Synthesis of 1-benzhydryl piperazine derivatives and evaluation of their ACE inhibition and antimicrobial activities

Article abstract of DOI:10.1007/s00044-013-0895-7

This study presents the synthesis of 14 new 1,4-disubstituted piperazine derivatives from allyl bromides of Baylis-Hillman adduct and 4,4-disubstituted benzhydryl piperazines. All the synthesized compounds were further screened for in vitro ACE inhibitor and antimicrobial activities. Among the synthesized piperazine derivatives, compound 12h showed moderate ACE inhibitor activity as compared to the standard, angiotensin-converting enzyme inhibitor (Sigma). The kinetic data (K _m, K _i and V _max values) of enzyme inhibition for compound 12h and ACE inhibitor standard were also determined. Similarly, all compounds were screened against different bacterial strains. Compounds 12a, 12b, 12d, 12h and 12i showed excellent to moderate activity against various tested bacterial strains. Compounds 12b and 12i showed broad spectrum of antibacterial activity against Pseudomonas aeruginosa, Staphylococcus aureus, S. aureus MLS 16, Escherichia coli, Bacillus subtilis and Klebsiella planticola, while compounds 12a, 12d and 12i showed promising activity against P. aeruginosa (MIC value of 8.96 μM), S. aureus (MIC value of 42.2 μM) and S. aureus MLS 16 (MIC value of 81.3 μM), respectively. The remaining compounds showed activity at a concentration of >491 μM.

Full text of DOI:10.1007/s00044-013-0895-7

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0895-7
ORIGINAL RESEARCH
Synthesis of 1-benzhydryl piperazine derivatives and evaluation
of their ACE inhibition and antimicrobial activities
•
Amlipur Santhoshi Singam Naveen Kumar Pombala Sujitha
•
•
•
•
•
Yedla Poornachandra Partha Sarathi Sadhu C. Ganesh Kumar
Vaidya Jayathirtha Rao
Received: 20 June 2013 / Accepted: 13 December 2013
Ó Springer Science+Business Media New York 2014
Abstract This study presents the synthesis of 14 new
1,4-disubstituted piperazine derivatives from allyl bro-
mides of Baylis–Hillman adduct and 4,4-disubstituted
benzhydryl piperazines. All the synthesized compounds
were further screened for in vitro ACE inhibitor and anti-
microbial activities. Among the synthesized piperazine
derivatives, compound 12h showed moderate ACE inhib-
itor activity as compared to the standard, angiotensin-
converting enzyme inhibitor (Sigma). The kinetic data (Km,
Ki and Vmax values) of enzyme inhibition for compound
12h and ACE inhibitor standard were also determined.
Similarly, all compounds were screened against different
bacterial strains. Compounds 12a, 12b, 12d, 12h and 12i
showed excellent to moderate activity against various tes-
ted bacterial strains. Compounds 12b and 12i showed
broad spectrum of antibacterial activity against Pseudo-
monas aeruginosa, Staphylococcus aureus, S. aureus MLS
16, Escherichia coli, Bacillus subtilis and Klebsiella
planticola, while compounds 12a, 12d and 12i showed
promising activity against P. aeruginosa (MIC value of
8.96 lM), S. aureus (MIC value of 42.2 lM) and S. aureus
MLS 16 (MIC value of 81.3 lM), respectively. The
remaining compounds showed activity at a concentration of
[491 lM.
Keywords Benzhydryl piperazine ꢀ
Baylis–Hillman bromides ꢀ ACE inhibitor ꢀ Antimicrobial
Introduction
In recent years, there has been an increased concern to
explore new angiotensin-converting enzyme inhibitors for
treatment of cardiovascular diseases like hypertension,
congestive heart failure, diabetic nephropathy, chronic
renal failure, systemic sclerosis, left ventricular systolic
dysfunction and acute myocardial infarction (Acharaya
¨
et al., 2003; Skidgel and Erdos, 2004). Angiotensin
I-converting enzyme (ACE, dipeptidyl carboxy peptidase
I) is a multifunctional, zinc-containing enzyme that is
located in different tissues mainly in the lungs, including
vascular endothelial cells, epithelial kidney cells, testicu-
lar Leydig cells and brain neuron cells (Jain et al., 2008).
The physiological action of ACE includes conversion of
inactive decapeptide, angiotensin I, to a potent vasopres-
sor octapeptide, angiotensin II, which is involved in
increasing blood pressure through G-protein-coupled sig-
nalling mechanisms and also inactivates bradykinin (Ahn
et al., 1998; Ehlers et al., 1989). Hence, inhibition of
ACE leads to a decrease in concentration of angiotensin II
and consequently reduces blood pressure. Several natural
and synthetic compounds are reported as ACE inhibitors
such as prazosin, doxazosin, captopril, enalapril, lisino-
pril, benazepril, trandolapril, etc. In the field of chemo-
therapeutic drugs, the search for new, safer, more
selective and low-cost ACE inhibitors is necessary for
prevention of hypertension. Rao et al. (1987) reported that
the compound 1-[3-(4-acetamidophenylthio)propyl]-4-[3-
methyl phenyl] piperazine acted as a potential antihy-
pertensive agent. Further, a series of 1-[3-(4-substituted
A. Santhoshi ꢀ S. N. Kumar ꢀ P. S. Sadhu ꢀ V. J. Rao (&)
Crop Protection Chemicals Division, CSIR-Indian Institute of
Chemical Technology, Uppal Road, Hyderabad 500007, India
e-mail: jrao@iict.res.in
P. Sujitha ꢀ Y. Poornachandra ꢀ C. G. Kumar (&)
Medicinal Chemistry and Pharmacology Division, CSIR-Indian
Institute of Chemical Technology, Uppal Road,
Hyderabad 500007, India
e-mail: cgkumar5@gmail.com; cgkumar@iict.res.in
123

Med Chem Res
phenylthio) propyl]-4-(substituted phenyl) piperazines and
related compounds including 1-(aryl-piperazin-1-yl)-1-
oxo-2-(thioaryloxy/aryloxy) propanes were earlier
screened for their hypotensive activity (Saxena et al.,
2007).
2010; Singh and Batra, 2008). BH adducts have been used
for the synthesis of several bioactive molecules. Several
BH adducts have been reported to be bioactive. We have
reported the antimalarial activity of BH adducts of
2-chloronicotinaldehydes (Narender et al., 2006) and
antimicrobial activity of Tetrazolone derivatives containing
BH nuclei (Santhoshi et al., 2013). So, the presence of BH
scaffold in molecules played a significant role towards its
bioactivity. Based on these observations and in follow-up
of our research on application of BH chemistry (Ravinder
et al., 2009, 2010; Narender et al., 2005; Santhoshi et al.,
2013), we report herein the synthesis of 14 new 1,4-
disubstituted piperazine derivatives from allyl bromides of
BH adduct and 4,4-disubstituted benzhydryl piperazines
derivatives and their further evaluation for in vitro ACE
inhibitor and antimicrobial activities.
A majority of bioactive molecules are heterocyclic
compounds. Among them, piperazine and its derivatives
have played a significant role in medicinal chemistry.
Piperazine is a small molecule with a rigid backbone with
numerous biological activities such as anticancer (Guo
et al., 2004; Kumar et al., 2009), calcium channel blockers
(Borzenko et al., 2013; Pajouhesh et al., 2012), antima-
larial (Ryckebusch et al., 2003), antihistamine (Choo and
Chung, 2000; Gillard et al., 2002), antimicrobial (Cunico
et al., 2009), antidepressant (Dorsey et al., 2004), antiox-
idant (Kimura et al., 2002), antiallergic (Kawasaki et al.,
1999), antiviral (McCombie et al., 2003), antipsychotic
(Yevich et al., 1986) and antiparasitic (Becker et al., 2006;
Mayence et al., 2004). Many potent marketed drugs like
flunarizine, cinnarizine, lomerizine, fluphenazine, cipro-
floxacin, Merck HIV proteus, crixivan, etc. have a piper-
azine nucleus. Some typical examples of bioactive
piperazine derivatives are shown in Fig. 1. The various
biological activities of piperazine nuclei are due to easy
modification, proper alkalinity, water solubility, capacity
for formation of hydrogen bonds and adjustment of
molecular physicochemical properties (Foye et al., 1995).
A simple 1(1-naphthylmethyl)-piperazine derivative is as
potent as tetracycline and ciprofloxacin drugs (C¸ oban et al.,
2009; Chaudhary et al., 2006). Similarly, benzotriazole-
based piperazine derivatives and N,N’-bis(alkyloxymeth-
yl)piperazines possess moderate antibacterial and anti-
fungal activities (Farzaliev et al., 2009). Recently, Sharath
Chandra et al. demonstrated the synthesis and significant
in vitro antibacterial activity of N-alkyl and N-sulfonyl
derivatives of 1-[bis(4-fluorophenyl)-methyl]piperazine as
compared to the standard drug Streptomycin (Sharath
Chandra et al., 2006).
Experimental
Melting points were determined using the Barnstead Elec-
trothermal digital melting point apparatus and were uncor-
rected. IR spectra were recorded using a Thermo Nicolet
1
Nexus 670 FTIR spectrometer. H NMR and 13C NMR
spectra were recorded on either a Bruker Avance 300 MHz
or a Varian Inova 400 MHz FT spectrometer, using TMS as
an internal standard (chemical shifts in d values, J in Hz).
HRMS (ESI) was recorded on QSTAR XL High resolution
mass spectrometer. All reagents and solvents used are
commercially available. Column chromatography was
performed using silica gel (60–120 mesh).
General procedure for the preparation of BH bromides
To a stirred solution of BH adduct (41.1 mmol) in DCM,
concentrated HBr (48 %, 13.5 ml) was added dropwise
followed by concentrated H2SO4 (11.9 ml) at 0 °C. After
stirring overnight at room temperature, the reaction mix-
ture was carefully diluted with DCM (40 ml) and H2O
(20 ml). The aqueous phase was then extracted with DCM
Baylis–Hillman (BH) chemistry has been the subject of
intense research for the last three decades (Basavaiah et al.,
Fig. 1 Bioactive piperazine
derivatives
NH2
N
O
N
O
H3CO
H3CO
F
F
F
OH
N
N
O
N
N
HN
N
N
O
Prozosin 1
AntiHypertensive Drug
Ciprofloxacin 3
Antibiotic
Flunarizine 2
Calcium Channel Blocker
123

Med Chem Res
(30 ml 9 2). The combined organic phase was washed
with H2O (15 ml 9 2), dried over Na2SO4, concentrated
under vacuum and the residual oil was purified by column
chromatography (eluent hexane/ethyl acetate (10:1).
61.32 (OCH2), 53.09 (C14), 52.71 (C3, C5), 51.86 (C2,
C6), 14.25 (CH3); ESIMS (m/z) 522 [M?H]?; Elemental
analysis calculated for C29H29F2N3O4, C 66.78, H 5.60, N
8.06; found C 66.79; H 5.66; N 8.07.
General procedure for synthesis of
1-benzhydrylpiperazine
(Z)-methyl 2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-
yl)methyl)-3-p-tolylacrylate (12b) Cream solid; mp
114–116 °C; IR: 1696, 1597, 1506, 1456, 1225, 1157,
1096, 958 cm-1; H1 NMR (300 MHz, CDCl3): 7.76 (s,
1H), 7.54–7.51 (d, J = 7.93 Hz, 2H), 7.32–7.28 (m, 4H),
7.15–7.12 (d, J = 7.93 Hz, 2H), 6.95–6.89 (m, 4H), 4.15
(s, 1H), 3.78 (s, 3H), 3.31 (s, 2H), 2.48–2.36 (m, 11H); C13
NMR (75 MHz, CDCl3): d 169.14 (C=O), 163.29 (C11),
160.03 (C11’), 143.54 (C8, C8’), 139.09 (C16), 138.36
(C20), 132.51 (C17), 130.62 (C15), 129.19 (C9, C13),
129.11 (C9’, C13’), 129.05 (C18, C22), 128.47 (C19, C21),
115.40 (C10, C12), 115.12 (C10’, C12’), 74.45 (C7), 53.18
(C14), 52.68 (OCH3), 51.96 (C3, C5), 51.86 (C2, C6),
21.29 (CH3); ESIMS (m/z) 477 [M?H]?; Elemental ana-
lysis calculated for C29H30F2N2O2, C 73.09, H 6.35, N 5.
88; found C 73.12; H 6.38; N 5.92.
To a stirred solution of piperazine dihydrochloride (10.0 g,
62.86 mmol) in dimethyl formamide, anhydrous potassium
carbonate (43.44 g, 314.3 mmol) was added. After 10 min,
benzhydryl chloride (11.46 g, 56.58 mmol) was added and
the reaction mixture was heated at 80 °C for 8 h. The
solvent was removed under reduced pressure and residue
was taken in water (70 ml) and extracted with ethyl acetate
(50 ml 9 3). The organic layer was washed with brine
(100 ml) and dried over anhydrous sodium sulphate. The
solvent was evaporated and the crude product obtained was
purified by column chromatography over silica gel
(60–120 mesh) using chloroform: methanol (9:1) as an
eluent.
General procedure for synthesis of piperazine
derivatives
(Z)-ethyl-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)
methyl)-3-(4-fluorophenyl)acrylate (12c) Cream solid;
mp 112–116 °C; IR: 1696, 1597, 1506, 1456,1225,1157,
1096, 958 cm-1; H1 NMR (300 MHz, CDCl3): d 7.73
(s, 1H), 7.71–7.65 (m, 2H), 7.33–7.26 (m, 4H), 7.02–6.98
(m, 2H), 6.95–6.90 (m, 4H), 4.24 (q, J = 7.18 Hz, 2H), 4.
16 (s, 1H), 3.28 (s, 2H), 2.56–2.30 (m, 8H), 1.33 (t, J = 7.
18 Hz, 3H); C13 NMR (75 MHz, CDCl3): d 168.47 (C=O),
164.63 (C20), 163.36 (C11’), 160.11 (C11), 142.08 (C8,
C8’), 138.36 (C16), 132.72 (C17), 132.61 (C15), 129.49
(C18, C22), 129.23 (C9, C13), 129.13 (C9’, C13’), 115.47
(C10, C12), 115.18 (C10’, C12’), 74.52 (C7), 60.92
(OCH2), 53.14 (C14), 52.70 (C3, C5), 51.95 (C2, C6), 14.
30 (CH3); ESIMS (m/z) 495 [M?H]?; Elemental analysis
calculated for C29H29F3N2O2, C 70.43, H 5.91, N 5.66;
found C 70.50, H 5.98, N 5.72.
To a stirred solution of 1-substituted benzhydryl piperazine
(1 mmol) and potassium carbonate (2 mmol) in DMF, a
solution of BH bromide (1 mmol) in DMF (3 ml) was added
and stirred at room temperature for 6 h. After completion of
the reaction (monitored by TLC), ice-cold water (5 ml) was
added to the reaction mixture and allowed to stir for 15 min.
The compound solidified and excess DMF was removed
again and again by repeatedly washing with cold water. The
organic layer was extracted with ethyl acetate and dried over
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 hex-
ane: ethyl acetate (7:3) as an eluent.
Characterization data
(E)-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)
methyl)-3-p-tolylacrylonitrile (12d) White solid; mp
122–126 °C; IR: 1696,1597, 1506, 1456,1225,1157,
1096 cm-1; H1 NMR (300 MHz, CDCl3): d 7.63–7.60 (d,
J = 8.12 Hz, 2H), 7.36 (d, J = 7.37 Hz, 4H), 7.24–7.09
(m, 6H), 6.96 (s, 1H), 4.20 (s, 1H), 3.24 (s, 2H), 2.55–2.40
(m, 8H), 2.38 (s, 3H); C13 NMR (75 MHz, CDCl3): 163.00
(C11), 159.74 (C11’), 143.59 (C16), 137.91 (C8, C8’), 131.82
(C20), 130.73(C17), 129.62 (C9, C13), 129.22 (C9’, C13’),
128.98 (C18, C22), 128.87 (C19, C21), 115.72 (CN), 115.
58 (C10, C12), 115.42 (C10’, C12’), 107.88 (C15), 73.94
(C7), 61.85 (C14), 52.71 (C3, C5), 51.17 (C2, C6), 21.29
(CH3); ESIMS (m/z) 444 [M?H]?; Elemental analysis
(Z)-ethyl-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)
methyl)-3-(4-nitrophenyl)acrylate (12a) Yellow solid;
mp 126–129 °C; IR: 1726, 1635, 1560, 1510 cm-1; H1
NMR (300 MHz, CDCl3): d 8.21 (d, J = 8.69 Hz, 2H),
7.87 (d, J = 8.69 Hz, 2H), 7.78 (s, 1H), 7.37–7.10 (m, 5H),
6.97–6.90 (m, 3H), 4.27 (q, J = 7.18 Hz, 2H), 4.16 (s, 1H),
3.28 (s, 2H), 2.58–2.30 (m, 8H), 1.36 (t, J = 7.18 Hz,3H);
C13 NMR (75 MHz, CDCl3): 167.74 (C=O), 163.36 (C11),
160.10 (C11’), 147.47 (C20), 142.70 (C17), 141.84 (C8),
140.29 (C8’), 138.22 (C16), 133.41 (C15), 131.19 (C18,
C22), 129.21 (C9, C13), 129.11 (C9’, C13’), 123.38 (C19,
C21), 115.48 (C10, C12), 115.19 (C10’, C12’),74.45 (C7),
123

Med Chem Res
(E)-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)methyl)-
3-phenylacrylonitrile (12h) White solid, mp 107–110 °C;
IR: 1696, 1597, 1506, 1456, 1225, 1157, 1096, cm-1; H1
NMR (300 MHz, CDCl3): d 7.78–7.70 (m, 2H), 7.43–7.36
(m, 3H), 7.36–7.27 (m, 4H), 7.04 (s, 1H), 6.99–6.91 (m,
4H), 4.22 (s, 1H), 3.28 (s, 2H), 2.56–2.31 (m, 8H); C13
NMR (75 MHz, CDCl3): d 163.23 (C11), 159.98 (C11’),
145.17 (C16), 137.99 (C8, C8’), 133.07 (C17), 130.22 (C9,
C13) 129.02 (C9’, C13’), 129.14 (C19, C21), 128.73 (C18,
C22), 128.67 (C20), 118.52 (CN), 115.35 (C10, C12),
115.09 (C10’, C12’), 108.08 (C15), 74.18 (C7), 62.17
(C14), 52.59 (C3, C5), 52.38 (C2, C6); ESIMS (m/z) 430
[M?H]?; Elemental analysis calculated for C27H25F2N3, C
75.50, H 5.87, N 9.78; found C 75.54, H 5.92, N 9.79.
calculated for C28H27F2N3, C 75.82, H 6.14, N 9.47; found
C 75.90; H 6.20; N 9.44.
(Z)-methyl 2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-
yl)methyl)-3-(4-methoxyphenyl)acrylate
(12e) White
solid, m.p. 114–117 °C; IR: 1696,1597, 1506, 1456,
1225,1157, 1096, 958 cm-1; H1 NMR (300 MHz, CDCl3):
d 7.75 (s, 1H), 7.63–7.62 (d, J = 8.88 Hz, 2H), 7.34–7.26
(m, 4H), 6.94–6.89 (m, 4H), 6.84 (d, J = 8.68 Hz, 2H),
4.16 (s, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.31(s, 2H), 2.57–
2.28 (m, 8H); C13 NMR (75 MHz, CDCl3): d 169.26 (C=O),
163.32 (C11), 160.29 (C11’), 160.06 (C20), 143.51 (C8,
C8’), 138.36 (C16), 132.63 (C15), 129.23 (C9, C13), 129.11
(C9’ C13’),128.03 (C18, C22), 126.89 (C17), 115.41 (C10,
C12), 115.11 (C10’, C12’), 113.75 (C19, C21), 74.47 (C7),
55.19 (C14), 53.27 (C20–OCH3), 52.68 (COOCH3), 51.93
(C3, C5), 51.83 (C2, C6); ESIMS (m/z) 493 [M?H]?;
Elemental analysis calculated for C29H30F2N2O3, C 70.71,
H 6.14, N 5.69; found C 70.76; H 6.12; N 5.68.
(Z)-methyl-2-((4-benzhydrylpiperazin-1-yl)methyl)-3-(4-
chlorophenyl)acrylate (12i)
White solid; mp 112–
116 °C; IR: 1696, 1597, 1506, 1456,1225,1157, 1096,
958 cm-1; H1 NMR (300 MHz, CDCl3): d 7.73 (s, 1H), 7.63
(d, J = 8.30 Hz, 2H), 7.40–7.28 (m, 6H), 7.26–7.18 (m, 4H),
7.16–7.08 (m, 2H), 4.15 (s, 1H), 3.77 (s, 3H), 3.27 (s, 2H), 2.
54–2.30 (m, 8H); C13 NMR (75 MHz, CDCl3): d 168.82 (C=
O), 142.83 (C8, C8’), 142.11 (C16), 134.98 (C20), 133.85
(C17), 131.94 (C15), 130.15 (C18, C22), 128.54 (C10, C12,
C10’, C12’), 128.39 (C9, C13, C9’, C13’), 127.89 (C19,
C21), 126.83 (C11, C11’), 76.25 (C7), 53.19 (C14), 52.78
(OCH3), 52.06 (C3, C5), 52.09 (C2, C6); ESIMS (m/z) 461
[M?H]?; Elemental analysis calculated for C28H29ClN2O2,
C 72.95, H 6.34, N 6.08; found C 72.90, H 6.39, N 6.12.
(Z)-methyl-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-
yl)methyl)-3-(4-chlorophenyl) acrylate (12f) Cream
solid, mp 112–115 °C; IR: 1696,1597, 1506, 1456,1225,
1157, 1096, 958 cm-1; H1 NMR (300 MHz, CDCl3): d 7.
73 (s, 1H), 7.63 (d, J = 8.49 Hz, 2H), 7.35–7.27 (m, 6H),
6.98–6.88 (m, 4H), 4.15 (s, 1H), 3.78 (s, 3H), 3.27 (s, 2H),
2.53–2.37 (m, 8H); C13 NMR (75 MHz, CDCl3): d 168.81
(C=O), 163.40 (C11), 160.14 (C11’), 142.24 (C8, C8’),
138.35 (C16), 135.03 (C20), 133.84 (C17), 131.89 (C15),
130.03 (C9, C13), 129.28 (C9’, C13’), 129.18 (C18, C22),
128.57 (C19, C21), 115.48 (C1O, C12), 115.23 (C10’,
C12’),74.49 (C7), 53.18 (C14), 52.73 (OCH3), 52.16 (C3,
C5), 51.93 (C2, C6); ESIMS (m/z) 497 [M?H]?; Ele-
mental analysis calculated for C28H27ClF2N2O2, C 67.67,
H 5.48, N 5.64; found C 67.71, H 5.52, N 5.69.
(Z)-ethyl 2-((4-benzhydrylpiperazin-1-yl)methyl)-3-(4-fluo-
rophenyl)acrylate (12j) White solid; mp 124–127 °C;IR:
1696, 1597, 1506, 1456, 1225, 1157, 1096 cm-1; H1 NMR
(300 MHz, CDCl3): d 7.76 (s, 1H), 7.73–7.65 (m, 2H), 7.39
(d, J = 7.18 Hz, 4H), 7.29–7.19 (m, 4H), 7.18–7.10 (m,
2H), 7.09–7.00 (m, 2H), 4.24 (q, J = 7.18 Hz, 2H), 4.17 (s,
1H), 3.30 (s, 2H), 2.57–2.32 (m, 8H), 1.34 (t, J = 7.18 Hz,
3H); C13 NMR (75 MHz, CDCl3): d 168.47 (C=O), 162.23
(C20), 142.84 (C8, C8’), 141.99 (C16), 132.60 (17), 131.58
(C15), 129.57 (C18, C22), 128.38 (C10, C12, C10’, C12’),
127.86 (C9, C13, C9’, C13’), 126.81 (C11, C11’), 115.44
(C19, C21), 76.28 (C7), 60.89 (OCH2), 53.12 (C14), 52.74
(C3, C5), 52.06 (C2, C6), 14.25 (CH3); ESIMS (m/z) 459
[M?H]?; Elemental analysis calculated for C29H31FN2O2,
C 75.96, H 6.81, N 6.11; found C 75.92, H 6.88, N 6.18.
(Z)-ethyl-2-((4-(bis(4-fluorophenyl)methyl)piperazin-1-yl)me-
thyl)-3-(4-chlorophenyl) acrylate (12g) Cream solid, mp
115–117 °C; IR: 1696,1597, 1506, 1456,1225,1157,
1096 cm-1; H1 NMR (300 MHz, CDCl3): d 7.72 (s, 1H), 7.
63 (d, J = 8.49 Hz, 2H), 7.36–7.26 (m, 6H), 6.98–6.90 (m,
J = 8.49 Hz, 4H), 4.24 (q, J = 6.98 Hz, 2H), 4.15 (s, 1H),
3.27(s, 2H), 2.48–2.35 (m, 8H), 1.36–1.32 (t, J = 7.18 Hz,
3H); C13 NMR (75 MHz, CDCl3): d 168.32 (C=O), 163.35
(C11), 160.09 (C11’), 141.82 (C8, C8’), 138.29 (C16), 134.
89 (C20), 133.88 (C17), 131.90 (C15), 130.36 (C9, C13),
129.23 (C9’, C13’), 129.14 (C18, C22), 128.49 (C19, C21),
115.46 (C10, C12), 115.18 (C10’, C12’), 74.51 (C7), 60.98
(OCH2), 53.12 (C14), 52.69 (C3, C5), 51.93 (C2, C6), 14.
28 (CH3); ESIMS (m/z) 511 [M?H]?; Elemental analysis
calculated for C29H29ClF2N2O2, C 68.16, H 5.72, N 5.48;
found C 68.20, H 5.81, N 5.67.
(E)-2-((4-benzhydrylpiperazin-1-yl)methyl)-3-(4-fluoro-
phenyl)acrylonitrile (12k) White solid; mp 110–113 °C;
IR: 1696, 1597, 1506, 1456, 1225,1157, 1096 cm-1; H1
NMR (300 MHz, CDCl3): d 7.77–7.72 (m, 2H), 7.40 (d,
4H), 7.28–7.22 (m, 4H), 7.18–7.14 (m, 2H), 7.11–7.06 (m,
2H), 7.03 (s, 1H), 4.24 (s, 1H), 3.27 (s, 2H), 2.60–2.41 (m,
123

Med Chem Res
8H); C13 NMR (75 MHz, CDCl3): d 165.21 (C20), 143.83
(C8, C8’), 142.62 (C16), 130.98 (C17), 130.86 (18, C22),
128.43 (C9, C13, C9’ C13’), 127.86 (C10, C12, C10’,
C12’), 126.89 (C11, C11’), 118.57 (CN), 116.10 (C19),
115.81 (C21), 107.99 (C15), 76.09 (C7), 62.23 (C14), 52.
89 (C3, C5), 51.69 (C2, C6); ESIMS (m/z) 412 [M?H]?;
Elemental analysis calculated for C27H26FN3, C 78.80; H
6.37; N 10.21; found C 78.84; H 6.39; N 10.28.
(OCH2), 53.23 (C14), 52.81 (C3, C5), 52.13 (C2, C6),
21.35 (C20–CH3), 14.32 (OCH2CH3); ESIMS (m/z) 455
[M?H]?; Elemental analysis calculated for C30H34N2O2,
C 79.26, H 7.54, N 6.16; found C 79.36; H 7.57; N 6.23.
Biological activities
Antimicrobial activity assay
(E)-2-((4-benzhydrylpiperazin-1-yl)methyl)-3-p-tolylacry-
lonitrile (12l) Cream solid, mp 116–118 °C; IR:
1696,1597, 1506, 1456,1225,1157, 1096 cm-1; H1 NMR
(300 MHz, CDCl3): d 7.61 (d, J = 8.12 Hz, 2H), 7.36 (d,
J = 7.37 Hz, 4H), 7.26–7.08 (m, 8H), 6.96 (s, 1H), 4.20 (s,
1H), 3.24 (s, 2H), 2.59–2.40 (m, 8H), 2.38 (s, 3H); C13
NMR (75 MHz, CDCl3): d 145.29 (C8, C8’), 142.68 (C16),
140.81 (C20), 130.52 (C17), 129.50 (C10, C12, C10’
C12’), 128.88 (C9, C13, C9’, C13’), 128.43 (C19, 21), 127.87
(C18, C22), 126.88 (C11, C11’), 118.95 (CN), 106.88
(C15), 76.09 (C7), 62.44 (C14), 52.85 (C3, C5), 51.71 (C2,
C6), 21.47 (CH3); ESIMS (m/z) 408 [M?H]?; Elemental
analysis calculated for C28H29N3, C 82.52, H 7.17, N 10.
31; found C 82.55; H 7.12; N 10.38.
The antimicrobial activity of the synthesized compounds
was determined using the well diffusion method (Amster-
dam, 1996) against different pathogenic reference strains
procured from the Microbial Type Culture Collection and
Gene Bank (MTCC), CSIR, the Institute of Microbial
Technology, Chandigarh, India. The pathogenic reference
strains were seeded on the surface of the media Petri plates,
containing Muller-Hinton agar with 0.1 ml of previously
prepared microbial suspensions individually containing
1.5 9 108 cfu mL-1 (equal to 0.5 McFarland). Wells of
6.0 mm diameter were prepared in the media plates using a
cork borer, and the synthesized compounds at a dose range
of 300–1.4 lg well-1 (equivalent amount in molar con-
centrations of each compound) were added in each well
under sterile conditions in a laminar air flow chamber.
Standard antibiotic solutions of Levofloxacin, Ciprofloxa-
cin and Flucanozole at a dose range of 300–1.4 lg well-1
(equivalent amount in molar concentrations of each anti-
biotic solution) were used as positive controls and the well
containing methanol served as negative control. The plates
were incubated for 24 h at 30 °C and the well containing
the least concentration showing the inhibition zone is
considered as the minimum inhibitory concentration. All
experiments were carried out in duplicates and the mean
values are represented.
(Z)-ethyl-2-((4-benzhydrylpiperazin-1-yl)methyl)-3-(4-methoxy-
phenyl)acrylate (12m) Colourless solid; mp 118–120 °C;
IR: 1696,1597, 1506, 1456,1225,1157, 1096 cm-1; H1
NMR (300 MHz, CDCl3): d 7.73 (s, 1H), 7.64 (d, J = 9.07
Hz, 2H), 7.37 (d, J = 6.79 Hz, 4H), 7.27–7.17 (m, 4H), 7.
16–7.07 (m, 2H), 6.83 (d, J = 9.07 Hz, 2H), 4.23 (q, J =
7.55 Hz, 2H), 4.15 (s, 1H), 3.81 (s, 3H), 3.30 (s, 2H), 2.
58–2.32 (m, 8H), 1.33 (t, J = 7.55 Hz, 3H); C13 NMR
(75 MHz, CDCl3): d 168.82 (C=O), 160.18 (C20), 143.03
(C8, C8’),142.90 (C16), 132.56 (C15), 128.36 (C10, C12,
C10’, C12’), 128.18 (C18, C22), 127.87 (C9, C13, C9’,
C13’), 126.78 (C11, C11’), 113.73 (C19, C21), 76.30 (C7),
60.67 (OCH2), 55.19 (C14), 53.30 (C2O–OCH3), 52.78 (C3,
C5), 52.15 (C2, C6), 14.32 (CH3); ESIMS (m/z) 471
[M?H]?; Elemental analysis calculated for C30H34N2O3,
C 76.57, H 7.28, N 5.95; found C 76.59; H 7.35; N 6.01.
Assay for ACE inhibitor activity and enzyme inhibition
kinetics
The ACE extracted from rabbit lung acetone powder was
used in the present study, obtained from Sigma Chemical
Co., St. Louis, MO, USA. The ACE inhibitor activity was
assayed by a modification of the method of Cushman et al.
(1977). The mixture containing 100 mM sodium borate
buffer (pH 8.3), 300 mM NaCl, 3 units of ACE from rabbit
lung and an appropriate amount of the inhibitors solution
was preincubated for 10 min at 37 °C. The reaction was
initiated by adding 50 ll of Hip-His-Leu substrate at a final
concentration of 5 mM and was terminated after 30 min of
incubation by adding 250 ll of 1.0 M HCl. The liberated
hippuric acid was extracted with 1 ml of ethyl acetate, and
0.8 ml of the extract was evaporated by a SpeedVac con-
centrator (EYELA Co., Japan). The residue was then
(Z)-ethyl 2-((4-benzhydrylpiperazin-1-yl)methyl)-3-p-tolyl-
acrylate (12n) Cream solid; mp 114–115 °C; IR:
1696,1597, 1506, 1456, 1225, 1157, 1096 cm-1; H1 NMR
(300 MHz, CDCl3): d 7.73 (s, 1H), 7.53 (d, J = 7.93 Hz,
2H), 7.36 (d, J = 7.37 Hz, 4H), 7.24–7.16 (m, 4H), 7.15–7.08
(d, J = 7.74 Hz, 4H), 4.22 (q, J = 7.18 Hz, 2H), 4.15
(s, 1H), 3.29 (s, 2H), 2.54–2.33 (m, 11H), 1.33 (t, J = 7.
18 Hz, 3H); C13 NMR (75 MHz, CDCl3): d 168.76 (C=O),
142.92 (C8, C8’), 143.08 (C16), 138.97 (C20), 132.97
(C17), 132.66 (C15), 130.61 (C19, C21), 129.04 (C10,
C12, C10’, C12’), 128.38 (C9, C13, C9’ C13’), 127.87
(C18, C22), 126.79 (C11, C11’), 76.30 (C7), 60.76
123

Med Chem Res
dissolved in 1 ml of sodium borate buffer. Absorbance at
228 nm was measured to estimate the ACE inhibitor
activity. The IC50 value (mM) is the concentration of ACE
inhibitor required for 50 % inhibition of the ACE activity
under the above assay conditions. The statistical analysis
was performed using GraphPad PRISM software version
3.0 (GraphPad Software, Inc, La Jolla, CA, USA). Data
were expressed as the mean S.D. The unpaired Student
t test was adopted to determine the statistical significance.
In all the comparisons, p \ 0.05 was considered statisti-
cally significant.
30 min, with less than 5 % total substrate hydrolysed for
all substrate concentrations. The mean Km, Ki and Vmax
values were determined from the mean of the Km, Ki and
Vmax values found from the Lineweaver–Burk plot from
three independent experiments.
Results and Discussion
BH adducts 6a–6k were prepared by treating aromatic
aldehydes 4a–4f with acrylonitriles/acrylates 5a–5c and
DABCO as catalyst (20–50 mol%) under neat conditions at
room temperature in very good yields. Thus, the prepared BH
adducts were then treated with conc. H2SO4 and HBr (48 %
in acetic acid) at 0 °C in DCM to get corresponding BH
bromides 7a–7k in high yields (Scheme 1). (Z)-stereo-
chemistry was assigned to BH bromides of esters and (E)-
stereochemistry to BH bromides of nitriles on the basis of
chemical shift of b-vinylic H as per the literature (Basavaiah
et al., 1994). 1-benzhydrylpiperazine and 4,4’-difluoro-1-
benzhydrylpiperazine were prepared as shown in Scheme 2.
Reduction of benzophenones 8a–8b by sodium borohydride
provided benzhydryl compounds 9a–9b in high yield
Kinetic studies
The compounds 12h, ACE inhibitor and without inhibitor
were assayed with Hip-His-Leu as a substrate in 50 mM
Tris–HCl buffer (adjusted to pH 7.5 with NaOH) contain-
ing 300 mM NaCl (Galardy, 1980) by the fluorometric
reaction of histidyl-leucine with O-phthalaldehyde (Che-
ung and Cushman, 1973) with a single 30-min time point.
The Km value observed was 0.5 mM. The reaction was
initiated by the addition of enzyme to a final concentration
of 0.05 nm. Hydrolysis was linear with time to well beyond
Scheme 1 Reagents and
conditions: (i) DABCO
(20–50 mol%), neat, rt,
60–90 %; (ii) Conc. H2SO4,
HBr, 0 °C-rt, overnight,
80–90 %
OH
CHO
EWG
(ii)
COOR
Br
Br
EWG
(i)
+
CN
R1
R1
R1
R1
7d,h,i
6a-k
7a-c, e-g, j,k
4a-f
5a-c
EWG = COOR, CN
7a R = Et, R1 = NO2, 7b R = R1 = Me, 7c R = Et, R1 = F, 7d R1 = Me, 7e R = Me, R1 = OMe, 7f R = Me,
R1 = Cl, 7g R = Et, R1 = Cl, 7h R1 = H, 7i R1 = F, 7j R = Et, R1 = OMe, 7k R = Et, R1 = Me
Scheme 2 Reagents and
conditions: (i) NaBH4, MeOH,
0-rt, 5 h; (ii) SOCl2, 0-rt, 12 h;
(iii) anh. piperizine, AcCN,
reflux, 24 h; (iv) 4, DMF,
K2CO3, rt, 78–93 %
F/H
(i)
F/H
F/H
F/H
(iii)
(ii)
O
OH
Cl
N
NH
F/H
9a-b
F/H
8a-b
F/H
10a-b
F/H
11a-b
8a R = H
8b R = F
F/H
F/H
(iv)
NC
H
N
R1
N
O
OR
N
N
F/H
R
F/H
H
12a-c, e-g, i-j, m-n
12d,h,k,l
123

Med Chem Res
Table 1 Synthesis of various benzhydryl piperazine derivatives
S. no.
1
BH bromides
1-Sub benzhydryl piperazine
Products
Yield (%)
89
7a
11b
2
7b
11b
91
3
7c
11b
93
4
7d
11b
87
123

Med Chem Res
Table 1 continued
S. no.
5
BH bromides
7e
1-Sub benzhydryl piperazine
Products
Yield (%)
78
11b
6
7f
11b
85
7
7g
11b
80
8
7h
11a
84
123

Med Chem Res
Table 1 continued
S. no.
9
BH bromides
1-Sub benzhydryl piperazine
Products
Yield (%)
89
7f
11a
10
7c
11a
82
11
7i
11a
84
12
7d
11a
86
123

Med Chem Res
Table 1 continued
S. no.
13
BH bromides
1-Sub benzhydryl piperazine
Products
Yield (%)
86
7j
11a
14
7k
11a
90
(90 %). Subsequently, these benzhydryl derivatives were
treated with thionyl chloride at ambient temperature to give
corresponding benzhydryl chlorides 10a–10b. Treatment of
these chlorides with piperazine and anhydrous potassium
carbonate in DMF under heating furnished key intermediate
1-benzhydrylpiperazine 11a–11b. Nucleophilic substitution
reaction between 1-benzhydrylpiperazines and different BH
bromides was carried out in the presence of anhydrous
potassium carbonate in DMF to give the products 12a–12n
with excellent yields (Scheme 2; Table 1).
Table 2 ACE inhibitor analysis of the piperazine derivative, 12h
Compounds
Concentration ACE inhibition (%) IC50 (mM)
(mM)
Compound 12h 0.232
14.4 0.19
29.90 0.25
54.10 0.19
66.75 0.24
39.12 0.13
54.18 0.22
79.09 0.15
91.984 0.2
0.874 0.19
0.465
0.930
1.860
ACE inhibitor 0.091
(standard)
0.182
0.162 0.25
0.363
0.726
Biological activities
Evaluation of ACE inhibitor activity
Table 3 Kinetic analysis of ACE enzyme inhibition for piperazine
derivative, 12h
All the synthesized piperazine derivatives were screened
for ACE inhibitor activity. The inhibitory activity is dose
dependent and the results are summarized in Table 2. All
the synthesized piperazine derivatives were checked up to
the highest concentration of 200 mM. Among these tested
synthesized piperazine derivatives, only compound 12h
showed good ACE inhibitory activity with IC50 value of
0.874 mM as compared to the standard Sigma ACE
inhibitor (IC50 value of 0.162 mM), while the remaining
compounds did not exhibit ACE inhibition activity upto the
maximum tested concentration of 200 mM. The structure–
activity relationship (SAR) study revealed that there are no
substituents present on the allyl phenyl nitryl ring of the
Test compound
Km (mM) Vmax (lM/min) Ki (mg/mL)
No inhibitor
0.5049
0.9790
59.70
59.97
60.01
–
12h
0.1576
0.2077
ACE inhibitor (standard) 1.1353
scaffold which contributed to the promising ACE inhibi-
tion activity. The data on the different kinetic parameters of
ACE enzyme inhibition such as Km, Ki and Vmax for
compound 12h, standard Sigma ACE inhibitor and without
inhibitor are shown in Table 3.
123

Med Chem Res
Table 4 Antimicrobial activity of piperazine derivatives
Test
compound
Minimum inhibitory concentration (MIC, in lM)
Staphylococcus Bacillus
aureus MTCC subtilis
S. aureus
MLS16
MTCC 121 MTCC 2940 2470
Micrococcu
Klebsiella
luteus MTCC planticola
Escherichia Pseudomonas
coli MTCC aeruginosa
Candida
albicans
MTCC 3018
96
MTCC 530
739
MTCC 2453
12a
12b
12c
12d
12e
12f
[383.1
78.6 0.05
[404.0
42.2 0.048
[405.6
[402.4
[391.3
[465.1
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
[383.1
[419.2
[404.0
[450.4
[405.6
[402.4
[391.3
[383.1
[383.1
[383.1
[419.2
[404.0
[450.4
[405.6
[402.4
[391.3
21.7 0.05
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
[383.1
8.96 0.05
[383.1
[419.2
[404.0
[450.4
[405.6
[402.4
[391.3
[465.1
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
539.9
19.6 0.03 [419.2
38.5 0.05 [419.2
[404.0
[450.4
[405.6
[402.4
[391.3
[404.0
[450.4
[405.6
[402.4
[391.3
[404.0
[450.4
[405.6
[402.4
[391.3
[404.0
[450.4
[405.6
[402.4
[391.3
12g
12h
12i
10.8 0.05 43.6 0.05 [465.1
10.8 0.05 [465.1
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
81.3 0.05 [433.8
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
[433.8
[435.7
[485.4
[490.1
[424.6
[439.5
12j
[435.7
[485.4
[490.1
[424.6
[439.5
[435.7
[485.4
[490.1
[424.6
[439.5
12k
12l
12m
12n
Levofloxacin 50.6 0.05
Ciprofloxacin 56.5 0.05
25.2 0.05 50.6 0.05 101.2 0.05 50.6 0.05
50.6 0.05 50.6 0.05
56.5 0.05 56.5 0.04 56.5 0.05
56.5 0.05
–
56.5 0.04 56.5 0.05
603.6
Fluconazole
–
–
–
–
–
–
104.4
Evaluation of antimicrobial activity
against S. aureus MTCC 96 with MIC value equal to that of
the standards. None of the compounds showed activity
against C. albicans. Based on these results, it could be
specified that the presence of BH scaffold on molecules led
to these excellent findings as we had also observed in our
previous studies (Narender et al., 2005; Santhoshi et al.,
2013). Compounds with substitutions like p-nitro, p-chloro
and p-methyl on the phenyl rings of BH part showed sig-
nificant activity, whereas methoxy-substituted compounds
showed no activity. Compounds 12b, a BH ester com-
pound, and 12h, a BH nitrile compound, are the two most
active compounds. So, the nitrile or ester groups might
have similar effects towards activity. Among the five
compounds, 12j with p-chloro substitution showed mod-
erate activity. The remaining compounds showed activity
at a concentration of [491 lM.
The synthesized piperazine derivatives were then tested for
in vitro antibacterial activity against Staphylococ-
cus aureus MTCC 96, Bacillus subtilis MTCC 121,
Staphylococcus aureus MLS 16 MTCC 2940, Micrococcus
luteus MTCC 2470, Klebsiella planticola MTCC 530,
Escherichia coli MTCC 739 and Pseudomonas aeruginosa
MTCC 2453, and antifungal activity against Candida
albicans MTCC 3018 by the well diffusion method. Lev-
ofloxacin and Ciprofloxacin were employed as standard
drugs for antibacterial evaluation, whereas Fluconazole
was used as a standard for antifungal evaluation.
Among all the synthesized piperazines derivatives, five
compounds, 12a, 12b, 12d, 12h and 12i, showed excellent
to moderate activities against the different tested bacterial
strains (Table 4). Compound 12h emerged as the lead
compound from the study. It showed remarkable activity
against B. subtilis MTCC 121, S. aureus MLS16 MTCC
2940, K. planticola MTCC 530 and E. coli MTCC 739
with MIC values much less than or equal to that of the
tested standard drugs, Levofloxacin and Ciprofloxacin.
Compound 12a was highly active against P. aeruginosa
with MIC value of 8.96 lM, whereas the standards had
MIC values of 50.6 and 56.5 lM. Compound 12b showed
excellent activity against S. aureus MLS 16 MTCC 2940
and E. coli MTCC 739, and was moderately active against
S. aureus MTCC 96. Compound 12d was highly active
Conclusions
A series of 1-benzhydryl piperazine derivatives were syn-
thesized by a convenient and efficient method and were
screened for in vitro ACE inhibition and antibacterial
activities. Compound 12h showed moderate ACE inhibi-
tion activity as compared to the standard angiotensin-con-
verting enzyme inhibitor (Sigma). Compounds 12a, 12b,
12d, 12h and 12i showed good to moderate antibacterial
activity. The presence of BH scaffold at the fourth position
123

Med Chem Res
potentially improved adverse reaction profile. Bioorg Med Chem
12:1483–1491
of benzhydryl piperazine ring played a significant role
towards bioactivity.
Ehlers MR, Fox EA, Strydom DJ, Riordan JF (1989) Molecular
cloning of human testicular angiotensin-converting enzyme: the
testis isozyme is identical to the C-terminal half of endothelial
angiotensin-converting enzyme. Proc Natl Acad Sci USA
86:7741–7745
Farzaliev VM, Abbasova MT, Ashurova AA, Babaeva GB, Ladokh-
ina NP, Kerimova YM (2009) Synthesis of N, N0-bis(alkyl-
oxymethyl)piperazines and examination of their antimicrobial
properties. Russ J Appl Chem 82:928–930
Acknowledgements The authors thank the Director, CSIR, Indian
Institute of Chemical Technology, for the facilities and encourage-
ment. AS, SNK, PS and PSS acknowledge the CSIR, New Delhi, for
research fellowships.
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