) bis(1-isobutoxycarbonyloxy-methyl-2,6-piperazinedione)] was recently approved as an oral anticancer drug for clinical use in Japan (Yoshida, 1999). The piperazine analogues have been shown to have potent antiproliferative activity against colon, prostate, breast, lung, and leukemia tumors; additional studies have demonstrated the ability of the lead piperazines to suppress and eliminate experimental tumors in small-animal models performed by the National Cancer Institute (NCI). Mechanistic evaluations have shown that piperazines inhibit microtubule synthesis by a unique mechanism, inhibit cell cycle progression, and inhibit angiogenesis, which is critical to a tumor cell’s ability to grow and metastasize. The piperazines kill tumor cells directly through the induction of apoptosis. The anti-tumor mode of action of the piperazines is quite distinct from that of Taxol and in addition they are significantly more potent, active against a variety of different tumor types, and are orally bioavailable, when compared to Taxol. In the literature, we also found that diaryl piperazine derivatives were identified as potent and selective dopamine D4 receptor antagonists (Mark et al., 2004; Glase et al., 1997; Perrone et al., 2000), enterovirus inhibitors (Chern et al., 2004), and inhibitors of dopamine uptake in the central nervous system (Kimura et al., 2003a, b; Guo et al., 2004; Kimara et al. 2004). Piperazine sulfonamides exhibit diverse pharmacological activity such as MMP-3 enzyme inhibition and carbonic anhydrase inhibition (Chern et al., 2004; Amin and Welsh, 2003). In continuation of our research on the synthesis of bioactive heterocycles and their biological evaluation (Nanjunda Swamy et al., 2006; Narendra Sharath Chandra et al., 2006, 2007; Priya et al., 2005; Thimmegowda et al., 2007; Anil Kumar et al., 2007, 2007), we describe here the synthesis of 1-benzhydryl-sulfonyl-piperazine derivatives and their effect on inhibition of proliferation of MDA-MB-231 human breast cancer cells.

Chemistry

1-benzhydryl-piperazine derivatives 7(a-e) were prepared by the method summarized in Scheme 1. Initially compound 3, benzhydrol, was synthesized by a Grignard reaction under nitrogen with benzaldehyde (1) and phenyl magnesium bromide (2); the obtained yield was found to be 60%. Finally, we synthesized benzhydrol by reduction of benzophenone using sodium borohydride and achieved a 90% yield. Herein, we report the synthesis of 1-benzhydryl-piperazine from benzophenone for the first time. Compound 3 was subsequently treated with thionyl chloride to give the corresponding benzhydryl chloride (5), which was directly reacted with piperazine and anhydrous potassium carbonate using dimethyl formamide as a solvent at 80°C to give the target key intermediate 1-benzhydryl-piperazine (6). The nucleophilic substitution reactions of (6) with different sulfonyl chlorides (R-SO2-Cl) were carried out in the presence of triethylamine and dichloromethane as the solvent with a good yield of 76–85% and good purity. Synthesized molecules 7(a-e) were structurally characterized by 1H NMR, LC/MS, IR and elemental analysis. The chemical structures, physical data and purity of all the synthesized compounds are given in Table 1.

Scheme 1.
scheme 1

Reagents and conditions: (a) dry THF, room temperature, 4 hrs, under N2; (b) NaBH4, methanol, room temperature, 5 hrs; (c) thionyl chloride, methylene dichloride (MDC), 0–5°C, 4 hrs; (d) piperazine, K2CO3, DMF, 80°C, 8 hrs; (e) R-SO2Cl, 6(a-e), MDC, triethylamine, room temperature, 5–6 hr, where R-SO2Cl is:, 6a: Methanesulfonyl chloride, 6b: 4-methyl-benzenesulfonyl chloride, 6c: 4-chloro-benzenesulfonyl chloride, 6d: 4-tert-butyl-benzenesulfonyl chloride, 6e: 3,5-dimethyl-isoxazole-4-sulfonyl-chloride

Full size image
Table 1 Chemical structures, physical data, and purities of synthesized compounds
Full size table

Results and discussion

Chemistry

The N-substitution of 1-benzhydryl-piperazine with different sulfonyl chlorides was confirmed by the disappearance of the N-H group in IR and 1H NMR data. Compounds 7(a-e) were also confirmed by IR data, which showed asymmetric stretching frequency of O=S=O at 1350 cm-1 and symmetric stretching frequency at 1280 cm-1. Several new derivatives of 1-benzhydryl-piperazine 7(a-e) were synthesized and evaluated for their anti-cancer activities. The obtained products were purified by column chromatography using hexane:ethyl acetate (8:2) as an eluent.

Biology: in vitro cell viability assay – MTS assay

To check the anticancer activity, we carried out the reactions of 1-benzhydryl-piperazine with different sulfonyl chlorides containing aliphatic, substituted aromatic, and heterocyclic groups. Our results reveal that the title compounds showed inhibition of proliferation of MDA-MB-231 human breast cancer cells (Table 2). Among the tested compounds, the inhibitory activity was observed in the following order 7d > 7e > 7b > 7a > 7c. Compounds 7d and 7e exhibited 28.6 ± 0.0003% (P = 0.00030) and 20.6 ± 0.001% (P = 0.001) inhibition of proliferation of MDA-MB-231 breast cancer cells, respectively. Similarly compounds 7a, 7b and 7c showed 3.79 ± 0.2552% (P = 0.2552), 10 ± 0.0086% (P = 0.0086), and 1 ± 0.8673% (P = 0.8673) inhibition, respectively. Compound 7d exhibited good inhibition compared to compound 7b; the inhibition of the compound 7d may be due to the presence of a tertiary butyl group, a strong electron-donating group. Compound 7c showed less inhibition (1%), which might be due to the presence of an electronegative chloro group. Similarly compound 7e showed 20.6% inhibition, which might due to the isoxazole ring containing methyl groups. Compound 7a showed 3.79% inhibition with a methyl group only. From our results, it could be concluded that inhibition increases with the number of electron-donating groups, whereas in the presence of electron-withdrawing groups the observed inhibition is not significant. Thus, we conclude that the presence of electron-donating groups may be responsible for the observed inhibition. Further modification of the groups in the basic scaffold to increase efficacy and in vivo work is under progress.

Table 2 Percentage inhibition of MDA-MB-231 cell proliferation
Full size table

Conclusion

Currently, a large variety of chemotherapeutic drugs are used to treat cancer. Unfortunately, many compounds have limited efficacy due to problems of delivery and penetration and a moderate degree of selectivity for cancer cells. From our studies, it is clear that compound 7d inhibits MDA-MB-231 human breast cancer cell proliferation. The 1-benzhydryl-sulfonyl-piperazine derivatives were obtained with good yield and purity. This study sheds light on the identification of this new series of agents for cancer therapy.

Experimental

Melting points were determined using a SELACO-650 hot-stage melting-point apparatus and were used uncorrected. Infrared (IR) spectra were recorded using a Jasco FTIR-4100 device. Nuclear magnetic resonance (1H NMR) spectra were recorded on a Shimadzu AMX 400-Bruker, 400 MHz spectrometer using dimethyl sulfoxide (DMSO) as a solvent and trimethylsilyl (TMS) as an internal standard (chemical shift in δ ppm). Spin multiplets are given as s (singlet), d (doublet), t (triplet), dd (doublet of doublet), and m (multiplet). Mass and purity were recorded on a LC-MSD-Trap-XCT. Elemental (CHNS) analyses were obtained on a Vario EL III Elementar device. Silica gel column chromatography was performed using Merck 7734 silica gel (60-120 mesh) and Merck thin-layer chromatography (TLC) plates.

General procedure for the synthesis of 1-benzhydryl-piperazine (6)

A solution of piperazine dihydrochloride (10.0 g, 62.86 mmol) in dimethyl formamide was taken, anhydrous potassium carbonate (43.44 g, 314.3 mmol) was added to the solution and stirred for 10 min, and then benzhydryl chloride (11.46 g, 56.58 mmol) was added. The reaction mixture was heated to 80°C for 8 hrs, and monitored by TLC. Upon completion, the solvent was removed under reduced pressure and the residue was taken in water and extracted with ethyl acetate. Finally a water wash was given to the organic layer, followed by drying with anhydrous sodium sulphate. The solvent was evaporated to obtain the crude product, which was purified by column chromatography over silica gel (60-120 mesh) using chloroform:methanol (9:1) as the eluent.

General procedure for the synthesis of 1-benzhydryl-sulfonyl-piperazine derivatives 7(a-e)

A solution of 1-benzhydryl-piperazine 6 (1.0 eq) in dry dichloromethane was taken and cooled to 0–5°C in an ice bath. Triethylamine (3.0 eq) was added to the cold reaction mixture and stirred for 10 min, and then different sulfonyl chlorides (1.0 eq) were added. The reaction mixture was stirred for 5–6 hrs at room temperature, and monitored by TLC. Upon completion, the solvent was removed under reduced pressure and residue was taken in water and extracted with ethyl acetate. The organic layer was washed with 10% ammonium chloride solution and finally a water wash was given to the organic layer and dried with anhydrous sodium sulphate. The solvent was evaporated to obtain the crude product, which was purified by column chromatography over silica gel (60-120 mesh) using hexane:ethyl acetate (8:2) as the eluent.

Synthesis of 1-benzhydryl-4-methanesulfonyl-piperazine (7a)

This was obtained from 1-benzhydryl-piperazine (6) (0.5 g, 1.98 mmol), methanesulfonyl chloride (0.226 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product obtained was a white crystalline solid (0.556 g, 85%). IR (KBr, cm−1): 3029, 2959, 2850, 1346, 1285. 1H NMR (DMSO, 400 MHz) δ: 7.38 (d, 4H, Ar-H), 7.27 (t, 4H, Ar-H), 7.15 (t, 2H, Ar-H), 4.25 (s, 1H, -CH-), 2.92 (br s, 4H, -CH2-), 2.50 (br s, 4H, -CH2-), 2.7 (s, 3H, -CH3-). MS: 331.56. Anal. calcd. for C18H22N2O2S (in %): C-65.43, H-6.71, N-8.48, S-9.70. Found C-65.40, H-6.68, N-8.45, S-9.66.

Synthesis of 1-benzhydryl-4-(toluene-4-sulfonyl)-piperazine (7b)

This was obtained from 1-benzhydryl-piperazine (6) (0.5 g, 1.98 mmol), 4-methyl-benzenesulfonyl chloride (0.377 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product obtained was a white crystalline solid (0.644 g, 82%). IR (KBr, cm−1): 3029, 2962, 1398, 1346, 1280. 1H NMR (DMSO, 400 MHz) δ: 7.63-7.7 (m, 4H, Ar-H), 7.35 (d, 4H, Ar-H), 7.25 (t, 4H, Ar-H), 7.15 (t, 2H,Ar-H), 4.3 (s, 1H, -CH), 2.9 (br s, 4H, -CH2-), 2.4 (br s, 4H, -CH2-), 2.4 (s, 3H, -CH3-). MS: 407.3. Anal. calcd. for C24H26N2O2S (in %): C-70.91, H-6.45, N-6.89, S-7.89. Found C-70.87, H-6.41, N-6.86, S-7.87.

Synthesis of 1-benzhydryl-4-(4-chloro-benzenesulfonyl)-piperazine (7c)

This was obtained from 1-benzhydryl-piperazine (6) (0.5 g, 1.98 mmol), 4-chlorobenzene sulfonyl chloride (0.417 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product obtained was an off-white crystalline solid (0.676 g, 80%). IR (KBr, cm−1): 2961, 2889, 1350, 1279, 707. 1H NMR (DMSO, 400 MHz) δ: 7.7-7.8 (m, 4H, Ar-H), 7.4 (d, 4H, Ar-H), 7.28 (t, 4H, Ar-H), 7.16 (t, 2H, Ar-H), 4.3 (s, 1H, -CH), 3.0 (br s, 4H, -CH2-), 2.41 (br s, 4H, -CH2-). MS: 427.90. Anal. calcd. for C23H23ClN2O2S (in %): C-64.70, H-5.43, N-6.56, S-7.51. Found C-64.66, H-5.40, N-6.54, S-7.50.

Synthesis of 1-benzhydryl-4-(4-tert-butyl-benzenesulfonyl)-piperazine (7d)

This was obtained from 1-benzhydryl-piperazine (6) (0.5 g, 1.98 mmol), 4-tert-butyl-benzenesulfonyl chloride (0.460 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product obtained was an off-white amorphous solid (0.746 g, 84%). IR (KBr, cm−1): 3028, 2852, 1346, 1279, 1399. 1H NMR (DMSO, 400 MHz) δ: 7.63-7.7 (m, 4H, Ar-H), 7.35 (d, 4H, Ar-H), 7.25 (t, 4H, Ar-H), 7.15 (t, 2H,Ar-H), 4.3 (s, 1H, -CH), 2.94 (br s, 4H, -CH2-), 2.42 (br s, 4H, -CH2-), 1.3 (s, 9H, (-CH3)3-). MS: 449.58. Anal. calcd. for C27H32N2O2S (in %): C-72.29, H-7.19, N-6.24, S-7.15. Found C-72.25, H-7.15, N-6.20, S-7.12.

Synthesis of 1-benzhydryl-4-(3,5-dimethyl-isoxazole-4-sulfonyl)-piperazine (7e)

This was obtained from 1-benzhydryl-piperazine (6) (0.5 g, 1.98 mmol), 3,5-dimethyl-isoxazole-4-sulfonyl-chloride (0.387 g, 1.98 mmol), triethylamine (0.601 g, 5.94 mmol). The product obtained was a white crystalline solid (0.619 g, 76%). IR (KBr, cm−1): 3055, 2956, 2831, 1345, 1296. 1H NMR (DMSO, 400 MHz) δ: 7.38 (d, 4H, Ar-H), 7.27 (t, 4H, Ar-H), 7.17 (t, 2H, Ar-H), 4.25 (s, 1H, -CH-), 3.05 (br s, 4H, -CH2-), 2.55 (br s, 4H, -CH2-), 2.4 (s, 6H, -CH3). MS: 412.1. Anal. calcd. for C22H25N3O3S (in %): C-64.21, H-6.12, N-10.21, S-7.79. Found C-64.19, H-6.08, N-10.20, S-7.76.

Biology

Culturing mammalian cell lines

MDA-MB-231 human breast cancer cells were grown in Roswell Park Memorial Institute (RPMI) medium (Sigma), supplemented with 10% fetal bovine serum (FBS). The cells were maintained at 37°C in a 5% CO2 incubator. They were subsequently dislodged from the substratum by 1X trypsin treatment for 5 min followed by inactivation using FBS.

Cell proliferation assay

The MTS assay was performed using the Promega CellTiter 96® aqueous nonradioactive cell proliferation assay as previously described (Zou et al., 2004). Briefly, MB-MB-231 cells were added to the wells of a 96-well plate at a density of 2000 cells/well. The test compounds at 2 mM concentration dissolved in 1% DMSO were added, and the cultures were continued for 72 hrs. Replenishment with fresh compounds was done after the first 48 hrs. At the end of 72 hrs of treatment, the cultured medium was removed and 20 μl/well of combined MTS/PMS solution was added and incubated for 4 hrs. The absorbance at 490 nm was recorded using an ELISA plate reader.