Design, synthesis, and preliminary in vitro and in vivo pharmacological evaluation of 2-{4-[4-(2,5-disubstituted thiazol-4-yl)phenylethyl]piperazin-1- yl}-1,8-naphthyridine-3-carbonitriles as atypical antipsychotic agents

Article abstract of DOI:10.3109/14756366.2010.537658

A series of 2-{4-[4-(2,5-disubstituted thiazolyl)phenylethyl] piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles were synthesized in an effort to prepare novel atypical antipsychotic agents. The compounds were synthesized either by microwave irradiation technique or by conventional synthesis and were characterized by spectral data (IR, ¹H NMR, and MS) and the purity was ascertained by microanalysis. The D₂ and 5-HT_2A affinity of the synthesized compounds was screened in vitro by radioligand displacement assays on membrane homogenates isolated from rat striatum and rat cortex, respectively. Furthermore, all the synthesized compounds were screened for their in vivo pharmacological activity in Swiss albino mice. The D ₂ antagonism studies were performed using climbing mouse assay model and 5-HT_2A antagonism studies were performed using quipazine-induced head twitches in mice. It was observed that none of the new chemical entities exhibited catalepsy and 10f is the most active among the synthesized compounds with 5-HT_2A/D₂ ratio of 1.1286 although the standard drug risperidone exhibited 5-HT_2A/D₂ ratio of 1.0989.

Full text of DOI:10.3109/14756366.2010.537658

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(4): 561–568
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.537658
ORIGINAL ARTICLE
Design, synthesis, and preliminary in vitro and in vivo
pharmacological evaluation of 2-{4-[4-(2,5-disubstituted
thiazol-4-yl)phenylethyl]piperazin-1-yl}-1,8-naphthyridine-3-
carbonitriles as atypical antipsychotic agents
Kondapalli Venkata Gowri Chandra Sekhar1, Vajja Samabasiva Rao1, Winnie Deuther-Conrad2,
Aravalli Satish Reddy1, Peter Brust2, and Mutyala Murali Krishna Kumar1
1Chemistry Group, Birla Institute of Technology & Science, Pilani, Rajasthan, India, and 2Forschungszentrum Dresden–
Rossendorf, Interdisciplinary Isotope Research, Institute of Radiopharmacy, Leipzig, Germany
Abstract
A series of 2-{4-[4-(2,5-disubstituted thiazolyl)phenylethyl] piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles were
synthesized in an effort to prepare novel atypical antipsychotic agents. The compounds were synthesized either by
1
microwave irradiation technique or by conventional synthesis and were characterized by spectral data (IR, H NMR,
and MS) and the purity was ascertained by microanalysis. The D2 and 5-HT2A affinity of the synthesized compounds
was screened in vitro by radioligand displacement assays on membrane homogenates isolated from rat striatum and
rat cortex, respectively. Furthermore, all the synthesized compounds were screened for their in vivo pharmacological
activity in Swiss albino mice. The D antagonism studies were performed using climbing mouse assay model and
5-HT2A antagonism studies were pe2rformed using quipazine-induced head twitches in mice. It was observed that
none of the new chemical entities exhibited catalepsy and 10f is the most active among the synthesized compounds
with 5-HT2A/D2 ratio of 1.1286 although the standard drug risperidone exhibited 5-HT2A/D2 ratio of 1.0989.
Keywords: Schizophrenia, atypical antipsychotics, D2 antagonists, 5-HT2A antagonists
Introduction
extrapyramidal syndrome (EPS), tardive dyskinesia, and
Schizophrenia is a devastating mental disorder with
high morbidity and mortality, affecting about 1% of the
population worldwide.1 Symptoms of schizophrenia
emerge during adolescence and are classified into posi-
tive (hallucinations, delusions, and severe thought disor-
ganization), negative (alogia, anhedonia, avolition, and
flattened affect), and cognitive symptoms (slow think-
ing, poor concentration, poor memory, and difficulty in
understanding).2
Classical antipsychotics, which act as D2 antagonists
in the limbic forebrain, are useful for the treatment of
the positive symptoms, but failed to manage the nega-
tive symptoms of schizophrenia3 and their use is fre-
quently associated with serious side effects, such as
hyperprolactinaemia.4 is led to the development of
improved “atypical” antipsychotic agents, like the proto-
type antipsychotic drug clozapine, which are in general
effective for positive, negative, and cognitive symptoms
of schizophrenia. ese drugs are more efficacious than
classical antipsychotics in treatment-refractory patients
and have a low incidence of EPS.5,6
Over the past two decades, much attention regarding
the treatment for schizophrenia has focused on this new
class of antipsychotic medications and led to the prolifer-
ation of “atypical” antipsychotics, including risperidone,
olanzapine, quetiapine, ziprasidone, sertindole, iloperi-
done, aripiprazole, amisulpride, and zotepine.7 Meltzer
et al. suggested that in the efficacy of clozapine and other
Address for Correspondence: Kondapalli Venkata Gowri Chandra Sekhar, Chemistry Group, Birla Institute of Technology & Science, Pilani–
333 031, Rajasthan, India. E-mail: kvgcs@yahoo.com
(Received 15 October 2009; revised 08 October 2010; accepted 02 November 2010)
561

562 K.V.G.C. Sekhar et al.
atypical antipsychotics such as risperidone and olanzap-
ine the most important factor is their relative affinities
for the D2 and 5-HT2A receptors.8–10 Antagonism at 5-HT2A
and D2 receptors by these molecules is responsible for
alleviating the negative and positive symptoms of the
disorder, respectively.11,12 But these compounds are also
not completely devoid of side effects. Side effects caused
by “atypical antipsychotics” are a result of their signifi-
cant binding affinity to numerous receptors other than
required for atypical antipsychotic activity. Side effects
associated with these drugs include weight gain (sero-
tonergic 5-HT2C and histaminic H1 receptors blockade),13
postural or orthostatic hypotension, sedation, dizzi-
ness (α1-adrenergic blockade), somnolence (histaminic
H1 receptor blockade), seizures (muscarinic receptor
blockade),14 new-onset type 2 diabetes mellitus,15 hyper-
lipidaemia, atropine-like side effects such as dry mouth,
constipation, urinary retention (muscarinic M1 receptor
blockade), cardiac ventricular arrhythmias (prolonga-
tion of QT interval due to the blockade of Ikr channels),
myocarditis, insomnia, headache, and other possible
secondary cardiovascular complications.16
this strategy, the current marketed drug ziprasidone was
developed.19 We adopted this strategy and employed
2-(piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile,
which has affinity towards serotonin receptors21 as one
portion of the molecule and chloroethylphenylthiazoles
was selected as other portion as Pfizer group has come
up with potent atypical antipsychotics using this hetero-
cyclic system.22
We synthesized compounds in which 2- and
5-substituted chloroethylphenylthiazoles (1–7) have
been incorporated at the piperazinyl nitrogen atom of
2-(piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile
looking for 5-HT2A and D2 antagonism. Seven new com-
pounds have been synthesized and the synthetic schemes
are illustrated in Schemes 1–3. Scheme 1 illustrates the
synthesis of 2- and 5-substituted chloroethylphenylthi-
azoles (1–7), whereas Scheme 2 illustrates the synthesis
of 2-(piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile
(9) and Scheme 3 depicts the coupling of these two frag-
ments to yield the title compounds.
Experimental
e rationale for development of the antipsychotic
drugs recently introduced, and currently under devel-
opment is predominantly based on dopamine and
serotonin hypotheses of schizophrenia. In continuation
of our quest for novel atypical antipsychotics,17,18 we fol-
lowed the strategy employed by Pfizer group,19 synthe-
sized the title compounds, and evaluated for the atypical
antipsychotic activity in animal models.
Materials
e starting compounds, 2- and 5-substituted chloroeth-
ylphenylthiazoles (1–7) and 2-(piperazin-1-yl)-1,8-naph-
thyridine-3-carbonitriles (9), were prepared according
to the reported method.21,22 All other starting materials
and solvents were obtained from commercially available
sources and used without additional purification.
e strategy of Ariens has been employed for the
design of the compounds.20 Ariens strategy, in brief,
involves modification of the structure of a receptor
agonist, in this case dopamine, with a large lipophilic
group on the amino position, which binds to the acces-
sory binding site adjacent to the agonist binding site
and transforms the agonist into an antagonist. Using
Methods
Melting points were determined in open capillaries using
Büchi 530 melting point apparatus and are uncorrected.
e reactions were monitored and the purity of the com-
pounds checked by ascending thin-layer chromatogra-
phy (TLC) on silica gel-coated aluminium plates (Merck
Cl
ClCH(R)COCl, DCM
Cl
R
AlCl3 (0–5°C)
Cl
R = H, CH3
O
KSCN, reflux
ZCSNH2, reflux
Z = NH2, CH3, NHCH3
Cl
Cl
N
OH
S
R
N
Z
S
R
4
1, 2, 3, 5, 6, 7
Scheme 1. Synthesis of chloroethylphenylthiazoles (1–7).
Journal of Enzyme Inhibition and Medicinal Chemistry

2-{4-[4-(2,5-Disubstituted thiazol-4-yl)phenylethyl]piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles 563
60 F254, 0.25 mm) using mixture of chloroform and
with water, and recrystallized in DMF–water mixture to
afford the pure final compounds 10a–g.
methanol and the spots were visualized under ultraviolet
light at 254 and 366 nm. e microwave-assisted proce-
dures were carried out in a LG microwave oven specially
designed for organic synthesis operating at a maximum
power of 1000 W. Infrared (IR) spectra were recorded in
KBr pellets on Schimadzu IR Prestige-21 FT-IR spectro-
photometer (cm−1). 1H NMR spectra were obtained from
Bruker DRX300 spectrometer using tetramethylsilane as
internal standard [chemical shifts (δ) in parts per million
(ppm)], mass spectra on a VG-70-S mass spectrometer,
and elemental analysis on a Perkin Elmer 2400 CHN
elemental analyzer.
2-{4-[4-(2-Aminothiazol-4-yl)phenethyl]piperazin-
1-yl}-1,8-naphthyridine-3-carbonitrile (10a). Yield:
77% (0.17 g); m.p.: 260–262ºC. 1H NMR (CDCl3) (δ)
ppm: 2.65–2.69 (m, 4H, (CH2)2); 2.76 (t, 4H, J = 4.8 Hz,
N4(CH2)2); 3.17 (t, 4H, J = 4.8 Hz, N1(CH2)2); 3.87 (s, 2H,
NH2); 6.92 (s, 1H, thiazole); 7.15–7.29 (m, 4H, Ph); 7.34
(dd, 1H, J = 8.2 Hz, J = 1.4 Hz, C6-H; 1,8-naphthyridine);
7.84 (d, 1H, J = 5.1 Hz, C5-H; 1,8-naphthyridine); 8.35
(s, 1H, C4-H; 1,8-naphthyridine); 9.09 (d, 1H, J = 5.1 Hz,
C7-H; 1,8-naphthyridine). IR (KBr) cm−1: 3428 and 3400
(NH2 stretch); 3037, 3018 (aromatic C-H stretch); 2955,
2890 (aliphatic C-H stretch); 2243 (C-N stretch); 1651
(C=N ring stretch); 1602 (aromatic C=C stretch); 1264
(aliphatic C-N stretch); 808 (para disubstituted ben-
zene); 706 (C-S-C stretch). FAB-MS m/z: 441.1710 calcu-
lated: 441.1712; Anal. calculated for C24H23N7S: C 65.28,
H 5.25, N 22.21, S 7.26; found: C 65.26, H 5.21, N 22.17,
S 7.19.
2-Hydroxy-1,8-naphthyridine-3-carbonitrile
(7a):
Literature procedure21 was used to synthesize 7a. Yield:
90% (1.54 g); m.p.: > 300ºC (Lit. m.p. > 300ºC).21
2-Chloro-1,8-naphthyridine-3-carbonitrile
(8):
Compound 8 was prepared according to the reported
method.21 Yield: 79% (1.5 g); m.p.: > 300ºC (Lit.
m.p. > 300ºC).21
2-Piperazin-1-yl-1,8-naphthyridine-3-carbonitrile
(9): Compound 9 was prepared using the literature pro-
tocol.21 Yield: 74% (0.90 g); m.p.: 225–226ºC (Lit. m.p.
226–227ºC).21
Cl
CN
N
Z
NH
N
N
N
S
1–7
General procedure for 2-{4-[4-(2,5-disubstituted
thiazolyl)phenylethyl] piperazin-1-yl}-1,8-
naphthyridine-3-carbonitriles (10a–g)
R
9
Na2CO3, Kl
DMF, reflux
e procedure described by Lowe and coworkers22 was
adapted for this preparation. In a 10-mL round bottom
flask equipped with a reflux condenser and N2 inlet were
placed equimolar amounts (0.05 mM) of 2-piperazin-1-
yl-1,8-naphthyridine-3-carbonitrile (9) and respective
chloroethylphenylthiazoles (1–7), 0.1174 g (1.11 mM) of
sodium carbonate, and 2 mg of potassium iodide in 2 mL
of dimethylformamide (DMF). e reaction mixture
was refluxed for 2 days. After completion of reaction, as
indicated by TLC (9:1 chloroform:methanol as mobile
phase), the cooled reaction mixture was poured into ice–
water mixture and the precipitate was filtered, washed
CN
N
N
N
N
N
Z = NH2, CH3, NHCH3, OH
R = H, CH3
Z
S
R
10 a–g
Scheme 3. Synthesis of 2-{4-[4-(2,5-disubstituted thiazolyl)
phenylethyl]piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles
(10a–g).
CN
CN
CN
Cl
CHO
Piperidine
Solid state
POCl3
Reflux
+
N
N
OH
OC2H5
N
N
N
NH2
O
7a
8
NH
K2CO3/DMF
MWI
HN
CN
N
NH
N
N
9
Scheme 2. Synthesis of 2-(piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile (9).
© 2011 Informa UK, Ltd.

564 K.V.G.C. Sekhar et al.
2-{4-[4-(2-(Methylamino)thiazol-4-yl)phenethyl]
J=1.4Hz, C6-H; 1,8-naphthyridine); 7.91 (d, 1H, J=5.1Hz,
C5-H; 1,8-naphthyridine); 8.28 (s, 1H, C4-H; 1,8-naphthy-
ridine); 9.14 (d, 1H, J=5.1Hz, C7-H; 1,8-naphthyridine). IR
(Neat) cm−1: 3070, 3045 (aromatic C-H stretch); 2980, 2955
(aliphatic C-H stretch); 2249 (C-N stretch); 1648 (C=N
ring stretch); 1620, 1602 (aromatic C=C stretch); 1256
(aliphatic C-N stretch); 820 (para disubstituted benzene);
710 (C-S-C stretch). FAB-MS m/z: 454.1920, calculated:
454.1926; Anal. calculated for C26H26N6S: C 68.69, H 5.76,
N 18.49, S 7.05; found: C 68.62, H 5.71, N 18.46, S 7.02.
2-{4-[4-(5-Methyl-2-(methylamino)thiazol-4-yl)
phenethyl]piperazin-1-yl}-1,8-naphthyridine-3-carbo-
nitrile (10f). Yield: 41% (0.16 g); m.p.: 198–200°C. 1H NMR
(CDCl3) (δ) ppm: 2.36 (s, 3H,CH3); 2.51 (s, 3H, NHCH3);
2.62 (t, 4H, J= 4.9 Hz, N4(CH2)2); 2.66–2.69 (m, 4H, (CH2)2);
3.18 (t, 4H, J= 4.9 Hz, N1(CH2)2); 4.21 (s, 1H,NHCH3);
7.15–7.40 (m, 4H, Ph); 7.45 (dd, 1H, J= 8.4 Hz, J= 1.2 Hz,
C6-H; 1,8-naphthyridine); 7.87 (d, 1H, J= 5.3 Hz, C5-H;
1,8-naphthyridine); 8.47 (s, 1H, C4-H; 1,8-naphthyri-
dine); 9.09 (d, 1H, J= 5.3 Hz, C7-H; 1,8-naphthyridine). IR
(KBr) cm−1: 3400 (NH stretch); 3025, 3010 (aromatic C-H
stretch); 2917, 2895 (aliphatic C-H stretch); 2245 (C-N
stretch); 1652 (C=N ring stretch); 1618, 1586 (aromatic
C=C stretch); 1256 (aliphatic C-N stretch); 828 (para
disubstituted benzene); 705 (C-S-C stretch). FAB-MS
m/z: 469.2138, calculated: 469.2142; Anal. calculated for
C26H27N7S: C 66.50, H 5.80, N 20.88, S 6.83; found: C 66.43,
H 5.69, N 20.79, S 6.86.
2-{4-[4-(2-Methylthiazol-4-yl)phenethyl]piperazin-
1-yl}-1,8-naphthyridine-3-carbonitrile (10g). Yield: 57%
(0.13g, oil). 1H NMR (CDCl3) (δ) ppm: 2.59 (t, 4H, J=4.9Hz,
N4(CH2)2); 2.65-2.71 (m, 4H, (CH2)2); 2.74 (s, 3H, CH3); 3.17
(t, 4H, J=4.9Hz, N1(CH2)2); 7.11–7.28 (m, 4H, Ph); 7.43 (s,
1H, thiazole); 7.51 (dd, 1H, J=8.4Hz, J=1.6Hz, C6-H; 1,8-
naphthyridine); 7.82 (d, 1H, J=5.2Hz, C5-H; 1,8-naphthy-
ridine); 8.32 (s, 1H, C4-H; 1,8-naphthyridine); 9.21 (d, 1H,
J=5.2Hz, C7-H; 1,8-naphthyridine). IR (Neat) cm−1: 3065,
3015 (aromatic C-H stretch); 2976, 2952 (aliphatic C-H
stretch); 2247 (C-N stretch); 1638 (C=N ring stretch); 1616,
1591 (aromatic C=C stretch); 1261 (aliphatic C-N stretch);
827 (para disubstituted benzene); 708 (C-S-C stretch).
FAB-MS m/z: 440.1831, calculated: 440.1836; Anal. calcu-
lated for C25H24N6S: C 68.16, H 5.49, N 19.08, S 7.28; found:
C 68.12, H 5.48, N 18.99, S 7.26.
piperazin-1-yl}-1,8-naphthyridine-3-carbonitrile (10b).
1
Yield: 66% (0.15 g); m.p.: 148–150ºC. H NMR (CDCl3)
(δ) ppm: 2.47 (s, 3H, NHCH3); 2.68-2.71 (m, 4H, (CH2)2);
2.78 (t, 4H, J= 4.8 Hz, N4(CH2)2); 3.17 (t, 4H, J= 4.8 Hz,
N1(CH2)2); 4.08 (s, 1H, NHCH3); 6.79 (s, 1H, thiazole);
7.18–7.34 (m, 4H, Ph); 7.46 (dd, 1H, J= 8.4 Hz, J= 1.6 Hz,
C6-H; 1,8-naphthyridine); 7.87 (d, 1H, J= 5.2 Hz, C5-H;
1,8-naphthyridine); 8.44 (s, 1H, C4-H; 1,8-naphthyri-
dine); 9.11 (d, 1H, J= 5.2 Hz, C7-H; 1,8-naphthyridine). IR
(KBr) cm−1: 3400 (NH stretch); 3030, 3005 (aromatic C-H
stretch); 2985, 2875 (aliphatic C-H stretch); 2250 (C-N
stretch); 1640 (C=N ring stretch); 1258 (aliphatic C-N
stretch); 1610, 1590 (aromatic C=C stretch); 815 (para
disubstituted benzene); 706 (C-S-C stretch). FAB-MS
m/z: 455.1670, calculated: 455.1677; Anal. calculated for
C25H25N7S: C 65.91, H 5.33, N 21.52, S 7.04; found: C 65.81,
H 5.31, N 21.47, S 7.01.
2-{4-[4-(2-Amino-5-methylthiazol-4-yl)phenethyl]
piperazin-1-yl}-1,8-naphthyridine-3-carbonitrile (10c).
Yield: 81% (0.184 g); m.p.: 210–212ºC. 1H NMR (CDCl3) (δ)
ppm: 2.29 (s, 3H, CH3); 2.57 (t, 4H, J= 4.8 Hz, N4(CH2)2);
2.61–2.66 (m, 4H, (CH2)2); 3.21 (t, 4H, J= 4.8 Hz, N1(CH2)2);
3.92 (s, 2H, NH2); 7.19–7.31 (m,4H, Ph); 7.37 (dd, 1H,
J= 8.3 Hz, J= 1.5 Hz, C6-H; 1,8-naphthyridine); 7.89 (d, 1H,
J= 5.1 Hz, C5-H; 1,8-naphthyridine); 8.52 (s, 1H, C4-H; 1,8-
naphthyridine); 9.31 (d, 1H, J= 5.1 Hz, C7-H; 1,8-naph-
thyridine). IR (KBr) cm−1: 3430 and 3415 (NH2 stretch);
3045, 3020 (aromatic C-H stretch); 2980, 2875 (aliphatic
C-H stretch); 2240 (C-N stretch); 1637 (C=N ring stretch);
1608 (aromatic C=C stretch); 1255 (aliphatic C-N stretch);
825 (para disubstituted benzene); 712 (C-S-C stretch).
FAB-MS m/z: 455.2111, calculated: 455.2114; Anal. calcu-
lated for C25H25N7S: C 65.91, H 5.33, N 21.52, S 7.04; found:
C 65.94, H 5.31, N 21.47, S 6.98.
2-{4-[4-(2-Hydroxythiazol-4-yl)phenethyl]piperazin-
1-yl}-1,8-naphthyridine-3-carbonitrile (10d). Yield: 76%
(0.167 g); m.p.: 232–234ºC. 1H NMR (CDCl3) (δ) ppm: 2.62
(t, 4H, J= 4.8 Hz, N4(CH2)2); 2.68–2.79 (m, 4H, (CH2)2);
3.19 (t, 4H, J= 4.8 Hz, N1(CH2)2); 5.42 (br s, 1H, OH);
7.04–7.19 (m, 4H, Ph); 7.23 (s, 1H, thiazole); 7.43 (dd, 1H,
J= 8.0 Hz, J= 1.4 Hz, C6-H; 1,8-naphthyridine); 7.87 (d,
1H, J= 5.4 Hz, C5-H; 1,8-naphthyridine); 8.37 (s, 1H, C4-H;
1,8-naphthyridine); 9.14 (d, 1H, J= 5.4 Hz, C7-H; 1,8-
naphthyridine). IR (KBr) cm−1: 3600 (OH stretch, broad);
3030, 3010 (aromatic C-H stretch); 2960, 2925 (aliphatic
C-H stretch); 2241 (C-N stretch); 1645 (C=N ring stretch);
1587 (aromatic C=C stretch); 1260 (aliphatic C-N stretch);
818 (para disubstituted benzene); 705 (C-S-C stretch).
FAB-MS m/z: 442.1413, calculated: 442.1415; Anal. cal-
culated for C24H22N6OS: C 65.14, H 5.01, N 18.99, S 7.25;
found: C 65.10, H 4.99, N 18.91, S 7.19.
In vitro radioligand displacement studies
e affinity and specificity of the NCEs were estimated
in radioligand displacement studies on rat 5-HT2A and D2
receptors obtained from rat cortical (5-HT receptors) and
striatal (D2 receptors) membrane preparations. Test com-
pounds were dissolved in dimethyl sulphoxide (DMSO)
(10 mM stock solution), aliquoted, and stored at −25°C.
For competitive binding experiments, the membrane
preparations were thawed, diluted with assay buffer,
50 mM Tris–HCl, pH 7.4, and washed twice. e particu-
lar receptor preparation was incubated with the respec-
tive radioligand (5-HT2A: [3H]ketanserine, Perkin Elmer
Life and Analytical Sciences, Inc., Rodgau - Jügesheim,
2-{4-[4-(2,5-Dimethylthiazol-4-yl)phenethyl]piper-
azin-1-yl}-1,8-naphthyridine-3-carbonitrile (10e). Yield:
1
57% (0.13g, oil). H NMR (CDCl3) (δ) ppm: 2.31 (s, 3H,
5-CH3); 2.64 (t, 4H, J=4.9Hz, N4(CH2)2); 2.69–2.75 (m,
4H, (CH2)2); 2.79 (s, 3H, 2-CH3); 3.16 (t, 4H, J=4.9Hz,
N1(CH2)2); 7.07-7.26 (m, 4H, Ph); 7.45 (dd, 1H, J=8.2Hz,
Journal of Enzyme Inhibition and Medicinal Chemistry

2-{4-[4-(2,5-Disubstituted thiazol-4-yl)phenylethyl]piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles 565
Germany and GE Healthcare (former Amersham): GE
“2”, when two feet were placed on the cage floor
“3”, when one foot was placed on the cage floor
“4”, when all the four feet were off the cage floor.
Healthcare Europe GmbH, Freiburg, Germany Aspec
:
:
67 Ci/mmol; D2: [3H]spiperone, Amersham GE, Aspec
101 Ci/mmol) and up to six concentrations of the NCEs.
Dilutions of the NCEs were made with assay buffer.
Nonspecific binding of the radioligands was determined
with 100 µM mianserin for the 5-HT2A ligand [3H]ketan-
serine and 100 µM haloperidol for D2 ligand [3H]spiper-
one. To block the 5-HT affinity of the D2 radioligand [3H]
spiperone, 10 µM ketanserin was added to the respective
assays. e assay samples were incubated at ambient
temperature for 60 min (D2) or 90 min (5-HT2A), rapidly
filtered through Whatman GF/B glass-fibre filters, and
washed four times with ice-cold assay buffer. Filter-
bound radioactivity was determined by liquid scintil-
lation counting. All test compounds were assayed in at
least three independent experiments. e IC50 values
were estimated using iterative nonlinear curve fitting.
e percentage inhibition or reversal of climbing behav-
iour of apomorphine hydrochloride was calculated by
the difference from the score of treated subjects to the
score of control animals and referring it to score of con-
trol group set to 100%. Haloperidol (1.0 mg/kg, i.p.) was
used as standard as it completely inhibited the climbing
induced by apomorphine.
5-HT2A receptor antagonism studies (quipazine-induced head
twitches)
Quipazine maleate (5 mg/kg) solution (as per the base
calculations) was prepared in triple distilled water con-
taining 0.1% w/v sodium metabisulphite and was injected
i.p. 30 min before testing. Risperidone (0.6 mg/kg) and
NCEs (10 mg/kg) were prepared as suspension in 0.25%
w/v sodium carboxymethylcellulose in distilled water
and were also injected i.p. 30 min before testing.
In vivo pharmacological studies
e Institutional Animal Ethics Committee of the Birla
Institute of Technology and Science, Pilani, Rajasthan,
India approved experimentation on animals (Protocol
No. IAEC/RES/11/2). Swiss albino mice (25–30 g) of either
sex obtained from Hissar Agricultural University, Hissar,
Haryana, India were used for the pharmacological stud-
ies. Pharmacokinetic studies showed that the exposure
at 10 mg/kg dose was similar to the exposure of several
atypical antipsychotics at therapeutically relevant doses,
and hence this dose was chosen to carry out the in vivo
pharmacological tests.
Inhibition or reversal of quipazine-induced head
twitches in mice by the test molecule is an indication
of central serotonergic 5-HT2A receptor antagonism.24
During the experimentation, mice were placed indi-
vidually in separate plastic translucent cages, measur-
ing 20 × 15 × 15 cm3. ey were placed in the above cages
30 min for adaptation before the experiment. Groups of
mice were administered i.p. with either the test molecule
(10 mg/kg) or vehicle or risperidone 1 h prior to the qui-
pazine maleate challenge (5 mg/kg, i.p.). Risperidone
(0.6 mg/kg, i.p.) was used as standard as it completely
inhibits quipazine-induced head twitches in mice. e
head twitches were then counted between 30 and 40 min.
e percentage inhibition or reversal of head twitches
was calculated by the difference from the count of treated
subjects to the count of control animals and referring it to
count of control group set to 100%.
D2 receptor antagonism studies in nigrostriatal pathway
(climbing mouse assay)
Apomorphine hydrochloride (1 mg/kg) solution (as per
the base calculations) was prepared in triple distilled
water containing 0.1% w/v sodium metabisulphite and
was injected s.c. 1 h before testing.
Risperidone (0.6 mg/kg) and new chemical entities
(NCEs) (10 mg/kg) were prepared as suspension in 0.25%
w/v sodium carboxymethylcellulose in triple distilled
water and were injected i.p. 30 min before testing.
Inhibition or reversal of apomorphine-induced
cage-climbing behaviour in mice by a test molecule is
an indication of mesolimbic dopaminergic D2 receptor
antagonism.21,23 During the experimentation, mice were
placed individually in separate aluminium cages, mea-
suring 20 × 15 × 15 cm3, with walls lined with 1cm2 alu-
minium wire mesh (diameter 2 mm). ey were placed in
the above cages 30 min for adaptation before the experi-
ment. Groups of mice were administered with either the
test molecule (10 mg/kg) or vehicle or risperidone i.p. 1h
prior to the apomorphine challenge (1 mg/kg, s.c.). Mice
were then observed for the climbing behaviour after 10,
20, and 30 min and the scoring was done as below.
D2 receptor antagonism studies in nigrostriatal pathway
(catalepsy test)
NCEs (10 mg/kg) were prepared as suspension in 0.25%
w/v sodium carboxymethylcellulose in triple distilled
water and were injected i.p. 30 min before testing.
Induction of catalepsy by the test molecules is an
indication of antagonism at nigrostriatal dopaminergic
D2 receptors leading to EPS.25,26 During the experimenta-
tion, mice were placed individually in separate plastic
translucent cages, measuring 20 × 15 × 15 cm3. ey were
placed in the above cages 30 min for adaptation before
the experiment. Groups of mice were administered i.p.
with either the test molecule (10 mg/kg) or vehicle. e
mice were then tested for catalepsy by placing both the
front paws on a 4-cm high wooden block (6 × 4× 4 cm3)
and measuring the time taken for it to come back to the
normal posture. e scoring was done in accordance
with literature.26 If the animal maintained the imposed
posture for at least 20 sec, then it was said to be cataleptic
“0”, when all the four feet were placed on the cage floor
“1”, when three feet were placed on the cage floor
© 2011 Informa UK, Ltd.

566 K.V.G.C. Sekhar et al.
and given one point. For every further 20 sec it continued
to maintain the imposed posture, an extra point was
given, thus the animal was given a score of 2 points if it
maintained the posture for 40 sec, 3 points for 60 sec, and
so on. e mice were tested for cataleptic behaviour 1.0,
2.0, 3.0, 4.0, and 5.0 h after treatment with the test mole-
cule. Average cataleptic times and scores were calculated
at each time of measurement of cataleptic behaviour per
molecule. e maximum of all average cataleptic scores/
times were noted per molecule and then conclusions
were drawn with respect to which test molecule is cata-
leptic and the degree of catalepsy.
[3H]ketanserine or the D2 ligand [3H]spiperone in vitro
(Table 1), in vivo pharmacological data clearly indicate
an atypical antipsychotic efficacy of the NCEs. Because
the compounds obviously possess efficacy at micromo-
lar concentrations in vivo, we assume that the observed
slight increase in specific binding of the two radioligands
in in vitro assays reflects cooperativity, that is, the bind-
ing of one ligand to one binding site changes the affinity
of another ligand, because G-protein-coupled receptors
such as 5-HT2A and D2 are characterized by this phenom-
enon. In particular, this has been shown for the binding
of [3H]ketanserine in rabbit retina23 and in [3H]spiper-
one in cell culture.24,25 Furthermore, the inconsistency
between in vitro and in vivo data might be attributed to
the difference in the extent of uptake or distribution of
NCEs into the target cells. e difference in the uptake
or distribution into the cells could be because of the
mechanism involved in the transportation of the NCEs
such as carrier-mediated transport in the in vivo studies,
although such mechanism might be absent in case of in
vitro studies.
e effect of pretreatment with 10 mg/kg dose of the
test compounds on apomorphine (0.5 mg/kg s.c.)-in-
duced cage-climbing behaviour was studied by the liter-
ature method.26 Haloperidol (1.0 mg/kg i.p.) was used as
standard as it completely inhibited the climbing induced
by apomorphine. Inhibition or reversal of quipazine-in-
duced head twitches in mice by the test molecule (10 mg/
kg dose) is an indication of central serotonergic 5-HT2A
receptor antagonism and this behaviour was studied by
the literature method.27 Risperidone (0.6 mg/kg i.p.) was
used as standard as it completely inhibits quipazine-
induced head twitches in mice. Cataleptic effect of NCEs
was evaluated by the literature method28 and scoring was
done as per literature.29
Results and discussion
Substituted chloroethylphenylthiazoles were prepared
as per the literature protocol with modifications in
some steps.22 2-(Piperazin-1-yl)-1,8-naphthyridine-3-
carbonitrile was also prepared as defined using micro-
wave irradiation technique.21 Equimolar amounts of
2-(piperazin-1-yl)-1,8-naphthyridine-3-carbonitrile (9)
and 2- and 5-substituted chloroethylphenylthiazoles (17)
along with 2.125 equivalents of anhydrous Na2CO3 and
catalytic amount of KI (2 mg) in DMF as solvent when
refluxed for 48 h afforded the title compounds. All the syn-
thesized compounds were characterized by spectral (IR,
1H NMR, and MS) and elemental analysis data. IR spectral
analysis of the final compounds (10a–g) showed strong
peaks at ~ 2245, ~ 1650, and ~ 825 cm−1 due to C≡N, C=N,
and para disubstituted benzene functions, respectively.
1
In H NMR spectra, methylene protons (cyclic) adjacent
to N1 nitrogen of piperazine showed triplet in the range of
δ 3.16–3.22, whereas methylene protons (cyclic) adjacent
to N4 nitrogen of piperazine showed triplet in the range of
δ 2.55–2.81. e final compounds showed the following
1H NMR signals for 1,8-naphthyridinyl moiety; C4-H: δ
~ 8.28–8.52 (s), C5-H: δ ~ 7.80–7.96 (d), C6-H: δ ~ 7.30–7.55
(dd), C7-H: δ ~ 9.07–9.34 (d). PMR signal corresponding
to four protons of the ethyl linker was observed at δ 2.65–
2.79 as multiplet. Elemental (CHNS) analysis indicated
that the calculated and observed values were within the
acceptable limits ( 0.4%).
e initial trial compound, 10a, mimicking both the
position and acidity of one of the phenol groups in dop-
amine, gratifyingly possesses the desired antagonistic
activity at D2 receptor as it exhibited ≥60% inhibition in
the in vivo studies. Among the synthesized compounds,
10c and 10d exhibited highest D2 antagonistic activity.
On increasing the steric bulk of amino group (N-methyl
derivatives), as seen in 10b and 10f, the D2 antagonis-
tic activity decreased. Compound 10e, 2,5-dimethyl
Although up to 1 µM, none of the NCEs tested inhib-
ited either the specific binding of the 5-HT2A ligand
Table 2. Results of D2 and 5-HT2A antagonism studies of final
compounds (10a–g).
Table 1. Summary of in vitro radioligand displacement studies of
final compounds (n=3) (10a–g).
% D2 Inhibition (mean SEM)
% 5-HT2A
% Specific binding of
5-HT2A radioligand [3H]
ketanserine at 1µM
% Specific binding of
D2 radioligand [3H]
spiperone at 1µM
Inhibition
S. No. Code 10th min 20th min
30th min (mean SEM)
S. No. Code
1
2
3
4
5
6
7
10a 60 10.00 60 10.00 70 12.25
10b 70 12.25 55 05.00 45 02.00
10c 85 06.12 85 10.00 95 05.00
10d 85 06.12 95 05.00 80 12.25
10e 55 05.00 70 12.25 80 12.25
10f 70 12.25 60 04.49 70 12.25
10g 60 10.00 60 10.00 60 10.00
31 02.37
49 08.33
51 01.67
42 02.37
36 06.26
79 03.74
58 07.25
100 0.00
1
2
3
4
5
6
7
10a
10b
10c
10d
10e
10f
128 23
136 22
112 13
115 22
n.d.
163 20
148 12
127 12
134 05
n.d.
109 01
125 14
119 04
120 06
10g
Risperidone 91 05.00 91 05.00 90 05.00
n.d. =not determined.
Journal of Enzyme Inhibition and Medicinal Chemistry

2-{4-[4-(2,5-Disubstituted thiazol-4-yl)phenylethyl]piperazin-1-yl}-1,8-naphthyridine-3-carbonitriles 567
Table 3. Results of D2 and 5-HT2A antagonism studies, catalepsy test of final compounds (10a–g).
% D2 Inhibition (mean SEM)
20th min
Maximum average
Code cataleptic time (sec)
% 5-HT2A Inhibition
(mean SEM)
S. No.
10th min
30th min
5-HT2A/D2 ratio
1
10a
10b
10c
10d
10e
10f
3.12
4.12
6.84
4.24
12.72
8.75
9.32
5.48
60 10.00
70 12.25
85 06.12
85 06.12
55 05.00
70 12.25
60 10.00
91 05.00
60 10.00
55 05.00
85 10.00
95 05.00
70 12.25
60 04.49
60 10.00
91 05.00
70 12.25
45 02.00
95 05.00
80 12.25
80 12.25
70 12.25
60 10.00
90 05.00
31 02.37
49 08.33
51 01.67
42 02.37
36 06.26
79 03.74
58 07.25
100 0.00
0.44286
0.70000
0.53684
0.44211
0.45000
1.12857
0.96667
1.09890
2
3
4
5
6
7
10g
Risperidone
derivative, exhibited better D2 antagonistic activity than
the monosubstituted derivative 10g. Compound 10f,
N,5-dimethylthiazol-2-amine derivative, exhibited high-
est 5-HT2A antagonism. Except 10a, the remaining amino
derivatives (10b, 10c, 10f) exhibited moderate to good
5-HT2A antagonism.
Declaration of interest
e authors report no conflicts of interest. e authors
alone are responsible for the content and writing of the
paper.
Overall seven compounds have been synthesized
in the present series and 10f was the most active com-
pound showing 79% and 70% 5-HT2A and D2 inhibition,
respectively.
References
1. Jablensky, A. e 100-year epidemiology of schizophrenia.
Schizophr. Res. 1997, 28, 111–125.
2. Mueser, K.T., McGurk, S.R. Schizophrenia. Lancet 2004, 363,
2063–2072.
3. Lewine, R.R., Fogg, L., Meltzer, H.Y. Assessment of negative and
positive symptoms in schizophrenia. Schizophr. Bull. 1983, 9,
368–376.
4. Marder, S.R., Wirshing, W.C., Van Putten, T. Drug treatment of
schizophrenia. Overview of recent research. Schizophr. Res. 1991,
4, 81–90.
5. Tandon,R.,Jibson,M.D.Efficacyofnewergenerationantipsychotics
in the treatment of schizophrenia. Psychoneuroendocrinology
2003, 28(Suppl. 1), 9–26.
6. Miyamoto, S., Duncan, G.E., Marx, C.E., Lieberman, J.A.
Treatments for schizophrenia: a critical review of pharmacology
and mechanisms of action of antipsychotic drugs. Mol. Psychiatry
2005, 10, 79–104.
7. Horacek, J., Bubenikova-Valesova, V., Kopecek, M., Palenicek, T.,
Dockery, C., Mohr, P., Höschl, C. Mechanism of action of atypical
antipsychotic drugs and the neurobiology of schizophrenia. CNS
Drugs 2006, 20, 389–409.
8. Meltzer, H.Y., Matsubara, S., Lee, J.C. e ratios of serotonin2 and
dopamine2affinitiesdifferentiateatypicalandtypicalantipsychotic
drugs. Psychopharmacol. Bull. 1989, 25, 390–392.
9. Roth, B.L., Meltzer, H.Y., Khan, N. Binding of typical and atypical
antipsychotic drugs to multiple neurotransmitter receptors. Adv.
Pharmacol. 1998, 42, 482–485.
10. Lowe, J.A., III. Atypical antipsychotics based on the D2/5-HT2 ratio
hypothesis. Curr. Med. Chem. 1994, 1, 50–60.
Conclusions
In summary, we have demonstrated the synthesis and
pharmacological activity of novel 2-{4-[4-(2,5-disubsti-
tutedthiazolyl)phenylethyl]piperazin-1-yl}-1,8-naphthy-
ridine-3-carbonitriles (10a–g) as atypical antipsychotic
agents. e results (Table 2) clearly indicate that all the
NCEs have the capability of antagonizing mesolimbic
dopaminergic D2 receptors with % inhibition varying
between 45% and 95% at the dose level studied (10 mg/
kg). A maximum of 95% inhibition was observed in 10c
and 10d, whereas a minimum of 45% inhibition was
noticed in 10b. e capability of antagonizing central
serotonergic 5-HT2A receptors varied between 31% and
79% at the dose level studied (10 mg/kg). A maximum
of 79% inhibition was evidenced in 10f, whereas a mini-
mum of 31% inhibition was evidenced in 10a. e results
tabulated in Table 3 clearly indicate that the maximum
average cataleptic score observed is 0 (as maximum
average cataleptic time is <20 sec) for the NCEs at dose
level studied (10 mg/kg) indicating that all compounds
are noncataleptic. From Table 3, it is evident that 10f is
the most active among the synthesized compounds with
5-HT2A/D2 ratio of 1.1286 and an average cataleptic score
of zero (Risperidone exhibited 5-HT2A/D2 ratio of 1.0989).
Hence 10f satisfies all the criteria required for a com-
pound to be atypical antipsychotic according to Meltzer’s
classification.30
11. Meltzer, H.Y., Li, Z., Kaneda, Y., Ichikawa, J. Serotonin
receptors: their key role in drugs to treat schizophrenia. Prog.
Neuropsychopharmacol. Biol. Psychiatry 2003, 27, 1159–1172.
12. Meltzer, H.Y. What’s atypical about atypical antipsychotic drugs?
Curr. Opin. Pharmacol. 2004, 4, 53–57.
13. Wirshing, D.A., Wirshing, W.C., Kysar, L., Berisford, M.A.,
Goldstein, D., Pashdag, J., Mintz, J., Marder, S.R. Novel
antipsychotics: comparison of weight gain liabilities. J. Clin.
Psychiatry 1999, 60, 358–363.
14. Owens, D.G. Adverse effects of antipsychotic agents. Do newer
agents offer advantages? Drugs 1996, 51, 895–930.
15. Cohen, D. Atypical antipsychotics and new onset diabetes mellitus.
An overview of the literature. Pharmacopsychiatry 2004, 37, 1–11.
16. Tamminga, C.A. e promise of new drugs for schizophrenia
treatment. Can. J. Psychiatry 1997, 42, 265–273.
Acknowledgements
Sincere thanks are due to UGC, New Delhi, India for
providing financial assistance. e authors are grateful
to Head, RSIC, CDRI, Lucknow for providing H NMR,
1
17. Chandra Sekhar, K.V.G., Rao, V.S., Vyas, D.R.K., Kumar, M.M.K.
Synthesis and preliminary pharmacological evaluation of N-2-(4-(4-
elemental analysis, and mass spectral data.
© 2011 Informa UK, Ltd.

568 K.V.G.C. Sekhar et al.
(2-substituted thiazol-4-yl)piperazin-1-yl)-2-oxoethyl)acetamides as
novel atypical antipsychotic agents. Bioorg. Med. Chem. Lett. 2008,
18, 6054–6057.
23. Schmeer, C., Lima, L. Modulation of outgrowth from goldfish
retinal explants by a 5-HT2 receptor agonist and [3H]ketanserin
binding sites in goldfish and rabbit retina. Vision Res. 2000, 40,
33–40.
24. Vivo, M., Lin, H., Strange, P.G. Investigation of cooperativity in the
binding of ligands to the D(2) dopamine receptor. Mol. Pharmacol.
2006, 69, 226–235.
25. Armstrong, D., Strange, P.G. Dopamine D2 receptor dimer
formation: evidence from ligand binding. J. Biol. Chem. 2001, 276,
22621–22629.
26. Costall, B., Naylor, R.J., Nohria, V. Climbing behaviour induced
by apomorphine in mice: a potential model for the detection of
neuroleptic activity. Eur. J. Pharmacol. 1978, 50, 39–50.
27. Malick, J.B., Doren, E., Barnett, A. Quipazine-induced head-twitch
in mice. Pharmacol. Biochem. Behav. 1977, 6, 325–329.
28. Costall, B., Naylor, R.J. Mesolimbic involvement with behavioural
effects indicating antipsychotic activity. Eur. J. Pharmacol. 1974,
27, 46–58.
18. Chandra Sekhar, K.V.G., Rao, V.S., Vyas, D.R.K., Kumar, M.M.K.
Synthesis and preliminary screening of novel N-{2-[4-(substituted)
piperazin-1-yl]-2-oxoethyl}acetamides as potential atypical
antipsychotic agents. J. Enzyme Inhib. Med. Chem. 2009, 24,
871–875.
19. Howard, H.R., Lowe, J.A. 3rd, Seeger, T.F., Seymour, P.A., Zorn,
S.H., Maloney, P.R., Ewing, F.E., Newman, M.E., Schmidt, A.W.,
Furman, J.S., Robinson, G.L., Jackson, E., Johnson, C., Morrone,
J. 3-Benzisothiazolylpiperazine derivatives as potential atypical
antipsychotic agents. J. Med. Chem. 1996, 39, 143–148.
20. Ariens, E.J., Beld, A.J., Rodrigues, J.M.F., Simonis, A.M. In e
Receptors: A Comprehensive Treatise. O’Brien (Ed.), Plenum
Press, New York, 1979, Vol. 1, p. 33.
21. Mahesh, R., Perumal, R.V., Pandi, P.V. Microwave assisted synthesis
of
2-(4-substituted
piperazin-1-yl)-1,8-naphthyridine-3-
carbonitrile as a new class of serotonin 5-HT3 receptor antagonists.
Bioorg. Med. Chem. Lett. 2004, 14, 5179–5181.
29. Joshi, V.V., Muley, M.P., Balsara, J.J., Chandorkar, A.G. Effect of
l-histidine pretreatment on haloperidol induced catalepsy and
methamphetamine stereotypy in mice. Indian J. Pharmacol. 1979,
11, 293–300.
30. Meltzer, H.Y., Matsubara, S., Lee, J.C. e ratios of serotonin2 and
dopamine2affinitiesdifferentiateatypicalandtypicalantipsychotic
drugs. J. Pharmacol. Exp. er. 1989, 251, 238–246.
22. Lowe, J.A. 3rd, Seeger, T.F., Nagel, A.A., Howard, H.R., Seymour,
P.A., Heym, J.H., Ewing, F.E., Newman, M.E., Schmidt, A.W.,
Furman, J.S. 1-Naphthylpiperazine derivatives as potential
atypical antipsychotic agents. J. Med. Chem. 1991, 34, 1860–
1866.
Journal of Enzyme Inhibition and Medicinal Chemistry