Identification of novel 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo- 3-phenylpropan-2-yl)carbamoyl)benzoic acid analogues as BMP-2 stimulators

Article abstract of DOI:10.1021/jm300985d

The synthesis and SAR studies of 10 new chemical entities (NCEs) that have shown BMP-2 stimulation and osteoblast differentiation are reported. Among these, 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl) carbamoyl)benzoic acid (11) was the most effective while its analogue 13 also showed good activity in inducing osteoblast BMP-2 production. Compound 11 induced osteoblast differentiation in vitro, and this effect was abrogated by a physiological BMP-2 inhibitor, noggin. It also exhibited dose dependent increase in nascent bone formation (2.16- and 3.12-fold more than the control at 1 and 5 mg/kg dose, respectively) at the fracture site in rats. At the maximum osteogenic concentration, compound 11 significantly inhibited osteoblastic proteosomal activity. This compound was safe, as it had no effect on BMP synthesis in cardiovascular tissue.

Full text of DOI:10.1021/jm300985d

Article
pubs.acs.org/jmc
Identification of Novel 2‑((1-(Benzyl(2-hydroxy-2-
phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic
Acid Analogues as BMP‑2 Stimulators
Vishal M. Balaramnavar,† Imran A. Khan,† Jawed Akhtar Siddiqui,‡ Mohd Parvez Khan,‡
Bandana Chakravarti,‡ Kunal Sharan,‡ Gaurav Swarnkar,‡ Namrata Rastogi,‡ H. H. Siddiqui,§
Durga Prasad Mishra,‡ Naibedya Chattopadhyay,‡ and Anil K. Saxena*,†
‡
†Division of Medicinal and Process Chemistry and Division of Endocrinology, CSIRCentral Drug Research Institute, Lucknow,
226001, India
§Department of Pharmacy, Integral University, Lucknow, India
S
* Supporting Information
ABSTRACT: The synthesis and SAR studies of 10 new chemical entities
(NCEs) that have shown BMP-2 stimulation and osteoblast differentiation
are reported. Among these, 2-((1-(benzyl(2-hydroxy-2-phenylethyl)amino)-
1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid (11) was the most
effective while its analogue 13 also showed good activity in inducing
osteoblast BMP-2 production. Compound 11 induced osteoblast differ-
entiation in vitro, and this effect was abrogated by a physiological BMP-2
inhibitor, noggin. It also exhibited dose dependent increase in nascent bone
formation (2.16- and 3.12-fold more than the control at 1 and 5 mg/kg dose,
respectively) at the fracture site in rats. At the maximum osteogenic
concentration, compound 11 significantly inhibited osteoblastic proteosomal
activity. This compound was safe, as it had no effect on BMP synthesis in
cardiovascular tissue.
only anabolic agent available for clinical use, which not only
increase bone mass but also bone quality and strength by
improving microarchitecture and geometry.11,12
Recently, PTH (1−84, Preos) has been launched in Europe,
including U.K., and is awaiting FDA approval in the U.S.
However, PTH is the therapy of last resort and is
recommended for a maximum of only 24 months in the U.S.
and in Europe.13 Major drawbacks associated with PTH are its
parenteral route of administration and the risk of developing
osteogenic sarcoma.
The bone morphogenetic proteins (BMPs) are secreted
signaling molecules belonging to the transforming growth
factor β (TGF-β) superfamily. They have been shown to
augment fracture healing in several animal models and clinical
cases.14,15 BMPs are typically delivered to the fracture site as
recombinant proteins, but there are several limitations with
their use. Recombinant proteins are rapidly resorbed and
redistributed from the fracture site. Their synthesis and
purification are expensive, and their requirement in large
doses for treatment increases the probability of side effects.16,17
Moreover, multiple treatments are required to obtain sustained
concentrations at the fracture site that are inconvenient and
may reduce treatment adherence.
INTRODUCTION
■
Osteoporosis is a leading cause of fractures in adults. It is
characterized by progressive bone loss with deterioration of
bone microarchitecture.1−3 In pathophysiological terms,
increased bone loss in osteoporosis is coupled with reduced
formation of new bone.4 Osteoporosis associated with estrogen
deficiency after menopause is the most common cause of age-
related bone loss,1 affecting at least one-quarter of all
postmenopausal women (70% of women in United States)
with increased proportion to women older than 80 years.
Hormone replacement therapy (HRT) effectively prevents
postmenopausal osteoporosis and reduces the incidence of
fractures.2 However, HRT also increases the risk of breast and
endometrial cancer, in addition to other undesirable side
effects.
Agents for the treatment of osteoporosis are classified as
antiresorptive and anabolic. The first line of treatment for
osteoporosis in the clinic is by antiresorptive agents, which
prevent bone loss by inhibiting the activity of osteoclasts.
Anabolic agents act by stimulating the formation of new bone
and thus provide an additional option for osteoporosis patients
and represent a major advancement in the treatment of
osteoporosis. Anabolic agents are capable of increasing bone
mass to a greater degree than antiresorptive agents with the
capacity to improve both the bone quality and strength.5−10
Parathyroid hormone PTH (1−34) and PTH (1−84) are the
Received: February 13, 2012
Published: September 14, 2012
© 2012 American Chemical Society
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a
Scheme 1
a
Reagents and conditions: (a) dried DCM, TEA (1.2 equiv) (0 °C to rt) for 12 h; (b) LiOH (4 equiv), THF/MeOH/H2O (4:4:1) for 2 h; (c) SiO2,
N2 atm for 24 h; (d) 1.6 equiv of Boc anhydride, 1.2 equiv of TEA, DCM, for 6 h; (e) dry DCM, TEA (1.2 equiv), benzyl chloride, rt for 12 h; (f)
dry DCM, TEA (1.2 equiv), benzoyl chloride for 12 h; (g) TsIm/TBAI/TEA, DMF, reflux, 3−6 h; (h) Ac2O/pyridine (2:6) for 12 h; (i) dry DCM,
TEA (1.2 equiv), tosyl chloride, rt, for 12 h; (j)TFA/DCM (50%:50%) for 12 h; (k) COCl2, THF, 0 °C, 1 h, TEA, 2 equiv for 2 h; (l) DCC
coupling; (m) BOP coupling; (n) debenzylation.
BMP-2, a member of the BMP family, is synthesized by
osteoblasts and stimulates osteoblast differentiation and bone
generation and regeneration in different animal models.18
Recombinant adenoviral transfer of BMP-2 c-DNA in
osteoporotic sheep model induces fracture healing.19 This
approach represents a significant advancement in bone repair
and received FDA approvals for use in orthopedics.20 However,
systemic infusion of BMP-2 has no significant osteogenic effect
because of poor bioavailability, and considering the cost of
synthesis and purification of the recombinant protein,21 the
selective augmentation of BMP-2 in osteoblast by small
molecules that are orally efficacious may be considered as a
significant advancement in this field. Such molecules may
provide new therapeutic agents for the treatment of fracture
healing, osteoporosis, and bone loss conditions in the future.
Statins have been shown to stimulate bone formation, and
the effect is associated with increased expression of BMP-2 in
bone cells.22 Previous reports have shown that the BMP-2 gene
is a transcriptional target of estrogen receptor, and estrogens
including various phytoestrogens stimulate BMP-2 synthesis in
osteoblasts.23,24 Piceatannol stimulates osteoblast differentia-
tion and is associated with the increase in BMP-2 production.25
Recently, some proteasome inhibitors, viz. epoximycin (a
natural product) and bortezomib, showed increased bone
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Figure 1. Strategy employed for the design of NCEs based on known reference compound.
a
Table 1. Structures and Effect of Compounds on BMP-2 Secretion in RCOs
BMP-2 level in the conditioned medium
compd
R1
R2
0.1 nM
1 nM
10 nM
100 nM
1000 nM
11
-CH2C6H5
-CH2C6H5
-CH2C6H5
-CH2C6H5
-CH2C6H5
-CH2C6H5
H
-OH
140 3.4**
89.8 3.5
98.4 2.3
83.6 3.6
88.7 4.2
122 3.2
106 2.4
119 3.1
87.8 1.9
111 1.6
159.5 2.3***
100.66 2.2
107.69 1.9
97.35 2.6
155.6 4.1**
98.34 3.1
103.15 3.1
89.28 3.7
83.45 3.1
109.39 3.1
98.33 4.2
111.78 1.3
101.32 6.2
114.24 5.2
137.2 3.2**
103.27 2.2
103.15 3.1
96.69 4.1
133 3.1*
92.6 2.2
112 3.5
92.6 1.6
100.6 1.4
111 2.5
103 1.6
104 1.8
93.6 2.3
132 4.4*
12a
12b
12c
12d
12e
12f
12g
12h
13
-OCH2C6H5
-OCOC6H5
-CN
-OCOCH3
89.49 4.2
98.76 3.7
-OSO2C6H5CH3
-OCOCH3
141.67 3.2**
98.67 3.1
138.25 1.4**
96.03 2.6
H
-OSO2C6H5CH3
-OCH2C6H5
131.75 2.9**
107.27 1.1
122.8 4.2
114.88 2.8
117.2 5.2
H
H
166.11 7.3**
a
Each assay was performed in triplicate, and results are expressed as the mean SEM of percentage of control (100%) after normalization of total
protein content/well: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001 vs control.
formation rates in vitro, with the participation of BMP-2
signaling in osteoblasts.26−29 Recently two series of substituted
benzothiophene and benzofuran derivatives have been
reported.30,31 as novel classes of BMP-2 up-regulators.
Ten compounds belonging to the prototype incorporating
pharmacophoric features of the bortezomib core were
prioritized with the aid of preliminary modeling studies that
are not presented in the manuscript. The synthesis and
biological evaluation of these compounds as BMP-2 promoters
are presented in this study.
7, 10a−e through amide coupling. In view of the high cost of
BOP/PyBOP and sluggish working up procedure for DCC the
discriminated reactivity of aliphatic acid over aromatic acid was
used for the activation of aliphatic acid selectively through in
situ generation of acyl chloride.
SAR STUDY
■
The series of the compounds represented by the prototype was
designed in such a manner that structural modifications were in
parts A, B, and C of the template/reference molecule
bortezomib (Figure 1). All the compounds were tested on
BMP-2 secretion assay using primary calvarial osteoblast
cultures from rats (RCO) (Table 1) with respect to different
R2 substitutions. Among all these compounds, compound 11
(R2 = OH) showed maximum effect on BMP-2 secretion at
lower concentrations (up to 0.1 nM). The substitution of the
−OH functionality by different bulkier groups starting from
acetyl (12d), cyano (12c), benzyl (12a), benzoyl (12b)
resulted in the reduction in the activity. The tosyl derivative
(12e) seemed to have reasonable activity showing that the free
−OH at the R2 position is optimum for the activity. The next
important variation was carried out at the R1 portion in the
compounds, viz. 12f, 12g, 12h, and 13 where the activity
improved in case of N-debenzylated analogue of acetylated
derivative 12f compared to the benzyl substituted acetylated
analogue 12d. However, this trend was not observed in the
debenzylated analogue 12e or 12d. The substitution of the
bulky groups at the R2 position by hydrogen in the
debenzylated analogue (12f−h) led to the compound 13
CHEMISTRY
■
The synthesis of designed molecules (11, 12a−h, 13) was
accomplished by the method outlined in Scheme 1 from two
key intermediates 4 (2-((1-carboxy-2-phenylethyl)carbamoyl)-
benzoic acid) and 7 (2-(benzylamino)-1-phenylethanol). The
key intermediate 4 was synthesized by the hydrolysis of 3 by
lithium hydroxide. The 3 was prepared by the reaction of
phthalic anhydride (l) with phenylalanine methyl ester
hydrochloride (2) in dry DCM using TEA as acid scavenger.
The other key intermediate 7 was synthesized by silica
catalyzed regioselective epoxide opening of ( )-styrene oxide
(6) by nucleophilic attack of benzylamine (5) to give other key
intermediate (7) in 88% along with its corresponding
regioisomer 2-(benzylamino)-2-phenylethanol (7a).32 The
desired β-aminol (7) was used for the synthesis of the
prioritized molecule through other intermediates (10a−e). The
target molecules (11, 12a−h, 13) were prepared by the
reaction of the key intermediate 4 with other key intermediates
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Figure 2. Compound 11 increases osteoblast function. (A) RCOs were cultured in osteoblast differentiation medium as described above in the
presence of compound 11 (10 nM) or vehicle (control). Cells were stained with Alizarin Red S. Photomicrographs show increased formation of
mineralized nodules by compound 11 treatment of osteoblasts compared with vehicle treated osteoblasts (upper panel). Quantification of
mineralization by extraction of Alizarin Red S dye is shown (lower panel). (B) qPCR analysis of various osteogenic genes, Runx-2, osteocalcin, BMP-
2 and type I collagen in osteoblasts treated with compound 11 (10 nM) at different time intervals as indicated. Each assay was performed in
triplicate, and results are represented as the mean SEM of percentage/fold change: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.
which showed significant increase in BMP-2 secretion. Most of
the compounds did not show an increase in BMP-2 secretion in
a dose/concentration dependent manner, possibly because of
copious production of BMP-2 by osteoblasts at the basal level
that negatively impacts the assay sensitivity with respect to the
determination of concentration−response relation by ELISA.
Hence, we confirmed the effect of the most effective
compounds 11 and 13 at BMP-2 mRNA levels in osteoblasts
at 48 h. The qPCR quantification revealed that relative to the
control RCOs, cells exposed to increasing concentrations of
compounds 11 and 13 responded with increased BMP-2
mRNA levels (Table 1, Supporting Information). The absolute
BMP-2 levels (pg/mL) in the conditioned medium were
included in Table 2 in Supporting Information.
Figure 3. Effect of BMP-2 neutralization on osteoblast differentiation
by compound 11. Cells were treated for 72 h with compound 11 (10
nM), BMP-2 (100 ng/mL), and noggin (50 ng/mL) either alone or in
combination as indicated. mRNA analysis of Runx-2 and osteocalcin
DETAILED BIOLOGICAL EVALUATION OF THE
LEAD
■
genes were performed by qPCR. Data represent the mean
SEM
As the ability of any agent/compound to induce BMP-2
production in osteoblast has the potential to promote
osteoblast differentiation, we assessed the effect of compound
11 on RCO differentiation. As BMP-2 stimulatory effect of
compound 11 was maximum at 10 nM, we used this
concentration to study its impact on osteoblast mineralization.
In comparison to the control RCOs, compound 11 stimulated
the formation of mineralized nodules (P < 0.001) (Figure 2A).
Furthermore, the effect of compound 11 was investigated on
the expression of various osteogenic genes including Runx-2,
BMP-2, and osteocalcin, and type I collagen by qPCR and the
data showed a time-dependent increase in their mRNA levels
over control (Figure 2B). Out of these four genes, Runx-2 and
BMP-2 mRNA levels were significantly elevated over control at
as early as 24 h. Stimulation of mRNA levels of Runx-2 and
BMP-2 genes by compound 11 continued up to 72 h. Since
BMP-2 is a potent stimulator of osteoblast differentiation, we
investigated whether stimulation of osteoblast differentiation by
compound 11 was mediated via the increased production of
BMP-2. To that effect, RCOs were treated with compound 11
with or without noggin. Figure 3 shows that the induction of
Runx-2 and osteocalcin mRNA levels in the RCOs by
compound 11 was brought back to the basal (unstimulated)
from three independent experiments: ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P
< 0.001.
levels when noggin was present in the culture medium.
Together, these data indicate that compound 11 exerts an
osteogenic effect in an autocrine/paracrine mode by stimulating
osteoblastic BMP-2 production.
Effect of Compound 11 on Fracture Healing in the
Drill-Hole Fracture Model in Rats. BMPs have been shown
to enhance fracture healing in several animal models and
clinical cases.33,34 BMP-2 and -7 are usually delivered to the
fracture site as recombinant proteins to accelerate healing.
Because compound 11 stimulated BMP-2 production to
promote osteoblast function, we tested whether it could
enhance fracture healing. Figure 4 shows that compared to
the control (rats receiving vehicle), compound 11 dose-
dependently increased osteoblasts recruitment, callus regener-
ation, and osteoblasts formation (measured from the intensity
of calcein labeling) in the femur drill-hole. Compared to
control rats, new bone formation was 2.16- and 3.12-fold more
at 1.0 and 5.0 mg/kg dose, respectively.
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treatment to rats at both doses resulted in higher bone volume/
trabecular volume (BV/TV), trabecular number (Tb.N),
trabecular thickness (Tb.Th), connection density (conn.D),
lower trabecular spacing (Tb.sp), and structure model index
(SMI) compared to the vehicle treated groups (Figure 5).
These data indicated that compound 11 treatment promoted
fracture healing by stimulating the formation of new bone at
the site of the drill-hole.
Assessment of Off-Target Effect of Compound 11. The
osteoinductive potential of BMP-2 could lead to not only
stimulated bone formation in the skeleton but also extraskeletal
tissues such as heart and aorta.35 Therefore, we investigated
whether compound 11 has an off-target effect, i.e., induction of
BMP-2, -4, and -7 mRNAs in cardiovascular tissues. While
compound 11 at 1.0 and 5.0 mg/kg doses enhanced fracture
healing, the expression levels of BMP-2, -4, and -7 in the aorta
(Figure 6A) and cuspid valves of the heart (Figure 6B) of these
rats were comparable to controls.
Proteosome Inhibition Assay. On the basis of the close
structural analogy to bortezomib, compound 11 was analyzed
for its possible mechanism of action through proteosomal
inhibition. The proteosomal inhibition of this compound was
evaluated using the 20S proteasome activity assay kit
(Chemicon International, U.S.). Compound 11 significantly
inhibited proteosomal activity in a concentration dependent
fashion from 0.1 to 10 nM. However, at 100 nM, compound 11
had no proteosome inhibitory action. Lactacystin (25 μM), a
standard proteosome inhibitor, was used for comparison
Figure 4. Compound 11 promotes fracture healing in the drill-hole in
rats. Representative confocal images of calcein labeling are shown in
the drill-hole of various groups 2 weeks after drill-hole injury. Lower
panel shows quantification of the mean intensity of calcein label.
Values are expressed as the mean SEM (n = 10 rats/group): ∗∗, P <
0.01; ∗∗∗, P < 0.001.
Assessment of internal microstructure of the mineralized
tissue in the bony hole was analyzed by 3-D μCT (micro-
computed tomography). At the drill-hole site, compound 11
Figure 5. Representative μCT images from the center of the bony hole (upper panel). μCT analysis shows BV/TV, bone volume/tissue volume (%);
Tb.N, strut number (1/mm); Tb.Th, strut thickness (mm); Tb.Sp, strut spacing (mm); Conn.Dens, connection density (1/mm3); SMI, structure
model index. All values are expressed as the mean SEM (n = 10 rats/group): ∗, P < 0.05; ∗∗, P < 0.01; ∗∗∗, P < 0.001.
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osteoblasts accumulates in the extracellular matrix and acts as
a factor promoting osteoblast function.37 BMP-2 signals via
BMP-2 receptors and promotes osteoblast differentiation by an
autocrine/paracrine mechanism.38,39 Our data showing aboli-
tion of osteoblast differentiation by compound 11 in the
presence of noggin suggest that its osteogenic effect is mediated
via the activation of autocrine/paracrine loop of BMP-2.
Enhanced calcification, a major risk factor for cardiovascular
diseases, is associated with the emergence of osteoblast-like
cells in the vascular tissues due to increased BMP production.40
Despite having a BMP-2 stimulatory effect in osteoblasts,
compound 11 has no effect on the expression of BMPs in
cardiovascular tissues, indicating cardiovascular safety.
BMP-2 is required for normal fracture healing, as its absence
results in the failure of mesenchymal progenitors to differ-
entiate at the fracture site leading to a failed healing
response.41Since compound 11 stimulated osteoblast differ-
entiation via BMP-2 production, we studied its effect on the
bone healing process in a drill-hole injury model of long
bone.42−44 This model seems suitable for analyzing the healing
process quantitatively and useful for investigating osteoblast
differentiation in vivo.42,44 Our data showed that treatment of
rats with compound 11 significantly increased the fracture
healing process by stimulating new bone formation at the drill-
hole injury site compared to the vehicle treated group. This
finding was complemented by μCT analysis showing greater
newly generated bone in the drill-hole of compound 11 treated
groups compared with vehicle. Increased BV/TV, Tb.Th, and
Tb.N by the treatment of compound 11 indicated structurally
more robust growth of bone and compact assembly of
trabecular bones at the drill-hole site compared to controls.
The stability of the newly formed bone is importantly
dependent on structural parameters determined by Conn.D,
SMI, and Tb.pf.45 Higher Conn.D, preferred platelike structure
(lower SMI), and more concave trabecular surface (lower
Tb.pf) presented a more compact bone in rats treated with
compound 11 compared to control. These results suggested
that the impact of compound 11 treatment on the structure
(connectivity) and microarchitecture (bone geometry) of new
bone in the drill-hole was better than control. Our results
suggest that compound 11 stimulates BMP-2 production in
osteoblasts and promotes new bone formation at the fracture
site. Being orally efficacious, compound 11 could be an
attractive strategy to shorten the healing period of fracture in
the clinical setting.
Figure 6. Compound 11 has no effect on BMP expression in
cardiovascular tissues (A, B). Total RNA was extracted from aorta and
cuspid valves of heart following 2 weeks of 11 treatment and qPCR
was performed for the analysis of BMP-2, BMP-4, and BMP-7 mRNA
levels. Data represent the mean SEM (n = 10 rats/group).
(Figure 7). Because inhibition of proteosome activity by
bortezomib has been reported to induce osteoblast differ-
Figure 7. Compound 11 showed reduction in the proteasome activity.
Mouse calvarial cell line MC3T3-E1 was incubated with compound 11
for 48 h, and proteasome activity was measured. Lac is lactacystin (25
μM). Data represent the mean
experiments: ∗∗, P < 0.01.
SEM from three independent
EXPERIMENTAL SECTION
■
Biological Methods. Reagents and Chemicals. Cell culture
media and supplements were purchased from Invitrogen (Carlsbad,
CA). All fine chemicals were purchased from Sigma Aldrich (St. Louis,
MO). High-performance liquid chromatography grade acetonitrile was
obtained from Merck India Ltd. (Mumbai, India). Heparin sodium
injection (1000 IU/mL IP) was purchased from Gland Pharma
(Hyderabad, India).
entiation by augmenting BMP-2 production,36 compound 11
may serve to act similarly and in the process accelerate BMP-2
mediated fracture healing in vivo.
CONCLUSION
■
There is no oral drug available for accelerating fracture repair.
Human recombinant BMP-2 and BMP-7 have been approved
by U.S. FDA for local application at the site of fracture,
including compound fracture (spinal fusion or open tibial
surgery). Clearly, this is an interventional approach having
limited use.
In Vitro Studies. Culture of Calvarial Osteoblasts. Rat calvarial
osteoblasts (RCOs) were obtained following previously published
protocol of sequential digestion.46−48 Briefly, calvaria from 1- to 2-day-
old Sprague−Dawley rats (both sexes) were pooled. After surgical
isolation from the skull and the removal of sutures and adherent
mesenchymal tissues, calvaria were subjected to five sequential (10−15
min) digestions at 37 °C in a solution containing 0.1% Dispase and
0.1% collagenase P. Cells released from the second to fifth digestions
were pooled, centrifuged, resuspended, and plated in T-25 cm2 flasks
In our studies, compound 11 was found to potently stimulate
BMP-2 and osteoblast differentiation from a series of
structurally similar compounds. BMP-2 synthesized by
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in α-MEM containing 10% FCS and 1% penicillin/streptomycin
(complete growth medium).
described before.45 The front skin of the mid-femur in rats was incised
longitudinally in a straight manner at 1 cm in length under anesthesia.
After splitting the muscle, we stripped the periosteum to expose the
femoral bone surface. A drill-hole injury was made by inserting a drill
bit with a diameter of 0.8 mm in the anterior portion of the diaphysis
of the bilateral femurs, 2 cm above the knee joint. Treatments started
from the next day of injury and continued for 2 weeks. For the various
treatments, rats were divided into three equal groups (10 rats/group)
as follows: vehicle (gum acacia in distilled water), 11 (1.0
mg·kg−1·day−1) and 11 (5.0 mg·kg−1·day−1). Each animal received
intraperitoneal administration of fluorochrome calcein (20 mg kg−1) 2
days before autopsy. After 2 weeks of various treatments described
above, all rats were euthanized and autopsied to collect their heart and
aorta for safety measurement and to collect the femur for the
measurement of bone microarchitectural parameters in the drill-hole as
described below. Bones were embedded in an acrylic material. Sections
of 50 μm were made using an Isomet bone cutter, and photographs
were taken under a confocal microscope (Carl Zeiss LSM 510 Meta)
aided with appropriate filters. The intensity of calcein binding, which is
an indication of the amount of new mineral depositions, was calculated
using Carl Zeiss AM 4.2 image analysis software.
Statistical Analysis. Data are expressed as the mean SEM. The
data obtained in experiments with multiple treatments were subjected
to one-way ANOVA followed by post hoc Newman−Keuls multiple
comparison test of significance using GraphPad Prism 5.04 software.54
Proteasome Activity Assay. Isolated 20S proteasome activity was
evaluated using proteasome activity assay kit (Chemicon International,
U.S.) according to the manufacturer’s instructions. Mouse calvarial cell
line MC3T3-E1, having a phenotypic response comparable to that of
its rat counterpart, was seeded in six-well plates at a density of 2 × 106
per well and treated with compound 11 for 48 h. Both treated and
untreated cells were harvested by scraping followed by washing twice
with ice cold PBS and resuspending in lysis buffer (50 mM HEPES
(pH 7.5), 5 mM EDTA, 150 mM NaCl, and 1% Triton X-100). Cells
were incubated for 30 min on ice with regular vortexing for 15 s after
every 10 min of incubation. Lysate was centrifuged for 15 min at 4 °C
at 14000g, and protein was estimated using the Bradford assay.
Aliquots of 10 μg of isolated proteasome were incubated with
fluorogenic substrate in 1× assay buffer provided with the kit for 1 h in
the dark at 37 °C. Fluorescence intensity was measured by fluorimeter
(BMG Fluorostar Omega) with a 380/460 nm filter set.
Osteoblast Differentiation. For the measurement of alkaline
phosphatase (ALP) activity, RCOs at ∼80% confluence were
trypsinized and 2 × 103 cells/well were seeded in 96-well plates.
Cells were treated with different concentration of compound for 48 h
in α-MEM supplemented with 5% charcoal treated FCS, 10 mM β-
glycerophosphate, 50 μg/mL ascorbic acid, and 1% penicillin/
streptomycin (osteoblast differentiation medium). At the end of the
incubation period, total ALP activity was measured using p-
nitrophenylphosphate (PNPP) as substrate and absorbance was read
at 405 nm.49
BMP-2 ELISA. For measurement of BMP-2 production from
osteoblasts, 5 × 103 cells/well were seeded in 24-well plates. Cells
were exposed to given concentrations of compounds for 48 h in α-
MEM medium supplemented with 10% charcoal-stripped FCS, 10
mM β-glycerophosphate, and 50 μg/mL ascorbic acid. At the end of
incubation, supernatants were collected for determination of BMP-2
by ELISA according to the manufacturer’s instructions (Quantikine,
R&D Systems).
Mineralization of Calvarial Osteoblasts. Mineralization of
RCOs was performed following a previously published protocol.50
Briefly, RCOs were cultured until 80% confluence and were
trypsinized and plated in the differentiation medium (25 000 cells/
well in a 12-well plate), consisting of complete growth medium with
ascorbic acid (50 μg/mL) and β-glycerophosphate (100 mM). The
medium was changed every other day up to 21 days. The treatment
group contained a similar medium with 11 (10nM). 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 40 mM (pH 4.5) Alizarin Red S for 30 min followed by washing
with water.51
For quantification of Alizarin Red S staining, 800 μL of 10% (v/v)
acetic acid was added to each well, and plates were incubated at room
temperature for 30 min with shaking. The monolayer, now loosely
attached to the plate, was then scraped with a cell scraper and
transferred with 10% (v/v) acetic acid to a 1.5 mL tube. After the
mixture was vortexed for 30s, the slurry was overlaid with 500 μL of
mineral oil (Sigma−Aldrich), heated to exactly 85 °C for 10 min, and
transferred to ice for 5 min. The slurry was then centrifuged at 20000g
for 15 min, and 500 μL of the supernatant was removed to a new tube.
Then 200 μL of 10% (v/v) ammonium hydroxide was added to
neutralize the acid. In some cases, the pH was measured at this point
to ensure that it was between 4.1 and 4.5. OD (405 nm) and 150 μL
aliquots of the supernatant were measured in 96-well format using
opaque-walled, transparent-bottomed plate.46,48,52
Quantitative Real-Time Polymerase Chain Reaction (qPCR).
qPCR reaction was performed for quantitative comparative measure-
ment of the expression of osteoblast specific genes Runx-2, BMP-2,
osteocalcin, and collagen type-1 following our optimized proto-
col.45,48,53 The housekeeping gene GAPDH was used as the internal
control in this study. Primers were designed using the Universal
ProbeLibrary (Roche Applied Sciences) for the following genes: BMP-
2, 5′-CGG CTGCGGTCTCCTAA-3′ (sense), 5′-GGGAAGCAG-
CAACACTAGA-3′ (antisense); osteocalcin, 5′-ATA-
GACTCCGGCGCTACCTC-3′ (sense), 5′-CCAGGGGATCTGGG-
TAGG-3′ (antisense); Runx-2, 5′- CCACAGAGCTATTAAAGTGA-
CAGTG-3′ (sense), 5′-AACAAACTAGGTTTAGAGTCATCAAGC-
3′ (antisense); GAPDH, 5′-CAGCAAGGATAC TGAGAGCAAGAG-
3′ (sense), 5′-GGATGGAATTGTGAGGGAGATG-3′ (antisense).
For real-time PCR, cDNA was synthesized with a Revert Aid cDNA
synthesis kit (Fermentas, Austin, TX, U.S.) using 2.0 μg of total RNA.
SYBR green chemistry was used to perform quantitative determination
of the relative expression of transcripts for all genes. All genes were
analyzed using the Light Cycler 480 (Roche Molecular Biochemicals,
Indianapolis, IN, U.S.) real time PCR machine.
Synthetic Methods. General Chemistry. All the chemicals were
purchased from different chemical suppliers. All the reactions were
monitored by TLC (thin layer chromatography). The purity of all the
compounds was determined by a high-pressure liquid chromatography
(HPLC) by lachrome and was >95% using a diode array detector. The
eluting process was performed on Chirapak 18 columns using an
1
acetonitrile/methanol (90:10% v/v for compounds) system. H NMR
spectra of all the synthesized compounds were recorded by using a
Bruker Spectrospin spectrometer at 300 MHz with tetramethylsilane
used as internal standard. Mass spectra were recorded on a Jeol
(Japan) SX 102/DA-6000 mass spectrometer. IR spectroscopy was
carried out using a Perkin-Elmer 881 spectrophotometer, and the
values are expressed as vmax, cm−1. Elemental analyses were performed
on a Carlo Erba model EA-1108 elemental analyzer, and data for C, H,
and N are indicated.
Synthesis of 2-((1-Methoxy-1-oxo-3-phenylpropan-2-yl)-
carbamoyl)benzoic Acid (3). Anhydrous phthalic anhydride
(0.495 mg, 3.352 mmol, 1.2 equiv) was added to a stirred mixture
of methyl ester of phenylalanine (500 mg, 2.791 mmol) and
triethylamine (0.3384 mg, 3.35 mmol, 1.2 equiv) at 0 °C in dry
DCM (dichloromethane). The mixture was stirred for 12 h. After the
completion of the reaction the reaction mixture was treated with 1 N
HCl (5 mL) and then diluted with water (10 mL) and extracted with
ethyl acetate (2 × 25 mL). The extracts layer of ethyl acetate was dried
by using sodium sulfate and was concentrated in vacuo to give the title
1
product 3 (1.04 g, 95.41%). H NMR (CDCl3, 300 MHz), δ 11.586
(1H, s,COOH), 7.8325−7.6548 (4H, m, CH), 7.4984 (5H, m,
CH),5.822 (1H, bs, NH), 4.91725 (1H, d, J = 2.13 Hz, CH), 3.702
(3H, s, CH3), 3.4285 (2H, m, CH2). Chemical formula: C18H17NO5.
In Vivo Studies. Fracture Healing in Drill-Hole Injury at
Femur. Thirty adult female Sprague−Dawley rats (200 20 g each)
were taken for the study. Drill-hole injury was created in femur as
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FTIR (KBr): cm−1 3415, 3312, 3031, 2829, 2620, 1715, 1480, 1320,
1200, 1120, 760, 751, 669. EIMS: m/z 328.2 (M + 1). Elemental
analysis: C, 65.90; H, 5.21; N, 4.27.
1654, 1562, 1335, 1229, 1258, 1087, 824, 784, 745, 699. EIMS: m/z
418.2 (M + 1). Elemental analysis: C, 77.60; H, 7.40; N, 3.34.
Synthesis of tert-Butyl Benzyl(2-cyano-2-phenylethyl)-
carbamate (9c). To a stirred solution of tert-butyl benzyl(2-
hydroxy-2-phenylethyl)carbamate (0.4 g, 1.222 mmol) in dry
dichloromethane and triethylamine (0.247 g, 2.445 mmol, 2 equiv)
were added TsIm (0.325 g, 1.466 mmol, 1.2 equiv), NaCN (0.119 g,
2.42 mmol, 2 equiv), and a catalytic amount of TBAI (0.045 g, 0.1222
mmol) in DMF (30 mL). The reaction mixture was refluxed for 6 h.55
The solvent was evaporated under vacuum, and the remainder was
diluted with DCM and washed with water (2 × 100 mL). The organic
layer was dried over sodium sulfate and evaporated. The crude
compound 9c was purified by using column chromatography by silica
Synthesis of 2-((1-Carboxy-2-phenylethyl)carbamoyl)-
benzoic Acid (4). 2-((1-Methoxy-1-oxo-3-phenylpropan-2-yl)-
carbamoyl)benzoic acid (500 mg,1.53 mmol) acid was stirred in a
solvent mixture of tetrahydrofuran/methanol/water (4:4:1). To the
stirring solution was subsequently added lithium hydroxide (0.1464
mg, 6.11 mmol, 4 equiv), and the mixture was stirred for 2 h at room
temperature. The milky suspension was then treated with 1 N HCl (5
mL) and diluted with water (10 mL) and extracted with ethyl acetate.
The ethyl acetate layer was dried over sodium sulfate and concentrated
in vacuo to give the title product 4 (0.4 g, 85.10%). 1H NMR (CDCl3,
300 MHz), δ 11.36 (2H, s, COOH), 7.7984 (4H, m, CH), 7.3725−
7.1328 (5H, m, CH), 5.6312 (1H, bs, NH), 4.9251 (1H, d, J = 2.10
Hz, CH), 3.1245(2H, m, CH2). Chemical formula: C17H15NO5. FTIR
(KBr): cm−1 3415, 3312, 3031, 2829, 2620, 1715, 1480, 1320, 1200,
1120, 760, 751, 669. EIMS: m/z 314.2 (M + 1). Elemental analysis: C,
65.10; H, 4.81; N, 4.46.
1
gel, eluting with n-hexane/EtOAc (10:1). Yield (0.283 g, 69.02%). H
NMR (CDCl3, 300 MHz), δ 7.3725−7.1328 (10H, m, CH), 4.4322
(1H, dd, J = 2.16 Hz, CH), 4.3070 (2H, s, CH2), 3.75−3.59 (2H, m,
CH2), 1.52 (9H, s, CH3). Chemical formula: C21H24N2O2. FTIR
(KBr): cm−1 3050, 2347, 1741, 1721, 1688, 1360, 1290, 1141, 1030,
786, 734, 634. EIMS: m/z 337.2 (M + 1). Elemental Analysis: C,
74.90; H, 7.17; N, 8.30.
Synthesis of 2-(Benzylamino)-1-phenylethanol (7). Benzyl-
amine (0.5 g, 4.66 mmol) was added to a stirred solution of styrene
oxide (0.56 g, 4.67 mmol) and SiO2 (30%) in nitrogen atmosphere for
24 h. To the resulting suspension was added dichloromethane to
dissolve the solid product. The solution was filtered and concentrated
under pressure to obtain a solid product to give the title product 7
(1.02 g, 97.14%). 1H NMR (CDCl3, 300 MHz), δ 7.21−7.33 (10H, m,
CH), 4.9825 (1H, d, J = 2.23 Hz, CH), 3.824 (2H, bs, CH2), 3.2413
(2H, m, CH2), 1.78 (1H, s, NH). Chemical formula: C15H17NO. FTIR
(KBr): cm−1 3445, 2314, 1336, 1218, 1148, 771, 669. EIMS: m/z
228.2 (M + 1). Elemental analysis: C, 79.19; H, 7.52; N, 6.14.
Synthesis of tert-Butyl Benzyl(2-hydroxy-2-phenylethyl)-
carbamate (8). tert-Butyl formate (0.66 g, 3.522 mmol, 1.6 equiv)
was added to a stirred mixture of 2-(benzylamino)-1-phenylethanol
(0.5 g, 2.201 mmol) and triethylamine (0.266 g, 2.633 mmol, and 1.2
equiv) and stirred for 6 h. After 6 h, the reaction mixture was diluted
with water (10 mL) and extracted with ethyl acetate (3 × 25 mL) and
was concentrated under vacuum and kept at 0 °C for crystallization to
give the title product 8 (0.65 g, 90.27%). 1H NMR (CDCl3, 300
MHz), δ 7.23−7.38 (10H, m, CH), 5.2625 (1H, bs, CH), 4.24 (2H,
bs, CH2), 3.37 (2H, m, CH2), 1.52 (9H, s, CH3). Chemical formula:
C20H25NO3. FTIR (KBr): cm−1 3462, 2362, 1654, 1303, 1147, 1057,
1398, 1346, 1302, 1147, 1057, 738, 697. EIMS: m/z 328.2 (M + 1).
Elemental analysis: C, 73.34; H, 7.68; N, 4.27.
Synthesis of 2-(Benzyl(tert-butoxycarbonyl)amino)-1-phe-
nylethyl Benzoate (9d). To a stirred solution of tert-butyl
benzyl(2-hydroxy-2-phenylethyl)carbamate (0.4 mg,1.222 mmol) in
dry dichloromethane and triethylamine (0.148 mg, 1.467 mmol, 1.2
equiv) was added benzoyl chloride (0.179 g, 1.22 mmol, 1 equiv), and
the mixture was stirred for 12 h. The reaction was monitored by TLC
and after completion of reaction worked up with water (10 mL) and
extracted in DCM (2 × 25 mL), dried over sodium sulfate, and
concentrated under vacuum to give the title product 9d (0.49 g,
1
94.23%). H NMR (CDCl3, 300 MHz), δ 7.96−7.92 (2H, d, J = 7.5),
7.3725−7.1328 (13H, m, CH), 5.9239 (1H, d, J = 7.18,CH), 4.1633
(2H, m, CH), 3.4156 (2H, m, CH2), 1.52 (9H, s, CH3). Chemical
formula: C27H29NO4. FTIR (KBr): cm−1 3020, 2927, 1731, 1741,
1698, 1340, 1230, 1161, 1040, 789, 735, 635. EIMS: m/z 432.2 (M +
1). Elemental analysis: C, 75.10; H, 6.75; N, 3.24.
Synthesis of 2-(Benzyl(tert-butoxycarbonyl)amino)-1-phe-
nylethyl 4-Methylbenzenesulfonate (9e). To a stirred solution
of tert-butyl benzyl(2-hydroxy-2-phenylethyl)carbamate (0.4 mg, 1.222
mmol) in dry dichloromethane and triethylamine (0.148 mg, 1.467
mmol, 1.2 equiv) was added 4-methylbenzene-1-sulfonyl chloride
(0.2322 g. 1.222 mmol, 1 equiv). The reaction was monitored by TLC
and after completion of reaction worked up with water (10 mL) and
extracted in DCM (2 × 25 mL), dried over sodium sulfate, and
concentrated under vacuumto give the title product 9e (0.55 g,
1
Synthesis of 2-(Benzyl(tert-butoxycarbonyl)amino)-1-phe-
nylethyl Acetate (9a). tert-Butyl benzyl(2-hydroxy-2- phenylethyl)-
carbamate (0.4 g, 1.222 mmol) was added to a stirred solution of
acetic anhydride and pyridine in a proportion of 2:6 and stirred for 12
h. After completion of reaction the reaction mixture was worked up
with 1 N HCl (5 mL) and diluted with water (10 mL) and extracted
with DCM (2 × 25 mL). The organic layer was dried over sodium
sulfate and concentrated under vacuum to give the title product 9a
94.82%). H NMR (CDCl3, 300 MHz), δ 7.76−7.74 (2H, d, J = 7.5,
CH), 7.4725−7.232 (12H, m, CH), 6.75 (1H, m, CH), 4.22 (2H, s,
CH2), 3.2659 (2H, m, CH2), 2.37 (3H, s, CH3), 1.52 (9H, s, CH3).
Chemical formula: C27H31NO5S. FTIR (KBr): cm−1 3485, 1658, 1504,
1453, 1204, 1130. EIMS: m/z 482.2 (M + 1). Elemental analysis: C,
67.39; H, 6.47; N, 2.90.
Synthesis of 2-(Benzylamino)-1-phenylethyl Acetate (10a).
The compound was synthesized by stirring 2-(benzyl(tert-
butoxycarbonyl)amino)-1-phenylethyl acetate (0.3 g, 0.8134 mmol)
in trifluoroacetic acid (0.1111 g, 0.9745 mmol, 1.2 equiv) and
dichloromethane (50:50%) and for 2 h. After the completion of the
reaction, to the reaction mixture was added sodium bicarbonate until
the solid was separated out to give the title product 10a (0.18 g,
1
(0.39 g, 86.66%). H NMR (CDCl3, 300 MHz), δ 7.3725−7.1328
(10H, m, CH), 5.7414 (1H, dd, J = 2.16 Hz, CH), 4.3070 (2H, s,
CH2), 3.6539 (2H, m, CH2), 2.1985 (3H, s, CH3), 1.52 (9H, s, CH3).
Chemical formula: C22H27NO4. FTIR (KBr): cm−1 3062, 2933, 1654,
1562, 1335, 1229, 1258, 1087, 824, 784, 745, 699. EIMS: m/z 370.2
(M + 1). Elemental analysis: C, 71.49; H, 7.35; N, 3.78.
1
76.1%). H NMR (CDCl3, 300 MHz), δ 7.23−7.38 (10H, m, CH),
Synthesis of tert-Butyl Benzyl(2-(benzyloxy)-2-phenylethyl)-
carbamate (9b). To a stirred solution of tert-butyl benzyl(2-hydroxy-
2-phenylethyl)carbamate (0.4 g, 1.222 mmol) in dry dichloromethane
and triethylamine (0.148 g, 1.467 mmol, 1.2 equiv) was added benzyl
chloride (0.154 g, 1.222 mmol, 1 equiv), and the mixture was stirred
for 12 h. The reaction was monitored by TLC and after completion of
reaction worked up with water (10 mL) and extracted in DCM (2 ×
25 mL), dried over sodium sulfate, and concentrated under vacuum to
5.7414 (1H, dd, J = 2.16 Hz, CH), 3.90 (2H, s, CH2), 3.2519 (2H, m,
CH2), 2.1985 (3H, s, CH3), 1.84 (1H, s, NH). Chemical formula:
C17H19NO2. FTIR (KBr): cm−1 3412, 2363, 2101, 1740, 1637, 1320,
1219, 771, 671, 552. EIMS: m/z 270.2 (M + 1). Elemental analysis: C,
75.79; H, 7.09; N, 5.18.
Synthesis of N-Benzyl-2-(benzyloxy)-2-phenylethanamine
(10b). The compound was synthesized by stirring tert-butyl
benzyl(2-(benzyloxy)-2-phenylethyl)carbamate (0.3 g, 0.9458 mmol)
and trifluoroacetic acid (0.123 g, 1.135 mmol, 1.2 equiv) and
dichloromethane (50:50%) for 2 h, using the same protocol used
1
give the title product 9b (0.48 g, 94.12%). H NMR (CDCl3, 300
MHz), δ 7.3725−7.1328 (15H, m, CH), 4.7211 (1H, m,CH), 4.6213
(2H, s, CH2), 4.5808 (2H, s, CH2), 3.7494 (2H, s, CH2), 1.52 (9H, s,
CH3). Chemical formula: C27H31NO3. FTIR (KBr): cm−1 3062, 2933,
1
for compound 10a to give the title product 10b (0.18 g, 81.81%). H
NMR (CDCl3, 300 MHz), δ 7.23−7.48 (15H, m, CH), 4.60 (2H, s,
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CH2), 4.34 (1H, m, CH), 3.13 (2H, m, CH2), 3.82 (2H, s, CH2), 1.87
(1H, s, NH). Chemical formula: C22H23NO. FTIR (KBr): cm−1 3430,
2930, 1400, 1340, 1240, 1100, 780, 769. EIMS: m/z 318.2 (M + 1).
Elemental analysis: C, 83.20; H, 7.28; N, 4.39.
Synthesis of 2-((1-(Benzyl(2-(benzyloxy)-2-phenylethyl)-
amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid
(12a). This product was synthesized by using the same method
described for compound 11 by stirring a suspension of 2-((1-carboxy-
2-phenylethyl)carbamoyl)benzoic acid (4) (0.5 g, 1.5959 mmol) with
4 equiv of oxylyl chloride (0.8102 g, 6.38 mmol, 4 equiv) to form 2-
((1-chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid. Then
N-benzyl-2-(benzyloxy)-2-phenylethanamine (0.607 g, 1.192 mmol,
1.2 equiv) in THF and 4 equiv of triethylamine (0.6447 g, 6.383
mmol) were added at 0 °C. After completion of the reaction the final
product was treated with 1 N HCl (10 mL) and extracted by the same
protocol described for compound 11 to give the title product 12a
(0.82 g, 83.93%). 1H NMR (CDCl3, 300 MHz), δ 11.36 (1H, s,
COOH), 7.9812−7.8111 (2H, m, CH), 7.4984 (4H, m, CH), 7.3725−
7.1328 (18H, m, CH), 5.022 (1H, bs, NH), 4.3808 (1H, d, J = 10.02
Hz, CH), 4.1271 (1H, m, CH), 3.9494 (2H, s, CH2), 3.5395 (2H, s,
CH2), 3.5051 (2H, m, CH2), 3.0094 (2H, t, J = 5.79 Hz, 6.21 Hz,
CH2). 13C NMR (CDCl3, 300 MHz), δ 171.51, 167.55, 167.32,
158.15, 146.12, 145.21, 138.50, 137.65, 137.42, 136.99, 135.55, 134.04,
129.4, 128.87, 128.51, 128.16, 128.38, 127.36, 127.30, 127.26, 127.34,
127.21, 127.14, 127.10, 126. 76, 126.54, 126.32, 125.55, 124.54,
124.37,123.39, 74.66, 53.32, 51.54, 48.40, 45.99, 34.53, 30.33, 27.70.
Chemical formula: C39H36N2O5. FTIR (KBr): cm−1 3755, 3460, 3090,
2925, 2368, 1686, 1643, 1447, 1346, 1158, 1062, 707, 697. EIMS: m/z
613.2 (M + 1). Elemental analysis: C, 76.40; H, 5.90; N, 4.55.
Synthesis of 2-((1-((2-(Benzoyloxy)-2-phenylethyl)(benzyl)-
amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid
(12b). This product was synthesized by using the same method
described for compound 11 by stirring a suspension of 2-((1-carboxy-
2-phenylethyl)carbamoyl)benzoic acid (0.5 g, 1.5959 mmol) with 4
equiv of oxylyl chloride (0.8102 g, 6.38 mmol, 4 equiv) to form 2-((1-
chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid. Then 2-
(benzylamino)-1-phenylethyl benzoate (0.6347 g, 1.9150 mmol, 1.2
equiv) in THF and 4 equiv of triethylamine (0.6447 g, 6.383 mmol)
were added at 0 °C. After completion of the reaction the final product
was treated with 1 N HCl (10 mL) and extracted by the same protocol
described for compound 11 to give the title product 12b (0.86 g,
86%). 1H NMR (CDCl3, 300 MHz), δ 11.48 (1H, s, COOH), 8.0278
(1H, s, NH), 7.7593 (1H, d, J = 1.94 Hz, CH), 7.74215 (1H, d, CH, J
= 3.45 Hz), 7.6766−7.1197 (22H, m, CH), 5.6014 (1H, dd, J = 4.89
Hz, CH), 5.4727 (1H, t, J = 8.43 Hz, J = 7.7 Hz, CH), 4.4321 (2H, s,
CH2), 3.775 (2H, d, J = 6.8 Hz, J = 8.87 Hz, CH2), 3.598 (2H, t, J =
6.87 Hz, CH2). 13C NMR (CDCl3, 300 MHz), δ 170.24, 167.42,
166.34, 138.21, 137.67, 136.64, 136.50, 134.32, 133.56, 132.45, 130.63,
130.34, 128.42, 129.86, 129.75, 129.40, 128.67, 128.63, 128.55, 128.46,
128.39, 128.33, 128.30, 128.25,128.13, 127.82, 127.78, 127.74, 127.65,
127.60, 127.52, 127.43, 127.37, 125.43, 72.03, 56.62, 52.67, 49.03,
37.17. Chemical formula: C39H34N2O6. FTIR (KBr): cm−1 3781, 3426,
3034, 2982, 1773, 1716, 1630, 1448, 1381, 1336, 1235, 1088,875, 746,
718, 699. EIMS: m/z 627.2 (M + 1). Elemental analysis: C, 74.70; H,
5.48; N, 4.46.
Synthesis of 3-(Benzylamino)-2-phenylpropanenitrile (10c).
The compound was synthesized by stirring tert-butyl benzyl(2-cyano-
2-phenylethyl)carbamate (0.3 g, 0.813 mmol), trifluoroacetic acid
(0.110 g, 0.975 mmol, 1.2 equiv), and dichloromethane (50:50%) for 2
h, using the same protocol used for compound 10a to give the title
product 10c (0.16gm, 76.19%). 1H NMR (CDCl3, 300 MHz), δ 7.23−
7.38 (10H, m, CH), 4.2334 (1H, dd, J = 2.16 Hz, CH), 4.1002 (2H, s,
CH2), 3.7559 (2H, m, CH2), 1.89 (1H, s, NH). Chemical formula:
C17H16N2O2. FTIR (KBr): cm−1 3409, 2353, 2121, 1730, 1622, 1310,
1229, 765, 651, 562. EIMS: m/z 237.2 (M + 1). Elemental analysis: C,
81.25; H, 6.80; N, 11.86.
Synthesis of 2-(Benzylamino)-1-phenylethyl Benzoate (10d).
The compound was synthesized by stirring 2-(benzyl(tert-
butoxycarbonyl)amino)-1-phenylethyl benzoate (0.3 g, 0.6952
mmol) and trifluoroacetic acid (0.0951 g, 0.8342 mmol, 1.2 equiv)
and dichloromethane (50:50%) for 2 h using the same protocol used
1
for compound 10a to give the title product 10d (0.17 g, 73.91%). H
NMR (CDCl3, 300 MHz), δ 8.02 (2H, d, J = 7.5, CH), 7.56−7.23
(13H, m, CH), 5.49 (1H, d, J = 7.18, CH), 3.91 (2H, s, CH), 3.23
(2H, m, CH2), 1.96 (1H, s, NH). Chemical formula: C22H21NO2.
FTIR (KBr): cm−1 3442, 3023, 1640, 1492, 1341, 1215, 1031, 762,
669, 559. EIMS: m/z 332.2 (M + 1). Elemental analysis: C, 79.69; H,
6.37; N, 4.23.
Synthesis of 2-(Benzylamino)-1-phenylethyl 4-Methylben-
zenesulfonate (10e). The compound was synthesized by stirring 2-
(benzyl(tert-butoxycarbonyl)amino)-1-phenylethyl 4-methylbenzene-
sulfonate (0.3 g, 0.6229 mmol), trifluoroacetic acid (0.085 g, 0.7454
mmol, 1.2 equiv), and dichloromethane (50:50%) for 2 h using the
same protocol used for compound 10a to give the title product 10e
1
(0.19 g, 82.60%). H NMR (CDCl3, 300 MHz), δ 7.74−7.232 (14H,
m, CH), 6.40 (1H, m, J = 6.39 Hz, CH), 3.92 (2H, s, CH2), 3.2659
(2H, m, CH2), 2.34 (3H, s, CH3), 1.82 (1H, s, NH). Chemical
formula: C22H23NO3S. FTIR (KBr): cm−1 3429, 3022, 1638, 1337,
1217, 1152, 769,671. EIMS: m/z 382.2 (M + 1). Elemental analysis: C,
69.20; H, 6.05; N, 3.65.
Synthesis of 2-((1-(Benzyl(2-hydroxy-2-phenylethyl)amino)-
1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid (11). To a
stirring suspension of 2-((1-carboxy-2-phenylethyl)carbamoyl)benzoic
acid (4) (0.5 g, 1.5959 mmol) was added 4 equiv of oxylyl chloride
(0.8102 g, 6.38 mmol, 4 equiv) at 0 °C to form clear solution. The
mixture was stirred for 30 min to form 2-((1-chloro-1-oxo-3-
phenylpropan-2-yl)carbamoyl)benzoic acid, which was then concen-
trated in vacuum to remove excess of oxylyl chloride. Then in another
flask of stirred 2-(benzylamino)-1-phenylethanol (7) (0.434 g, 1.195
mmol, 1.2 equiv) in THF was added 4 equiv of triethylamine (0.644 g,
6.3836 mmol, 4 equiv) at 0 °C, and the mixture was stirred for 30 min.
Finally the 2-((1-chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)-
benzoic derivative was added to this system and mixture was allowed
to come to room temperature and stirred for 5−6 h. After the
completion of reaction the reaction was worked up with 1 N HCl (10
mL) and extracted with dichloromethane (4 × 25 mL). After vigorous
shaking, the organic layer was separated and dried over sodium sulfate
and concentrated over vacuum to give the title product 11 (0.79 g,
Synthesis of 2-((1-(Benzyl(2-cyano-2-phenylethyl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid (12c). This
product was synthesized by using the same method described for
compound 11 by stirring a suspension of 2-((1-carboxy-2-
phenylethyl)carbamoyl)benzoic acid (0.5 g,1.5959 mmol) with 4
equiv of oxylyl chloride (0.8102 g, 6.38 mmol, 4equiv) to form 2-((1-
chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid. Then 3-
(benzylamino)-2-phenylpropanenitrile (0.4522 g, 1.915 mmol, 1.2
equiv) in THF and 4 equiv of triethylamine (0.6447 g, 6.383 mmol)
were added at 0 °C. After completion of the reaction the final product
was treated with 1 N HCl (10 mL) and extracted by the same protocol
described for compound 11 to give the title product 12c (0.67 g,
1
94.78%). H NMR (CDCl3, 300 MHz), δ 9.8921 (1H, brs, COOH),
7.5465 (1H, d, J = 3.33 Hz, CH), 7.5274 (1H, d, J = 3.06 Hz, CH),
7.0561 (17H, m, CH), 5.022 (1H, bs, NH), 4.9091 (1H, d, J = 18 Hz,
CH), 4.6442 (1H, d, J = 18 Hz, CH), 3.9494 (2H, s, CH2), 3.6502
(1H, s, OH), 3.7804 (2H, m, CH2), 3.2177 (2H, m, CH2) . 13C NMR
(CDCl3, 300 MHz): 170.65, 167.25, 167.20, 142.06, 137.02, 136.90,
135.62, 133.67,131.10, 129.25, 128.70, 128.61, 128.49, 128.32,
128.07,128.02, 128.04, 127.71, 126.95, 126.84, 125.82, 125.65,
125.47, 125.39, 125.29, 125.26, 123.19, 73.46, 56.42, 52.55, 52.41,
35.27. C32H30N2O5. FTIR (KBr): cm−1 3708, 3426, 3246, 2928, 1690,
1625, 1520, 1472, 1472, 1322, 1230, 1162, 1001,775, 739. EIMS: m/z
523.2 (M + 1). Elemental analysis: C, 73.50; H, 5.77; N, 5.38.
1
78.98%). H NMR (CDCl3, 300 MHz), δ 11.4820 (1H, s, COOH),
9.2741 (1H, s, NH), 8.3159 (1H, d, J = 7.7 Hz CH), 7.866 (2H, d, J =
8.34 Hz, CH), 7.6594 (2H, dd, J = 7.56, 9.9 Hz, CH), 7.4347 (14H, m,
CH), 5.2878 (1H, m, CH), 5.0953 (1H, m, CH), 4.4321 (2H, s, CH2),
3.5101 (2H, t, J = 7.27 Hz, CH2), 3.1221 (2H, t, J = 7.6 Hz, CH2). 13
C
NMR (CDCl3, 300 MHz), δ 173.21, 168.12, 168.01, 137.84, 137.63,
137.45, 134.56, 132.48, 131.56, 130.42, 130.02, 128.76, 128.63, 128.57,
8256
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Journal of Medicinal Chemistry
Article
128.43, 128.32, 127.91, 127.88, 127.84, 127.80, 127.72, 127.69, 127.67,
127.59, 127.43, 127.39, 127.21, 127.11, 57.21, 51.09, 49.03, 37.89,
29.84. Chemical formula: C33H29N3O4. FTIR (KBr): cm−1 3624, 3326,
3054, 2382, 1763, 1716, 1640, 1438, 1281, 1236, 1225, 1088, 875, 746,
718, 699. EIMS: m/z 532.2 (M + 1). Elemental analysis: C, 74.35; H,
5.48; N, 7.89.
procedure as described above to give the title product 12f (0.13 g,
81.25%). H NMR (CDCl3, 300 MHz), δ 10.8630 (1H, s, 1COOH),
1
8.2062 (1H, d, J = 9.03 Hz, CH), 7.9827 (1H, bs, NH), 7.3915 (10H,
m, CH), 7.1410 (3H, m, CH), 5.0206 (1H, dd, J = 2.19, 2.29 Hz, CH),
4.91725 (1H, d, J = 2.13 Hz, CH), 3.7967 (2H, dd, J = 5.43, 5.07 Hz,
CH2), 3.2413 (2H, m, CH2), 2.0968 (3H, s, CH3). 13C NMR (CDCl3,
300 MHz), δ 171.71, 170.22, 167.60, 167.53, 130.11, 137.90, 138.52,
134.0,136.67, 132.10, 128.63, 129.23, 128.92, 128.65, 128.69, 128.56,
128.54, 128.50, 127.26,127.24, 127.22, 125.69, 74.91, 57.80, 44.13,
37.44, 21.50. Chemical formula: C27H26N2O6. FTIR (KBr): cm−1
3779, 3656, 3379, 2927, 1755, 1698, 1599, 1420, 1389, 1309, 1163,
1083, 838, 766, 722, 699, 660. EIMS: 475.2 (M + 1). Elemental
analysis: C, 68.30; H, 5.50; N, 5.88.
Synthesis of 2-((1-((2-Acetoxy-2-phenylethyl)(benzyl)-
amino)-1-oxo-3-phenylpropan-2-Yl)carbamoyl)benzoic Acid
(12d). This product was synthesized by using same method described
for compound 11 by stirring a suspension of 2-((1-carboxy-2-
phenylethyl)carbamoyl)benzoic acid (0.5 g, 1.5959 mmol) with 4
equiv of oxylyl chloride (0.8102 g, 6.38 mmol, 4equiv) to form 2-((1-
chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid. Then 2-
(benzylamino)-1-phenylethyl acetate (0.5158 g, 1.915 mmol, 1.2
equiv) in THF and 4 equiv of triethylamine (0.6447 g, 6.383 mmol)
were added at 0 °C. After completion of the reaction the final product
was treated with 1 N HCl (10 mL) and extracted by the same protocol
described for compound 11 to give the title product 12d (0.76 g,
Synthesis of 2-((1-Oxo-3-phenyl-1-((2-phenyl-2-(tosyloxy)-
ethyl)amino)propan-2-yl)carbamoyl)benzoic Acid (12g). The
product was synthesized by using 2-((1-(benzyl(2-phenyl-2-
(tosyloxy)ethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)-
benzoic acid (0.2 g, 0.2955 mmol) as the initial compound and was
debenzylated and converted to its corresponding product by a
debenzylation procedure as described above to give the title product
1
86.44%). H NMR (CDCl3, 300 MHz), δ 11.24 (1H, s, COOH),
7.70515 (2H, dd, J = 2.7 Hz, CH), 7.88 (1H, bs, NH), 7.7898 (2H, m,
J = 3.1587, CH), 7.6998 (2H, t, J = 3.51 Hz, J = 2.95 Hz, CH) 7.5753
(1H, t, J = 3.51 Hz, CH), 7.3291−7.1821 (12H, m, CH), 5.8770 (1H,
bs, NH), 5.7414 (1H, dd, J = 2.16 Hz, CH), 4.9361 (1H, dd, J = 2.22
Hz, CH), 4.4070 (2H,s, CH2), 3.8403 (2H, dd, J = 5.43, 5.07, CH2),
3.2832 (2H, m, CH2), 2.1985 (3H, s, CH3). 13C NMR, δ 170.22,
170.01, 167.63, 167.55, 138.52, 137.97, 136. 43, 136.67, 134.01,
132.08, 130.24, 130.12,129.24, 128.81,128.69, 128.65, 128.66, 128.60,
128.55, 128.54, 127.95, 127.76, 127.16, 127.39, 127.22, 127.16, 127.10,
125.9, 72.7,55.6, 52.5, 49.6, 37.7, 21.0. Chemical formula:
C34H32N2O6. FTIR (KBr): cm−1 3781, 3426, 3035, 2927, 1960,
1773, 1716, 1629, 1447, 1381, 1235, 1093, 874, 717. EIMS: 565.2 (M
+ 1). Elemental analysis: C, 72.29; H, 5.69; N, 4.94.
1
12g (0.14 g, 82.35%). H NMR (CDCl3, 300 MHz), δ 10.820 (1H, s,
1COOH), 9.2741 (1H, s, NH), 8.3159 (1H, s, NH), 7.8664 (4H, m,
CH), 7.6594 (4H, m, CH), 7.4479 (10H, m, CH), 6.4739 (1H, d, J =
7.38, CH), 4.8333 (1H, m, CH), 3.4156 (2H, dd, J = 5.8, 4.99 Hz,
CH2), 3.2638 (2H, dd, J = 6.5, 6.49 Hz, CH2), 2.1929 (3H, s, CH3).
13C NMR (CDCl3, 300 MHz), δ 171.72, 167.64, 167.52, 144.47,
140.65, 140.43, 140.26, 137.59, 136.66, 136.56, 134.80, 132.10,130.31,
130.75, 129.42, 128.79, 128.46, 128.23, 128.16, 127.71, 127.67, 127.61,
128.59, 128.56, 127.47, 127.36, 125.29, 74.82, 57.80, 44.01, 37.14,
21.53. Chemical formula: C32H30N2O6S. FTIR (KBr): cm−1 3441,
3022, 1740, 1637, 1543, 1630, 1455, 1367, 1217, 1160, 768, 701, 669,
607. EIMS: 587.2 (M + 1). Elemental analysis: C, 65.49; H, 5.13; N,
4.76.
Synthesis of 2-((1-(Benzyl(2-phenyl-2-(tosyloxy)ethyl)-
amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid
(12e). This product was synthesized by using same method described
for compound 11 by stirring a suspension of 2-((1-carboxy-2-
phenylethyl)carbamoyl)benzoic acid (0.5 g,1.5959 mmol) with 4
equiv of oxylyl chloride (0.8102 g, 6.38 mmol, 4 equiv) to form 2-((1-
chloro-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid. Then 2-
(benzylamino)-1-phenylethyl 4-methylbenzenesulfonate (0.0.73 g,
1.9150 mmol,1.2 equiv) in THF and 4 equiv of triethylamine
(0.6447 g, 6.383 mmol) were added at 0 °C. After completion of the
reaction the final product was treated with 1 N HCl (10 mL) and
extracted by the same protocol described for compound 11 to give the
Synthesis of 2-((1-((2-(Benzyloxy)-2-phenylethyl)amino)-1-
oxo-3-phenylpropan-2-yl)carbamoyl)benzoic Acid (12h). The
product was synthesized by using 2-((1-(benzyl(2-(benzyloxy)-2-
phenylethyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic
acid (0.2 g, 0.3167 mmol) as initial compound and debenzylated to its
corresponding product by a debenzylation procedure as described
above to give the title product 12h (0.12gm, 70.58%). 1H NMR
(CDCl3, 300 MHz), δ 12.7012 (1H, s, COOH), 9.2741 (1H, s, NH),
8.0670−8.0544 (1H, J = 1.83 Hz,1.95 Hz, CH), 8.0382 (1H,t, J = 8.43,
2.3 Hz, CH), 7.9078−7.1965 (17H, m, CH), 5.4727 (1H, s, NH),
5.0627 (1H, d, J = 7.17 Hz, CH), 4.2136 (1H, t, J = 15.93 Hz, CH),
4.6321 (2H, s, CH2), 3.8241 (2H, dd, J = 7.83, 7.56 Hz, CH2),3.775
(2H, m, CH2). 13C NMR, δ 171.72, 167.72, 167.64, 147.06, 142.11,
137.9, 136.67, 134.40, 132.30, 130.11, 129.63, 128.56, 128.46, 128.40,
128.36, 128.34, 128.29, 128.25, 127.87, 127.80, 127.76, 127.74,127.64,
127.60, 127.54, 127.51, 125.50, 86.31, 72.10, 57.84, 43.92, 37.41.
Chemical formula: C32H30N2O5. FTIR (KBr): cm−1 3751, 3506, 3027,
2926, 1605, 1531, 1651, 1349, 1217, 1159, 1095, 937, 764, 699. EIMS:
m/z 523.2 (M + 1). Elemental analysis: C, 73.50; H, 5.77; N, 5.36.
Synthesis of 2-((1-Oxo-1-(phenethylamino)-3-phenylpro-
pan-2-yl)carbamoyl)benzoic Acid (13). This product was
synthesized by using same method as described for compound 11
by stirring a suspension of 2-((1-carboxy-2-phenylethyl)carbamoyl)-
benzoic acid (0.5 g, 1.5959 mmol) with 4 equiv of oxylyl chloride
(0.8102 g, 6.38 mmol, 4 equiv) to form 2-((1-chloro-1-oxo-3-
phenylpropan-2-yl)carbamoyl)benzoic acid. Then 2-phenylethanamine
(0.2320 g, 1.5959 mmol, 1.2 equiv) in THF and 4 equiv of
triethylamine (0.6447 g, 6.383 mmol) were added at 0 °C, and
extraction was by the same protocol as described for compound 11 to
1
title product 12e (0.85 g, 78.70%). H NMR (CDCl3, 300 MHz), δ
10.824 (1H, s, COOH), 9.2741 (1H, s, NH), 8.7712 (d, 1H, 8.67 Hz,
CH), 7.9501 (1H, d, 4.5 Hz, CH), 7.4907 (21H, m, CH), 6.7947 (1H,
m, J = 6.39 Hz, CH), 4.912 (1H, t, J = 6.66, 4.86 Hz, CH), 4.6691
(2H,s, CH2), 3.8324 (2H, m, CH2), 3.2659 (2H, m, CH2), 2.1929
(3H, s, CH). 13C NMR (CDCl3, 300 MHz): 170.01, 167.63, 167.57,
144.43, 140.60, 140.53, 134.0, 132.0, 137.91, 137.82 136.44, 136.36,
130.10, 130.51, 130.45, 128.42, 128.40, 128.36, 129.28, 128.19,128.15,
128.12, 128.10, 128.09,128.03, 128.02, 127.1, 127.9, 127.7,127.1,
127.9, 127.0, 125.9, 72.7, 49.9, 55.6, 52.2, 37.7, 21.3. Chemical
formula: C39H36N2O6S. FTIR (KBr): cm−1 3393, 3064, 1750, 1740,
1493, 1451, 1373, 1336, 1157, 1241, 1102, 1041, 903, 824, 760,
700,616. EIMS: m/z 677.2 (M + 1). Elemental analysis: C, 69.16; H,
5.35; N, 4.14.
Debenzylation of Compounds 12f−h. The title products 12f,
12g, and 12h were synthesized by debenzylation using palladium on
carbon (10%) in methanol with 12 h of stirring in a Parr assembly with
a pressure of 40 par. The title products were residues there were
chromatographed on silica gel column using ethyl acetate/hexane
(20:80).
1
give the title product 13 (0.52 g, 78.79%). H NMR (CDCl3, 300
MHz), δ 12.7012 (1H, s, COOH), 7.7054 (4H, m, CH), 7.2805 (1H,
s, NH), 7.1339 (10H, m, CH), 6.1225 (1H, m, CH), 5.0698 (1H, dd, J
= 6.96 Hz, 6.99 Hz, CH), 3.283 (2H, t, CH2), 2.8134 (2H, t, J = 6.81,
6.87, CH2), 2.938 (2H, d, J = 6.816, CH2). 13C NMR (CDCl3, 300
MHz), δ 171.94, 168.46, 167.93, 151.80, 138.58, 136.71, 134.28,
131.34, 130.21, 129.13, 128.81, 128.77, 128. 64, 128.53, 126.93,
126.46, 123.51, 119.27, 117.80, 117.50, 116.87, 55.93, 40.94, 35.37,
Synthesis of 2-((1-((2-Acetoxy-2-phenylethyl)amino)-1-oxo-
3-phenylpropan-2-yl)carbamoyl)benzoic Acid (12f). The prod-
uct was synthesized by using 2-((1-((2-acetoxy-2-phenylethyl)-
(benzyl)amino)-1-oxo-3-phenylpropan-2-yl)carbamoyl)benzoic acid
(0.2 g, 0.3546 mmol) as the initial compound and was debenzylated
and converted to its corresponding product by a debenzylation
8257
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Journal of Medicinal Chemistry
Article
34.69. Chemical formula: C25H24N2O4. FTIR (KBr): cm−1 3440, 3023,
2923, 2821, 1491, 1299, 1031, 747, 687, 636. EIMS: 417.2 (M + 1).
Elemental analysis: C, 72.10; H, 5.81; N, 6.73.
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ASSOCIATED CONTENT
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Cierny, G.; Muschler, G. F.; Zych, G. A.; Calhoun, J. H.; LaForte, A. J.;
Yin, S. Osteogenic protein-1 (bone morphogenetic protein-7) in the
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S
* Supporting Information
MTT assay for cell viability at 1, 10, and 100 nM compounds
and ALP production by osteoblasts for the selected compound
11 at 10 nM (Figure 1); relative BMP-2 transcript level in
RCOs (Table 1); absolute BMP-2 levels (pg/mL) in the
conditioned medium (Table 2). This material is available free
of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +91 522 2624273. Fax: +91 522 2623405. E-mail:
anilsak@gmail.com.
Notes
The authors declare no competing financial interest.
(15) Govender, S.; Csimma, C.; Genant, H. K.; Valentin-Opran, A.;
Amit, Y.; Arbel, R.; Aro, H.; Atar, D.; Bishay, M.; Borner, M. G.;
̈
ACKNOWLEDGMENTS
Chiron, P.; Choong, P.; Cinats, J.; Courtenay, B.; Feibel, R.; Geulette,
B.; Gravel, C.; Haas, N.; Raschke, M.; Hammacher, E.; van der Velde,
D.; Hardy, P.; Holt, M.; Josten, C.; Ketterl, R. L.; Lindeque, B.; Lob,
G.; Mathevon, H.; McCoy, G.; Marsh, D.; Miller, R.; Munting, E.;
Oevre, S.; Nordsletten, L.; Patel, A.; Pohl, A.; Rennie, W.; Reynders,
P.; Rommens, P. M.; Rondia, J.; Rossouw, W. C.; Daneel, P. J.; Ruff,
S.; Ruter, A.; Santavirta, S.; Schildhauer, T. A.; Gekle, C.; Schnettler,
R.; Segal, D.; Seiler, H.; Snowdowne, R. B.; Stapert, J.; Taglang, G.;
Verdonk, R.; Vogels, L.; Weckbach, A.; Wentzensen, A.; Wisniewski,
T. BMP-2 evaluation in surgery for tibial trauma (BESTT) study
group. Recombinant human bone morphogenetic protein-2 for
treatment of open tibial fractures: a prospective, controlled,
randomized study of four hundred and fifty patients. J. Bone Jt. Surg.
Am. 2002, 84A, 2123−2134.
(16) Baltzer, A. W.; Lattermann, C.; Whalen, J. D.; Ghivizzani, S.;
Wooley, P.; Krauspe, R.; Robbins, P. D.; Evans, C. H. Potential role of
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■
V.M.B. and M.P.K. are thankful to the Indian Council of
Medical Research, New Delhi, for financial assistance in the
form of a fellowship. Fellowship grants were given by the
University Grant Commission, Government of India (to J.A.S.),
Department of Biotechnology, Government of India (to K.S.),
Council of Scientific and Industrial Research, Government of
India (to B.C. and G.S.). The authors are thankful to the
Sophisticated Analytical Instrument Facility (SAIF) of CSIR
Central Drug Research Institute, Lucknow, for the character-
ization of compounds. The technical assistance of A. S.
Kushwaha, Mahesh Chandra Tiwari, and Z. Ali is also
acknowledged. The CSIR-CDRI communication number to
this manuscript is 8323.
ABBREVIATIONS USED
■
BMP-2, bone morphogenetic protein 2; HRT, hormone
replacement therapy; PTH, parathyroid hormone; TGF-β,
transforming growth factor β; SAR, structure−activity relation-
ship
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