Synthesis and cytotoxicity studies of novel benzhydrylpiperazine carboxamide and thioamide derivatives

Article abstract of DOI:10.3109/14756366.2013.765416

Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives with carboxamide and thioamide moieties were reported. In vitro cytotoxic activities of compounds were screened against hepatocellular (HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell lines by sulphorhodamine B assay. In general, 4-chlorobenzhydrylpiperazine derivatives were more cytotoxic than other compounds. In addition, thioamide derivatives (6a-g) have higher growth inhibition than their carboxamide analogs.

Full text of DOI:10.3109/14756366.2013.765416

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ISSN: 1475-6366 (print), 1475-6374 (electronic)
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
2014 Informa UK Ltd. DOI: 10.3109/14756366.2013.765416
!
Synthesis and cytotoxicity studies of novel benzhydrylpiperazine
carboxamide and thioamide derivatives
Enise Ece Gurdal1, Irem Durmaz2, Rengul Cetin-Atalay2, and Mine Yarim1
2
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University, Kayisdagi, Istanbul, Turkey, and Department of Molecular
Biology and Genetics, BilGen, Genetics and Biotechnology Research Center, Faculty of Science, Bilkent University, Bilkent, Ankara, Turkey
Abstract
Keywords
Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives with carboxamide and
thioamide moieties were reported. In vitro cytotoxic activities of compounds were screened
against hepatocellular (HUH-7), breast (MCF-7) and colorectal (HCT-116) cancer cell lines by
sulphorhodamine B assay. In general, 4-chlorobenzhydrylpiperazine derivatives were more
cytotoxic than other compounds. In addition, thioamide derivatives (6a–g) have higher growth
inhibition than their carboxamide analogs.
Benzhydrylpiperazine, cytotoxicity,
isocyanate, isothiocyanate,
sulphorhodamine B
History
Received 28 November 2012
Revised 2 January 2013
Accepted 6 January 2013
Published online 8 February 2013
Introduction
derivatives of benzhydrylpiperazines were discussed for their
cytotoxicities against HUH-7, MCF-7 and HCT-116 cancer cell
lines52.
Cancer is the disease resulting from abnormal cells with abilities
of uncontrolled dividing and invasion to other tissues through
blood and lymph systems. Recently advanced treatment opportu-
nities are unable to overcome the major problems of chemother-
apy such as drug resistance and severe side effects due to the lack
of specificity. Regarding issues lead the researchers to develop
varying drug-like compounds targeting cancer.
In this study, we reported the synthesis, purification and
characterization of some novel compounds bearing benzhydrylpi-
perazine backbone. Those compounds were tested for their
cytotoxic activities against hepatocellular (HUH-7), breast
(MCF-7) and colorectal (HCT-116) cancer cell lines with
sulphorhodamine B (SRB) assay. We aimed to develop a structure
activity relationship for benzhydrylpiperazine derivatives in
accordance with their cytotoxic activity results.
Piperazine-1-carboxamides have diverse actions such as antag-
onism of CB1, human CCR2 chemokine, androgen and vanilloid
receptors or inhibition of PDGFR phosphorylation1–5
Benzhydrylpiperazine scaffold is well known for its antihista-
.
Materials and methods
minic activity6–11. Furthermore calcium channel blocking12–19
,
dopaminergic20–23, antimicrobial24–41 and antiviral42,43 activities Chemistry
are often mentioned in literature.
All chemicals and reagents used in the current study were of
Anticancer activity of benzhydrylpiperazines has recently
advanced44–51. Kumar et al. have performed cytotoxicity assays
to several 1-benzhydrylpiperazine derivatives substituted with
variable sulfonyl chlorides, acid chlorides and isothiocyanates.
These derivatives have potent cytotoxicity over breast cancer
(MCF-7), hepatocellular (HepG-2), cervix (HeLa) and colon
carcinoma (HT-29) cell lines44. Yarim et al., also performed
cytotoxicity screenings for some 4-chlorobenzhydrylpiperazines
substituted with variable benzoyl chloride derivatives and
reported their high activities against liver (HUH-7, FOCUS,
MAHLAVU, HepG-2, Hep-3B), breast (MCF-7, BT20, T47D,
CAMA-1), colon (HCT-116), gastric (KATO-3) and endometrial
(MFE-296) cancer cell lines45. In addition, our work group has
recently reported a study in which sulfonamide and benzamide
analytical grade. The reactions were monitored by thin layer
chromatography (TLC) on Merck pre-coated silica GF254 plates
(Merck KGaA, Darmstadt, Germany). Melting points (ꢀC) of the
compounds were determined by using a Mettler Toledo FP62
capillary melting point apparatus (Mettler-Toledo, Greifensee,
Switzerland) and are uncorrected. Ultraviolet spectra were
recorded with Agilent 8453 UV-Visible Spectrophotometer
(Agilent Technologies, Santa Clara, CA). Infrared spectra were
recorded on a Perkin-Elmer Spectrum One series FT-IR apparatus
(Version 5.0.1, Perkin Elmer, Norwalk, CT), using potassium
bromide pellets, the frequencies were expressed in cmꢁ1. The 1H-
and 13C-NMR spectra were recorded with a Varian Mercury-400
FT-NMR spectrometer (Varian Inc., Palo Alto, CA), using
tetramethylsilane (TMS) as the internal reference, with dimethyl-
sulfoxide (DMSO-d6) as solvent, the chemical shifts were
reported in parts per million (ppm). Coupling constants were
recorded in Hertz (Hz). The mass spectra were recorded with a
Waters 2695 Alliance Micromass ZQ LC/MS instrument (Waters
Corp., Milford, MA). Elemental analyses were performed on
Address for correspondence: Enise Ece Gurdal, Department of
Pharmaceutical Chemistry, Faculty of Pharmacy, Yeditepe University,
Kayisdagi, Istanbul, Turkey. Tel: þ90-216-578-0000 (ext. 3293). Fax:
þ90-216-578-0068. Email: egurdal@yeditepe.edu.tr

206 E. E. Gurdal et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
LECO 932 CHNS (LECO-932, St. Joseph, MI) instrument and and dried with anhydrous sodium sulfate. Solvent was evaporated
were within ꢂ0.4% of the theoretical values.
under vacuo and solid product was recrystallized with ethanol/
water.
General procedure for preparation of benzhydrole
derivatives
N-sec-Butyl-4-(diphenylmethyl)piperazine-1-carboxamide (5a,
CAS No: 1071382-92-7)
Ten millimoles (2.2 g) of benzophenone was dissolved in
10 ml of ethanol. In a separate flask, 11 mmol (0.4 g) of sodium White, opaque, needle-shaped crystals, 68% (0.240 g), m.p.
borohydride (NaBH4) was dissolved in 2 ml of ethanol. Sodium 198.4 ꢀC. UV (MeOH, ꢀmax, nm); 205 (log ": 5.17), 224 (log ":
borohydride solution was slowly added to benzophenone solu- 4.69). FT-IR (KBr, cmꢁ1); 3342 (N–H), 3022 (C–H, aromatic),
tion with a Pasteur pipette. Reaction mixture was allowed 2959 (C–H, aliphatic), 1619 (C¼O, amide), 1540 (C¼C,
to continue stirring for a further 30 min. For the work up of aromatic), 1246 (C–N). 1H-NMR (DMSO, ppm); 0.78 (t, 3H,
reaction, 2 ml of concentrated HCl was added to a 20 ml ice-water –CH2–CH3, J ¼ 7.6 Hz); 0.98 (d, 3H, –CH–CH3, J ¼ 6.8 Hz); 1.35
solution. Reaction mixture was poured into this ice cold (m, 2H, –CH2–CH3); 2.23 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz);
solution slowly with stirring. White solid product was collected 3.28 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 3.53 (m, 1H, –NH–
with vacuum filtration and washed twice with distilled water. CH–); 4.29 (s, 1H, (Ar)2CH–); 6.02 (d, 1H, –CONH–, J ¼ 7.6 Hz);
4-Chlorobenzophenone and 4,40-difluorobenzophenone were also 7.20 (m, 2H, diphenyl H4, H4 ); 7.30 (t, 4H, diphenyl H3, H5, H3 ,
0
0
0
0
0
reacted with sodium borohydride to give 4-chlorobenzhydrole H5 , J ¼ 7.6 Hz); 7.43 (t, 4H, diphenyl H2, H6, H2 , H6 ,
and 4,40-difluorobenzhydrole, respectively, according to above J ¼ 7.2 Hz). 13C-NMR (DMSO, ppm); 11.43 (C21); 21.45 (C22);
procedure.
29.90 (C20); 44.25 (C14,16); 47.82 (C19); 52.06 (C15,17); 75.59
(C7); 127.56 (C4,11); 128.29 (C2,6,9,13); 129.20 (C3,5,10,12); 143.30
(C1,8); 157.76 (C18). MS (m/z); 352.8 (Mþ); 253.7 ((C6H5)2
CHN(C2H4)2NHeþ); 167.5 ((C6H5)2CHeþ). Elemental analysis of
C22H29N3O (MW: 351.49 g/mol); C 75.18, H 8.32, N 11.96
(Calcd.); C 75.12, H 8.27, N 11.85 (Found).
General procedure for preparation of benzhydryl chloride
derivatives
Ten millimoles (1.84 g) of benzhydrole was added to 15 ml
of concentrated HCl. 10 mmol (1.1 g) of anhydrous calcium
chloride was added to the mixture to be refluxed at 85 ꢀC for 4 h
with stirring. After the reaction is completed, the flask was cooled
N-tert-Butyl-4-(diphenylmethyl)piperazine-1-carboxamide (5b)
to room temperature and extracted twice with 20 ml of ethyl White, opaque, needle-shaped crystals, 62% (0.436 g), m.p.
acetate. Organic layers were combined together, washed with 192.4 ꢀC. UV (MeOH, ꢀmax, nm); 206 (log ": 5.13), 227 (log ":
brine and water, then dried over anhydrous sodium sulfate. 4.62). FT-IR (KBr, cmꢁ1); 3322 (N–H), 3023 (C–H, aromatic),
Followed by the concentration under vacuo, the product 2970 (C–H, aliphatic), 1621 (C¼O, amide), 1536 (C¼C,
was collected as brown liquid. 4-Chlorobenzhydryl chloride and aromatic), 1260 (C–N). 1H-NMR (DMSO, ppm); 1.22 (s, 9H,
4,40-difluorobenzhydryl chloride were also synthesized from –C(CH3)3); 2.23 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.25
4-chlorobenzhydrole and 4,40-difluorobenzhydrole according to (t, 4H, piperazine H2, H6, J ¼ 4.4 Hz); 4.29 (s, 1H, (Ar)2CH–);
0
5.68 (s, 1H, CONH); 7.19 (m, 2H, diphenyl H4, H4 ); 7.30 (t, 4H,
above procedure.
0
0
diphenyl H3, H5, H3 , H5 , J ¼ 7.6 Hz); 7.43 (t, 4H, diphenyl H2,
0
0
General procedure for preparation of
benzhydrylpiperazine derivatives
H6, H2 , H6 , J ¼ 7.2 Hz). Elemental analysis of C22H29N3O (MW:
351.49 g/mol); C 75.18, H 8.32, N 11.96 (Calcd.); C 74.60,
H 8.21, N 11.84 (Found).
Nine millimoles (0.78 g) of piperazine was dissolved in
dimethylformamide. Anhydrous potassium carbonate was added
to the solution and stirred for 10 min. Followed by the addition of
N-Isopropyl-4-(diphenylmethyl)piperazine-1-carboxamide (5c)
9 mmol (1.82 g) of benzhydryl chloride, reaction mixture was White, opaque, clustered crystals, 94% (0.318 g), m.p. 220.4 ꢀC.
heated at 80 ꢀC for 8 h. After completion, dimethylformamide UV (MeOH, ꢀmax, nm); 207 (log ": 5.21), 227 (log ": 4.81). FT-IR
was removed under vacuo, then the residue was taken in water (KBr, cmꢁ1); 3367 (N–H), 3060 (C–H, aromatic), 2964 (C–H,
and extracted with ethyl acetate. Organic layer was washed with aliphatic), 1611 (C¼O, amide), 1538 (C¼C, aromatic), 1254
water and dried over anhydrous sodium sulfate. The solvent (C–N). 1H-NMR (DMSO, ppm); 0.98 (d, 6H, –CH(CH3)2,
was evaporated and white solid product was obtained. 1-[(4- J ¼ 6.8 Hz); 2.19 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.25
Chlorophenyl)(phenyl)methyl]piperazine and 4,40-benzhydrylpi- (t, 4H, piperazine H2, H6, J ¼ 5.2 Hz); 3.68 (m, 1H, –CH(CH3)2);
perazine were also synthesized from 4-chlorobenzhydryl chloride 4.25 (s, 1H, (Ar)2CH–); 6.05 (d, 1H, CONH, J ¼ 7.6 Hz); 7.15
and 4,40-difluorobenzhydryl chloride consecutively according to (m, 2H, diphenyl H4, H4 ); 7.26 (t, 4H, diphenyl H3, H5, H3 ,
0
0
0
0
0
above procedure.
H5 , J ¼ 7.2 Hz); 7.39 (t, 4H, diphenyl H2, H6, H2 , H6 ,
J ¼ 6.8 Hz). Elemental analysis of C21H27N3O (MW: 337.46 g/
mol); C 74.74, H 8.06, N 12.45 (Calcd.); C 74.89, H 7.73, N 12.30
(Found).
General procedure for preparation of N-alkyl-4-
[benzhydryl/4-chlorobenzhydryl/4,40-
difluorobenzhydryl]piperazine-1-carboxamides
N-Ethyl-4-(diphenylmethyl)piperazine-1-carboxamide (5d)
Two millimoles (0.515 g) of 1-benzhydrylpiperazine or 1.7 mmol
(0.515 g) of 1-(4,40-difluorobenzhydryl)piperazine or 0.872 mmol White, shiny, flat crystals, 84% (0.294 g), m.p. 208.9 ꢀC. UV
(0.2632 g) of 1-(4-chlorobenzhydryl)piperazine was dissolved in (MeOH, ꢀmax, nm); 203 (log ": 5.11), 221 (log ": 4.58). FT-IR
20 mL of dry dichloromethane. Reaction flask was taken into ice (KBr, cmꢁ1); 3365 (N–H), 3024 (C–H, aromatic), 2978 (C–H,
bath and triethylamine (1:3 moles) was added to the solution. Ice aliphatic), 1622 (C¼O, amide), 1545 (C¼C, aromatic), 1259
bath was removed after 10 min and appropriate isocyanate (C–N).1H-NMR (DMSO, ppm); 0.98 (t, 3H, –CH3, J ¼ 7.6 Hz);
derivative (1:1 mole) was added. Reaction was mixed overnight 2.23 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.01 (m, 2H, –CH2–);
at room temperature. After the reaction is completed, solution was 3.28 (t, 4H, piperazine H2, H6, J ¼ 5.2 Hz); 4.28 (s, 1H,
extracted with water and ammonium chloride solution (10%), (Ar)2CH–); 6.41 (t, 1H, CONH, J ¼ 5.2 Hz); 7.20 (m, 2H,
0
0
0
respectively. Dichloromethane layer was washed with water again diphenyl H4, H4 ); 7.29 (t, 4H, diphenyl H3, H5, H3 , H5 ,

DOI: 10.3109/14756366.2013.765416
Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives 207
0
0
J ¼ 8 Hz); 7.43 (t, 4H, diphenyl H2, H6, H2 , H6 , J ¼ 7.2 Hz). N-sec-Butyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
Elemental analysis of C22H29N3O (MW: 351.49 g/mol); C 74.27, carboxamide (5i)
H 7.79, N 12.99 (Calcd.); C 73.77, H 7.46, N 12.93 (Found).
White, opaque, powdered crystals, 54% (0.208 g), m.p. 157.7 ꢀC.
UV (MeOH, ꢀmax, nm); 207 (log ": 5.24), 225 (log ": 4.71). FT-IR
N-(2,6-Dichlorophenyl)-4-(diphenylmethyl)piperazine-1-
(KBr, cmꢁ1); 3310 (N–H), 3076 (C–H, aromatic), 2965 (C–H,
carboxamide (5e)
aliphatic), 1615 (C¼O, amide), 1548 (C¼C, aromatic), 1247
White, opaque, powdered crystals, 88% (0.386 g), m.p. 234.6 ꢀC. (C–N), 1223 (C–F). 1H-NMR (DMSO, ppm); 0.8 (t, 3H,
UV (MeOH, ꢀmax, nm); 207 (log ": 5.32), 226 (log ": 4.72). FT-IR –CH2CH3, J ¼ 7.2 Hz); 0.98 (d, 3H, –CH–CH3, J ¼ 6.8 Hz);
(KBr, cmꢁ1); 3237 (N–H,), 3025 (C–H, aromatic), 2967 (C–H, 2.24 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 2.5 (m, 2H, –CH–
aliphatic), 1638 (C¼O, amide), 1528 (C¼C, aromatic), 1255 CH2–CH3); 3.28 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 3.54 (m,
1
(C–N). H-NMR (DMSO, ppm); 2.29 (t, 4H, piperazine H3, H5, 1H, –NH–CH–); 4.38 (s, 1H, (Ar)2CH–); 6.04 (d, 1H, CONH,
0
0
J ¼ 4.8 Hz); 3.44 (t, 4H, piperazine H2, H6, J ¼ 4 Hz); 4.33 (s, 1H, J ¼ 7.6 Hz); 7.10–7.16 (m, 4H, diphenyl H2, H6, H2 , H6 ); 7.41–
þ
0
0
(Ar)2CH–); 7.15–7.3 (m, 10H, diphenyl); 7.40–7.47 (m, 3H, 2,6- 7.45 (m, 4H, diphenyl H3, H5, H3 , H5 ). MS (m/z); 388.95 (M );
dichlorophenyl); 8.34 (s, 1H, CONH). Elemental analysis of 290.00 ((4-F-C6H5)2CH[N(C2H4)2N]Heþ); 203.5 (100%, (4-F-
C24H23Cl2N3O (MW: 440.36 g/mol); C 65.46, H 5.26, N 9.54 C6H5)2CHeþ). Elemental analysis of C22H27F2N3O (MW:
(Calcd.); C 65.37, H 5.36, N 9.62 (Found).
387.46 g/mol); C 68.20, H 7.02, N 10.84 (Calcd.); C 67.44,
H 7.01, N 10.89 (Found).
N-(2-Benzylphenyl)-4-(diphenylmethyl)piperazine-1-
carboxamide (5f)
N-tert-Butyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-carboxa-
mide (5j)
White, opaque, feather-like crystals, 89% (0.412 g), m.p. 192.1 ꢀC.
UV (MeOH, ꢀmax, nm); 203 (log ": 5.12), 224 (log ": 4.26). FT-IR White, opaque, feather-like crystals, 82% (0.317 g), m.p. 162.4 ꢀC.
(KBr, cmꢁ1); 3251 (N–H), 3060 (C–H, aromatic), 2954 (C–H, UV (MeOH, ꢀmax, nm); 208 (log ": 5.32), 227 (log ": 4.78). FT-IR
aliphatic), 1637 (C¼O, amide), 1524 (C¼C, aromatic), 1253 (KBr, cmꢁ1); 3332 (N–H), 3046 (C–H, aromatic), 2968 (C–H,
1
(C–N). H-NMR (DMSO, ppm); 2.24 (t, 4H, piperazine H3, H5, aliphatic), 1623 (C¼O, amide), 1537 (C¼C, aromatic), 1259
J ¼ 4.8 Hz); 3.37 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 3.91 (C–N), 1219 (C–F). 1H-NMR (DMSO, ppm); 1.22 (s, 9H,
(s, 2H, –CH2–); 4.29 (s, 1H, (Ar)2CH–); 7.05–7.25 (m, 10H, C(CH3)3); 2.20 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.24
diphenyl); 7.30 (m, 5H, phenyl); 7.44 (m, 4H, N-phenyl); 7.97 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 4.38 (s, 1H, (Ar)2CH–);
0
(s, 1H, CONH). Elemental analysis of C31H31N3O (MW: 5.68 (s, 1H, CONH); 7.10–7.16 (m, 4H, diphenyl H2, H6, H2 ,
0
0
0
461.60 g/mol); C 80.66, H 6.77, N 9.10 (Calcd.); C 80.90, H6 ); 7.41–7.45 (m, 4H, diphenyl H3, H5, H3 , H5 ). MS (m/z);
H 6.48, N 9.13 (Found).
388.88 (100%, Mþ); 203.51 ((4-F-C6H5)2CHeþ). Elemental
analysis of C22H27F2N3O (MW: 387.46 g/mol); C 68.20, H 7.02,
N 10.84 (Calcd.); C 67.96, H 7.32, N 10.87 (Found).
Ethyl 2-[4-(diphenylmethyl)piperazino]carbamoyl]acetate
(5g, CAS No: 1350123-57-7)
N-Butyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carboxamide (5k)
White, opaque, powdered crystals, 69% (0.263 g), m.p. 150 ꢀC.
UV (MeOH, ꢀmax, nm); 202 (log ": 4.87), 223 (log ": 4.35). FT-IR
(KBr, cmꢁ1); 3360 (N–H), 3026 (C–H, aromatic), 2986 (C–H, White, opaque, flat crystals, 45% (0.174 g), m.p. 132.9 ꢀC. UV
aliphatic), 1755 (C¼O, ester), 1636 (C¼O, amide), 1531 (C¼C, (MeOH, ꢀmax, nm); 209 (log ": 5.43), 226 (log ": 4.83). FT-IR
aromatic), 1192 (C–O), 1147 (C–N). 1H-NMR (DMSO, ppm); (KBr, cmꢁ1); 3402 (N–H), 3073 (C–H, aromatic), 2962 (C–H,
1.17 (t, 3H, –CH2–CH3, J ¼ 6.8 Hz); 2.25 (t, 4H, piperazine H3, aliphatic), 1629 (C¼O, amide), 1531 (C¼C, aromatic), 1251
1
H5, J ¼ 4.4 Hz); 3.31 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 3.68 (C–N), 1217 (C–F). H-NMR (DMSO, ppm); 0.85 (t, 3H, –CH3,
(d, 2H, –NH–CH2–, J ¼ 5.6 Hz); 4.05 (q, 2H, –O–CH2–); 4.30 J ¼ 7.2 Hz) 1.20-1.27 (m, 2H, –CH2–CH3); 1.31–1.37 (m, 4H,
(s, 1H, (Ar)2CH–); 6.93 (t, 1H, CONH, J ¼ 6 Hz); 7.19 (t, 2H, –CH2CH2CH3–); 2.21 (t, 4H, piperazine H3, H5, J ¼ 4.4 Hz);
0
diphenyl H4, H4 , J ¼ 7.2 Hz); 7.29 (t, 4H, diphenyl H3, H5, 2.95–3.06 (q, 2H, –NH–CH2–); 3.27 (t, 4H, piperazine H2, H6,
0
0
0
H3 , H5 , J ¼ 7.2 Hz); 7.44 (d, 4H, diphenyl H2, H6, H2 , J ¼ 5.2 Hz); 4.38 (s, 1H, (Ar)2CH–); 6.38 (t, 1H, CONH); 7.1–
0
0
0
H6 , J ¼ 7.6 Hz). Elemental analysis of C22H27N3O3 (MW: 7.15 (m, 4H, diphenyl H2, H6, H2 , H6 ); 7.41–7.45 (m, 4H,
þ
0
0
381.47 g/mol); C 69.27, H 7.13, N 11.02 (Calcd.); C 69.24, diphenyl H3, H5, H3 , H5 ). MS (m/z); 388.93 (100%, M ); 203.55
H 6.96, N 10.96 (Found).
((4-F-C6H5)2CHeþ). Elemental analysis of C22H27F2N3O (MW:
387.46 g/mol); C 68.20, H 7.02, N 10.84 (Calcd.); C 67.92,
H 6.82, N 10.85 (Found).
N-Allyl-4-(diphenylmethyl)piperazine-1-carboxamide (5h, CAS
No: 1349487-56-4)
N-Ethyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carboxamide (5l)
White, shiny, flat crystals, 96% (0.323 g), m.p. 213.6 ꢀC. UV
(MeOH, ꢀmax, nm); 207 (log ": 5.32), 226 (log ": 4.75). FT-IR
(KBr, cmꢁ1); 3343 (N–H), 3027 (C–H, aromatic), 2954 (C–H, White, opaque, cotton-like crystals, 83% (0.297 g), m.p. 175 ꢀC.
aliphatic), 1625 (C¼O, amide), 1546 (C¼C, aromatic), 1255 UV (MeOH, ꢀmax, nm); 207 (log ": 5.39), 225 (log ": 4.81). FT-IR
1
(C–N). H-NMR (DMSO, ppm); 2.23 (t, 4H, piperazine H3, H5, (KBr, cmꢁ1); 3349 (N–H), 3060 (C–H, aromatic), 2972 (C–H,
J ¼ 4.8 Hz); 3.30 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 3.63 aliphatic), 1617 (C¼O, amide), 1544 (C¼C, aromatic), 1253
1
(t, 2H, –CH2–, J ¼ 5.2 Hz); 4.29 (s, 1H, (Ar)2CH-); 5.0 (dd, 2H, (C–N), 1216 (C–F). H-NMR (DMSO, ppm); 0.98 (t, 3H, –CH3,
–CH¼CH2, J1¼17.2 Hz, J2 ¼ 8 Hz, J3 ¼ 1.6 Hz); 5.78 (m, 1H, J ¼ 6.8 Hz); 2.21 (t, 4H, piperazine H3, H5, J ¼ 4 Hz); 3.05–2.98
–CH¼CH2); 6.61 (t, 1H, CONH, J ¼ 5.2 Hz); 7.17 (t, 2H, (m, 2H, –CH2–); 3.28 (t, 4H, piperazine H2, H6, J ¼ 4 Hz); 4.38
0
0
diphenyl H4, H4 , J ¼ 7.6 Hz); 7.29 (t, 4H, diphenyl H3, H5, H3 , (s, 1H, (Ar)2CH–); 6.42 (t, 1H, CONH, J ¼ 5.2 Hz); 7.11–7.15
0
0
0
0
0
H5 , J ¼ 7.6 Hz); 7.43 (d, 4H, diphenyl H2, H6, H2 , H6 , (m, 4H, diphenyl H2, H6, H2 , H6 ); 7.42–7.45 (m, 4H, diphenyl
J ¼ 8.8 Hz). Elemental analysis of C21H25N3O (MW: H3, H5, H3 , H5 ). 13C-NMR (DMSO, ppm); 16.26 (C20); 35.44
0
0
335.44 g/mol); C 75.19, H 7.51, N 12.53 (Calcd.); C 75.09, H (C19); 44.03 (C14,16); 51.84 (C15,17); 73.48 (C7); 115.91–116.12
7.25, N 12.46 (Found).
(C3,5,10,12); 130.03–130.12 (C2,6,9,13); 139.20, 139.17 (C1,8);

208 E. E. Gurdal et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
157.96–160.52 (C4,11); 162.95 (C18). MS (m/z); 360.85 (Mþ); C 68.47, H 7.31, N 10.89 (Calcd.); C 68.65, H 7.20, N 10.93
203.53 (100%, (4-F-C6H5)2CHeþ). Elemental analysis of (Found).
C20H23F2N3O (MW: 359.41 g/mol); C 66.84, H 6.45, N 11.69
(Calcd.); C 66.44, H 6.28, N 11.68 (Found).
N-tert-Butyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carboxamide (5q)
N-Isopropyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carboxamide (5m)
White, shiny, flat crystals, 36% (0.137 g), m.p .190.3 ꢀC. UV
(MeOH, ꢀmax, nm); 207 (log ": 5.29), 225 (log ": 4.52). FT-IR
(KBr, cmꢁ1); 3371 (N–H), 3027 (C–H, aromatic), 2968 (C–H,
aliphatic), 1629 (C¼O, amide), 1538 (C¼C, aromatic), 1257
(C–N), 1092 (C–Cl). 1H-NMR (DMSO, ppm); 1.19 (s, 9H,
–C(CH3)3); 2.19 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.21
(t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 4.31 (s, 1H, (Ar)2CH–);
5.65 (s, 1H, CONH); 7.17–7.42 (m, 9H, diphenyl). Elemental
analysis of C22H28ClN3O (MW: 385.93 g/mol); C 68.47, H 7.31,
N 10.89 (Calcd.); C 68.67, H 7.23, N 10.93 (Found).
White, opaque, powdered crystals, 92% (0.345 g), m.p. 169.9 ꢀC.
UV (MeOH, ꢀmax, nm); 205 (log ": 5.25), 223 (log ": 4.47). FT-IR
(KBr, cmꢁ1); 3331 (N–H), 3074 (C–H, aromatic), 2976 (C–H,
aliphatic), 1615 (C¼O, amide), 1547 (C¼C, aromatic), 1252
(C–N), 1215 (C–F). 1H-NMR (DMSO, ppm); 1.01 (d, 6H,
–CH(CH3)2, J ¼ 6.8 Hz); 2.21 (t, 4H, piperazine H3, H5,
J ¼ 4.4 Hz); 3.28 (t, 4H, piperazine H2, H6, J ¼ 4.4 Hz); 3.68–
3.76 (m, 1H, –CH(CH3)2); 4.38 (s, 1H, (Ar)2CH–); 6.10 (d, 1H,
0
0
CONH, J ¼ 7.6 Hz); 7.11–7.15 (m, 4H, diphenyl H2, H6, H2 , H6 );
0
0
7.42–7.45 (m, 4H, diphenyl H3, H5, H3 , H5 ). MS (m/z); 374.87
(Mþ); 289.72 ((4-F-C6H5)2CHN(C2H4)2NHeþ); 203.54 (100%,
(4-F-C6H5)2CHeþ). Elemental analysis of C21H25F2N3O (MW:
373.44 g/mol); C 67.54, H 6.75, N 11.25 (Calcd.); C 67.87,
H 6.64, N 11.20 (Found).
N-Ethyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carboxamide (5r)
White, shiny, clustered crystals, 17% (0.06 g), m.p. 288.6 ꢀC. UV
(MeOH, ꢀmax, nm); 205 (log ": 5.24), 224 (log ": 4.46). FT-IR
(KBr, cmꢁ1); 3363 (N–H), 3020 (C–H, aromatic), 2970 (C–H,
aliphatic), 1620 (C¼O, amide), 1539 (C¼C, aromatic), 1254
(C–N), 1090 (C–Cl). 1H-NMR (DMSO, ppm); 0.98 (t, 3H, –CH3,
J ¼ 7.2 Hz); 2.22 (t, 4H, piperazine H3, H5, J ¼ 4.4 Hz); 3.00–3.03
(m, 2H, –CH2–); 3.27 (t, 4H, piperazine H2, H6, J ¼ 5.2 Hz); 4.34
(s, 1H, (Ar)2CH–); 6.41 (t, 1H, CONH, J ¼ 5.6 Hz); 7.18–7.46 (m,
9H, diphenyl). Elemental analysis of C20H24ClN3O (MW:
357.88 g/mol); C 67.12, H 6.76, N 11.74 (Calcd.); C 67.22,
H 6.69, N 11.79 (Found).
Ethyl 2-[bis(4-fluorophenyl)methyl]piperazino]
carbamoylacetate (5n)
White, opaque, powdered crystals, 20% (0.08 g), m.p. 152.3 ꢀC.
UV (MeOH, ꢀmax, nm); 203 (log ": 4.89), 221 (log ": 4.29). FT-IR
(KBr, cmꢁ1); 3359 (N–H), 3070 (C–H, aromatic), 2978 (C–H,
aliphatic), 1748 (C¼O, ester), 1640 (C¼O, amide), 1602 (C¼C,
aromatic), 1224 (C–O), 1198 (C–N), 1153 (C–F). 1H-NMR
(DMSO, ppm); 1.17 (t, 3H, –CH3, J ¼ 7.2 Hz); 2.23 (t, 4H,
piperazine H3, H5, J ¼ 5.2 Hz); 3.11 (t, 4H, piperazine H2, H6,
J ¼ 4.8 Hz); 3.68 (d, 2H, –NH–CH2–, J ¼ 6 Hz); 4.03–4.08 (q, 2H,
–O–CH2–); 4.39 (s, 1H, (Ar)2CH–); 6.93 (t, 1H, CONH,
N-Isopropyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carboxamide (5s)
White, shiny, flat crystals, 34% (0.128 g), m.p. 198.6 ꢀC. UV
(MeOH, ꢀmax, nm); 205 (log ": 5.15), 223 (log ": 4.45). FT-IR
(KBr, cmꢁ1); 3390 (N–H), 3020 (C–H, aromatic), 2969 (C–H,
aliphatic), 1617 (C¼O, amide), 1532 (C¼C, aromatic), 1252
(C–N), 1092 (C–Cl). 1H-NMR (DMSO, ppm); 1.01 (d, 6H,
–CH(CH3)2, J ¼ 6.8 Hz); 2.22 (t, 4H, piperazine H3, H5,
J ¼ 4.4 Hz); 3.27 (t, 4H, piperazine H2, H6, J ¼ 5.2 Hz); 3.68–
3.75 (m, 1H, –CH(CH3)2); 4.34 (s, 1H, (Ar)2CH–); 6.08 (d, 1H,
CONH, J ¼ 7.6 Hz); 7.18–7.46 (m, 9H, diphenyl). Elemental
analysis of C21H26ClN3O (MW: 371,9 g/mol); C 67.82, H 7.05,
N 11.30 (Calcd.); C 67.88, H 7.11, N 11.35 (Found).
0
0
J ¼ 6 Hz); 7.11–7.16 (m, 4H, diphenyl H2, H6, H2 , H6 ); 7.42–
0
0
7.46 (m, 4H, diphenyl H3, H5, H3 , H5 ). Elemental analysis of
C22H25F2N3O3 (MW: 417.45 g/mol); C 63.30, H 6.04, N 10.07
(Calcd.); C 63.46, H 6.05, N 10.02 (Found).
N-(4-Bromophenyl)-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carboxamide (5o)
White, opaque, powdered crystals, 67% (0.325 g), m.p. 210.9 ꢀC.
UV (MeOH, ꢀmax, nm); 202 (log ": 4.31), 237 (log ": 4.15) , 246
(log ": 4.12). FT-IR (KBr, cmꢁ1); 3290 (N–H), 3044 (C–H,
aromatic), 2999 (C–H, aliphatic), 1646 (C¼O, amide), 1506
(C¼C, aromatic), 1246 (C–N), 1224 (C–F). 1H-NMR (DMSO,
ppm); 2.29 (t, 4H, piperazine H3, H5, J ¼ 4.4 Hz); 3.45 (t, 4H,
piperazine H2, H6, J ¼ 4.8 Hz); 4.44 (s, 1H, (Ar)2CH–); 7.12–7.47
(m, 12H, aromatic H’s); 8.61 (s, 1H, CONH). Elemental analysis
of C24H23BrClN3O (MW: 484.82 g/mol); C 59.27, H 4.56, N 8.64
(Calcd.); C 59.02, H 4.38, N 8.73 (Found).
N-Allyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carboxamide (5t)
White, opaque, powdered crystals, 27% (0.1 g), m.p. 172.7 ꢀC. UV
(MeOH, ꢀmax, nm); 204 (log ": 5.11), 225 (log ": 4.38). FT-IR
(KBr, cmꢁ1); 3356 (N–H), 3027 (C–H, aromatic), 2981 (C–H,
aliphatic), 1622 (C¼O, amide), 1543 (C¼C, aromatic), 1252
(C–N), 1094 (C–Cl). 1H-NMR (DMSO, ppm); 2.23 (t, 4H,
piperazine H3, H5, J ¼ 4.8 Hz); 3.30 (t, 4H, piperazine H2, H6,
J ¼ 4.8 Hz); 3.63 (t, 2H, NH–CH2–CH¼, J ¼ 4.8 Hz); 4.34 (s, 1H,
(Ar)2CH–); 4.97–5.08 (dd, 2H, –CH¼CH2, J1 ¼ 16 Hz,
J2 ¼ 10 Hz, J3 ¼ 1.6 Hz); 5.75–5.82 (m, 1H, –CH¼CH2); 6.62
(t, 1H, CONH); 7.18–7.46 (m, 9H, diphenyl). Elemental analysis
of C21H24ClN3O (MW: 371.9 g/mol); C 68.19, H 6.54, N 11.36
(Calcd.); C 68.52, H 6.43, N 11.43 (Found).
N-sec-Butyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carboxamide (5p)
White, shiny, clustered crystals, 62% (0.240 g), m.p. above
300 ꢀC. UV (MeOH, ꢀmax, nm); 207 (log ": 5.32), 226 (log ":
4.51). FT-IR (KBr, cmꢁ1); 3393 (N–H), 3027 (C–H, aromatic),
2970 (C–H, aliphatic), 1618 (C¼O, amide), 1533 (C¼C,
1
aromatic), 1246 (C–N), 1091 (C–Cl). H-NMR (DMSO, ppm);
0.78 (t, 3H, –CH2–CH3, J ¼ 7.6 Hz); 1.00 (d, 3H, –CH–CH3,
J ¼ 6.8 Hz); 1.32–1.40 (m, 2H, –CH–CH2–CH3); 2.22 (t, 4H,
piperazine H3, H5, J ¼ 4.4 Hz); 3.28 (t, 4H, piperazine H2, H6,
J ¼ 4.4 Hz); 3.51–3.55 (m, 1H, –NHCH); 4.35 (s, 1H, (Ar)2CH–);
N-(2,6-Dichlorophenyl)-4-[(4-chlorophenyl)(phenyl)methyl]
piperazine-1-carboxamide (5u)
6.03 (d, 1H, CONH, J ¼ 8 Hz); 7.18–7.46 (m, 9H, diphenyl). White, shiny, powdered crystals, 38% (0.178 g), m.p. 224.6 ꢀC.
Elemental analysis of C22H28ClN3O (MW: 385.93 g/mol); UV (MeOH, ꢀmax, nm); 205 (log ": 4.47), 245 (log ": 4.12).

DOI: 10.3109/14756366.2013.765416
Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives 209
FT-IR (KBr, cmꢁ1); 3316 (N–H), 3020 (C–H, aromatic), 2963 2.32 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.48 (t, 4H, piperazine
(C–H, aliphatic), 1645 (C¼O, amide), 1519 (C¼C, aromatic), H2, H6, J ¼ 4.8 Hz); 4.41 (s, 1H, (Ar)2CH–); 7.21–7.47
1
1254 (C–N), 1089 (C–Cl). H-NMR (DMSO, ppm); 2.31 (t, 4H, (m, 9H, diphenyl); 7.61–7.67 (m, 4H, 4-cyanophenyl); 8.97
piperazine H3, H5, J ¼ 4.8 Hz); 3.46 (t, 4H, piperazine H2, H6, (s, 1H, CONH). Elemental analysis of C25H23ClN4O (MW:
J ¼ 4.4 Hz); 4.41 (s, 1H, (Ar)2CH–); 7.21–7.49 (m, 12H, aromatic 430.93 g/mol); C 69.68, H 5.38, N 13.00 (Calcd.); C 69.74,
H’s); 8.37 (s, 1H, CONH). Elemental analysis of C24H22Cl3N3O H 5.50, N 13.06 (Found).
(MW: 474.81 g/mol); C 60.71, H 4.67, N 8.85 (Calcd.); C 60.70,
H 4.77, N 9.18 (Found).
General procedure for preparation of N-Alkyl-4-
[4-chlorobenzhydryl/4,40-difluorobenzhydryl]piperazine-
N-(2-Phenylethyl)-4-[(4-chlorophenyl)(phenyl)methyl]piperazine- 1-carbothioamides
1-carboxamide (5v)
A total of 1.7 mmol (0.515 g) 1-[Bis(4-fluorophenyl)methyl]
White, opaque, feather-like crystals, 49% (0.212 g), m.p. 147.8 ꢀC. piperazine or 0.872 mmol (1 mol, 0.2632 g) 1-[(4-chlorophenyl)
UV (MeOH, ꢀmax, nm); 205 (log ": 4.52), 245 (log ": 4.07). FT-IR (phenyl)methyl]piperazine was dissolved in 20 ml dry dichlor-
(KBr, cmꢁ1); 3307 (N–H), 3022 (C–H, aromatic), 2955 (C–H, omethane. Reaction flask was taken into ice bath and triethyla-
aliphatic), 1617 (C¼O, amide), 1543 (C¼C, aromatic), 1256 mine (1:3 moles) was added to the solution. After 10 min, ice bath
(C–N), 1091 (C–Cl). 1H-NMR (DMSO, ppm); 2.22 (t, 4H, was removed and suitable isothiocyanate derivative (1:1 mole)
piperazine H3, H5, J ¼ 4.4 Hz); 2.69 (t, 2H, –CH2–C6H5, was added. Reaction was stirred overnight at room temperature.
J ¼ 6.8 Hz); 3.19 (q, 2H, –NHCH2); 3.28 (t, 4H, piperazine H2, After the reaction was completed, solution was extracted in
H6, J ¼ 5.2 Hz); 4.34 (s, 1H, (Ar)2CH–); 6.55 (t, 1H, CONH, order with water and ammonium chloride solution (10%).
J ¼ 5.6 Hz); 7.15–7.46 (m, 14H, aromatic H’s). Elemental analysis Dichloromethane layer was washed with water again and dried
of C26H28ClN3O (MW: 433.97 g/mol); C 71.96, H 6.50, N 9.68 with anhydrous sodium sulfate. Solvent was evaporated under
(Calcd.); C 72.04, H 6.72, N 9.70 (Found).
vacuo and solid product was recrystallized with ethanol/water.
N-(4-Bromophenyl)-4-[(4-chlorophenyl)(phenyl)methyl]
piperazine-1-carboxamide (5w)
N-tert-Butyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carbothioamide hydrochloride (6a)
White, opaque, feather-like crystals, 37% (0.180 g), m.p. 195.5 ꢀC. Yellowish white, opaque, powdered crystals, 14% (0.06 g), m.p.
UV (MeOH, ꢀmax, nm); 203 (log ": 4.33), 236 (log ": 4.11). FT-IR 176.8 ꢀC. UV (MeOH, ꢀmax, nm); 205 (log ": 4.23), 223 (log ":
(KBr, cmꢁ1); 3316 (N–H), 3028 (C–H, aromatic), 2966 (C–H, 4.11). FT-IR (KBr, cmꢁ1); 3258 (N–H), 3057 (C–H, aromatic),
aliphatic), 1634 (C¼O, amide), 1537 (C¼C, aromatic), 1243 2972 (C–H, aliphatic), 1606 (C¼C, aromatic), 1288 (C–N), 1236
(C–N), 1089 (C–Cl). 1H-NMR (DMSO, ppm); 2.30 (t, 4H, (C¼S, thioamide), 1189 (C–F). 1H-NMR (DMSO, ppm); 1.45
piperazine H3, H5, J ¼ 4.8 Hz); 3.45 (t, 4H, piperazine H2, H6, (s, 9H, –C(CH3)3); 2.96–3.15 (m, 4H, piperazine H3, H5); 3.65
J ¼ 4.8 Hz); 4.39 (s, 1H, (Ar)2CH–); 7.21–7.47 (m, 13H, aromatic (t, 4H, piperazine H2, H6); 4.59 (d, 1H, (Ar)2CH–, J ¼ 14.4 Hz);
H’s); 8.6 (s, 1H, CONH). Elemental analysis of C24H23BrClN3O 5.75 (d, 1H, CSNH, J ¼ 8.8 Hz); 7.17–7.33 (m, 4H, diphenyl H2,
0
0
0
0
(MW: 484.82 g/mol); C 59.46, H 4.78, N 8.67 (Calcd.); C 59.43, H6, H2 , H6 ); 7.95 (bs, 4H, diphenyl H3, H5, H3 , H5 ); 12.55
H 4.97, N 8.84 (Found).
(bs, 1H, N–H salt). MS (m/z); 404.90 (100%, Mþ – Cl); 205.3
((4-F-C6H5)2CHeþ). Elemental analysis of C22H28ClF2N3S (MW:
439.99 g/mol); C 60.05, H 6.41, N 9.55, S 7.29 (Calcd.); C 59.55,
H 6.45, N 9.47, S 6.64 (Found).
N-(2-Benzylphenyl)-4-[(4-chlorophenyl)(phenyl)methyl]
piperazine-1-carboxamide (5x)
White, shiny, needle-shaped crystals, 44% (0.219 g), m.p.
174.6 ꢀC. UV (MeOH, ꢀmax, nm); 205 (log ": 4.57), 224 (log ":
4.21). FT-IR (KBr, cmꢁ1); 3332 (N–H), 3026 (C–H, aromatic),
N-Cyclohexyl-4-[bis(4-fluorophenyl)methyl]piperazine-1-
carbothioamide (6b)
2967 (C–H, aliphatic), 1626 (C¼O, amide), 1523 (C¼C, White, shiny, needle-shaped crystals 50%, (0.214 g), m.p.
1
aromatic), 1251 (C–N), 1089 (C–Cl). H-NMR (DMSO, ppm); 198.2 ꢀC. UV (MeOH, ꢀmax, nm); 202 (log ": 4.10), 224 (log ":
2.23 (bs, 4H, piperazine H3, H5); 3.36 (bs, 4H, piperazine H2, H6); 4.02), 248 (log ": 3.88). FT-IR (KBr, cmꢁ1); 3328 (N–H), 3060
3.91 (s, 2H, –CH2–); 4.34 (s, 1H, (Ar)2CH–); 7.05–7.47 (m, 18H, (C–H, aromatic), 2996 (C–H, aliphatic), 1603 (C¼C, aromatic),
aromatic H’s); 7.96 (s, 1H, CONH). 13C-NMR (DMSO, ppm); 1299 (C–N), 1221 (C¼S, thioamide), 1104 (C–F). 1H-NMR
37.74 (C25); 44.46 (C14,16); 51.91 (C15,17); 74.48 (C7); 125.46 (DMSO, ppm); 1.13–1.21 (m, 5H, cyclohexyl); 1.53–1.79 (m, 6H,
(C20); 126.52 (C22); 126.97 (C29); 127.23 (C21); 127.79 (C27,31); cyclohexyl); 2.22 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.71
128.31 (C11); 128.91 (C9,13); 129.23 (C23); 129.33 (C28,30); (t, 4H, piperazine H2, H6, J ¼ 4.4 Hz); 4.12 (s, 1H, CSNH); 4.40
0
0
129.42 (C10,12); 130.11 (C3,5); 130.64 (C2,6); 132.09 (C4); 136.87 (s, 1H, (Ar)2CH–); 7.09–7.14 (m, 4H, diphenyl H2, H6, H2 , H6 );
(C19); 138.23 (C1); 141.05 (C26); 142.26 (C8); 142.64 (C24); 7.39–7.43 (m, 4H, diphenyl H3, H5, H3 , H5 ). 13C-NMR (DMSO,
156.06 (C18). MS (m/z); 496.9 (Mþ, 100%); 498.9 (M þ 2, 33%); ppm); 24.99 (C21,23); 25.18 (C22); 31.97 (C20,24); 47.06 (C14,16);
287.8 ((4-Cl-C6H5)(C6H5)CH-N(C2H4)2Neþ); 201.6 ((4-Cl- 50.85 (C15,17); 54.28 (C19); 72.32 (C7); 115.14 (C10,12); 115.35
C6H5)(C6H5)CHeþ). Elemental analysis of C31H30ClN3O (MW: (C3,5); 129.35 (C9,13); 129.43 (C2,6); 138.12 (C8); 138.15 (C1);
496.04 g/mol); C 75.06, H 6.10, N 8.47 (Calcd.); C 75.13, H 6.28, 159.79 (C11); 162.21 (C4); 180.14 (C18). MS (m/z); 430.95
0
0
N 8.54 (Found).
(100%, Mþ); 203.65 ((4-F-C6H5)2CHeþ). Elemental analysis
of C24H29F2N3S (MW: 429.57 g/mol); C 67.10, H 6.80, N 9.78,
S 7.46 (Calcd.); C 66.94, H 6.94, N 9.89, S 7.42 (Found).
N-(4-Cyanophenyl)-4-[(4-chlorophenyl)(phenyl)methyl]
piperazine-1-carboxamide (5y)
N-Ethyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carbothioamide (6c)
White, shiny, powdered crystals, 26% (0.114 g), m.p. 196.8 ꢀC.
UV (MeOH, ꢀmax, nm); 202 (log ": 4.82), 270 (log ": 4.59). FT-IR
(KBr, cmꢁ1); 3278 (N–H), 3027 (C–H, aromatic), 2951 (C–H, White, opaque, powdered crystals, 15% (0.056 g), m.p. 150.6 ꢀC.
aliphatic), 2221 (CꢃN), 1653 (C¼O, amide), 1513 (C¼C, UV (MeOH, ꢀmax, nm); 204 (log ": 4.25), 225 (log ": 4.13).
1
aromatic), 1245 (C–N), 1089 (C–Cl). H-NMR (DMSO, ppm); FT-IR (KBr, cmꢁ1); 3294 (N–H), 3020 (C–H, aromatic), 2966

210 E. E. Gurdal et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
1
(C–H, aliphatic), 1531 (C¼C, aromatic), 1255 (C–N), 1229 (C¼S, thioamide), 1201 (C–N), 1001 (C–Cl). H-NMR (DMSO,
1
(C¼S, thioamide), 1289 (C–N), 1091 (C–Cl). H-NMR (DMSO, ppm); 0.87 (t, 3H, –CH2CH2CH3, J ¼ 7.2 Hz); 1.20–1.29 (m, 2H,
ppm); 1.06 (t, 3H, –CH3, J ¼ 6.8 Hz); 2.27 (t, 4H, piperazine –CH2CH2CH3); 1.44–1.52 (m, 2H, –CH2CH2CH3); 2.27 (t, 4H,
H3, H5, J ¼ 5.2 Hz); 3.52–3.45 (m, 2H, –CH2–); 3.75 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.42–3.47 (q, 2H, –NHCH2–);
piperazine H2, H6, J ¼ 4.8 Hz); 4.39 (s, 1H, (Ar)2CH–); 7.19–7.46 3.75 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 4.39 (s, 1H, (Ar)2CH–
(m, 9H, diphenyl); 7.61 (t, 1H, CSNH). Elemental analysis ); 7.19–7.46 (m, 9H, diphenyl); 7.58 (t, 1H, CSNH, J ¼ 5.6 Hz).
of C20H24ClN3S (MW: 373.94 g/mol); C 64.24, H 6.47, N 11.24, Elemental analysis of C22H28ClN3S (MW: 401.17 g/mol); C
S 8.57 (Calcd.); C 64.44, H 6.19, N 11.35, S 8.67 (Found).
65.73, H 7.02, N 10.45, S 7.98 (Calcd.); C 66.06, H 7.07, N 10.56,
S 8.05 (Found).
N-Isopropyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carbothioamide (6d)
Cytotoxicity studies
White, shiny, needle-shaped crystals, 39% (0.15 g), m.p. 252.4 ꢀC. The cytotoxic activity of the synthesized compounds was
UV (MeOH, ꢀmax, nm); 203 (log ": 4.31), 223 (log ": 4.15). FT-IR investigated initially on liver (HUH-7), breast (MCF-7) and
(KBr, cmꢁ1); 3371 (N–H), 3059 (C–H, aromatic), 2967 (C–H, colon (HCT-116) cancer cell lines, by means of SRB assays
aliphatic), 1539 (C¼C, aromatic), 1270 (C–N), 1232 (C¼S, in triplicate. Serial dilutions from 100 mM to 2.5 mM were used,
1
thioamide), 1232 (C–N), 1001 (C–Cl). H-NMR (DMSO, ppm); 5-fluorouracil (5-FU) was the reference compound and camp-
1.09 (d, 6H, –(CH3)2, J ¼ 6.8 Hz); 2.27 (t, 4H, piperazine H3, H5, tothecin (CPT) was the positive control for the cytotoxic effect.
J ¼ 4.8 Hz); 3.76 (t, 4H, piperazine H2, H6, J ¼ 4.8 Hz); 4.39
(s, 1H, (Ar)2CH–); 4.44–4.53 (m, 1H, –CH(CH3)2); 7.19–7.46
(m, 10H, diphenyl H’s þ NH). 13C-NMR (DMSO, ppm); 38.79–
40.05 (C20,21); 46.93–47.01 (C14,15,16,17); 50.93 (C19); 73.36 (C7);
127.05 (C11); 127.57 (C9,13); 128.42 (C10,12); 128.52 (C3,5);
129.35 (C2,6); 131.32 (C4); 141.28 (C8); 141.64 (C1); 180.17
(C18). MS (m/z); 388.8 (Mþ, 100%); 390.8 (M þ 2, 33%); 201.5
(4-Cl-C6H5)(C6H5)CHþ). Elemental analysis of C21H26ClN3S
(MW: 387.97 g/mol); C 65.01, H 6.75, N 10.83, S 8.26
(Calcd.); C 64.88, H 6.88, N 10.87, S 8.29 (Found).
Cell culture
The human cancer cell lines were grown in Dulbecco’s Modified
Eagle’s Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS) and 1% penicillin. Each cell line was maintained in
an incubator at 37 ꢀC supplied with 5% CO2 and 95% air.
NCI-60 SRB assay
Cancer cells (range of 2000 cell/well to 5000 cell/well) were
inoculated into 96-well plates in 200 ml of media and incubated in
37 ꢀC incubators containing 5% CO2 and 95% air. After a 24 h
incubation period, one plate for each cell line was fixed with
100 ml of 10% ice-cold trichloroacetic acid (TCA). This plate
represents the behavior of the cells just prior to the drug treatment
and is accepted as the time-zero plate. The compounds to be
tested were solubilized in DMSO to a final concentration of
40 mM and stored at þ4 ꢀC. While treating the cells with the
compounds, the corresponding volume of the compound was
applied to the cell to achieve the desired drug concentration and
diluted through serial dilution. After the drug treatment, the cells
were incubated in 37 ꢀC incubators containing 5% CO2 and 95%
air for 72 h. Following the termination of the incubation period
after the drug treatment, the cells were fixed with 100 ml of 10%
ice-cold TCA and incubated in the dark at þ4 ꢀC for 1 h. Then the
TCA was washed away with ddH2O five times and the plates were
left to air dry. For the final step, the plates were stained with
100 ml of 0.4% SRB solution in 1% acetic acid solution. Following
staining, the plates were incubated in dark for 10 min at room
temperature. The unbound dye was washed away using 1% acetic
acid and the plates were left to air dry. To measure the absorbance
results, the bound stain was then solubilized using 200 ml of
10 mM Tris-Base. The OD values were obtained at 515 nm.
N-Allyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carbothioamide (6e)
White, opaque, powdered crystals, 10% (0.040 g), m.p. 139.4 ꢀC.
UV (MeOH, ꢀmax, nm); 204 (log ": 4.27), 225 (log ": 4.13). FT-IR
(KBr, cmꢁ1); 3296 (N–H), 3023 (C–H, aromatic), 2960 (C–H,
aliphatic), 1528 (C¼C, aromatic), 1252 (C–N), 1224 (C¼S,
1
thioamide), 1223 (C–N), 1090 (C–Cl). H-NMR (DMSO, ppm);
2.28 (t, 4H, piperazine H3, H5, J ¼ 5.2 Hz); 3.79 (t, 4H, piperazine
H2, H6, J ¼ 4 Hz); 4.15 (t, 2H, –CH2–CH¼CH2, J ¼ 5.6 Hz); 4.39
(s, 1H, (Ar)2CH–); 5.01–5.11 (dd, 2H, –CH¼CH2, J1 ¼ 17.2 Hz,
J2 ¼ 8.6 Hz, J3 ¼ 1.6 Hz); 5.80–5.90 (m, 1H, –CH¼CH2);
7.19–7.46 (m, 9H, diphenyl); 7.80 (t, 1H, CSNH, J ¼ 5.6 Hz).
Elemental analysis of C21H24ClN3S (MW: 385.95 g/mol);
C 65.35, H 6.27, N 10.89, S 8.31 (Calcd.); C 65.71, H 6.44,
N 11.01, S 8.28 (Found).
N-Benzyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carbothioamide (6f)
White, opaque, featherlike crystals, 23% (0.1 g), m.p. 157.2 ꢀC.
UV (MeOH, ꢀmax, nm); 203 (log ": 4.51), 226 (log ": 4.33). FT-IR
(KBr, cmꢁ1); 3236 (N–H), 3020 (C–H, aromatic), 2813 (C–H,
aliphatic), 1539 (C¼C, aromatic), 1246 (C–N), 1211 (C¼S,
1
thioamide), 1246 (C–N), 1001 (C–Cl). H–NMR (DMSO, ppm);
Results and discussion
2.31 (t, 4H, piperazine H3, H5, J ¼ 4.8 Hz); 3.83 (t, 4H, piperazine
H2, H6, J ¼ 4.4 Hz); 4.41 (s, 1H, (Ar)2CH–); 4.79 (d, 2H, –CH2–,
J ¼ 5.2 Hz); 7.19–7.47 (m, 14H, aromatic H’s); 8.19 (t, 1H,
CSNH, J ¼ 5.6 Hz). Elemental analysis of C25H26ClN3S (MW:
436.01 g/mol); C 68.87, H 6.01, N 9.64, S 7.35 (Calcd.); C 69.02,
H 5.98, N 9.80, S 7.46 (Found).
Chemistry
The synthesis of the benzhydrylpiperazine derivatives (5a–y)
and (6a–g) is outlined in Figure 1. Reduction with sodium
borohydride of benzophenone, 4-chlorobenzophenone and 4,40-
difluorobenzophenone afforded benzhydrole derivatives which
were chlorinated with HCl and anhydrous calcium chloride.
Resulting benzhydryl chloride derivatives were used for
N-alkylation of piperazine to give 1-benzhydrylpiperazine,
4-chlorobenzhydrylpiperazine and 4,40-difluorobenzhydrylpiper-
azine. The final step was nucleophilic addition to isocyanates
or isothiocyanates in order to obtain benzhydrylpiperazine
derivatives (5a–y) and (6a–g).
N-Butyl-4-[(4-chlorophenyl)(phenyl)methyl]piperazine-1-
carbothioamide hydrochloride (6g)
White, opaque, feather-like crystals, 20% (0.080 g), m.p. 125.5 ꢀC.
UV (MeOH, ꢀmax, nm); 204 (log ": 4.43), 227 (log ": 4.35). FT-IR
(KBr, cmꢁ1); 3261 (N–H), 3028 (C–H, aromatic), 2958
(C–H, aliphatic), 1541 (C¼C, aromatic), 1298 (C–N), 1201

DOI: 10.3109/14756366.2013.765416
Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives 211
R
R
R
1
1
1
HCl / CaCl2
NaBH4
HCl
Cl
OH
O
R
R
2
R
2
2
R
1
HN
NH
K CO
2
3
N
NH
R
2
a.
b.
R
R
1
1
O
S
N
N
N
N
HN
R
HN
R
3
3
R
R
2
2
5a-y
a. isocyanates, TEA, DCM, b. isothiocyanates, TEA, DCM
Figure 1. Synthesis of compounds 5a–y and 6a–g.
6a-g
Synthesized compounds were identified with IR, UV and (t, 4H, H1, J ¼ 4 Hz) ppm approximately. Diphenylmethyl C–H
1H-NMR spectra. In addition, some compounds were selected gives a singlet nearly at 4.5 ppm. Protons of aromatic rings
for LC-MS and 13C-NMR spectral evaluation. In UV spectra give multiplets at 7–7.5 ppm. Thioamide N–H is observed
of carboxamide derivatives there are two significant bands at 205 approximately at 7.5 ppm. The 13C-NMR spectrum of 5x
and 224 nm, which represent ꢁ ! ꢁ* and n ! ꢁ* transitions. shows characteristic peaks of the carboxamide derivatives
In UV spectra of thioamide derivatives there are three significant approximately at 45 and 50 ppm for piperazine ring, 75 ppm
bands nearly at 202, 224 and 248 nm, which represent ꢁ ! ꢁ* for diphenylmethyl carbon and 150 ppm for carbonyl group. The
and n ! ꢁ* transitions. In IR spectrum of carboxamide deriva- 13C-NMR spectrum of 6b shows characteristic peaks of the
tives, chꢁa1racteristic N–H stretching band is observed nearly at thioamide derivatives nearly at 45 and 50 ppm for piperazine ring,
3332 cm . Other stretching bands are observed approximately 70 ppm for diphenylmethyl carbon and 180 ppm for thiocarbonyl
at 3020ꢁc1mꢁ1 (C–H; aromatic), 2965 cmꢁ1 (C–H; aliphatic), group.
1625 cm
(C¼O; amide), 1520 cmꢁ1 (C¼C; aromatic) and
Structures of the prepared benzhydrylpiperazine derivatives
1250 cmꢁ1 (C–N). In IR spectrum of thioamide derivatives, are illustrated in Table 1.
characteristic N–H stretching band is observed nearly at
3330 cmꢁ1. Other stretching bands are observed approximately
Cytotoxicity
at 3060 cmꢁ1 (C–H; aromatic), 2995 cmꢁ1 (C–H; aliphatic),
1600 cmꢁ1 (C¼C; aromatic), 1300 cmꢁ1 (C–N) and 1220 cmꢁ1 The cytotoxic activity of the synthesized compounds 5a–y and
(C¼S) and 1100 cmꢁ1 (C–F). In H1-NMR spectra of carboxamide 6a–g was investigated on liver (HUH-7), breast (MCF-7) and
derivatives the protons of piperazine are seen approximately colon (HCT-116) cancer cell lines, by means of SRB assays
at 2.23 and 3.36 ppm as broad singlets. Diphenylmethyl C–H in triplicate. As shown in Table 2, all tested compounds were
gives a singlet nearly at 4 ppm. Aromatic rings give multiplets screened with mean 50% growth inhibition concentration (GI50)
at 7–7.5 ppm. Amide N–H gives a singlet nearly at 8 ppm. in micromolar concentration range.
In H1-NMR spectra of thioamide derivatives, the protons
of piperazine are seen at 2.5 (t, 4H, J ¼ 5.2 Hz) ppm and 3.5 are inactive or they have low activities against all cancer
Most of the nonsubstituted benzhydrylpiperazine derivatives

212 E. E. Gurdal et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
Table 1. Structural and physical information of compounds 5a–y Table 2. Cytotoxic activity data for compounds 5a–y and 6a–g.
and 6a–g.
Cancer cell line GI50 (mM)
R
1
Sample
HUH-7
R2
MCF-7
R2
HCT-116
R2
5a
5b
5c
5d
5e
NI*
NI
NI
NI
NI
NI
NI
NI
13.85
29.96
13.39
34.84
36.57
NI
9.46
13.03
10.88
20.92
15.36
16.29
6.44
13.18
8.54
17.22
1.29
5.97
25.8
10.81
6.20
9.95
22.59
8.10
30.66
0.15
–
–
–
–
–
–
–
–
0.94
0.88
0.91
0.79
0.79
–
0.98
0.98
0.93
0.92
0.86
0.96
0.97
0.97
0.94
0.74
0.88
0.87
0.98
0.66
0.86
0.93
0.81
0.93
0.98
0.89
NI
NI
NI
25.7
NI
NI
NI
10.91
NI
NI
19.03
NI
45.23
36.14
8.68
11.39
8.77
60.24
13.16
9.12
6.14
8.51
9.28
16.91
6.34
10.62
NI
–
–
–
0.86
–
NI
1.01
NI
NI
NI
NI
NI
NI
24.48
28.4
16.24
17.98
20.94
NI
8.87
9.33
9.33
10.78
17.12
10.14
8.93
5.72
7.34
4.76
1.81
13.09
NI
–
0.78
–
–
–
–
–
–
0.77
0.85
0.86
0.88
0.91
–
0.97
0.95
0.94
0.99
0.95
0.99
0.96
0.98
0.99
0.98
0.99
0.86
0.67
0.91
0.84
0.98
0.89
0.79
0.98
0.91
X
N
N
HN
R
3
5f
–
–
5g
5h
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
0.78
0.79
0.77
0.84
0.64
0.76
0.55
0.85
0.71
0.98
0.24
0.74
0.77
0.93
0.93
0.92
0.71
0.92
0.91
0.83
–
R
2
Melting
point (ꢀC)
Yield
(%)
Sample
X
R1
R2
R3
5a*
5b
5c
5d
5e
5f
5g*
5h*
5i
5j
5k
5l
5m
5n
5o
5p
5q
5r
5s
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
O
S
- H - H
- H - H
- H - H
- H - H
sec-Butyl
tert-Butyl
Isopropyl
Ethyl
198.4
192.4
220.4
208.9
234.6
192.1
150.0
213.6
157.7
162.4
132.9
175
169.9
152.3
210.9
68
62
94
84
88
89
69
96
54
82
45
83
92
20
67
62
36
17
34
27
38
49
37
44
26
14
50
15
39
10
23
20
- H - H 2,6-Dichlorophenyl
5s
5t
- H - H
- H - H
- H - H
- F
- F
- F
- F
- F
- F
- F
- Cl - H
- Cl - H
- Cl - H
- Cl - H
- Cl - H
2-Benzylphenyl
Ethylacetato
Allyl
5u
5v
5w
5x
5y
6a
6b
6c
6d
6e
6f
6g
5-FU
CPT
- F
- F
- F
- F
- F
- F
- F
sec-Butyl
tert-Butyl
Butyl
Ethyl
Isopropyl
Ethylacetato
4-Bromophenyl
sec-Butyl
tert-Butyl
Ethyl
NI
13.75
14.98
8.85
12.68
13.91
18.67
50.1
11.47
4.94
23.00
14.80
3.51
50.1
0.89
0.82
0.59
0.88
0.96
0.87
4300 (dec.)
190.3
288.6 (dec.)
198.6
172.7
Isopropyl
Allyl
5t
5u
5v
5w
5x
5y
6a
6b
6c
6d
6e
6f
- Cl - H 2,6-Dichlorophenyl
224.6
147.8
195.5
174.6
196.8
176.8
198.2
150.6
- Cl - H
- Cl - H
- Cl - H
- Cl - H
- F
- F
- Cl - H
- Cl - H
- Cl - H
- Cl - H
- Cl - H
2-Phenylethyl
4-Bromophenyl
2-Benzylphenyl
4-Cyanophenyl
tert-Butyl
Cyclohexyl
Ethyl
(*): Compounds active above 100 mM concentration were considered to
have no inhibition.
- F
- F
these cancer cell lines. However, 5x has good cytotoxicity against
all the cancer cell lines.
S
S
S
S
S
S
Compounds 5h, 5t and 6e have the same substituents on
NH group (R3 ¼ allyl). 5h has no cytotoxicity against HUH-7 and
HCT-116 cell lines nevertheless it has good cytotoxicity against
MCF-7 cell line. However, 5t has elevated cytotoxicity and 6e has
the highest cytotoxicity against all three cancer cell lines.
In general, nonsubstituted benzhydryl derivatives are inactive
or have low inhibition whereas 4-chlorobenzhydryl derivatives are
more active than other compounds against HUH-7 cell line.
The most active compounds against HUH-7 cell line are 5y
Isopropyl
Allyl
Benzyl
Butyl
252.4 (dec.)
139.4
157.2
6g
125.5
(*) 5a, CAS No: 1071382-92-7; 5g, CAS No: 1350123-57-7; 5h, CAS
No: 1349487-56-4.
cell lines. It should also be noted that, in general, 4-chlorobenz-
hydrylpiperazine derivatives have higher activities becoming (GI50 ¼ 1.29 mM) and 6a (GI50 ¼ 5.97 mM). Additionally, most
superior over their 4,40-difluoro and nonsubstituted counterparts. of the compounds have higher cytotoxicity against HUH-7 than
Moreover, thioamide derivatives are more potent than carbox- reference compound 5-fluorouracil.
amide derivatives against all cancer cell lines. Corresponding
compound groups representing these findings are detailed in electron withdrawing substituents on phenyl ring such as 5o
Table 3.
Among the carboxamide derivatives, compounds bearing
(GI50 ¼ 9.46 mM), 5u (GI50 ¼ 6.44 mM), 5w (GI50 ¼ 8.54 mM)
Compounds 5c, 5m, 5s and 6d have the same substituents and 5y (GI50 ¼ 1.29 mM) are highly active against HUH-7 cell
on NH group (R3 ¼ isopropyl). Compound 5c has no cytotoxicity line. In addition, alkyl substituted derivatives, except thioamide
against any of these cancer cell lines. However, 5m has slight derivatives, have no (5a–d, 5h, 5n) or low inhibition (5i–m, 5p–t).
cytotoxicity, 5s has good cytotoxicity and 6d has the highest
cytotoxicity against all three cancer cell lines.
Thioamide derivatives are generally cytotoxic against
HUH-7 cell line. It can be noted that thioamides show higher
Compounds 5e and 5u have the same substituents on NH group activity than their carboxamide derivatives, which can be
(R3 ¼ 2,6-dichlorophenyl). 5e has no cytotoxicity against any of exemplified by compounds 5j (GI50 ¼ 29.96 mM) compared
the cancer cell lines. Interestingly, 5u has increased cytotoxicity with 6a (GI50 ¼ 5.97 mM), 5r (GI50 ¼ 20.92 mM) compared with
against all the cancer cell lines.
6c (GI50 ¼ 10.81 mM), 5s (GI50 ¼ 15.36 mM) compared with 6d
Compounds 5f and 5x have the same substituents on NH group (GI50 ¼ 6.20 mM) and 5t (GI50 ¼ 16.29 mM) compared with 6e
(R3 ¼ 2-benzylphenyl). 5f has no cytotoxicity against none of (GI50 ¼ 9.95 mM).

DOI: 10.3109/14756366.2013.765416
Synthesis and cytotoxic activities of 32 benzhydrylpiperazine derivatives 213
Table 3. GI50 (mM) values of some carboxamide and thioamide derivatives for detailed discussion of SAR.
Samples
X
R1
R2
R3
HUH-7
MCF-7
HCT-116
5c
5m
5s
6d
5e
5u
5f
5x
5h
5t
O
O
O
S
O
O
O
O
O
O
S
- H
- F
- H
- F
Isopropyl
Isopropyl
Isopropyl
–
–
–
36.57
15.36
6.20
–
6.44
–
17.22
–
16.29
9.95
45.23
13.16
11.47
–
6.14
–
16.91
10.91
9.12
4.94
20.94
17.12
14.98
–
8.93
–
4.76
–
10.14
8.85
- Cl
- Cl
- H
- Cl
- H
- Cl
- H
- Cl
- Cl
- H
- H
- H
- H
- H
- H
- H
- H
- H
Isopropyl
2,6-Dichlorophenyl
2,6-Dichlorophenyl
2-Benzylphenyl
2-Benzylphenyl
Allyl
Allyl
Allyl
6e
Table 4. MCF-7 (breast cancer cell line) and MCF-12A (normal-like
breast epithelial cell line) cytotoxicity comparison of compound 6e (mM).
chiral compounds, further exploration of chiral separation
methods will be performed. The primary ambition regarding
future research is to evaluate the mechanism of cytotoxicity.
Compound
MCF-7
R2
MCF-12A
R2
6e
CPT
4.94
50.1
0.8
0.9
8.5
50.1
0.9
0.8
Declaration of interest
The authors have declared no conflicts of interest.
The most active compounds against MCF-7 cell line are 5u
(GI50 ¼ 6.14 mM) and 6e (GI50 ¼ 4.94 mM). Furthermore, we
observed that compound 6e was less toxic in MCF-12A
(GI50 ¼ 8.5 mM), which is a normal-like breast epithelial cell
line (Table 4).
Against MCF-7 cell line, nonsubstituted benzhydryl carbox-
amide derivatives (except 5d and 5h) and 5i, 5j, 5l, 6b, 6c show
no inhibition. Alkyl-substituted carboxamide derivatives have
References
1. Gao L, Li M, Meng T, et al. Asymmetric synthesis and biological
evaluation of N-cyclohexyl-4-[1-(2,4-dichlorophenyl)-1-(p-tolyl)-
methyl]piperazine-1-carboxamide as hCB1 receptor antagonists.
Eur J Med Chem 2011;46:5310–16.
2. Cumming JG, Bower JF, Waterson D, et al. The design and synthesis
of novel, potent and orally bioavailable N-aryl piperazine-1-
carboxamide CCR2 antagonists with very high hERG selectivity.
Bioorg Med Chem Lett 2012;22:3895–9.
3. Kinoyama I, Taniguchi N, Toyoshima A, et al. (þ)-(2R,5S)-4-[4-
cyano-3-(trifluoromethyl)phenyl]-2,5-dimethyl-N-[6-(trifluoromethyl)
pyridin-3-yl]piperazine-1-carboxamide (YM580) as an orally potent
and peripherally selective nonsteroidal androgen receptor antagonist.
J Med Chem 2006;49:716–26.
4. Sun Q, Tafesse L, Islam K, et al. 4-(2-Pyridyl)piperazine-1-
carboxamides: potent vanilloid receptor 1 antagonists. Bioorg Med
Chem Lett 2003;13:3611–16.
5. Matsuno K, Nakajima T, Ichimura M, et al. Potent and selective
inhibitors of PDGF receptor phosphorylation. 2. Synthesis, structure
activity relationship, improvement of aqueous solubility, and
biological effects of 4-[4-(N-substituted(thio)carbamoyl)-1-piperazi-
nyl]-6,7-dimethoxyquinazoline derivatives. J Med Chem 2002;
45:4513–23.
6. Albro LP, Baltzly R, Phillips AP. Unsymmetrically disubstituted
piperazines II. Histamine antagonists. J Org Chem 1949;14:771–4.
7. Baltzly R, DuBreuil S, Ide WS, Lorz E. Unsymmetrically disubsti-
tuted piperazines III. N-methyl-N0-benzhydrylpiperazines as hista-
mine antagonists. J Org Chem 1949;14:775–82.
low activity values such as 5d (GI50 ¼ 25.7 mM), 5k (GI50
¼
19.03 mM), 5m (GI50 ¼ 45.23 mM), 5n (GI50 ¼ 36.14 mM),
5r (GI50 ¼ 60.24 mM). However, compounds such as 5u
(GI50 ¼ 6.14 mM) and 5y (GI50 ¼ 6.34 mM) that contain phenyl
ring with electron withdrawing substituents are highly cytotoxic.
Against HCT-116 cell line, 5b (GI50 ¼ 1.01 mM) and 5y
(GI50 ¼ 1.81 mM) are the most active derivatives. In addition,
most of the compounds have higher cytotoxicity against HCT-116
than reference compound 5-fluorouracil.
With the exception of 5b, nonsubstituted benzhydryl carbox-
amide derivatives present no inhibition against HCT-116 cell
line. 4-Chlorobenzhydryl carboxamide derivatives are higher
in activity than 4,40-difluorobenzhydryl carboxamide derivatives
demonstrated with compounds 5i (GI50 ¼ 24.48 mM) and 5p
(GI50 ¼ 9.33 mM) or compounds 5j (GI50 ¼ 28.4 mM) and 5q
(GI50 ¼ 9.33 mM). Thioamides generally show good activity
values considering HCT-116 cell line.
8. Iemura R, Kawashima T, Fukuda T, et al. Synthesis of 2-(4-
substituted-1-piperazinyl)benzimidazoles as
agents. J Med Chem 1986;29:1178–83.
9. Beck K, Hamlin K, Weston A. Histamine antagonists. IV. C-methyl
derivatives of 1,4-disubstituted piperazines. J Am Chem Soc
1952;74:605–8.
10. Vandenberk J, Kennis L, Van der Aa M, et al. Piperazine derivatives.
Janssen Pharmaceutica, US 4250176; February 1981.
11. Wang L, Wang T, Yang B, et al. Design, synthesis, and anti-allergic
activities of novel (R)(-)-1-[(4-chlorophenyl)phenylmethyl]pipera-
zine derivatives. Med Chem Res 2010;21:124–32.
12. Zamponi G, Feng Z, Zhang L, et al. Scaffold-based design and
synthesis of potent N-type calcium channel blockers. Bioorg Med
Chem Lett 2009;19:6467–72.
13. Alps B. Drugs acting on calcium channels – potential treatment for
ischemic stroke. Brit J Clin Pharmaco 1992;34:199–206.
14. Miyake N, Fujita R, Ishikawa M, et al. Reversal of multidrug
resistance in human leukemia K562 by tamolarizine, a novel
calcium antagonist. Jpn J Pharmacol 2000;82:265–8.
Conclusion
H1-antihistaminic
In this study, 32 benzhydrylpiperazine derivatives with carbox-
amide and thioamide moieties were prepared. In vitro cytotoxic
activities were screened against hepatocellular (HUH-7),
breast (MCF-7) and colorectal (HCT-116) cancer cell lines by
SRB assay. Most of the compounds presented higher cyto-
toxicity against HUH-7 and HCT-116 cancer cell lines in com-
parison with reference compound 5-fluorouracil. Interestingly,
4-chlorobenzhydrylpiperazine derivatives were more active than
benzhydrylpiperazine and 4,40-difluorobenzhydrylpiperazine
derivatives. In addition, thioamide derivatives were observed to
have markedly elevated cytotoxicity values opposed to their
carboxamide analogs. Future synthesis of similar derivatives will
take place to create a larger set of compounds, in order to produce
a rational quantitative structure-activity relationship (QSAR)
mapping. Since 4-chlorobenzhydrylpiperazine derivatives are

214 E. E. Gurdal et al.
J Enzyme Inhib Med Chem, 2014; 29(2): 205–214
15. Kimura M, Masuda T, Yamada K, et al. Novel diphenylalkyl
piperazine derivatives with dual calcium antagonistic and antiox-
idative activities. Bioorg Med Chem Lett 2002;12:1947–50.
16. Pajouhesh H, Feng Z, Ding Y, et al. Structure-activity relationships
of diphenylpiperazine N-type calcium channel inhibitors. Bioorg
Med Chem Lett 2010;20:1378–83.
reductase from Mycobacterium tuberculosis. J Mol Model 2008;
14:923–35.
34. Lu X, Chen Y, Jiang Y, You Q. Discovery of potential new InhA
direct inhibitors based on pharmacophore and 3D-QSAR analysis
followed by in silico screening. Eur J Med Chem 2009;44:3718–30.
35. Muddassar M, Jang J, Gon H, et al. Identification of novel
antitubercular compounds through hybrid virtual screening
approach. Bioorg Med Chem 2010;18:6914–21.
17. Kam Y, Rhee H, Rhim H, et al. Synthesis and T-type calcium
channel blocking activity of novel diphenylpiperazine compounds,
and evaluation of in vivo analgesic activity. Bioorg Med Chem 36. Chandra J, Sadashiva C, Kavitha C, Rangappa K. Synthesis and
2010;18:5938–44.
18. Kurokawa M, Sato F, Hatano N, et al. A new class of calcium
in vitro antimicrobial studies of medicinally important novel N-alkyl
and N-sulfonyl derivatives of 1-[bis(4-fluorophenyl)methyl]pipera-
zine. Bioorg Med Chem. 2006;14:6621–7.
antagonists. Synthesis and biological activity of 11-[(o-
aminoalkanoyl)amino]-6,6a,7,8,9,10,10a,11-octahydrodibenzo[b,e] 37. Verderame M. 1,4-Disubstituted piperazines. 3. Piperazinylben-
thiepin derivatives. J Med Chem 1991;34:593–9. zothiazoles. J Med Chem 1972;15:693–4.
19. Doddareddy M, Choo H, Cho Y, et al. 3D pharmacophore based 38. Aytemir M, Ozcelik B. A study of cytotoxicity of novel chlorokojic
virtual screening of T-type calcium channel blockers. Bioorg Med acid derivatives with their antimicrobial and antiviral activities.
Chem 2007;15:1091–105. Eur J Med Chem 2010;45:4089–95.
20. Sasse B, Mach U, Leppaenen J, et al. Hybrid approach for the design 39. Patel I, Parmar S. Synthesis and studies of novel optically active
of highly affine and selective dopamine D-3 receptor ligands using
privileged scaffolds of biogenic amine GPCR ligands. Bioorg Med
Chem 2007;15:7258–73.
Schiff’s base derivatives and their antimicrobial activities. E-J Chem
2010;7:617–23.
40. Srinivasan S, Gupta S, Marwah R, et al. Synthesis, characterization
and in vitro biological studies of novel N-aryl piperazinyl fluoro-
quinolones. Res J Pharm Biol Chem Sci 2010;1:208–18.
41. Shivakumara K, Prakasha K, Gowda D. Synthesis and antimicrobial
activity of amino acids conjugated diphenylmethylpiperazine
derivatives. E-J Chem 2009;6:473–9.
21. Jung J, Jung S, Koh H. Asymmetric synthesis of chiral piperazinyl-
propylisoxazoline ligands for dopamine receptors. Eur J Med Chem
2007;42:1044–8.
22. Kimura M, Masuda T, Yamada K, et al. Synthesis of novel diphenyl
piperazine derivatives and their activities as inhibitors of dopamine
uptake in the central nervous system. Bioorg Med Chem 42. Chern J, Shia K, Hsu T, et al. Design, synthesis, and structure-
2003;11:1621–30.
activity relationships of pyrazolo[3,4-d]pyrimidinies: a novel
class of potent enterovirus inhibitors. Bioorg Med Chem Lett
2004;14:2519–25.
23. Abou-Gharbia M, Patel UR, Moyer JA, Muth EA. Psychotropic
agents: synthesis and antipsychotic activity of substituted b-carbi-
noles. J Med Chem 1987;30:1100–5.
43. Curreli F, Zhang H, Zhang X, et al. Virtual screening based
identification of novel small-molecule inhibitors targeted to the
HIV-1 capsid. Bioorg Med Chem 2011;19:77–90.
44. Kumar C, Prasad S, Vinaya K, et al. Synthesis and in vitro
antiproliferative activity of novel 1-benzhydryl-piperazine deriva-
24. Okachi R, Niino H, Kitaura K, et al. Synthesis and antibacterial
derivatives
activity
of
2,20-dithiobis(benzamide)
Mycobacterium species. J Med Chem 1985;28:1772–9.
against
25. Punkvang A, Saparpakorn P, Hannongbua S, et al. Insight into
crucial inhibitor-enzyme interaction of arylamides as novel direct
inhibitors of the enoyl ACP reductase (InhA) from Mycobacterium
tives against human cancer cell lines. Eur
2009;44:1223–9.
J Med Chem
tuberculosis: computer-aided molecular design. Monatsh Chem 45. Yarim M, Koksal M, Durmaz I, Atalay R. Cancer cell cytotoxi-
2010;141:1029–41.
26. Dinakaran M, Senthilkumar P, Yogeeswari P, et al.
cities of 1-(4-Substitutedbenzoyl)-4-(4-chlorobenzhydryl)piperazine
derivatives. Int J Mol Sci 2012;13:8071–85.
Antimycobacterial activities of novel 2-(sub)-3-fluoro/nitro-5,12- 46. Huang W, Liu M, Li Y, et al. Design, synthesis and antitumor
dihydro-5-oxo-benzothiazolo[3,2-a]quinoline-6-carboxylic
Bioorg Med Chem 2008;16:3408–18.
27. Senthilkumar P, Dinakaran M, Banerjee D, et al. Synthesis and 47. Huang W, Ding Y, Miao Y, et al. Synthesis and antitumor activity
acid.
activity of novel chromone and aurone derivatives. Bioorg Med
Chem 2007;15:5191–7.
antimycobacterial evaluation of newer 1-cyclopropyl-1,4-dihydro- of novel dithiocarbamate substituted chromones. Eur J Med Chem
6-fluoro-7-(substituted secondary amino)-8-methoxy-5-(sub)-4- 2009;44:3687–96.
oxoquinoline-3-carboxylic acids. Bioorg Med Chem 2008;16: 48. Gan L, Fang B, Zhou C. Synthesis of azole-containing
2558–69.
piperazine derivatives and evaluation of their antibacterial,
antifungal and cytotoxic activities. B Kor Chem Soc 2010;31:
3684–92.
28. Upadhayaya R, Vandavasi J, Kardile R, et al. Novel quinoline and
naphthalene derivatives as potent antimycobacterial agents. Eur J
Med Chem 2010;45:1854–67.
29. Kumar A, Siddiqi MI. Receptor based 3D-QSAR to identify putative
binders of Mycobacterium tuberculosis Enoyl acyl carrier protein
reductase. J Mol Model 2010;16:877–93.
49. Kumar C, Swamy S, Thimmegowda N, et al. Synthesis and
evaluation of 1-benzhydryl-sulfonyl-piperazine derivatives as inhibi-
tors of MDA-MB-231 human breast cancer cell proliferation.
Med Chem Res 2007;16:179–87.
30. Lu XY, Chen YD, You QD. 3D-QSAR studies of arylcarboxamides 50. Wang W, Xu X, Chen Y, et al. Apoptosis of human Burkitt’s
with inhibitory activity on InhA using pharmacophore-based
alignment. Chem Biol Drug Des 2010;75:195–203.
31. Sriram D, Senthilkumar P, Dinakaran M, et al. Antimycobacterial
lymphoma cells induced by 2-N,N-diethylaminocarbonyloxymethyl-
1-diphenyl-methyl-4-(3,4,5-trimethoxybenzoyl)piperazine
chloride (PMS-1077). Arch Pharm Res 2009;32:1727–36.
hydro-
activities of novel 1-(cyclopropyl/tert-butyl/4-fluorophenyl)- 51. Demma M, Maxwell E, Ramos R, et al. SCH529074, a small
1,4-dihydro-6-nitro-4-oxo-7-(substituted secondary amino)-1,8-
naphthyridine-3-carboxylic acid. J Med Chem 2007;50:6232–9.
32. Senthilkumar P, Dinakaran M, Yogeeswari P, et al. Synthesis and
antimycobacterial activities of novel 6-nitroquinolone-3-carboxylic
acids. Eur J Med Chem 2009;44:345–58.
molecule activator of mutant p53, which binds p53 DNA binding
domain (DBD), restores growth-suppressive function to mutant p53
and interrupts HDM2-mediated ubiquitination of wild type p53.
J Biol Chem 2010;285:198–212.
52. Gurdal EE, Yarim M, Durmaz I, Cetin-Atalay R. Cytotoxic
activities of some novel benzhydrylpiperazine derivatives.
Arzneimittel-Forsch 2013; Accepted Manuscript.
33. Kumar A, Siddiqi M. CoMFA based de novo design of pyrroli-
dine carboxamides as inhibitors of enoyl acyl carrier protein