Benzofuran-dihydropyridine hybrids: A new class of potential bone anabolic agents

Article abstract of DOI:10.1016/j.bmc.2017.10.018

A series of novel benzofuran-dihydropyridine hybrids were designed by molecular hybridization approach and evaluated for bone anabolic activities. Among the screened library, ethyl 4-(7-(sec-butyl)-2-(4-methylbenzoyl)benzofuran-5-yl)-2-methyl-5-oxo-1,4,5,

Full text of DOI:10.1016/j.bmc.2017.10.018

Accepted Manuscript
Benzofuran-Dihydropyridine Hybrids: A new class of potential bone anabolic
agents
Ram K. Modukuri, Dharmendra Choudhary, Sampa Gupta, K. BhaskaraRao,
SulekhaAdhikary, Tanuj Sharma, Mohammad Imran Siddiqi, Ritu Trivedi,
Koneni V. Sashidhara
PII:
S0968-0896(17)31358-5
https://doi.org/10.1016/j.bmc.2017.10.018
BMC 14023
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
4 July 2017
4 October 2017
16 October 2017
Please cite this article as: Modukuri, R.K., Choudhary, D., Gupta, S., BhaskaraRao, K., SulekhaAdhikary, Sharma,
T., Siddiqi, M.I., Trivedi, R., Sashidhara, K.V., Benzofuran-Dihydropyridine Hybrids: A new class of potential bone
anabolic agents, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.10.018
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Benzofuran-Dihydropyridine Hybrids: A new class of potential bone anabolic agents
†
Ram K. Modukuri , DharmendraChoudhary
,
#
‡,
#
†
, Sampa Gupta , K. BhaskaraRao ,
†
‡
ɸ
ɸ
‡,
SulekhaAdhikary , Tanuj Sharma , Mohammad Imran Siddiqi ,RituTrivedi *and Koneni V.
†
Sashidhara *
,
†
‡
ɸ
Medicinal and Process Chemistry Division, Endocrinology Division, Molecular and
Structural Biology Division, CSIR-Central Drug Research Institute, (CSIR-CDRI), BS-10/1,
Sector 10, Jankipuram Extension, Sitapur Road, Lucknow, 226031, India
Phone: +91 9919317940; Fax: +91-518-2623405.
Correspondence and requests for materials should be addressed to K.V.S or R.T (e-mail:
sashidhar123@gmail.com, kv_sashidhara@cdri.res.in, ritu_trivedi@cdri.res.in)
th
Part 44 the series, “Advances in drug design and discovery”
#
These authors contributed equally

Abstract
A series of novel benzofuran-dihydropyridine hybrids were designed by molecular
hybridization approach and evaluated for bone anabolic activities. Among the screened
library,
,4,5,6,7,8-hexahydroquinoline-3-carboxylate (compound 21) significantly enhanced the
ALP production and mineralized nodule formation, which are primary requisites in the
ethyl
4-(7-(sec-butyl)-2-(4-methylbenzoyl)benzofuran-5-yl)-2-methyl-5-oxo-
1
-
1
-1
process of in-vitro osteogenesis. Oral administration of compound 21 at 10 mg.kg day for
two weeks led to restoration of trabecular bone microarchitecture in drill hole fracture model
by significantly increasing BV/TV and Tb.N. Furthermore, histological and molecular studies
showed compound 21 triggering the new bone regeneration in a drill hole defect site by
increasing BMP expression. Furthermore, molecular modeling studies were performed to
gain insight into the binding approach, which revealed that both benzofuran and
dihydropyridine moieties are essential to show similar binding interactions to fit into the
active site of BMP2 receptor, an important target of the osteogenic agents.Our results suggest
that compound 21 stimulates BMP2 synthesis in osteoblast cells that promotes new bone
formation (~40%) at the fracture site which helps in shorten the healing period.
Keywords: Benzofuran-dihydropyridine hybrids;Alkaline phosphatase; Bone morphogenic
protein-2; Molecular hybridization.
Abbreviation: Bone morphogenic protein-2 (BMP2), alkaline phosphatase (ALP), 3-(4, 5-
dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT), Quantitative Real-Time
Polymerase Chain Reaction (qRT-PCR), Runt-related transcription factor 2(RUNX2),
Alkaline phosphatase (ALP), Collagen type-I (COL1), Osterix(Osx), Trabecular bone
volume/tissue volume (BV/TV), Trabecular number (Tb.N).

1
. Introduction
Osteoporosis is a silent skeletal disease that weakens the bone microarchitecture and
increases the risk of fragility. It has been recognized as a global health threat and is more
prevalent in aged population, mainly postmenopausal women [1]. This disease is initiated
because the process of bone resorption overtakes that of new bone formation [2].
Epidemiological studies have estimated that ‘‘by the year 2015, osteoporosis will be
responsible for approximately 3 million fractures and $ 25.3 billion in costs each year’’ in the
USA [3,4]. At clinical level it is mainly characterized by low bone mineral density and
deterioration of the microarchitecture of bone tissue, thereby increasing the risk of fractures
[
5]. During the post menopause in elderly women, estrogen deprivation enhances osteoclast
recruitment and contributes to rapid skeletal loss and bone fragility [6, 7]. Current clinical
approaches for osteoporosis fall under two categories, namely anti-resorptive and anabolic
agents. Anti-resorptive medications includeestrogens, hormone replacement therapy (HRT),
selective estrogen receptor modulators (SERMs), bisphosphonates, and antibody therapy
(
calcitonin and denosumab) [8]. Although their sites of action may differ, these medications
reduce bone loss by decreasing osteoclastic bone resorption [9]. However, most of the
medications available in this class are associated with serious side effects (nausea,
esophageal, osteonecrosis of the jaw and cancer) and unsatisfactory results [10]. Anabolic
agents which stimulate the osteoblastic bone formation and increase the bone mineral density
have recently widened the therapeutic options [11]. Teriparatide (a recombinant version of
human Parathyroid hormone (PTH)) is the only FDA-approved anabolic agent which reverses
the disease by stimulating bone synthesis [12]. However, because of long-term safety and
efficacy are unknown, it is prescribed for only two years. Main limiting factors of PTH
therapy are its compliance (parenteral route of administration) and development of

osteosarcoma at higher doses, thereby highlighting the need for alternate bone anabolic
agents [13,14].
Bone morphogenetic protein 2 (BMP2) is mainly synthesized by bone forming osteoblast
cells and it stimulates osteoblasts differentiation, bone generation and regeneration at
defecated site [15]. Animal studies confirmed the positive role of recombinant BMP2 in
fracture healing [16-17]. In this direction synthesise of BMP2 up regulator like novel
molecules may provide new therapeutic agents for the treatment of fracture healing studies.
In current drug discovery, anabolic strategy is an attractive concept for the discovery of new
agents to advance the therapy of osteoporosis [18].With this new paradigm, there is a
tremendous opportunity to make a significant positive impact on the health and lives of
osteoporotic people through the discovery and development of new bone anabolic agents, that
enhances both new bone formation (rapid fracture healing property) and also increase the
bone mineral density with less adverse effects.
Benzofuran and their derivatives have been found to exhibit different biological and
pharmacological activities like anti-tubercular, antibacterial, anti-fungal, anti-inflammatory,
anti-depressant etc [19-20]. Owing to their beneficial effects on bone health, they have been a
subject of further studies [21-23]. Benzofuran derivatives were reported to exert beneficial
effects in the prevention of bone loss (Figure 1A) [24]. Interestingly, Kumar et al.
demonstrated that benzofuran containing non-peptide inhibitor SB-462795 prevents bone loss
in both in-vitro and in vivo animal models (Figure 1B) [25]. Recent remarkable work of
Guoet al. have established the potential of 2-substituted benzofuran derivatives as anovel
class of bone anabolic agents that were able to up-regulateBMP2 expression in
ovariectomized rats (Figure 1C) [26]. In the field of anabolic therapy a small molecule which
enhances bone strength and the growth factor could be crucial for the treatment of the
osteoporosis. Hence, 2-substituted benzofuran structural moiety was incorporated as one of

the pharmacophoricpartners in our hybridization approach. However, several reports suggest
that calcium channel blockers containing dihydropyridine in their molecular makeup
stimulate the osteoblast differentiation and have a beneficial effect by reducing fracture risk
[
(
27-30]. Interestingly, the studies by Wang et al. have suggested that this class of compounds
benidipine) promoted cell proliferation and exhibited osteogenic differentiation at potentially
lower concentrations (Figure 1D) [31]. Furthermore, Ushijima et al. have revealed the
protective role of amlodipine against osteoporosis in hypertensive rats (Figure 1E) [32]. We
have previously developed coumarin-containingdihydropyridine hybrids as a new class of
bone anabolic agents, which also underscores the importance of dihydropyridine scaffold
against osteoporosis (Figure 1F) [33].
Small molecules, which can modulate relevant multiple targets can enhance efficacy and
emerge as a better therapeutic candidate. [34-39]. In this study, following the medicinal
chemistry hybridization approach a series of compounds belonging to the prototype
incorporating pharmacophoric features of benzofuran and 1, 4- dihydropyridinewere
synthesized and evaluated in vitro and in vivo for their intended activity. Figure 1 shows
representative potent inhibitors that either contains benzofuran or dihydropyridine in their
molecular makeup, along with the structure of our designed prototype.

Figure 1: Strategy employed for the design of benzofuran-1,4-dihydropyridine hybrids

2
. Results and Discussion
2
.1 Chemistry
Interestingly, the anti-osteoporotic potential of benzofuran based 1, 4-dihydropyridines has
never been explored. The structural novelty and pharmacophoric importance led us to
synthesize 5-substituted benzofuran based hybrids from the benzofurancarbaldehyde key
intermediate, which in turn was prepared from our standard dicarbaldehyde substrate using
established protocol [40].The synthetic strategy followed is depicted in scheme 1. Initially,
the Duff formylation on ortho-substituted phenol (1-3) in the presence of
hexamethylenetetramine (HMTA) and trifluoroacetic acid (TFA) at 120 °C gave the
corresponding aromatic dicarbaldehydes (4-6) [41]. Next, the Rap-Stoermer condensation of
2 3
the intermediates with distinct phenacyl bromides in the presence of K CO provided
benzofurancarbaldehyde derivatives (7-12) in considerable to good yields [42]. Further, these
benzofuran derivatives were subjected to the Hantzsch reaction, involving benzofuran
aldehydes, active methylene ethyl/methyl acetoacetate, different 1,3-cyclohexadiones, and
ammonium acetate (nitrogen donor) in the presence of glacial acetic acid gave the desired
benzofuran-1, 4-dihydropyridine hybrids (13-32) in good yields (scheme 1a), while 1,4-
dihydropyridine derivatives (33, 34) were synthesised by the Hantzsch synthesis on
benzaldehyde, ethyl acetoacetate and different 1,3 cyclohexadiones (scheme 1b).

Scheme 1. (a) Synthesis outline of benzofuran-1, 4-dihydropyridine hybrids. (b) Synthesis outline of
1
, 4-dihydropyridine derivatives
O
R2
OH
OH
R1
CHO
O
R1
(i)
ii)
(iii)
R1
(
CHO
1
-3
CHO
4-6
7
-12
R1
1
2
3
, 4
, 5
, 6
CH CH2
3
(iv)
-butyl
-butyl
O
R2
R1
R2
O
R1
O
7
8
9
CH3CH2
OCH3
-butyl OCH3
-butyl
-butyl
-butyl
CH3
Cl
NO2
O
1
0
R3
1
1
1
2
O
-butyl OCH3
R4
R4
N
H
1
3-32
R1
R2
R3
R4
R1
R2
R3
R4
CH CH2 OCH3 CH3
CH3
H
-butyl Cl
-butyl Cl
-butyl Cl
CH3
H
H
CH3
3
CH CH2 OCH3 CH CH2
CH CH2
3
3
3
CH CH2 OCH3 CH CH2 CH3
CH3
3
3
-
-
butyl OCH3 CH CH2
butyl OCH3 CH3
H
H
-butyl NO2 CH3
H
H
3
-butyl NO2 CH CH2
3
-
-
butyl OCH3 CH3
CH3
-butyl NO2 CH3
-butyl OCH3 CH3
CH3
H
butyl OCH3 CH CH2 CH3
3
-
-
butyl CH3
butyl CH3
CH3
H
H
CH3
-butyl OCH3 CH CH2
-butyl OCH3 CH3
H
CH3
3
CH CH2
3
-
butyl CH3
CH3
-butyl OCH3 CH CH2 CH3
3
O
O
R'
(
i)
H
CH3
O
CHO
R'
R'
N
H
Benzaldehyde
1,4-Dihydropyridine
Reagents and conditions (a):(i) HMTA, TFA, 120 °C, 4 h. (ii) aq H
bromides, K CO /CH CN, 110 °C, 3 h, (iv) Ethyl/methyl acetoacetate, different 1,3-cyclohexadiones, NH
EtOH, reflux, 5 h. (b):(i) Ethyl acetoacetate, different 1,3-cyclohexadiones, NH OAc, AcOH, EtOH, reflux, 5 h.
2
SO
4
, 100 °C, 2 h (iii) Appropriate substituted phenacyl
2
3
3
4
OAc, AcOH,
4

2
.2. ALP and mineralization activity in primary osteoblasts
Bone specific alkaline phosphatase (ALP) activity represents the proliferation of osteoblasts
during bone formation. It is widely recognised biomarker to identify the lead candidate in a
hit to lead optimization. We screened the racemic hybrids 13 to 32, as well as their precursors
(
benzofurans) (7 to 12), and dihydropyridines (33 and 34) for stimulation of bone alkaline
phosphatase (ALP) production in rat calvarial osteoblasts cells at different concentrations.
Among the benzofuran and dihydropyridine parent compounds 7,8, 9,
and33showedsignificant activity at 10 nM and 100 pM which was comparatively less
effective than the newly synthesized hybrid compounds. Compound 21 exhibited robust ALP
activity at the lowest concentrations of 1 pM whereas, compound 12, 14 and 16 exhibited
activity at 10 nM concentration compared to that of control. Among all screened compounds,
eight compounds i.e, 7, 8, 9, 12, 14, 16, 21 and 33 were found to stimulate the ALP activity
significantly (Table 1) and these compounds with their respective active concentration were
selected for mineralization assay.
Table 1.Alkaline phosphatase (ALP) activity of all the screened compounds at different
a
concentrations in calvarial osteoblast cells
Compound
Control
1 pM
100 pM
10 nM
1 µM
100 µM
7
8
9
1.01±0.05
1.03±0.12
1.01±0.07
1.06±0.07
1.06±0.06
1.21±0.07
1.11±0.09
1.04±0.10
1.12±0.28
1.03±0.07
1.09±0.09
1.35±0.01
1.23±0.07
0.98±0.06
1.04±0.11
1.33±0.10
1.26±0.25
1.32±0.19
1.24±0.20
0.94±0.09
1.11±0.06
1.49±0.10*
1.16±0.07
0.78±0.05
0.66±0.06
1.50±0.16
0.73±0.08
1.49±0.15
1.45±0.23
0.99±0.07
1.24±0.03*
1.59±0.12**
1.41±0.11**
0.96±0.06
0.73±0.08
1.70±0.19
1.19±0.14
1.64±0.20*
1.65±0.28
1.40±0.13*
1.03±0.05
1.21±0.12
1.42±0.09**
0.77±0.05
1.08±0.09
1.26±0.05
1.20±0.15
1.80±0.15**
1.72±0.26
1.37±0.08*
0.29±0.01
0.58±0.08
0.30±0.01
0.26±0.01
0.31±0.01
0.59±0.02
0.18±0.01
0.41±0.04
0.50±0.16
0.25±0.01
1
1
1
0
1
2
1
3
1
1
1
4
5
6

1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
1.02±0.09
1.06±0.09
1.06±0.11
1.07±0.04
1.0±0.03
1.37±0.08
0.97±0.09
1.08±0.04
0.99±0.07
1.14±0.03*
1.08±0.05
1.34±0.13
0.64±0.07
0.82±0.05
1.12±0.05
1.32±0.07***
1.16±0.08
1.48±0.20
1.02±0.07
0.85±0.04
0.72±0.06
0.82±0.07
1.03±0.06
1.01±0.12
1.14±0.20
1.15±0.13
1.53±0.14
1.25±0.07
1.41±0.26
1.09±0.12
1.14±0.12
1.30±0.13
1.17±0.07
1.19±0.03**
1.11±0.05
1.34±0.10
1.04±0.09
0.86±0.09
0.82±0.03
0.89±0.04
1.06±0.04
1.07±0.15
1.17±0.15
1.10±0.11
1.47±0.20
1.43±0.1*
1.41±0.16
1.34±0.13
1.26±0.09
1.13±0.12
0.84±0.05
1.16±0.02*
1.13±0.08
1.31±0.12
0.79±0.06
0.23±0.01
0.82±0.06
0.69±0.04
0.76±0.03
1.17±0.15
1.77±0.28
1.25±0.29
1.15±0.17
1.44±0.10*
1.23±0.12
0.37±0.03
0.29±0.03
0.51±0.11
0.25±0.01
0.36±0.01
0.37±0.01
0.50±0.01
0.31±0.01
0.23±0.01
0.25±0.01
0.37±0.01
0.30±0.01
0.32±0.03
0.29±0.04
0.36±0.03
0.36±0.04
0.74±0.03
0.58±0.03
1.05±0.05
1.09±0.08
1.02±0.08
1.03±0.07
1.01±0.08
1.02±0.04
1.04±0.05
1.14±0.13
1.04±0.23
1.07±0.14
1.06±0.14
1.07±0.08
1.1±0.14
3
9
1. ±0.07
0.90±0.05
1.08±0.11
0.65±0.05
1.12±0.14
0.98±0.03
1.19±0.07
1.14±0.13
0.79±0.11
1.04±0.7
1.26±0.11
1.03±0.15
Data Values are expressed as Mean ± SEM. (n = 8).*p< 0.05, **p< 0.01 and ***p< 0.001 as compared with
untreated cells taken as control only when ALP activity was higher.
Mineralization of bone is essential for bone hardness, strength and involves a well-organized
process in which crystals of calcium phosphate is developed by bone-forming cells thatlay
down in precise amounts within the bone matrix. In mineralization, we found that compounds
1
6 and 14 exhibited significantly higher mineralization at 10 nM and 1 µM concentrations
respectively. Interestingly, compound 21 showed greater mineralizing ability at all
concentrations ranging from 100 pM to 10 nM. Although, highest mineralizing effect of
compound 21as compared to other compounds were observed at 100 pM concentration
(
Figure 2).This experimentnoticeably demonstrated that benzofuran-dihydropyridine hybrids
were found to be more potent than their individuals.

Figure 2 :- Mineralizing potential of compounds 8, 9, 14, 16 and 21 as compared to the
control (non-treated) cells in calvarial osteoblasts cells after 21 days of treatment in growth
medium. Data represented as (n=8)Mean ± SEM. *p< 0.05, **p< 0.01 and ***p< 0.001
compared to controls.
2
.3. Transcriptional analysis of osteogenic genes
We next assessed the effect of active compounds on osteogenic genes at the molecular level
and the expression of alkaline phosphatase (Alp), osterix (Osx), collagen type-1 (Col1), runt-
related transcription factor 2 (Runx2) and bone morphogenic protein-2 (Bmp2), regulating
bone differentiation were assessed by qRT-PCR. 48-hour treatment revealed that the potent
hybrids effectively increased the expression of osteogenic genes ranging from ∼2.0 to ∼5.5-
folds over the control cells (Figure 3). At 10 nM concentration, compound 21 demonstrated
significantly increased expressions of Col1, Runx2 and Bmp2 genes that were in the order of
3
.7, 3.4 and 3.1 folds respectively. However, 100 pM concentration of compound 21
exhibited maximum expression of all tested osteogenic genes; Alp, Osx, Col1, Runx2 and
Bmp2 osteogenic genes approximately 4.5, 3.7, 5.0, 3.9, and 4.5 folds respectively (Figure

3
). This study shows that benzofuran-dihydropyridine hybrid compound 21 have potential to
up regulate the expression of various key osteogenic genes.
Figure 3 : Effect of compounds 9, 14, 16 and 21 on the expression of osteogenic genes (Alp,
Osx, Col1, Runx-2and Bmp2) in calvarial osteoblasts as compared to control by qRT-PCR
(n=5). Data represented as n=5 Mean ± SEM. *p< 0.05, **p< 0.01 and ***p< 0.001
compared to control.
2
.4. BMP-2 elisa experiment
The synthesized compound 21(100 pM) was evaluated by BMP2 Elisa assay. After treatment
with the compound 21, BMP2 expression was up-regulated (p<0.01) as compared to control
group. From this study, it is evident that leads compound 21 increased expression of BMP2 in
bone cells which plays an important role in osteoblast differentiation and bone regeneration
(
Figure 4).

Figure 4: BMP2 elisa after 48 hours treatment. incalvarial osteoblasts as compared to
control. Data represented as n=5 Mean ± SEM. **p< 0.01 compared to control.
2
.5. Cell viability study
Preliminary cytotoxicity of the compound 21 by MTT assay was studied in calvarial
osteoblasts cells. Data from the MTT assay suggested that compound 21 did not exhibit any
toxicity to the cells at concentrations ranging from 1 pM to 1 µM compared to the control
cells (Figure 5) corresponding to a significantly lower value of the ALP activity, compound
2
1 at 100 µM showed a cytotoxic effect on the osteoblast cells. From MTT studies we
concluded that compound 21 was safe for osteoblasts cells and benzofuran-dihydropyridine
hybrids could be novel agents for bone cells.The potential of compound 21 was also
corroborated by its EC50 value (Supporting Table S1) and western blot analysis of
osteogenic marker protein expression of RUNX2, BMP2 and COL1 (SupportingFig. S2)
.

Figure 5 : Cells were cultured in differentiation medium and treated with various
concentrations of the compound ranging from 1 pM to 100 µM for 24 h and cell viability
assessed by MTT assay. Data represented as n=8 Mean ± SEM. compared to control.
2
.6. Subcutaneous injection of compound 21 for selecting best possible dose
On the basis of promising in vitro potency and the expression of osteogenic genes compound
1was selected for further in in-vivoassessment for osteogenesis. Compound 21 was
administrated subcutaneously to skull region in 2 days old pups at doses of 0.1, 0.5, 1.0, and
2
-
.0, 10.0 and 20 mg.kg day following three consecutive days. After 3 days injection pups
1
-1
5
were sacrificed and calvarial tissue was isolated for mRNA expression studies of osteogenic
marker genes Runx2 and Bmp2. Overall, on the basis of qRT-PCR data out of 6 doses of
-
1
compound 21, we selected 1.0, 5.0 and 10 mg.kg day doses for in-vivo studies. On the basis
-1
-1
-1
of qRT-PCR data, we found that 1.0, 5.0, and 10 mg.kg day doses showing good response
-1
-1
but maximum responses of Runx2 and Bmp2 was shown by compound 21 at 10 mg.kg day
-
dose. Further compound 21 had no significantly osteogenic responses and above10 mg.kg
1
-1
day dose shows no dose-dependent response as represented in figure (Figure6).Therefore,

we took compound 21 and its active doses for further detailed in vivo evaluation in drill hole
defect rat models.
Figure 6 : Subcutaneous injection of compound 21 for selecting best possible doses. Data
represented as n=5 Mean ± SEM. *p< 0.05, **p< 0.01 and ***p< 0.001 compared to control.
2
.7. Effect of the compound 21 in regeneration of bone at fracture site
In order to quantitatively determine the bone healing process, we studied the effect of
compound 21 on rapid fracture healing in the 0.8 mm drill hole defect fracture model in rats.
After creation of 0.8 mm defect in femur bones of rodents we started treatment the next day
-1
-1
with compound 21at 10 mg.kg day dose for 14 days regularly. Representative images of
rapid fracture healing at different doses in Figure7a, upper panel shows 3D images and lower
panel shows 2D images. Data shows that compound 21 had higher bone volume/trabecular
volume (BV/TV), trabecular thickness (Tb.Th), connection density (conn.D) and trabecular
number (Tb.N) compared to the vehicle treated control group (Figure 7b-7e).These data
indicated that compound 21had significant effects that could restore bone-

microarchitecturewithpositive effects on bone formation parameters by expansion of new
bone forming cells at the drill-hole injury site.
Figure 7 :Figure shows effect of compound 21 on drill hole bone defect. Figure a shows 3D
and lower panelshows 2D images of 0.8 mm size defect, which was made at 2 cm above the
knee joint of distal femur side. Figure c-f shows µ-CT analysis at defect site of the control
and compound 21 treated groups. Lead compound 21 treatments had better micro
architectural at defect site bt improving callus regeneration. (b) µ-CT analysis showing
-1
-3
BV/TV (%), (c)Tb.Th (mm), (d)Tb.N (mm ), (e)Conn.D, (mm ). All values are expressed as

#
mean ± SEM (n = 6 rats/group); *p< 0.05; **p< 0.01; ***p< 0.001 compared to 0 day and p
#
0.05, p < 0.01, p < 0.001 compared with 14 day of control group.
#
###
<
Data revealed that the lead compound increased the bone healing by enhancing callus
regeneration at defect site. Histological changes with compound 21 showimproved callus
regeneration. Enhanced callus regeneration at 50 µm size section of femur bone was observed
(
(
Figure8a), this finding was further validated by intensity of calcein labeling at defect site
Figure8b) and H&E staining of the callus (Figure8c). Analysis of mean intensity of calcein
at the defect site by confocal microscope shows that treatment of compound 21 at a dose of
-
1
-1
0 mg.kg day increased mineral deposition by enhancing callus regeneration in the defect
1
site. Data of calcein labeling intensity showed that new bone formation was significantly
-1
-1
improved ~40% (p< 0.01) at a dose of 10 mg.kg day treatment group compared to control
(
Figure 8d). Further, checked the qRT-PCR expression of Bmp2 at defect site and found that
compound 21 significantly increased the Bmp2 expression (Figure 8e). From these studies, it
conclude that compound 21 stimulates BMP2 production in bone forming cells that promotes
bone formation at defect site. Benzofuran-dihydropyridine hybrid compound 21has efficiency
to upsurge fracture healing process as compare to their respective untreated control.
Therefore compound 21 could be an attractive strategy to enhance fracture healing to shorten
the healing period.

Figure 8 : Lead compound 21 treatments promote rapid fracture healing that was measured
by calcein labeling intensity. (a) Representative images of bones with fracture healing of
5
0µm section sizes (4×). (b) Representative images of calcein labeling at defect site by
confocal (20×) after two weeks of treatments. (c) Representative images of callus after H&E
staining (10×). (d) Quantification of the mean intensity of calcein label per pixel.
(e)Quantitative expression of Bmp2 by qRT-PCR at defect site in control and treated group.
All values are expressed as mean ± SEM (n = 6 rats/group); *p< 0.05; **p< 0.01; ***p<
0
.001 compared to control group.
2
.8. Effect of compound 21 on liver histomorphology for in vivo toxicity
The safety assessment of the compound 21 was also carried out in liver tissue. Treatment
with this compound orally for 14 days on rats was done and at the end of treatment livers
from different groups of rats was collected and a histological analysis was performed using 5
µm thick sections. The liver histomorphology of the treated and untreated animals were
similar and no morphological alterations were observed (Figure9).This study suggested that
compound 21 to be safe and devoid of liver toxicity.

Figure 9 : Sections of livers (20x) of SD rat after 12 weeks of lead compound 21 treatment with
-
different doses (10 mg kg dayorally) and different control groups shows no significant changes in
1
-1
liver histology. Section observed from liver of 5 animals in each group
2
.9. Structure-activity relationship (SAR) study
SAR was inferred from their activities in ALP and mineralization assay. For clear
understanding of SAR, we have divided the hybrid molecules into northern hemisphere and
southern hemisphere (Figure 10). In northern hemisphere, compounds containing methoxy
and methyl substituted benzofuran compounds 7, 8, 9, 14, 16 and 21 were active for ALP and
activity and compounds 14, 16 and 21 were active for mineralization. At C-7 position sec-
butyl group (8, 9, 16 and 21) and ethyl group (7, 14) are more preferred than tert-butyl group
(
29, 30, 31 and 32), for activity. Compounds containing electron withdrawing groups like
chlorine and nitro (10, 11, 23, 24, 25, 26, 27 and 28) were not favorable for the ALP activity.
It is important to mention that though in the initial ALP activities benzofuran parent
compounds 7, 8, 9 and 12 (non-dihydropyridine compound) showed significant activity, the
presence of toxicophore (free aldehyde group) precluded us to proceed with this class and
thus we proceeded to synthesize our designed hybrids that brought this free aldehyde into the
reaction. In the case of southern hemisphere, compounds containing the
unsubstitutedcyclohexanone and ethyl ester groups were found to be more potent (14, 16 and
2
1) than compounds containing substituted cyclohexanone and methyl esters (13, 15, 18, 19

and 22). Interestingly, incorporation of the dihydropyridine moiety enhances activity in some
cases (14, 16 and 21).
Figure 10:- SAR of the synthesized hybrids
3
. Molecular modelling studies
Docking analysis was done to find the molecular interactions of active hybrid 21 on BMP-2.
Due to the dimeric nature BMP-2 protein, dimeric structure of 3BK3 was used for interaction
studies as functionality [43-45]. For the agonist crucial binding pocket was identified by the
MetaPocket 2.0 as a putative binding site and was docked using AutoDock 4.2 [46].The
binding pocket (Site-1) identified by the MetaPocket 2.0 tool was a deep seated cavity and
was different from the binding sites of the 4 receptors (Figure 11a, 11b) as suggested by
previous mutational studies [47]. Compound 21 is racemic and to get a preliminary insight
we performed interactions studies for the major two isomers. For compound 21(S) docking
studies revealed a large cluster of 11 poses having lowest Kb (binding constant) of 6.96 µM
and binding energy-7.05 kcal/mol. Compound 21(S) at this pose shows hydrogen bond of

1
.76 Ǻ with amide group of Cys79, 2.07 Ǻ and 2.67 Ǻ with thiol group of Cys78, 3.24 Ǻ
forming hydrogen bond with amide of Cys47 (BMP2 of chain-A) and 2.03Ǻ forming
hydrogen bond with imidazole ring of His44 (BMP2 of chain-B) by its keto group of
benzofuran ring (Figure 11d). For Compound 21(R), large cluster of 18 poses with lowest Kb
1
0.58 µM and binding energy -6.79kcal/mol was observed at same binding pocket. At this
pose, compound 21(R) indicated possibility of hydrogen bond of 2.20Ǻ with amide of Cys47
BMP2 of chain-A) by its keto group of benzofuran ring; 1.92Ǻ with imidazole ring of His44
and 2.26Ǻ with carbonyl of Glu47 (BMP2 of chain-B) (Figure 11e).
(

Figure 11: Figure 11a indicates the surface view model of dimer BMP2 along with the bound
inhibitory CV-2 peptide (dark gray). The binding site predicted by MetaPocket 2.0 (Site-1)
which was utilized for docking was different from this inhibitory peptide. Figure 11b
indicated the dimer of BMP2 protein with BMPR-1 (green) having PDBid-2QJA.
Interestingly it was observed that the interaction site of BMPR-1 to BMP2 is different from
Site-1 predicted by MetaPocket 2.0. Figure 11c indicating slight variation in the interaction of
compound 21(S) w.r.t. compound 21(R) at same binding pocket. Figure 11d and 11e
indicating molecular interaction of compound 21(S) (green) and compound 21(R) (yellow)
with BMP2 protein respectively.

Docking results (Table 2) and experimental results are in agreement with respect to the order
of activity of compounds 21, 16, 14, 9, 8 and DHP-1. Binding studies indicated that presence
of ethyl ester group of dihydropyridine ring imparts the compound highest activity due to
hydrophobic interaction, followed by methyl ester group. Methyl substituent at para-position
of benzene ring has lower Kb value than methoxy group as presence of methoxy group
destabilizes hydrophobic interaction. Interaction studies do indicate the importance of
backbone amide (Cys47 and Cys79) and carbonyl groups (Glu47) that are completely buried
in the hydrophobic contact region as major binding determinants. Also, it has been observed
that presence of keto group of benzofuran ring imparts more activity to agonist possibly by
forming strong hydrogen bonds. Furthermore, molecular modelling studies that were
performed to gain insight into the binding mode revealed that both benzofuran and
dihydropyridine moieties are essential to show similar binding interactions to fit into the
binding site of BMP2 lead compound. Computational studies have indicated that this deep
seated cavity in the dimerfunctions as binding site for our lead compounds. The increased
level of BMP-2 in presence of our lead compounds(observed by Western blot assay) possibly
indicate that our lead compounds may stabilize the BMP-2 dimer. This stabilized dimer, as
computational studies suggest may still be free for interaction with BMPR-1 and BMPR-2
simultaneously and form hetero-tetrameric complex [48-51]. Molecular modelling studies not
only revealed the structural insights of the potent hybrid, but also inspired us to synthesize
enantiomerically pure compounds, which will be our future priority.
Table 2.Computational data docking results: Binding energy values of active compounds
Name of
Compound
Binding Energy
(Kcal/mol)
Kb Value
(µM)
Number of poses in
cluster
2
1(R)
1(S)
6(R)
6(S)
-6.79
-7.05
-5.89
-6.93
10.58
6.78
18
11
16
14
2
1
48.16
8.31
1

1
4(R)
-6.35
-6.36
-5.50
-5.71
-5.52
-5.46
21.84
21.65
92.57
65.38
89.54
98.95
13
13
25
28
36
13
1
4(S)
9
8
3
3(R)
3(S)
3
Legends: The table summarizes the binding energies of various compounds along with BMP-2 dimer along with
the number of poses observed in the cluster.
4
. Conclusion
In our studies, compound 21 was found to significantly stimulate BMP2 and osteoblast
differentiation from a series of structurally similar compounds of benzofuran-dihydropyridine
hybrids. Compound 21 was found to increase alkaline phosphatase activity, which is the
essential step in the process of evaluation of osteogenic compounds. It also enhanced
osteoblasts by improve mineralization activity in extra cellular matrix.It also enhanced the
mRNA expression of osteogenic markers genes expression of RUNX2, BMP2 and COL1 that
are the hallmark in process of new bone formation acts as a factor promoting osteoblast
function. BMP2 plays an important role in fracture healing by stimulation and differentiation
of mesenchymal progenitor cells in osteoblasts at fracture site. In this direction compound 21
increased BMP2 formations. Further, we studied the in-vivo efficacy of compound 21 in a
drill-hole fracture (defect) model for rapid bone healing process in femoral bone. Data
showed that compound 21 triggers the regeneration and healing properties in bone compared
to the vehicle treated group. From by µCT analysis data, it is can be verified that oral
administration of lead compound 21mitigatesmicro-architectural loss of bone tissue by
increasing trabecular number and bone volume that indicate recovery of trabecular bone
-1
-1
micro-architecture with better connectivity at 10 mg.kg day dose in drill hole rats. These in-
vitro and in-vivo results suggested that the compound 21 stimulate new bone formation at
fracture site by increasing BMP2 production that leads to improve in structure of bone

trabecular bone microarchitecture at drill hole site. Through computational studies, we
concluded that our lead compound possible bind to a deep seated cavity in dimeric
conformation of BMP2 protein and this site stabilize the BMP2 dimer, which further induce
osteogenic responses. Due to the absence of in-vitro and in-vivo toxicity, compound 21 could
be developed as alternative for shorten the healing period. Lastly, this compound provides a
scaffold and platform for the development of new bone forming agents against bone related
disorders.
5
5
. Experimental Section
.1. General Procedure: All reagents were commercial available and were used without
further purification. Chromatography was carried on silica gel (100-200 and 230-400 mesh).
All reactions were monitored by thin-layer chromatography (TLC), silica gel plates with
fluorescence F254 were used. Melting points were taken in open capillaries on Stuart-SMP30
melting point apparatus and are presented uncorrected. Infrared spectra were recorded on a
1 13
Perkin-Elmer FT-IR RXI spectrophotometer. H NMR and C NMR spectra were recorded
1
using BrukerSupercon Magnet DRX-300 spectrometer (operating at 300 and 400 MHz for H
1
and 75 and 100 MHz for C) using CDCl
3
3 6
and DMSO-d as solvent whereas
tetramethylsilane (TMS) taken as internal standard. Chemical shifts were reported in parts per
million and multiplicity (s = singlet, brs = broad singlet, d = doublet, brd = broad doublet, dd
=
double doublet, t = triplet, q = quartet, m = multiplet). Electro spray ionization mass spectra
(
(
ESI-MS) were recorded on Thermo Lcq Advantage Max-IT. High resolution mass spectra
HRMS) were recorded on 6520 Agilent Q Tof LC MS/MS (Accurate mass).High
performance liquid chromatography analyses for checking purity of targeted compounds were
performed on Shimadzu SCL 20 Avp, which was equipped with an Shimadzu SIL-HTC
autosampler on a Agilent Zorbax C18 column (100 mm x 4.6 mm, having particle size 5 µm).

Isocratic elution was carried out using mobile phase consisting of 10 mM ammonium acetate
containing pH 5.0) and acetonitrile at 10: 90 % (v/v). All targeted final compounds were
found to have >95% purity by HPLC.
.2. Typical method for the preparation of Compounds 1 to 3
(
5
Starting materials (1-3) were commercially available and were used without further
purification.
5
.3. Typical method for the preparation of Compounds 4 to 6
Synthesis of dicarbaldehyde substrates (Compounds 4 to 6) were achieved by our previously
reported protocol [33].
5
-Ethyl-4-hydroxyisophthalaldehyde (4)
White solid, yield: 69%; mp: 173-174 °C; 1H NMR (400 MHz, CDCl
3
) δ: 11.85 (s, 1H), 9.98
s, 1H), 9.91 (s, 1H), 7.97-7.95 (m, 2H), 2.78-2.72 (m, 2H), 1.26 (t, 3H, J = 7.5 Hz); IR
neat): 3259, 2870, 1700, 1628, 1013 cm-1; ESI-MS: m/z: 179 (M+H)+; HRMS m/z:calcd for
(M+H) + 179.0708, found 179.0711.
(
(
10 10 3
C H O
5
-Sec-butyl-4-hydroxybenzene-1,3-dicarbaldehyde (5)
1
Oily; Yield 55%; H NMR (400 MHz, CDCl
3
) δ: 11.99 (s, 1H), 10.05 (s, 1H), 9.97 (s, 1H),
8
.09 (brs, 1H), 8.00 (brs, 1H), 3.27−3.10 (m, 1H), 1.74−1.57 (m, 2H), 1.26 (d, 3H, J = 7.0
1
Hz), 0.86 (t, 3H, J = 7.3Hz); C NMR (75 MHz, CDCl
3
3
); 190.1, 163.3, 156.5, 155.8, 149.0,
1
1
2
5
37.5, 133.0, 131.9, 129.9, 118.9, 118.0, 53.2, 33.4, 29.6, 20.4, 12.0; IR (neat): 3267, 2862,
−
1
+
+
14 3
709, 1622, 1018 cm . ESI-MS m/z: 207 (M + H) ; HRMS m/z:calcd for C12H O (M+H)
07.1021, found 207.1016.
-Tert-butyl-4-hydroxyisophthalaldehyde (6)
1
Oily; Yield: 65%; H NMR (400 MHz, CDCl
3
) δ: 12.39 (s, 1H), 9.99 (s, 1H), 9.93 (s, 1H),
1
.07 (brs, 1H), 7.99 (brs, 1H), 1.46 (s, 9H); C NMR (100 MHz, CDCl
3
8
1
3
); 196.4, 190.0,
66.1, 140.0, 135.4, 133.9, 128.6, 120.4, 35.2, 29.1; IR (neat): 3252, 2865, 1703, 1626, 1013

-
1
+
+
14 3
cm ; ESI-MS m/z: 207 (M+H) ; HRMS m/z:calcd for C12H O (M+H) 207.1021, found
2
07.1018.
.4 Typical method for the preparation of compounds 7 to 12
5
General synthetic procedure for preparation of 7-(sec-butyl)-2-(4-methoxybenzoyl)
benzofuran-5-carbaldehyde (8)
To
methoxyphenacylbromide (1.2 mmol) and K
added. The reaction mixture was refluxed for 3h. After completion of the reaction, K
a
mixture of 5-(sec-butyl)-4-hydroxyisophthalaldehyde (5) (1.0 mmol), 4-
CO (0.6 mmol), acetonitrile (20 mL) was
CO
2
3
2
3
was removed in a sintered funnel, and the filtrate was concentrated under vacuum and
subjected to column chromatography with ethylacetate-hexane (15:85, v/v) to give pure
compound 8 as a white solid.
The compounds (7-12) were prepared in a way similar to the procedure described above.
7
-(Sec-butyl)-2-(4-methoxybenzoyl) benzofuran-5-carbaldehyde (8)
1
White solid, yield: 68 %; mp: 74-75 °C; H NMR (400 MHz, CDCl
3
) δ: 10.07 (s, 1H), 8.14
(
d, 2H, J = 8.9 Hz), 8.09 (d, 1H, J = 1.6 Hz), 7.87 (d, 1H, J = 1.2 Hz), 7.63 (s, 1H), 7.03 (d,
2
0
1
H, J = 8.9 Hz), 3.92 (s,3H), 3.39-3.30 (m, 1H), 1.89-1.78 (m, 2H), 1.43 (d, 3H, J = 6.9 Hz),
1
3
.89 (t, 3H, J = 7.3 Hz); C NMR (100 MHz, CDCl
3
) : 191.7, 182.2, 163.9, 157.4, 154.2,
33.6, 133.4, 132.1, 129.5, 127.3, 125.4, 124.8, 115.5, 114.0, 55.6, 35.9, 29.7, 20.3, 12.2; IR
-
KBr): 3050, 1680, 1654, 1651, 1210, 768 cm ; ESI-MS m/z: 337 (M+H) ; HRMS m/z:calcd
1
+
(
+
(M+H) 337.1440, found 337.1434.
for C21
H
20
O
4
7
-(Sec-butyl)-2-(4-methylbenzoyl)benzofuran-5-carbaldehyde (9)
1
Oily; yield: 70 %; H NMR (400 MHz, CDCl
3
) δ: 10.07 (s, 1H), 8.09 (d, 1H, J = 1.5 Hz),
.00 (d, 2H, J = 8.2 Hz), 7.87 (d, 1H, J = 1.2 Hz), 7.63 (s, 1H), 7.36 (d, 2H, J = 8.0 Hz), 3.38-
.32 (m,1H), 2.47 (s, 3H), 1.88-1.80 (m, 2H), 1.43 (d, 3H, J = 7.0 Hz), 0.89 (t, 3H, J = 7.4
8
3
1
3
3
Hz); C NMR (100 MHz, CDCl ) : 191.6, 183.5, 157.5, 153.9, 144.3, 134.3, 133.8, 133.4,

1
1
3
7
29.8, 129.4, 127.3, 125.6, 124.9, 35.9, 29.7, 21.8, 20.3, 12.3; IR (KBr): 3043, 1684, 1659,
-
1
+
+
20 3
629, 1204, 755 cm ; ESI-MS m/z: 321 (M+H) ; HRMS m/z:calcd for C21H O (M+H)
21.1491, found 321.1500.
-(Sec-butyl)-2-(4-chlorobenzoyl)benzofuran-5-carbaldehyde (10)
1
Oily; yield: 70 %; H NMR (400 MHz, CDCl
3
) δ: 10.07 (s, 1H), 8.10 (d, 1H, J = 1.5 Hz),
8
3
.05 (d, 2H, J = 8.6 Hz), 7.89 (d, 1H, J = 1.2 Hz), 7.66 (s, 1H), 7.54 (d, 2H, J = 8.6 Hz), 3.36-
1
3
.30 (m,1H), 1.88-1.78 (m, 2H), 1.43 (d, 3H, J = 7.0 Hz), 0.89 (t, 3H, J = 7.3 Hz); C NMR
) : 182.6, 171.9, 157.4, 153.3, 139.9, 135.2, 133.0, 131.1, 129.1, 127.7,
27.0, 125.8, 124.4, 116.7, 36.1, 29.8, 20.4, 12.3; IR (KBr): 3020, 1691, 1646, 1600, 1217,
(
100 MHz, CDCl
3
1
7
-
1
+
+
3
77 cm ; ESI-MS m/z: 341 (M+H) ; HRMS m/z:calcd for C20H17ClO (M+H) 341.0944,
found 341.0939.
7
-(Sec-butyl)-2-(4-nitrobenzoyl)benzofuran-5-carbaldehyde (11)
1
White solid, yield: 80%; mp: 117-118 °C; H NMR (400 MHz, CDCl
3
) δ: 10.09 (s, 1H), 8.41
(
d, 2H, J = 8.9 Hz), 8.24 (d, 2H, J = 8.9 Hz), 8.13 (d, 1H, J = 1.5 Hz), 7.92 (d, 1H, J = 1.2
Hz), 7.73 (s, 1H), 3.35-3.30 (m,1H), 1.87-1.81 (m, 2H), 1.43 (d, 3H, J = 7.0 Hz), 0.89 (t, 3H,
1
J = 7.4 Hz); C NMR (100 MHz, CDCl
3
3
) : 191.4, 182.0, 157.8, 152.9, 150.4, 141.7, 134.0,
1
1
33.8, 130.6, 127.1, 126.5, 125.2, 123.9, 117.5, 36.0, 29.7, 20.3, 12.3;IR (KBr): 3030, 1670,
-1
+
665, 1615, 1537, 1229, 768 cm ; ESI-MS m/z: 352 (M+H) ; HRMS m/z:calcd for
+
(M+H) 352.1185, found 352.1184.
C
20
H17NO
5
5
.5 Typical method for the preparation of compounds 13 to 32
General synthetic procedure for preparation of
Methyl-4-(7-ethyl-2-(4-
methoxybenzoyl)benzofuran-5-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-hexahydroquinoline-3-
carboxylate (13)
To a solution of 7-ethyl-2-(4-methoxybenzoyl)benzofuran-5-carbaldehyde (1.0 mmol) (7) in
5
mL ethanol were added methyl acetoacetate (1.0 mmol), 1,3-cyclohexadione (1.0 mmol),

ammonium acetate (3.0 mmol), and 5 mL glacial acetic acid. The reaction mixture was
continued under reflux condition for 8 h. After completion of reaction (monitored by TLC),
the reaction mixture was cooled to room temperature. The solid thus obtained was filtered
and subjected to column chromatography with ethylacetate-hexane (40:60, v/v) furnishing
compound 13 as a white solid in good yield.
Methyl-4-(7-ethyl-2-(4-methoxybenzoyl)benzofuran-5-yl)-2,7,7-trimethyl-5-oxo-1,4,5,6,7,8-
hexahydroquinoline-3-carboxylate (13)
1
White solid, yield: 80%; mp: 129-130 °C; H NMR (400 MHz, DMSO-d
6
) δ: 9.13 (s, 1H),
8
8
1
.03 (d, 2H, J = 8.9 Hz), 7.68 (s, 1H), 7.36 (d, 1H, J = 1.4 Hz), 7.23 (s, 1H), 7.11 (d, 2H, J =
.9 Hz), 4.98 (s, 1H), 3.87 (s, 3H), 3.53 (s, 3H), 2.91-2.79 (m, 2H), 2.50-2.42 (m, 5H), 2.31-
1
3
.96 (m, 2H), 1.26 (t, 3H, J = 7.5 Hz), 1.01 (s, 3H), 0.84 (s, 3H); C NMR (100 MHz,
): 195.8, 183.1, 168.1, 163.6, 153.6, 152.5, 148.9, 144.2, 143.5, 132.1, 130.1, 128.1,
27.9, 126.5, 119.4, 116.7, 113.9, 112.1, 105.9, 55.6, 51.1, 40.9, 36.5, 32.7, 29.6, 27.0, 23.0,
CDCl
3
1
1
-1
+
9.4, 14.2; IR (KBr): 3252, 3069, 1491, 1217, 1683 cm ; ESI-MS: m/z: 528 (M+H) ; HRMS
+
(M+H) 528.2386, found 528.2385.
m/zcalcd for C32H33NO
6
Ethyl-4-(7-ethyl-2-(4-methoxybenzoyl)benzofuran-5-yl)-2-methyl-5-oxo-1,4,5,6,7,8-hexa
hydroquinoline-3-carboxylate (14)
1
White solid, yield: 78%; mp: 210-211 °C; H NMR (500 MHz, DMSO-d
6
) δ: 9.16 (s, 1H),
8
7
2
3
1
5
.03 (d, 2H, J = 8.9 Hz), 7.68 (s, 1H), 7.36 (s, 1H), 7.36 (s, 1H), 7.26 (d, 1H, J = 1.4 Hz),
.11 (d, 2H, J = 8.9 Hz), 5.00 (s, 1H), 4.00-3.96 (m, 2H), 3.87 (s, 3H), 2.91-2.82 (m, 2H),
.51-2.49 (m, 2H), 2.30-2.15 (m, 2H), 1.93-1.74 (m, 2H), 1.29 (t, 3H, J = 7.5 Hz), 1.13 (t,
1
3
3
H, J = 7.0 Hz); C NMR (100 MHz, CDCl ) : 196.1, 183.1, 167.6, 163.7, 153.7, 152.5,
50.7, 143.9, 143.8, 132.1, 130.1, 128.4, 127.9, 126.6, 119.6, 116.5, 113.9, 113.4, 106.2,
9.9, 55.6, 37.1, 36.6, 27.4, 23.1, 21.1, 19.3, 14.4, 14.1; IR (KBr): 3350, 3020, 1670, 1469,