Synthesis and identification of β-aryloxyquinoline based diversely fluorine substituted N-aryl quinolone derivatives as a new class of antimicrobial, antituberculosis and antioxidant agents

Article abstract of DOI:10.1016/j.ejmech.2013.03.017

A new class of β-aryloxyquinoline based diversely fluorine substituted N-aryl quinolone derivatives 8a-x have been synthesized via a one-pot multicomponent reaction. In vitro antimicrobial activity of the synthesized compounds was investigated against a r

Full text of DOI:10.1016/j.ejmech.2013.03.017

European Journal of Medicinal Chemistry 63 (2013) 675e684
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and identification of b-aryloxyquinoline based diversely
fluorine substituted N-aryl quinolone derivatives as a new class of
antimicrobial, antituberculosis and antioxidant agents
*
Harshad G. Kathrotiya, Manish P. Patel
Department of Chemistry, Sardar Patel University, Vallabh Vidyanagar 388120, Gujarat, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of b-aryloxyquinoline based diversely fluorine substituted N-aryl quinolone derivatives 8aex
Received 9 August 2012
Received in revised form
28 December 2012
Accepted 8 March 2013
Available online 18 March 2013
have been synthesized via a one-pot multicomponent reaction. In vitro antimicrobial activity of the
synthesized compounds was investigated against a representative panel of pathogenic strains. Com-
pounds 8g, 8h, 8m, 8q and 8v exhibited excellent antimicrobial activity compared with first line drugs.
In vitro antituberculosis activity was evaluated against Mycobacterium tuberculosis H37Rv and com-
pounds 8h and 8q emerged as the promising antimicrobial member with better antituberculosis activity.
The brine shrimp bioassay was carried out to study the in vitro cytotoxic properties of the synthesized
compounds. In vitro antioxidant activity was evaluated by ferric-reducing antioxidant power method.
Compounds 8e, 8k, 8l, 8s, 8u and 8w showed highest antioxidant potency.
Keywords:
b
-Aryloxyquinoline
N-Aryl quinolone
Multicomponent reaction
Antimicrobial activity
Antituberculosis activity
Cytotoxicity
Ó 2013 Elsevier Masson SAS. All rights reserved.
FRAP assay
1. Introduction
antimycobacterial compounds. So this spot of research is accorded
an immense meaning and keeps on attracting much attention of
Fluorine has played a pivotal role in novel drug discovery for
modulating physical and biological properties of the molecule. Due
to its higher electronegativity, incorporation of fluorine atom(s)
within the molecule can enhance their biopotency, bioavailability,
metabolic stability and lipophilicity [1]. Trifluoromethylation is one
of the most significant strategies to improve pharmacological ac-
tivities of the molecule due to its high lipophilicity, thereby
enhancing in vivo uptake and transport of the candidate [2].
Emerging infectious diseases and the increasing number of
multi-drug resistant microbial pathogens still make the treatment
of infectious diseases an important and pressing global health
problem. Therefore, there is a vital need to discover new antimi-
crobial agents to avert the emergence of resistance and ideally
shorten the duration of therapy [3,4]. Furthermore, the ever-
increasing drug resistance, toxicity and side effects of currently
used antituberculosis drugs, and the absence of their bactericidal
activity highlight the need for new, safer and more effective
growing number of medicinal chemists.
Nowadays free metals become more harmful which play an
important role in the pathogenesis of many diseases, accounting for
continuing interest in the identification and development of novel
antioxidants that prevent metal-induced damages. Compounds
with antioxidant activity have been found to posses anticancer, anti-
cardiovascular, anti-inflammatory and many other activities [5].
Over the past few years, we have been principally engrossed in
the synthesis of quinoline incorporating structures for biological
evaluations [6e10] on the premise that the quinoline moiety is
found in a large variety of naturally occurring compounds and also
chemically useful synthons bearing diverse bioactivities [5,11e19].
It has been well-established that presence of aryl ring appended by
ether linkage at 2nd position of quinoline moiety is highly active
against bacterial pathogen and plays a pivotal role in development
of new antimicrobial drugs [6] and in consequence, emerged as a
validated molecular target.
On the other hand, since the discovery of nalidixic acid,
numerous quinolone derivatives have been prepared in search of
improved antibacterial activities [20e22]. In the past decade, the
so-called new fluorine substituted quinolones such as norfloxacin,
* Corresponding author. Tel.: þ91 02692 226856.
E-mail address: patelmanish1069@yahoo.com (M.P. Patel).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.03.017

676
H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
ofloxacin, ciprofloxacin, temafloxacin, defloxacin, sparfloxacin,
delafloxacin, lomefloxacin etc. which possess potent antibacterial
activity has been discovered and used clinically [23] (Fig. 1).
Although their structureeactivity relationship (SAR) has been
extensively studied [20e26], the SAR for N-1 and C-4 of quinolone
ring has not reached a clear conclusion. Studies by Koga et al.
indicated that the antibacterial activity of quinolones is greatly
influenced by the steric bulk of the N-1 substituent [24], Later, Chu
et al. discovered that quinolones with fluorophenyl at the N-1 po-
sition also possess excellent activity [25,26].
amount of acetic acid [28]. Subsequently, the one-pot three
component cyclocondensation of a series of -aryloxyquinoline-3-
carbaldehydes 3aed, malononitrile 7 and -enaminones 6aef in
ethanol containing a catalytic amount of piperidine afforded the
target compounds 8aex (Scheme 1) in good to excellent yields
(Supplementary Table 1).
b
b
3. Pharmacology
3.1. Antimicrobial activity
In this study, we introduce substituted b-aryloxyquinolines at the
C-4 position and diversely fluorine substituted aromatic ring at N-1
position on 5-quinolone for probing biological activity. An attempt
has been through to undertake the synthesis of N-aryl quinolone
The antimicrobial activity of synthesized compounds was carried
out by broth microdilution method according to National Commit-
tee for Clinical Laboratory Standards (NCCLS) [29]. Antibacterial
activity was screened against three Gram positive (B. subtilis MTCC
441, C. tetani MTCC 449, S. pneumonia MTCC 1936) and three Gram
negative (E. coli MTCC 443, S. typhi MTCC 98, V. cholerae MTCC 3906)
bacteria by using ampicillin, ciprofloxacin, norfloxacin and chlor-
amphenicol as a standard antibacterial drug. Antifungal activity was
screened against two fungal species (A. fumigatus MTCC 3008 and
C. albicans MTCC 227) where griseofulvin and nystatin used as
standard antifungal drugs. The antimicrobial screening data are
shown in Table 1. All MTCC cultures were collected from Institute of
Microbial Technology, Chandigarh and tested against above
mentioned known drugs. Mueller Hinton broth was used as nutrient
medium to grow and dilute the drug suspension for the test.
bearing b-aryloxyquinoline derivatives with the assumption that the
incorporation of more than one bioactive moieties into a single
scaffold may produce novel heterocycles with fascinating antimi-
crobial activity along with antitubercular and antioxidant activity.
2. Chemistry
The synthetic approach adopted to obtain the target compounds
is depicted in Scheme 1. The starting material 2-chloro-3-
formylquinolines 1aed were prepared according to literature pro-
cedure [27] by VilsmeiereHaack reaction and conveniently con-
verted to b-aryloxyquinoline-3-carbaldehydes 3aed by nucleophilic
displacement of chloro group at C2 in 1aed with p-cyano phenol 2
in refluxing dimethylformamide using anhydrous potassium car-
3.2. Antituberculosis activity
bonate as a base. The required
by the reaction of -diketones 4a, b with fluorinated aromatic
amines 5aec in methanol under reflux in the presence of a catalytic
b-enaminones 6aef were prepared
b
In vitro antituberculosis activity of all the newly synthesized
compounds against Mycobacterium tuberculosis H37Rv strain was
Fig. 1. Representative of fluorine substituted quinolone drugs.

H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
677
Scheme 1. General synthetic route for the synthesis of title compounds 8aex.
determined by using Lowenstein-Jensen medium (conventional
method) as described by Rattan [30] and the observed results are
presented in Table 2 in the form of % inhibition, relative to that of
standard antitubercular drugs isoniazid and rifampicin. Of the
compounds studied, five compounds those exhibited highest %
inhibition, were again screened to get their MIC values (Table 3).
were expressed as ascorbic equivalent (mmol/100 gm compound)
and are listed in Table 5.
4. Results and discussion
4.1. Biological results
3.3. Cytotoxicity (brine shrimp lethality bioassay)
4.1.1. Antimicrobial activity
Upon investigation of antimicrobial activity data (Table 1), it has
been observed that compounds 8g, 8h and 8q showed excellent
The in vitro lethality test was carried out using brine shrimp eggs
i.e. Artemia cysts. The Brine shrimp lethality bioassay is considered
as a useful tool for preliminary toxicity assessment of bioactive
compounds. Five compounds those exhibited highest % inhibition,
were again screened for their cytotoxicity (Table 4) by using the
protocol of Meyer et al. [31].
activity MIC ¼ 62.5
against gram negative bacteria V. cholerae as compared to ampicillin
MIC ¼ 100 g/mL, chloramphenicol 50 g/mL and ciprofloxacin
25 g/
g/mL. In case of inhibiting S. typhi, compounds 8g MIC ¼ 50
mL and 8m 62.5 g/mL were found to be exceedingly potent upon
comparison with reference drugs. Towards E. coli, compounds 3b,
8h and 8v MIC ¼ 62.5 g/mL have shown outstanding inhibitory
effect as well as compound 8q MIC ¼ 62.5 g/mL displayed strong
mg/mL, 25 mg/mL and 62.5 mg/mL respectively
m
m
m
m
m
3.4. Antioxidant activity (FRAP assay)
m
m
Ferric reducing antioxidant power (FRAP) of newly synthesized
compounds was measured by a modified method of Benzie and
Strain [32]. The antioxidant potentials of the compounds 3aed and
8aex were estimated as their power to reduce the TPTZ-Fe(III)
complex to TPTZ-Fe(II) complex. Absorbance of intensive blue
colour [Fe(II)-TPTZ] complex was measured at 593 nm. The ascorbic
acid was used as a standard antioxidant compound. The results
inhibition against S. pneumonia as compared to the standard drugs.
In case of inhibiting gram positive bacteria B. subtilis and C. tetani, all
the compounds have shown good to excellent activity than that of
standard drugs except 8y and 8z. Some of the compounds were
found to be equipotent upon comparison with standard drugs.
Antifungal study revealed that all the synthesized
b-arylox-
yquinolines and their quinolyl quinoline derivatives have poor

678
H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
Table 1
In vitro antimicrobial activity of
b
-aryloxyquinolines 3aed and their N-aryl quinolone derivatives 8aex (MICs,
Gram negative bacteria
mg/mL).
Entry
Gram positive bacteria
Fungi
B.S. MTCC 441
C.T.MTCC 449
S.P. MTCC 1936
E.C. MTCC 443
S.T. MTCC 98
V.C. MTCC 3906
A.F. MTCC 3008
C.A. MTCC 227
3a
3b
3c
3d
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
8l
8m
8n
8o
8p
8q
8r
8s
8t
8u
8v
8w
8x
200
100
125
250
100
250
250
200
100
250
125
200
200
250
200
100
200
100
125
200
200
250
200
250
200
250
200
250
250
50
250
100
125
200
125
200
100
100
250
200
200
100
100
250
125
250
100
125
200
125
200
250
125
200
200
250
500
500
250
50
250
200
125
200
250
100
100
200
125
200
250
100
250
200
250
125
100
250
200
100
62.5
125
125
250
200
250
125
200
100
50
100
62.5
250
250
100
250
100
125
200
250
100
62.5
200
200
250
100
100
250
200
125
250
125
250
200
500
62.5
200
125
100
50
100
125
200
100
250
250
125
200
250
200
50
200
250
250
250
100
250
125
200
250
200
250
100
100
250
200
500
250
250
1000
250
1000
>1000
>1000
1000
>1000
500
1000
>1000
500
1000
1000
>1000
>1000
500
1000
>1000
1000
1000
>1000
>1000
500
1000
1000
1000
>1000
1000
>1000
1000
500
250
500
1000
1000
250
>1000
250
>1000
250
>1000
250
1000
1000
500
1000
>1000
1000
>1000
1000
>1000
500
62.5
25
200
250
100
125
100
250
100
250
62.5
125
250
200
200
125
100
250
500
200
125
200
100
50
62.5
100
250
200
250
100
100
250
100
50
25
10
n.t.
n.t.
1000
n.t.
n.t.
n.t.
n.t.
Ampicillin
Chloramphenicol
Ciprofloxacin
Norfloxacin
Griseofulvin
Nystatin
n.t.a
n.t.
n.t.
n.t.
100
100
50
100
n.t.
100
50
n.t.
50
10
n.t.
n.t.
25
10
n.t.
n.t.
25
10
n. t.
n.t.
500
100
n.t.
n.t.
B.S.: Bacillus subtilis, C.T.: Clostridium tetani, S.P.: Streptococcus pneumoniae, E.C.: Escherichia coli, S.T.: Salmonella typhi, V.C.: Vibrio cholerae, A.F.: Aspergillus fumigatus, C.A.:
Candida albicans.
MTCC: Microbial Type Culture Collection.
a
n. t. Not tested.
activity against A. fumigatus. In comparison with standard fungi-
cidal griseofulvin MIC ¼ 500 g/mL, compounds 8c, 8g, 8i, 8k and
g/mL were found to posses better inhibitory results
8aex have found to be increase antimicrobial activity upto two, three
or four fold. The data indicate that a change in the substituent might
also affect the antibacterial activity of title compounds 8aex. The
parent compounds with R2 ¼ H showed strong inhibitory effect
against most of the employed strains than R2 ¼ CH3 i.e. 8c, 8g, 8i, 8k
m
8m MIC ¼ 250
m
towards fungal pathogen C. albicans.
The anti-microorganism tests with all four b-aryloxyquinolines
and their twenty four fluorine substituted N-aryl quinolone
derivatives (Table 1) have established some interesting structure-
activity relationships. Upon cyclocondensation of compounds 3ae
and 8m. Compounds having R1 ¼ CH3 on
b-aryloxyquinoline and
fluorine substitution at R3 and R6 position on N-aryl rings displayed
highest antibacterial activity against gram negative bacteria i.e. 8g
and 8h. It is interesting to point out that compound 8q carrying
d withmalononitrileand variousb-enaminones, resultedcompounds
electron negative groups on both b-aryloxyquinoline and N-aryl rings
Table 2
showed excellent antibacterial potency towards S. pneumonia and
V. cholerae. This excellent inhibition could be attributed to the strong
electron negativity combination of Cl and CF3 groups. Surprisingly,
replacement of methyl group by H at R2 in compound 8g (compound
8h) showed superior inhibition against C. tetani, E. coli and V. cholerae
but observed remarkable reduction in antimicrobial spectrum
In vitro antituberculosis activity (% Inhibition) of
N-aryl quinolone derivatives 8aex against M. tuberculosis H37Rv (at concentration
250 g/mL).
b-aryloxyquinolines 3aed and their
m
Entry
% Inhibition
Entry
% Inhibition
3a
3b
3c
3d
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
42
26
61
11
10
21
75
64
52
45
80
99
27
32
38
8l
93
84
87
95
84
99
98
35
54
88
24
23
62
98
99
8m
8n
8o
8p
Table 3
In vitro antituberculosis activity of title compounds exhibiting higher % inhibition
against M. tuberculosis H37Rv (MICs, mg/mL).
8q
8r
8s
8t
Entry
% Inhibition
MIC
62.5
125
100
25
62.5
40
0.20
8h
8l
8o
8q
99
93
95
99
98
98
99
8u
8v
8w
8x
Rifampicin
Isoniazid
8r
Rifampicin
Isoniazid

H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
679
Table 4
Effect of compounds on brine shrimp lethality bioassay.
4.1.2. Antituberculosis activity
The encouraging results from the antimicrobial studies
impelled us to go for the preliminary screening of the synthesized
compounds for their in vitro antituberculosis activity against
M. tuberculosis H37Rv. Of the compounds screened for antitu-
berculosis activity, compound 8q displayed excellent activity
against M. tuberculosis H37Rv. This excellent potency could be
credited to the strong electron negativity combination of Cl and
CF3 groups. Whereas compounds 8h and 8r which are having
inductively electron withdrawing but mesomerically electron
releasing methyl groups on both quinoline and quinolone rings
with fluorine substituent on N-aryl ring found to possess better
activity against M. tuberculosis H37Rv. Antitubercular potency of
compounds 3b (26%) and 3c (61%) were found to get intensified,
upon cyclocondensation with malononitrile and
which results compounds 8h (99%), 8l (93%), 8o (95%), 8q (99%)
and 8r (98%).
Compounds 8h and 8q have been emerged as the promising
antimicrobial member along with better antitubercular activity
(Tables 1e3) of this series of compounds. Unfortunately, majority of
compounds showed poor inhibition of M. tuberculosis growth.
Entry Concentration Log No. of No. of
% of
LC50
g mLꢀ1
)
(m
g mLꢀ1
(Conc.) nauplii nauplii mortality
)
(
m
taken
dead
8h
8l
5
0.699
1
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
0
1
2
5
7
8
1
3
5
7
8
9
2
4
6
7
8
9
0
1
2
4
5
7
1
2
4
5
7
8
0
10
20
50
70
80
10
30
50
70
80
90
20
40
60
70
80
90
0
10
20
40
50
70
10
20
40
50
70
80
27.50
17.14
14.10
37.78
23.34
10
20
30
40
50
5
10
20
30
40
50
5
10
20
30
40
50
5
10
20
30
40
50
5
1.301
1.477
1.602
1.699
0.699
1
1.301
1.477
1.602
1.699
0.699
1
1.301
1.477
1.602
1.699
0.699
1
1.301
1.477
1.602
1.699
0.699
1
b-enaminones
8o
8q
8r
4.1.3. Cytotoxicity
The LC50 values obtained for the five compounds those exhibited
highest % inhibition are shown in Table 4. As can be seen for the five
compounds, there was no significant toxicity observed in case of
compounds 8q, 8h and 8r after 24 h incubation. Among the com-
pounds tested, compounds 8l and 8o showed greater toxicity.
10
20
30
40
50
1.301
1.477
1.602
1.699
4.1.4. Antioxidant activity
The examination of the data (Table 5) revealed that compounds
8l and 8u showed relatively high antioxidant power while, com-
pounds 8e, 8k, 8s and 8w found to have better ferric reducing
power. Compounds 8c, 8g and 8i displayed promising antioxidant
potency.
From the ferric reducing power results it can be stated that,
compounds carrying electron withdrawing Cl group at R1 position
exhibited very poor antioxidant activity, while compounds having
electron donating CH3 and OCH3 groups at R1 position exhibited
excellent ferric reducing power. In addition, it should be noted that
the compounds with unsubstituted quinolone ring (R2 ¼ H) gives
better results than methyl substituted quinolone ring (R2 ¼ CH3) i.e.
compounds 8c, 8e, 8g, 8i, 8k, 8s, 8u and 8w except 8l. In case of
compound 8l, CH3 group at R2 position improves the antioxidant
power, it may be due to the combination effect of R1 ¼ CH3,
R2 ¼ CH3 and R3 ¼ CF3.
towards B. subtilis, S. pneumonia, S. typhi and C. albicans. In addition,
it should be noted that the fluorine substitution at R3 and R6 position
on N-aryl ring are better than substitution at R4 and R5 i.e. 8g, 8h,
8m and 8q. This revealed that the position of fluorine substitutions
on N-aryl ring has an impact on the antimicrobial activity. The
Order of functional groups basis on activity (higher to lower),
R1 ¼ CH3 > Cl> H > OCH3, R2 ¼ H > CH3 and R3, R6 ¼ F > R4 ¼ CF3 > R4,
R5 ¼ F. A close examination of the structures of the active compounds
depicted in Table 1 revealed that, their antimicrobial activity is
strongly bound to the nature of the substituent on b-aryloxyquino-
line, together with the substituent linked to the quinolone and N-aryl
rings of the structure.
Table 5
In vitro antioxidant activity of the compounds 3aed and their 8aex derivatives.
5. Conclusion
Entry
OD (593 nm)
Frap valuea
Entry
OD (593 nm)
Frap valuea
3a
3b
3c
3d
8a
8b
8c
8d
8e
8f
8g
8h
8i
8j
8k
0.097
0.081
0.176
0.237
0.083
0.426
0.976
0.099
1.365
0.102
1.208
0.070
1.226
0.617
1.276
19.21
16.04
34.87
46.95
16.44
84.40
193.37
19.61
270.44
20.20
239.33
13.86
8l
1.712
0.506
0.011
0.015
0.017
0.026
0.036
1.401
0.128
1.864
0.772
1.310
0.072
2.511
339.19
100.25
2.17
2.97
3.36
In conclusion, the aim of the present investigation was to design
a new series of quinolone derivatives by introduction of substituted
8m
8n
8o
8p
8q
8r
b-aryloxyquinolines at the C-4 position and diversely fluorine
substituted aromatic ring at N-1 position for probing antimicrobial,
antituberculosis and antioxidant activity. Modification of sub-
stituents on both b-aryloxyquinolines ring and N-aryl quinolone
5.15
7.13
8s
8t
277.57
25.36
369.31
152.95
259.54
14.26
e
ring with various electron releasing and electron withdrawing
groups improved the activity. Compounds 8g, 8h, 8m, 8q and 8v
exhibited excellent antimicrobial inhibition. While, compounds 8e,
8k, 8l, 8s, 8u and 8w showed highest ferric reducing power.
Compounds 8h and 8q emerged as the promising antimicrobial
member with better antitubercular activity and lower toxicity.
Consequently, such type of compounds would represent a fertile
matrix for further development of more biologically potent agents
that deserve further investigation and derivatization in order to
discover the scope and limitation of its biological activities.
8u
8v
8w
8x
A.A.
242.90
122.24
252.81
A.A. ¼ Ascorbic acid.
Concentration of compounds used ¼ 200
m
g/mL.
mg/mL.
Concentration of Standard (A.A.) ¼ 176
a
A.A. mm/100 gm sample.

680
H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
6. Experimental
6.1.2.1. 20-Amino-2-(4-cyanophenoxy)-10-(2,5-difluorophenyl)-50-
oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-carbonitrile (8a).
IR (KBr, nmax, cmꢀ1): 3445 and 3350 (asym. and sym. str. of eNH2),
2175 (C^N str.), 1650 (C]O str.), 1225 (CeOeC ether str.). 1H NMR
6.1. Chemistry
All reactions were performed with commercially available
reagents and they were used without further purification.
Organic solvents were purified by standard methods [33] and
stored over molecular sieves. All reactions were monitored by
thin-layer chromatography (TLC, on aluminium plates coated
with silica gel 60 F254, 0.25 mm thickness, Merck) carried on
fluorescent coated plates and detection of the components was
made by exposure to iodine vapours or UV light. Melting points
were determined in open capillaries and the declared values are
not corrected. Infrared spectra were recorded on Shimadzu FTIR
8401 spectrophotometer using potassium bromide pellets in the
range 4000e400 cmꢀ1 and frequencies of only characteristic
(400 MHz, DMSO-d6): d 1.78e1.85 (m, 2H, CH2eCH2), 1.97e2.04 (m,
2H, CH2eCO), 2.11e2.30 (m, 2H, CH2eCH2eCO), 5.04 (s, 1H, CH),
5.71 (s, 2H, NH2), 7.29e8.21 (m, 12H, AreH). 13C NMR (100 MHz,
DMSOed6) d: 21.12 (CH2), 28.31 (CH2), 32.45 (C4), 36.49 (CH2eCO),
59.01 (CeCN), 107.45, 111.21, 119.01, 119.17, 119.92, 120.10, 120.72,
121.98, 122.75, 123.18, 125.88, 127.17, 128.35, 130.31, 130.92, 133.22,
134.61, 138.43, 144.61, 152.12, 154.19, 158.33, 159.29, 162.77 (24C,
AreC), 195.48 (C]O); MS (m/z): 545.7 [M þ 1]þ.
6.1.2.2. 20-Amino-2-(4-cyanophenoxy)-10-(2,5-difluorophenyl)-70,70-
dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-car-
bonitrile (8b). IR (KBr, nmax, cmꢀ1): 3460 and 3355 (asym. and sym.
str. of eNH2), 2165 (C^N str.), 1670 (C]O str.), 1230 (CeOeC ether
peaks are expressed in cmꢀ1 1H and 13C Nuclear Magnetic
.
Resonance spectra were recorded in DMSO-d6 on a Bruker Avance
400F (MHz) spectrometer (Bruker Scientific Corporation Ltd.,
Switzerland) using residual solvent signal as an internal standard
at 400 MHz and 100 MHz respectively. Chemical shifts are re-
ported in parts per million (ppm). Splitting patterns are desig-
nated as s, for singlet; d, for doublet; m, for multiplet. UV spectra
were recorded on Shimadzu Type 160-A spectrometer. The ESI
mass spectra were scanned on a Shimadzu LCMS 2010 spec-
trometer (Shimadzu, Tokyo, Japan) at Oxygen Healthcare
Research Pvt. Ltd., Ahmedabad. Elemental analyses were per-
formed by PerkineElmer 2400 series-II elemental analyzer (Per-
kineElmer, USA) at Sophisticated Instrumentation Centre for
Applied Research and Training (SICART), Vallabh Vidyanagar and
all compounds are within ꢁ0.4% of the theoretical compositions.
Yields are not optimized. Ampicillin, ciprofloxacin, norfloxacin,
chloramphenicol, griseofulvin, nystatin, isoniazid, rifampicin and
str.). 1H NMR (400 MHz, DMSO-d6):
d 0.91 (s, 3H, CH3), 0.93 (s, 3H,
CH3), 1.81 (d, 1H, J ¼ 16.0 Hz, CH2), 2.01 (d, 1H, J ¼ 16.0 Hz, CH2),
2.10e2.24 (m, 2H, CH2eCO), 5.01 (s,1H, CH), 5.69 (s, 2H, NH2), 7.35e
8.21 (m, 12H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 27.19 (CH3),
29.09 (CH3), 32.61 (C4), 34.10 (C(CH3)2), 41.12 (CH2), 49.68 (CH2e
CO), 58.89 (CeCN), 106.98, 110.93, 119.15, 119.47, 119.81, 120.11,
120.82, 121.85, 122.68, 123.43, 125.51, 127.15, 128.12, 130.35, 130.65,
133.29, 135.01, 138.30, 144.62, 151.93, 154.15, 158.39, 159.40, 161.64
(24C, AreC), 195.29 (C]O); MS (m/z): 573.9 [Mþ1]þ.
6.1.2.3. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-50-
oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-carbonitrile
(8c).
IR (KBr, nmax, cmꢀ1): 3470 and 3350 (asym. and sym. str. of eNH2),
2175 (C^N str.), 1675 (C]O str.), 1245 (CeOeC ether str.). 1H NMR
(400 MHz, DMSO-d6): d 1.79e1.88 (m, 2H, CH2eCH2), 1.98e2.03 (m,
2H, CH2eCO), 2.17e2.32 (m, 2H, CH2eCH2eCO), 5.06 (s, 1H, CH),
L
-ascorbic acid were commercial.
5.66 (s, 2H, NH2), 7.18e8.25 (m, 12H, AreH). 13C NMR (100 MHz,
DMSO-d6) d: 21.19 (CH2), 28.37 (CH2), 32.44 (C4), 36.44 (CH2eCO),
6.1.1. General procedure for the synthesis of 4-(6-(un)substituted-3-
formylquinolin-2-yloxy)benzonitrile 3aed
58.75 (CeCN), 107.69, 111.18, 118.79, 118.98, 119.17, 119.43, 120.60,
121.92, 122.89, 123.36, 125.69, 127.02, 128.29, 130.07, 130.87, 133.04,
134.67, 138.24, 144.58, 152.06, 154.08, 158.27, 159.28, 162.61 (24C,
AreC), 195.59 (C]O); MS (m/z): 545.8 [M þ 1]þ.
A 100 mL round bottomed flask, fitted with a reflux condenser,
was charged with a mixture of 2-chloro-3-formylquinoline 1ae
d (1 mmol), p-cyano phenol 2 (1 mmol) and anhydrous potassium
carbonate (2 mmol) in dimethylformamide (10 mL). The mixture
was heated at 90 ꢂC for 3.0 h and the progress of the reaction was
monitored by TLC. After the completion of reaction (as evidenced
by TLC), the reaction mixture was cooled to room temperature and
then poured into chilled water (100 mL) with continuous stirring
followed by neutralization with 1.5 N HCl until pH 7 resulted. The
solid mass separated was collected by filtration, washed well with
water, dried and crystallized from ethyl acetate.
6.1.2.4. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-70,70-
dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-car-
bonitrile (8d). IR (KBr, nmax, cmꢀ1): 3435 and 3360 (asym. and sym.
str. of eNH2), 2185 (C^N str.), 1660 (C]O str.), 1220 (CeOeC ether
str.). 1H NMR (400 MHz, DMSO-d6):
d 0.89 (s, 3H, CH3), 0.92 (s, 3H,
CH3), 1.77 (d, 1H, J ¼ 16.2 Hz, CH2), 1.98 (d, 1H, J ¼ 16.2 Hz, CH2),
2.08e2.26 (m, 2H, CH2eCO), 5.08 (s, 1H, CH), 5.81 (s, 2H, NH2),
7.31e8.26 (m, 12H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 27.21
(CH3), 29.13 (CH3), 32.29 (C4), 34.09 (C(CH3)2), 41.30 (CH2), 49.78
(CH2eCO), 58.71 (CeCN), 107.37, 111.35, 118.62, 118.90, 119.38,
119.62, 120.25, 121.63, 122.38, 123.40, 125.75, 127.09, 128.46, 130.12,
130.91, 133.25, 134.69, 137.90, 144.65, 152.12, 154.45, 158.35, 159.31,
162.54 (24C, AreC), 195.63 (C]O); MS (m/z): 574.0 [M þ 1]þ.
6.1.2. General procedure for the synthesis of 20-amino-6-(un)
substituted-2-(4-cyanophenoxy)-10-(2,5/3,4/4-substitutedphenyl)-
70,70-(un)substituted-50-oxo-10,40,50,60,70,80-hexahydro-3,40-
biquinoline-30-carbonitrile 8aex
A 100 mL round bottomed flask, fitted with a reflux con-
denser, was charged with a mixture of
b-aryloxyquinoline-3-
6.1.2.5. 20-Amino-2-(4-cyano0phenoxy)-50-oxo-10-[3-(tri-
fluoromethyl)phenyl]-10,40,50,60,70,8 -hexahydro-3,40-biquinoline-30-
carbonitrile (8e). IR (KBr, nmax, cmꢀ1): 3430 and 3345 (asym. and
sym. str. of eNH2), 2160 (C^N str.), 1655 (C]O str.), 1235 (CeOeC
carbaldehydes 3aed (1 mmol), malononitrile 7 (1 mmol),
b
-
enaminone 6a,b (1 mmol) and a catalytic amount of piperidine
(0.2 mmol) in ethanol (10 mL). The reaction mixture was heated
under reflux for 1e1.5 h and the progress of the reaction was
monitored by TLC. After the completion of reaction (as evidenced
by TLC), the reaction mixture was cooled to room temperature and
stirred magnetically for further 20 min, the solid mass separated
was collected by filtration, washed well with ethanol (15 mL) and
purified by leaching in equal volume ratio of chloroform and
methanol (15 mL) to obtain pure solid sample.
ether str.). 1H NMR (400 MHz, DMSO-d6):
d 1.76e1.84 (m, 2H, CH2e
CH2), 1.95e2.07 (m, 2H, CH2eCO), 2.15e2.35 (m, 2H, CH2eCH2e
CO), 5.02 (s, 1H, CH), 5.79 (s, 2H, NH2), 7.26e8.24 (m, 12H, AreH).
13C NMR (100 MHz, DMSO-d6)
d: 21.31 (CH2), 28.42 (CH2), 32.35
(C4), 36.38 (CH2eCO), 59.11 (CeCN), 106.83, 111.04, 118.43, 118,77,
119.25, 119.63, 120.33, 121.81, 122.78, 123.45, 125.66, 127.20, 128.47,
130.35, 130.98, 131.28, 133.09, 134.42, 138.11, 144.53, 152.25, 154.63,

H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
681
158.44, 159.19, 160.48 (25C, AreC), 195.51 (C]O); MS (m/z): 578.1
ether str.). 1H NMR (400 MHz, DMSO-d6):
3H, CH3), 1.82 (d, 1H, J ¼ 16.2 Hz, CH2), 2.03 (d, 1H, J ¼ 16.2 Hz, CH2),
2.13e2.27 (m, 2H, CH2eCO), 2.50 (s, 3H, AreCH3), 5.05 (s, 1H, CH),
5.65 (s, 2H, NH2), 7.26e8.28 (m, 11H, AreH). 13C NMR (100 MHz,
d
0.90 (s, 3H, CH3), 0.93 (s,
[M þ 1]þ.
6.1.2.6. 20-Amino-2-(4-cyanophenoxy)-70,70-dimethyl-50-oxo-10-[3-
(trifluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-biquinoline-
30-carbonitrile (8f). IR (KBr, nmax, cmꢀ1): 3450 and 3330 (asym. and
sym. str. of eNH2), 2175 (C^N str.), 1650 (C]O str.), 1240 (CeOeC
DMSO-d6) d: 21.41 (AreCH3), 27.09 (CH3), 29.20 (CH3), 32.38 (C4),
34.13 (C(CH3)2), 41.22 (CH2), 49.66 (CH2eCO), 59.61 (CeCN), 107.43,
110.84, 111.67, 119.10, 119.57, 120.69, 120.77, 121.86, 123.47, 123.59,
126.48, 126.90, 127.15, 130.78, 131.53, 132.25, 134.55, 134.70, 135.07,
137.24, 142.86, 151.16, 151.92, 160.18 (24C, AreC), 195.61 (C]O); MS
(m/z): 588.0 [M þ 1]þ.
ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.93 (s, 3H, CH3), 0.96 (s,
3H, CH3), 1.75 (d, 1H, J ¼ 16.0 Hz, CH2), 1.99 (d, 1H, J ¼ 16.0 Hz, CH2),
2.12e2.25 (m, 2H, CH2eCO), 5.05 (s, 1H, CH), 5.72 (s, 2H, NH2),
7.38e8.29 (m, 12H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 27.15
(CH3), 29.18 (CH3), 32.40 (C4), 33.87 (C(CH3)2), 41.19 (CH2), 49.81
(CH2eCO), 58.86 (CeCN), 107.26, 111.31, 118.44, 118.85, 119.37,
115.43, 120.44, 121.70, 122.93, 123.39, 125.78, 127.26, 128.53, 130.47,
131.07, 131.62, 133.12, 134.47, 138.27, 144.62, 152.38, 154.73, 158.40,
159.36, 162.70 (25C, AreC), 195.42 (C]O); MS (m/z): 605.9
[M þ 1]þ.
6.1.2.11. 20-Amino-2-(4-cyanophen0oxy)-6-methyl-50-oxo-10-[3-(tri-
fluoromethyl)phenyl]-10,40,50,60,70,8 -hexahydro-3,40-biquinoline-30-
carbonitrile (8k). IR (KBr, nmax, cmꢀ1): 3455 and 3325 (asym. and
sym. str. of eNH2), 2180 (C^N str.), 1655 (C]O str.), 1235 (CeOeC
ether str.). 1H NMR (400 MHz, DMSO-d6):
d 1.75e1.86 (m, 2H, CH2e
CH2), 1.93e2.06 (m, 2H, CH2eCO), 2.15e2.33 (m, 2H, CH2eCH2e
CO), 2.44 (s, 3H, AreCH3), 5.09 (s, 1H, CH), 5.73 (s, 2H, NH2), 7.38e
6.1.2.7. 20-Amino-2-(4-cya0nophenoxy)-10-(2,5-difluorophenyl)-6-
methyl-50-oxo-10,40,50,60,70,8 -hexahydro-3,40-biquinoline-30-carbon-
itrile (8g). IR (KBr, nmax, cmꢀ1): 3445 and 3360 (asym. and sym. str.
of eNH2), 2160 (C^N str.),1670 (C]O str.),1235 (CeOeC ether str.).
8.27 (m, 11H, AreH). 13C NMR (100 MHz, DMSOed6)
d: 21.25 (CH2),
21.33 (AreCH3), 28.38 (CH2), 32.35 (C4), 36.40 (CH2eCO), 58.39 (Ce
CN), 107.51, 110.82, 116.52, 119.30, 119.68, 120.12, 120.75, 121.92,
123.09, 123.41, 126.39, 126.85, 127.11, 127.54, 130.78, 131.67, 132.24,
134.63, 134.70, 135.04, 137.15, 142.81, 151.12, 151.93, 159.08 (25C,
AreC), 195.42 (C]O); MS (m/z): 592.1 [M þ 1]þ.
1H NMR (400 MHz, DMSO-d6):
d 1.80e1.88 (m, 2H, CH2eCH2), 1.94e
2.07 (m, 2H, CH2eCO), 2.17e2.32 (m, 2H, CH2eCH2eCO), 2.49 (s,
3H, AreCH3), 5.05 (s, 1H, CH), 5.77 (s, 2H, NH2), 7.33e8.25 (m, 11H,
AreH). 13C NMR (100 MHz, DMSO-d6)
d
: 21.35 (CH2), 21.42 (Are
6.1.2.12. 20-Amino-2-(4-cyanophenoxy)-6,70,70-trimethyl-50-oxo-10-
[3-(trifluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-biquino-
line-30-carbonitrile (8l). IR (KBr, nmax, cmꢀ1): 3475 and 3330 (asym.
and sym. str. of eNH2), 2160 (C^N str.), 1670 (C]O str.), 1240 (Ce
CH3), 28.37 (CH2), 32.41 (C4), 36.45 (CH2eCO), 59.25 (CeCN),
107.34, 111.02, 111.81, 119.28, 119.73, 120.46, 120.90, 121.49, 123.15,
123.46, 126.52, 126.89, 127.12, 130.66, 131.50, 132.25, 134.48, 134.68,
135.23, 136.82, 142.16, 152.15, 153.28, 159.38 (24C, AreC), 195.47
(C]O); MS (m/z): 560.3 [M þ 1]þ.
OeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.88 (s, 3H, CH3),
0.90 (s, 3H, CH3), 1.70 (d, 1H, J ¼ 16.2 Hz, CH2), 2.02 (d, 1H,
J ¼ 16.2 Hz, CH2), 2.13e2.22 (m, 2H, CH2eCO), 2.47 (s, 3H, AreCH3),
5.01 (s, 1H, CH), 5.56 (s, 2H, NH2), 7.40e8.20 (m, 11H, AreH). 13C
6.1.2.8. 20-Amino-2-(4-cyanophenoxy)-10-(2,5-difluorophenyl)-
6,70,70-trimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-
30-carbonitrile (8h). IR (KBr, nmax, cmꢀ1): 3465 and 3365 (asym. and
sym. str. of eNH2), 2180 (C^N str.), 1660 (C]O str.), 1220 (CeOeC
NMR (100 MHz, DMSO-d6) d: 21.40 (AreCH3), 27.13 (CH3), 29.33
(CH3), 32.50 (C4), 33.96 (C(CH3)2), 41.74 (CH2), 49.71 (CH2eCO),
59.04 (CeCN), 107.82, 110.23, 119.13, 121.85, 123.42, 125.32, 126.75,
126.84, 126.99, 127.12, 127.37, 130.07, 131.27, 131.77, 132.12, 134.75,
135.06, 135.17, 137.19, 138.24, 143.03, 151.49, 152.15, 158.29, 159.10
(25C, AreC), 195.47 (C]O); MS (m/z): 619.8 [M þ 1]þ.
ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.92 (s, 3H, CH3), 0.98 (s,
3H, CH3), 1.86 (d, 1H, J ¼ 16.2 Hz, CH2), 2.00 (d, 1H, J ¼ 16.2 Hz, CH2),
2.09e2.26 (m, 2H, CH2eCO), 2.45 (s, 3H, AreCH3), 5.08 (s, 1H, CH),
5.80 (s, 2H, NH2), 7.43e8.20 (m, 11H, AreH). 13C NMR (100 MHz,
DMSO-d6)
d
: 21.35 (AreCH3), 27.16 (CH3), 29.41 (CH3), 32.46 (C4),
6.1.2.13. 20-Amino-6-chloro-2-(4-cyanophenoxy)-10-(2,5-difluoro-
phenyl)-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-carbon-
itrile (8m). IR (KBr, nmax, cmꢀ1): 3470 and 3345 (asym. and sym. str.
of eNH2), 2175 (C^N str.),1675 (C]O str.),1225 (CeOeC ether str.).
34.28 (C(CH3)2), 40.91 (CH2), 49.78 (CH2eCO), 60.37 (CeCN),107.53,
110.83, 111.78, 119.23, 119.52, 120.64, 120.89, 121.58, 123.26, 123.37,
126.36, 126.79, 127.03, 130.71, 131.47, 132.12, 134.49, 134.69, 135.12,
137.06,142.96,151.09,151.78,158.26 (24C, AreC),195.40 (C]O); MS
(m/z): 587.9 [M þ 1]þ.
1H NMR (400 MHz, DMSO-d6):
d 1.78e1.90 (m, 2H, CH2eCH2), 1.98e
2.08 (m, 2H, CH2eCO), 2.19e2.36 (m, 2H, CH2eCH2eCO), 5.07 (s,1H,
CH), 5.76 (s, 2H, NH2), 7.31e8.22 (m,11H, AreH). 13C NMR (100 MHz,
6.1.2.9. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-6-
methyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-carbon-
itrile (8i). IR (KBr, nmax, cmꢀ1): 3470 and 3360 (asym. and sym. str.
of eNH2), 2175 (C^N str.), 1640 (C]O str.),1215 (CeOeC ether str.).
DMSO-d6) d: 21.28 (CH2), 28.41 (CH2), 32.60 (C4), 36.47 (CH2eCO),
59.12 (CeCN), 107.91, 110.90, 118.68, 119.25, 120.92, 121.63, 122.47,
123.52, 126.91, 127.78, 128.44, 129.31, 129.82, 130.47, 132.21, 132.79,
134.78, 137.55, 143.10, 149.78, 151.97, 152.44, 158.20, 159.91 (24C,
AreC), 195.53 (C]O); MS (m/z): 580.1 [M þ 1]þ, 581.1 [M þ 2].
1H NMR (400 MHz, DMSO-d6):
d 1.72e1.83 (m, 2H, CH2eCH2), 1.91e
2.02 (m, 2H, CH2eCO), 2.12e2.31 (m, 2H, CH2eCH2eCO), 2.42 (s,
3H, AreCH3), 5.00 (s, 1H, CH), 5.61 (s, 2H, NH2), 7.42e8.21 (m, 11H,
6.1.2.14. 20-Amino-6-chloro-2-(4-cyanophenoxy)-10-(2,5-difluoro-
phenyl)-70,70-dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquino-
line-30-carbonitrile (8n). IR (KBr, nmax, cmꢀ1): 3445 and 3360 (asym.
and sym. str. of eNH2), 2185 (C^N str.), 1660 (C]O str.), 1240 (Ce
AreH). 13C NMR (100 MHz, DMSO-d6)
d: 21.18 (CH2), 21.39 (Are
CH3), 28.12 (CH2), 32.29 (C4), 36.51 (CH2eCO), 59.74 (CeCN),
106.84, 111.18, 119.44, 121.67, 123.65, 125.34, 126.77, 126.89, 127.12,
127.52, 130.44, 131.60, 131.94, 132.4, 134.67, 135.58, 135.96, 137.55,
138.51,143.12,151.62,158.07,158.74,161.07 (24C, AreC),195.39 (C]
O); MS (m/z): 560.0 [M þ 1]þ.
OeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.91 (s, 3H, CH3),
0.94 (s, 3H, CH3), 1.85 (d, 1H, J ¼ 16.0 Hz, CH2), 2.01 (d, 1H,
J ¼ 16.0 Hz, CH2), 2.14e2.27 (m, 2H, CH2eCO), 5.06 (s, 1H, CH), 5.58
(s, 2H, NH2), 7.36e8.25 (m, 11H, AreH). 13C NMR (100 MHz, DMSO-
6.1.2.10. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-
6,70,70-trimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-
30-carbonitrile (8j). IR (KBr, nmax, cmꢀ1): 3465 and 3355 (asym. and
sym. str. of eNH2), 2155 (C^N str.), 1645 (C]O str.), 1220 (CeOeC
d6) d: 27.29 (CH3), 29.25 (CH3), 32.56 (C4), 34.18 (C(CH3)2), 41.43
(CH2), 49.61 (CH2eCO), 59.36 (CeCN), 106.83, 111.07, 118.72, 119.11,
120.82, 121.25, 122.51, 123.24, 126.47, 127.90, 128.46, 129.14, 129.78,
130.51, 132.27, 132.90, 134.81, 137.46, 143.37, 149.59, 151.82, 152.47,

682
H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
158.34, 158.28 (24C, AreC), 195.60 (C]O); MS (m/z): 608.1
[M þ 1]þ, 609.0 [M þ 2].
(s, 3H, AreOCH3), 5.07 (s, 1H, CH), 5.73 (s, 2H, NH2), 7.28e8.21 (m,
11H, AreH). 13C NMR (100 MHz, DMSO-d6)
: 21.28 (CH2), 28.44
d
(CH2), 32.31 (C4), 36.39 (CH2eCO), 55.88 (AreOCH3), 59.67 (CeCN),
106.68, 111.17, 118.77, 119.10, 120.68, 121.36, 122.48, 123.44, 126.51,
127.92, 128.30, 129.09, 129.80, 130.55, 132.44, 132.81, 134.78, 137.41,
143.64, 149.65, 151.84, 152.56, 158.80, 159.94 (24C, AreC), 195.44
(C]O); MS (m/z): 576.2 [M þ 1]þ.
6.1.2.15. 20-Amino-6-chloro-2-(4-cyanophenoxy)-10-(3,4-difluoro-
phenyl)-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-carbon-
itrile (8o). IR (KBr, nmax, cmꢀ1): 3465 and 3330 (asym. and sym. str.
of eNH2), 2195 (C^N str.), 1640 (C]O str.), 1235 (CeOeC ether str.).
1H NMR (400 MHz, DMSO-d6):
d 1.81e1.90 (m, 2H, CH2eCH2), 1.99e
2.08 (m, 2H, CH2eCO), 2.17e2.30 (m, 2H, CH2eCH2eCO), 5.08 (s, 1H,
6.1.2.20. 20-Amino-2-(4-cyanophenoxy)-10-(2,5-difluorophenyl)-6-
methoxy-70,70-dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biqui-
noline-30-carbonitrile (8t). IR (KBr, nmax, cmꢀ1): 3450 and 3345
(asym. and sym. str. of eNH2), 2190 (C^N str.), 1655 (C]O str.),
CH), 5.62 (s, 2H, NH2), 7.23e8.30 (m,11H, AreH). 13C NMR (100 MHz,
DMSO-d6) d: 21.16 (CH2), 28.19 (CH2), 32.42 (C4), 36.52 (CH2eCO),
60.09 (CeCN), 107.64, 111.28, 117.45, 119.34, 120.77, 121.27, 122.35,
123.51, 126.40, 127.88, 128.54, 129.17, 129.92, 130.64, 132.21, 132.77,
134.55, 137.05, 143.46, 149.62, 151.83, 152.54, 157.87, 162.25 (24C,
AreC), 195.41 (C]O); MS (m/z): 579.7 [M þ 1]þ, 580.7 [M þ 2].
1235 (CeOeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.93 (s,
3H, CH3), 0.97 (s, 3H, CH3), 1.86 (d, 1H, J ¼ 16.2 Hz, CH2), 2.00 (d, 1H,
J ¼ 16.2 Hz, CH2), 2.16e2.25 (m, 2H, CH2eCO), 3.81 (s, 3H, Are
OCH3), 5.01 (s, 1H, CH), 5.60 (s, 2H, NH2), 7.20e8.20 (m, 11H, AreH).
6.1.2.16. 20-Amino-6-chloro-2-(4-cyanophenoxy)-10-(3,4-difluoro-
phenyl)-70,70-dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquino-
line-30-carbonitrile (8p). IR (KBr, nmax, cmꢀ1): 3460 and 3335 (asym.
and sym. str. of eNH2), 2200 (C^N str.), 1645 (C]O str.), 1220 (Ce
13C NMR (100 MHz, DMSO-d6)
d: 27.31 (CH3), 29.08 (CH3), 32.85
(C4), 34.17 (C(CH3)2), 41.38 (CH2), 49.76 (CH2eCO), 55.91 (Are
OCH3), 58.92 (CeCN), 107.34, 109.55, 11.48, 119.27, 121.68, 122.45,
122.80, 123.07, 126.40, 127.18, 127.45, 127.88, 128.13, 130.17, 131.62,
134.75, 135.44, 137.35, 137.68, 140.15, 152.11, 157.15, 158.17, 159.28
(24C, AreC), 195.45 (C]O); MS (m/z): 604.0 [M þ 1]þ.
OeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.90 (s, 3H, CH3),
0.93 (s, 3H, CH3), 1.88 (d, 1H, J ¼ 16.2 Hz, CH2), 2.03 (d,1H, J ¼ 16.0 Hz,
CH2), 2.18e2.26 (m, 2H, CH2eCO), 5.06 (s, 1H, CH), 5.69 (s, 2H, NH2),
7.22e8.26 (m, 11H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 27.23
6.1.2.21. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-6-
methoxy-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-car-
bonitrile (8u). IR (KBr, nmax, cmꢀ1): 3465 and 3360 (asym. and sym.
str. of eNH2), 2165 (C^N str.), 1675 (C]O str.), 1225 (CeOeC ether
(CH3), 29.27 (CH3), 32.53 (C4), 32.72 (C(CH3)2), 41.48 (CH2), 49.77
(CH2eCO), 58.59 (CeCN),108.05,110.31,118.96,119.10,120.81,121.81,
122.09, 123.47, 126.89, 127.77, 128.25, 129.15, 129.96, 130.43, 132.38,
132.88, 134.76, 137.82, 143.07, 149.73, 151.89, 152.20, 157.97, 159.56
(24C, AreC),195.44 (C]O); MS (m/z): 608.2 [M þ 1]þ, 609.2 [M þ 2].,
str.). 1H NMR (400 MHz, DMSO-d6):
d 1.77e1.86 (m, 2H, CH2eCH2),
1.96e2.07 (m, 2H, CH2eCO), 2.13e2.29 (m, 2H, CH2eCH2eCO), 3.79
(s, 3H, AreOCH3), 5.09 (s, 1H, CH), 5.68 (s, 2H, NH2), 7.25e8.27 (m,
6.1.2.17. . 20-Amino-6-chloro-2-(4-cyanophenoxy)-50-oxo-10-[3-(tri-
fluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-
carbonitrile (8q). IR (KBr, nmax, cmꢀ1): 3445 and 3340 (asym. and
sym. str. of eNH2), 2190 (C^N str.), 1680 (C]O str.), 1245 (CeOeC
11H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 21.16 (CH2), 28.51
(CH2), 32.45 (C4), 36.52 (CH2eCO), 55.82 (AreOCH3), 59.12 (CeCN),
108.01, 110.12, 111.49, 119.14, 121.85, 122.49, 122.91, 123.28, 126.56,
127.47, 127.49, 127.72, 128.30, 130.28, 131.75, 134.82, 135.47, 137.48,
137.92, 140.17, 152.24, 157.43, 158.09, 161.26 (24C, AreC), 195.56
(C]O); MS (m/z): 576.2 [M þ 1]þ.
ether str.). 1H NMR (400 MHz, DMSO-d6):
d
1.73e1.85 (m, 2H, CH2e
CH2), 1.95e2.07 (m, 2H, CH2eCO), 2.12e2.29 (m, 2H, CH2eCH2e
CO), 5.02 (s,1H, CH), 5.59 (s, 2H, NH2), 7.19e8.25 (m,11H, AreH). 13
NMR (100 MHz, DMSO-d6) : 21.34 (CH2), 28.37 (CH2), 32.81 (C4),
C
d
6.1.2.22. 20-Amino-2-(4-cyanophenoxy)-10-(3,4-difluorophenyl)-6-
methoxy-70,70-dimethyl-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biqui-
noline-30-carbonitrile (8v). IR (KBr, nmax, cmꢀ1): 3455 and 3355
(asym. and sym. str. of eNH2), 2160 (C^N str.), 1650 (C]O str.),
36.43 (CH2eCO), 59.43 (CeCN),107.25,110.98,116.28,118.45,119.15,
120.77, 121.80, 122.70, 123.48, 126.90, 127.81, 128.54, 129.35, 129.71,
130.49, 132.82, 133.04, 134.68, 137.88, 143.15, 149.79, 151.60, 152.27,
158.12,160.75 (25C, AreC), 195.48 (C]O); MS (m/z): 611.9 [M þ 1]þ,
612.9 [M þ 2].
1220 (CeOeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.90 (s,
3H, CH3), 0.94 (s, 3H, CH3), 1.74 (d, 1H, J ¼ 16.0 Hz, CH2), 1.99 (d, 1H,
J ¼ 16.0 Hz, CH2), 2.14e2.24 (m, 2H, CH2eCO), 3.83 (s, 3H, Are
OCH3), 5.05 (s, 1H, CH), 5.64 (s, 2H, NH2), 7.34e8.28 (m, 11H, AreH).
6.1.2.18. 20-Amino-6-chloro-2-(4-cyanophenoxy)-70,70-dimethyl-50-
oxo-10-[3-(trifluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-
biquinoline-30-carbonitrile (8r). IR (KBr, nmax, cmꢀ1): 3440 and 3370
(asym. and sym. str. of eNH2), 2175 (C^N str.), 1675 (C]O str.),
13C NMR (100 MHz, DMSO-d6)
d: 27.24 (CH3), 29.14 (CH3), 32.46
(C4), 33.88 (C(CH3)2), 41.68 (CH2), 49.69 (CH2eCO), 55.97 (Are
OCH3), 59.75 (CeCN), 107.41, 111.13, 119.98, 119.24, 121.79, 122.35,
122.80, 123.40, 126.55, 127.56, 127.52, 127.80, 128.37, 130.42, 131.88,
135.04, 135.58, 137.63, 137.89, 140.35, 152.34, 157.56, 158.12, 162.10
(24C, AreC), 195.62 (C]O); MS (m/z): 604.5 [M þ 1]þ.
1230 (CeOeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.88 (s,
3H, CH3), 0.92 (s, 3H, CH3), 1.80 (d, 1H, J ¼ 16.0 Hz, CH2), 1.98 (d, 1H,
J ¼ 16.0 Hz, CH2), 2.15e2.23 (m, 2H, CH2eCO), 5.03 (s, 1H, CH), 5.64
(s, 2H, NH2), 7.30e8.23 (m, 11H, AreH). 13C NMR (100 MHz, DMSO-
d6)
d: 27.20 (CH3), 29.15 (CH3), 32.70 (C4), 33.95 (C(CH3)2), 41.53
6.1.2.23. 20-Amino-2-(4-cyanophenoxy)-6-methoxy-50-oxo-10-[3-
(trifluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-biquinoline-
30-carbonitrile (8w). IR (KBr, nmax, cmꢀ1): 3450 and 3325 (asym.
and sym. str. of eNH2), 2175 (C^N str.), 1640 (C]O str.), 1235 (Ce
(CH2), 49.75 (CH2eCO), 59.71 (CeCN), 108.01, 111.22, 116.37, 118.40,
119.09, 120.45, 121.91, 122.77, 123.53, 126.91, 127.75, 128.67, 129.31,
129.65, 130.54, 132.88, 133.45, 134.71, 137.93, 143.27, 149.56, 151.82,
152.34, 158.28, 160.18 (25C, AreC), 195.52 (C]O); MS (m/z): 640.1
[Mþ1]þ, 641.1 [Mþ2].
OeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 1.79e1.88 (m, 2H,
CH2eCH2), 1.98e2.08 (m, 2H, CH2eCO), 2.15e2.31 (m, 2H, CH2e
CH2eCO), 3.89 (s, 3H, AreOCH3), 5.02 (s, 1H, CH), 5.58 (s, 2H, NH2),
6.1.2.19. 20-Amino-2-(4-cyanophenoxy)-10-(2,5-difluorophenyl)-6-
methoxy-50-oxo-10,40,50,60,70,80-hexahydro-3,40-biquinoline-30-car-
bonitrile (8s). IR (KBr, nmax, cmꢀ1): 3435 and 3355 (asym. and sym.
str. of eNH2), 2180 (C^N str.), 1660 (C]O str.), 1240 (CeOeC ether
7.26e8.20 (m, 11H, AreH). 13C NMR (100 MHz, DMSO-d6)
d: 21.17
(CH2), 28.56 (CH2), 33.16 (C4), 36.40 (CH2eCO), 55.96 (AreOCH3),
59.16 (CeCN), 106.78, 107.49, 111.14, 119.21, 121.93, 122.67, 122.88,
123.15, 126.53, 127.05, 127.56, 127.85, 128.40, 130.54, 131.60, 134.71,
135.13, 137.27, 137.51, 140.10, 152.05, 153.85, 157.04, 157.94, 158.57
(25C, AreC), 195.61 (C]O); MS (m/z): 608.2 [M þ 1]þ.
str.). 1H NMR (400 MHz, DMSO-d6):
d
1.76e1.87 (m, 2H, CH2eCH2),
1.97e2.09 (m, 2H, CH2eCO), 2.17e2.35 (m, 2H, CH2eCH2eCO), 3.86

H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
683
6.1.2.24. 20-Amino-2-(4-cyanophenoxy)-6-methoxy-70,70-dimethyl-
50-oxo-10-[3-(trifluoromethyl)phenyl]-10,40,50,60,70,80-hexahydro-3,40-
biquinoline-30-carbonitrile (8x). IR (KBr, nmax, cmꢀ1): 3460 and 3330
(asym. and sym. str. of eNH2), 2180 (C^N str.), 1655 (C]O str.),
taken as MIC concentration of test compound. The screening results
are summarized as % inhibition (Table 2) relative to standard drugs
isoniazid and rifampicin. MIC values of five compounds with
highest % inhibition i.e. 8h, 8l, 8o, 8q and 8r are shown in Table 3.
The standard strain M. tuberculosis H37Rv was tested with standard
drugs isoniazid and rifampicin for comparison purpose.
1230 (CeOeC ether str.). 1H NMR (400 MHz, DMSO-d6):
d 0.93 (s,
3H, CH3), 0.97 (s, 3H, CH3), 1.81 (d, 1H, J ¼ 16.0 Hz, CH2), 2.02 (d, 1H,
J ¼ 16.0 Hz, CH2), 2.15e2.26 (m, 2H, CH2eCO), 3.85 (s, 3H, Are
OCH3), 5.04 (s, 1H, CH), 5.74 (s, 2H, NH2), 7.24e8.19 (m, 11H, AreH).
6.2.3. Cytotoxicity e brine shrimp lethality bioassay
13C NMR (100 MHz, DMSO-d6)
d: 27.30 (CH3), 29.17 (CH3), 32.94
Brine shrimp lethality bioassay technique was applied for the
determination of general toxic property of compounds. The in vitro
lethality test has been carried out using brine shrimp eggs i.e.
Artemia cysts. Brine shrimp eggs were hatched in a shallow rect-
angular plastic dish (22 ꢃ 32 cm), filled with artificial seawater,
which was prepared with commercial salt mixture and double
distilled water. An unequal partition was made in the plastic dish
with the help of a perforated device. Approximately 50 mg of eggs
were sprinkled into the large compartment, which was darkened
while the minor compartment was opened to ordinary light. After
two days nauplii were collected by a pipette from the lighter side.
A stock solution of the test complex was prepared in DMSO. From
this stock solution, solutions were transferred to the vials to make
(C4), 33.98 (C(CH3)2), 41.52 (CH2), 49.79 (CH2eCO), 55.83 (Are
OCH3), 59.40 (CeCN), 107.52, 107.62, 111.17, 119.43, 121.94, 122.61,
123.10, 123.62, 126.64, 127.24, 127.71, 127.91, 128.47, 130.62, 131.77,
132.18, 134.88, 135.25, 137.81, 140.26, 152.27, 153.91, 157.45, 157.81,
159.88 (25C, AreC), 195.44 (C]O); MS (m/z): 636.4 [M þ 1]þ.
6.2. Biological assay
6.2.1. In vitro evaluation of antimicrobial activity
The MICs of synthesized compounds were carried out by broth
microdilution method. DMSO was used as diluents to get desired
concentration of compounds to test upon standard bacterial strains.
Serial dilutions were prepared in primary and secondary screening.
The control tube containing no antibiotic was immediately sub-
cultured (before inoculation) by spreading a loopful evenly over a
quarter of plate of medium suitable for the growth of the test or-
ganism and put for incubation at 37 ꢂC overnight. The tubes were
then incubated overnight. The MIC of the control organism was
read to check the accuracy of the compound concentrations. The
MIC was defined as the lowest concentration of the antibiotic or
test sample allowing no visible growth. All the tubes not showing
visible growth (in the same manner as control tube described
above) was subcultured and incubated overnight at 37 ꢂC. The
amount of growth from the control tube before incubation (which
represents the original inoculum) was compared. Subcultures
might show: similar number of colonies indicating bacteriostatic; a
reduced number of colonies indicating a partial or slow bactericidal
activity and no growth if the whole inoculum has been killed. The
test must include a second set of the same dilutions inoculated with
an organism of known sensitivity. Each synthesized compound was
final concentration 5, 10, 20, 30, 40, 50 mg/mL (dilutions were used
in triplicate for each test sample and LC50 is the mean of three
values) and three vial was kept as control having of DMSO only.
After two days, when the of nauplii were ready, 1 mL of seawater
and 10 of nauplii were added to each vial and the volume was
adjusted with seawater to 2.5 mL per vial [31]. After 24 h each vial
was observed using a magnifying glass and the number of survi-
vors in each vial was counted and noted. Data were analysed by
simple logit method to determine the LC50 values, in which log of
dose concentration of samples were plotted against percent of
mortality of nauplii [34].
6.2.4. In vitro evaluation of antioxidant activity
FRAP assay was employed to measure total antioxidant capacity
of the compounds. It measure a reduction power of the compounds,
converting ferric tripyridyl triazine (Fe (III)-TPTZ) complex into a
blue colour ferrous tripyridyl triazine (Fe (II)-TPTZ) complex at low
pH, measurable at 593 nm [32].
diluted obtaining 2000
primary screening 500, 250 and 200
m
g/mL concentration as a stock solution. In
g/mL concentrations of the
Reagents:
m
Buffer solution: 0.187 gm sodium acetate and 1.6 mL acetic acid
dissolved in double distilled water to make 100 mL.
TPTZ: 0.155 gm TPTZ was dissolved in 100 mL 40 mM HCl.
FeCl3 solution: 0.324 gm FeCl3 was dissolved in 100 mL distilled
water.
synthesized compounds were taken. The active synthesized com-
pounds found in this primary screening were further tested in a
second set of dilution against all microorganisms. The compounds
found active in primary screening were similarly diluted to obtain
100, 62.5, 50, 25 and 12.5
tion showing at least 99% inhibition is taken as MIC.
m
g/mL concentrations. The highest dilu-
Standard Ascorbic acid: 0.176 gm of standard ascorbic acid was
dissolved in 100 mL distilled water.
Fe(II)-TPTZ(2,4,6-tripyridyl-s-triazine) reagent was prepared by
mixing a 10.0 mL TPTZ solution, 10 mL FeCl36H2O solution and
6.2.2. In vitro evaluation of antituberculosis activity
Drug susceptibility and determination of antituberculosis ac-
tivity of the test compounds against M. tuberculosis H37Rv were
performed by LowensteineJensen method [30] with slight modi-
100 mL acetate buffer at pH 3.6. A mixture of 200.0 mL sample so-
lution and 3 mL of Fe(II)TPTZ reagent was incubated at 37 ꢂC for
15 min. The absorbance of colour complex Fe(II)TPTZ was measured
at 593 nm using ascorbic acid as standard. The results were
expressed as ascorbic equivalent (mmol/100 gm compound).
Ascorbic acid taken ¼ 1.99 ꢃ 10ꢀ4 mm.
fication where 250
mg/mL dilution of each test compound were
added liquid Lowenstein-Jensen Medium and then media were
sterilized by inspissation method. A culture of M. tuberculosis
H37Rv growing on Lowenstein-Jensen medium was harvested in
0.85% saline in bijou bottles. All test compound make solution of
Sample taken ¼ 0.04 mg.
The Ferric Reducing Antioxidant Power (FRAP) can be calculated
using the following equation:
250 mg/mL concentration of compounds was prepared in DMSO.
These tubes were then incubated at 37 ꢂC for 24 h followed by
streaking of M. tuberculosis H37Rv (5 ꢃ 104 bacilli per tube). These
tubes were then incubated at 37 ꢂC. Growth of bacilli was seen after
12 days, 22 days and finally 28 days of incubation. Tubes having the
compounds were compared with control tubes where medium
alone was incubated with M. tuberculosis H37Rv. The concentration
at which no development of colonies occurred or < 20 colonies was
D
OD593nm of test sample
OD593nm of standard
FRAPvalue
¼
D
ðmmA:A:=100gm sampleÞ
standard ðmmÞ
sample ðmgÞ
ꢃ
ꢃ 105

684
H.G. Kathrotiya, M.P. Patel / European Journal of Medicinal Chemistry 63 (2013) 675e684
[12] K.D. Thomas, A.V. Adhikari, S. Telkar, I.H. Chowdhury, R. Mahmoode, N.K. Pal,
Acknowledgements
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The authors are thankful to Head, Department of Chemistry,
Sardar Patel University for providing 1H NMR and 13C NMR spec-
troscopy and research facilities. We are also thankful to Oxygen Bio
Research Pvt. Ltd., Ahmedabad, for providing mass spectroscopy
facilities, Vaibhav Laboratories, Ahmedabad, Gujarat, India for the
FT-IR, SICART, Vallabh Vidyanagar, for elemental analysis and
Dhanji P. Rajani, Microcare Laboratory, Surat, Gujarat, India for
antimicrobial and antimycobacterial screening of the compounds
reported herein. One of the authors is grateful to UGC, New Delhi
for Research Fellowship in Sciences for Meritorious Students.
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Appendix A. Supplementary data
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