A facile and single pot strategy for the synthesis of novel naphthyridine derivatives under microwave irradiation conditions using ZnCl2 as catalyst, evaluation of AChE inhibitory activity, and molecular modeling studies

Article abstract of DOI:10.1007/s00044-011-9695-0

A series of novel naphthyridine derivatives 3 and 4 was prepared from substituted pyridine 2 and ketones using ZnCl₂ as catalyst under microwave irradiation conditions. All the compounds were evaluated for AChE inhibitory activity and promising compounds 3d, 3e, 4b, and 4g was identified. Representative compounds 3d and 3e were found to show insignificant THLE-2 liver cell viability/toxicity. The binding mode between X-ray crystal structure of human AChE and compounds was studied using molecular docking method and fitness scores were found to be in good correlation with the activity data.

Full text of DOI:10.1007/s00044-011-9695-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:1785–1795
DOI 10.1007/s00044-011-9695-0
ORIGINAL RESEARCH
A facile and single pot strategy for the synthesis of novel
naphthyridine derivatives under microwave irradiation conditions
using ZnCl2 as catalyst, evaluation of AChE inhibitory activity,
and molecular modeling studies
•
C. Kurumurthy P. Sambasiva Rao B. Veeraswamy G. Santhosh Kumar
•
•
•
•
P. Shanthan Rao Srigiridhar Kotamraju Sathish Babu Vasamsetti
•
•
•
Chinmayee Choudhury B. Narsaiah
Received: 21 February 2011 / Accepted: 8 June 2011 / Published online: 22 June 2011
Ó Springer Science+Business Media, LLC 2011
Abstract A series of novel naphthyridine derivatives 3
and 4 was prepared from substituted pyridine 2 and ketones
using ZnCl2 as catalyst under microwave irradiation con-
ditions. All the compounds were evaluated for AChE
inhibitory activity and promising compounds 3d, 3e, 4b,
and 4g was identified. Representative compounds 3d and
3e were found to show insignificant THLE-2 liver cell
viability/toxicity. The binding mode between X-ray crystal
structure of human AChE and compounds was studied
using molecular docking method and fitness scores were
found to be in good correlation with the activity data.
Acetylcholinesterase, also known as AChE, is an enzyme
that degrades the neurotransmitter acetylcholine, producing
choline and an acetate group. It is mainly found at neuro-
muscular junctions and cholinergic nervous system, where
its activity serves to terminate synaptic transmission.
In order to inhibit the acetylcholinesterase enzyme,
some organic compounds have been identified that can
block the function of AChE and thus cause excessive
acetylcholine to accumulate in the synaptic cleft. Among
the most common acetylcholinesterase inhibitors, phos-
phorus-based compounds are designed to bind to the active
site of the enzyme. Further developments realized several
new generation acetylcholinesterase inhibitors which have
additional advantages. A rational strategy to treat the
patients with acetylcholinesterase inhibitors (AChEI) can
increase the acetylcholine levels in brain as a result the
decrease in metabolic rate of released neurotransmitter
thereby enhanced neurotransmission (Bartus et al., 1982).
Tacrine (1) was the first AchE inhibitor approved in 1953;
however, its clinical usefulness was limited due to hepa-
totoxicity (Balson et al., 1995; Stachlewitz et al., 1997). In
recent past, the ortho amino nitriles are used as important
building blocks for the synthesis of tacrine analogues via
Friedlander reaction mainly using AlCl3 as catalyst (Marco
et al., 2001; Marco et al., 2004; Barreiro et al., 2003; Leon
et al., 2005). Similarly Lewis acids (Da Costa et al., 2009;
Proctor and Harvey, 2000; Gamba et al., 2008) like InCl3,
BF3ꢀEt2O, FeCl3, BiCl3, SbCl3, SnCl2ꢀ2H2O, RuCl3, CeCl3,
NiCl2, CoCl2ꢀ2H2O, and CsCl are also used as catalysts for
the synthesis of tacrine analogues. Apart from tacrine
analogues, more recently, chiral tetrahydroacridine ana-
logues (Pisoni et al., 2010) was also reported as AchE
inhibitors. Based on the importance, the interest is con-
tinuously growing on the synthesis and identification of
potential AChE inhibitors. In this study, we have
Keywords Acyclic ketones ꢀ Cyclic ketones ꢀ
Microwave irradiation ꢀ Catalyst ꢀ Tacrine ꢀ Naphthyridine ꢀ
AChE inhibitory activity ꢀ Liver cell viability ꢀ
Molecular modelling
Introduction
Alzheimer’s disease (AD) is a neurodegenerative disorder
caused due to progressive loss of several cognitive abilities.
C. Kurumurthy ꢀ P. Sambasiva Rao ꢀ B. Veeraswamy ꢀ
G. Santhosh Kumar ꢀ P. Shanthan Rao ꢀ B. Narsaiah (&)
Fluoroorganic Division, Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500607, India
e-mail: narsaiah_1984@yahoo.co.in
S. Kotamraju ꢀ S. B. Vasamsetti
Chemical Biology, Indian Institute of Chemical Technology,
Tarnaka, Hyderabad 500607, India
C. Choudhury
Molecular Modeling Group, Indian Institute of Chemical
Technology, Tarnaka, Hyderabad 500607, India
123

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Med Chem Res (2012) 21:1785–1795
synthesized a series of novel naphthyridine derivatives,
evaluated for AChE inhibitory activity and identified
promising compounds 3d, 3e, 4b, and 4g.
chloride under microwave irradiation conditions even
after running for a long time, i.e., 20 min. The reaction
CF3 NH2
CF3 NH2
NH2
X
R'
Ph
N
N
Ph
N
N
N
R
Tacrine 1
3d, R = Cl, R’= H
3e, R = H, R’= Br
4b, X = H
4g, X = CH3
Chemistry
sequence is outlined in Scheme 1 and products are tab-
ulated in Table 1.
In continuation of our efforts (Sridhar et al., 2009;
Chandra Shekhar et al., 2009; Shanthan Rao and Ven-
kataratnam, 1991), we used ZnCl2 as an inexpensive and
environment friendly catalyst for the synthesis of novel
tacrine analogues, isosteres of azaquinolines in single pot
under microwave irradiation conditions. In a typical
reaction, the 2-amino-3-cyano-4-trifluoromethyl-6-phe-
nylpyridine 2 (Narsaiah et al., 1994; Guttsait et al., 1987)
was reacted with acetophenone in DMF at 120–130°C for
18 h and found several products. To improve the selec-
tivity, the same reaction was conducted under microwave
irradiation conditions using synthos 3000 microwave
reactor at 540 W, 130°C and 15 bar pressure using ZnCl2
as catalyst and on subsequent workup furnished the
desired products 3 in high yields. In order to see the
versatility of the reaction, the compound 2 was further
reacted with diverse substituted acetophenones/methyl
heteroaryl/alkyl ketones in 10–14 min under the same
conditions and obtained products in the range of 80–96%
yield. In reaction of methyl isobutyl ketone with com-
pound 2, two products are formed in definite proportions.
The participation of methyl protons in cyclization resul-
ted major product 3h, while methylene protons gave
minor product 3i in the ratio of 85:15, respectively. The
ratio of the products was established after separation of
each product and identification based on their intensity in
1H NMR, CH2 appeared as doublet at d 2.75 in com-
pound 3h and CH3 as singlet at d 2.8 in compound 3i.
However, the reaction of compound 2 with b-ketoesters,
b-1, 3-diketones, cyclic alkene, or pentafluorophenyl
methylketone could not give the product and recovered
the starting material. This may be attributed to hydrogen
bonding due to chelation as a result no reaction. Further,
the reaction did not proceed in the absence of zinc
The mechanism of the reaction is mainly the formation
of Schiff’s base followed by intramolecular cyclization of
enamine over to nitrile carbon which was complexed with
zinc chloride. The complex on quenching with water
resulted product as indicated in Scheme 2.
Similarly, when the compound 2 was reacted with
diverse cyclic ketones in 6–14 min, tricyclic products 4 are
formed (Scheme 1; Table 2). Ring strain cyclic ketones
such as pentanone, octanone, indanone, and tetralone took
longer reaction times, whereas 2,6-dimethylcyclohexanone
could not give product even after 15 min. The possible
reason assumed to be steric hindrance as a result no
reaction.
CF3 NH2
ArCOCH3
ZnCl2
MW,
10-14 min.
540W
Ph
N
N
Ar
CF3
3 a-h
CN
O
Ph
N
NH2
( )n
CF3 NH2
2
n=1-4
( )n
ZnCl2
MW, 540W
6-14 min.
Ph
N
N
4 a-j
Scheme 1 Synthesis of 2-substituted-4-amino/2,3-fused-5-amino
naphthyridine derivatives (3a–i/4a–j)
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Med Chem Res (2012) 21:1785–1795
1787
Table 1 Preparation of
2-substituted-4-amino
naphthyridine derivatives (3a–i)
O
O
CF3NH2
CF3
N
CN
Ph
Ph
N
N
Ph
NH2
CF3NH2
N
N
F
F
O
CF3NH2
F
Ph
N
N
F
O
CF3NH2
Cl
Ph
N
N
Cl
O
CF3NH2
Br
Ph
N
N
Br
O
CF3NH2
MeO
Ph
N
N
OMe
CF3NH2
S
O
S
Ph
N
N
O
CF3 NH2
CF3 NH2
+
Ph
N
N
Ph
N
N
:
15
85
123

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Med Chem Res (2012) 21:1785–1795
Scheme 2 A possible
mechanistic pathway
Mechanism:
ZnCl2
CF3
N
N
CF3
CN
CF3
N
CN
N
:
:
O
ZnCl2
ZnCl2
+
..
-
H2O
Ar
Ph
N
Ar
Ph
N
NH2
Ph
Ar
H
-
ZnCl2
CF3
N
N
CF3 NH2
H2O
+
N
Ph
Ar
Ph
N
N
Ar
H
Results and discussion
AChE was studied using molecular docking with GOLD
3.2. The crystal structure of human AChE that was used
contains two molecules of N-acetyl glucosamine (NAG).
The active site of the protein analyzed with PDBSum
reveals that the important active site residues are Ser347
and Asn350. The important active site residues along with
the co-crystalized ligand are shown in Figs. 2 and 3. The
co-crystal has close contact with the residue Asn350. The
GOLD fitness function is made up of four components:
protein–ligand hydrogen bond energy, protein–ligand van
der Waals energy, ligand internal vdw energy, and ligand
torsional strain energy and the fitness score is taken as the
negative of the sum of the component energy terms (Jones
et al., 1997). The docked poses of each ligand were visu-
alized and the interactions were analyzed with silver. The
best Fitness Scores for each ligand are tabulated along with
the details of H-bonds and other interactions in Table 5.
Compound 3d has a fitness score 15.96 and it makes
hydrogen bond with the active site residue Ser347. Com-
pound 3e has a fitness score of 18.78 and both 3d and 3e
make close contacts with the active site residues Phe346
and Asp349. The fitness scores are in good correlation with
the activity data as indicated in Fig. 4.
AChE inhibition evaluation
To determine the potency of novel naphthyridine deriva-
tives, AChE inhibitory activity was measured at different
concentrations in vitro by the principle of Ellman method
(Ellman et al., 1961) and tacrine 1 was used as the refer-
ence compound. Compounds 3d, 3e, 4b, and 4g were found
to show high activity and on par with the standard tacrine.
All other compounds show moderate activity and com-
pound 4i show least activity. The structure activity rela-
tionship analysis of compounds show that the fusion of
cyclopentane, cycloheptane, or cyclooctane at 2,3-position
of naphthyridine has no additional advantage in promoting
activity. However, in compound 4b with cyclohexane ring
at 2,3-position and in compound 4g methyl at a specific
position of cyclohexane ring influenced the activity. Fur-
ther fusion of phenyl ring over cyclopentane or cyclohex-
ane and thiophene on 2nd position was found to be
detrimental to the activity as experienced in compounds 3g
and 4h–i. Further studies are underway to identify a lead
compound and the activity data are tabulated in Table 3.
THLE-2 liver cell viability/toxicity
Conclusion
The promising AChE inhibitors 3d and 3e identified above
were further screened for THLE-2 normal liver cell via-
bility/toxicity at different concentrations using tacrine as
standard. Either tacrine or compound 3e did not show any
significant cell death; however, compound 3d at 10 lm
caused a marginal 16% cell death as indicated in Table 4
and Fig. 1.
We have developed a novel and an efficient method for
1,8-naphthyridine derivatives in presence of inexpensive
ZnCl2 catalyst under microwave irradiation conditions.
All the compounds were screened for AchE inhibitory
activity at different concentrations and promising com-
pounds were identified. Further, representative compounds
3d and 3e were found to show insignificant THLE-2 liver
cell viability/toxicity. The fitness scores for all the com-
pounds are calculated through molecular docking studies
and are found to be in good correlation with the activity
data.
Molecular modelling studies
In order to revalidate the activity data, the binding mode of
the compounds along with X-ray crystal structure of human
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Med Chem Res (2012) 21:1785–1795
1789
Table 2 Preparation of 2,3-
fused-5-amino naphthyridine
derivatives (4a–j)
Entry
1
Compound
Substrate
Product 4
Reaction
time (min)
Yield (%)
85
O
CF3
CF3NH2
8
CN
Ph
N
N
Ph
N
NH2
4a
2
O
CF3NH2
2
2
4
98
Ph
N
N
4b
O
O
O
O
O
O
CF3NH2
3
4
5
6
7
8
2
2
2
2
2
4
90
90
90
98
98
Ph
N N
4c
CF3NH2
14
6
Ph
N
N
4d
CF3NH2
Ph
N N
4e
CF3NH2
6
Ph
N N
4f
CF3NH2
6
Ph
N N
4g
CF3NH2
2
8
85
80
Ph
N
N
4h
O
CF3NH2
9
2
2
14
Ph
N
N
4i
O
10
12
85
CF3NH2
Ph
N
N
4j
123

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Med Chem Res (2012) 21:1785–1795
Table 3 IC50 (lM) values for the AChE inhibition
Entry
Compound no.
IC50 (lM)
1
Tacrine (1)
0.14
7.10
7.03
6.58
0.52
0.89
8.24
10.36
19.09
8.05
0.35
10.13
7.69
9.37
3.25
0.51
17.56
23.14
2
3a
3b
3b
3d
3e
3f
3
4
5
6
7
8
3g
3h
4a
4b
4c
4d
4e
4f
9
Fig. 1 THLE-2 liver cell viability/toxicity graph
10
11
12
13
14
15
16
17
18
4g
4h
4i
Experimental section
Melting points were recorded on Casia-Siamia (VMP-AM)
melting point apparatus and are uncorrected. All reactions
were carried out in a synthos 3000 microwave reactor. IR
spectra were recorded on a Perkin-Elmer FT-IR 240-C
spectrophotometer using KBr optics. 1H NMR spectra were
recorded on Bruker AV 300 MHz in CDCl3 using TMS as
internal standard. Electron impact (EI) and chemical ioni-
zation mass spectra were recorded on a VG 7070 H
instrument at 70 eV. All the reactions were monitored by
thin layer chromatography (TLC) on precoated silica gel 60
F254 (mesh); spots were visualized with UV light. Merck
silica gel (60–120 mesh) was used for column chroma-
tography. CHN analyses were recorded on a vario EL
analyzer. AchE inhibitory activity was measured by Ell-
man’s method and THLE-2 liver cell viability/toxicity was
measured by MTT assay method.
Fig. 2 The co-crystallized compound N-acetyl glucosamine (green)
in the active site of human AChE. The key residues Ser347 and
Asn350 are highlighted (Color figure online)
Preparation of 2-substituted-4-amino naphthyridine
derivatives (3a–i) and 2,3-fused-5-amino naphthyridine
derivatives (4a–j)
General procedure
The 2-amino-3-cyano-4-trifluoromethyl-6-phenylpyridine
2
(1.9 mmol) was reacted with cyclic ketones or
Table 4 THLE-2 liver cell viability/toxicity at 48 h time (%)
Compound No.
Micromolar concentration verses percentage of cell viability
0.5 lm
SD
1.0 lm
SD
10 lm
SD
Tacrine (standard)
100.73
97.09
98.66
1.67
3.01
4.01
96.59
98.08
95.92
5.00
2.35
4.35
98.45
84.25
99.94
6.13
2.40
2.94
3d
3e
SD standard deviation (values in percentage)
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Med Chem Res (2012) 21:1785–1795
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Fig. 3 Snapshots of the
interaction of the ligands with
human AChE ligand is show in
green, the active site residues
are highlighted, and the
hydrogen bonds are shown in
green dotted lines (Color figure
online)
2-(3-Fluorophenyl)-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3b) Yield: 0.69 g (95%); mp 210–
212°C; I.R. (KBr, cm-1): 3538, 3381 (–NH2), 1634
(–C=N), 1323 (–C–F); 1H NMR (CDCl3, 300 MHz): d 5.38
(br., s, 2H, –NH2), 7.10 (m, 2H, Ar–H), 7.4 (s, 1H, Ar–H–
C(3)), 7.5 (m, 3H, Ar–H), 7.9 (m, 2H, Ar–H), 8.15 (s, 1H,
Ar–H–C(6)), 8.3 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 383
(M?), 367 (M?–NH2), 314 (M?–CF3); Anal. Calcd. for
C21H13F4N3: C, 65.80; H, 3.42; N, 10.96%. Found: C,
65.45; H, 3.32; N, 10.76%.
acetophenones (5.7 mmol) in presence of ZnCl2 (0.73
mmol) under microwave irradiation conditions with 540 W
power during 4–14 min and products were obtained in high
yield. The product was purified by passing through a col-
umn packed with silica gel and n-hexane:ethylacetate (3:2)
as eluents
2,7-Diphenyl-5-(trifluoromethyl)-1,8-naphthyridin-4-amine
(3a) Yield: 0.65 g (93%); mp 203–205°C; I.R. (KBr,
cm-1): 3550, 3320 (–NH2), 1652 (–C=N), 1323 (–C–F); 1H
NMR (CDCl3, 300 MHz): d 5.4 (br., s, 2H, –NH2), 7.18 (s,
1H, Ar–H–C(3)), 7.4–7.6 (m, 6H, Ar–H, Ar–H–C(6)),
8.1–8.2 (m, 3H, Ar–H), 8.3 (m, 2H, Ar–H); MS (EI,
70 eV): m/z 365 (M?), 349 (M?–NH2), 296 (M?–CF3);
Anal. Calcd. for C21H14F3N3: C, 69.04; H, 3.86; N,
11.50%. Found: C, 68.81; H, 3.76; N, 11.30%.
2-(4-Fluorophenyl)-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3c) Yield: 0.70 g (96%); mp 204–
206°C; I.R. (KBr, cm-1): 3554, 3326 (–NH2), 1657
(–C=N), 1327 (–C–F); 1H NMR (CDCl3, 300 MHz): d 5.30
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Med Chem Res (2012) 21:1785–1795
Table 5 Docking results obtained with GOLD, indicating Fitness Score, its hydrogen bonding and Van der Waals components
Compound no.
Fitness
S (HB_ext)
S (vdw_ext)
S (int)
H-bond with residues
Close contacts with residues
Tacrine (1)
29.94
29.89
15.16
14.70
15.96
18.78
18.82
19.11
15.30
3.84
3.63
0.17
0.03
0.69
0.79
0.00
1.13
0.84
0.00
0.00
0.00
0.00
0.00
5.59
0.00
0.70
0.00
0.94
0.00
0.00
19.53
24.73
23.95
23.39
24.25
26.27
26.86
25.27
23.71
22.53
23.70
25.57
25.99
19.96
27.59
21.00
24.54
24.49
24.99
27.34
0.00
-4.28
Tyr77,Val340
Tyr77
3a
3b
3c
3d
3e
3f
Phe346
Phe346,Val340
Phe346, Asp349, Leu76
Phe346, Asp349
Phe346, Asp349
Leu76, Tyr77
Phe346, Asp349,Ala343
Ser347, Leu76
Leu76, Pro78
Gly342,Phe346,Glu358
Val340
-17.81
-18.15
-18.17
-17.35
-19.23
-16.47
-17.30
-27.15
-20.13
-23.86
-23.36
-21.61
-17.56
-20.80
-21.29
-17.13
-20.84
-21.86
Phe346
Ser347
Ser347
–
Ser347, Leu76,Gly342
3g
3h
3i
Ser347
–
Gly345
4a
4b
4c
4d
4e
4f
12.46
11.30
13.37
11.42
20.38
8.78
–
–
Val340, Gly342
Phe346, Gly345
Val340, Phe346
Val340
–
Ser346
–
Ser347
Gly342
Ser347
–
Phe346,Leu76
–
4g
4h
4i
12.45
17.48
13.53
15.73
Phe346,Leu76
–
4j
–
Phe346,Gly342,Val340
S (HB_ext) protein–ligand hydrogen bond energy, S (vdw_ext) protein–ligand Van der Waals (vdw) energy (external vdw), S (int) (internal
torsion ? internal vdw)
Fig. 4 Superposition of the
synthesized drugs a 3e and b 4b
with NAG
2-(4-Chlorophenyl)-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3d) Yield: 0.69 g (92%); mp
218–220°C; I.R. (KBr, cm-1): 3552, 3415 (–NH2), 1655
(–C=N), 1326 (–C–F); 1H NMR(CDCl3, 300 MHz): d 5.35
(br., s, 2H, –NH2), 7.18 (s, 1H Ar–H–C(3)), 7.4–7.6 (m, 5H
Ar–H), 8.10–8.20 (m, 3H Ar–H), 8.3 (m, 2H Ar–H); MS
(br., s, 2H, –NH2), 7.15–7.20 (m, 2H, Ar–H), 7.28 (s, 1H,
Ar–H–C(3)), 7.5–7.6 (m, 3H, Ar–H), 8.10–8.20 (m, 3H,
Ar–H), 8.35 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 383
(M?), 367 (M?–NH2), 314 (M?–CF3); Anal. Calcd. for
C21H13F4N3: C, 65.80; H, 3.42; N, 10.96%. Found: C,
65.27; H, 3.32; N, 11.10%.
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Med Chem Res (2012) 21:1785–1795
1793
1
(EI, 70 eV): m/z 399 (M?), 383 (M?–NH2), 364 (M?–Cl),
330 (M?–CF3); Anal. Calcd. For C21H13ClF3N3: C, 63.09;
H, 3.28; N, 10.51%. Found: C, 62.55; H, 3.20; N, 10.18%.
(–C=N), 1321 (–C–F); H NMR (CDCl3, 300 MHz): d 1.5
(d, J = 7.365 Hz, 6H, –2CH3), 2.8 (s, 3H, –CH3), 3.6 (m,
1H, –CH), 5.25 (br., s, 2H, –NH2), 7.5 (m, 3H, Ar–H), 8.15
(s, 1H, Ar–H–C(6)), 8.3 (m, 2H, Ar–H); MS (EI, 70 eV):
m/z 345(M?), 330 (M?–CH3), 275 (M?–CF3); Anal.
Calcd. for C19H18F3N3: C, 66.08; H, 5.25; N, 12.17%.
Found: C, 65.89; H, 5.15; N, 12.10%.
2-(2-Bromophenyl)-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3e) Yield: 0.71 g (85%); mp 139–
141°C; I.R. (KBr, cm-1): 3552, 3325 (–NH2), 1657
1
(–C=N), 1321(C–F); H NMR(CDCl3, 300 MHz): d 5.30
(br., s, 2H, –NH2), 7.05 (m, 1H, Ar–H–C(3)), 7.25 (m, 1H,
Ar–H), 7.4 (m, 1H, Ar–H), 7.5 (m, 3H, Ar–H), 7.65 (d,
J = 9.065 Hz, 1H, Ar–H), 7.75 (dd, J = 9.065 Hz, 1H,
Ar–H), 8.20 (s, 1H, Ar–H–C(6)), 8.3 (m, 2H, Ar–H); MS
(EI, 70 eV): m/z 443 (M?), 445 (M??2), 427 (M?–NH2),
376 (M?–CF3), 364(M?–Br); Anal. Calcd. for
C21H13BrF3N3: C, 56.78; H, 2.95; N, 9.46%. Found: C,
56.55; H, 3.08; N, 9.82%.
2-Phenyl-4-(trifluoromethyl)-7,8-dihydro-6H-cyclopenta[b]
[1,8]naphthyridin-5-amine (4a) Yield: 0.53 g (85%); mp
208–210°C; I.R. (KBr, cm-1): 3540, 3339 (–NH2), 1661
(–C=N), 1351 (–C–F); 1H NMR (CDCl3, 300 MHz): d 2.38
(m, 2H, –CH2–), 2.98 (t, J = 7.554 Hz, 2H, –CH2–), 3.25
(t, J = 7.743 Hz, 2H, –CH2–), 4.95 (br., s, 2H, –NH2), 7.5
(m, 3H, Ar–H), 8.15 (s, 1H, Pyridine-H), 8.3–8.4 (m, 2H,
Ar–H); MS (EI, 70 eV): m/z 329 (M?), 260 (M?–CF3);
Anal. Calcd. for C18H14F3N3: C, 65.65; H, 4.28; N,
12.76%. Found: C, 65.68; H, 4.15; N, 12.56%.
2-(4-Methoxyphenyl)-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3f) Yield: 0.60 g (80%); mp 194–
196°C; I.R. (KBr, cm-1): 3550, 3327 (–NH2), 1657 (–C=N),
2-Phenyl-4-(trifluoromethyl)-6,7,8,9-tetrahydrobenzo[b]
[1,8]-naphthyridin-5-amine (4b) Yield: 0.64 g (98%);
mp 202–204°C; I.R. (KBr, cm-1): 3468, 3386 (–NH2),
1
1328 (–C–F); H NMR(CDCl3, 300 MHz): d 3.85 (s, 3H,
–OCH3), 5.25 (br., s, 2H, –NH2), 6.95 (d, J = 8.687 Hz,
2H, Ar–H), 7.15 (s, 1H, Ar–H–C(3)), 7.5 (m, 3H, Ar–H),
8.1–8.2 (m, 3H, Ar–H), 8.3 (m, 2H, Ar–H); MS (EI,
70 eV): m/z 395 (M?), 379 (M?–NH2), 364 (M?–OCH3),
326 (M?–CF3); Anal. Calcd. for C22H16F3N3O: C, 66.83;
H, 4.08; N, 10.63%. Found: C, 66.43; H, 4.00; N, 10.45%.
1
1624 (–C=N), 1323 (–C–F); H NMR (CDCl3, 300 MHz):
d 2.0 (m, 5H, –CH2-), 2.5–2.7 (m, 3H, –CH2–), 5.7 (br., s,
2H, –NH2), 7.5 (m, 3H, Ar–H), 8.1 (s, 1H, Pyridine-H), 8.3
(m, 2H, Ar–H); MS (EI, 70 eV): m/z 343 (M?), 274 (M?–
CF3); Anal. Calcd. For C19H16F3N3: C, 66.46; H, 4.70; N,
12.24%. Found: C, 65.80; H, 4.50; N, 12.04%.
7-Phenyl-2-(thiophin-2-yl)-5-(trifluoromethyl)-1,8-naphthyri-
din-4-amine (3g) Yield: 0.63 g (90%); mp 219–221°C;
I.R. (KBr, cm-1): 3550, 3333 (–NH2), 1650 (–C=N), 1326
2-Phenyl-4-(trifluoromethyl)-7,8,9,10-tetrahydro-6H-
cyclohepta[b][1,8]-naphthyridin-5-amine
(4c) Yield:
1
(–C–F); H NMR (CDCl3, 300 MHz): d 5.25 (br., s, 2H,
0.61 g (90%); mp 210–212°C; I.R. (KBr, cm-1): 3534,
3334 (–NH2), 1671 (–C=N), 1324 (–C–F); 1H NMR
(CDCl3, 300 MHz): d 1.8–2.0 (m, 6H, –CH2–), 2.8–2.9 (m,
2H, –CH2–), 3.3 (m, 2H–CH2–), 5.1 (br., s, 2H, –NH2),
7.5–7.6 (m, 3H, Ar–H), 8.15 (s, 1H, Pyridine-H), 8.3 (m,
2H, Ar–H); MS (EI, 70 eV): m/z 357 (M?), 342 (M?–
NH2), 288 (M?–CF3); Anal. Calcd. for C20H18F3N3: C,
67.22; H, 5.08, N, 11.76%. Found: C, 67.30; H, 5.10; N,
11.46%.
–NH2), 7.1 (m, 2H, thiophine-H), 7.5 (m, 4H, Ar–H), 7.78
(d, J = 3.588 Hz, 1H, thiophine-H), 8.1 (s, 1H, Ar–H–
C(6)), 8.3 (m, 2H Ar–H); MS (EI, 70 eV): m/z 371 (M?),
302 (M?–CF3); Anal. Calcd. for C19H12F3N3S: C, 61.45;
H, 3.26; N, 11.31%. Found: C, 61.15; H, 3.19; N, 11.09%.
2-Isobutyl-7-phenyl-5-(trifluoromethyl)-1,8-naphthyridin-
4-amine (3h) Yield: 0.40 g (61%); mp 151–153°C; I.R.
(KBr, cm-1): 3556, 3315 (–NH2), 1661 (–C=N), 1322 (–C–
1
F); H NMR (CDCl3, 300 MHz): d 1.0 (d, J = 6.610 Hz,
2-Phenyl-4-(trifluoromethyl)-6,7,8,9,10,11-hexahydrocyclo-
octa[b][1,8]-naphthyridin-5-amine (4d) Yield: 0.63 g
(90%); mp 208–210°C; I.R. (KBr, cm-1): 3561, 3328
(–NH2), 1657 (–C=N), 1329 (–C–F); 1H NMR(CDCl3,
300 MHz): d 1.4–2.0 (m, 8H, –CH2-), 2.9–3.0 (t,
J = 6.592 Hz, 2H, –CH2–), 3.2 (t, J = 6.592 Hz, 2H,
–CH2–), 5.1 (br., s, 2H, –NH2), 7.5 (m, 3H, Ar–H), 8.15 (s,
1H, Pyridine-H), 8.3 (m, 2H, Ar–H); MS (EI, 70 eV): m/
z 371 (M?), 356 (M?–NH2), 302 (M?–CF3); Anal. Calcd.
For C21H20F3N3: C, 67.91; H, 5.43; N, 11.31%. Found: C,
67.51; H, 5.33; N, 11.15%.
6H, –2CH3), 2.3 (m, 1H, –CH), 2.75 (d, J = 7.176 Hz, 2H,
–CH2), 5.25 (br., s, 2H, –NH2), 6.65 (s, 1H, Ar–H–C(3)),
7.5 (m, 3H, Ar–H), 8.15 (s, 1H, Ar–H–C(6)), 8.3 (m, 2H,
Ar–H); MS (EI, 70 eV): m/z 345 (M?), 330 (M?–CH3),
275 (M?–CF3); Anal. Calcd. For C19H18F3N3: C, 66.08; H,
5.25; N, 12.17%. Found: C, 65.75; H, 5.19; N, 12.01%.
2-Isopropyl-3-methyl-7-phenyl-5-(trifluoromethyl)-1,8-naph-
thyridin-4-amine (3i) Yield: 0.08 g (12%); mp 150–
152°C; I.R. (KBr, cm-1): 3551, 3312 (–NH2), 1620
123

1794
Med Chem Res (2012) 21:1785–1795
7-Methyl-2-phenyl-4-(trifluoromethyl)-7,8-dihydro-6H-
10-Phenyl-8-(trifluoromethyl)-5,6-dihydronaphtho[1,2-b]
[1,8]-naphthyridin-5-amine (4i) Yield: 0.59 g (80%);
mp 150–152°C; I.R. (KBr, cm-1): 3526, 3425 (–NH2),
cyclopenta[b][1,8]-naphthyridin-5-amine
(4e) Yield:
0.58 g (90%); mp 148–150°C; I.R. (KBr, cm-1): 3524,
3420 (–NH2), 1648 (–C=N), 1328 (–C–F); 1H
NMR(CDCl3, 300 MHz): d 1.25 (d, J = 6.830 Hz, 3H,
–CH3), 2.47–2.53 (dd, J = 5.855 Hz, J = 6.830 Hz, 1H,
–CH-), 2.7–2.8 (m, 1H, –CH–), 2.8–2.9 (dd, J = 6.830 Hz,
J = 7.806 Hz, 1H, –CH–), 3.05–3.10 (dd, J = 7.806 Hz,
J = 8.782 Hz, 1H, CH–), 3.35–3.42 (dd, J = 7.806 Hz,
J = 8.782 Hz, 1H, –CH–), 4.9 (br., s, 2H, –NH2),
7.45–7.55 (m, 3H, Ar–H), 8.15 (s, 1H, Pyridine-H),
8.25–8.35 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 343 (M?),
328 (M?–CH3), 274 (M?–CF3); Anal. Calcd. For
C19H16F3N3: C, 66.46; H, 4.70; N, 12.24%. Found: C,
66.50; H, 4.58; N, 12.07%.
1
1641 (–C=N), 1342 (–C–F); H NMR (CDCl3, 300 MHz):
d 2.9 (t, J = 6.987 Hz, 2H, –CH2–), 3.1 (t, J = 6.987 Hz,
2H, –CH2–), 5.2 (br., s, 2H, –NH2), 7.15 (m, 1H, Ar–H),
7.25–7.35 (m, 2H, Ar–H), 7.50–7.55 (m, 3H, Ar–H), 8.15
(s, 1H, Pyridine-H), 8.2–8.3 (m, 2H, Ar–H), 8.6–8.7 (m,
1H, Ar–H); MS (EI, 70 eV): m/z 391 (M?), 322 (M?–CF3);
Anal. Calcd. for C23H16F3N3: C, 70.58; H, 4.12; N,
10.74%. Found: C, 70.38; H, 4.02; N, 10.64%.
9-Phenyl-11-(trifluoromethyl)-5,6-dihydronaphtho[2,1-b]
[1,8]-naphthyridin-5-amine (4j) Yield: 0.63 g (85%);
mp 160–162°C; I.R. (KBr, cm-1): 3522, 3421 (–NH2),
1642 (–C=N), 1344 (–C–F); 1H NMR(CDCl3, 300 MHz): d
3.0(t, J = 6.421 Hz, 2H, –CH2–), 3.3 (t, J = 6.421 Hz,
2H, –CH2–), 5.3 (br., s, 2H, –NH2), 7.2 (m, 1H, Ar–H),
7.3–7.4 (m, 2H, Ar–H), 7.45–7.55(m., 3H, Ar–H), 8.1 (s,
1H, Pyridine-H), 8.2–8.3 (m, 2H, Ar–H), 8.6–8.7 (m, 1H,
Ar–H); MS (EI, 70 eV): m/z 391 (M?), 322 (M?–CF3);
Anal. Calcd. for C23H16F3N3: C, 70.58; H, 4.12; N,
10.74%. Found: C, 70.28; H, 4.10; N, 10.54%.
8-Methyl-2-phenyl-4-(trifluoromethyl)-6,7,8,9-tetrahydrobenzo
[b][1,8]-naphthyridin-5-amine (4f) Yield: 0.66 g (98%);
mp 160–162°C; I.R. (KBr, cm-1): 3502, 3404 (–NH2),
1
1625 (–C=N), 1317 (–C–F); H NMR (CDCl3, 300 MHz):
d 1.1 (d, J = 6.043 Hz, 3H, –CH3), 1.9–2.1 (m, 2Hm,
–CH2–), 2.5–2.7 (m, 4H, –CH2–), 3.2–3.3 (dd,
J = 1.511 Hz, J = 2.266 Hz, 1H, –CH–), 5.35 (br., s, 2H,
–NH2), 7.4–7.5 (m, 3H, Ar–H), 8.1 (s, 1H, Pyridine-H),
8.2–8.3 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 357 (M?),
342 (M?–CH3), 288 (M?–CF3); Anal. Calcd. for
C20H18F3N3: C, 67.22; H, 5.08, N, 11.76%. Found: C,
67.30; H, 4.99; N, 11.46%.
AChE inhibitory activity
Acetylcholine esterase (AChE) inhibitory activity was
measured according to Ellman et al. (1961). AChE (5 mU)
in 10 mM phosphate buffer at pH 7.4 was incubated with
200 ll of 100 mM phosphate buffer in the presence or
absence of test compounds (0.1–10 lM) along with
0.8 mM acetylcholine iodide and 10 mM 5,50-dithiobis-(2-
nitrobenzoic acid) (DTNB) at 37°C and continuous
absorbance was measured for 10 min at an interval of
every 30 s at 412 nm using Tecan multimode reader Infi-
nite M200 series.
7-Methyl-2-phenyl-4-(trifluoromethyl)-6,7,8,9-tetrahydrobenzo
[b][1,8]-naphthyridin-5-amine (4g) Yield: 0.66 g (98%);
mp 200–202°C; I.R. (KBr, cm-1): 3508, 3420 (–NH2),
1638 (–C=N), 1327 (–C–F); 1H NMR(CDCl3, 300 MHz): d
1.2 (d, J = 6.043 Hz, 3H, –CH3), 2.0–2.2 (m, 4H, –CH2–),
2.68–2.74 (dd, J = 3.777 Hz, J = 5.288 Hz, 1H, –CH–),
3.0–3.1 (m, 1H, –CH–), 3.2–3.3 (m, 1H, –CH–), 5.1 (br., s,
2H, –NH2), 7.4–7.5 (m, 3H, Ar–H), 8.1 (s, 1H, Pyridine-
H), 8.2–8.3 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 357 (M?),
342 (M?–CH3), 288 (M?–CF3); Anal. Calcd. For
C20H18F3N3: C, 67.22; H, 5.08; N, 11.76%. Found: C,
67.28; H, 4.99; N 11.46%.
THLE-2 liver cell viability/toxicity assay
To assess the cytotoxic effects of compound 3d, 3e, and
Tacrine, normal liver cells (THLE-2) from ATCC were
grown in bronchial epithelial growth medium bullet kit
(Lonza) and treated with 0–10 lM of either Tacrine,
compound 3d or 3e for a period of 48 h and cytotoxicity
was measured by standard MTT assay. At the end of the
treatments, medium was removed and cells were washed
with Dulbecco’s phosphate buffered saline (DPBS) and
10 ll of 5 mg/ml MTT solution in 200 ll of culture
medium was added and incubated for 1 h at 37°C. Cells
were solubilized with 200 ll of DMSO and absorbance
was measured at 562 nm in a spectrophotometer.
2-Phenyl-4-(trifluoromethyl)-6H-indeno[1,2-b][1,8]-naph-
thyridin-5-amine (4h) Yield: 0.61 g (85%); mp
145–147°C; I.R. (KBr, cm-1): 3520, 3424 (–NH2), 1664
1
(–C=N), 1344 (–C–F); H NMR (CDCl3, 300 MHz): d 4.0
(s, 2H, –CH2–), 6.35 (br., s, 2H, –NH2), 7.3–7.4 (m, 1H,
Ar–H), 7.45–7.55 (m, 2H, Ar–H), 7.6–7.7 (m, 3H, Ar–H),
7.9 (d, J = 7.365 Hz, 1H, Ar–H), 8.2 (s, 1H, Pyridine-H),
8.3–8.4 (m, 2H, Ar–H); MS (EI, 70 eV): m/z 377 (M?),
308 (M?–CF3); Anal. Calcd. For C22H14F3N3: C, 70.02; H,
3.74; N, 11.14%. Found: C, 69.68; H, 3.65; N, 11.01%.
123

Med Chem Res (2012) 21:1785–1795
Molecular modelling studies
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reference compound (Tacrine) was constructed and pre-
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Compound minimization was done using OPLS 2005 Force
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˚
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Acknowledgments Authors are thankful to Dr. J.S. Yadav, Direc-
tor, IICT for his constant encouragement and authors (C. Kurumurthy,
P. Sambasiva Rao and B. Veeraswamy, G. Santhosh kumar) are
thankful to Council of Scientific and Industrial Research (CSIR),
India for providing financial assistance in the form of Junior Research
Fellowship. Authors also thank Dr. G. Narahari Sastry for his help in
molecular modeling studies.
Sridhar M, Ramanaiah B, Narsaiah C, Mahesh B, Kumaraswamy M,
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