Synthesis, docking studies and anti-inflammatory activity of some 2-amino-5,6,7,8-tetrahydroquinoline-3-carbonitriles and related compounds

Article abstract of DOI:10.1691/ph.2009.8703

A series of 2-amino-5,6,7,8-tetrahydroquinoline-3-carbonitriles 4a-c has been synthesized and reacted with various reagents. Docking studies into the catalytic site of p38 mitogen activated protein kinase (MAPK) were used to identify potential anti-inflammatory lead compounds. The anticipated lead derivative 2-(pyridin-3-yl)-5-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrimido[4,5- b]quinolin-4-amine 9b was endowed with good binding energy. Also, the obtained products were evaluated for their in vivo antiinflammatory activity as prostaglandin inhibitiors (% inhibition of edema and % inhibition of plasma PGE2 at the two dose levels were determined). The tested compounds exhibited significant anti-inflammatory activity and minor ulcerogenic effects, when compared to the reference standard indomethacin, with product 9b being of particular interest.

Full text of DOI:10.1691/ph.2009.8703

ORIGINAL ARTICLES
Applied Organic Chemistry Department1, National Research Centre, Dokki; Pharmaceutical Chemistry Department2, Faculty
of Pharmacy, Ain-Shams University; Research Units3, Hi-Care Pharmaceutical Co., Cairo, Egypt
Synthesis, docking studies and anti-inflammatory activity of some
2-amino-5,6,7,8-tetrahydroquinoline-3-carbonitriles and related compounds
O. I. Abd El-Salam1, D. A. Abou El Ella2, N. S. M. Ismail2, M. Abdullah3
Received August 15, 2008, accepted October 3, 2008
Ass. Prof. Dr. Osama I. Abd El-Salam, Applied Organic Chemistry Department, National Research Centre,
Dokki, Cairo – 12622, Egypt
oiel_salam@yahoo.com
Pharmazie 64: 147–155 (2009)
doi: 10.1691/ph.2009.8703
A series of 2-amino-5,6,7,8-tetrahydroquinoline-3-carbonitriles 4a–c has been synthesized and reacted
with various reagents. Docking studies into the catalytic site of p38 mitogen activated protein kinase
(MAPK) were used to identify potential anti-inflammatory lead compounds. The anticipated lead deriva-
tive 2-(pyridin-3-yl)-5-(4-methoxyphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinolin-4-amine 9b was en-
dowed with good binding energy. Also, the obtained products were evaluated for their in vivo anti-
inflammatory activity as prostaglandin inhibitiors (% inhibition of edema and % inhibition of plasma
PGE2 at the two dose levels were determined). The tested compounds exhibited significant anti-inflam-
matory activity and minor ulcerogenic effects, when compared to the reference standard indomethacin,
with product 9b being of particular interest.
1. Introduction
cytokines such as tumor necrosis factor-a (TNF-a) and in-
terleukin-1b (IL-1b) at both transcription and translation
levels while the roles of p38-b, p38-g, p38-d have to be
identified critically (Ravindra et al. 2008).
Non-steroidal anti-inflammatory drugs (NSAIDs) are
among the most widely used therapeutics, primarily for the
treatment of pain and inflammation in arthritis for decades.
However, long-term clinical usage of NSAIDs is associated
with significant side effects of gastrointestinal lesions,
bleeding, and nephrotoxicity. Therefore the discovery of
new safer anti-inflammatory drugs represents a challenging
goal for such a research area (Kontogiorgis and Hadjipav-
lou-Litina 2002; van Ryn et al. 2000; Beuck 1999).
In the late 1990s, the introduction of selective cyclooxy-
genase-2 (COX-2) inhibitors, such as celecoxib and rofe-
coxib, provided novel anti-inflammatory- analgesic agents
with reduced gastrointestinal side effects (Praveen et al.
2005; Bombardier et al. 2000). However, the recent market
withdrawal of rofecoxib and valdecoxib due to their ad-
verse cardiovascular side effects clearly delineates the need
to develop alternative anti-inflammatory agents with re-
duced toxicity (Dogne et al. 2005). Intracellular signaling
pathways involving p38 mitogen activated protein kinase
(MAPK) regulate the production of several pro-inflamma-
tory cytokines and are considered as a focal point in the
development of new therapeutic agents to treat inflamma-
tory diseases such as rheumatoid arthritis (RA) (Kulkarni
et al. 2006). p38 is a member of Ser/Thr kinases family of
the mitogen-activated protein (MAP) kinase super family
(Sandra et al. 2006). The four-p38 isoforms, p38-a, (also
known as p38), p38-b, p38-g, p38-d varies based on their
substrate specificity. Several reports indicate that p38a has
a profound role in RA and is involved in the expression of
The interest in the development of p38 MAP kinase inhibi-
tors is based on the expectations that p38 inhibiting drugs
will treat the underlying cause of chronic inflammatory dis-
ease and cease their progression (Revesz et al. 2002).
Although a number of structurally different inhibitors have
been reported to inhibit p38 with varying degrees of selectiv-
ity, none has reached commercial status (Nayan et al. 2008).
The pyridinyl-imidazoles represented by SB203580 1
were the first reported class of p38a inhibitors (Gallagher
et al. 1995; Wilson et al. 1997). Following high through-
out screening of the Bayer compound library, the commer-
cially available thienyl urea 2 (Maybridge GK 00687) was
identified as a reversible p38 inhibitor.
Many naturally occurring as well as synthetic compounds
containing the quinoline scaffold exhibit interesting biolo-
gical properties (Kouznetsov et al. 2005; Fadel et al. 2004;
Crousse et al. 2000; Kobayashi et al. 2004). It has been
reported that compounds containing quinoline, pyrimidine
and tetrazole functionality exhibit anti-inflammatory activ-
ity (Marco-Contelles et al. 2006; Bekhit et al. 2004; Cal-
houn et al. 1995). Recently Gholap et al. (2007) synthe-
sized a series of 5,6,7,8-tetrahydroquinoline analogues,
based on the interesting pharmacological properties exhib-
ited by compounds containing the pyridine scaffold. On
the basis of these results and as a continuation of our on-
going effort in the field of pyridine derivatives (Abd-El-
Pharmazie 64 (2009) 3
147

ORIGINAL ARTICLES
F
O
O
F
H
N
H
N
H
N
N
O
O
S
S
O
2
N
N
N
N
N
1
3
Salam and Mohamed 2005; Abd-El-Salam 2000; Attia
et al. 1995), we are herein reporting the synthesis of a
new series of 5,6,7,8-tetrahydroquinoline analogues and
their anti-inflammatory evaluation, with the aim of devel-
oping novel anti-inflammatory lead compounds.
acetate furnished the maximum yield for 1 mmol of the reac-
tants. Cyclization of 4 with chloroformamidine hydrochlor-
ide in diglyme (Harris et al. 1990), was proceeded to give
5-(4-substitutedphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]-
quinoline-2,4-diamine 5, as white powder (Scheme 1). The
methaneimidates 6, as intermediates for further reactions,
were obtained by the reaction of 4 with triethylorthoformate
in the presence of a catalytic amount of acetic anhydride
(Swelam et al. 1999). The obtained methanimidates 6 and 4-
pyridinecarbohydrazide were refluxed in dioxane, to give
12-(4-substitutedphenyl)-2-(pyridin-4-yl)-8,9,10,11-tetra-
hydro-quinoline[2,3-d]-1,2,4-triazolo[5,1-f]pyrimidines 7.
Their 1H NMR spectra showed the CH-pyrimidine signal at
9.3, 9.25 and 9.32 ppm for 7a, 7b and 7c, respectively. The
IR spectra of the products 7 showed the absence of bands
corresponding to a CN group present in 6. Further, when a
well-stirred solution of 6 in ethanol hydrazine hydrate was
added, the mixture was stirred at room temperature to
afford 5-imino-6-substitutedphenyl-7,8,9,10-tetrahydropyr-
imido-[4,5-b]quinolin-4-amine 8. When a mixture of 4 and
pyridine-3-carbonitrile and sodium methoxide in isopropa-
nol was refluxed for 48 and 60 h, furnished the formation of
2-(pyridin-4-yl)-5-substitutedphenyl-6,7,8,9-tetrahydropyri-
mido[4,5-b]quinolin-4-amine 9 (Taylor and McKillop 1970).
Diazonium ion condensation with an adjacent nucleophilic
function to form a five- or six-membered ring has proved
valuable for synthesizing various nitrogen heterocycles.
Among these are numerous 1,2,3-triazines that are formed
via intramolecular attack of an electrophilic nitrogen func-
tion. Thus, 1,2,3-triazin-4-chloro or 4-hydroxy derivatives
are obtained from the condensation of diazonium ion with
an adjacent cyanide (Beck and Yahner 1976) or carbox-
amide (Ellis 1987), respectively. Thus, 1,2,3-triazines are
obtained from the condensation of diazonium ion with an
adjacent cyanide or carboxamide, respectively. When a
suspension of 4a, c in 20% alcoholic KOH solution was
refluxed for 10 h, the products 2-amino-4-(4-substituted-
2. Investigations, results and discussion
2.1. Synthesis of the compounds
The synthesis of 2-amino-3-cyanopyridines was performed
from arylidene-malononitriles and cyclohexanone in the pre-
sence of ammonium acetate using ethanol as a solvent (Attia
et al. 1995; Elkholy and Morsy 2006). The amount of ammo-
nium acetate was adjusted to get the maximum yield of the
products 4, it has been found that 8 mmol of the ammonium
Scheme 1 Reagents and conditions: (a) chloroformamidine hydrochlor-
ide, diglyme; (b) triethylorthoformate, acetic anhydride, reflux; (c) 4-pyri-
dinecarbohydrazide, dioxane, reflux; (d) hydrazine hydrate, ethanol, rt; (e)
3-pyridinecarbonitrile, sodium methoxide, 2-propanol, reflux
R
R
N
N
N
NH2
N
N
N
N
N
N
NH2
5a, R= Cl; b, R= F; c, R= OCH
7a, R= Cl; b, R= F; c, R= OCH
3
3
c
a
R
R
phenyl)-5,6,7,8-tetrahydroquinoline-3-carboxamides
10,
were obtained as white crystalline material. Their IR spec-
tra showed the absence of bands corresponding to CN
group present in 4 and the amide CO bands are well re-
presented by bands at 1669 and 1675 cmꢀ1 for 10a and
10b, respectively. Diazotization of the product 10 with so-
dium nitrite in concentrated H2SO4––HOAc gave the triazi-
no[4,5-b]quinolin-4-ol 11 (Scheme 2). Preparation of the
required the 4-chloro-[1,2,3]triazino[4,5-b]quinolines 12
using standard reagents such as thionyl chloride or phos-
phorous oxychloride led to decomposition of 11, which
could be attributed to the instability of the triazine ring
under these conditions. Nevertheless, the 4-chlorotriazines
12 were easily obtained from diazotization of an acetic
acid solution of 4 with sodium nitrite in concentrated hy-
drochloric acid. Treatment of 12 with 2-aminopyridine led
to normal halide displacement to give N-(pyridin-2-yl)-
[1,2,3]-triazino[4,5-b]quinolin-4-amines 13. Treatment of a
mixture of 4 and ethylene diamine with carbon disulfide
CN
CN
N
b
N
NH2
N
OEt
4a, R= Cl; b, R= F; c, R= OCH
6a, R= Cl; b, R= F; c, R= OCH
3
3
e
d
R
R
NH2
NH
N
N NH2
N
N
N
N
8a, R= Cl; b, R= F; c, R= OCH
9a, R= Cl; b, R= OCH
N
3
3
148
Pharmazie 64 (2009) 3

ORIGINAL ARTICLES
Scheme 2 Reagents and conditions: (a) 20% alcoholic KOH, reflux, 10 h; (b) sodium nitrite, H2SO4, acetic acid, ꢀ5 ꢁC; (c) thionyl chloride or phos-
phorous oxychloride, reflux; (d) sodium nitrite, conc. HCl, acetic acid, ꢀ5 ꢁC; (e) 2-aminopyridine, TEA, reflux; (f) ethylene diamine, carbon disulfide,
reflux; (g) sodium nitrite, 5% HCl, ꢀ5 ꢁC
a
f
d
R
N
R
N
R
N
O
Cl
N
N
H
NH2
NH2
NH2
N
N
10a,R= Cl; b, R= OCH
14a,R= Cl; b, R= F;c, R= OCH
12a,R= Cl; b, R= F;c, R= OCH
3
3
3
b
e
g
c
R
R
R
N
OH
HN
N
N
N
N
N
N
N
N
N
N
N
N
N
11a,R= Cl; b, R= OCH
13a,R= Cl; b, R= F;c, R= OCH
15a,R= Cl; b, R= F; c, R= OCH
3
3
3
and heating the reaction mixture on a steam bath for 8 h
afforded compound 14. The synthetic application of con-
densation of diazonium ion with an adjacent nitrogen
function was extended to the synthesis of the tetracyclic
15, which was prepared by the diazotization of the inter-
mediate 14. Thus, when a cold suspension of 14 in hydro-
chloric acid (5%) was treated with a solution of sodium
nitrite in water and stirring the reaction at room tempera-
ture furnished products 15.
docked compound was assigned a score according to its
fit in the ligand binding pocket (LBP)
The predicted binding energies of the compounds and the
corresponding experimental values are also listed in the
Table. The docking of ligand 3 and highly active molecule
9b in to the active site of p38 was performed (Fig. 1).
The docking results were indicative that; firstly, the para-
substitution of phenyl ring at position-5 by a methoxy
group will increase the hydrophobic binding interaction
with the deep hydrophobic pocket created by Thr106, Lys
53, Ala51 and Leu108. Secondly, the hydrogen bonding
interactions have been found, between the crucial features
of compounds with high docking scores and N––H group
of Lys53, C¼O of Tyr35 and Asp168. Moreover, replace-
ment of the methoxy group on para-position of phenyl
ring by halogen (electron withdrawing group) like Cl and
F will decrease the docking value.
Alignment study of docked compound 9b and ligand 3
with the binding pocket of p38 kinase protein (Fig. 1c)
revealed that (i) tetrahydroquinolin ring system was per-
fectly aligned with triazolopyridine nucleus of ligand 3 (ii)
p-methoxyphenyl side chain superimposed with p-fluro-
phenyl side chain of ligand 3 (iii) additionally, both the
ligand 3 and compound 9b make same hydrogen bonding
interaction with Asp 168.
The products obtained were well characterized by their MS,
IR and 1H NMR spectral analysis; additionally the elemental
analyses were in conformity with the respective structures.
2.2. Molecular docking studies of p38 kinase inhibitors
and binding conformation
This technique considered as direct molecular modeling
where the 3D structure of the enzyme was known which used
to detailed intermolecular interactions between the ligand
and the target protein. An automated docking study was car-
ried out using the crystal structure of inhibitor (3)/p38 kinase
complex obtained from protein data bank website (pdb) en-
ꢀ
try 1ZZL; having resolution of 2.0 A (Wilson et al. 1997).
This regularized protein complex structure was used in deter-
mination of the active site that is mentioned in the literature
(Wilson et al. 1997). The performance of the docking meth-
od on p38 inhibitors was evaluated by re-docking crystal li-
gand with 0.00621 RMSD value. Docking process was car-
ried out for the test set of compounds 4–15.
In the flexible-ligand-rigid enzyme docking, the enzyme
was represented by six potential energy maps, namely,
electrostatic, hydrogen bond, hydrophobic, and three van
der Waals. Interactive docking using Mol table ligand was
carried out for all the conformers of each compound of
the test set (4–15) to the selected active site of p38. Each
Docking results provide useful information in understand-
ing the structural features of the target and chemical fea-
tures of the ligands. This was extended to the successful
designing of highly active analogs of tetrahydroquinoline
derivatives against p38 MAP kinase.
2.3. Pharmacology
All the tested compounds showed potent anti-inflamma-
tory activities with potent prostaglandin inhibition at the
Pharmazie 64 (2009) 3
149

ORIGINAL ARTICLES
Table: Results of docking score and in vivo ulcerogenic and anti-inflammatory activity
Compd.
Acute toxicity
(LD50 mg
Ulcerogenic
Docking
score
(kcal/mol)
Anti-inflammatory activity
activity (UD50
kg––1 p.o.)
mg kg––1 i.p.)
Dose
% Inhibition of
% inhibition of
plasma PGE2
mg kg––1 p.o.
oedema
4a
4b
1786.28
1265.34
1237.45
1765.98
1398.34
1378.56
1247.45
1287.45
1675.38
1246.45
1295.56
1293.33
1234.67
1342.23
1782.87
1354.98
1753.99
1145.67
1245.33
1345.98
1298.98
1782.98
1565.78
2874.98
1765.98
1357.56
2345.87
234.5
287.6
213.6
223.9
209.7
234.7
245.6
235.7
214.87
243.5
236.5
236.8
214.6
223.76
245.6
254.8
200.56
234.16
234.5
238.8
265.8
243.6
245.7
211.4
213.4
213.4
66.7
ꢀ70.10
ꢀ78.88
ꢀ84.25
ꢀ68.93
ꢀ74.10
ꢀ61.00
ꢀ65.61
ꢀ66.76
ꢀ72.7
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
2.5
5
76.12c
87.00c
86.77b
97.88c
87.00a
88.87b
78.99c
88.99c
84.11a
94.60c
70.45c
81.00c
71.12c
81.46c
71.89c
81.50c
83.22a
93.11b
86.00a
96.00c
71.00c
81.00c
75.11b
85.09c
82.16b
92.11a
88.19a
99.78c
75.66b
86.66c
73.78a
83.55a
85.00a
95.00b
87.99b
98.99b
71.16c
81.84c
72.98b
82.98b
74.00b
84.33a
85.77b
95.00a
70.00c
81.00c
81.22c
91.14c
79.58c
89.99c
80.56c
90.12b
63.54c
73.43c
59.78c
72.00a
70.00c
82.00c
70.18c
82.16c
61.00c
73.00c
66.56a
79.87a
53.98c
71.00c
54.00b
73.56c
54.33b
74.32a
65.12a
78.54c
69.65c
81.50c
54.00b
72.00c
57.87c
70.63b
64.58c
77.76c
71.17c
83.40c
58.45c
71.09b
55.11b
68.65b
67.45c
80.65c
71.00c
83.00c
54.60c
75.00c
55.00b
76.09c
56.88a
69.55a
68.77b
81.00b
52.45b
70.12b
63.77c
76.98c
61.67b
74.30a
62.12c
75.39a
49.16a
65.23a
4c
5a
5c
6a
6b
6c
7b
7c
ꢀ76.72
ꢀ64.43
ꢀ69.17
ꢀ71.46
ꢀ91.87
ꢀ67.8
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13a
13c
14a
14b
15a
15b
Indomethacin
ꢀ69.21
ꢀ75.12
ꢀ85.30
ꢀ67.41
ꢀ67.92
ꢀ70.10
ꢀ75.40
ꢀ65.12
ꢀ71.32
ꢀ70.25
ꢀ70.40
ꢀ
2.5
5
2.5
5
2.5
5
a P < 0.05; b P < 0.01; c P < 0.001
two dose levels tested. They are more potent than indo-
methacin and the degree of potency in descending order is
9b, 11b, 4b, 7c, 13c, 11a, 5c, 7b, 9a, 14b, 15b, 15a, 5a,
4c, 4a, 10a, 8b, 10b, 12b, 12a, 6c, 6b, 6a, 8a and 14a.
The tested compounds of this series showed LD50 values
above 1100 mg kg––1 p.o., with maximum in compound
14b (2875 mg kg––1 p.o). All the results are depicted in
the Table. Random screening of compounds and reference
drug, indomethacin, was performed at two dose levels 2.5
and 5 mg kg––1 p.o.
The ulcerogenic activity (UD50 mg kg––1 i.p.) of the tested
compounds and indomethacin are listed in the Table. All
compounds are safer than indomethacin due to high ul-
cerogenic causing doses that were above 200 mg kg––1 i.p.
3. Experimental
3.1. Chemistry
Melting points were determined in open glass capillaries using an Electro-
thermal IA 9000 SERIES digital melting point apparatus (Electrothermal,
150
Pharmazie 64 (2009) 3

ORIGINAL ARTICLES
(a)
(b)
Fig. 1:
(c)
(a) and (b): the proposed binding mode of li-
gand 3 and compound 9b inside the active site
of p38 MAP kinase resulting from docking,
respectively. The most important amino acids
are shown together with their respective num-
bers. Compound 9b form three hydrogen
bonds with Lys53, Tyr35 and Asp168. (c):
Alignment of docked compound 9b and li-
gand 3 with the binding pocket
UK) and are uncorrected. Microanalyses were performed with all final
compounds on an Elementar-Vario EL (Elementar-Vario EL, Germany)
(Micro-analytical Unit, Central Services Laboratory, National Research
Centre, Cairo; Egypt). Analyses of C, H, N were found to be within
acceptable limits of theoretical values. The 1H NMR spectra were re-
corded on a Varian Mercury VX-300 NMR spectrometer (Varian, USA).
1H NMR spectra were run at 300 MHz in DMSO-d6 as solvent. Chemi-
cal shifts d are quoted in ppm and were related to that of the solvents.
Mass spectra were recorded on a Shimadzu GCMS-QP 1000EX (EI,
70 eV) (Shimadzu, Japan) and Hewlett-Packard (EI, 70 eV) (Hewlett-
Packard, USA). IR spectra were obtained with a Bruker-Vector 22 (Bru-
ker, Rheinstetten, Germany). All the reactions were monitored using thin
layer chromatography (TLC) using silica gel aluminum sheets 60F254
(Merck).
3.1.1.2. 2-Amino-4-(4-fluorophenyl)-5,6,7,8-tetrahydroquinoline-3-carboni-
trile (4b)
Yield: 14.7 g (55%), m.p. 262–263 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 3410, 3300
(NH2), 2220 (CN). 1H NMR (DMSO-d6, ppm): 1.55–1.76 (m, 4 H, 2 CH2),
2.27–2.35 (m, 2 H, CH2), 2.61–2.7 (m, 2 H, CH2), 6.6 (s, 2 H, NH2, exchange-
able with D2O), 7.2–7.45 (m, 4 H, Ar––H). MS, m/z (%): 268 [Mþ þ 1] (100).
C
16H14FN3 (267.30)
3.1.1.3. 2-Amino-4-(4-methoxyphenyl)-5,6,7,8-tetrahydroquinoline-3-car-
bonitrile (4c)
Yield: 12 g (43%); m.p. 232–233 ꢁC (Dioxane/H2O). IR (KBr, cmꢀ1):
3437, 3315 (NH2), 2210 (CN). 1H NMR (DMSO-d6, ppm): 1.58–1.75 (m,
4 H, 2CH2), 2.31–2.38 (m, 2 H, CH2), 2.69–2.75 (m, 2 H, CH2), 3.85 (s,
3 H, CH3), 6.45 (s, 2 H, NH2, exchangeable with D2O), 7.0 (d, 2 H,
Ar––H), 7.2 (d, 2 H, Ar––H). MS, m/z (%): 279 [Mþ] (100).
C17H17N3O (279.34)
3.1.1. General procedure for the synthesis of compounds 4a–c
A mixture of malononitrile (6.6 g, 100 mmol) and the proper aldehyde
(100 mmol) in absolute ethanol (250 ml) was refluxed for 3 h. After addi-
tion of cylohexanone 10.3 ml (9.8 g, 100 mmol), and ammonium acetate
(30.8 g, 400 mmol) the reaction mixture was refluxed for 5 h. The reaction
mixture was concentrated to its volume and left to cool. The separated
solid was filtered off, washed with aqueous ethanol (1 : 1), dried and crys-
tallized from the proper solvent to yield compounds 4a–c.
3.1.2. General procedure for the synthesis of compounds 5a–c
A mixture of 4a–c (5 mmol) and chloroformamidine hydrochloride (0.7 g,
6 mmol) in diglyme (5 ml) was stirred at 130–140 ꢁC for 5 h. The reaction
mixture was left to cool to room temperature. The obtained solid mass was
diluted with dioxane and filtered. The crude hydrochloride was suspended
in water, neutralized with TEA, filtered off, washed with water, dried, and
crystallized from DMF/H2O to give 5a–c, as a white powder.
3.1.1.1. 2-Amino-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-3-carboni-
trile (4a)
3.1.2.1. 5-(4-Chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline-
2,4-diamine (5a)
Yield: 17.6 g (62%), m.p. 258–259 ꢁC (Dioxane/H2O). IR (KBr, cmꢀ1):
3422, 3303 (NH2), 2212 (CN). 1H NMR (DMSO-d6, ppm): 1.55–1.7 (m,
4 H, 2 CH2), 2.29–2.38 (m, 2 H, CH2), 2.56–2.74 (m, 2 H, CH2), 6.6 (s,
2 H, NH2, exchangeable with D2O), 7.35 (d, 2 H, Ar––H), 7.55 (d, 2 H,
Ar––H). MS, m/z (%): 283 [Mþ] (100).
Yield: 0.82 g (50%), m.p. 283–285 ꢁC. IR (KBr, cmꢀ1): 3422, 3395, 3255,
3117 (NH2). 1H NMR (DMSO-d6, ppm): 1.54–1.7 (m, 4 H, 2 CH2), 2.34–
2.39 (m, 2 H, CH2), 2.69–2.74 (m, 2 H, CH2), 6.2 (s, 2 H, NH2, exchange-
able with D2O), 7.18 (s, 2 H, NH2, exchangeable with D2O), 7.25–7.32
(m, 4 H, Ar––H); MS, m/z (%): 326 [Mþ þ 1] (100).
C
16H14ClN3 (283.76)
C17H16ClN5 (325.80)
Pharmazie 64 (2009) 3
151

ORIGINAL ARTICLES
3.1.2.2. 5-(4-Fluorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline-2,4-
3.1.4.3. 12-(4-Methoxyphenyl)-2-(pyridin-4-yl)-8,9,10,11-tetrahydro-quino-
diamine (5b)
line[2,3-d]-1,2,4-triazolo[5,1-f]pyrimidine (7c)
Yield: 0.65 g (42%), m.p. 304 ꢁC (decomp.). IR (KBr, cmꢀ1): 3410, 3300,
3125 (NH2). 1H NMR (DMSO-d6, ppm): 1.55–1.76 (m, 4 H, 2 CH2),
2.28–2.33 (m, 2 H, CH2), 2.63–2.71 (m, 2 H, CH2), 6.1 (s, 2 H, NH2,
exchangeable with D2O), 6.9 (s, 2 H, NH2, exchangeable with D2O), 7.4
(m, 4 H, Ar––H). MS, m/z (%): 310 [Mþ þ 1] (100).
Yield: 0.35 g (43%); mp: 263–265 ꢁC (decomp., dioxane/H2O). IR (KBr,
cmꢀ1): 2925, 2850 (CH2), 1612 (H¼CN). 1H NMR (DMSO-d6, ppm):
1.57–1.75 (m, 4 H, 2 CH2), 2.31–2.39 (m, 2 H, CH2), 2.66–2.73 (m, 2 H,
CH2), 3.85 (s, 3 H, CH3), 7.13 (d, 2 H, Ar––H), 7.38 (d, 2 H, Ar––H), 7.67
(d, 2 H, pyridine-H), 8.65 (d, 2 H, pyridine-H), 9.32 (s, 1 H, pyrimidine-H).
MS, m/z (%) 409 [Mþ þ 1] (100).
C
17H16FN5 (309.34)
C24H20N6O (408.46)
3.1.2.3. 5-(4-Methoxyphenyl)-6,7,8,9-tetrahydropyrimido[4,5-b]quinoline-
2,4-diamine (5c)
3.1.5. General procedure for the synthesis of compounds 8a–c
Yield: 0.63 g (39%), m.p. 296 ꢁC (decomp.). IR (KBr, cmꢀ1): 3400, 3336,
To a well-stirred solution of methaneimidates 6a–c (3 mmol) in C2H5OH
(25 ml), hydrazine hydrate (3 ml) was added, and then the mixture was
stirred at room temperature until a solid started to be formed. Stirring was
continued for additional 2 h; the solid formed was filtered off and crystal-
lized from the proper solvent to afford 8a–c, as a white powder.
1
3217, 3100 (NH2). H NMR (DMSO-d6, ppm): 1.57–1.74 (m, 4 H, 2 CH2),
2.32–2.37 (m, 2 H, CH2), 2.67–2.73 (m, 2 H, CH2), 3.68 (s, 3 H, CH3), 5.92 (s,
2 H, NH2, exchangeable with D2O), 7.0 (s, 2 H, NH2, exchangeable with D2O),
7.05 (d, 2 H, Ar––H), 7.32 (d, 2 H, Ar––H). MS, m/z (%): 322 [Mþ þ 1] (100).
C
18H19N5O (321.38)
3.1.5.1. 5-Imino-6-(4-chlorophenyl)-7,8,9,10-tetrahydropyrimido[4,5-b]-
quinolin-4-amine (8a)
3.1.3. General procedure for the synthesis of compounds 6a–c
A mixture of o-aminonitriles 4a–c (10 mmol), triethylorthoformate (25 ml)
and acetic anhydride (0.3 ml) was refluxed for 5 h. The solvent was re-
moved under reduced pressure nearly to half volume and left to cool at
room temperature. The resulting solid was filtered off, dried and crystal-
lized to yield compounds 6a–c, as white crystals.
Yield: 0.9 g (92%), m.p. 248–250 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3420, 3144, 3100 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.52–1.69 (m,
4 H, 2 CH2), 2.37–2.44 (m, 2 H, CH2), 2.71–2.78 (m, 2 H, CH2), 6.66 (s,
2 H, NH2, exchangeable with D2O), 7.35–7.42 (m, 4 H, Ar––H), 8.48 (s,
1 H, pyrimidine-H), 8.85 (s, 1 H, NH, exchangeable with D2O). MS, m/z
(%): 326 [Mþ þ 1] (100).
3.1.3.1. 2-Ethoxymethyleneamino-4-(4-chlorophenyl)-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (6a)
C
17H16ClN5 (325.8)
Yield: 2.65 g (78%), m.p. 145–147 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 2940,
2830 (CH2), 2210 (CN), 1597 (N¼CH). 1H NMR (DMSO-d6, ppm): 1.17
(t, 3 H, CH3), 1.52–1.69 (m, 4 H, 2 CH2), 2.38 (t, 2 H, CH2), 2.73 (t, 2 H,
CH2), 4.25 (q, 2 H, CH2), 7.15–7.3 (m, 4 H, Ar––H), 7.48 (s, 1 H, N¼CH).
MS, m/z (%): 340 [Mþ þ 1] (37), 294 [Mþ––OC2H5] (78), 284 (17), 77
(100).
3.1.5.2. 5-Imino-6-(4-fluorophenyl)-7,8,9,10-tetrahydropyrimido[4,5-b]-qui-
nolin-4-amine (8b)
Yield: 0.8 g (86%), m.p. 285–287 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3400, 3132, 3112 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.53–1.69 (m,
4 H, 2 CH2), 2.39–2.47 (m, 2 H, CH2), 2.66–2.75 (m, 2 H, CH2), 6.75 (s,
2 H, NH2, exchangeable with D2O), 7.17–7.32 (m, 4 H, Ar––H), 8.27 (s,
1 H, pyrimidine-H), 8.9 (s, 1 H, NH, exchangeable with D2O). MS, m/z
(%): 310 [Mþ þ 1] (100).
C
19H18ClN3O (339.82)
3.1.3.2. 2-Ethoxymethyleneamino-4-(4-fluorophenyl)-5,6,7,8-tetrahydro-q-
uinoline-3-carbonitrile (6b)
C17H16FN5 (309.34)
Yield: 2.1 g (64%), m.p. 173–175 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 2925,
2852 (CH2), 2220 (CN), 1610 (N¼CH). 1H NMR (DMSO-d6, ppm): 1.22
(t, 3 H, CH3), 1.53–1.72 (m, 4 H, 2CH2), 2.36 (t, 2 H, CH2), 2.75 (t, 2 H,
CH2), 4.25 (q, 2 H, CH2), 6.95–7.2 (m, 4 H, Ar––H), 7.81 (s, 1 H, N¼CH).
MS, m/z (%): 324 [Mþ þ 1] (100).
3.1.5.3. 5-Imino-6-(4-methoxyphenyl)-7,8,9,10-tetrahydropyrimido[4,5-b]
quinolin-4-amine (8c)
Yield: 0.72 g (75%), m.p. 255–257 ꢁC (decomp.; C2H5OH). IR (KBr,
cmꢀ1): 3452, 3128, 3098 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.57–
1.75 (m, 4 H, 2CH2), 2.31–2.39 (m, 2 H, CH2), 2.66–2.73 (m, 2 H, CH2),
3.87 (s, 3 H, CH3), 6.35 (s, 2 H, NH2, exchangeable with D2O), 7.13 (d,
2 H, Ar––H), 7.45 (d, 2 H, Ar––H), 8.27 (s, 1 H, pyrimidine-H), 8.65 (s,
1 H, NH, exchangeable with D2O). MS, m/z (%): 322 [Mþ þ 1] (100).
C18H19N5O (321.38)
C19H18FN3O (323.36)
3.1.3.3. 2-Ethoxymethyleneamino-4-(4-methoxyphenyl)-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (6c)
Yield: 2.1 g (63%), m.p. 138–140 ꢁC (decomp.; 2-propanol). IR (KBr, cmꢀ1):
2925, 2850 (CH2), 2210 (CN), 1595 (N¼CH). 1H NMR (DMSO-d6, ppm): 1.17
(t, 3 H, CH3), 1.57–1.74 (m, 4 H, 2 CH2), 2.3–2.37 (m, 2 H, CH2), 2.68–2.73
(m, 2 H, CH2), 3.85 (s, 3 H, CH3), 4.25 (q, 2 H, CH2), 7.14 (d, 2 H, Ar––H), 7.42
(d, 2 H, Ar––H), 7.87 (s, 1 H, N¼CH). MS, m/z (%): 336 [Mþ þ 1] (100).
3.1.6. General procedure for the synthesis of compounds 9a, b
A mixture of 4a, c (10 mmol) and pyridine-3-carbonitrile (1.05 g, 10 mmol)
and sodium methoxide (1.08 g, 20 mmol) in 2-propanol (50 ml) was refluxed
for 48 h or 60 h, respectively. The solvent was removed under reduced pres-
sure and the residue was stirred with ice water. The crude product was filtered
off, washed thoroughly with water, C2H5OH (50%), dried and crystallized
from the proper solvent to yield compounds 9a, b, as white powders.
C
20H21N3O2 (335.4)
3.1.4. General procedure for the synthesis of compounds 7a–c
A mixture of methanimidates 6a–c (2 mmol) and 4-pyridinecarbohydra-
zide (2 mmol) in dioxane (25 ml) was refluxed for 10 h. After cooling, the
obtained solid was filtered off, dried and crystallized from the proper sol-
vent to afford compounds 7a–c, as yellowish white powders.
3.1.6.1. 2-(Pyridin-3-yl)-5-(4-chlorophenyl)-6,7,8,9-tetrahydropyrimido[4,5-
b]-quinolin-4-amine (9a)
Yield: 2.9 g (75%), m.p. 284–286 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3477, 3137 (NH2), 2945 (CH2), 1626 (HC¼N). 1H NMR (DMSO-d6,
ppm): 1.53–1.71 (m, 4 H, 2CH2), 2.37–2.44 (m, 2 H, CH2), 2.71–2.78 (m,
2 H, CH2), 6.2 (s, 2 H, NH2, exchangeable with D2O), 7.35–7.42 (m, 5 H,
Ar––H þ pyridine-H), 7.97 (m, 1 H, pyridine-H), 8.47 (d, 1 H, pyridine-H),
8.92 (s, 1 H, pyridine-H). MS, m/z (%): 388 [Mþ þ 1] (7), 171 (100).
C22H18ClN5 (387.86)
3.1.4.1. 12-(4-Chlorophenyl)-2-(pyridin-4-yl)-8,9,10,11-tetrahydroquino-
line-[2,3-d]-1,2,4-triazolo[5,1-f]pyrimidine (7a)
Yield: 0.41 g (50%), m.p. 227–229 ꢁC (dioxane). IR (KBr, cmꢀ1): 2940,
2830 (CH2), 1620 (H¼CN). 1H NMR (DMSO-d6, ppm): 1.52–1.69 (m,
4 H, 2 CH2), 2.37–2.44 (m, 2 H, CH2), 2.71–2.78 (m, 2 H, CH2), 7.36–
7.55 (m, 6 H, Ar––H þ pyridine-H), 8.65 (d, 2 H, pyridine-H), 9.3 (s, 1 H,
Pyrimidine-H). MS, m/z (%): 413 [Mþ þ 1] (100).
C
23H17ClN6 (412.87)
3.1.6.2. 2-(Pyridin-3-yl)-5-(4-methoxyphenyl)-6,7,8,9-tetrahydropyri-
mido[4,5-b]quinolin-4-amine (9b)
3.1.4.2. 12-(4-Fluorophenyl)-2-(pyridin-4-yl)-8,9,10,11-tetrahydroquino-
line-[2,3-d]-1,2,4-triazolo[5,1-f]pyrimidine (7b)
Yield: 0.72 g (75%), m.p. 255–257 ꢁC (dec.; C2H5OH). IR (KBr, cmꢀ1):
3409, 3112 (NH2), 2929 (CH2), 1619 (H¼CN). 1H NMR (DMSO-d6,
ppm): 1.57–1.75 (m, 4 H, 2 CH2), 2.31–2.39 (m, 2 H, CH2), 2.66–2.73
(m, 2 H, CH2), 3.67 (s, 3 H, CH3), 5.6 (s, 2 H, NH2, exchangeable with
D2O), 7.15 (d, 2 H, Ar––H), 7.41 (m, 3H, Ar––H þ pyridine-H), 7.95 (m,
1 H, pyridine-H), 8.45 (d, 1 H, pyridine-H), 8.9 (s, 1 H, Pyridine-H). MS,
m/z (%): 384 [Mþ þ 1] (100).
Yield: 0.33 g (42%), m.p. 241–243 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
2925, 2852 (CH2), 1627 (H¼CN). 1H NMR (DMSO-d6, ppm): 1.53–1.67
(m, 4 H, 2 CH2), 2.37–2.46 (m, 2 H, CH2), 2.66–2.75 (m, 2 H, CH2),
7.14–7.62 (m, 6 H, Ar––H þ pyridine-H), 8.62 (d, 2 H, pyridine-H), 9.25
(s, 1 H, pyrimidine-H). MS, m/z (%): 397 [Mþ þ 1] (100).
C
23H17FN6 (396.42)
C23H21N5O (383.45)
152
Pharmazie 64 (2009) 3

ORIGINAL ARTICLES
3.1.7. General procedure for the synthesis of compounds 10a, b
3.1.9.3. 4-Chloro-5-(4-methoxyphenyl)-6,7,8,9-tetrahydro-[1,2,3]-triazi-
no[4,5-b]quinoline (12c)
A suspension of 4a, c (10 mmol) in 20% alcoholic KOH solution (100 ml)
was refluxed for 10 h. After cooling, H2O (30 ml) was added to the reac-
tion mixture and the whole was left to stand overnight. The separated
white crystalline product was filtered off, dried and crystallized from
DMF/H2O to afford 10a, b.
Yield: 1.8 g (56%), m.p. 188–190 ꢁC. IR (KBr, cmꢀ1): 3090 (CH-aro-
matic), 2986 (CH-aliphatic), 1637 (C¼N). 1H NMR (DMSO-d6, ppm):
1.57–1.73 (m, 4 H, 2 CH2), 2.29–2.35 (m, 2 H, CH2), 2.69–2.75 (m, 2 H,
CH2), 3.87 (s, 3 H, CH3), 7.23 (d, 2 H, Ar––H), 7.57 (d, 2 H, Ar––H). MS,
m/z (%): 327 [Mþ þ 1] (100).
C
17H15ClN4O (326.78)
3.1.7.1. 2-Amino-4-(4-chlorophenyl)-5,6,7,8-tetrahydroquinoline-3-carbox-
amide (10a)
3.1.10. General procedure for the synthesis of compounds 13a–c
Yield: 1.97 g (64%), m.p. 292–294 ꢁC. IR (KBr, cmꢀ1): 3594, 3335
(NH2), 1669 (C¼O). 1H NMR (DMSO-d6, ppm): 1.53–1.71 (m, 4 H,
2 CH2), 2.37–2.44 (m, 2 H, CH2), 2.71–2.78 (m, 2 H, CH2), 5.4 (s, 2 H,
NH2, exchangeable with D2O), 6.34 (s, 2 H, NH2, exchangeable with
D2O), 7.28–7.34 (m, 4 H, Ar––H). MS, m/z (%): 302 [Mþ þ 1] (100).
A mixture of 12a–c (3 mmol), 2-aminopyridine (0.28 g, 3 mmol) and TEA
(3 ml) in dioxane (50 ml) was refluxed for 4 h. The reaction mixture was
left to cool, poured onto ice-water and then treated with few drops of HCl.
The crude precipitate was filtered off, washed thoroughly with H2O, dried
and crystallized from the proper solvent to afford 13a–c, as a yellowish
white powder.
C
16H16ClN3O (301.77)
3.1.7.2. 2-Amino-4-(4-methoxyphenyl)-5,6,7,8-tetrahydroquinoline-3-carb-
oxamide (10b)
3.1.10.1. 5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-N-(pyridin-2-yl)[1,2,3]-tri-
azino[4,5-b]quinolin-4-amine (13a)
Yield: 1.6 g (54%), m.p. 272–274 ꢁC. IR (KBr, cmꢀ1): 3535, 3290 (NH2),
1675 (C¼O). 1H NMR (DMSO-d6, ppm): 1.57–1.75 (m, 4 H, 2 CH2),
2.31–2.39 (m, 2 H, CH2), 2.66–2.73 (m, 2 H, CH2), 3.67 (s, 3 H, CH3),
5.6 (s, 2 H, NH2, exchangeable with D2O), 6.37 (s, 2 H, NH2, exchange-
able with D2O), 7.15 (d, 2 H, Ar––H), 7.41 (m, 2 H, Ar––H). MS, m/z (%):
298 [Mþ þ 1] (100).
Yield: 0.65 g (56%), m.p. 319–321 ꢁC (DMF/H2O). IR (KBr, cmꢀ1): 3345
(NH). 1H NMR (DMSO-d6, ppm): 1.54–1.68 (m, 4 H, 2 CH2), 2.27–2.37
(m, 2 H, CH2), 2.56–2.73 (m, 2 H, CH2), 6.7 (d, 1 H, pyridine-H), 6.81 (d,
1 H, pyridine-H), 7.3–7.48 (m, 5 H, Ar––H þ pyridine-H), 8.21 (d, 1 H,
pyridine-H), 8.39 (s, 1 H, NH, exchangeable with D2O). MS, m/z (%): 389
[Mþ þ 1] (100).
C17H19N3O2 (297.35)
C21H17ClN6 (388.85)
3.1.8. General procedure for the synthesis of compounds 11a, b
3.1.10.2. 5-(4-Fluorophenyl)-6,7,8,9-tetrahydro-N-(pyridin-2-yl)[1,2,3]-tri-
azino[4,5-b]quinolin-4-amine (13b)
To a cold solution of NaNO2 (0.21 g, 3.1 mmol) in conc. H2SO4 (5 ml) a
suspension of the 2-amino-3-carbamoyl derivatives 10a, b (3 mmol) in
AcOH (20 ml) was slowly added. Stirring was continued for 1 h. It was
then filtered and the filtrate was poured onto ice. The crude product was
filtered, dissolved in NaOH (5%), treated with charcoal, filtered, and acid-
ified with AcOH, giving compound 11a, b.
Yield: 0.36 g (32%), m.p. 224–226 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3390 (NH). 1H NMR (DMSO-d6, ppm): 1.54–1.73 (m, 4 H, 2 CH2), 2.26–
2.33 (m, 2 H, CH2), 2.6–2.68 (m, 2 H, CH2), 6.65 (d, 1 H, pyridine-H),
6.79 (t, 1 H, pyridine-H), 7.3–7.43 (m, 3 H, Ar––H þ pyridine-H), 8.21 (d,
1 H, pyridine-H), 7.51 (d, 2 H, Ar––H), 8.54 (s, 1 H, NH, exchangeable
with D2O). MS, m/z (%): 373 [Mþ þ 1] (100).
C
21H17FN6 (372.4)
3.1.8.1. 5-(4-Chlorophenyl)-6,7,8,9-tetrahydro-[1,2,3]-triazino[4,5-b]
quinolin-4-ol (11a)
3.1.10.3. 5-(4-Methoxyphenyl)-6,7,8,9-tetrahydro-N-(pyridine-2-yl)[1,2,3]-
triazino-[4,5-b]quinolin-4-amine (13c)
Yield: 0.32 g (34%), m.p. 183–185 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3227 (br, OH). 1H NMR (DMSO-d6, ppm): 1.53–1.71 (m, 4 H, 2 CH2),
2.37–2.44 (m, 2 H, CH2), 2.71–2.78 (m, 2 H, CH2), 7.26 (d, 2 H, Ar––H),
7.35 (d, 2 H, Ar––H), 9.1 (br s, 1 H, OH, exchangeable with D2O). MS, m/z
(%): 313 [Mþ þ 1] (100).
Yield: 0.35 g (59%), m.p. 218–220 ꢁC (dioxane/H2O). IR (KBr, cmꢀ1):
3298 (NH). 1H NMR (DMSO-d6, ppm): 1.57–1.73 (m, 4 H, 2 CH2), 2.29–
2.35 (m, 2 H, CH2), 2.69–2.75 (m, 2 H, CH2), 3.87 (s, 3 H, CH3), 6.65 (d,
1 H, Pyridine-H), 6.79 (t, 1 H, Pyridine-H), 7.23 (d, 2 H, Ar––H), 7.3–7.43
(m, 3 H, Ar––H þ pyridine-H), 8.19 (d, 1 H, Pyridine-H), 8.41 (s, 1 H, NH,
exchangeable with D2O). MS, m/z (%): 385 [Mþ þ 1] (100).
C
16H13ClN4O (312.75)
3.1.8.2. 5-(4-Methoxyphenyl)-6,7,8,9-tetrahydro-[1,2,3]-triazino[4,5-b]-
quinolin-4-ol (11b)
C
22H20N6O (384.43)
Yield: 0.5 g (54%), m.p. 146–148 ꢁC (2-propanol/H2O). IR (KBr, cmꢀ1):
3214 (br, OH). 1H NMR (DMSO-d6, ppm): 1.57–1.75 (m, 4 H, 2 CH2),
2.31–2.39 (m, 2 H, CH2), 2.66–2.73 (m, 2 H, CH2), 3.67 (s, 3 H, CH3),
7.15 (d, 2 H, Ar––H), 7.41 (m, 2 H, Ar––H), 9.24 (s, 1 H, OH, exchange-
able with D2O). MS, m/z (%): 309 [Mþ þ 1] (100).
3.1.11. General procedure for the synthesis of compounds 14a–c
To a mixture of 4a–c (10 mmol) and ethylene diamine (1.19 g, 1.3 ml;
20 mmol) was added drop-wisely carbon disulfide (2.28 g, 1.8 ml,
30 mmol). The reaction mixture was heated on a steam bath for 8 h. After
cooling, it was triturated with water and the separated solid was filtered off,
dried and crystallized from the proper solvent to yield compounds 14a–c.
C
17H16N4O2 (308.33)
3.1.9. General procedure for the synthesis of compounds 12a–c
3.1.11.1. 4-(4-Chlorophenyl)-5,6,7,8-tetrahyro-3-(4,5-dihydro-1H-imidazo-
lin-2-yl)quinolin-2-amine (14a)
To a well-stirred cold suspension (ꢀ5 ꢁC) of 4a–c (10 mmol) in conc. HCl
(20 ml) and AcOH (20 ml) a solution of NaNO2 [1 g, 12 mmol/water
(10 ml)] was added drop-wise. After addition, the ice bath was removed
and stirring was continued at room temperature for 2 additional hours. The
crude products were crystallized from dioxane/H2O to give 12a–c.
Yield: 2.1 g (64%), m.p. 234–236 ꢁC (2-propanol). IR (KBr, cmꢀ1): 3435,
3292, 3200 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.55–1.7 (m, 4 H,
2 CH2), 2.29–2.38 (m, 2 H, CH2), 2.56–2.74 (m, 2 H, CH2), 3.54 (m, 4 H,
2 CH2), 6.63 (s, 2 H, NH2, exchangeable with D2O), 7.34 (d, 2 H, Ar––H),
7.54 (d, 2 H, Ar––H), 8.39 (s, 1 H, NH, exchangeable with D2O). MS, m/z
(%): 327 [Mþ þ 1] (100).
3.1.9.1. 4-Chloro-5-(4-chlorophenyl)-6,7,8,9-tetrahydro-[1,2,3]-triazi-
no[4,5-b]-quinoline (12a)
C
18H19ClN4 (326.82)
Yield: 1.85 g (56%), m.p. 297–299 ꢁC. IR (KBr, cmꢀ1): 3088 (CH-aro-
matic), 2987 (CH-aliphatic), 1638 (C¼N). 1H NMR (DMSO-d6, ppm):
1.53–1.67 (m, 4 H, 2 CH2), 2.25–2.34 (m, 2 H, CH2), 2.55–2.73 (m, 2 H,
CH2), 7.35 (d, 2 H, Ar––H), 7.55 (d, 2 H, Ar––H). MS, m/z (%): 332
[Mþ þ 1] (100).
3.1.11.2. 4-(4-Fluorophenyl)-5,6,7,8-tetrahyro-3-(4,5-dihydro-1H-imidazo-
lin-2-yl)quinolin-2-amine (14b)
Yield: 2 g (67%), m.p. 245–247 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 3439,
3288, 3230 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.55–1.76 (m, 4 H,
2 CH2), 2.27–2.35 (m, 2 H, CH2), 2.61–2.7 (m, 2 H, CH2), 3.55 (m, 4 H,
2 CH2), 6.45 (s, 2 H, NH2, exchangeable with D2O), 7.36 (m, 4 H, Ar––H),
8.35 (s, 1 H, NH, exchangeable with D2O). MS, m/z (%): 310 [Mþ] (100).
C18H19FN4 (310.37)
C
16H12Cl2N4 (331.2)
3.1.9.2. 4-Chloro-5-(4-fluorophenyl)-6,7,8,9-tetrahydro-[1,2,3]-triazi-
no[4,5-b]-quinoline (12b)
Yield: 1.7 g (55%), m.p. 275–277 ꢁC. IR (KBr, cmꢀ1): 3090 (CH-aromatic),
2991 (CH-aliphatic), 1641 (C¼N). 1H NMR (DMSO-d6, ppm): 1.54–1.73
(m, 4 H, 2 CH2), 2.26–2.33 (m, 2 H, CH2), 2.6–2.68 (m, 2 H, CH2), 7.43 (d,
2 H, Ar––H), 7.52 (d, 2 H, Ar––H). MS, m/z (%): 315 [Mþ þ 1] (100).
3.1.11.3. 4-(4-Methoxyphenyl)-5,6,7,8-tetrahyro-3-(4,5-dihydro-1H-imida-
zolin-2-yl)quinolin-2-amine (14c)
Yield: 2.2 g (70%), m.p. 267–269 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 3420,
C
16H12ClFN4 (314.74)
3278, 3112 (NH2, NH). 1H NMR (DMSO-d6, ppm): 1.58–1.75 (m, 4 H,
Pharmazie 64 (2009) 3
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ORIGINAL ARTICLES
2 CH2), 2.31–2.38 (m, 2 H, CH2), 2.69–2.75 (m, 2 H, CH2), 3.52 (m, 4 H,
paw and served as control. Four hours after drug administration the animal
was decapitated, blood was collected and the paws were rapidly excised.
The average weight of edema was estimated for the treated as well as the
control group and the percentage inhibition of weight of edema was also
evaluated; then percentage protection against edema was estimated (Table).
Indomethacin (2.5 and 5 mg/kg) was employed as standard reference
against which the test compounds were compared.
2 CH2), 3.85 (s, 3 H, CH3), 6.45 (s, 2 H, NH2, exchangeable with D2O),
7.11 (d, 2 H, Ar––H), 7.36 (d, 2 H, Ar––H), 8.47 (s, 1 H, NH, exchangeable
with D2O). MS, m/z (%): 322 [Mþ] (100).
C
19H22N4O (322.4)
3.1.12. General procedure for the synthesis of compounds 15a–c
To an ice cold stirred suspension of 14a–c (5 mmol) in 5% HCl
(20 ml) a solution of NaNO2 (0.37 g, 5.5 mmol) in H2O (5 ml) was
slowly added. After addition was completed, the reaction mixture was
stirred at room temperature for 1 h. It was then neutralized with
1N NaHCO3 and kept in a freezer. The solid formed was collected by
filtration, washed with water and dried. It was dissolved in CHCl3, trea-
ted with charcoal, filtered and the solvent was removed under reduced
pressure. The obtained residue was solidified by trituration with ether,
and the obtained solid was crystallized from the proper solvent to af-
ford compounds 15a–c.
3.3.3. Estimation of plasma prostaglandin E2 (PGE2)
Heparinized blood samples were collected from rats (n ¼ 8), plasma was
separated by centrifugation at 12 000 ꢂ g for 2 min at 4 ꢁC and immedi-
ately stored frozen 20 ꢁC until use.
The designs correlate-EIA prostaglandin in E2 (PGE2) kit is a competitive
immune assay for the quantitative determination of PGE2 in biological
fluids. The kit uses a monoclonal antibody to PGE2 to bind, in a competi-
tive manner, the PGE2 in the sample. After a simultaneous incubation at
room temperature the excess reagents were washed away and the substrate
was added. After a short incubation time the enzyme reaction was stopped
and the yellow color generated was read on a micro plate reader (DY-
NATCH, MR 5000) at 405 nm. The intensity of the bound yellow color is
inversely proportional to the concentration of PGE2 in either standard or
samples. The percentage inhibition of plasma PGE2 for each compound
was estimated (Table) (Herrmann et al. 1990; Weithmann et al. 1994).
3.1.12.1. 8-Chlorophenyl-5,6,9,10,11,12-hexahydroimidazo[20,10 : 6,5]
[1,2,3]-triazino[4,5-b]quinoline (15a)
Yield: 0.7 g (42%), m.p. 183–185 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 3112
1
(CH-aromatic), 2933 (CH-aliphatic). H NMR (DMSO-d6, ppm): 1.55–1.7
(m, 4 H, 2 CH2), 2.29–2.38 (m, 2 H, CH2), 2.56–2.74 (m, 2 H, CH2), 3.54
(m, 4 H, 2 CH2), 7.33 (d, 2 H, Ar––H), 7.52 (d, 2 H, Ar––H). MS, m/z (%):
338 [Mþ þ 1] (100).
3.3.4. Evaluation of acute toxicity study
The test compounds were administered orally at different dose levels in
separate groups of animals. After 24 h of drug administration percent mor-
tality in each group was observed. From the data obtained, the lethal dose
(LD50) was calculated by the method of Smith (1960).
C18H16ClN5 (337.81)
3.1.12.2. 8-Fluorophenyl-5,6,9,10,11,12-hexahydroimidazo[20,10 : 6,5]
[1,2,3]-triazino[4,5-b]quinoline (15b)
Yield: 0.56 g (36%), m.p. 155–157 ꢁC (2-propanol). IR (KBr, cmꢀ1): 3075
(CH-aromatic), 2932 (CH-aliphatic). 1H NMR (DMSO-d6, ppm): 1.55–
1.76 (m, 4 H, 2 CH2), 2.27–2.35 (m, 2 H, CH2), 2.61–2.7 (m, 2 H, CH2),
3.55 (m, 4 H, 2 CH2), 7.24 (d, 2 H, Ar––H), 7.44 (d, 2 H, Ar––H). MS, m/z
(%): 321 [Mþ] (100).
3.3.5. Evaluation of ulcerogenic activity
Ulcerogenic activity was determined according to the method of Verma
et al. (1981). In this method, adult albino rats, fasted 24 h prior to the
administration of drugs, were divided into groups of ten animals each.
Water was allowed ad libitum to the animals. The test compounds and
standard drugs were given intraperitoneally and the animals sacrificed 8 h
after drugs treatment. The stomach, duodenum and jejunum were removed
and examined with a hand lens for any evidence of (a) shedding of epithe-
lium (b) petechial and frank haemorrhage and (c) erosion or discrete ul-
ceration with or without perforation. The presence of any one of these
criteria was considered to be an evidence of ulcerogenic activity.
C
18H16FN5 (321.35)
3.1.12.3. 8-Methoxydphenyl-5,6,9,10,11,12-hexahydroimidazo[20,10 : 6,5]-
[1,2,3]-triazino[4,5-b]quinoline (15c)
Yield: 0.62 g (37%), m.p. 168–170 ꢁC (C2H5OH). IR (KBr, cmꢀ1): 3100
(CH-aromatic), 2944, 2835 (CH-aliphatic). 1H NMR (DMSO-d6, ppm):
1.58–1.75 (m, 4 H, 2 CH2), 2.31–2.38 (m, 2 H, CH2), 2.69–2.75 (m, 2 H,
CH2), 3.55 (m, 4 H, 2 CH2), 3.84 (s, 3 H, CH3), 7.12 (d, 2 H, Ar––H), 7.34
(d, 2 H, Ar––H). MS, m/z (%): 333 [Mþ] (100).
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