Ultrasound mediated catalyst free synthesis of 6H-1-benzopyrano[4,3-b] quinolin-6-ones leading to novel quinoline derivatives: Their evaluation as potential anti-cancer agents

Article abstract of DOI:10.1016/j.bmc.2011.12.001

A facile and catalyst free synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6- ones has been accomplished via the reaction of 4-chloro-2-oxo-2H-chromene-3- carbaldehyde with various aromatic amines in the presence of ultrasound. Some of these compounds were c

Full text of DOI:10.1016/j.bmc.2011.12.001

Bioorganic & Medicinal Chemistry 20 (2012) 759–768
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Ultrasound mediated catalyst free synthesis of 6H-1-benzopyrano
[4,3-b]quinolin-6-ones leading to novel quinoline
derivatives: Their evaluation as potential anti-cancer agents
Naveen Mulakayala a, D. Rambabu a,b, Mohan Rao Raja a, Chaitanya M. c, Chitta Suresh Kumar c,
Arunasree M. Kalle a, G. Rama Krishna d, C. Malla Reddy d, M.V. Basaveswara Rao e, Manojit Pal a,
⇑
a Institute of Life Sciences, University of Hyderabad Campus, Hyderabad 500 046, India
b Department of Chemistry, K L University, Vaddeswaram, Guntur 522502, AP, India
c Department of Biochemistry, Sri Krishnadevaraya University, Anantapur 515003, India
d Department of Chemical Sciences, Indian Institute of Science Education and Research, Kolkata, West Bengal 741252, India
e Department of chemistry, Krishna University, Machilipatnam 521001, AP, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A facile and catalyst free synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-ones has been accomplished
via the reaction of 4-chloro-2-oxo-2H-chromene-3-carbaldehyde with various aromatic amines in the
presence of ultrasound. Some of these compounds were converted to the corresponding 2-(3-(hydroxy-
methyl)quinolin-2-yl)phenols and further structure elaboration of a representative quinoline derivative
is presented. Molecular structure of two representative compounds was confirmed by single crystal
X-ray diffraction study. Many of these compounds were evaluated for their anti-proliferative properties
in vitro against four cancer cell lines and several compounds were found to be active. Further in vitro
studies indicated that inhibition of sirtuins could be the possible mechanism of action of these molecules.
Ó 2011 Elsevier Ltd. All rights reserved.
Received 10 November 2011
Revised 30 November 2011
Accepted 1 December 2011
Available online 8 December 2011
Keywords:
Quinoline
Amine
Ultrasound
Cancer
1. Introduction
transformation of a number of 6H-1-benzopyrano[4,3-b]quinolin-6-
one derivatives along with the in vitro anticancer properties of some
Coumarins are well known aromatic lactones isolated from a
variety of plant sources1 and possess diverse pharmacological activ-
ities. For example, the coumarin framework is present in a number of
promising drug candidates such as nonpeptidic HIV protease inhib-
itors,2 topoisomerase II3 and tyrosine kinase inhibitors.4 Quinoline
derivatives on the other hand are known to have wide applications
as drugs and pharmaceuticals.5 A combination of chromen or benz-
opyran with a quinoline moiety in a single molecule, for example,
6H-chromeno[4,3-b]quinoline6 or 1-benzopyrano[3,4-f]quinoline7
have also been explored for the identification of promising bioactive
molecules. While combination of coumarin and quinoline in a single
molecule, for example, 6H-1-benzopyrano[4,3-b]quinolin-6-one8 is
known as a separate class of heterocycle its use as a template for the
identification of bioactive molecules is not common. In view of
known cytotoxicities of coumarins and chromeno[4,3-b]quinoline
derivatives9 we hypothesized that design of small molecules based
on 6H-1-benzopyrano[4,3-b]quinolin-6-one (A, Fig. 1) or converting
them into the corresponding quinoline derivatives (B, Fig. 1) might
show anticancer properties. Herein we report synthesis and further
of the compounds synthesized.
2. Results and discussion
2.1. Chemistry
The 6H-1-benzopyrano[4,3-b]quinolin-6-ones were synthe-
sized previously from 4-chloro-2-oxo-2H-chromene-3-carbalde-
hyde using AlCl3 as
a
catalyst10 or via other methods.8
X
O
N
O
Ar
O
O
N
N
A
B
Coumarin
Quinoline
⇑
Corresponding author. Tel.: +91 40 6657 1500; fax: +91 40 6657 1581.
E-mail addresses: manojitpal@rediffmail.com, palmanojit@yahoo.co.uk (M. Pal).
Figure 1. Design of benzopyranoquinolinone (A) and quinoline derivatives (B) as
potential anticancer agents.
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.12.001

760
N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
(NMR, MS and IR) data. Additionally, the molecular structure of a
representative compound 3a was established unambiguously by
single crystal X-ray diffraction (Fig. 2).13
O
N
O
O
O
NH2
Sonication
rt, MeOH
We have developed a mild and catalyst free direct synthesis of
6H-1-benzopyrano[4,3-b]quinolin-6-ones in good yields. To dem-
onstrate the potential of this methodology a number of compounds
prepared were converted to quinoline derivatives (4, Table 3). Thus
treatment of compound 3 with NaBH4 in THF at room temperature
gave 2-(3-(hydroxymethyl)quinolin-2-yl)phenols in good yields.
All these compounds (4a–d) were characterized by spectral
(NMR, MS and IR) data and the molecular structure of a represen-
tative compound 4d was confirmed by X-ray analysis (Fig. 3).14
Further structure elaboration of a quinoline derivative 4a was car-
ried out by converting it into an azide derivative 5a (Scheme 2).
Treating the azide 5a with a terminal alkyne in the presence of
CuI provided the corresponding triazole derivative 5b (Scheme
2). Further, on reduction the azide 5a provided the primary amine
5c which on reaction with 4-chloro-3-nitro-5-sulfamoylbenzoic
acid afforded a novel sulfonamide based compound 5d (Scheme 2).
+
CHO
R
R
Cl
1
2
3
Scheme 1. Synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-ones from 4-chloro-2-
oxo-2H-chromene-3-carbaldehyde.
However, we were particularly interested in developing a catalyst
free method for the synthesis of this class of compounds. This
prompted us to develop an ultrasound-promoted catalyst free
synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-ones (3) from
4-chloro-2-oxo-2H-chromene-3-carbaldehyde (1) and anilines
(2) under ambient conditions. In general the ultrasound-pro-
moted chemical reactions offer several advantages over conven-
tional process especially with respect to the reaction time and
product yields.11 The required starting material 1 was prepared
from 4-hydroxy coumarin under Vilsmeier–Haack reaction condi-
tions12 and then reacted with aromatic amines in methanol under
ultrasonication at room temperature to give 3 in good yields
(Scheme 1). Initially the reaction of 1 with aniline (2a) was exam-
ined in a range of solvents to identify the best solvent for this
process (Table 1). The use of MeOH was found to be effective as
the reaction was completed within 5 min providing the product
3a in 94% yield (entry 1, Table 1). The use of water increased
the reaction time with significant decrease in product yield (entry
2, Table 1). The use of dichloromethane and toluene was found to
be ineffective (entries 3 and 4, Table 1) and the reaction did not
proceed in DMF (entry 5, Table 1). Thus MeOH was identified as
the solvent for the present condensation reaction.
Having the optimized reaction condition for the preparation of
6H-1-benzopyrano[4,3-b]quinolin-6-ones in hand we then exam-
ined the generality and scope of this methodology. Thus a variety
of aromatic amines (2) were reacted with chloro aldehyde (1) in
MeOH at room temperature under ultrasonication and results are
presented in Table 2. The reaction proceeded well in all these cases
and the substituents like F, Cl, Br, OMe and Me present in the aro-
matic amines (2) were well tolerated. The reaction appeared to be
clean as no formation of side product was observed and the desired
product 3 was isolated in good to excellent yield in each case. All
the compounds synthesized were well characterized by spectral
2.2. Pharmacology
Many of the compounds synthesized, for example, 3, 4 and 5
were tested for their anticancer properties in vitro. Cancer is the
second leading cause of death15 worldwide after cardiovascular
diseases, according to WHO. We evaluated our compounds for
their anti-proliferative properties in vitro against a number of can-
cer cell lines, for example, human chronic myeloid leukemia cells
(K562), human colon carcinoma cells (Colo-205), breast cancer
cells (MDA-MB 231), and human neuroblastoma cells (IMR32).
The test compounds were examined at various concentrations in
a MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-
mide) assay and the IC50 values obtained for each compounds are
summarized in Table 4. Harmine, a member of beta-carboline fam-
ily of compounds showed cytotoxicity against HL60 and K562 cell
lines16 was used as a reference compound. While most of the com-
pounds showed inhibition of leukemia cell growth as reflected by
their IC50 values the best results however were obtained using
compounds 4b, 4c, 4d, 5a, 5b and 5d (IC50 ꢀ10–13
lM, Table 4).
All these compounds are derivatives of 2-aryl substituted quinoline
indicating that the 2-arylquinoline framework played a key role in
the inhibition of leukemia cell growth. Compounds 3a, 3c, 3g, 3j,
5a, 5b and 5d (IC50 ꢀ11–18
against colon carcinoma cells whereas 3a, 3b, 3c, 3g, 3h, 3i, 3j,
M, Table 4) showed promising activi-
lM, Table 4) were found to be active
5a, 5b and 5d (IC50 ꢀ8–16
l
ties against breast cancer cells. While many of these compounds
were found to be active against human neuroblastoma cells none
Table 1
of them however showed IC50 <20
lM except compound 5d (IC50
Effect of solvents on the reaction of chloroaldehyde (1) and aniline (2a) under
ꢀ18 M, Table 4). In order to understand the mechanism of action
l
ultrasonicationa
some of the compounds were tested for their inhibitory potential
against sirtuins. Being considered as important targets for cancer
therapeutics sirtuins (class III NAD-dependent deacetylases) are
shown to up-regulated in various types of cancer.17 Inhibition of
sirtuins allows re-expression of silenced tumor suppressor genes,
leading to reduced growth of cancer cells. The activity of test com-
pounds was determined using Sirt1 fluorescence activity assay18
using suramin, a known inhibitor of Sirt1 as a reference compound.
O
O
Sonication
N
PhNH2
1
+
rt, MeOH
2a
3a
At the concentration of 10 lM compounds 3d, 4c, 5a, 5b, 5c and 5d
Entry
Solvent
Time (min)
Yieldb (%)
showed 48%, 48%, 53%, 62%, 59% and 61% inhibition, respectively, in
compared to suramin’s 79% inhibition indicating that the antican-
cer properties of these molecules are possibly due to their sirtuin
inhibiting properties. To understand the nature of interactions
between these compounds and the Sir1 protein a molecular dock-
ing simulation study was carried out using a representative com-
pound 5b (Fig. 4). The three dimensional model of hSirt1 (NCBI
gi no: 7555471, 200–500 amino acid residues) was developed by
1
2
3
4
5
MeOH
H2O
DCM
Toluene
DMF
5
94
65
46
35
15
20
30
45
No reaction
a
All the reactions were carried out using 1 (1.0 mmol) and 2a (1.5 mmol) at room
temperature.
b
Isolated yield.

N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
761
Table 2
Catalyst free synthesis of 6H-1-benzopyrano[4,3-b]quinolin-6-ones (3) from 1 (Scheme 1)a
Entry
2; R=
Product (3)
Time (min)
Yieldb (%)
O
O
958
3a
1
2a; H
5
N
O
O
3b
2
3
2b; 2-OCH3
7
8
928
N
H3CO
O
O
3c
2c; 3,4-F
N
O
90
92
89
94
F
F
O
3d
4
5
6
2d; 2,3-F
2e; 3-Br
2f; 3-F
N
F
9
7
8
F
O
O
3e
N
Br
O
O
N
3f
F
O
O
3g
7
8
9
2g; 4-F
6
7
5
93
N
O
F
O
O
2h; 4-OCH3
918
3h
N
OCH3
O
N
2i; 4-CH3
898
3i
CH3
(continued on next page)

762
N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
Table 2 (continued)
Entry
2; R=
Product (3)
Time (min)
Yieldb (%)
O
O
938
3j
10
2j; 4-Cl
6
N
Cl
a
All the reactions were carried out using 1 (1.0 mmol) and 2 (1.5 mmol) under ultrasonication at room temperature.
Isolated yield.
b
Figure 2. X-ray crystal structure of 3a (ORTEP diagram). Displacement ellipsoids are drawn at 50% probability level for non-hydrogen atoms.
homology modeling using the templates PDB: 2HJH and PDB: 1J8F
in the Modeller9v6. Four amino acid residues, for example, Arg274,
Asn348, Cys362 and Pro293 were found to play key roles in this
interaction with the overall binding energy of À10.6 Kcal/mol indi-
cating that molecule 5b interacts well with this protein.
medicinal importance and the basic framework of both these clas-
ses of heterocycles could be an attractive template for the identifi-
cation of novel and potential anticancer agents.
4. Experimental section
3. Conclusions
4.1. Chemistry—general methods
In conclusion, 6H-1-benzopyrano[4,3-b]quinolin-6-ones and
various functionalized quinoline derivatives have been explored
as new and potential anti cancer agents. Synthesis of 6H-1-benzo-
pyrano[4,3-b]quinolin-6-ones were carried out by reacting
4-chloro-2-oxo-2H-chromene-3-carbaldehyde with various aro-
matic amines via a catalyst free method in the presence of ultra-
sound. To the best of our knowledge ultrasound mediated
synthesis of this class of compounds was not known in the litera-
ture. Some of these compounds were converted to the correspond-
ing 2-(3-(hydroxymethyl)quinolin-2-yl)phenols and further
structure elaboration of a representative quinoline derivative was
carried out. The single crystal X-ray diffraction study was used to
confirm the molecular structure of two representative compounds
unambiguously. Many of these compounds were evaluated for
their anti-proliferative properties in vitro against four cancer cell
lines, for example, human chronic myeloid leukemia cells (K562),
human colon carcinoma cells (Colo-205), breast cancer cells
Unless stated otherwise, reactions were monitored by thin layer
chromatography (TLC) on silica gel plates (60 F254), visualizing
with ultraviolet light or iodine spray. Column chromatography
was performed on silica gel (60–120 mesh) using distilled petro-
leum ether and ethyl acetate. 1H and 13C NMR spectra were deter-
mined in CDCl3 and DMSO solutions using 400 and 100 MHz
spectrometers, respectively. Proton chemical shifts (d) are relative
to tetramethylsilane (TMS, d = 0.0) as internal standard and
expressed in parts per million. Spin multiplicities are given as s
(singlet), d (doublet), t (triplet), and m (multiplet) as well as b
(broad). Coupling constants (J) are given in hertz. Infrared spectra
were recorded on a FTIR spectrometer. Melting points were deter-
mined by using a Buchi melting point B-540 apparatus. MS spectra
were obtained on a mass spectrometer. HRMS was determined
using waters LCT premier XETOF ARE-047 apparatus.
4.2. General procedure for the preparation of 6H-chromeno[4,3-
b]quinolin-6-one (3a–j)
(MDA-MB 231), and human neuroblastoma cells (IMR32).
A
number of compounds showed promising anticancer properties.
Further in vitro studies indicated that inhibition of sirtuins could
be the possible mechanism of action of these molecules and was
supported by a docking study. Overall, our study suggests that both
benzopyranoquinolinones and quinolines presented here have
A
solution of 4-chloro-2-oxo-2H-chromene-3-carbaldehyde
(1 mmol) and aromatic amine (1.5 mmol) in methanol (5 ml) was
taken in a 10 mL reaction vial. After being capped, the vial containing
the reaction mixture was kept under continuous ultrasound

N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
763
Table 3
purified by flash chromatography (n-hexane/ethylacetate 3:1) to af-
ford the desired product.
Synthesis of 2-(3-(hydroxymethyl)quinolin-2-yl)phenols (4)a
O
O
OH OH
NaBH4,THF
Room temp
4.3. Spectral data of 6H-chromeno[4,3-b]quinolin-6-one (3a–j)
4.3.1. 6H-Chromeno[4,3-b]quinolin-6-one (3a)
N
N
R
R
3
4
O
O
Entry
1
3; R =
Product (4)
OH OH
Time (h)
Yieldb (%)
N
4a
white solid; yield (95%); Rf = 0.60 (30% EtOAc-n-Hexane); mp 226–
228 °C; 1H NMR (400 MHz, DMSO-d6): d 9.36 (s, 1H), 8.67 (dd, J = 7.6
and 1.6 Hz, 1H), 8.31 (d, J = 8.0 Hz, 1H), 8.20 (d, J = 8.8 Hz, 1H), 8.02–
8.03 (m, 1H), 7.66–7.76 (m, 2H), 7.46–7.50 (m, 2H); 13C NMR
(100 MHz, DMSO-d6): d 160.9, 152.9, 150.5, 149.6, 141.4, 134.2,
133.1, 130.4, 129.2, 128.0, 127.6, 125.4, 125.1, 119.8, 117.7, 116.5;
IR (cmÀ1): 3070, 2928, 1740, 1599, 1465, 1190; MS (ES mass): m/
z 248 (M+1, 100%); HRMS: calcd for C16H10NO2: 248.0712, found
248.0710.
H
2
85
N
OH
N
OH
4b
2
3
4-CH3
2.5
1.5
88
82
OH OH
4.3.2. 11-Methoxy-6H-chromeno[4,3-b]quinolin-6-one (3b)
4c
3,4-F
N
O
O
F
F
N
OH OH
H3CO
4
4-Cl
4d
2
84
N
white solid; yield (95%); Rf = 0.35 (10% EtOAc-n-Hexane); mp
236–238 °C; 1H NMR (400 MHz, DMSO-d6): d 9.2 (s, 1H), 8.86
(dd, J = 7.6 and 1.2 Hz, 1H), 7.57–7.63 (m, 3H), 7.39–7.46 (m,
2H), 7.26 (d, J = 5.6 Hz, 1H), 4.17 (s, 3H); 13C NMR (100 MHz,
CDCl3): d 160.7, 153.3, 152.4, 150.1, 136.0, 129.8, 129.7, 127.8,
127.0, 126.6, 125.9, 124.4, 122.1, 110.0, 114.9, 112.3, 56.2; IR
(cmÀ1): 3063, 2836, 1735, 1604, 1380, 1177, 751; MS (ES mass):
m/z 278 (M+1, 10%); HRMS: calcd for C17H12NO3: 278.0817, found
278.0819.
Cl
a
All the reactions were carried out using compound 3 (4.05 mmol) and NaBH4
(8.1 mmol) in dry THF (5 mL).
b
Isolated yield.
irradiation. After completion of the reaction (confirmed by TLC), the
reaction mixture was concentrated under vacuum. The residue was
Figure 3. X-ray crystal structure of 4d (ORTEP diagram). Displacement ellipsoids are drawn at 50% probability level for non-hydrogen atoms.

764
N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
Ms
I
OH
N
OH N3
Ms
N
I
N
N
N
NaN3,PPh3,
N
4a
N
CCl4-DMF, 90 oC
CuI, DIPEA, DMF
5b
5a
HOOC
SO2NH2
NH
10% Pd/C, EtOAc
COOH
O2N
NH2
N
OH
O2N
SO2NH2
OH
Cl
N
n-BuOH, 90 °C
5d
5c
Scheme 2. Structure elaboration of quinoline derivative 4a.
Table 4
Cytotoxic properties of compounds 3, 4 and 5 against various cancer cell lines
Compound
IC50
(l
M)a,b
K562
Colo-205
MDA-MB 231
IMR32
3a
3b
3c
3d
3e
3f
3g
3h
3i
3j
4a
4b
4c
4d
5a
5b
5c
5d
Harmine
43
35
41
28
23
35
37
39
23
32
74
12
12
11
11
13
23
10
45
18
24
11
29
38
21
17
41
29
16
40
>100
>100
24
19
12
32
19
12
16
08
23
19
29
11
09
12
28
46
25
36
23
41
43
36
33
14
46
>100
>100
>100
>100
12
08
19
08
54
>100
>100
63
65
24
38
46
18
68
46
a
IC50 represent the concentration of compound that causes a 50% growth inhi-
bition to untreated cells using the MTT assay.
b
Data represent the mean values of three independent determinations.
4.3.3. 9,10-Difluoro-6H-chromeno[4,3-b]quinolin-6-one (3c)
O
O
N
H3CO
Figure 4. Docking of compound 5b into the active site of hSirt1.
white solid; yield (90%); Rf = 0.4 (10% EtOAc-n-Hexane); mp 225–
254 °C; 1H NMR (400 MHz, CDCl3): d 9.24 (s, 1H), 8.85 (dd, J = 8.0
and 1.6 Hz, 1H), 7.82–7.86 (m, 1H), 7.62–7.66 (m, 1H), 7.40–7.57
(m, 3H); 13C NMR (100 MHz, CDCl3): d 160.8, 152.7, 150.0, 140.1,
140.0, 132.7, 125.2, 125.1, 119.1, 117.4, 115.8, 115.6, 114.4, 114.3,
114.2, 114.1; IR (cmÀ1): 3070, 2930, 1740, 1610, 1470, 1185; MS
(ES mass): m/z 284 (M+1, 100%). HRMS: calcd for C16H8F2NO2:
284.0467, found: 284.0465.

N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
765
white solid; yield (93%); Rf = 0.55 (20% EtOAc-n-Hexane); mp
225–227 °C; 1H NMR (400 MHz, CDCl3): d 9.19 (s, 1H), 8.77 (dd,
J = 8.0 and 1.6 Hz, 1H), 8.25–8.28 (m, 1H), 7.59–7.74 (m, 3H),
4.3.4. 10,11-Difluoro-6H-chromeno[4,3-b]quinolin-6-one (3d)
O
O
7.40–7.47 (m, 2H); 13C NMR (100 MHz, DMSO-d6):
d 160.9,
152.8, 150.4, 149.5, 141.4, 134.2, 133.1, 130.3, 129.2, 127.9,
127.5, 125.3, 125.0, 119.7, 117.7, 116.4; IR (cmÀ1): 3100, 2990,
2836, 1737, 1601, 1496, 1237, 835; MS (ES mass): m/z 266.3
N
(M+1, 100%); HRMS: calcd for
266.3028.
C16H9FNO2: 266.3042, found
F
F
white solid; yield (92%); Rf = 0.42 (20% EtOAc-n-Hexane); mp
258–260 °C; 1H NMR (400 MHz, DMSO-d6): d 9.47 (s, 1H), 8.63
(dd, J = 7.6 and 1.2 Hz, 1H), 8.25–8.29 (m, 1H), 7.85–7.92 (m,
1H), 7.76–7.71 (m, 1H), 7.42–7.50 (m, 2H); 13C NMR (100 MHz,
CDCl3): d 160.8, 152.9, 150.6, 141.1, 133.2, 125.7, 125.5, 125.4
(2C), 125.3, 125.2, 124.9, 119.1, 118.8, 118.6, 117.4; IR (cmÀ1):
3065, 2836, 1735, 1604, 1380, 1177; MS (ES mass): m/z 284
4.3.8. 9-Methoxy-6H-chromeno[4,3-b]quinolin-6-one (3h)
O
O
(M+1, 100%); HRMS: calcd for
284.0476.
C16H7F2NO2: 284.0488, found
N
OCH3
white solid; yield (96%); Rf = 0.71 (20% EtOAc-n-Hexane); mp
228–230 °C; 1H NMR (400 MHz, CDCl3): d 9.1 (s, 1H), 8.76 (dd,
J = 10 and 1.6 Hz, 1H), 8.14 (d, J = 9.2 Hz, 1H), 7.55–7.60 (m, 2H),
7.39–7.45 (m, 2H), 7.23 (d, J = 2.8 Hz, 1H), 3.99 (s, 3H); 13C NMR
(100 MHz, TFA): d 159.6, 153.8, 151.6, 147.6, 142.2, 138.9, 134.7,
134.6, 127.2, 124.8, 124.0, 122.1, 121.8, 118.9, 105.9, 105.6,
58.8; IR (cmÀ1): 3090, 2980, 2845, 1745, 1601, 1490, 1245, 840;
MS (ES mass): m/z 278 (M+1, 10%); HRMS: calcd for C17H12NO3:
278.0819, found 278.0817.
4.3.5. 10-Bromo-6H-chromeno[4,3-b]quinolin-6-one (3e)
O
O
N
Br
White solid; yield (89%); mp 246–248 °C; Rf = 0.47 (10% EtOAc-n-
Hexane); 1H NMR (400 MHz, CDCl3): d: 9.20 (s, 1H), 8.75 (dd, J = 8
and 1.6 Hz, 1H), 8.45 (s, 1H), 7.89 (d, J = 8.8 Hz, 1H), 7.73 (dd,
J = 8.8 and 1.6 Hz, 1H), 7.60–7.64 (m, 1H),7.39–7.46 (m, 2H); 13C
NMR (100 MHz, DMSO-d6): d 161.0, 152.8, 150.4, 141.1, 132.9,
132.8, 131.8, 131.7, 131.2, 130.3, 125.8, 125.4, 125.1, 117.4, 115.9,
110; IR (cmÀ1): 3070, 2840, 1740, 1604, 1370, 1185; MS (ES mass):
m/z 327.1 (M+2, 100%); HRMS: calcd for C16H9BrNO2: 327.2546,
found 327.2462.
4.3.9. 9-Methyl-6H-chromeno[4,3-b]quinolin-6-one (3i)
O
O
N
white solid; yield (89%); Rf = 0.60 (30% EtOAc-n-Hexane); mp 233–
235 °C; 1H NMR (400 MHz, CDCl3): d 9.12 (s, 1H), 8.77 (dd, J = 8 and
1.6 Hz, 1H), 8.14 (d, J = 8.4 Hz, 1H), 7.74–7.76 (m, 2H), 7.56–7.58
(m, 1H), 7.38–7.45 (m, 2H), 2.59 (s, 3H); 13C NMR (100 MHz,
DMSO-d6): d 160.9, 152.8, 150.8, 149.6, 145.1, 140.9, 132.9, 130.3,
129.9, 128.0, 125.8, 125.3, 124.9, 119.8, 117.7, 115.6, 22.3; IR
(cmÀ1): 3125, 2995, 2840, 1740, 1595, 1480, 1250; MS (ES mass):
m/z 262 (M+1, 100%); HRMS: calcd, for C17H12NO2: 262.0879, found
262.0868.
4.3.6. 10-Fluoro-6H-chromeno[4,3-b]quinolin-6-one (3f)
O
O
N
F
white solid; yield (94%); Rf = 0.45 (20% EtOAc-n-Hexane); mp 233–
236 °C; 1H NMR (400 MHz, CDCl3): d 9.21 (s, 1H), 8.77 (dd, J = 8.0
and 1.6 Hz, 1H), 8.02–8.06 (m, 1H), 7.86 (dd, J = 10 and 2.4 Hz,
1H), 7.59–7.64 (m, 1H), 7.39–7.47 (m, 3H); 13C NMR (100 MHz,
4.3.10. 9-Chloro-6H-chromeno[4,3-b]quinolin-6-one (3j)
O
O
DMSO-d6):
d 160.7, 153.0, 151.6, 150.5, 141.5, 133.4, 125.4,
125.1, 124.9, 119.4, 118.8, 118.5, 117.7, 116.0, 112.7, 112.5; IR
(cmÀ1): 3065, 2836, 1735, 1599, 1380, 1170; MS (ES mass): m/z
266.2 (M+1, 10%); HRMS: calcd for C16H9FNO2: 266.2022, found
266.2016.
N
Cl
white solid; yield (93%); Rf = 0.4 (20% EtOAc-n-Hexane); mp 243–
245 °C; 1H NMR (400 MHz, CDCl3): d 9.1 (s, 1H), 8.77 (dd, J = 7.6
and 1.6 Hz, 1H), 8.18–8.20 (d, J = 9.2 Hz 1H), 8.0 (s, 1H), 7.85 (dd,
J = 9.2 and 2.4 Hz, 1H), 7.59–7.62 (m, 1H), 7.39–7.46 (m, 2H); 13C
NMR (100 MHz, CDCl3): d 160.9, 152.6, 149.7, 149.3, 139.9, 134.2,
133.3, 132.6, 131.0, 127.7, 127.6, 125.2, 125.0, 119.2, 117.4, 116.4;
IR(cmÀ1): 3063, 2836, 1735, 1604, 1380, 1177; MS (ES mass): m/z
282 (M+1, 100%); HRMS: calcd for C16H9ClNO2, 282.0246, found
282.0242.
4.3.7. 9-Fluoro-6H-chromeno[4,3-b]quinolin-6-one (3g)
O
O
N
F

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N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
8.05(m, 1H), 7.84–7.87 (m, 1H), 7.20–7.31(m, 2H), 6.90–6.96(m, 2H),
5.38 (t, J = 5.6 Hz,1H), 4.52 (d, J = 5.2 Hz, 2H); 13C NMR (100 MHz,
DMSO-d6): d 157.4, 154.5(2C), 143.2, 135.8, 131.9, 130.1, 129.8,
126.4, 124.2, 119.0, 115.5, 114.9, 114.6, 113.4, 59.6; IR (cmÀ1):
3143, 3074, 2924; MS (ES mass): m/z 288.1(M+1)+, 100%).
4.4. General procedure for the Preparation of 2-(3-
(hydroxymethyl)quinolin-2-yl)phenol derivatives (4a–d)
A suspension of NaBH4 (0.3 g, 8.1 mmol) in dry THF (5 mL) was
stirred at 0 °C. To this was added a solution of 6H-chromeno[4,3-
b]quinolin-6-one (1 g, 4.05 mmol) in THF (5 mL) and mixture was
stirred at room temperature for 3–4 h. After completion of the reac-
tion (monitored by TLC) the mixture was cooled to 0 °C and
quenched by adding saturated NH4Cl solution (5 mL). The mixture
was then poured in ethyl acetate (50 mL) and stirred for 10 min.
The organic layer was collected, washed with water (30 mL) and
brine (30 mL), dried over anhydrous Na2SO4, filtered and concen-
trated under low vacuum. The residue isolated was purified by flash
chromatography on silica gel using EtOAc-n-hexane (2:8) as eluent.
The desired product was isolated as a white solid (80%, 0.81 g).
4.5.4. 2-(6-Chloro-3-(hydroxymethyl)quinolin-2-yl)phenol (4d)
OH
N
Cl
OH
white solid; yield (84%); mp: 213–214 °C; Rf = 0.6 (20%
EtOAc-n-Hexane); 1H NMR (400 MHz, DMSO-d6): d 9.89 (s, 1H),
8.38 (s, 1H), 8.17 (s, 1H), 7.98 (d, J = 8.8 Hz, 1H), 7.69 (d, J = 9.2 Hz,
1H), 7.19–7.29 (m, 2H), 6.88–6.95 (m, 2H), 5.38 (t, J = 5.6 Hz, 1H),
4.50 (s, 2H); 13C NMR (100 MHz, DMSO-d6): d 157.9, 155.1, 144.9,
137.0, 131.9, 131.1, 131.0, 130.6, 130.2, 129.7, 128.4, 127.0, 126.7,
119.4, 116.0, 60.2; IR (KBr, cmÀ1) 2930, 2210, 1718, 1274; MS (ES
mass): m/z 285.9 (M+1, 100%).
4.5. Spectral data of 2-(3-(hydroxymethyl)quinolin-2-yl)phenol
derivatives (4a–d)
4.5.1. 2-(3-(Hydroxymethyl)quinolin-2-yl)phenol (4a)
OH
4.6. Preparation of 2-(3-(Azidomethyl)quinolin-2-yl)phenol (5a)
N
N3
OH
White solid; yield (85%); mp 155–157 °C; Rf = 0.65 (20% EtOAc-
n-Hexane); 1H NMR (400 MHz, CDCl3): d 9.89(s, 1H), 8.51 (s, 1H),
8.01 (d, J = 8.4 Hz, 1H), 7.84 (d, J = 8.4 Hz, 1H), 7.74 (t, J = 8.2 Hz,
1H), 7.55 (dd, J = 7.4 and 1.6 Hz, 1H), 7.26 (s, 1H), 7.33 (t,
J = 8.4 Hz, 1H), 7.10 (d, J = 7.6 Hz, 1H), 6.97 (t, J = 7.6 Hz, 1H), 5.58
(t, J = 5.6 Hz, 1H), 5.02 (s, 2H); 13C NMR (100 MHz, DMSO-d6); d
157.9, 157.2, 144.7, 138.3, 132.5, 131.2, 130.4, 129.8, 127.6,
127.5, 127.1, 126.7, 121.3, 119.1, 118.2, 62.9; IR (cmÀ1): 3130,
3067, 2954; MS (ES mass): m/z 251.9 (M+1, 100%).
N
OH
To a mixture of 2-(3-(hydroxyl methyl)quinolin-2-yl)phenol
(0.8 g, 3.2 mmol), sodium azide (0.25 g, 3.84 mmol) and PPh3 (1.0 g,
3.84 mmol) in 1:4 CCl4–DMF (5 mL) was stirred at 90 °C for 2–3 h.
After disappearance of starting material (monitored by TLC), the reac-
tion mixture was cooled to room temperature and quenched with
water (5 mL). After stirring the mixture for 5 min it was diluted with
ethylacetate (50 mL). The organic layer was collected, washed thor-
oughly with water (30 mL), dried over anhydrous sodium sulfate, fil-
tered and concentrated under reduced pressure. The residue was
purified by column chromatography using ethyl acetate–hexane
(1:9) as eluant. The desired product was isolated as a white solid
(90%, 0.79 g); mp: 124–126 °C; Rf = 0.4 (30% EtOAc-n-Hexane); 1H
NMR (400 MHz, CDCl3): d 12.2 (s, 1H), 8.39 (s, 1H), 8.05 (d J = 8.8 Hz,
1H), 7.88 (d, J = 8.4 Hz, 1H), 7.77 (t, J = 7.6 Hz, 1H), 7.60–7.64 (m,
2H), 7.36 (t, J = 7.6 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 6.98 (t, J = 7.6 Hz,
1H), 4.7 (s, 2H); 13C NMR (100 MHz, CDCl3): d 157.6, 145.0, 139.4,
131.8, 131.4, 130.9, 129.7, 127.8, 127.5, 127.3, 125.5, 122.5, 119.2,
118.2, 117.3, 52.9; IR (KBr, cmÀ1) 3041, 1667, 1487, 1345; MS (ES
4.5.2. 2-(3-(Hydroxymethyl)-6-methylquinolin-2-yl)phenol (4b)
OH
N
OH
Brown solid; yield (88%); mp 145–146 °C; Rf = 0.65 (20% EtOAc-
n-Hexane); 1H NMR (400 MHz, DMSO-d6): d 9.79 (s, 1H), 8.34 (s,
1H), 7.84–7.87 (m, 2H), 7.74 (dd, J = 8.8 and 1.6 Hz, 1H), 7.20–
7.32 (m, 2H), 6.91–6.97 (m, 2H), 5.37 (t, J = 5.6 Hz, 1H), 4.50 (d,
J = 5.2 Hz, 2H), 2.59 (s, 3H); 13C NMR (100 MHz, DMSO-d6): d
154.8, 144.4, 135.8, 135.2, 131.9, 131.1, 130.3, 129.6(2C), 128.1,
127.1, 126.7, 126.2, 119.0, 115.6, 60.0, 21.2; IR(cmÀ1): 3090,
3041, 2957; MS(ES mass): m/z 266.2 (M+1, 10%).
mass): m/z 276.9 (M+1, 100%); HRMS: calcd for
C16H13N4O:
277.2692, found 277.2688.
4.7. Preparation of N-((1-((2-(2-hydroxyphenyl)quinolin-3-
yl)methyl)-1H-1,2,3-triazol-4-yl)methyl)-N-(2-iodophenyl)
methanesulfonamide (5b)
4.5.3. 2-(6,7-Difluoro-3-(hydroxymethyl)quinolin-2-yl)phenol (4c)
Ms
N
I
OH
F
N
OH
N
N
N
N
F
OH
white solid; yield (82%); mp: 232–233 °C; Rf = 0.3 (20% EtOAc-n-Hex-
ane); 1H NMR (400 MHz, DMSO-d6): d 9.77 (s, 1H), 8.40 (s, 1H), 8.02–

N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
767
2 mmol) in n-butanol (5 mL) was stirred at 80–90 °C for 3–4 h. The
progress of the reaction was monitored by TLC. After completion of
the reaction the mixture was concentrated under vacuum and the
residue was purified by column chromatography on silica gel using
6:4 EtOAc–hexane as eluant. The desired product was isolated as a
yellow solid (400 mg, 41%); mp: 292–294 °C; Rf = 0.4 (30% EtOAc-
n-Hexane); 1H NMR (400 MHz, DMSO-d6 + acetone-d6): d 8.61
(d, J = 7.6 Hz, 1H), 8.56 (d, J = 8.2 Hz, 1H), 8.51 (s, 1H), 8.33 (d,
J = 8.0 Hz, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.96 (d, J = 8 Hz, 1H), 7.8 (t,
J = 7.2 Hz, 1H), 7.63 (t, J = 7.2 Hz, 1H), 7.43 (t, J = 4.4 Hz, 1H), 7.38
(t, J = 6.4 Hz, 1H), 7.32 (bs, 1H), 7.2 (t, J = 8.4 Hz, 1H), 7.14 (bs, 2H),
6.95 (d, J = 8.4 Hz, 1H), 6.82 (t, J = 7.2 Hz, 1H), 4.52 (s, 2H); 13C NMR
(100 MHz, Acetone-d6): d 164.4, 158.0, 155.0, 146.7, 136.6, 134.0,
133.8, 132.3, 131.7, 130.6, 130.0, 128.5(2C), 128.2(2C), 127.7,
127.1, 126.9, 125.2, 119.4, 116.1, 110.0, 48.9; IR (KBr, cmÀ1) 2927,
1671, 1484, 1362; EI-MS: m/z 494.8 (M+1, 100%); HRMS: calcd for
To
a stirred solution of the N-(2-iodophenyl)-N-(prop-2-
ynyl)methanesulfonamide (0.12 g, 0.33 mmol) in DMF (5 mL) was
added copper iodide (30 mol %), 2-(3-(azidomethyl)quinolin-2-
yl)phenol (0.092 mg) and DIPEA (3 equiv) under a nitrogen atmo-
sphere. The mixture was stirred at 70 °C for 6 h. The progress of
the reaction was monitored by TLC. After consumption of the reac-
tion the mixture was cooled to room temperature and diluted with
ethylacetate (50 mL). The mixture was filtered through celite pad.
The filtrate was collected and concentrated under reduced pressure.
The residue was purified with silica gel column chromatography
using 4:6 EtOAc–hexane as eluant. The desired product was isolated
as a white solid (0.12 g, 60%); mp: 198–199 °C; Rf = 0.4 (30% EtOAc-
n-Hexane); 1H NMR (400 MHz, CDCl3): d 8.02 (d, J = 8.4 Hz, 1H), 7.83
(d, J = 10.8 Hz, 1H), 7.75 (d, J = 7.6 Hz, 1H), 7.69 (d, J = 8 Hz, 1H), 7.59
(t, J = 7.6 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.42 (s, 1H), 7.32–7.39 (m,
2H), 7.17 (t, J = 7.2 Hz, 1H), 7.07–7.13 (m, 2H), 6.97–7.00 (m, 2H),
5.87 (s, 2H), 5.08 (d, J = 1.6, 1H), 4.64 (d, J = 1.6, 1H), 3.13 (s, 3H);
13C NMR (100 MHz, DMSO-d6): d 156.8, 156.4, 145.5, 143.8, 140.6,
140.3, 138.1, 132.2, 131.5, 131.1, 130.4, 129.6, 129.1, 128.0, 127.6,
127.5, 127.1, 126.5, 124.1, 121.8, 119.7, 118.4, 101.5, 53.4, 46.2,
41.4; IR (KBr, cmÀ1) 3365, 2956, 1654; EI-MS: m/z 611.8 (M+1,
100%); HRMS: calcd for C26H23IN5O3S: 612.0522, found 612.0499.
C23H19N4O7S: 495.0942, found 495.0940.
4.10. Single crystal X-ray data for compound 3a, 4d
Single crystals suitable for X-ray diffraction of 3a, 4d were
grown from methanol. The crystals were carefully chosen using a
stereo zoom microscope supported by a rotatable polarizing stage.
The data was collected at room temperature on Bruker’s KAPPA
4.8. Preparation of 2-(3-(aminomethyl)quinolin-2-yl)phenol (5c)
APEX II CCD Duo with graphite monochromated Mo-Ka radiation
(0.71073 Å). The crystals were glued to a thin glass fibre using
FOMBLIN immersion oil and mounted on the diffractometer. The
intensity data were processed using Broker’s suite of data process-
ing programs (SAINT), and absorption corrections were applied
using SADABS.19 The crystal structure was solved by direct meth-
ods using SHELXS-97 and the data was refined by full matrix
least-squares refinement on F2 with anisotropic displacement
parameters for non-H atoms, using SHELXL-97.20
OH NH2
N
To a solution of 2-(3-(azidomethyl)quinolin-2-yl)phenol (0.7 g,
2.53 mmol) in EtOAc (5 mL) was added 10% Pd/C (0.026 g,
0.25 mmol) carefully. The reaction mixture was stirred under
hydrogen atmosphere (filled in a balloon) at room temperature
for 4 h. The progress of the reaction was monitored by TLC (9:1
EtOAc–MeOH, UV). After completion of the reaction the mixture
was filtered through celite. The filtrate was collected and concen-
trated under vacuum. The residue was purified by flash chroma-
tography on silica gel using 6:4 EtOAc–Hexane as eluant to give
Crystal data of 3a: Molecular formula = C16H9NO2, Formula
weight = 247.24, Crystal system = Monoclinic, space group =
P2(1)/n, a = 16.772 (11) Å, b = 3.832 (3) Å, c = 18.299 (11) Å,
V = 1117.72 (13) Å3, T = 296 K, Z = 4, Dc = 1.389 Mg mÀ3
,
l
(Mo-
) = 0.71073 mmÀ1, 7702 reflections measured, 1251 indepen-
dent reflections, 1123 observed reflections [I >2.0 (I)],
Ka
r
R1_obs = 0.042, Goodness of fit = 1.003. Crystallographic data
(excluding structure factors) for 3a have been deposited with the
Cambridge Crystallographic Data Center as supplementary publi-
cation number CCDC 840017.
the desired product as
171–174 °C; Rf = 0.2 (40% EtOAc-n-Hexane); 1H NMR (400 MHz,
DMSO-d6): 8.38 (s, 1H), 8.05 (d, J = 8.8 Hz, 1H), 7.83 (d,
a white solid (0.6 g, 95%). mp:
Crystal data of 4d: Molecular formula = C16H12ClNO2, Formula
weight = 285.72, Crystal system = Orthorhombic, space group =
Pca21, a = 10.568 (4) Å, b = 7.241 (3) Å, c = 17.2358 (6) Å,
d
J = 7.6 Hz, 1H), 7.69–7.74 (m, 2H), 7.51–7.61 (m, 3H), 7.34 (t,
J = 8.0 Hz, 1H), 7.12–6.97 (m, 3H), 4.2 (s, 2H); 13C NMR (100 MHz,
DMSO-d6): d 158.5, 155.6, 146.7, 134.5, 133.9, 131.1, 130.1,
129.6, 128.9, 128.2, 127.8, 127.4, 126.9, 119.3, 116.7, 42.3; IR
(KBr, cmÀ1) 3041, 1667, 1487, 1345; EI-MS: m/z 251.2 (M+1, 100%).
V = 1319.02 (9) Å3, T = 296 K, Z = 4, Dc = 1.434 Mg mÀ3
,
l
(Mo-K
7596 reflections measured, 2665 independent
reflections, 2651 observed reflections [I >2.0 (I)], R1_obs =
a) =
0.71073 mmÀ1
,
r
0.039, Goodness of fit = 0.876, Crystallographic data (excluding
structure factors) for 4d have been deposited with the Cambridge
Crystallographic Data Center as supplementary publication num-
ber CCDC 840016.
4.9. Preparation of 4-((2-(2-hydroxyphenyl)quinolin-
3-yl)methylamino)-3-nitro-5-sulfamoylbenzoic acid (5d)
COOH
4.11. Pharmacology
Cell lines and culture conditions: Human chronic myeloid leuke-
mia cells (K562), human colon carcinoma cells (Colo-205), breast
cancer cells (MDA-MB 231), and human neuroblastoma cells
(IMR32) were procured from National Center for Cell Sciences,
Pune, India. All cells were grown in RPMI-1640 supplemented with
10% heat inactivated fetal bovine serum (FBS), 100 IU/ml penicillin,
100 mg/ml streptomycin and 2 mM-glutamine. Cultures were
maintained in a humidified atmosphere with 5% CO2 at 37 °C.
The cells were subcultured twice each week, seeding at a density
of about 2 Â 103 cells/ml.
O2N
OH NH
SO2NH2
N
A mixture of 2-(3-(aminomethyl)quinolin-2-yl)phenol (0.5 g,
2 mmol) and 4-chloro-3-nitro-5-sulfamyl benzoic acid (0.56 g,

768
N. Mulakayala et al. / Bioorg. Med. Chem. 20 (2012) 759–768
MTT Assay: Cell viability was determined by 3-(4,5-dimethylthi-
and C.M. thank CSIR, New Delhi, India for awarding fellowships.
M.P. thanks DBT, New Delhi, India for financial support (Grant
No. BT/PR13997/Med/30/310/2010).
azol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Cells
(5 Â 103 cells/well) were seeded to 96-well culture plate and cul-
tured with or without compounds at 1 and 10 lM concentration
for 24 h in a final volume of 200
was removed and 20 l of MTT (5 mg/ml in PBS) was added to
the fresh medium. After 2 h incubation at 37 °C, 100 l of DMSO
ll. After treatment, the medium
Supplementary data
l
l
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.12.001.
was added to each well and plates were agitated for 1 min. Absor-
bance was read at 570 nm on a multi-well plate reader (Victor3,
Perkin Emler). In all of these experiments, three replicate wells
were used to determine each point.
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4.12. Docking study
The three dimensional model of hSirt1 (NCBI gi no: 7555471,
200–500 amino acid residues) was developed by homology model-
ing using the templates PDB: 2HJH and PDB: 1J8F in the Model-
ler9v6. To the developed model hydrogens were added and
subjected to restrained minimization using the OPLS-AA force field
in GROMACS 3.0 package to RMSD of 0.4 Å.
Molecular docking: The compound 5b was sketched by using
Chemdraw and converted them to their 3D representation using
Dundee Prodrg server. The compound 4B and protein (homology
model of hSIRT1) were prepared for docking (i.e., adding hydro-
gens, gasteiger charges, Kollman charges) by using AutoDock Tools
(ADT). AutoDOck 4.0 program was used for docking. The best mod-
el of hSirt1 was predicted and validated. The receptor hSirt1 with
gird coordinates X: 60; Y:60; Z:60 was used for docking with the
compound 5b. The best 5 poses and corresponding scores have
been evaluated by AutoDock 4.0 program.21
21. Morris, G. M.; Goodsell, D. S.; Halliday, R. S.; Huey, R.; Hart, W. E.; Belew, R. K.;
Olson, A. J. J. Comput. Chem. 1998, 19, 1639.
Acknowledgments
The authors thank Professor J. Iqbal for encouragement and
support. R.D. thanks DBT, New Delhi, India for a fellowship. N.M.