Synthesis and evaluation of antifungal properties of a series of the novel 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and its analogues

Article abstract of DOI:10.1016/j.bmc.2007.08.009

A series of 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and various analogues have been synthesized in excellent isolated yields starting from various arylidenemalononitrile and 3-amino-2-cyclohexen-1-one in 1-propanol as solvent at reflux temperature in the absence of any added catalyst. All the synthesized compounds were evaluated for their antifungal activity. The relationship between functional group variation and biological activity of the evaluated compounds is discussed in the article.

Full text of DOI:10.1016/j.bmc.2007.08.009

Bioorganic & Medicinal Chemistry 15 (2007) 6705–6715
Synthesis and evaluation of antifungal properties of a series
of the novel 2-amino-5-oxo-4-phenyl-5,6,7,8-
tetrahydroquinoline-3-carbonitrile and its analogues
Atul R. Gholap,a Kiran S. Toti,a Fazal Shirazi,b Ratna Kumari,c Manoj Kumar Bhat,c
Mukund V. Deshpandeb and Kumar V. Srinivasana,*
aDivision of Organic Chemistry; Technology, National Chemical Laboratory, Pune 411008, India
bBiochemical Sciences Division, National Chemical Laboratory, Pune 411008, India
cNational Center for Cell Science, Pune University Campus, Pune 411007, India
Received 20 June 2007; revised 3 August 2007; accepted 4 August 2007
Available online 15 August 2007
Abstract—A series of 2-amino-5-oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile and various analogues have been synthe-
sized in excellent isolated yields starting from various arylidenemalononitrile and 3-amino-2-cyclohexen-1-one in 1-propanol as sol-
vent at reflux temperature in the absence of any added catalyst. All the synthesized compounds were evaluated for their antifungal
activity. The relationship between functional group variation and biological activity of the evaluated compounds is discussed in the
article.
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
acetate in conventional heating mode or under micro-
wave irradiation and by other methods.10 The 2-ami-
no-3-cyanopyridines are versatile intermediates for the
synthesis of nitrogen heterocycles.11
Many naturally occurring as well as synthetic com-
pounds containing the pyridine scaffold exhibit interest-
ing pharmacological properties.1 As a consequence
many efficient procedures have been reported in the lit-
erature for the synthesis of functionalized pyridines.2
Pyridine is one of the most popular N-heteroaromatics
incorporated into the structure of many pharmaceuti-
cals. Among these, cyanopyridines with different alkyl
and aryl groups were found to have antimicrobial,3 anti-
hypertensive,4 cardiovascular,5 anti-inflammatory, anal-
gesic, antipyretic properties6 as well as 1KK-b inhibitor
properties.7 The preparation of the 2-amino-3-cyano-
pyridine derivatives has been reported in the literature
from chalcones on treatment with ammonium acetate
via Michael-type condensation,8 as well as via a one-
pot coupling reaction of four components9 acetophe-
none, benzaldehyde, malononitrile, and ammonium
As part of our ongoing development of efficient proto-
cols for the preparation of biologically active heterocy-
cles from common intermediates12 and keeping in view
the biological activity of the cyanopyridine derivatives
along with the versatility of the organic synthon viz. cyc-
lic enaminones and the cyano olefins, we herein report
for the first time the synthesis of the novel 2-amino-5-
oxo-4-phenyl-5,6,7,8-tetrahydroquinoline-3-carbonitrile
R
R
R
O
O
n-propanol
+
CN
N
+
N
Reflux
NH2
N
NH2
N
Keywords: Arylidenemalononitriles; Michael addition; Arylquinoline
N
3 (a-i)
carbonitrile; Antifungal activity; Antiproliferative activity.
*
4 (a-i)
2 (a-i)
1
Corresponding author. Tel.: +91 20 25902098; fax: +91 20 25893616;
e-mail: kv.srinivasan@ncl.res.in
Scheme 1.
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.08.009

6706
A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
O
and its analogues 3a–i in excellent isolated yields. All the
compounds 3a–i were synthesized by Michael addition
of various arylidenemalononitriles 2a–i with 3-amino-
2-cyclohexen-1-one 1 followed by aromatization in the
absence of any added catalyst (Scheme 1). They were
then evaluated for their antifungal activity.
Ar
H
H
CN
1 + 2 i
NH2
N
Ar = C6H5-NO2
NO2
NO2
2. Chemistry
O
O
CN
CN
The starting arylidenemalononitriles were synthesized
by reacting the corresponding aromatic aldehyde and
malononitrile in methanol in the presence of a catalytic
amount of piperidine at room temperature, and the
product arylidenemalononitriles 2a–i formed were used
as such for the reaction with the enaminone 1 in reflux-
ing n-propanol for an appropriate time to afford the
arylquinoline carbonitrile 3a–i in excellent isolated
yields. The results are recorded in Table 1. The products
thus obtained 3a–i were well characterized by their IR
and 1H NMR spectral analysis, additionally the elemen-
tal analyses were in conformity with the respective
structures.
N
H
NH2
N
NH2
3 i
5
NO2
NO2
CN
CN
4 i
CN
CN
2 i
Figure 1. Mechanism.
The IR spectra of 3a showed sharp bands at 3422 and
3306 cmÀ1 (NH2), 2215 cmÀ1 (CN), and 1685 cmÀ1
(C@O). The 1H NMR spectrum showed triplet at d
2.63, d 2.13 and quintet at d 1.72 for methylene protons
of cyclohexanone ring. The by-product 2-(4-nitrobenzyl)
malononitrile 4i was isolated after the reaction of 1 with
2-(4-nitrobenzyledine) malononitrile 2i and was com-
pletely characterized by spectral analysis. The formation
of the reduced by-product 4i indicates the dual role of
arylidenemalononitrile as reactant as well as an oxidiz-
ing agent. The formation of hexahydroquinoline inter-
mediate 5 was observed as a major product in the
reaction mixture when equimolar amounts of 1 and 2i
were allowed to reflux in n-propanol. A reasonable
mechanism for the formation of 3i could be explained
via initial Michael addition of 1 to 2i followed by cycli-
zation to hexahydroquinoline intermediate 5 which sub-
sequently underwent oxidation in the presence of two
molar excess of the cynoolefine 2i to the fully aroma-
tized product 3i. The formation of nearly equimolar
proportion of compound 4i as compared to 3i confirms
the participation of 2i in the hydrogen transfer process
(Fig. 1). Additional evidence for the formation of fully
aromatized product 3 was obtained by the X-ray crystal-
lographic studies on 3f. The ORTEP diagram of 3f
C8'
O3
O2
C4'
C1'
O4
C7'
C9'
C3'
C2'
C5'
O1
C6'
N2
C4
C3
C10
C5
C9
C2
C6
C1
C8
C7
N1
N3
Figure 2. X-ray crystal structure of compound 3f.
(Fig. 2) shows the formation of a fully aromatized
product.
Table 1. Synthesis of compounds 3(a–i)
Compound
R
Time
Yielda
To study the significance of each group on core structure
`
3a
3b
3c
3d
3e
3f
4-Cl
4-F
4
4
4
5
7
5
6
7
4
92
94
95
87
82
89
90
81
90
vis-a-vis the antifungal activity, the respective modifica-
tions of amino and cyano functional groups were carried
out. Compound 3d was selectively monoacetylated to 6
by using acetyl chloride in dry pyridine at reflux temper-
ature for 3 h. The preparation of the diacylated deriva-
tive 7 was achieved by refluxing 3d in acetic anhydride
for 12 h. The conversion of the nitrile group in 3d to
the amide 8 was carried out by hydrolysis using 60%
H2SO4 at 80 ꢁC for 24 h (Scheme 2).
3-NO2
4-CH3
2-Cl
3,4,5-OMe
4-OMe
H
3g
3h
3i
4-NO2
a Isolated yields after column chromatography.

A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
6707
CH3
CH3
CH3
HOOC
H
N
NH2
O
O
O
O
a
b
O
CN
CN
CN
O
b
a
O
O
CN
CN
N
N
CH3
CH3
N
3d
NH2
N
N
H
CH3
N
NH2
N
NH2
6
7
O
9
14
c
O
N
O
CH3
N
O
O
O
O
O
O
CN
CN
NH2
NH2
+
O
N
N
N
H
CH3
N
NH2
8
15
16
Scheme 2. Reagents and conditions: (a) pyridine, acetyl chloride, rt to
reflux 3 h, 85%; (b) acetic anhydride, reflux 12 h, 90%; (c) 60% H2SO4,
80 ꢁC 24 h, 88%.
Scheme 4. Reagents and conditions: (a) maleic anhydride, THF, rt,
12 h, 95%; (b) acetic anhydride, NaOAc, 60 ꢁC, 1 h, (15: 60%, 16:
32%).
To modify the functionality in the aryl ring so as to
incorporate groups which can enhance biological activ-
ity, the nitro derivative 3c (Table 1) was reduced to
2-amino-5-oxo-4-(3-aminophenyl)-5,6,7,8-tetrahydro-
quinoline-3 carbonitrile 9 using SnCl2Æ2H2O in absolute
ethanol. N-Acylation of 9 with 3-chloropropionyl chlo-
ride in THF and in the presence of NaHCO3 gave chlo-
ropropionyl derivative 10. Amination of 10 was carried
out by refluxing it in morpholine for 4 h to afford 11.
Compound 12 was obtained by refluxing compound 9
and carbonyldiimidazole in dry THF for 16 h. The urea
derivative 13 was obtained by the reaction of compound
9 with 4-methoxyphenylisocyanate in dry THF for 12 h
at reflux temperature as shown in Scheme 3.
shown in Scheme 4. The products 15 and 16 were sepa-
rated by column chromatography and completely char-
acterized by spectral and elemental analyses.
3. Antifungal activity
All the synthesized compounds were screened for their
in vitro antifungal activities. For preliminary screening
the antifungal tests were carried out by disc-diffusion
method as described earlier.13 Amphotericin B (ergos-
terol synthesis inhibitor) and cycloheximide (protein
synthesis inhibitor) were used as a positive control in
the disc diffusion method. The yeast cell suspension
for Candida albicans strains 1 and 2 (human pathogen),
mycelial suspension for Fusarium oxysporum strains 1
and 2 (plant pathogen), and Mucor sp. (saprophyte)
(100 ll) were spread on sterile YPG (yeast extract,
0.3%, peptone, 0.5%, and glucose, 1%) and PDA (pota-
to, 20% dextrose, 2%) agar plates separately. Sterile
Whatman filter paper No. 1 discs were placed on each
Likewise, reaction of 9 with maleic anhydride gave male-
anilic acid derivative 14 which was used as such for the
next step, without any further purification and charac-
terization, to obtain a mixture of the desired maleimide
derivative 15 and N-acylated product 16 (in 60% and
32% yield, respectively) by heating it with a mixture of
fused NaOAc and acetic anhydride at 60 ꢁC for 1 h as
O
H
N
H
N
Cl
NO2
NH2
N
O
O
O
O
O
O
b
c
a
CN
CN
CN
CN
N
NH2
N
NH2
N
NH2
N
NH2
11
9
3c
10
H
N
H
H
N
H
N
N
NH2
O
O
O
O
O
OMe
O
e
d
NC
CN
CN
CN
N
H N
N
NH2
N
NH2
2
N
NH2
9
12
13
Scheme 3. Reagents and conditions: (a) SnCl2Æ2H2O, ethanol, reflux 3 h, 97%; (b) 3-chloropropionyl chloride, THF, 0 ꢁC–rt, 2 h, 92%; (c)
morpholine reflux 4 h, 87%; (d) carbonyldiimidazole, THF, reflux 16 h, 80%; (e) 4-methoxyphenyl isocyanate, THF, reflux 12 h, 86%.

6708
A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
Table 2. In vitro antifungal activity detection by using disc-diffusion method
Sr. No.
Compound (lg/disc)
Zone of inhibition (mm)
C. albicans 1
C. albicans 2
F. oxysporum 1
F. oxysporum 2
Mucor
40
60
100
40
60
100
40
60
100
40
60
100
40
60
100
a
1
2
3a
3b
2
3
4
4
4
2
—
3
2
3
3
4
2
3
2
3
3
5
2
4
7
5
6
7
7
4
7
4
6
5
7
6
4
6
7
7
6
7
7
7
12
2
3
4
4
4
2
—
3
2
3
3
4
2
3
2
3
3
5
2
4
7
5
6
7
7
4
7
4
6
5
7
6
4
6
7
7
6
7
7
7
12
—
—
3
4
7
7
7
7
6
4
6
6
3
5
4
4
5
5
6
2
7
6
7
9
—
—
3
4
3
7
7
7
7
6
4
6
6
3
5
4
4
5
5
6
2
7
6
7
9
—
—
—
—
—
—
—
—
—
—
3
7
10
12
15
14
10
—
—
—
—
—
10
—
10
—
—
—
—
11
—
10
2
2
3
9
3
3c
3d
3
3
4
4
9
4
3
3
2
3
2
3
10
7
5
3e
—
—
—
—
—
—
—
—
—
2
—
—
—
—
—
—
—
—
—
2
2
3
2
3
6
3f
3g
2
2
2
2
—
—
—
—
—
7
7
—
—
—
—
—
—
2
—
3
3
—
3
8
3h
3i
3
9
2
—
—
—
—
2
2
10
11
12
13
14
15
16
17
18
19
20
6
3
3
7
2
2
8
—
3
2
—
5
—
7
9
11
3
2
3
2
3
—
—
—
—
8
—
—
—
—
9
12
13
3
3
—
—
3
2
—
—
3
2
2
2
—
3
—
3
15
16
3
3
2
2
2
2
2
2
Cycloheximide
Amphotericin B
2
2
3
5
3
5
—
5
—
7
4
4
2
6
3
6
Positive control, cycloheximide and Amphotericin B.
Negative control, DMSO (50% v/v).
a No inhibition.
plate. Different concentrations of the compounds were
added on the filter paper discs. DMSO (50% v/v) with-
out inhibitor was used as a control. The plates were then
incubated at 28 ꢁC for 24 h and antifungal activity was
evaluated by measuring the diameter of zone of inhibi-
tion against that of the test organisms (Table 2).
activity against C. albicans 1 and 2, F. oxysporum 1
and no activity against Mucor, but showed comparable
activity with those of the standards against F. oxyspo-
rum 2. The compound with halogen substituents showed
good to moderate activity. Among the halogen substi-
tuted compounds 3e showed the better activity than 3a
and 3b against F. oxysporum species emphasizing the
importance of the positioning of the functional group
on the aryl ring. It was observed that compounds 3a,
3b, 3c, and 3d were effective against C. albicans 1 and
2 whereas compounds 3c, 3d, 3e, and 3f were more effec-
tive against F. oxysporum 1 and 2 and compounds 3a–e
were effective against Mucor sp. at higher concentration,
the results are comparable with those of the standards.
The studied compounds could be divided into two
groups. One group includes compounds 3a to 3i which
are generic and the second group includes compounds
6–16 with modification of functional groups present in
parent compounds. N-Diacetylation of compound 3d
gave compound 7, which showed poor activity as com-
pared to 3d in the case of C. albicans 1, 2 and F. oxysporum
1, 2. But in the case of Mucor compound 7 showed good
activity, while compounds 6 and 7 which were acylated
derivatives of compound 3d had less activity than parent
compound having free NH2 group.
Before discussing the structure–activity relationship it
must be pointed out that the ratio between the highest
and the lowest concentrations of compounds is only
2:3. The effective concentrations sometimes alter decep-
tively in the biological experiments because of condi-
tions of living things. Moreover, since the antifungal
assays are done using paper discs the structure–activity
relationship is described purely on a qualitative basis
with respect to substituents incorporated in the molecu-
lar skeleton.
It was observed that activity of the synthesized com-
pounds was dependent on the substituent present at 3
or 4 position of the phenyl ring in 2-amino-3-cyano-4-
arylpyridine moiety. In terms of structure–activity rela-
tionship, 2-amino-5-oxo-4-(3-nitro-phenyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitrile, 3c with a nitro group
substituent in the meta position showed, in general, a
more potent antifungal activity than other compounds
synthesized (Table 1). Thus the compound with meta
substituted –NO2 group 3c had prominent activity
against all the species studied than the compound with
para substituted –NO2 group 3i. The compound 3i did
not have any activity against Mucor but compound 3c
showed good activity against Mucor. Compound 3h
without any substitution on phenyl ring showed poor
All the synthesized compounds 11–16 from Schemes 3
and 4 were effective against both the species of C. albicans
and F. oxysporum, but compounds 11–15 did not show
activity against Mucor. Compound 9 and its monoacy-
lated maleimide derivative showed good activity against
Mucor. Compounds 3c, 3d, and 12 showed activity com-
parable to that of cycloheximide. It was compound 16,

A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
6709
Table 4. Effect of compounds on yeast-hypha transition
Group Compounds (20 lg/ml)
the N-acetylated maleimide derivative, which showed
the best activity against all the fungal species studied
and greater activity than Amphotericin B against
Mucor.
Inhibition
(%)
1
2
3
4
3a, 3b, 3e, 3f, 3g, 3i, 8, 9, 11, 12, 13, 15, 16 95–99
3d, 3h, 7, 6
3c
90–95
85–90
>90
3.1. Yeast-hypha transition experiment
Nikkomycin Z (4 lg/ml)
Most of the pathogenic fungi change their morphology
viz. unicellular yeast or filamentous hypha for survival
and proliferation in the host. The yeast-hypha transition
experiment was carried out in a model non-pathogenic
fungus Benjaminiella poitrasii to check the effect of com-
pounds as described earlier.14,15
Standard used was Nikkomycin Z (4 lg/ml).
3.2. Non-radioactive chitin synthase assay
Chitin synthase activity from B. poitrasii was estimated
with and without compounds (4 lg/ml) using a non-
radioactive chitin synthase assay according to Lucero
et al.17
Yeast inoculum was grown in YPG medium for 24 h at
37 ꢁC and the transition was studied in YP medium at
28 ꢁC. The yeast cells were inoculated in YP broth (with
and without compounds) at 28 ꢁC for 4 h and the per-
centage of cells forming germ tubes was assessed as de-
scribed earlier.16
It was seen that compounds 3d, 12, 15, and 16 inhibited
91–95% chitin synthase activity, 3a–3c, 3e–3g exhibited
80–90% of inhibition. The rest of the compounds exhib-
ited substantial percentage of inhibition in the range of
60–80% (Table 5).
Nikkomycin Z (chitin synthase inhibitor) was used as a
positive control in the yeast-hypha transition experiment
(Table 4). It was seen that compounds 3a, 3b, 3e–3g, 3i,
8, 9, 11–13, 15, and 16 exhibited 95–99% of inhibition
while 3c, 3d, 3h, 7, and 6 showed 85–95% of inhibition
at 20 lg/ml. Minimum inhibitory concentration (MIC)
of the compounds was estimated by disc-diffusion meth-
od. Cycloheximide showed 2–7 mm inhibition in case of
C. albicans and 3–7 mm inhibition for F. oxysporum at
40 lg/ml. While Amphotericin B showed 4–12 mm inhi-
bition in C. albicans, 2–9 mm inhibition in F. oxyspo-
rum, and 5–10 mm inhibition in Mucor at 40 lg/ml.
Therefore for other compounds the zone of inhibition
2 mm at 28 ꢁC for 24 h was considered as minimum
inhibitory concentration (MIC), Table 3.
3.3. Antiproliferative activity: materials and methods
Human hepatocellular carcinoma Hep3B and human
mammary adenocarcinoma (MCF-7) cell lines were ob-
tained from American Type Culture Collection (Manas-
sas, VA, USA), and maintained in our in-house
National Cell Repository. Cells were maintained as a
monolayer in culture medium consisting of nutrient med-
ia MEM supplemented with heat-inactivated fetal bovine
serum (10%), penicillin (100 lg/ml), and streptomycin
(100 lg/ml) (Invitrogen Life Technologies, MD, USA).
The cells were grown at 37 ꢁC in 5% CO2 and humidified
air atmosphere. Stock solutions of all compounds were
Table 3. Minimum inhibitory concentration values of the compounds against the fungal species using disc diffusion method
Sr. No.
Compound
Concentration of compounds (lg/disc)
Candida albicans 1
Candida albicans 2
F. oxysporum 1
F. oxysporum 2
Mucor
1
2
3a
3b
40
40
40
40
60
100
60
60
60
60
60
60
60
40
40
40
40
40
40
40
40
40
40
40
60
100
60
60
60
60
60
60
60
40
40
40
40
40
40
40
60
40
20
40
40
20
100
60
60
60
60
100
40
40
60
100
40
40
20
40
60
40
20
40
40
20
100
60
60
60
60
100
40
40
60
100
40
40
20
40
60
60
60
60
60
3
3c
3d
4
5
3e
a
6
3f
3g
—
7
—
—
—
—
40
—
40
—
—
—
—
40
—
40
8
3h
3i
9
10
11
12
13
14
15
16
17
18
19
20
6
7
8
9
11
12
13
15
16
Cycloheximide
Amphotericin B
Standard used were cycloheximide and Amphotericin B.
a Not detected.

6710
A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
Table 5. In vitro antifungal activity of selected compounds for the
inhibition of chitin synthase enzyme from B. poitrasii
140
120
100
80
Amphotericin B
Compound 3c
Compound 16
Group
Compounds (4 lg/ml)
Inhibition (%)
1
2
3
4
5
6
7
8
3d, 12, 15, 16
3e
3a, 3b, 3c, 3f, 3g
91–95
86–90
81–85
76–80
71–75
66–70
61–65
>90
9
13
60
3h, 6, 8
3i, 7, 11
Nikkomycin Z
40
20
0
prepared in DMSO (concentration of 4 mg/ml) and after-
wards diluted to the required concentration in cell culture
media. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltet-
razolium bromide (MTT) was dissolved (1 mg/ml) in
MEM (without phenol red) and filtered through a Milli-
pore filter, 0.22 lm, before use. The generic compound
3c and the functionalized derivative 16 prepared from 3c
which showed the highest antifungal activity were taken
up for the determination of their antiproliferative activity.
0
500
1000
1500
2000
2500
Concentration (µg/ml)
Figure 3. Antiproliferative activities of compounds in MCF-7 cells.
140
Amphotericin B
Compound 3c
Compound 16
120
100
80
60
40
20
0
3.4. Antiproliferative activity-MTT cell proliferation
assay
Hep3B and MCF-7 cells were plated at a density of
10,000 cells per well in 96-well tissue culture plates. Cells
were allowed to adhere for 24 h at 37 ꢁC and then trea-
ted with various concentrations (0, 1, 10, 100, 1000, and
2000 lg/ml) of compounds diluted in culture medium,
for additional 48 h. In the cells in control wells a culture
medium consisting of corresponding concentration of
DMSO only was added. After 48 h of drug treatment
growth medium was removed from each well containing
cells and fresh culture medium was added to each well.
Cells were allowed to grow for another 24 h.
0
500
1000
1500
2000
2500
Concentration (µg/ml)
Figure 4. Antiproliferative activities of compounds in Hep3B cells.
compounds was toxic to the Hep3B cells up to a concen-
tration of 400 lg/ml. At a concentration of 1 mg/ml or
more all the three compounds were toxic to both the
cells and inhibited growth.
Thereafter, cell proliferation was assessed by replacing
culture medium with 50 ll MEM containing 1 mg/ml
MTT and subsequently incubated for additional 4 h at
37 ꢁC. The medium was then aspirated off and formazan
crystals were solubilized in 50 ll of 2-propanol. The
optical density was read on a microplate reader at
570 nm using 630 nm as a reference filter against a blank
prepared from cell-free wells. Absorbance given by cells
treated with the carrier DMSO alone was taken as 100%
cell growth. All assays were performed in triplicate.
4. Conclusion
A series of 2-amino-5,6,7,8-tetrahydro-5-oxo-4-phenyl-
quinoline-3-carbonitrile and its various analogues have
been synthesized in excellent isolated yields in the
absence of any added catalyst by the reaction of 3-ami-
no-cyclohexen-1one 1 with various arylidenemalononitr-
iles 2 and their biological activities were evaluated.
The dual role of 2 as a reactant in the Michael addition
step and as an oxidizing agent in the aromatization con-
tributing to the mechanism has been established with
experimental evidence. The antifungal activity of com-
pounds 3c, 3d, and 12 (40–100 lg/ml) showed activity
comparable to that of cycloheximide (40–100 lg/ml) un-
der similar experimental conditions. Compound 16
exhibited (2–11 mm) activity against all the test fungal
strains. However a greater zone of inhibition with
compound 16 was observed against Mucor. All the
compounds have profound suppressive effect on yeast-
hypha transition, exhibiting greater than 85% inhibition
3.5. Antiproliferative activity
The antiproliferative activity of all the three compounds,
3c and 16 and Amphotericin B, was tested against hu-
man cancer cells MCF-7 and Hep3B, which were grown
in vitro. Compounds 3c and 16 as well as Amphotericin
B at high doses were toxic to both MCF-7 (Fig. 3) and
Hep3B (Fig. 4) cells. However, at lower doses MCF-7
cells were resistant to compound 3c and the IC50 value
was found to be 293.4 lg/ml. Both compound 16 and
Amphotericin were similar in their toxicity to MCF-7
cells and their IC50 values were between 50 and 60 lg/ml.
All the three compounds were identical in their effect
on the survival of Hep3B cells. In fact none of the three

A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
6711
at concentration 20 lg/ml. Compounds 3d, 13, 15, 16,
and 17 exhibited more than 90% inhibition of chitin syn-
thase activity comparable to that of Nikkomycin. All
other compounds exhibited 61–90% inhibition of chitin
synthase activity at concentration 4 lg/ml. The generic
compound 3c and the functionalized derivative 16 pre-
pared from 3c showed the most antifungal activity.
The presence of halogen and nitrogen containing sub-
stituents on aryl ring seems to generate compounds with
good antifungal activities. The antiproliferative activi-
ties of compounds 3c and 16 were similar to that of
Amphotericin B though only slight differences in the lev-
els of toxicities to the two cell lines were observed. It was
observed that at low doses the mammary adenocarci-
noma cells MCF-7 were less sensitive to all the three
compounds whereas hepatocellular carcinoma cells
Hep-3B were more resistant to all the compounds. These
results indicate that compounds 3c and 16 do not have
any profound effect on proliferation of human cancer
cell lines at lower concentrations and their activity is
comparable to that of Amphotericin B.
200 MHz) d 6.89–6.73 (m, 4H), 6.51 (br s, 2H), 2.66 (t,
J = 6.5 Hz, 2H), 2.17 (t, J = 6.5 Hz, 2H), 1.75 (quin,
J = 6.5 Hz, 2H); Anal. Calcd for C16H12FN3O: C,
68.32; H, 4.30; F, 6.75; N, 14.94. Found: C, 68.45; H,
4.54; N, 14.82.
5.1.1.3. 2-Amino-5-oxo-4-(3-nitro-phenyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitrile (3c). White solid; mp
243 ꢁC; IR (cmÀ1) 3483, 3323, 2211, 1674, 1622, 1552,
1346, 755; 1H NMR (CDCl3+DMSO-d6, [11:1]
200 MHz) d 8.34–8.28 (m, 1H), 8.05 (t, J = 1.9 Hz,
1H), 7.64 (t, J = 7.9 Hz, 1H), 7.55–7.50 (m, 1H) 5.74
(br s, 2H), 3.04 (t, J = 6.2 Hz, 2H), 2.58–2.51 (m, 2H),
2.13 (quin, J = 6.2 Hz, 2H); 13C NMR (CDCl3+
DMSO-d6, [6:1] 125 MHz) d: 192.8, 168.2, 158.6,
153.1, 146.1, 138.3, 132.2, 127.9, 121.3, 120.8, 115.2,
113.5, 90.2, 37.6, 32.2, 19.1; Anal. Calcd for
C16H12N4O3: C, 62.34; H, 3.92; N, 18.17. Found: C,
62.40; H, 3.80; N, 18.02.
5.1.1.4. 2-Amino-5-oxo-4-(4-methyl-phenyl)-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (3d). White solid; mp
258 ꢁC; IR (cmÀ1) 3423, 3296, 2208, 1682, 1642, 1555,
1535, 1459; 1H NMR (CDCl3, 200 MHz) d 7.24 (d,
J = 8.0 Hz, 2H), 7.08 (d, J = 8.0 Hz, 2H), 5.74 (br s,
2H), 2.99 (t, J = 6.2 Hz, 2H), 2.53 (t, J = 6.2 Hz, 2H),
2.40 (s, 3H), 2.09 (quin, J = 6.2 Hz, 2H); 13C NMR
(CDCl3+DMSO-d6, [6:1] 50 MHz) d: 192.1, 167.3,
158.3, 155.6, 135.4, 133.3, 126.6, 125.4, 115.3, 113.7,
89.9, 37.5, 31.9, 19.3, 18.9; Anal. Calcd for
C17H15N3O: C, 73.63; H, 5.45; N, 15.15. Found: C,
73.77; H, 5.62; N, 15.32.
5. Experimental
5.1. General experimental procedures
All chemicals were of research grade and were used as ob-
tained from Aldrich or Fluka. IR spectra were recorded
on an ATIMATT-SON RS-1 Research Series FT-IR
spectrometer. NMR spectra were recorded on Bruker
AC-200, AC-400, and AC-500 spectrometers in CDCl3/
DMSO-d6. The melting points were uncorrected and re-
corded on the BUCHI melting point instrument model
B-540. Column chromatography was performed using
silica gel (60–120 mesh size) purchased from Thomas
Baker and TLC was carried out using aluminum sheets
pre-coated with silica gel 60F254 purchased from Merck.
5.1.1.5.
2-Amino-5-oxo-4-(2-chloro-phenyl)-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (3e). White solid; mp
286 ꢁC; IR (cmÀ1) 3392, 3308, 2218, 1680, 1647, 1543,
1462, 1377; 1H NMR (CDCl3+DMSO-d6, [11:1]
200 MHz) d 7.11–6.93 (m, 3H), 6.77–6.73 (m, 1H),
6.33 (br s, 2H), 2.64 (t, J = 6.0 Hz, 2H), 2.17–2.09 (m,
2H), 1.72 (quin, J = 6.0 Hz, 2H); Anal. Calcd for
C16H12ClN3O: C, 64.54; H, 4.06; Cl, 11.91; N, 14.11.
Found: C, 64.41; H, 4.16; Cl, 11.72; N, 14.30.
5.1.1. General procedures for the preparation of the
compounds 3(a–i). A solution of 3-amino-2-cyclohexen-
1-one 1 (2 g, 10 mmol) and arylidenemalononitrile 2
(30 mmol) was refluxed in 40 ml of n-propanol for the
requisite time as shown in Table 1. After completion of
reaction 1-propanol was removed under reduced pres-
sure and the product was isolated by column chromato-
graphy using 60% ethyl acetate in pet ether as eluent.
5.1.1.6. 2-Amino-5-oxo-4-(3,4,5-trimethoxy-phenyl)-
5,6,7,8-tetrahydroquinoline-3-carbonitrile (3f). Pale
yellow solid; mp 219 ꢁC; IR (cmÀ1) 3417, 3308, 2213,
1681, 1651, 1555, 1459, 1093; 1H NMR (CDCl3,
200 MHz) d 6.38 (s, 2H), 5.73 (br s, 2H), 3.89 (s,
3H), 3.82 (s, 6H), 2.99 (t, J = 6.2 Hz, 2H), 2.55 (t,
J = 6.2 Hz, 2H), 2.11 (quin, J = 6.2 Hz, 2H); 13C
NMR (CDCl3, 50 MHz) d: 194.4, 169.1, 159.4,
157.6, 152.9, 138.0, 132.5, 118.3, 115.2, 104.4, 93.1,
60.8, 55.9, 39.4, 33.7, 20.7; Anal. Calcd for
C19H19N3O4: C, 64.58; H, 5.42; N, 11.89. Found: C,
64.73; H, 5.60; N, 11.70.
5.1.1.1. 2-Amino-5-oxo-4-(4-chloro-phenyl)-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (3a). White solid; mp
289 ꢁC; IR (cmÀ1) 3422, 3306, 2215, 1685, 1648, 1537,
1461, 1243; 1H NMR (CDCl3+DMSO-d6, [11:1]
200 MHz)
d 7.03 (d, J = 8.5 Hz, 2H), 6.77 (d,
J = 8.5 Hz, 2H), 6.63 (br s, 2H), 2.63 (t, J = 6.3 Hz,
2H), 2.13 (t, J = 6.3 Hz, 2H), 1.72 (quin, J = 6.3 Hz,
2H); Anal. Calcd for C16H12ClN3O: C, 64.54; H, 4.06;
Cl, 11.91; N, 14.11. Found: C, 64.32; H, 4.15; Cl,
11.89; N, 14.24.
5.1.1.7. 2-Amino-5-oxo-4-(4-methoxy-phenyl)-5,6,7,8-
tetrahydroquinoline-3-carbonitrile (3g). White solid; mp
278 ꢁC; IR (cmÀ1) 3393, 3307, 2217, 1680, 1651, 1512,
1459, 1243; 1H NMR (CDCl3+DMSO-d6, [11:1]
200 MHz) d 6.72 (d, J = 8.7 Hz, 2H), 6.53 (d,
J = 8.7 Hz, 2H), 6.44 (br s, 2H), 3.44 (s, 3H), 2.57 (t,
5.1.1.2. 2-Amino-5-oxo-4-(4-fluoro-phenyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitrile (3b). White solid; mp
286 ꢁC; IR (cmÀ1) 3397, 3309, 2215, 1684, 1649, 1511,
1460, 1217; 1H NMR (CDCl3+DMSO-d6, [11:1]
J = 6.2 Hz, 2H), 2.09 (t, J = 6.2 Hz, 2H), 1.67 (quin,

6712
A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
J = 6.2 Hz, 2H); Anal. Calcd for C17H15N3O2: C, 69.61;
H, 5.15; N, 14.33. Found: C, 69.76; H, 5.04; N, 14.45.
of reaction excess acetic anhydride was removed under
reduced pressure. The residue obtained was added in
water (15 ml) and extracted with ethyl acetate (3·
15 ml). The organic layer was separated, dried over
anhydrous sodium sulfate, and evaporated to obtain
the crude product. The pure product 7 was obtained
by column chromatography using 15% ethyl acetate in
pet ether as eluent. (0.325 g, 90%, yellow solid); mp
183–185 ꢁC; IR (cmÀ1) 2229, 1721, 1707, 1557, 1509,
1459, 1416, 1367, 1263, 1228, 1208, 1037, 979, 931,
5.1.1.8. 2-Amino-5-oxo-4-phenyl-5,6,7,8-tetrahydro-
quinoline-3-carbonitrile (3h). White solid; mp 257 ꢁC; IR
(cmÀ1) 3391, 3299, 2208, 1692, 1651, 1585, 1478, 1404,
1
1215, 1082, 930, 758, 669; H NMR (CDCl3+DMSO-
d6, [11:1] 200 MHz) d 6.90–6.87 (m, 3H), 6.69–6.61 (m,
2H), 2.45 (t, J = 6.2 Hz, 2H), 1.91 (t, J = 6.2 Hz, 2H),
1.56 (quin, J = 6.2 Hz, 2H); Anal. Calcd for
C16H13N3O: C, 72.99; H, 4.98; N, 15.96. Found: C,
72.80; H, 5.05; N, 15.80.
823; 1H NMR (CDCl3, 200 MHz)
d
7.28 (d,
J = 8.2 Hz, 2H), 7.12 (d, J = 8.2 Hz, 2H), 3.26 (t,
J = 6.3 Hz, 2H), 2.70 (t, J = 6.3 Hz, 2H), 2.42 (s, 3H),
2.37 (s, 6H) 2.24 (quin, J = 6.3 Hz, 2H); 13C NMR
(CDCl3, 50 MHz) d 195.0, 171.2, 167.9, 157.8, 155.6,
139.3, 132.0, 129.0, 127.2, 126.6, 113.2, 110.9, 39.4,
33.3, 26.0, 21.2, 20.5; Anal. Calcd for C21H19N3O3: C,
69.79; H, 5.30; N, 11.63. Found: C, 69.68; H, 5.11; N,
11.44.
5.1.1.9. 2-Amino-5-oxo-4-(4-nitro-phenyl)-5,6,7,8-tet-
rahydroquinoline-3-carbonitrile (3i). White solid; mp
260 ꢁC; IR (cmÀ1) 3407, 3323, 2216, 1680, 1612, 1555,
1523, 1430, 1349, 1215, 929, 777, 669; 1H NMR (CDCl3,
200 MHz) d 8.31 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz,
2H), 5.76 (br s, 2H), 3.03 (t, J = 6.5 Hz, 2H), 2.54 (t,
J = 6.5 Hz, 2H), 2.12 (quin, J = 6.5 Hz, 2H); Anal.
Calcd for C16H12N4O3: C, 62.34; H, 3.92; N, 18.17.
Found: C, 62.48; H, 3.79; N, 18.26.
5.1.1.13. 2-Amino-5,6,7,8-tetrahydro-5-oxo-4-p-tolyl-
quinoline-3-carboxamide (8). A solution of 60% aqueous
H2SO4 (10 ml) and compound 3d (1.2 g, 4.33 mmol) was
heated at 80 ꢁC for 24 h with stirring. The reaction mix-
ture was cooled to room temperature, diluted with
water, and neutralized by 10% aqueous sodium hydrox-
ide solution. The resulting precipitate was filtered, dried,
and purified by column chromatography by using 70%
ethyl acetate in pet ether to obtain 8 (1.124 g, 88% yield,
white solid); mp 250 ꢁC; IR (cmÀ1) 3400, 3300, 3250,
5.1.1.10. Isolation of 2-(4-nitrobenzyl) malononitrile
(4i). Compound 4i was isolated after the reaction of 1
(1.8 mmol) with 2-(4-nitrobenzyledine) malononitrile 2i
(5.4 mmol) by column chromatography using 5% ethyl
acetate in pet ether as eluent. (white solid 2.54 g, 80%);
mp 156 ꢁC; IR (cmÀ1) 2256, 1526, 1444, 1421, 1350,
1215, 758, 669; 1H NMR (CDCl3, 200 MHz) d: 8.29
(d, J = 8.5 Hz, 2H), 7.54 (d, J = 8.5 Hz, 2H), 4.02 (t,
J = 6.5 Hz, 1H), 3.40 (d, J = 6.5 Hz, 2H); 13C NMR
(CDCl3+DMSO-d6, [6:1] 50 MHz) d 147.0, 140.2,
129.9, 123.2, 111.9, 34.7, 23.3; Anal. Calcd for
C10H7N3O2: C, 59.70; H, 3.51; N, 20.89. Found: C,
59.74; H, 3.55; N, 20.95.
1
3150, 1678, 1655, 1605, 1551, 1215; H NMR (CDCl3,
200 MHz) d 7.22 (d, J = 8.2, 2H), 7.06 (d, J = 8.2 Hz,
2H), 6.43 (br s, 2H), 5.39 (s, 1H), 4.95 (s, 1H), 2.95 (t,
J = 6.2 Hz, 2H), 2.49 (t, J = 6.2 Hz, 2H), 2.39 (s, 3H),
2.07 (quin, J = 6.2 Hz, 2H); 13C NMR (CDCl3,
50 MHz) d 193.0, 166.5, 163.4, 155.2, 146.8, 134.1,
134.0, 126.0, 115.2, 115.1, 37.8, 31.5, 19.5, 19.2; Anal.
Calcd for C17H17N3O2: C, 69.14; H, 5.80; N, 14.23.
Found: C, 69.25; H, 5.69; N, 14.15.
5.1.1.11. N-(3-Cyano-5,6,7,8-tetrahydro-5-oxo-4-
p-tolylquinolin-2-yl) acetamide (6). To a solution of com-
pound 3d (0.5 g, 1.80 mmol) in dry pyridine (3 ml), acet-
yl chloride (0.16 ml, 2.16 mmol) was added dropwise at
0 ꢁC in an inert atmosphere. The temperature was al-
lowed to rise to reflux for 3 h. After completion of reac-
tion pyridine was removed under reduced pressure to
obtain a white solid. The solid obtained was washed
with water, dried, and purified by column chromatogra-
phy by using 30% ethyl acetate in pet ether as eluent to
obtain 6 (0.489 g, 85% yield, white solid); mp 208–
210 ꢁC; IR (cmÀ1) 3270, 2224, 1694, 1546, 1516, 1262,
1215, 766, 668; 1H NMR (CDCl3, 200 MHz) d 8.08
(br s, 1H), 7.26 (d, J = 8.0 Hz, 2H), 7.09 (d,
J = 8.0 Hz, 2H), 3.16 (t, J = 6.2 Hz, 2H), 2.62 (t,
J = 6.2 Hz, 2H), 2.52 (s, 3H), 2.41 (s, 3H), 2.17 (quin,
J = 6.2 Hz, 2H); 13C NMR (CDCl3, 50 MHz) d 194.9,
169.8, 167.7, 158.0, 152.9, 139.0, 133.1, 129.0, 127.1,
122.8, 114.0, 100.5, 39.5, 33.7, 25.0, 21.3, 20.7; Anal.
Calcd for C19H17N3O2: C, 71.46; H, 5.37; N, 13.16.
Found: C, 71.62; H, 5.49; N, 13.10.
5.1.1.14. 2-Amino-4-(3-aminophenyl)-5,6,7,8-tetrahy-
dro-5-oxoquinoline-3-carbonitrile (9). A mixture of nitro
derivative 3i (0.695 g, 1 mmol) and SnCl2 2H2O (2.54 g,
Æ
5 mmol) was heated up to reflux in absolute ethanol
(40 ml) for 3 h. After completion of reaction, ethanol
was removed under reduced pressure. Water (60 ml)
was added to the oily residue and the mixture was neu-
tralized to pH 7 –8 by adding aqueous saturated NaH-
CO3 solution followed by extraction with ethyl acetate
(3· 30 ml). The combined ethyl acetate extract was dried
over anhydrous sodium sulfate and evaporated under
reduced pressure to obtain the pure product 9 (0.608 g,
97% yield, yellow solid). mp 276–278 ꢁC; IR (cmÀ1
3397, 3363, 3313, 2219, 1655, 1457, 1285; 1H NMR
(CDCl3+DMSO-d6, [11:1] 200 MHz) 6.95 (t,
)
d
J = 8.0 Hz, 1H), 6.57–6.52 (m, 1H), 6.33–6.30 (m, 2H),
3.06 (br s, 2H), 2.73 (t, J = 6.2 Hz, 2H), 2.25 (t,
J = 6.2 Hz, 2H), 1.83 (quin, J = 6.2 Hz, 2H); 13C
NMR (CDCl3+DMSO-d6, [6:1] 100 MHz) d: 192.5,
167.6, 158.5, 156.4, 145.3, 137.4, 127.1, 115.9, 114.6,
114.1, 113.1, 112.0, 90.5, 37.9, 32.3, 19.3: Anal. Calcd
for C16H14N4O: C, 69.05; H, 5.07; N, 20.13; Found:
C, 69.27; H, 5.24; N, 20.11.
5.1.1.12. N-Acetyl-N-3-cyano-5,6,7,8-tetrahydro-4-(4-
methylphenyl-5-oxo-4-p-tolylquinolin-2-yl) acetamide (7).
A mixture of compound 3d (0.277 g, 1 mmol) and acetic
anhydride (5 ml) was refluxed for 12 h. After completion

A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
6713
5.1.1.15. N-(3-(2-Amino-3-cyano-5,6,7,8-tetrahidro-5-
oxoquinolin-4-yl)phenyl)-3-chloropropanamide (10). To a
solution of compound 9 (0.504 g, 1.8 mmol) and NaH-
CO3 (0.152 g, 1.8 mmol) in THF, chloropropionyl chlo-
ride (0.18 ml, 1.8 mmol) was added at 0 ꢁC over a period
of 10 min and then stirred for 2 h at room temperature.
After completion of reaction, THF was removed under
reduced pressure to obtain a yellow solid. The solid
was washed with excess water, dried and finally washed
with pet ether to obtain a pure product. (0.614 g, 92%
yield, yellow solid); mp 284 ꢁC (dec); IR (cmÀ1) 3440,
3315, 3219, 2216, 1666, 1626, 1586, 1553, 1447, 1421,
159.8, 157.1, 152.3, 139.1, 138.6, 128.3, 120.7, 117.5,
116.7, 116.5, 115.2, 91.0, 39.0, 33.3, 20.4; Anal. Calcd
for C33H26N8O3: C, 68.03; H, 4.50; N, 19.23. Found:
C, 68.25; H, 4.32; N, 19.42.
5.1.1.18. 1-(3-(2-Amino-3-cyano-5,6,7,8-tetrahydro-5-
oxoquinolin-4-yl)phenyl)-3-(4-methoxyphenyl) urea (13).
A solution of compound 10 (0.250 g, 0.89 mmol) and 4-
methoxyphenyl isocyanate (0.11 ml, 0.89 mmol) was re-
fluxed in dry THF (60 ml) for 6 h. After completion of
reaction THF was removed under reduced pressure
and the solid obtained was washed with water, dried,
and recrystallized from acetone to obtain a pure solid
13 (0.330 g, 86% yield, pale yellow); mp 225–227 ꢁC;
IR (cmÀ1) 3405, 3320, 3168, 2215, 1712, 1675, 1650,
1598, 1560, 1541, 1461, 1377, 1294, 1244; 1H NMR
(CDCl3+DMSO-d6, [11:1] 400 MHz); d 8.08 (s, 1H),
7.85 (s, 1H), 7.40 (s, 1H), 7.21–7.19 (m, 4H), 6.93 (br
s, 2H), 6.69–6.67 (m, 3H), 3.64 (s, 3H), 2.94 (t, J = 6.2
Hz, 2H), 2.38 (t, J = 6.2 Hz, 2H), 1.98 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz) d 193.8, 169.3, 159.9,
157.2, 154.5, 152.6, 139.4, 138.6, 132.6, 128.3, 120.4,
120.0, 117.3, 116.7, 116.4, 115.3, 113.9, 91.1, 55.0, 39.0,
33.4, 20.4; Anal. Calcd for C24H21N5O3: C, 67.44; H,
4.95; N, 16.38. Found: C, 67.19; H, 4.89; N, 16.45.
1
1320, 1284, 886, 774; H NMR (CDCl3, 200 MHz) d
10.31 (s, 1H), 7.86 (br s, 2H), 7.76 (d, J = 7.5 Hz, 1H),
7.59 (s, 1H), 7.49 (t, J = 7.5 Hz, 1H), 6.99 (d,
J = 7.5 Hz, 1H), 4.03 (t, J = 6.2 Hz, 1H), 3.07 (t,
J = 6.0 Hz, 2H), 2.99 (t, J = 6.2 Hz, 2H), 2.57 (t,
J = 6.0 Hz, 2H), 2.14 (t, J = 6.0 Hz, 2H); 13C NMR
(DMSO-d6, 125 MHz) d 193.8, 169.4, 167.9, 159.9,
156.9, 138.6, 128.3, 122.0, 118.3, 117.3, 116.7, 115.2,
91.0, 40.7, 40.0, 39.0, 33.4, 20.4; Anal. Calcd for
C19H17ClN4O2: C, 61.88; H, 4.65; Cl, 9.61; N, 15.19.
Found: C, 61.98; H, 4.60; Cl, 9.51; N, 15.08.
5.1.1.16. N-(3-(2-Amino-3-cyano-5,6,7,8-tetrahidro-5-
oxoquinolin-4-yl)phenyl)-3-morpholinopropanamide (11).
A solution of compound 10 (0.150 g, 0.4 mmol) and ex-
cess morpholine (15 ml) was heated to reflux for 4 h.
After completion of reaction, morpholine was removed
under reduced pressure to obtain a residue. Water
(30 ml) was added to the residue and neutralized by di-
luted HCl to obtain a yellow solid which was further
filtered and washed with 1:1 ethyl acetate and pet
ether mixture to get the pure product. (0.148 g, 87%
yield, yellow solid); mp 162–164 ꢁC; IR (cmÀ1) 3396,
3322, 3211, 2215, 1731, 1651, 1629, 1584, 1557,
1462, 1376; 1H NMR (CDCl3, 200 MHz) d 10.94 (s,
1H), 7.68 (s, 1H), 7.40–7.37 (m, 2H), 6.95–6.89 (m,
1H), 5.64 (s, 2H), 3.83 (t, J = 4.8 Hz, 4H), 3.01 (t,
J = 6.2 Hz, 2H), 2.74 (t, J = 6.2 Hz, 2H), 2.63 (t,
J = 4.8 Hz, 4H), 2.57–2.51(m, 4H), 2.11 (quin,
J = 6.2 Hz, 2H); 13C CMR (CDCl3, 100 MHz) d
193.1, 169.2, 168.2, 159.0, 156.2, 137.8, 137.5, 127.3,
121.0, 117.8, 116.9, 116.4, 114.4, 91.2, 65.7, 53.1,
51.9, 38.3, 32.8, 32.3, 19.8; Anal. Calcd for
C23H25N5O3: C, 65.86; H, 6.01; N, 16.70. Found: C,
65.78; H, 6.10; N, 16.58.
5.1.2. Synthesis of compounds 15 and 16. To a stirred
solution of maleic anhydride (0.177 g, 1.8 mmol) in dry
THF (15 ml) was added a solution of amine compound
9 (0.500 g, 1.8 mmol) in dry THF (60 ml) dropwise at
room temperature over a period of 20 min and the reac-
tion mixture was further stirred for 12 h. After comple-
tion of reaction, THF was removed under reduced
pressure to obtain maleanilic acid derivative 14 in
quantitive yield, which was used for the next step with-
out any further purification and characterization. A
mixture of maleanilic acid derivative 14, acetic anhy-
dride (10 ml), and fused sodium acetate (75 mg) was
heated in an oil bath at 60 ꢁC for 1h. When complete
consumption of starting material was observed along
with the formation of two new spots on TLC, the reac-
tion mixture was cooled to room temperature and acetic
anhydride was removed under reduced pressure to ob-
tain mixture of products, which were separated by flash
column chromatography using 15% ethyl acetate in pet
ether as eluent to get 15 (0.386 g, 60% yield) and 16
(0.230 g, 32% yield).
5.1.1.17. 1,3-Bis(3-(2-amino-3-cyano-5,6,7,8-tetrahy-
dro-5-oxoquinolin-4-yl)phenyl) urea (12). A solution of
compound 10 (0.200 g, 0.71 mmol) and carbonyldiimi-
dazole (0.058 g, 0.35 mmol) in dry THF (60 ml) was
heated to reflux and maintained at reflux for 18 h. After
completion of reaction, THF was removed under re-
duced pressure to obtain a pale yellow solid. The solid
was washed with water, dried, and then washed with
cold ethyl acetate to obtain the pure product 12
(0.335 g, 80% yield, pale yellow solid); mp 285–287 ꢁC;
IR (cmÀ1) 3377, 2219, 1646, 1589, 1542, 1460, 1377;
1H NMR (DMSO-d6, 200 MHz) d 8.80 (s, 2H), 7.70
(s, 4H), 7.38–7.25 (m, 6H), 6.78 (d, J = 7.5 Hz, 2H),
2.97–2.91 (m, 4H), 2.57–2.41 (m, 4H), 2.03–1.97 (m,
4H); 13C NMR (DMSO-d6, 100 MHz) d 193.7, 169.2,
5.1.2.1. 2-Amino-5,6,7,8-tetrahydro-5-oxo-4-(3-(2,5-
dioxo-2H-pyrrol-1-(5H)-yl)phenyl)quinoline-3-carbonitri-
le (15). Gray solid; mp 262–264 ꢁC (dec); IR (cmÀ1
)
3465, 3357, 2222, 1702, 1683, 1461, 1377, 833; 1H
NMR (CDCl3+DMSO-d6, [11:1] 200 MHz) d 7.20–
7.11 (m, 2H), 6.90–6.87 (m,1H), 6.85–6.80 (m, 1H),
6.55 (s, 2H), 6.47 (br s, 2H), 2.64–2.56 (m, 2H), 2.14
(t, J = 6.2 Hz, 2H), 1.80–1.67 (m, 2H); 13C NMR
(DMSO-d6, 100 MHz) d 194.1, 169.8, 169.6, 160.0,
156.1, 138.8, 134.7, 131.3, 128.5, 126.5, 125.8, 124.9,
116.8, 115.2, 91.3, 39.0, 33.5, 20.5; Anal. Calcd for
C20H14N4O3: C, 67.03; H, 3.94; N, 15.63. Found: C,
67.15; H, 3.80; N, 15.52.

6714
A. R. Gholap et al. / Bioorg. Med. Chem. 15 (2007) 6705–6715
2. (a) Winter, A.; Risch, N. Synthesis 2003, 2667–2670; (b)
5.1.2.2. N-3-Cyano-5,6,7,8-tetrahydro-5-oxo-4-(3-
Henry, G. D. Tetrahedron 2004, 60, 6043–6061; (c)
Movassaghi, M.; Hill, M. D. J. Am. Chem. Soc. 2006,
128, 4592–4593; (d) Suzuki, H.; Sakai, N.; Iwahara, R.;
Fujiwaka, T.; Satoh, M.; Kakehi, A.; Konakahara, T.
J. Org. Chem. 2007, 72, 5878–5881; (e) Trost, B. M.;
Gutierrez, A. C. Org. Lett. 2007, 9, 1473–1476; (f) Hajbi,
Y.; Suzenet, F.; Khouili, M.; Lazar, S.; Guillaumet, G.
Tetrahedron 2007, 63, 8286–8297.
(2,5-dioxo-2,4-pyrrol-(5H)phenyl)quinolin-2-yl) acetam-
ide (16). White solid; mp 242–245 ꢁC; IR (cmÀ1) 3359,
2229, 1717, 1693, 1557, 1537, 1492, 1462, 1378, 1245,
1
992, 827, 701; H NMR (CDCl3, 200 MHz) d 8.11 (br
s, 1H), 7.59–7.51 (m, 2H), 7.33–7.31 (m, 1H), 7.22–
7.17 (m, 1H), 6.83 (s, 2H), 3.17 (t, J = 6.4 Hz, 2H),
2.63 (t, J = 6.4 Hz, 2H), 2.52 (s, 3H), 2.17 (quin,
J = 6.4 Hz, 2H); 13C NMR (CDCl3, 125 MHz) d
192.9, 167.5, 167.1, 165.8, 153.5, 152.0, 135.7, 132.7,
129.7, 126.7, 124.9, 124.0, 123.1, 121.5, 112.2, 37.4,
31.4, 21.5, 18.7. Anal. Calcd for C22H16N4O4: C,
66.00; H, 4.03; N, 13.99. Found: C, 66.20; H, 4.19; N,
13.81.
3. (a) Mishriky, N.; Girgis, N. S.; Arnos, S.; Nawwar, G. A.
M. Egypt J. Chem. 1980, 23, 433–438; (b) Moussa, H. H.;
Chabaka, L. M.; Zaki, D. Egypt J. Chem. 1983, 26, 469–
477.
4. Baldwin, J. J.; Engelhardt, E. L.; Hirschmann, R.;
Ponticello, G. S.; Atkinson, J. G.; Wasson, B. K.; Sweet,
C. S.; Scriabine, A. J. Med. Chem. 1980, 23, 65–70.
5. Krauze, A.; Vitolina, R.; Zarins, G.; Pelcers, J.; Kalme, Z.;
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Acknowledgments
6. (a) Manna, F.; Chimenti, F.; Bolasco, A.; Filippelli, A.;
Palla, A.; Filippelli, W.; Lampa, E.; Mercantini, R. Eur.
J. Med. Chem. 1992, 27, 627–632; (b) Manna, F.;
Chimenti, F.; Bolasco, A.; Bizzarri, B.; Filippelli, W.;
Filippelli, A.; Gagliardi, L. Eur. J. Med. Chem. 1999,
34, 245–254.
A.R.G. thanks CSIR, New Delhi for the award of
Senior Research Fellowship (SRF). We are thankful to
Mr. H. B. Singh for his help in correcting some typo-
graphical and grammatical errors.
7. Murata, T.; Shimada, M.; Sakakibara, S.; Yoshino, T.;
Masuda, T.; Shintani, T.; Sato, H.; Koriyama, Y.;
Fukushima, K.; Nunami, N.; Yamauchi, M.; Fuchikami,
K.; Komura, H.; Watanabe, A.; Ziegelbauer, K. B.;
Bacon, K. B.; Lowinger, T. B. Bioorg. Med. Chem. Lett.
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Supplementary data
Single-crystal data and CIF file of compound 3f (CCDC
# 282502) have been deposited at the Cambridge Crys-
tallographic Data Centre and can be obtained free of
charge via www.ccdc.cam.ac.uk/conts/retrieving.html(or
from the Cambridge Crystallographic Data Center, 12
Union Road, Cambridge CB21EZ, UK; fax: (+44)
1123-336-033; or email: deposit@ccdc.ac.uk).1H and
13C NMR spectra of the synthesized compounds are
available as supporting information. Supplementary
data associated with this article can be found, in the
online version, at doi:10.1016/j.bmc.2007.08.009.
8. For the first report see: (a) Sakurai, A.; Midorikawa, H.
Bull. Chem. Soc. Jpn. 1968, 41, 430–432; (b) Dobaria, A.
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