Synthesis and evaluation of new coumarin-pyridine hybrids with promising anti-osteoporotic activities

Article abstract of DOI:10.1016/j.ejmech.2013.10.060

Anti-osteoporotic effects of the newly synthesized coumarin-pyridine hybrids were evaluated in primary cultures of rat calvarial osteoblasts in vitro. Compounds 6a, i, j and k were potent in stimulating osteoblast differentiation and mineralization as ass

Full text of DOI:10.1016/j.ejmech.2013.10.060

European Journal of Medicinal Chemistry 70 (2013) 802e810
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Preliminary communication
Synthesis and evaluation of new coumarinepyridine hybrids with
promising anti-osteoporotic activitiesq
,
a
b
a
Koneni V. Sashidhara a , Ram K. Modukuri , Dharmendra Choudhary , K. Bhaskara Rao ,
*
Manoj Kumar a, Vikram Khedgikar b, Ritu Trivedi b
a Medicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, (CSIR-CDRI), BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, P.O.
Box 173, Lucknow 226031, India
b Endocrinology Division, CSIR-Central Drug Research Institute, (CSIR-CDRI), BS-10/1, Sector 10, Jankipuram Extension, Sitapur Road, P.O. Box 173, Lucknow
226031, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Anti-osteoporotic effects of the newly synthesized coumarinepyridine hybrids were evaluated in
primary cultures of rat calvarial osteoblasts in vitro. Compounds 6a, i, j and k were potent in stim-
ulating osteoblast differentiation and mineralization as assessed by the alkaline phosphatase pro-
duction and alizarin red-S staining assay, respectively. These compounds were also found to be
nontoxic in osteoblast cells as compared to the control group in an MTT assay. Furthermore, the effect
of these compounds on the transcript levels of osteogenic genes revealed that the compound 6j
robustly enhanced mineralization of the osteogenic genes in calvarial osteoblasts. In this context,
compound 6j was selected as a potential lead for further structural optimization in the development
of new anti-osteoporotic agents.
Received 23 June 2013
Received in revised form
22 October 2013
Accepted 24 October 2013
Available online 1 November 2013
Keywords:
Osteoporosis
Synthesis
Coumarin
Pyridine
Ó 2013 Elsevier Masson SAS. All rights reserved.
Hybrid
1. Introduction
However, with the long-term usage of these drugs most of the
patients experience undesirable side effects, such as osteonecrosis
Osteoporosis is a progressive skeletal disorder, due to the un-
equal coupling between osteoclast mediated bone resorption and
osteoblast mediated bone formation [1]. It is a wide spread disease
in the elderly population and effects up to 50% of females [2]. It is
characterized by decrease in bone mineral density and consequent
increase in bone fragility, which leads to a high risk of fractures [3e
5]. The main risk factors for osteoporosis are ageing (decline in the
supply of osteoblasts), estrogen deficiency (excessive bone
resorption), calcium deficiency, Vitamin D deficiency and fractures
[6]. This has been widely recognized as a ‘silent disease’ and in the
event of complicated fracture it becomes clinically apparent [7].
The FDA-approved medical options include bisphosphonates
(alendronate, ibandronate, risedronate and zoledronic acid),
calcitonin, estrogen hormone therapy, parathyroid hormone (ter-
iparatide) and estrogen agonist/antagonist (raloxifene) [8].
of the jaw (ONJ), unusual thigh bone fractures and oesophageal
cancer, etc [9]. In 2012, The European Medicines Agency (EMA) has
withdrawn intranasal calcitonin due to risk of hypercalcaemia
followed by cancer [10]. Therefore, there is a constant need
for a new class of bone anabolic agents for the treatment of
osteoporosis.
Coumarins are an important class of plant secondary metabo-
lites which possess a diverse range of biological activities [11]. It is
also reported that coumarins posses bone anabolic effects [12].
Inspired from the medicinal chemistry hybridisation approach, we
recently identified compound 1 ethyl 4-(8-sec-butyl-3-(methox-
ycarbonyl)-2-oxo-2H-chromen-6-yl)-2-methyl-5-oxo-1,4,5,6, 7,8
hexahydroquinoline-3-carboxylate (at 100 pM) as a hit compound,
that showed approximately 65e70% increased mineralization
compared to control untreated cells (Fig.1) [13]. Further, compound
1 did not exhibit any toxicity after oral administration. A hit com-
pound may have a simpler pharmacophore moiety that exists
within its structure, which through modification could exhibit
improved activity and be more useful as a scaffold for further
analogue design [14]. The exquisite potency and structural novelty
encouraged us to further explore the chemical space for the
development of new class of bone anabolic agents.
q
Part XXVI in the series, “Advances in drug design and discovery”. CSIR-
CDRI#8559.
* Corresponding author. Tel.: þ91 9919317940; fax: þ91 0522 2771942.
E-mail
addresses:
sashidhar123@gmail.com,
kv_sashidhara@cdri.res.in
(K.V. Sashidhara).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.10.060

K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
803
3. Pharmacology
3.1. Culture of calvarial osteoblasts
Mice calvarial osteoblast cultures were performed using 25e30
calvariae from 1 to 2 day old mice pups (both sexes). Calvarias were
surgically isolated from skull; sutures were segregated and
adherent tissue material cleaned by gentle scrapping. Isolated cal-
veriae were pooled and kept for repeated digestion (15 min/
digestion) with 0.1% dispase and 0.1% collagenase-1 was used to
release cell. Supernatant of first digestion was discarded, cells from
the next three digestions were pooled and cultured in
a modified
essential medium ( -MEM) containing 10% FCS and 1% penicillin/
a
Fig. 1. Hit compound 1.
streptomycin (complete growth medium). For experiment 70e80%
confluent mice calvarial osteoblast were used [13,19].
Interestingly, recent report suggests that the pyridine ring
containing compounds may modulate anti-osteoporotic activity
[15]. Furthermore, Saito et al. reported substituted thienopyridine
derivatives as bone anabolic agents [16]. This prompted us to syn-
thesize new coumarin containing pyridine hybrids as potential
anti-osteoporotic agents (Fig. 2) [17].
3.2. Alkaline phosphatase assay
At 70e80% confluent mice calvarial osteoblast was used for
trypsinization and 2 ꢁ 103 cells were seeded onto 96-well plates for
alkaline phosphatase (ALP) measurement. Cells were treated with
or without compound (6a, i, j and k) or vehicle for 48 h in
a
-MEM
2. Chemistry
supplemented with 10% FCS, 10 mM -glycerophosphate, 50
b
mg/mL
ascorbic acid, and 1% penicillin/streptomycin (osteoblast differen-
tiation medium). At the end of incubation period, cells were
washed with phosphate-buffered saline (PBS) and kept in ꢂ80 ꢀC
for 30 min and then at 37 ꢀC for 30 min. ALP activity was measured
using p-nitrophenylphosphate (PNPP) as substrate and absorbance
was read at 405 nm [19].
The synthesis of target and intermediate compounds is outlined
in Scheme 1. The Duff reaction on ortho-substituted phenols (2ae
d) in the presence of hexamethylenetetraamine (HMTA) and TFA at
120 ꢀC gave aromatic dicarbaldehydes (3aed). These dicarbalde-
hyde intermediates were then engaged in the Knoevenagel-type
reaction with different active methylene compounds resulted in
the formation of coumarinic compounds (4aef) [13]. Further, these
coumarinic aldehyde compounds were subjected to Hantzsch
3.3. Mineralized nodule formation assay
dihydropyridine synthesis via
a multi-component reaction,
involving coumarinic aldehydes, active methylene compounds and
ammonium acetate (nitrogen donor) in the presence of acetic acid
glacial. For symmetrical Hantzsch dihydropyrimidines (5aec), two
equivalent of ethyl/methyl acetoacetate were used while for un-
symmetrical Hantzsch polyhydroquinolines (5dem), uniequivalent
of ethyl/methyl acetoacetate and different 1,3-cyclohexadiones
were employed. Finally, aromatisation with the 2,3-Dichloro-5,6-
dicyano-1,4-benzoquinone (DDQ) in the presence of tetrahydro-
furan (THF) as a solvent at room temperature gives the desired
coumarinepyridine hybrids (6aem) [18]. All the synthesized
compounds were characterized using 1H NMR, 13C NMR, IR spec-
troscopy and ESI-MS.
For mineralization mice calvarial osteoblast were seeded onto
12-well plates (10,000 cells/well) in osteoblast differentiation me-
dium. Mice calvarial osteoblasts were cultured with or without
compound 18 for 21 days and after every 48 h media were changed.
At the end of the experiment, cells were washed with PBS and fixed
with 4% paraformaldehyde in PBS for 15 min. The fixed cells were
stained with alizarin red-S (40 mM, pH 4.5) for 30 min followed by
washing with water. Stained cells were first photographed under
light microscope, and extraction of alizarin stain was carried out by
using 10% (v/v) acetic acid with shaking at room temperature for
30 min. Cells were scrapped out from wells, centrifuged (20,000 ꢁ g
for 15 min), and supernatant was collected. To the supernatant, 10%
Fig. 2. - Designing of coumarinepyridine hybrids based on coumarin and pyridine scaffolds showing anti-osteoporotic effects.

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K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
Scheme 1. Synthesis of coumarinepyridine hybrids. Reagents and conditions: (i) HMTA, TFA, 120 ꢀC, 4 h. (ii) aq H2SO4, 100 ꢀC, 2 h. (iii) diethyl/methyl malonate, EtOH, piperidine,
reflux, 30 min (iv) Glacial AcOH, rt. (v) ethyl/methyl acetoacetate, NH4OAc, AcOH, EtOH, reflux, 5 h. (vi) ethyl/methyl acetoacetate, different 1,3-cyclohexadiones, NH4OAc, AcOH,
EtOH, reflux, 5 h. (vii) DDQ, THF, rt, 1 h.
(v/v) ammonium hydroxide was added to bring the pH of the so-
lution to 4.5 for colour formation. Absorbance of the solution was
read at 405 nm [19,20].
showed significant increase of ALP activity at concentration as low
as 100 pM.
4.2. Mineralization ability of active compounds 6a, i, j and k in
3.4. Cytotoxicity assay
calvarial osteoblast cells
Mice calvarial osteoblast was used to test toxicity of synthesized
compounds (6a, i, j and k) 2 ꢁ 103 cells. Cells were seeded onto 96-
well plates and treated with or without compounds (6a, i, j and k)
After ALP activity, the most active compounds 6a, i, j and k, were
further evaluated for their osteogenic activity by mineralisation
assay. At the end of differentiation process of calvarial osteoblast
cells, with or without treatment with the compounds at active
concentration for 21 days were fixed and stained with alizarin red-S
stain. In this process calcium has been deposited for the newly
formed mineralized nodules. Interestingly compounds 6a, i, j and k
showed significant increase of mineralizing ability as compared to
the control not treated cells. From the extraction of alizarin red-S
stained cultures, quantification of mineralization is determined
by the measurement of optical density. Quantification data suggests
at various concentrations (100 pMe1
tion, the cells were washed with PBS and were treated with MTT
solution (5 g/10 mL in -MEM devoid of Phenol Red) for 4 h.
mM) for 48 h. After incuba-
m
a
Formazon crystals formed were dissolved in DMSO and OD was
taken at 540 nm.
4. Results and discussion
4.1. Preliminary screening of coumarinepyridine hybrids using
that compound 6a at 1 mM (P < 0.001 vs corresponding control
osteoblast differentiation assay
group) concentration, show 2 fold increase in mineralization,
compound 6i at 10 nM concentration, (P < 0.001, vs corresponding
control group) exhibited 3 fold increase in mineralization, com-
pound 6j at 100 pM (P < 0.001 vs corresponding control group)
concentration showed approximately 3.5 fold increased minerali-
zation, while 6k at 1 pM concentration showed 2 fold increase in
mineralization (P < 0.001, as compared to control) (Fig. 4).
The synthesized compounds (6aem) were screened for alkaline
phosphatase (ALP) activity in primary mice calvarial osteoblasts,
which is an important marker of osteoblast differentiation. Cells
were treated with hybrid compounds to evaluate ALP activity at
concentrations ranging from 1 pM to 1
determined at 405 nm. Among all compounds screened, compound
6a at 1 M and 10 nM, compound 6i at 10 nM, compound 6j at 1 M,
mM and optical density was
m
m
4.3. Active compounds 6a, i, j and k increase osteoblastogenesis
10 nM and 100 pM and compound 6k at 1 pM and 100 pM were
found to significantly increase ALP activity over control (Fig. 3).
Though most compounds exhibited ALP activity at much lower
concentrations (nM concentrations), the compounds 6j and 6k
related marker gene expressions
In order to further validate and identify the most potent oste-
ogenic agent, we further examined the effect of compounds on the

K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
805
Fig. 3. Activity of the compounds 6a, i, j and k as assessed by alkaline phosphatase (ALP) activity in calvarial osteoblast cells (for details see text). At the end of the experiment, ALP
activity was measured colorimetrically as described before. Data shows mean ꢃ S.D. of three independent experiments ***p < 0.001, **p < 0.01, and *p < 0.01 as compared with
untreated cells taken as control.
expression of osteogenic genes (Runx-2, BMP-2 and COL-1) in cal-
varial osteoblasts by quantitative PCR (qPCR) [21,22]. In this study
glyceraldehydes 3-phosphate dehydrogenase (GAPDH) a house
keeping gene was used as the internal control. Runx-2, a bone-
specific transcription factor and BMP-2 are key regulators of oste-
oblast differentiation [21,23]. Whereas, COL-1 is well known to be
involved in the matrix maturation and mineralization process [24].
The transcript levels of Runx-2, BMP-2 and COL-1 were assessed by
qPCR after treatment with compounds and results were expressed
as fold change over untreated cells. Our data shows that the
expression of Runx-2 gene was increased by both 6i (w5.2 fold) and
6j (w5.0 fold). In case of BMP-2, mRNA expression was increased by
6i (w4.8 fold), 6a (w2.5 fold) and 6j (w2.4 fold). In case of COL 1,
mRNA expression was increased by only 6j (w3.1 fold) (Fig. 5).
Fig. 4. - Mineralizing ability of compounds 6a, i, j and k as compared to the control not treated cells in calvarial osteoblasts cells. Calvarial osteoblasts were grown in osteoblast
differentiation medium as described before (see text for details). At the end of the experiments, cells were stained with alizarin red-S. Photomicrographs show increased formation
of mineralized nodules by compound treatment compared to the vehicle treated cells. Bar diagram in the lower panel show quantification of mineralization by extraction of alizarin
red-S dye. Data represents mean ꢃ S.E. of three independent experiments (*p < 0.05, **p < 0.001, and ***p < 0.001 as compared to control).

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K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
Fig. 5. - Effect of compounds 6a, i, j and k on the expression of osteogenic genes (Runx-2, BMP-2 and CoL-1) in calvarial osteoblasts as compared to control by qPC.
Thus, on the basis of qPCR data the compound 6j was the most
active that caused marked increase in expression of the osteogenic
genes (Runx-2, BMP-2 and COL-1). Furthermore, the active com-
pounds 6a, i, j and k, were found to be non-toxic, when assessed
using MTT assay in osteoblast cells as compared to the control
chain (6j, i and k) seems to enhance the activity, with the exception
of 6h and l. Most interestingly, this study has revealed that
hydrogen bonding modification of the nitrogen ring does not effect
the activity, as both coumarinedihydropyridine hybrids (hydrogen
bond donors) (Hit compound eFig. 1) and coumarinepyridine hy-
brids (hydrogen bond acceptors) retains the activity, suggesting
that the binding sites for both are very similar.
group at doses ranging from 1 pM to 1 mM (Fig. 6).
In terms of structure activity relationship (SAR), in general most
unsymmetrical coumarinepyridine hybrids (6dem), exhibited
significant activity (6j, i and k), than the symmetrical counterparts
(6aec), which underscores the importance of the unsubstituted
cylohexanone moiety in the lower part of the molecule. While, on
the upper part of the hybrid the presence of sec-butyl (R1) side
5. Conclusion
A series of coumarinepyridine hybrids were synthesized and
evaluated for their anti-osteoporotic activity in calvarial osteoblast
Fig. 6. - Bioactive compounds 6a, i, j and k were assessed for cell viability and toxicity. Cells were cultured in differentiation medium and treated with various concentrations of the
compound ranging from 100 pM to 1 M for 48 h and cell viability assessed by MTT assay. The percent viable cells were calculated as compared to untreated cells taken as control.
m

K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
807
cells. Out of all compounds screened, compounds 6a, i, j and k
significantly increased the ALP activity and potentially increased
the mineralization in calvarial osteoblast cells by alizarin red-S
staining. Furthermore, the compound 6j increased the expression
of osteogenic genes (RUNX2, BMP-2 and COL-1). Taken together,
this study has identified compound 6j as a new class of promising
lead among the series of coumarinepyridine hybrids. Further
detailed studies (in vivo and pharmacokinetic) on 6j are planned so
as to advance this molecule in preclinical trials.
pressure, and the residue was neutralized with acetic acid. To this
residue water (25 mL) was added and extracted 3-fold with 20 mL
of CHCl3. The combined organic layers were dried on Na2SO4,
filtered, and concentrated to dryness under reduced pressure. The
crude product thus obtained was purified over column chroma-
tography to furnish (0.44 g, 64% yield) of compound 4d.
Compounds 4aef was synthesized by following the above
method.
6.3.1. Methyl-8-sec-butyl-6-formyl-2-oxo-2H-chromene-3-
carboxylate (4d)
6. Experimental
White solid, yield: 64%; mp 122e123 ꢀC; IR (KBr): 3062, 1749,
6.1. General information
1609, 1576, 1055 cmꢂ1 1H NMR (CDCl3, 300 MHz)
; d: 10.04 (s, 1H),
8.62 (s, 1H), 8.05 (brs, 1H), 7.98 (brs, 1H), 3.98 (s, 3H), 3.51e3.40 (m,
1H), 1.79e1.69 (m, 2H), 1.32 (d, 3H, J ¼ 7 Hz), 0.88 (t, 3H, J ¼ 7.4 Hz);
All reagents were commercial and were used without further
purification. Chromatography was carried on silica gel (60e120 and
100e200 mesh). All reactions were monitored by TLC; silica gel
plates with fluorescence F254 were used. Melting points were
uncorrected. The 1H NMR, and 13C NMR spectra were determined
on 200, 300, 400 MHz and 50, 75, 100 MHz, respectively, using
CDCl3 and DMSO-d6 as solvents and TMS as internal standard. All
chemical shifts were given in ppm. IR spectra were recorded on in
the range of 500e4000 cmꢂ1 and multiplicity (s ¼ singlet,
brs ¼ broad singlet, d ¼ doublet, brd ¼ broad doublet, dd ¼ double
doublet, t ¼ triplet, q ¼ quartet, m ¼ multiplet).
13C NMR: (CDCl3, 75 MHz)
d: 190.2, 163.4, 156.6, 155.9, 149.0, 137.5,
133.0, 131.9, 129.9, 118.9, 118.1, 53.2, 33.5, 29.7, 20.5, 12.0; ESI-MS:
m/z: 289 (M þ H)þ.
6.3.2. Ethyl-8-sec-butyl-6-formyl-2-oxo-2H-chromene-3-
carboxylate (4e)
White solid, yield: 65%; mp 89e90 ꢀC; IR (KBr): 3042, 1747, 1601,
1589, 1065 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d 10.05 (s, 1H), 8.60 (s,
1H), 8.05 (brs, 1H), 8.01 (brs, 1H) 4.44 (q, 2H, J ¼ 7.1 Hz), 3.51e3.40
(m, 1H), 1.78e1.69 (m, 2H), 1.43 (t, 3H, J ¼ 7.1 Hz), 1.33 (d, 3H,
J ¼ 7 Hz), 0.88 (t, 3H, J ¼ 7.4 Hz); 13C NMR: (CDCl3, 75 MHz)
d: 190.2,
6.2. General synthetic procedure for preparation of 5-alkyl-4-
hydroxy-benzene-1,3-dicarbaldehyde (3aef)
162.7, 156.5, 155.8, 148.5, 137.3, 132.9, 131.7, 129.9, 119.2, 118.0, 62.2,
33.4, 29.6, 20.4, 14.2, 12.0; ESI-MS: m/z: 303 (M þ H)þ.
2-alkyl phenol (1.0 equiv.) and hexamethylenetetramine
(1.2 equiv.) were dissolved in TFA and the solution was heated at
120 ꢀC for 3 h. After cooling to room temperature 10% aq.H2SO4
(25 mL) was added and again the temperature maintained (at 90e
100 ꢀC) for two more hours. The solution was basified with NaHCO3
to pH 8 and extracted 3-fold with 50 mL of CHCl3. The combined
organic layers were dried on Na2SO4, filtered, and concentrated to
dryness under reduced pressure. The crude product was purified on
a silica gel column (100e200 mesh) using hexaneeethylacetate
(12:88, v/v) as eluent to afford compounds 3aef in good yields.
6.3.3. Methyl-8-tert-butyl-6-formyl-2-oxo-2H-chromene-3-
carboxylate (4f)
White solid, yield: 68%; mp 92e93 ꢀC; IR (KBr): 3048, 1741, 1611,
1584, 1061 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d
: 10.05 (s, 1H), 8.62 (s,
1H), 8.16 (brs, 1H), 8.02 (brs, 1H), 3.98 (s, 3H), 1.55 (brs, 9H); 13C
NMR: (CDCl3, 75 MHz) : 190.2, 163.3, 157.7, 155.3, 149.3, 139.7,
d
132.5, 131.7, 130.7, 118.6, 118.4, 53.1, 35.4, 29.6; ESI-MS: m/z: 289
(M þ H)þ.
6.4. General synthetic procedure for preparation of dimethyl4-(8-
sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-chromen-6-yl)-2,6-
dimethyl-1,4-dihydropyridine-3,5-dicarboxylate (5b)
6.2.1. 5-Methyl-4-hydroxy-benzene-1,3-dicarbaldehyde (3a)
White solid; yield: 60%; mp: 125e127 ꢀC; IR (neat):3262, 2865,
1703, 1626, 1013 cmꢂ1 1H NMR (CDCl3, 300 MHz)
; d: 11.82 (s, 1H),
9.97 (s, 1H), 9.90 (s, 1H), 7.97 (d, 1H, J ¼ 1.8 Hz), 7.93 (brs, 1H), 2.33
To a solution of ethyl 8-sec-butyl-6-formyl-2-oxo-2H-chromene-
3-carboxylate (1.0 equiv.) (4e) in ethanol, methyl acetoacetate
(2.0 equiv.), ammonium acetate (3.0 equiv.) and 3 mL acetic acid
was added. The reaction mixture was continued under reflux
condition for 8 h. After completion of reaction (monitor by TLC), the
reaction mixture was cooled to room temperature. The solid thus
obtained was filtered and chromatographed over EtOAc furnished
compound 5b as white solid.
(s, 3H); ESI-MS: m/z: 164 (M þ H)þ.
6.2.2. 5-Sec-butyl-4-hydroxy-benzene-1,3-dicarbaldehyde (3c)
Oily, yield 55%, IR (neat): 3267, 2862, 1709, 1622, 1018 cmꢂ1; 1H
NMR (CDCl3, 200 MHz) d: 11.99 (s, 1H), 10.05 (s, 1H), 9.97 (s, 1H),
8.09 (brs, 1H), 8.01 (brs, 1H), 3.27e3.10 (m, 1H), 1.74e1.57 (m, 2H),
1.26 (d, 3H, J ¼ 7.0 Hz), 0.86 (t, 3H, J ¼ 7.3 Hz); ESI-MS: m/z: 207
(M þ H)þ.
The compounds (5aec) were prepared in a manner similar to
the procedure described above.
6.2.3. 5-Tert-butyl-4-hydroxyisophthalaldehyde (3d)
Oily, yield 60%, IR (neat): 3262, 2865, 1703, 1626, 1013 cmꢂ1; 1H
6.4.1. Dimethyl-4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(5b)
NMR (CDCl3, 300 MHz) d: 12.39 (s,1H), 9.99 (s,1H), 9.93 (s,1H), 8.07
(brs, 1H), 7.99 (brs, 1H), 1.46 (s, 9H); ESI-MS: m/z: 207 (M þ H)þ.
White solid, yield: 81%; mp 247e248 ꢀC; IR (KBr): 3004, 1680,
6.3. General synthetic procedure for preparation of methyl-8-sec-
1596, 1045 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.48 (s, 1H), 7.51 (d,
butyl-6-formyl-2-oxo-2H-chromene-3-carboxylate (4d)
1H, J ¼ 1.7 Hz), 7.29 (d, 1H, J ¼ 2.1 Hz), 6.09 (s, 1H), 5.04 (s, 1H), 4. 40
(q, 2H, J ¼ 7.1 Hz), 3.64 (brs, 6H), 3.36 (q, 1H, J ¼ 6.8 Hz), 2.38 (s, 6H),
1.67 (t, 2H, J ¼ 7.5 Hz), 1.40 (t, 3H, J ¼ 7.2 Hz), 1.25 (d, 3H, J ¼ 6.9 Hz),
0.83 (t, 3H, J ¼ 7.3 Hz); 13C NMR (CDCl3, 75 MHz): 167.7, 163.3, 157.3,
151.4, 149.9, 144.8, 134.4, 132.7, 125.9, 117.7, 116.9, 103.4, 61.7, 50.9,
39.3, 33.0, 29.5, 20.6, 19.4, 14.2, 11.7; ESI-MS: (m/z): 498 (M þ H)þ.
A solution of 5-secbutyl-4-hydroxy-benzene-1,3-dicarbaldehyde
(0.5 g, 2.42 mmol), dimethyl malonate (0.32 g, 2.42 mmol) in
methanol (25 mL) was treated with piperidine (0.2 mL) and
refluxed. Most of the excess solvent was evaporated under reduced

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K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
6.4.2. Diethyl-4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-dicarboxylate
(5c)
126.3, 117.4, 116.2, 112.6, 105.1, 59.9, 52.7, 36.9, 36.3, 33.4, 29.5, 27.2,
21.1, 20.5, 19.1, 14.3, 11.8; ESI-MS: (m/z): 494 (M þ H)þ.
White solid, yield: 83%; mp 240e241 ꢀC; IR (KBr): 3011, 1677,
6.5.4. (ꢃ)Methyl-4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (5j)
1591, 1033 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.48 (s, 1H), 7.52 (d,
1H, J ¼ 1.8 Hz), 7.31 (d, 1H, J ¼ 2.0 Hz), 6.09 (s, 1H), 5.04 (s, 1H), 4.40
(q, 2H, J ¼ 7.1 Hz), 4.13e4.06 (m, 4H), 3.34 (q, 1H, J ¼ 7.0 Hz), 2.38
(brs, 6H), 1.69e1.64 (m, 2H), 1.40 (t, 3H, J ¼ 7.1 Hz), 1.26e1.20 (m,
9H), 0.83 (t, 3H, J ¼ 7.2 Hz); 13C NMR (CDCl3, 75 MHz): 167.4, 163.3,
157.3,155.7, 151.4,149.9,144.9, 144.5,134.4,132.8,126.2,117.6,116.9,
103.6, 103.5, 61.7, 61.5, 59.8, 39.3, 33.2, 29.6, 22.9, 20.5, 19.4, 14.3,
14.2, 13.8, 11.9; ESI-MS: (m/z): 526 (M þ H)þ.
White solid, yield: 86%; mp 229e230 ꢀC; IR (KBr): 3005, 1680,
1597, 1046 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.52 (s, 1H), 7.58 (s,
1H), 7.34 (d, 1H, J ¼ 1.7 Hz), 7.21 (s, 1H), 5.15 (d, 1H, J ¼ 5.3 Hz), 4.40
(q, 2H, J ¼ 7.1 Hz), 3.60 (s, 3H), 3.30 (p, 1H, J ¼ 6.6 Hz), 2.52 (brs, 2H),
2.40 (s, 3H), 2.34 (s, 2H), 2.04e1.95 (m, 2H), 1.71e1.63 (m, 2H), 1.40
(t, 3H J ¼ 7.1 Hz), 1.23 (t, 3H J ¼ 6.9 Hz), 0.84e0.77 (m, 3H); 13C NMR
(CDCl3, 75 MHz): 195.8, 167.7, 163.1, 157.6, 151.3, 150.3, 144.9, 134.6,
133.2, 126.1, 117.5, 116.5, 112.5, 104.7, 61.7, 51.0, 37.0, 36.1, 33.4, 29.4,
27.1, 21.1, 20.4, 19.1, 14.2, 11.8; ESI-MS: (m/z): 494 (M þ H)þ.
6.5. General synthetic procedure for preparation of (ꢃ)ethyl-4-(8-
sec-butyl-3-(methoxycarbonyl)-2-oxo-2H-chromen-6-yl)-2,7,7-
trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-carboxylate (5i)
6.5.5. (ꢃ)Ethyl-4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
To a solution of methyl-8-sec-butyl-6-formyl-2-oxo-2H-chro-
mene-3-carboxylate (1.0 mmol) (4d) in ethanol, ethyl acetoacetate
(1.0 mmol), 1,3-cyclohexadione (1.0 mmol), ammonium acetate
(3.0 mmol) and 3 mL acetic acid was added. The reaction mixture
was continued under reflux condition for 7e8 h. After completion
of reaction (monitored by TLC), the reaction mixture was cooled to
room temperature. The solid thus obtained was filtered and chro-
matographed over EtOAc:Hexane, provided compound (5i) in good
yield.
hexahydroquinoline-3-carboxylate (5k)
White solid, yield: 88%; mp 225e226 ꢀC; IR (KBr): 3011, 1686,
1590, 1045 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.50 (s, 1H), 7.56 (d,
1H, J ¼ 1.8 Hz), 7.37 (d, 1H, J ¼ 1.8 Hz), 6.75 (s, 1H), 5.13 (s, 1H), 4. 39
(q, 2H, J ¼ 7.1 Hz), 4.08e4.04 (m, 2H), 3.30 (p, 1H, J ¼ 6.6 Hz), 2.45 (s,
4H), 2.39e2.10 (m, 3H), 1.67e1.60 (m, 2H), 1.40 (t, 3H, J ¼ 7.1 Hz),
1.25e1.15 (m, 6H), 1.07 (s, 3H), 0.89e0.77 (m, 3H); 13C NMR (CDCl3,
75 MHz):195.6, 167.2, 163.1, 157.6, 151.3, 150.2, 149.3, 144.8, 134.7,
133.2, 126.2, 117.3, 116.6, 111.3, 105.1, 61.7, 59.9, 50.7, 40.7, 36.4, 33.4,
32.6, 29.7, 26.9, 20.5, 19.2, 14.3, 11.9; ESI-MS: (m/z): 536 (M þ H)þ.
Compounds 5dem was synthesized in a similar pathway.
6.5.1. (ꢃ)Ethyl 4-(3-(ethoxycarbonyl)-2-oxo-8-propyl-2H-
chromen-6-yl)-2,7,7-trimethyl-5-oxo 1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (5g)
6.5.6. (ꢃ)Ethyl-4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (5l)
White solid, yield: 75%; mp 238e239 ꢀC; IR (KBr): 3002, 1685,
1593, 1042 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 9.15 (s, 1H), 8.73
White solid, yield: 89%; mp 215e216 ꢀC; IR (KBr): 3003, 1681,
(s, 1H), 7.47 (s, 1H), 7.41 (s, 1H), 4.91 (s, 1H), 4.28 (q, 2H, J ¼ 6.6 Hz),
3.96 (q, 2H, J ¼ 6.8 Hz), 2.76e2.58 (m, 2H), 2.41e1.96 (m, 7H), 1.61e
1.51 (m, 2H), 1.30 (t, 3H, J ¼ 7.0 Hz), 1.12 (t, 3H, J ¼ 7.0 Hz), 1.01 (s,
3H), 0.89 (t, 3H, J ¼ 7.1 Hz), 0.84 (s, 3H); 13C NMR (DMSO-d6,
100 MHz): 195.6, 167.2, 163.3, 157.6, 151.8, 150.0, 148.8, 144.5, 144.1,
135.7, 130.1, 126.6, 117.3, 116.9, 111.5, 105.2, 61.8, 60.0, 50.8, 40.9,
36.4, 32.7, 31.3, 29.5, 27.1, 22.9, 19.4, 14.3, 13.9; ESI-MS: (m/z): 522
(M þ H)þ.
1597, 1040 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.52 (s, 1H), 7.59 (d,
1H, J ¼ 1.4 Hz), 7.35 (d, 1H, J ¼ 1.9 Hz), 7.27 (s, 1H), 5.15 (s, 1H), 4.41e
4.35 (m, 2H), 4.08e4.02 (m, 2H), 3.34e3.25 (m, 1H), 2.51 (s, 2H),
2.41 (s, 3H), 2.34e2.31 (m, 3H), 2.03e1.97 (m, 2H), 1.70e1.61 (m,
2H), 1.39 (t, 3H, J ¼ 7.1 Hz), 1.25 (t, 3H, J ¼ 6.8 Hz), 1.22e1.14 (m, 3H),
0.85e0.79 (m, 3H); 13C NMR (CDCl3, 75 MHz):195.8, 167.2, 163.1,
157.6, 151.2, 150.2,144.6,134.5,133.2,126.2,117.4, 116.5, 112.6,104.9,
61.7, 59.8, 37.1, 36.3, 33.4, 29.5, 27.2, 21.1, 20.5, 19.1, 14.3, 11.9; ESI-
MS: (m/z): 508 (M þ H)þ.
6.5.2. (ꢃ)Methyl-4-(8-sec-butyl-3-(methoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (5h)
6.6. General synthetic procedure for preparation of diethyl 4-(3-
(methoxycarbonyl)-8-methyl-2-oxo-2H-chromen-6-yl)-2,6-
dimethylpyridine-3,5-dicarboxylate (6a)
White solid, yield: 85%; mp 240e241 ꢀC; IR (KBr): 3006, 1694,
1594, 1044 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d: 8.55 (s, 1H), 7.59 (s,
1H), 7.34 (d, 1H, J ¼ 1.7 Hz), 7.24 (s, 1H), 5.15 (d, 1H, J ¼ 3.7 Hz), 3.92
(s, 3H), 3.60 (s, 3H), 3.35e3.26 (m,1H), 2.52 (brs, 2H), 2.39e2.34 (m,
5H), 2.03e1.88 (m, 2H), 1.66 (d, 2H, J ¼ 8.3 Hz), 1.25 (d, 3H,
J ¼ 6.8 Hz), 0.85e0.79 (m, 3H); 13C NMR (CDCl3, 75 MHz): 195.9,
167.7, 163.7, 157.6, 151.4, 150.9, 144.9, 134.7, 133.4, 126.1, 117.5, 116.1,
112.5,104.8, 52.7, 51.0, 37.0, 36.2, 33.4, 29.4, 27.1, 21.1, 20.4,19.1,11.8;
ESI-MS: (m/z): 480 (M þ H)þ.
To a solution of diethyl 4-(3-(methoxycarbonyl)-8-methyl-2-
oxo-2H-chromen-6-yl)-2,6-dimethyl-1,4-dihydropyridine-3,5-
dicarboxylate (1.0 mmol) (5a) with 2,3-dichloro-5,6-dicyano-1,4-
benzoquinone (DDQ) in the presence of tetrahydrofuran (THF) as
a solvent at room temperature to gave compound 6a. The reaction
mixture was continued under room temperature for 1 h. After
completion of reaction (monitored by TLC), the reaction mixture
solvent was evaporated under reduced pressure, and chromato-
graphed over EtOAc:Hexane, provided pure compound (6a) in good
yield.
6.5.3. (ꢃ)Ethyl-4-(8-sec-butyl-3-(methoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (5i)
Compounds 6aem were synthesized in similar pathway.
White solid, yield: 85%; mp 263e264 ꢀC; IR (KBr): 3013, 1679,
1599, 1016 cmꢂ1; 1H NMR (CDCl3, 300 MHz)
d
: 8.55 (s, 1H), 7.60 (d,
6.6.1. Diethyl4-(3-(methoxycarbonyl)-8-methyl-2-oxo-2H-
1H, J ¼ 1.8 Hz), 7.34 (d, 1H, J ¼ 2.1 Hz), 7.04 (s,1H), 5.15 (s,1H), 4.09e
4.01 (m, 2H), 3.93 (s, 3H), 3.35e3.27 (m, 1H), 2.50 (d, 2H, J ¼ 5.1 Hz),
2.41 (s, 3H), 2.35e2.32 (m, 2H), 2.05e1.88 (m, 2H), 1.68e1.63 (m,
2H), 1.26e1.16 (m, 6H), 0.86e0.79 (m, 3H); 13C NMR (CDCl3,
75 MHz): 195.9, 167.2, 163.8, 157.6, 151.4, 150.8, 144.6, 134.6, 133.5,
chromen-6-yl)-2,6-dimethyl pyridine-3,5-dicarboxylate (6a)
Oily; yield: 70%; IR (KBr): 3019, 1692, 1215, 768 cmꢂ1 1H NMR
;
(DMSO-d6, 300 MHz) d: 8.83 (s, 1H), 7.61 (s, 1H), 7.42 (s, 1H), 4.02 (q,
4H, J ¼ 7.0 Hz), 3.89 (s, 3H), 2.52 (brs, 6H), 2.39 (s, 3H), 0.91e0.86
(m, 6H); 13C NMR (DMSO-d6, 100 MHz): 166.6, 163.0, 155.6, 155.0,

K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
809
152.8, 148.6, 143.7, 134.7, 131.8, 127.1, 126.4, 125.3, 118.0, 117.3, 61.3,
NMR (DMSO-d6, 100 MHz): 196.7, 166.5, 163.3, 162.5, 157.6, 155.9,
148.6, 145.6, 134.5, 133.5, 129.3, 128.5, 126.6, 122.4, 117.6, 117.0, 61.2,
61.2, 52.7, 46.5, 32.0, 30.2, 27.7, 27.5, 22.8, 22.2, 14.0, 13.4; ESI-MS:
(m/z): 520 (M þ H)þ.
52.5, 22.5, 14.7, 13.4; ESI-MS: (m/z): 468 (M þ H)þ
6.6.2. Dimethyl 4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,6-dimethy lpyridine-3,5-dicarboxylate (6b)
White solid, yield: 73%; mp 96e97 ꢀC; IR (KBr): 3015, 1685,
6.6.8. Methyl 4-(8-sec-butyl-3-(methoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo 5,6,7,8-tetrahydroquinoline-3-
carboxylate (6h)
1200, 760 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.80 (s, 1H), 7.61
(s, 1H), 7.35 (s, 1H), 4.31 (q, 2H, J ¼ 7.0 Hz), 3.30e3.23 (m, 1H), 2.52
(brs, 6H), 1.66e1.59 (m, 2H), 1.31 (t, 3H, J ¼ 7.5 Hz), 1.22 (d, 3H,
J ¼ 6.8 Hz), 0.78 (t, 3H, J ¼ 7.5 Hz); 13C NMR (DMSO-d6, 100 MHz):
167.2, 162.5, 155.7, 155.3, 151.9, 148.5, 144.1, 134.2, 132.1, 131.0, 126.6,
126.3, 118.3, 117.6, 61.2, 52.4, 32.6, 29.0, 22.6, 20.2, 14.0, 11.6; ESI-
MS: (m/z): 496 (M þ H)þ.
White solid, yield: 72%; mp 130e131 ꢀC; IR (KBr): 3017, 1680,
1205, 771 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.74 (s, 1H), 7.48
(s, 1H), 7.36 (s, 1H), 3.84 (s, 3H), 3.43 (s, 3H), 3.34e3.22 (m, 1H), 3.14
(t, 2H, J ¼ 5.8 Hz), 2.59e2.50 (m, 5H), 2.12e2.06 (m, 2H), 1.66e1.61
(m, 2H), 1.22 (d, 3H, J ¼ 6.9 Hz), 0.79 (t, 3H, J ¼ 6.5 Hz); 13C NMR
(DMSO-d6, 75 MHz): 196.6, 167.1, 164.8, 163.0, 157.1, 155.8,
151.4, 149.1, 146.1, 133.8, 133.2, 131.8, 129.3, 126.3, 123.6, 117.2, 116.9,
52.4, 52.3, 32.8, 28.7, 22.8, 20.7, 20.3, 11.6; ESI-MS: (m/z): 478
(M þ H)þ.
6.6.3. Diethyl 4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,6-dimethyl pyridine-3,5-dicarboxylate (6c)
Oily, yield: 66%; IR (KBr): 3021, 1690, 1208, 770 cmꢂ1 1H NMR
;
(DMSO-d6, 300 MHz) d: 8.81 (s, 1H), 7.63 (s, 1H), 7.36 (s, 1H), 4.31 (q,
2H, J ¼ 7.0 Hz), 3.97 (q, 4H, J ¼ 6.8 Hz), 3.29e3.22 (m, 1H), 2.53 (brs,
6H), 1.65e1.61 (m, 2H), 1.31 (t, 3H, J ¼ 7.1 Hz), 1.22 (d, 3H, J ¼ 7.0 Hz),
0.87e0.77 (m, 9H); 13C NMR (DMSO-d6, 100 MHz): 166.6, 162.4,
155.6, 155.1, 148.4, 144.0, 134.2, 132.2, 131.2, 126.4, 117.5, 61.2, 32.8,
28.9, 22.5, 20.1, 14.0, 13.4, 11.7; ESI-MS: (m/z): 524 (M þ H)þ.
6.6.9. Ethyl 4-(8-sec-butyl-3-(methoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-
carboxylate (6i)
Oily, yield: 70%; IR (KBr): 3025, 1680, 1223, 765 cmꢂ1 1H NMR
;
(DMSO-d6, 300 MHz) d: 8.77 (s, 1H), 7.50 (s, 1H), 7.38 (s, 1H), 3.93e
3.84 (m, 5H), 3.27e3.20 (m, 1H), 3.14 (t, 2H, J ¼ 6.2 Hz), 2.57 (s, 2H),
2.51 (brs, 3H), 2.10e2.06 (m, 2H), 1.65e1.59 (m, 2H), 1.23 (brs, 3H),
0.84e0.77 (m, 6H); 13C NMR (DMSO-d6, 100 MHz): 196.7, 166.6,
164.8, 163.1, 157.1, 155.8, 151.8, 149.1, 146.0, 133.2, 132.2, 129.3, 126.5,
126.4, 123.6, 117.2, 117.0, 61.2, 52.4, 32.9, 28.9, 28.7, 22.7, 20.7, 20.3,
13.4, 11.6; ESI-MS: (m/z): 492 (M þ H)þ.
6.6.4. Ethyl 4-(3-(methoxycarbonyl)-2-oxo-8-propyl-2H-chromen-
6-yl)-2-methyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-carboxylate
(6d)
White solid, yield: 72%; mp 123e124 ꢀC; IR (KBr): 3013, 1683,
1219, 775 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.76 (s, 1H), 7.50
(s, 1H), 7.36 (s, 1H), 3.94e3.84 (m, 5H), 3.14 (t, 2H, J ¼ 5.4 Hz), 2.83e
2.69 (m, 2H), 2.57e2.51 (m, 5H),2.11e2.07 (m, 2H), 1.66e1.59 (m,
2H), 0.91 (t, 3H, J ¼ 7.1 Hz), 0.82 (t, 3H, J ¼ 7.2 Hz); ESI-MS: (m/z):
478 (M þ H)þ.
6.6.10. Methyl 4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-
carboxylate (6j)
White solid, yield: 75%; mp 158e159 ꢀC; IR (KBr): 3010, 1678,
6.6.5. Ethyl 4-(3-(methoxycarbonyl)-2-oxo-8-propyl-2H-chromen-
6-yl)-2,7,7-trimethyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-
carboxylate (6e)
1225, 774 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.71 (s, 1H), 7.48
(s, 1H), 7.35 (s, 1H), 4.30 (q, 2H, J ¼ 7.1 Hz), 3.43 (s, 3H), 3.34e3.24
(m, 1H), 3.14 (t, 2H, J ¼ 5.4 Hz), 2.59e2.50 (m, 5H), 2.12e2.07 (m,
2H),1.65e1.60 (m, 2H),1.31 (t, 3H, J ¼ 6.8 Hz),1.22 (d, 3H, J ¼ 7.2 Hz),
0.79 (t, 3H, J ¼ 6.8 Hz); 13C NMR (DMSO-d6, 75 MHz): 196.6, 167.1,
164.8, 162.5, 157.1, 155.8, 151.4, 148.7, 146.1, 133.8, 133.1, 131.7, 129.3,
126.4, 123.6, 123.5, 117.6, 117.0, 61.1, 52.2, 39.2, 32.8, 29.0, 28.7, 22.8,
20.7, 14.0, 11.6; ESI-MS: (m/z): 492 (M þ H)þ.
White solid, yield: 75%; mp 129e130 ꢀC; IR (KBr): 3011, 1676,
1210, 769 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.77 (s, 1H), 7.51
(s, 1H), 7.35 (s, 1H), 3.94e3.84 (m, 5H), 3.08 (s, 2H), 2.84e2.66 (m,
2H), 2.52 (brs, 5H), 1.66e1.59 (m, 2H), 1.06 (s, 3H), 1.04 (s, 3H), 0.91
(t, 3H, J ¼ 7.1 Hz), 0.83 (t, 3H, J ¼ 7.0 Hz); 13C NMR (DMSO-d6,
75 MHz): 196.7, 166.5, 164.8, 163.0, 157.1, 155.8, 151.9, 148.9, 145.8,
134.7, 133.7, 129.4, 128.5, 126.5, 123.5, 117.3, 116.9, 61.1, 52.4, 32.9,
30.2, 22.7, 22.2, 20.7, 13.3; ESI-MS: (m/z): 506 (M þ H)þ.
6.6.11. Ethyl 4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2,7,7-trimethyl-5-oxo-5,6,7,8-tetrahydroquinoline-
3-carboxylate (6k)
6.6.6. Ethyl 4-(3-(ethoxycarbonyl)-2-oxo-8-propyl-2H-chromen-6-
Oily, yield: 65%; IR (KBr): 3024, 1695, 1216, 758 cmꢂ1 1H NMR
;
yl)-2-methyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-carboxylate (6f)
(DMSO-d6, 300 MHz) d: 8.74 (s, 1H), 7.50 (s, 1H), 7.35 (s, 1H), 4.30 (q,
Oily, yield: 75%; IR (KBr): 3018, 1690, 1220, 773 cmꢂ1
;
1H NMR
2H, J ¼ 6.8 Hz), 3.91 (q, 2H, J ¼ 7.4 Hz), 3.34e3.24 (m, 1H), 3.08 (s,
1H), 2.52 (brs, 5H), 1.63e1.62 (m, 2H), 1.30 (t, 3H, J ¼ 7.0 Hz), 1.21 (d,
3H, J ¼ 6.5 Hz), 1.06 (s, 3H), 1.03 (s, 3H), 0.83e0.79 (m, 6H); ESI-MS:
(m/z): 534 (M þ H)þ.
(DMSO-d6, 300 MHz) d: 8.73 (s, 1H), 7.50 (s, 1H), 7.35 (s, 1H), 4.30 (q,
2H, J ¼ 6.7 Hz), 3.92 (q, 2H, J ¼ 5.0 Hz), 3.14 (s, 2H), 2.78e2.51 (m,
7H), 2.09 (brs, 2H), 1.66e1.56 (m, 2H), 1.31 (t, 3H, J ¼ 7.1 Hz), 0.91 (t,
3H, J ¼ 7.2 Hz), 0.82 (t, 3H, J ¼ 7.2 Hz); 13C NMR (DMSO-d6,
100 MHz): 196.7, 166.6, 164.8, 162.5, 157.1, 155.9, 151.9, 148.6, 145.8,
134.6, 133.7, 129.4, 128.5, 126.6, 123.5, 117.6, 117.0, 61.2, 61.2, 32.9,
30.3, 22.7, 22.2, 20.7, 13.4; ESI-MS: (m/z): 492 (M þ H)þ.
6.6.12. Ethyl 4-(8-sec-butyl-3-(ethoxycarbonyl)-2-oxo-2H-
chromen-6-yl)-2-methyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-
carboxylate (6l)
White solid, yield: 69%; mp 95e96 ꢀC; IR (KBr): 3028, 1676,
6.6.7. Ethyl 4-(3-(ethoxycarbonyl)-2-oxo-8-propyl-2H-chromen-6-
yl)-2,7,7-trimethyl-5-oxo-5,6,7,8-tetrahydroquinoline-3-
carboxylate (6g)
1209, 768 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.73 (s, 1H), 7.49
(s, 1H), 7.37 (s, 1H), 4.30 (q, 2H, J ¼ 7.0 Hz), 3.89 (q, 2H, J ¼ 7.5 Hz),
3.28e3.20 (m, 1H), 3.30 (t, 2H, J ¼ 5.5 Hz), 2.56e2.51 (m, 5H), 2.12e
2.06 (m, 2H), 1.65e1.60 (m, 2H), 1.31 (t, 3H, J ¼ 7.1 Hz), 1.21 (d, 3H,
J ¼ 5.4 Hz), 0.85e0.77 (m, 6H); 13C NMR (DMSO-d6, 50 MHz): 196.7,
166.6, 164.7, 162.5, 157.1, 155.8,151.5, 148.8,146.0, 133.9, 133.1, 132.0,
129.3, 126.4, 123.6, 117.5, 116.9, 61.2, 32.9, 29.0, 28.7, 22.7, 20.7, 20.1,
14.0, 13.4, 11.7, 11.5; ESI-MS: (m/z): 506 (M þ H)þ.
Oily, yield: 76%; IR (KBr): 3023, 1675, 1211, 778 cmꢂ1 1H NMR
;
(DMSO-d6, 300 MHz) d: 8.74 (s, 1H), 7.51 (s, 1H), 7.35 (s, 1H), 4.30 (q,
2H, J ¼ 7.3 Hz), 3.92 (q, 2H, J ¼ 6.5 Hz), 3.08 (s, 2H), 2.84e2.65 (m,
2H), 2.52 (brs, 5H), 1.69e1.56 (m, 2H), 1.31 (t, 3H, J ¼ 7.0 Hz), 1.06 (s,
3H), 1.04 (s, 3H), 0.91 (t, 3H, J ¼ 7.3 Hz), 0.83 (t, 3H, J ¼ 6.9 Hz); 13
C

810
K.V. Sashidhara et al. / European Journal of Medicinal Chemistry 70 (2013) 802e810
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White solid, yield: 68%; mp 145e146 ꢀC; IR (KBr): 3014, 1683,
1218, 772 cmꢂ1; 1H NMR (DMSO-d6, 300 MHz)
d: 8.76 (s, 1H), 7.58
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Acknowledgements
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The authors are grateful to the Director, CDRI, Lucknow, India for
constant encouragement in drug development program, S.P. Singh
for technical support, SAIF for NMR, IR, and Mass spectral data.
R.K.M., K.B.R. M.K. and D.C are thankful to CSIR, New Delhi, India for
financial support. This is CSIR-CDRI communication number 8559.
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Appendix A. Supplementary data
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