Synthesis and pharmacological evaluation of [(4-Arylpiperazin-1-yl)-alkyl]- carbamic acid ethyl ester derivatives as potential anxiolytic agents

Article abstract of DOI:10.1007/s12272-012-0704-8

On the basis of our earlier studies, a series of N-{4-[4-(aryl) piperazin-1-yl]-phenyl}-amine derivatives containing terminal carbamoyl fragment with alkyl spacer of different lengths (15-20) were synthesized as ligands, for 5-hydroxytryptamine-1A (5-HT1A

Full text of DOI:10.1007/s12272-012-0704-8

Arch Pharm Res Vol 35, No 7, 1143-1152, 2012
DOI 10.1007/s12272-012-0704-8
Synthesis and Pharmacological Evaluation of [(4-Arylpiperazin-1-yl)-
alkyl]-carbamic Acid Ethyl Ester Derivatives as Potential Anxiolytic
Agents
Manisha Khatri1, Santosh K Rai2, Ranjit Ranbhor3, Krishna Kishore4, and Manisha Tiwari1
2
1Dr. B. R. Ambedkar Center for Biomedical Research, University of Delhi, Delhi 110007, India, Sphaera Pharma Pvt. Ltd.,
3
4
IMT Manesar, Gurgaon 122051, India, Glenmark Research Center, Mahape, Navi Mumbai 400701, India, and Department
of Neurochemistry and Haematology, Defence Institute of Physiology and Allied Sciences, Delhi 110054, India
(Received December 14, 2011/Revised February 5, 2012/Accepted February 13, 2012)
On the basis of our earlier studies, a series of N-{4-[4-(aryl) piperazin-1-yl]-phenyl}-amine deri-
vatives containing terminal carbamoyl fragment with alkyl spacer of different lengths (15-20)
were synthesized as ligands, for 5-hydroxytryptamine-1A (5-HT1A) receptor. Molecular modeling
studies were undertaken to explain the influence of spacer length on ligands affinity towards
5-HT1A receptor. Compound 19 showed all the specific interactions responsible for recognition.
The protonated amine of the ligand forms an ionic hydrogen bond with the negatively charged
Asp116 of transmembrane3 helix (TM3), while the carbamoyl moiety interacts with Asn386
and Tyr390 of TM7. The aryl group is involved in forming a CH-π interaction with Phe362.
The strong interaction of compound 19 with 5-HT1A receptor in docking studies was confirmed
by radio ligand binding studies. Compound 19 showed high affinity for the receptor (Ki =
0.018 nM). In vivo pharmacological testing of compound 19 (3 mg/kg body weight) showed
increased open arm entries, as well as time spent in Elevated plus Maze test. Toxicological
analysis also revealed no significant biochemical or morphological alterations in the vital organs
of experimental animals. Furthermore our results suggest that these compounds share some
pharmacological effects with established anxiolytics and might prove to be effective compounds
for the treatment of anxiety.
Key words: Serotonin receptor ligands, Anxiolytic activity, Molecular modeling, Ligand bind-
ing, Elevated plus Maze
as well as in normal human functions such as sleep,
sexual activity and appetite. 5-HT attains such variety
INTRODUCTION
Psychiatric disorders such as anxiety and depres- of functions by acting on distinct receptor types (Buhot
sion are closely connected with disturbances in the et al., 2000). Among the 14 different types of serotonin
function of different neuromediator systems. Seroto- receptors belonging to G protein-coupled receptor (GPCR)
nin (5-hydroxytryptamine, 5-HT) is one of the important family (Hoyer et al., 2002), 5-HT1A subtype is a major tar-
neurotransmitters, which mediates a wide variety of phy- get for neurobiological research and drug development.
siological responses in both the peripheral and central Its activation leads to a number of physiological changes.
nervous systems. Alterations in serotonin neurotrans- The development of drugs that alter 5-HT neurotrans-
mission have been implicated in a number of human mission is thus an area of intense research.
disorders such as migraine, depression and anxiety,
The 5-HT1A receptor has been the most extensively
studied of all the serotonin receptors. The reason res-
ides in the early availability of the selective agonist 8-
hydroxy-2-(di-N-propylamino)tetralin (8-OH-DPAT) that
has allowed extensive biochemical, physiological, and
pharmacological characterization of the receptor (Gozlan
et al., 1983). Several structural classes of agents are known
Correspondence to: Manisha Tiwari, Dr. B. R. Ambedkar Centre
for Biomedical Research, University of Delhi, Delhi-110007,
India
Tel: 91-11-27666272, Fax: 91-11-27666248
E-mail: mtiwari07@gmail.com
1143

1144
M. Khatri et al.
to bind 5-HT1A receptor sites, such as aminotetralins, ed using molecular docking analysis. In vitro radio-
indolylalkylamines, ergolines, aporphines, aryloxyalkyl- ligand binding study was carried out to evaluate the
amines and arylpiperazines. Among these, the arylpi- binding affinity. An elevated plus maze test was per-
perazine derivatives represent one of the most important formed to assess potential antianxiety activity. Moreover
classes of 5-HT1A receptor ligands (Obniska et al., 2006). the best anxiolytic derivatives were further analysed
Long chain aryl piperazines (LCAPs), having an alkyl for their acute and subacute toxicity to evaluate the
chain attached to the N4 atom of the piperazine moiety safety of these new compounds.
and a terminal amide or an imide fragment, are the most
thoroughly studied of all the aryl piperazine derivatives.
The significance of the respective parts of LCAPs for
5-HT1A receptor affinity, intrinsic activity, and selectivity
has been the subject of many structure-activity relation-
ship studies (Rodriguez et al., 2002).
MATERIALS AND METHODS
Chemistry
All organic solvents and common reagents were pro-
cured from Merck India Ltd. All the arylpiperazines
Molecular modeling techniques provide useful infor- were procured from Aldrich Chemical Company, Inc.
mation on structural and functional aspects of GPCRs USA. TLC analysis was carried out on commercially
and their interactions with ligands. GPCRs are mem- available flexible TLC silica gel (silica gel 60 F254)
brane spanning receptors that mediate most of their plates (Merck). The purity of all organic compounds
intracellular actions through pathways involving activa- was confirmed by TLC, 1H-NMR, IR, and Mass spectro-
tion of G-proteins. Until late 2007, the only high-resolu- scopy. 1H-NMR spectra were recorded in Bruker Spec-
tion structures of GPCRs available were those of bovine trospin Avance 300 instrument operating at 300 MHz
rhodopsin (Okada et al., 2004), and GPCR homology in CDCl3 or DMSO using TMS as an internal standard.
models were necessarily based on this template (Kiss The chemical shifts are reported in parts per million
et al., 2008). Recently, several GPCR crystal structures
have been reported, and the structures of human β2
adrenergic receptor ( 2), (Rasmussen et al., 2007; Hanson on a Perkin-Elmer BX FT-IR instrument. Melting points
et al., 2008) squid rhodopsin, (Murakami and Kouyama, were determined on a Buchi melting point B-450 instru-
2008) turkey 1-adrenergic receptor ( 1) (Warne et al., ment. Mass spectra were recorded on a Qstar (Applied
(
δ
) downfield from TMS and coupling constants are
-
reported in hertz (Hz). IR spectra (KBr) were recorded
β
β
β
2008) have been solved providing a wealth of informa- biosystem) ESI-MS mass spectrometer. Elemental an-
tion and revealing important structural differences alysis was performed on Heraeus CHN rapid analyzer.
between rhodopsin and other class A GPCRs, parti-
cularly in transmembrane helices and in the structure
of loop regions (Michino et al., 2009). Very close evolu-
General procedure for synthesis of 3-8
Sodium carbonate (15 mM) was added to a stirred
tionary relationships between 5-HT receptors and beta solution of aryl piperazine (10 mM) in ethanol (20 mL).
adrenoreceptors provide a means of obtaining a better Then bromo alkyl nitrile (10 mM) was added dropwise
model for the serotonin receptors. Due to the absence of and the mixture was stirred for 2 h. The precipitated
3D structures of 5-HT receptors, a model of rat 5-HT1A inorganic salts were then filtered off, washed with
receptor was constructed from the X-ray structure of ethanol and the filtrate was concentrated on rotary
turkey beta 1 adrenergic receptor using molecular evaporator. Then the compound was partitioned with
modeling.
ethyl acetate and water. The organic layer was dried
We have previously concluded that aryl piperazine over anhydrous sodium sulfate and concentrated on a
compounds with a unsubstituted phenyl piperazine rotary evaporator. Finally after evaporation of the sol-
fragment (
phenyl piperazine fragment (
functionality at their terminal N-4 fragment have the The spectroscopic data for all these intermediate com-
1) or having an O-methoxy substituted vent the residue was purified by column chromatog-
2) along with carbamoyl raphy, using hexane/ethyl acetate (80:20) as eluent.
potential to be used as anxiolytic agents (Khatri et al., pounds (3-8) is given as supplementary material.
2009). We have therefore synthesized a new series of
(4-aryl-piperazin-1-yl) alkyl]carbamic acid ethyl ester
derivatives (15 20). The structural modifications consist
General procedure for synthesis of 9-14
A solution of ( , 5 mM) in tetrahydrofuran (15 mL)
-
3-8
of the replacement of phenyl ring, which connects the was added dropwise to a suspension of lithium alumi-
aryl piperazine fragment to the carbamoyl terminal, nium hydride (380 mg, 10 mM) in dry tetrahydrofuran
with a different length alkyl spacer. The interactions (20 mL) under nitrogen atmosphere at 0oC. The tem-
of aryl piperazine derivatives with selective amino perature of reaction mixture was then allowed to rise
acid residues of 5-HT1A receptor active site were studi- to room temperature and stirred for 2-3 h. The reac-

New Anxiolytic Carbamic Acid Ethyl Ester Derivatives
1145
tion was then quenched by addition of water (0.47 Hz), 4.08-4.15 (q, 2H, O-CH2,
mL), 15% aqueous NaOH (0.47 mL) and water (1.41 -NH), 6.83-7.2 (m, 5H, Ar-H); LC-MS
J
= 6.9 Hz), 5.18 (br, 1H,
m/z
278 (M+ +1);
mL) sequentially. The resulting granular precipitate Anal. Calcd. for C15H23N3O2: C, 64.95; H, 8.36; N,
was passed through the pad of celite and washed with 15.15. Found: C, 64.82; H, 8.31; N, 15.09.
ethyl acetate. The combined filtrate was evaporated
under reduced pressure, and the residue was chroma- [3-(4-Phenyl-piperazin-1-yl)-propyl]-carbamic acid
tographed on silica gel. (Eluent, MeOH-CH2Cl2 30:70). ethyl ester (16)
The spectroscopic data for all these intermediate com- Yield: 83%; m.p. 189-191oC; IR(KBr)
ν
cm−1: 1252.46
N), 2967.89 (C-H),
3339.45 (N-H); H-NMR (CDCl3, 300 MHz): 1.70-
1.74 (t, 3H, -CH3, = 6.3 Hz), 1.9-2.04 (m, 2H, CH2-
L, 5 mM) in CH2-CH2), 2.47-2.52 (t, 2H, CH2-CN, = 6.6 Hz), 2.61-
dry DCM (5 mL) was added dropwise to a solution of 2.63 (m, 4H, piperazine), 3.19-3.22 (m, 4H, piperazine),
14 (5 mM) and triethylamine (2 mmol) in dry DCM 3.27-3.31 (t, 2H, N-CH2, = 6 Hz), 4.10-4.17 (q, 2H, -O-
(20 mL). The mixture was allowed to stir at room CH2, = 7.2 Hz), 5.6 (br, 1H, -NH), 6.92-7.34 (m, 5H,
temperature for 3 h. The reaction mixture was poured Ar-H); LC-MS
292 (M+ +1); Anal. Calcd. for
pounds (9-14) is given as supplementary material.
(C-N), 1442.40 (C=C), 2282.36 (C≡
1
δ
J
General procedure for synthesis of 15-20
A solution of ethyl chloro formate (540
µ
J
9-
J
J
m/z
into water (30 mL) and extracted with CH2Cl2 (2 × 30 C16H25N3O2: C, 65.95; H, 8.65; N, 14.42. Found: C,
mL). The organic layer was dried and the solvent was 65.98; H, 8.72; N, 14.50.
evaporated under reduced pressure to yield an oil,
which was purified by silica gel column chromatog- [4-(4-Phenyl-piperazin-1-yl)-butyl]-carbamic acid
raphy using a mixture of CH2Cl2 and MeOH (9.8:0.2) ethyl ester (17)
as eluent to yield (65-70%) of the desired product Yield: 85%; m.p. 202-204oC; IR(KBr)
ν
cm−1: 1239.46
N), 2972.9 (C-H),
3345.45 (N-H); 1H-NMR (CDCl3, 300 MHz):
1.18-1.24
[2-(4-Phenyl-piperazin-1-yl)-ethyl]-carbamic acid (m, 2H, CH2-CH2-CH2-CH2), 1.57-1.61 (m, 5H, CH2-
(Scheme 1).
(C-N), 1459.40 (C=C), 2248.78 (C≡
δ
ethyl ester (15)
CH2-CH2-CH2, -CH3), 2.39-2.44 (t, 2H, N-CH2,
J = 6.9
Yield: 85%; m.p. 175-178oC; IR(KBr)
ν
cm−1: 1237.67 Hz), 2.60-2.63 (m, 4H, piperazine), 3.18-3.24 (m, 6H,
(C-N), 1448.57 (C=C), 2267.83 (C
≡
N), 2939.89 (C-H), piperazine, CH2-NH), 4.04-4.11 (q, 2H, -OCH2), 5.46 (br,
3346.45 (N-H); H-NMR (CDCl3, 300 MHz): 1.22- 1H, -NH), 6.8-7.2 (m, 5H, Ar-H); LCMS (C17H27N3O2):
1.27 (t, 3H, -CH3, = 7.8 Hz), 2.51-2.55 (t, 2H, N-CH2,
306 (M+ +1); Anal. Calcd. for C17H27N3O2: C,
= 6.0 Hz), 2.60-2.63 (m, 4H, piperazine), 3.08-3.23 65.95; H, 8.65; N, 14.42. Found: C, 65.98; H, 8.72; N,
(m, 4H, piperazine), 3.29-3.33 (t, 2H, CH2-N, = 5.7 14.50.
1
δ
J
m/z
J
J
Scheme 1. General procedure for synthesis of compounds. Reagents and conditions: (a) EtOH, Na2CO3, rt, 2 h; (b) LiAlH4,
EtOAc, 0oC, 2 h; (c) ClCOOC2H5, DCM, Et3N, 2 h.

1146
M. Khatri et al.
{2-[4-(2-Methoxy-phenyl)-piperazine-1-yl]-ethyl}- receptor.
carbamic acid ethyl ester (18)
The sequences of 5-HT1A and 2VT4 were aligned
Yield: 85%; m.p. 202-204oC; IR(KBr)
ν
cm−1: 1267.46 using MOE-Align tool making sure that the gaps were
(C-N), 1473.78 (C=C), 2238.91 (C
≡
N), 2948.92 (C-H), inserted into the loop regions between the helices, pre-
3338.45 (N-H); H-NMR (CDCl3, 300 MHz): 1.22- ferably where there are no steric clashes with other
1.27 (t, 3H, -CH3, = 7.2 Hz), 2.52-2.56 (t, 2H, N-CH2, parts of structure. 2VT4 has a longer loop than 5-HT1A
= 6.0 Hz), 2.65 (s, 4H, piperazine), 3.07 (s, 4H, pi- between helices IV and V (Cys192 and 198). This loop
perazine), 3.30-3.34 (t, 2H, CH2-N, = 5.4 Hz), 3.85 (s, has Cys-Cys bond. To build the homology model for 5-
3H, -OCH3), 4.08-4.15 (q, 2H, O-CH2, = 6.9 Hz), 5.24 HT1A, the atoms for residues 192-201 in the 2VT4
(br, 1H, NH), 6.8-6.9 (m, 4H, Ar-H); LC-MS 308 template structure were deleted, and AMBER99 force
(M+ +1); Anal. Calcd. for C16H25N3O3: C, 62.52; H, 8.20; field was used with solvation parameter set to R-field.
1
δ
J
J
J
J
m/z
N, 13.67. Found: C, 62.58; H, 8.12; N, 13.72.
The developed homology model was checked for quality
using MOE suite. The criteria used in the analysis in-
{3-[4-(2-Methoxy-phenyl)-piperazine-1-yl]-propyl}- clude bond lengths, bond angles, dihedrals, side chain
carbamic acid ethyl ester (19)
contacts and chirality of alpha carbon atoms. The phi-
Yield: 80%; m.p. 216-218oC; IR(KBr)
ν
cm−1: 1267.76 psi map, Ramachandran plot, Chi plot and distance
(C-N), 1448.89 (C=C), 2228.21 (C
3348.85 (N-H); 1H-NMR (CDCl3, 300 MHz):
(s, 3H, -CH3), 1.67-1.71 (m, 2H, CH2-CH2-CH2), 2.48-2.53 force field. Force field bonded, Van der Waals and elec-
(t, 2H, CH2-NH, = 6.6 Hz), 2.66 (s, 4H, piperazine), trostatics energy was calculated with non-bonded cut-
3.10 (s, 4H, piperazine), 3.25-3.29 (t, 2H, N-CH2,
off switched on at 8 Å and switched off at 10 Å. Distance
5.4 Hz), 3.86 (s, 3H, -OCH3), 4.06-4.09 (q, 2H, -OCH2, dependent dielectric (internal 1 and external 80) was
= 3.6 Hz), 5.24 (br, 1H, -NH), 6.89-7.19 (m, 4H, Ar-H); used to model implicit solvation. Receptor was kept
LC-MS
322 (M+ +1); Anal. Calcd. for C17H27N3O3: fixed during docking with pocket cut-off 6 Å.
≡
N), 2941.62 (C-H), matrix plot of the model were constructed. Docking of
δ
1.61-1.67 ligands was done using MOE 2009.10 with MMFF94x
J
J
=
J
m/z
C, 63.53; H, 8.47; N, 13.07. Found: C, 63.62; H, 8.52;
N, 13.14.
Experimental animals
Male Wistar rats (6-8 weeks old), weighing 120-150 g
each, were selected from the stock colony and maintain-
ed in our animal facility with free access to food and
{4-[4-(2-Methoxy-phenyl)-piperazine-1-yl]-butyl}-
carbamic acid ethyl ester (20)
Yield: 85%; m.p. 230-232oC; IR(KBr)
ν
cm−1: 1252.87 water. Animals were maintained in an air-conditioned
(C-N), 1468.89 (C=C), 2238.10 (C
3340.54 (N-H); 1H-NMR (CDCl3, 300 MHz):
(m, 5H, CH2-CH2-CH2-CH2, -CH3), 1.57-1.59 (m, 2H, experiments were performed during the light phase
CH2-CH2-CH2-CH2), 2.41-2.46 (t, 2H, N-CH2, = 6.6 according to the guidelines of the Institutional Animal
≡
N), 2945.72 (C-H), room. The room was maintained at 25 ± 2oC with natural
1.19-1.34 daytime and no light after 19:00 h, until morning. All the
δ
J
Hz), 2.67 (s, 4H, piperazine), 3.12-3.20 (m, 6H, pipera- Ethics committee.
zine, CH2-CN), 3.86 (s, 3H, -OCH3), 4.10-4.17 (q, 2H,
-OCH2), 5.54 (br, 1H, -NH), 6.8-6.99 (m, 4H, Ar-H);
5-HT1A binding assay
Radioligand binding assay was performed following
LC-MS
m/z
336 (M+ +1); Anal. Calcd. for C18H29N3O3:
C, 64.45; H, 8.71; N, 12.53. Found: C, 64.52; H, 8.63; a published procedure (Schlegel and Peroutka, 1986).
N, 12.48.
The cerebral cortex obtained from male Wistar rats
was homogenized in 20 volumes of ice cold Tris-HCl
buffer (50 mM, pH 7.7) and centrifuged at 50,000 g for
Molecular modeling
The comparative homology modeling of human 5-HT1A 10 min at 0oC. The resulting pellet was resuspended,
receptor was carried out with MOE2009.10 (Chemical incubated at 37oC for 10 min and centrifuged again at
Computing Group) on Quad-Core intel Xeon workstation 50,000 g for 10 min at 37oC. The final pellet was re-
(2.33 GHz processor). The amino acid sequence of 5- suspended in Tris-HCl buffer containing 10 µM pargy-
HT1A was obtained from SwissProt database. The 5- line, 4 mM CaCl2 and 0.1% ascorbate. 5-HT1A binding
HT1A receptor sequence (SP08908) has 422 amino was determined in the cortical preparation with or with-
acids. The X-ray structure of turkey beta1 adrenergic out 0.1 nM [3H] 8-OH-DPAT (GE healthcare-TRK850)
receptor bound to cyanopindolol (PDB code: 2VT4.pdb) as a competitive binding agent. Specific binding was
(Berman et al., 2002) with 2.7 Å resolution was down- defined as the difference between binding in the absence
loaded from Protein Data Bank and was used as a and presence of 5-HT (10
template for modeling the 3D structure of the 5HT1A
µM).

New Anxiolytic Carbamic Acid Ethyl Ester Derivatives
1147
coagulant, respectively.
Elevated plus Maze test
The elevated plus maze was comprised of two open
arms (50 cm × 10 cm) and two closed arms (50 cm × 10
cm × 30 cm). The arms extend from a central platform
Haematological and biochemical analysis
Haematological analysis was performed using an
(5 cm × 5 cm) that was elevated to a height of 50 cm automatic haematological analyzer (Coulter STKS,
above floor level. The experiments were performed Beckman). The parameters included: red blood cell
between 9:00 h and 15:00 h. Each rat was placed in (RBC) count, white blood cell (WBC) count, haemoglobin
the central area of the maze with its head facing an (Hb), haematocrit (HCt), mean corpuscular volume
open arm. The following behavioural parameters were (MCV), mean corpuscular haemoglobin (MCH), mean
recorded, during 5 min exposure using FW: Camera corpuscular haemoglobin concentration (MCHC) and
O: Fire - iBBW 1.3 interface (Columbus Instruments): platelets count were determined (Costa-Silva et al.,
(1) time in open arms; (2) time in closed arms; (3) time 2007). For biochemical analysis, blood was centrifuged
in central area; (4) number of open arm entries; (5) at 1480 ×
g for 10 min to obtain serum, which was stored
number of closed arm entries; (6) number of central at 20oC until determination of the following parameters
area entries. Any rat, which dropped off the plus maze, according to standard methods of Bergmeyer and Bernt
was excluded in the result (Walf and Frye, 2007).
(1974) using an assay kit: glucose, blood urea nitrogen
The experimental animals were treated with Buspir- (BUN), creatinine, Aspartate aminotransferase (AST),
one hydrochloride (Sigma-Aldrich Co.) (5 mg/kg, n = 8) alanine aminotransferase (ALT), total cholesterol, tri-
or the compounds (3 mg/kg) intraperitoneally in a glycerides, alkaline phosphatase (ALP), total and direct
volume of 2 mL/kg body weight of rats, 60 min. before bilirubin, total protein.
evaluation in the maze. The control group was given
saline with 1% tween 80 and 1% DMSO.
RESULTS AND DISCUSSION
The synthetic methodology employed to develop target
Acute toxicity
As per the Organization of Economic Co-operation compounds is summarized in Scheme 1. The reaction
and Development (OECD) guidelines for testing of of 1-aryl piperazine with bromoalkyl nitrile in ethanol
chemicals (OECD, 2001a), 12 rats were randomly chosen in the presence of sodium carbonate afforded the cor-
and divided into three groups. They were orally admin- responding nitrile derivatives (
istered compounds at a dose of 375, 750 and 1000 mg/ was reduced in tetrahydrofuran with lithium aluminium
kg body weight and the control group received vehicle. hydride to give (4-aryl-piperazin-1-yl)-alkylamines (
3-8). The nitrile group
9
-
Body weight, signs of toxicity and mortality were ob- 14). Subseqent condensation of the resulting with ethyl
served after the administration at the first, second, chloroformate in methylene chloride in the presence of
fourth and sixth hour and once daily for 14 days. On triethyl amine provided the target compounds [(4-
day 15, all rats were made to fast for 16-18 h, and then aryl-piperazin-1-yl)-alkyl]-carbamic acid ethyl ester
sacrificed for necropsy examination. The internal organs
were excised and weighed. The gross pathological ob-
servations of the tissues were performed.
(15-20) in an approximate yield of 80%.
Molecular modeling
To explain the interaction of aryl piperazine deriva-
tives with 5-HT1A receptor molecular docking analysis
Subacute toxicity
The experiments were performed according to the of the compounds and natural ligand of serotonin re-
OECD test guidelines with slightly modifications (OECD, ceptor was carried out using a homology model based
2001b). Male Wistar rats (n = 6) received water vehicle on a recently solved X-ray crystal structure of turkey
orally (control group) or compounds at the effective beta 1 adrenergic receptor (PDB ID: 2VT4). The binding
dose of 3 mg/kg/day for 15 consecutive days. The body cavity is composed of TM2 (Ala93, Tyr96), TM3 (Ile113,
weight was recorded weekly and their food and water Asp116, Cys120), TM5 (Cys187, Ile189, Ser199), TM6
intake was monitored daily. Animals were observed (Phe361, Phe362) and TM7 (Asn386, Trp387, Tyr390)
for signs of abnormalities during the treatment period. domains. Building a protein model requires several the-
At the end of the treatment, the animals were made to oretical assumptions and methodological approxima-
fast overnight, but allowed access to water ad libitum
.
tions. To validate the ligand receptor interactions, sero-
After the animals were anesthetized with ether, the tonin, the natural ligand, was docked in the developed
blood samples were obtained by retro-orbital puncture homology model of the 5HT1A receptor. The 5-HT was
(Waynforth, 1980) using capillary tubes for hematologi- correctly positioned into the inner part of the seven
cal and biochemical studies, with and without anti- helix bundle and the fundamental ligand-protein

1148
M. Khatri et al.
interactions were detected. The available free space binding (Liapakis et al., 2000). Introduction of a phenyl
between the helices 3, 5 and 6 accommodate the phe- ring ( and ) instead of alkyl spacer makes the com-
1
2
nolic oxygen of serotonin. The indole ring of serotonin pound more rigid and causes unfavourable steric in-
is close to the backbone of helix V. The 5-OH group teractions with the receptor, leading to the loss of the
was found to form hydrogen bond with Ser199. The essential anchoring interaction between the aromatic
negatively charged Asp116 forms an ionic hydrogen moiety and Asp116.
bond with the protonated nitrogen of serotonin. The
The length of the alkyl spacer was also optimized:
three dimensional model developed for serotonin bind- compounds with piperazinyl propyl chain (16 and 19
)
ing was used to observe the binding mode of the syn- showed good affinity for the 5-HT1A receptor, (Fig. 2
thesized compounds. The binding modes of these mole- and 3B) compared to compounds containing spacer
cules were consistent with the binding mode of seroto- with one atom shorter (15 and 18) or one atom longer
nin. Thus, this model can serve as a platform for de-
signing of selective ligands for 5HT1A receptor.
The 2-methoxyphenyl moiety of compounds
(
17 and 20). The formation of additional hydrogen
bonds with Tyr390 and Asn386 might be attributed to
2 and the high affinity of these compounds for the receptor.
18 to 20 was found between transmembrane helices 3, On the other hand shortening of the spacer hindered
5 and 6 interacting with the aromatic cluster of helix the interaction with polar residues on TM7 making
6, especially Phe362. In compound
2 this moiety was compounds with least affinity in the series at 5-HT1A
also involved in the formation of hydrogen bond with receptor site.
Ser199 (Fig. 3A). The carbamoyl domain interacted
These results explained the influence of the spacer
predominantly with the amino acids localized on helices between the aryl piperazine and the carbamoyl frag-
2, 3 and 7, forming both favourable Van der Waals ment and that the length of the spacer is of great im-
contact with hydrophobic residues as well as hydrogen portance for 5-HT1A affinity. Ligands with three carbon
bonds with polar sites such as Tyr390 (Fig. 1). Analysis alkyl spacer (16 and 19) possess all the essential in-
of the complexes between the ligands 15 to 20 and 5- teractions when docked to the 5HT1A receptor model,
HT1A receptor model showed that introduction of an whereas compounds with two carbon alkyl spacer (15
alkyl spacer allows the formation of a salt bridge between and 18) are poorly active. Notably reduction of the
the protonated piperazine nitrogen of the compounds spacer or replacement by the phenyl ring leads to
and Asp116 of the receptor which is generally consid- rigid compounds
1 and 2, maintaining the anxiolytic
ered crucial for all monoamine neurotransmitter recep- activity in the EPM model (Khatri et al., 2009) but
tors (Medina et al., 2009; Kowalski et al., 2011). Directed their interactions within the 5-HT1A receptor binding
mutagenesis experiments also suggested that Asp116, pocket were reduced explaining their lower activity as
Asn386 and Ser199 may be involved in the ligand compared to compounds 16 and 19
.
Fig. 1. Surface view of molecular model of the ligand re-
ceptor complex for compound studied by means of ligand-
receptor docking. Helical bundle is presented from the ex-
tracellular side. Sticks representation depicts residues used
as “active site” in docking.
Fig. 2. Surface view of molecular model of the ligand re-
ceptor complex for compound 19 studied by means of ligand-
receptor docking. Helical bundle is presented from the ex-
tracellular side. Sticks representation depicts residues used
as “active site” in docking.
2

New Anxiolytic Carbamic Acid Ethyl Ester Derivatives
1149
Fig. 3. Detailed view of molecular model of the ligand receptor complex studied by means of ligand-receptor docking. (A) for
compound
2
(
B
) for compound 19. In the detailed view for compound
2, dashed green lines represent H-bonds with Ser199
and Tyr390. A solid green line shows a CH-
lines represent H-bonds with Asn386 and Tyr390 and a salt bridge with Asp116. A solid green line shows a CH-
with Phe362.
π
interaction with Phe362. In the detailed view for compound 19, dashed green
π interaction
ing 5-HT1A receptor affinity of compound 19 (Ki =
5-HT1A receptor binding
The ability of aryl piperazine derivatives to displace 0.018 nM) and compound 16 (Ki = 0.04 nM), Ki values
[3H]-8-OH DPAT, a 5-HT1A receptor agonist from the were clustered in a relatively narrow range from 2.4
5-HT1A receptor was determined and the results are nM for compound 20 to 28.2 nM for compound 15. Re-
summarized in Table I. All the compounds showed placement of phenyl group with alkyl spacer had a
nanomolar or even subnanomolar affinities. All the significant effect on 5-HT1A binding, since its replace-
compounds showed better results as compared to ment caused a remarkable increase (compound 1, 15.04
Buspirone (Ki = 12 nM) (Garrattini et al., 1988) except nM vs compound 19, 0.018 nM). Moreover, regarding
compound 15 (Ki = 28.2 nM) and 18 (Ki = 21.7 nM) the influence of the alkyl spacer, compounds with
having two carbon alkyl spacer. Besides the outstand- piperazinyl propyl chain (16 and 19) had the highest
affinity for the 5-HT1A receptor. Instead, the compounds
Table I. In vitro binding affinities of compounds 1, 2 and
15 to 20 for 5-HT1A receptor
with piperazinyl ethyl chain (15 and 18) seem to show
a less favourable affinity profile as compared to 16
and 19. Compounds having O-methoxy substituted
phenyl piperazine fragment showed high affinity for
the 5-HT1A receptor, relative to the compounds in which
phenyl group attached to piperazine is unsubstituted.
The binding profile observed for the derivatives, is in
accordance with our molecular docking results.
Substituents
Com-
pound
Ki (5-HT1AR) nM
R
n
1
2
H
Replaced with C6H4
15.04 ± 0.870
9.25 ± 1.10
28.2 ± 2.20
00.04 ± 0.001
8.37 ± 0.26
21.7 ± 1.60
0.018 ± 0.001
02.4 ± 0.21
Elevated plus Maze test
OCH3 Replaced with C6H4
H
H
To validate the in vitro results, functional profile of
all the derivatives was determined in vivo; to find out
whether changes in the structure of the aryl fragment
and the length of alkyl chain influence their anxiolytic
activity. The Elevated plus Maze is one of the most
widely used animal models for screening putative
anxiolytics, in which rodents show an avoidance of
exposed open areas of the maze, which are presumed
to be the most aversive, and a preference for sections
enclosed by protective walls (Dawson and Tricklebank,
15
16
17
18
19
20
1
2
3
1
2
3
H
OCH3
OCH3
OCH3
For purpose of comparison, 8-OH-DPAT binds 5-HT1A receptor
with Ki value of 0.80 nM, under same assay conditions. Ki
values are mean ± S.D. of two to four experiments performed
in triplicate.

1150
M. Khatri et al.
1995). Conventional anxiety indices in the elevated obtained compounds 19 turned out to be the most
plus-maze test comprise percent open arm entries and active as seen from the results of behavioural test.
percent time spent in these areas in the maze, with Our results showed that compounds 16 and 19 are the
anxiolytics generally increasing and anxiogenics decreas- most anxiolytic and these compounds were further
ing these measures.
evaluated for their toxicity in rat model to establish
The effects of single administration of all the 6 aryl their safety and efficacy.
piperazine derivatives along with previously reported
compound
1 and 2, Diazepam, Buspirone and vehicle
Acute toxicity
on spent time and the number of entries into the open
Compound 16 and 19, the most potent anxiolytics
arms of EPM are shown in Table II. One way analysis were selected for acute toxicity testing. Clinical symptoms
of variance revealed a significant increase in percent- were measured for 14 days after the single oral gavage
age time spent on the open arms after administration administration of 1000 mg/kg. Acute toxicity describes
of both positive controls, Diazepam and Buspirone, as the adverse effects of a substance which result either
compared to control. The majority of tested compounds from a single exposure or from multiple exposures in a
produced a significant anxiolytic action and displayed short space of time (usually less than 24 h). The results
a considerable increase in the number of open arm showed that all the rats after administration of dose of
entries. Among all the 6 compounds, 16 (3 mg/kg bwt) 1000 mg/kg body weight/day did not show any mortality,
prolonged the percentage of entries in open arms with and the autopsy of the animals at the end of the ex-
~90 % (
p
< 0.001) and 19 (3 mg/kg bwt) with ~105% (
p
perimental period (14 days) revealed no apparent
< 0.001) as compared to control. These results are found changes in any organs. The results indicated that the
to be comparable with Buspirone. The total number of compounds were safe at dose of 1000 mg/kg or less in
arm entries was not significantly different for all treat- rats, which is according to the OECD guidelines cate-
ment groups.
gorised as unclassified and safe.
The results presented in Table II have conclusively
proven that the derivatives having alkyl chain with
three carbons significantly increased the entry as well
Subacute toxicity
In order to establish the safety and efficacy of a new
as time spent in open arms (16 and 19) compared to drug, an essential first step is to evaluate the toxicity
the corresponding derivatives having alkyl chain with of the new molecular entity in experimental animals
two (15 and 18) and four carbons (17 and 20) and the (Alam et al., 2006). Depending on the duration of ex-
previously reported compounds having hydrophobic posure of the animal to the drug; toxicological studies
phenyl ring instead of alkyl spacer (1 and 2). Within a may be classified as acute, subacute, or chronic. Rats
set of respective structural analogs, phenyl piperazines in sub-acute toxicity study received repeated doses of
with a methoxy group (-OCH3) at the ortho position dis- both the compounds at their effective dose of 3 mg/kg
played good anxiolytic activity, compared to unsubsti- (Khatri et al., 2009) intraperitoneally for 14 days.
tuted phenyl piperazines (18 to 20). From the results Changes in body weight and organ weight are used as
Table II. Effects of arylpiperazines (1, 2 and 15 to 20) on the relevant anxiolytic parameters in the elevated plus maze
test
Spent time into
Compound
Number of entries Number of entries
Total number
of entries
% Number of entries
into open arms
open arms
into open arms
into closed arms
1a
2a
15
16
17
18
19
20
87.60 ± 21.40***
84.36 ± 23.68***
60.30 ± 27.12*
89.92 ± 18.76***
74.47 ± 16.84**
65.46 ± 23.05
98.23 ± 17.87***
73.78 ± 18.37
85.56 ± 26.98***
105.36 ± 22.46***
42.84 ± 17.51
6.29 ± 2.40
6.41 ± 2.12
3.56 ± 1.24
6.72 ± 2.23
5.12 ± 1.00
3.97 ± 1.12
7.47 ± 1.87
5.04 ± 2.52
5.35 ± 1.42
8.34 ± 1.56
3.03 ± 1.20
9.19 ± 4.58
11.80 ± 3.57
10.94 ± 4.62
9.18 ± 2.65
10.35 ± 3.91
11.12 ± 3.51
8.90 ± 4.08
11.20 ± 2.08
8.91 ± 4.20
10.13 ± 3.76
10.23 ± 3.40
15.48 ± 5.28
18.21 ± 4.80
14.50 ± 5.91
15.90 ± 3.83
15.47 ± 4.08
15.09 ± 3.83
16.37 ± 4.23
16.24 ± 4.46
14.26 ± 5.35
18.47 ± 3.89
13.26 ± 4.20
40.64 ± 8.01***
35.23 ± 16.27***
24.55 ± 13.21*
42.26 ± 17.40***
33.09 ± 12.43**
26.30 ± 11.23*
45.63 ± 14.75***
31.03 ± 16.41**
37.52 ± 12.38***
45.16 ± 14.7***
22.86 ± 10.28
Bus
Dzp
Veh
Data represent the mean ± S.E.M.; n=8. Bus, Buspirone; Dzp, Diazepam; Veh, Tween 80 (1%), DMSO (1%). ***
** < 0.01, *
< 0.05 compared with vehicle. aRef. (Khatri et al., 2009)
p < 0.001,
p
p

New Anxiolytic Carbamic Acid Ethyl Ester Derivatives
1151
Table III. Effect of compounds 16 and 19 intraperitoneally on haematological and serum biochemical parameters in
Wistar rats treated for 14 consecutive days
Parameters
Control (saline)
Compound 16 (3 mg/kg)
Compound 19 (3 mg/kg)
Haematological Parameters
1
RBC (
×
106 (mL)− )
8.0 ± 0.1
14.8 ± 0.2
42.7 ± 0.6
53.7 ± 1.34
18.6 ± 0.8
34.4 ± 0.2
7.31 ± 0.36
14.20 ± 0.5
39.50 ± 1.34
53.90 ± 2.65
19.40 ± 0.65
36.32 ± 1.7
8.23 ± 0.35
14.90 ± 0.60
44.50 ± 1.34
54.32 ± 2.10
18.23 ± 0.80
33.80 ± 3.10
Hemoglobin (g/dL)
Hematocrit (%)
MCV (fL)
MCH (rg)
MCHC (g/dL)
Serum Biochemical Parameters
ALT (U/L)
AST (U/L)
ALP (U/L)
BUN (mg/dL)
Creatinine (mg/dL)
Total Bilirubin (mg/dL)
38.88 ± 3.30
155.60 ± 14.85
145.65 ± 13.7
5.38 ± 0.59
0.74 ± 0.03
0.28 ± 0.02
40.21 ± 4.31
148.41 ± 14.7
146.08 ± 16.7
5.27 ± 0.460
0.67 ± 0.041
0.32 ± 0.024
46.26 ± 3.84*
152.50 ± 17.8ns
140.21 ± 14.3
5.43 ± 0.69
0.97 ± 0.047*
0.35 ± 0.016ns
The values are expressed as mean ± S.E.M. (n = 6 animals/group). *p < 0.05 as compared with vehicle, ns, nonsignificant.
RBC: red blood cell, MCV: mean corpuscular volume, MCH: mean corpuscular hemoglobin, MCHC: mean corpuscular
hemoglobin concentration and WBC: white blood cell, ALT: alanin aminotransferase, AST: aspartate aminotransferase,
ALP: alkaline phosphatase and BUN: blood urea nitrogen
an indicator of adverse effect of drugs and chemicals further designing new potential anxiolytic agents.
(Raza et al., 2002). In the study, treatment resulted in However, additional information related to the activity
small, and insignificant weight gains suggesting that and receptor specificity of these compounds would be
these compounds are non-toxic in experimental rats. helpful in the elucidation of the mechanism of their
The status of bone marrow activity and intravascular anti anxiety activity, which will be reported at a later
effects were monitored by haematological examinations. date.
There were no significant changes observed in various
haematological parameters such as Hb, RBC, Hemat-
ocrit etc. compared to the control group, which indicates
that compound 16 and 19 may not be toxic and does
ACKNOWLEDGEMENTS
The author M. Khatri thanks the Indian Council of
not affect circulating blood cells, haematopoiesis and Medical Research (ICMR), India, for the award of JRF
leukopoeisis. The increase of serum transaminase and SRF. The author M. Tiwari thanks the University
enzymes (ALT and AST) levels is a good indicator of of Delhi, India, for financial support. The authors wish
hepatocyte damage (Latha et al., 1998). Also, there to acknowledge editorial assistance provided by Dr.
were no significant changes observed in any liver func- Shampa Roy, Department of English, Miranda House,
tion parameters, such as SGPT, SGOT and ALP com- University of Delhi.
pared to the control group (Table III). Increase in these
parameters would have indicated hepatocyte damage.
The normal levels of serum creatinine and urea indi-
cate that these compounds did not interfere with renal
function and that renal integrity was preserved.
REFERENCES
Alam, A. H. M. K., Islam, R., Salam, K. A., Manir, M. M., Baki,
M. A., and Hossain, M. A., Toxicological studies of N-trans-
ferloyl-4-methyldopamine isolated from Achranthes ferru-
ginea. Pak. J. Biol. Sci., 9, 1052-1055 (2006).
Berman, H. M., Westbrook, J., Feng, Z., Gilliland, G., Bhat,
T. N., Weissig, H., Shindyalov, I. N., and Bourne, P. E.,
The Protein Data Bank. Nucleic Acids Res., 28, 235-242
(2002).
Bergmeyer, H. U. and Bernt, E., In Methods of Enzymatic
Analysis, Bergmeyer, H. U. (ed.), Academic Press, New
York, pp. 727-733, (1974).
In conclusion, compounds 16 and 19, fitted well in the
5-HT1A receptor site showed all the principle interac-
tions in docking analysis and exhibited a high binding
affinity (0.018 nM) in the radioligand binding assay.
These compounds significantly increased number of
entries as well as time spent in open arms, in the be-
havioural test EPM and revealed no toxicity in toxi-
cological analysis. All these data suggest that these
compounds might serve as candidates for the treat-
ment of anxiety disorders and as lead compounds for
Buhot, M. C., Martin, S., and Segu, L., Role of serotonin in

1152
M. Khatri et al.
memory impairment. Ann
.
Med., 32, 210-221 (2000).
Medina, R. A., Sallander, J., Benhamú, B., Porras, E., Campillo,
M., Pardo, L., and López-Rodríguez, M. L., Synthesis of New
Serotonin 5-HT7 Receptor Ligands. Determinants of 5-
HT7/5-HT1A Receptor Selectivity. J. Med. Chem., 52, 2384
(2009).
Costa-Silva, J. H., Lyra, M. M., Lima, C. R., Arruda, V. M.,
Araújo, A. V., Ribeiro e Ribeiro, A., Arruda, A. C., Fraga, M.
C., Lafayette, S. S., and Wanderley, A. G., A toxicological
evaluation of the effect of Carapa guianensis Aublet on
pregnancy in Wistar rats. J. Ethnopharmacol., 112, 122-
126 (2007).
MOE version 2009.10, Chemical Computing Group: Montreal,
Canada. http://www.chemcomp.com
Dawson, G. R. and Tricklebank, M. D., Use of the elevated
plus maze in the search for novel anxiolytic agents. Trends
Pharmacol. Sci., 16, 33-36 (1995).
Garattini, S., Bizzi, A., Caccia, S., Mennini, T., and Samanin,
R., Progress in assessing the role of serotonin in the
control of food intake. Clin. Neuropharmacol., 11 Suppl 1,
S8-S32 (1988).
Murakami, M. and Kouyama, T., Crystal structure of squid
rhodopsin. Nature, 453, 363-367 (2008).
Obniska, J., Ko³aczkowski, M., Bojarski, A. J., and Duszyñska,
B., Synthesis, anticonvulsant activity and 5-HT1A, 5-HT2A
receptor affinity of new N-[(4-arylpiperazin-1-yl)-alkyl]
derivatives of 2-azaspiro[4.4]nonane and [4.5]decane-1,3-
dione. Eur. J. Med. Chem., 41, 874-881 (2006).
Gozlan, H., Mestikawy, S., Pichat, L., Glowinski, J., and Hamon,
M., Identification of presynaptic serotonin autoreceptors
using a new ligand: 3H-PAT. Nature, 305, 140-142 (1983).
Hanson, M. A., Cherezov, V., Grifth, M. T., Roth, C. B., Jaakola,
V. P., Chien, E. Y. T., Velasquez, J., Kuhn, P., and Stevens,
R. C., A specic cholesterol binding site is established by the
2.8 Å structure of the human beta2-adrenergic receptor.
Structure, 16, 897-905 (2008).
Hoyer, D., Hannon, J. P., and Martin, G. R., Molecular,
pharmacological and functional diversity of 5-H Trecep-
tors. Pharmacol. Biochem. Behav., 71, 533-554 (2002).
Khatri, M., Rai, S. K., Alam, S., Vij, A., and Tiwari, M., Synthe-
sis and pharmacological evaluation of new arylpiperazines
N-[4-[4-(aryl) piperazine-1-yl]-phenyl]-amine derivatives:
putative role of 5-HT1A receptors. Bioorg. Med. Chem., 17,
1890-1897 (2009).
Okada, T., Sugihara, M., Bondar, A.-N., Elstner, M., Entel, P.,
and Buss, V., The retinal conformation and its environ-
ment in rhodopsin in light of a new 2.2 Å crystal structure.
J. Mol. Biol., 342, 571-583 (2004).
Rasmussen, S. G. F., Choi, H. J., Rosenbaum, D. M., Kobilka,
T. S., Thian, F. S., Edwards, P. C., Burghammer, M.,
Ratnala, V. R. P., Sanishvili, R., Fischetti, R. F., Schertler,
G. F. X., Weis, W. I., and Kobilka, B. K., Crystal structure
of the human beta2 adrenergic G-protein-coupled receptor.
Nature, 450, 383-387 (2007).
Raza, M., Al-Shabanath, O. A., El-Hadiyah, T. M., and Al-
Majed, A. A., Effect of prolonged vigabatrin treatment on
hematological and biochemical parameters in plasma, liver
and kidney of Swiss albino mice. Sci. Pharm., 70, 135-145
(2002).
Schlegel, J. R. and Peroutka, S. J., Nucleotide interactions with
5-HT1A binding sites directly labelled by [3H]-8-hydroxy-
2-(di-n-propylamino)tetralin[3H]-8OH-DPAT). Biochem.
Pharmacol., 35, 1943-1949 (1986).
The Organisation of Economic Co-operation and Develop-
ment (OECD), The OECD Guideline for Testing of Chemical:
407 Repeated Dose Oral Toxicity-Rodent: 28-day or 14-day
Study, OECD, Paris, pp. 1-7, (2001a).
The Organisation of Economic Co-operation and Develop-
ment (OECD), The OECD Guideline for Testing of Chemical:
420 Acute Oral Toxicity, OECD, Paris, pp. 1-14, (2001b).
Walf, A. A. and Frye, C. A., The use of the elevated plus maze
as an assay of anxiety-related behavior in rodents. Nat.
Protoc., 2, 322-328 (2007).
Kiss, R., Noszál, B., Rácz, A., Falus, A., Eros, D., and Keseru,
G. M., Binding mode analysis and enrichment studies on
homology models of the human histamine H4 receptor.
Eur. J. Med. Chem., 43, 1059-1070 (2008).
Kowalski, P., Jas´kowska, J., Bojarski, A. J., Duszyn´ska, B.,
Bucki, A., and Kolaczkowski, M., Evaluation of 1-arylpi-
´
perazine derivative of hydroxybenzamides as 5-HT1A and
5-HT7 serotonin receptor ligands: An experimental and
molecular modeling approach. J. Heterocycl. Chem., 48,
192-198 (2011).
Latha, R. M., Geentha, T., and Varalakshmi, P., Effect of
Vernonia cinerea Less. flower extract in adjuvant-induced
arthritis. Gen. Pharmacol., 39, 601-606 (1998).
Liapakis, G., Ballesteros, J. A., Papachristou, S., Chan, W. C.,
Chen, X., and Javitch, J. A., The forgotten Serine: A critical
role for Ser-2035.42 in ligand binding to and activation of
the beta 2 adrenergic receptor. J. Biol. Chem., 275, 37779-
37788 (2000).
Lopez-Rodriguez, M. L., Ayala, D., Benhamu, B., Morcillo,
M. J., and Viso, A., Arylpiperazine derivatives acting at 5-
HT1A receptors. Curr. Med. Chem., 9, 443-469 (2002).
Warne, T., Serrano-Vega, M. J., Baker, J. G., Moukhametzianov,
R., Edwards, P. C., Henderson, R., Leslie, A. G. W., Tate,
C. G., and Schertler, G. F. X., Structure of a beta1-adrenergic
G-protein-coupled receptor. Nature, 454, 486-491 (2008).
Waynforth, B. H., Injection Techniques: Experimental and
Surgical Techniques in the Rat, Academic Press, London,
pp. 3-61, (1980).