Design and synthesis of dalbergin analogues and evaluation of anti-osteoporotic activity

Article abstract of DOI:10.1016/j.bmcl.2017.02.062

The chemical modifications of the hydroxyl group of dalbergin have been described via the introduction of cyclic amine, ester and amide groups. Among the twenty-three prepared novel analogues of dalbergin, compound 4d (EC₅₀ 2.3?μM) showed significantly increased proliferation as assessed by alkaline phosphatase activity and mineralization in calvarial osteoblast cells in vitro. Compound 4d, at a dose of 1.0?mg/kg body weight exhibited the significant osteoprotective effect. It showed a significant increase in osteogenic gene expression RunX2 (～4fold), ALP (～5fold), OCN (～4fold) and COL1 (～4fold) as compared to control group at the same dose in vivo assay.

Full text of DOI:10.1016/j.bmcl.2017.02.062

Accepted Manuscript
Design and synthesis of dalbergin analogues and evaluation of anti-osteoporotic
activity
Padam Kumar, Priyanka Kushwaha, Naseer Ahmad, Saransh Wales Maurya,
Kapil Dev, Vikram Khedgikar, Ibadur Rahman Siddiqui, Ritu Trivedi, Rakesh
Maurya
PII:
S0960-894X(17)30205-6
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.062
BMCL 24734
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
29 October 2016
8 February 2017
24 February 2017
Please cite this article as: Kumar, P., Kushwaha, P., Ahmad, N., Maurya, S.W., Dev, K., Khedgikar, V., Siddiqui,
I.R., Trivedi, R., Maurya, R., Design and synthesis of dalbergin analogues and evaluation of anti-osteoporotic
activity, Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.062
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.els evier.com
Design and synthesis of dalbergin analogues and evaluation of anti-osteoporotic
activity
Padam Kumara,#, Priyanka Kushwahab,c,#, Naseer Ahmadc, Saransh Wales Mauryaa,d, Kapil Deva,b, Vikram Khedgikarc, Ibadur Rahman Siddiquid, Ritu
Trivedic,*and Rakesh Maurya a,b,
*
aMedicinal and Process Chemistry Division, CSIR- Central Drug Research Institute, Lucknow 226031, India
bAcademy of Scientific and Innovative Research, CSIR- Central Drug Research Institute, Lucknow 226031, India
cEndocrinology Division, CSIR-Central Drug Research Institute, Lucknow 226031, India
dDepartment of Chemistry, University of Allahabad, Allahabad 211002, India
ARTICLE INFO
ABSTRACT
Article history:
Received
The chemical modifications of the hydroxyl group of dalbergin have been described
via the introduction of cyclic amine, ester and amide groups. Among the twenty three
prepared novel analogues of dalbergin, compound 4d (EC50 2.3 µM) showed
significantly increased proliferation as assessed by alkaline phosphatase activity and
mineralization in calvarial osteoblast cells in vitro. Compound 4d, at a dose of 1.0
mg/kg body weight exhibited the significant osteoprotective effect. It showed a
significant increase in osteogenic gene expression RunX2 (~4fold), ALP (~5fold),
OCN (~4fold) and COL1 (~4fold) as compared to control group at the same dose in
vivo assay.
Revised
Accepted
Available online
Keywords:
Dalbergin
Aminodalbergin
Ritter Reaction
Post-menopausal osteoporosis
Micro-CT
2016 Elsevier Ltd. All rights reserved
Osteoporosis,
a
progressive
skeletal
disease
Neoflavonoid is a group of naturally occurring
characterized by decreased bone mass with micro-architectural
deterioration, which leads to the risk of fractures in both men and
women.1 Clinically, it is called as a silent disease because it
progresses without pain and symptoms until the bone fracture
occurs. A variety of factors are responsible for osteoporosis e.g.
level of sex hormones, calcium supplements, dietary supplements,
lifestyle, medication and aging. During bone remodeling,
osteoblasts fail to completely refill the resorption pits2 and hence,
cause osteoporosis. Women lose about 50% of their cancellous
bone and about 35% of cortical bone over their life span.3
Estrogen is effective in inhibiting bone resorption and increasing
bone mineral density (BMD) by binding to the estrogen receptors
on bone and blocking production of specific cytokines that are
responsible for increasing the number of osteoclasts and prolong
osteoblasts lifespan.4 Currently available drugs used to treat
osteoporosis are very few such as estrogen receptor modulators,
calcitonin and bisphosphates, and their effects on increasing or
recovering bone mass are relatively small, generally less than
10% over three years. The use of these drugs is associated with
several severe side effects such as breast cancer, cardiovascular
disease and uterine bleeding.5,6
compounds of fifteen carbons and having a 4-phenylchroman type
of skeleton. These are structurally and biosynthetically related to
flavonoids and isoflavonoids.7 There are a number of publications
available which show the broad spectrum of activities of
neoflavonoids
such as
antimicrobial,8 antiplasmodial,9
osteogenic,10 antifungal,11 anti-inflammatory,12 antidiabetic,13
anticancer14 etc. The fundamental neoflavonoid dalbergin, has
attracted several research groups for chemical modifications due
15
to its enormous biological activities like anti mycobacterium,
16
17
antidiabetic, and cardiovascular. and cytotoxicity18. Due to
such interesting biological activities and development of several
new synthetic methodologies, make it easy to synthesize
derivatives of such precious natural products in good yield.
Therefore, herein, we were keenly interested in exploring the
chemical modifications of dalbergin and the biological evaluation
of synthesized analogues for their osteogenic activity.
Recently, we have reported anti-osteoporotic activity of
dalbergin, where it exhibited increased alkaline phosphatase
activity and mineralization at a lower concentrations.10 Thus,
these results and also our ongoing research of interest in search of
biologically active natural product derived compounds19 prompted
us to isolate dalbergin in large amounts for further chemical
modifications for the management of osteoporosis.
Therefore, there is an urgent need to develop new safe
and alternative bone-building agents which can stimulate bone
formation and reduce the imbalance between osteoclast and
osteoblast cells in osteoporosis.
Structurally, dalbergin (1) has a free hydroxyl group at
position 6 which could provide a wide window to explore it for
biological importance. Therefore, we designed our synthetic
approach for the synthesis of derivatives of dalbergin via the
chemical modifications of the hydroxyl group at position 6
(Schemes 1-3).
*Corresponding author.
Dr. Rakesh Maurya: mauryarakesh@rediffmail.com
Dr Ritu Trivedi: ritu_trivedi@cdri.res.in
#Authors contributed equally
Initially, we synthesized compound 2 by heating a
mixture of dalbergin (1.0 eq.) and 1-bromo-2-chloroethane (1.2
Tel.: +91-(522)-2772450; Fax: +91-(522)-2771941;
CDRI Communication number

eq.) in presence of base K2CO3 (1.5 eq.) in acetone (40 mL). The
compound 2 (1.0 eq.) was further refluxed with piperidine (1.1
eq.) in 10 mL ethanol in the presence of triethylamine for 24 h to
get the piperidine derivative of dalbergin (2a).20 Similar procedure
was utilized to the synthesis of other nitrogenous derivatives (2b-
2d) of dalbergin. Furthermore, to the evaluate the effect of linker
chain on the osteogenic activity, we synthesized a series of
compounds 3a-3d and 4a-4d containing cyclic amine group with
extended aliphatic linker chain (Scheme 1).
the synthesis of amide analogue of dalbergin, compound 7 was
treated with p-methoxy benzonitrile (3.0 eq.) and the dropwise
addition of BF3.OEt2 (0.5 eq.) at 0°C in dry CH2Cl2 and the
compound 8 afforded as amide after the aqueous work up and
chromatographic purification in 54% yield (Scheme 3).23 All the
synthesized compounds were characterized by extensive analysis
of 1D and 2D NMR as well as mass spectral analysis and the
purity of compounds was determined by HPLC analysis before
biological evaluation.
As our approach relied on the chemical modification of
All the synthesized compounds were evaluated for their
alkaline phosphatase activity in vitro. For the measurement of
alkaline phosphatase (ALP) enzyme activity (osteoblast
differentiation marker), calvarial osteoblast cells were trypsinized
and 2×103 cells were plated onto 96 well plates for ALP activity.
Treated cells were incubated for 48h in α-MEM supplemented
with 10% FBS, 10 mM β-glycerophosphate, 50 µg/mL ascorbic
acid, and 1% penicillin/streptomycin (osteoblast differentiation
medium). At the termination day, total ALP activity was
measured
hydroxyl group of dalbergin at position 6, hence we further
synthesized the ester analogues from substituted carboxylic acids.
The compound 1 was treated with different carboxylic acid
chlorides (1.0 eq.), pyridine (0.4 mL) and DMAP (cat.) in dry
CH2Cl2, and afforded their respective ester analogues 5a-5e in 74-
82% yield (Scheme 2).21 Taking the consideration of the effect of
aliphatic group at position 6, we methylated hydroxyl by the
reaction of methyl iodide (2.0 eq.), K2CO3 (1.5 eq.) in dry acetone
(20 mL), afforded methyl dalbergin 6 in 92% yield.
The hydroxyl group of dalbergin provides a wide
Using p-nitrophenyl phosphate (PNPP) as substrate and
absorbance was read at 405 nm.24-26
prospect for the synthesis of the amide derivative of dalbergin via
Ritter reaction (Scheme 3). In this consequence, we synthesized
compound 7 by stirring the mixture of epichlorohydrin (2.0 eq.),
K2CO3 (3.0 eq.) in dry acetone for 6h at 50°C.22 Furthermore, to
Scheme 1: Synthesis of amine analogues of dalbergin at position 6 (2, 2a-2d, 3, 3a-3d, 4 and 4a-4d). Reagents and Conditions: (a) Dihalo alkane (1.2 eq.), K2CO3
(1.5 eq.), Acetone (40 mL), 4h. (b) Cyclic amine (1.1 eq.), triethylamine, ethanol (10 mL), reflux, 24 h.
Scheme 2: Synthesis of ester and methoxy derivatives of dalbergin at position 6 (5a-5e and 6). Reagents and Conditions: (a) RCOCl (1.2 eq.), Pyridine (0.4 mL),
DMAP (cat.), dry CH2Cl2, 0°C-rt, 6–8 h. (b) Methyl iodide (1.2 eq.), K2CO3 (1.5 eq.), Acetone (20 mL), 4 h.

O
O
O
O
O
O
O
O
O
O
O
O
a
b
N
H
HO
Aqueous
workup
OH
MeO
O
8
7
1
Scheme 3: Synthesis of amide analogue (8) of dalbergin via Ritter reaction. Reagents and Conditions: (a) Epichlorohydrin (2.0 eq.), K2CO3 (3.0 eq.), Acetone (20
mL), 50°C, 6 h. (b) p-Methoxybenzonitrile (3.0 eq.), BF3.OEt2 (0.5 eq.), Dry CH2Cl2 (5 mL), 0°C-rt, 2 h.
Out of twenty three compounds, only fifteen compounds showed
ALP activity at different concentration [Fig.1]. EC50 and Emax
values were calculated on the basis of ALP activity (Table 1).
mice calvarial osteoblast cells. Osteoblast cells were treated with
or without active compounds for twelve days and further fixed in
4% formaldehyde. Fixed cells were stained with alizarin red-S
stain, extracted and quantified by using acetic acid method.27,28
Quantification of alizarin extraction showed that the compounds
2b, 2c, 2d, 3, 3a, 4, 4a, 4b and 4d having calcium nodule
formation activity [Fig. 2] but compound 2d, 3a, 4a and 4d
showed maximum calcium deposition at active concentration.
Active compounds 2d, 3a, 4a and 4d were further tested
Table 1: Emax and EC50 of compounds for alkaline phosphatase
activity:
Compound
Emax
1 pM
-
EC50
19.4 nM
-
2
2a
2b
2c
2d
3
for osteoblast differentiation and mineralization marker genes
BMP2, RunX2 and COL1 at the active concentration in calvarial
osteoblast cells by Q-PCR. BMP2 (Bone Morphogenetic Protein
2) is the potential inducer of osteoblast differentiation; RunX2 is
1 pM
1 pM
1 pM
100 pM
1 pM
-
11.7 nM
13.6 nM
14.8 nM
5.9 nM
7.6 nM
-
transcription factor and
a
key regulator of osteoblast
differentiation whereas COL1 is the key component during
mineralization. Our results showed that after treatment with active
concentration, compound 4d showed maximum expression of
BMP2 (~2fold), RunX2 (~2fold) and COL1 (~6fold) [Fig. 3].29,30
3a
3b
3c
3d
4
10 nM
100 pM
10 nM
1 pM
10nM
-
27.9 nM
9.42 nM
5.7 nM
10.8 nM
0.9 µM
12.4 µM
For ALP (alkaline phosphatase) activity, we tested
dalbergin and its derivative compounds on calvarial osteoblast
cells which are used to investigate the modulatory effects for
osteogenic characteristic. Osteoblast cells serve as an important
tool to study bone cell proliferation, differentiation and
mineralization at the time of bone formation and maturation. The
activity of ALP increases at the time of early differentiation and
have distinguished characteristic of osteogenic cells in culture
that possess the ability to promote bone matrix mineralization.
During this phase the osteoblast cells have high levels of the
membrane bound enzyme alkaline phosphatase that initiates pre-
osteoblast to differentiate into osteoblast lineage. By measuring
the ALP activity dalbergin and its derivatives were screened for
their ability to induce osteogenesis. In-vitro studies showed that
dalbergin and its derivatives such as compound 4d had cell
differentiation, proliferation ability in osteoblast cells and
compound 4d showed significant enhancement in ALP activity
compared with dalbergin as shown in figure 1.
4a
4b
4c
4d
5a
5b
5c
5d
5e
6
1pM
-
1µM
10nM
1µM
-
-
-
-
2.3 µM
-
0.8nM
7.4nM
1.1nM
-
-
-
-
7
8
Compounds showing increased alkaline phosphatase
were further tested for the ability to form mineralized nodule in

Fig. 1: Dalbergin analogues have osteoblast differentiation property in primary osteoblast cells. After 48h of treatment, ALP activity was measured at 405 O.D.
Data was represented as the means SE from three independent experiments: ∗∗∗p<0.001, ∗∗p<0.01 and ∗p<0.05 compared with untreated cells taken as control
and #p< 0.05 when compound 4d was compared with dalbergin.
Fig. 2: Measurement of mineralized nodule formation of active compounds by mineralization assay in osteoblast cells. Upper panel shows representative
photomicrographs of mineralized nodules formation in active compounds with or without treatment. Bar diagram in the lower panel shows spectrophotometric
quantification of mineralized nodule by extraction of Alizarin Red-S dye. O.D. has taken at 405 nm. Data are represented as the means SE from three
independent experiments: ∗∗∗p<0.001, ∗∗p<0.01 and ∗p<0.05 compared with untreated cells taken as control
.

Fig. 3: Relative m-RNA expression of osteogenic genes Bmp2, RunX2, and COL1 of active compounds 2b, 2d, 3a, 4a and 4d in mice calvarial osteoblast cells.
Calvarial osteoblasts were treated with compound 2b, 2d, 3a, 4a and 4d at 1 pM for 24h and compare with control. GAPDH is used as internal control. All data
represents the means SE from three independent experiments: ∗∗∗p<0.001, ∗∗p<0.01 and ∗p<0.05 compared with untreated cells taken as control.
To confirm the gene expression data and bone formation
property of compound 4d, we performed in vivo study. In this
study, three-week-old Balb/c mice were injected subcutaneously
over the calvarial surface with or without the treatment of lead
compound 4d for five consecutive days at the doses of 0.1, 0.5, 1
and 5 mg/kg per day (3 mice per group). All mice were
euthanized after treatment and calvaria were dissected, fixed in
4% formaldehyde, and embedded in paraffin. Histologic sections
were cut and stained with hematoxylin and eosin (H&E).31
For fluorochrome labeling and bone histomorphometry,
each mouse received intraperitoneal administration of
fluorochromes tetracycline (20 mg/kg) and calcein (20 mg/kg),
following a previously published protocol.25 Histomorphometric
analysis of bone formation, such as for the determination of
mineral appositional rate (MAR) and bone formation rate (BFR),
was performed using Leica-Qwin software (Leica Microsystems
Inc., Buffalo Grove, IL, U.S.) as described in our previously
published protocol.32,24
light/12h dark cycles. Normal chow diet and water were provided
ad libitum. Three groups were taken for the further study. The
animals were ovariectomized and left for 6 weeks for osteopenia
to develop. Thereafter, the animals were divided into groups as
follows: sham (ovary intact) + vehicle (gum acacia in distilled
water), OVx + vehicle, OVx + 1.0 (mg/kg)/day body weight dose
of 4d. Mice were treated with compound 4d or vehicle once daily
for 6 weeks by oral gavage. After the period of 6 weeks, animals
were sacrificed and bones were collected for analysis of trabecular
microarchitecture. µCT experiments were carried out using Sky
Scan 1076 micro-CT scanner (Aartselaar, Belgium) as previously
reported.32
It was observed that after subcutaneous injection of 4d
at 0.5 mg/kg/d and 1.0 mg/kg/d per day into mouse calvaria
significantly stimulated bone formation. Above result was
confirmed as shown by histological analysis of calvaria samples
harvested after the last injection of tetracycline and stained with H
and E. Histomorphometric analysis revealed that compound 4d
showed a significant effect on bone formation [Fig. 4(A)]. This
effect, however, was not observed when compound 4d was used
at a higher dose of 5.0 mg/kg per day. For further confirmation of
bone formation capability of compound 4d, we used dynamic
histomorphometric analysis followed by calcein double labeling at
a higher dose of 5.0 mg/kg per day. Results showed that there was
significant difference between control and compound 4d at 1.0
mg/kg dose which is demonstrated by an increased mineral
appositional rate (MAR) at 0.1 mg (p<0.01), 0.5 mg and 1.0 mg
(p<0.001) significantly and the bone-formation rate (BFR) at 0.1
mg (p<0.01), 0.5 mg and 1.0 mg (p<0.001) increased significantly
in calvarial osteoblast in vivo [Fig. 4(B) & 4(C)].
In addition, at 1.0 mg/kg dose of compound 4d showed
enhanced osteogenic gene expression RunX2 (~4fold), ALP
(~5fold), OCN (~4fold) and COL1 (~4fold) as compared to
control group and play role in bone formation [Fig. 4(D)].
Eight-week old adult female Balb/c mice were used for
the analysis of trabecular bone formation in ovariectomized post-
menopausal model of mice. Animals were housed at 21°C in 12h

loss in OVx mice. Treatment with 1.0 mg dose increased ~1.5
fold bone volume/ tissue volume (BV/TV) compared to OVx
group of femoral metaphysis [Fig. 5(B)]. µ-CT analysis of
femoral trabecular bone revealed that trabecular number (Tb.No)
(p<0.001) and trabecular thickness (Tb.Th) (p<0.05) also
significantly increased as compared to OVx group [Fig. 5C) &
5(D)]. At the same time, trabecular separation (Tb.Sp) (p<0.001)
and structural model index (SMI) (p<0.001) has decreased in
treated group [Fig. 5(E) & 4(F)]. Tibia and lumber 5 vertebrae
have the same effect (Table 2). Biomechanical strength analysis
revealed that femur breaking strength stiffness (p<0.01) and
energy (p<0.01) is significantly increased in 1.0 mg/kg treatment
group as compared to OVx. Analysis of vertebrae compressive
strength in treated group revealed that stiffness (p<0.01) and
energy (p<0.05) significantly increased as compared to OVx
group as shown in Table 2.
Dynamic histomorphometric analysis of femur diaphysis
indicated that supplementation of compound 4d at 1.0 mg/kg dose
increased mineral apposition rate (MAR) (p<0.01) and bone
formation rate (BFR) (p<0.05) in the femur bone as compared to
OVx. Above data indicate that lead compound 4d enhanced bone
formation at active dose in an osteopenic model as mentioned in
Table 2.
Fig. 4: Compound 4d promotes new bone formation in mice calvaria. (A)
Compound 4d was injected subcutaneously over the surface at 0.1, 0.5, 1.0
and 5.0 mg/kg/d dose of 3 week-old Balb/c mice for 5 consecutive days. The
left panel shows longitudinal histomorphometric section treated and control
mice calvaria with
H and E staining (5µM). The right panel shows
fluorochrome labeling with calcein. 1.0mg/kg/d dose showed significantly
induced bone formation (Yellow arrow). (B) Measurement of mineral
apposition rate (MAR) and (C) bone formation rate (BFR) in mice calvaria
after calcein injection (20 mg/kg). All data represents the Mean SE from
three independent experiments: ∗∗∗p<0.001, ∗∗p<0.01 and ∗p<0.05 compared
with untreated cells taken as control. (D) Relative m-RNA expression of
osteogenic genes RunX-2, ALP, OCN and COL-1 in mice calvaria after
injecting different dose concentration. Dose 1 mg/kg/d have significantly
promoted osteogenic genes expression compare to other doses. All data
represents the Mean SE from three independent experiments: ∗∗∗p<0.001,
∗∗p<0.01 and ∗p<0.05 compared with control.
Ovariectomy causes the reduction in estrogen, resulted
in decreased uterine weight, luminal area and epithelial cell height
of uterus shown in Table 2. Administration of compound 4d at 1.0
mg/kg dose results in no change in uterine parameters. Results
revealed that compound 4d has no estrogenic effect on the uterus.
µ-CT analysis of femur bone demonstrated an apparent
bone loss in the ovariectomized mice compared with those that
underwent sham surgery. Oral dose supplementation of compound
4d for 6 weeks at 1.0 mg/kg dose significantly reversed the bone
Table 2: Different parameters evaluated in this study with compound 4d.
Parameters
) (A) Tibia micro-CT
BV/TV (%)
Sham
OVx
OVx+1mg
14.70 1.16***
3.11 0.18***
0.053 0.001***
0.238 0.006***
1.84 0.04***
6.43 0.47
1.49 0.25
0.0475 0.0005
0.387 0.038
2.32 0.08
13.53 0.63***
2.70 0.11***
0.052 .0005***
0.294 0.015*
1.84 0.03***
Tb.No. (mm-1)
Tb.Th. (mm)
Tb.Sp. (mm)
SMI
(B) Vertebrae micro-CT
BV/TV (%)
15.91 0.61**
3.61 0.27**
0.053 0.001*
0.213 0.016**
1.46 0.07*
13.92 1.66
2.24 0.16
23.43 1.79***
4.63 0.33***
0.0522 0.0006*
0.167 0.005***
1.07 0.08***
Tb.No. (mm-1)
Tb.Th. (mm)
0.047 0.001
0.288 0.021
1.78 0.08
Tb.Sp. (mm)
SMI
(C) Bone Strength
(i) Femur breaking strength
Energy (mJ)
5.6 0.57***
2.37 4.18**
2.6 0.18
34.3 1.84
4.55 0.02**
53.55 2.04**
Stiffness (N/mm)
(ii) Vertebrae compressive strength
Energy (mJ)
Stiffness (N/mm)
418.25 6.86***
468.75 9.32***
224.75 1.06
345.7 5.08
262.55 9.12**
381.35 10.76*
(D) Bone Histomorphometric analysis
Mineral apposition rate (µm/day)
Bone formation rate (µm3µm2/day)
(E) Uterine Histomorphometric analysis
Uterine weight (mg)
Total uterine area (µm2)
Luminal area (µm2)
Luminal epithelial height (µm)
0.283 0.016***
0.32 0.01***
0.099 0.01
0.098 0.01
0.177 0.01**
0.163 0.009*
0.145 0.008
4809.6 610.9
431.76 11.61
1.39 0.06
0.025 0.002# # #
837.2 36.09# # #
57.5 1.19# # #
0.55 0.03# # #
0.032 0.007# # #
958.6 20.44# # #
75.75 4.06# # #
0.66 0.03# # #
Compound 4d treatment improves micro-architectural parameters in tibia (A) and vertebrae (B) of OVx mice. Bone volume/tissue volume (BV/TV),
Trabecular number (Tb.No), Trabecular thickness. (Tb.Th), Trabecular separation (Tb.Sp), Structure model index (SMI). All values are expressed as mean SE;
*P< 0.05; **P< 0.01 and ***P< 0.001 compared with OVx group. (C) Compound 4d treatment improves bone biomechanical strength of femur (i) and
vertebrae (ii) in OVx mice. Compound 4d treated group shows improved femoral and vertebral strength represented by energy and stiffness,
respectively. All values are expressed as mean SE; *P< 0.05; **P< 0.01 and ***P< 0.001 compared with OVx group. (D) Oral administration of Compound
4d has the ability to form new bone as assessed by MAR and BFR (Mineral apposition rate and Bone formation rate). All values are expressed as mean
SE; *P< 0.05; **P< 0.01 and ***P< 0.001 compared with OVx group. (E) Compound 4d treatment exhibited no uterine estrogenic effect in OVx mice as

shown by uterine weight and histomorhometric parameters (total uterine area, luminal area and luminal epithelial height), which show that compound
4d treated group had histological features comparable to those of OVx mice. All values are expressed as mean SE; # # #P< 0.001 compared with Sham group.
Extraction and quantification of the dye showed that compound
4d treated group enhanced mineralization about ~50% (P<0·01)
as compared to OVx group as shown in Fig. 6(C). Overall data
suggested that compound 4d has the ability to differentiate
BMC’s into osteoblast lineage cells.
We further assessed the osteogenic gene expression in
long bones. Lead compound 4d significantly increased BMP2
(p<0.001), RunX2 (p<0.001), Osx (p<0.001), ALP (p<0.01), OPG
(p<0.001), OCN (p<0.001) and COL1 (p<0.001) expression as
compared to OVx group [Fig. 6(D)].
Ovariectomy resulted in the increase of osteoclast genes
expressions (bone resorbing cells) which were reverted back after
treatment with compound 4d at 1.0 mg dose. Expression of
RANK (receptor activator of nuclear factor kappa B) (p<0.01),
RANKL (receptor activator of nuclear factor kappa B ligand)
(p<0.001), TRAP (tartrate-resistant acid phosphatase) (p<0.001)
was reduced by 4d at 1.0 mg dose as compared to OVx group
[Fig. 6 (E)].
Serum samples were isolated from blood of control and
compound 4d treated group after autopsy for analysis of bone
turnover marker. On the basis of our previously published
protocols, serum osteocalcin (mid-portion) was determined by
enzyme-linked immunosorbent assay kits by following the
manufacturer's protocols (Immunodiagnostic Systems Inc.).31
During bone remodeling, osteocalcin is produced by osteoblasts
Fig. 5: Compound 4d reversed bone loss induced by ovariectomy in mice
without uterine estrogenic effect. (A) 3D µ-CT image of trabecular micro-
architecture of femoral metaphysis bone (B) Assessment of bone
microarchitectural parameters including Bone volume/Tissue volume
and forms matrix component. Osteocalcin levels were increased
(BV/TV) (C) Trabecular number (Tb.no) (D) Trabecular thickness (Tb.Th),
during rapid bone turnover (OVx model). The OVx-induced bone
(E) Trabecular separation (Tb.Sp) and (F) Structure Model Index (SMI)
loss was characterized by high bone turnover rates, as represented
respectively in various groups. Treatment of 4d at a dose of 1.0 mg/kg/d to
by higher levels of serum osteocalcin (OCN) ~2 fold compared
with the sham group [Fig. 6(F)]. However, 6 weeks of compound
4d at 1.0 mg dose treatment to OVx group significantly lowered
the level of OCN, indicating that compound 4d was effective in
lowering the serum OCN levels and bone turnover.
OVx mice improved trabecular micro-architecture, as is clearly visible when
compared with OVx group in mice. Data were represented as the mean
SEM. ∗∗∗p < 0.001, ∗∗p < 0.01, and ∗p < 0.05 compared with OVx group.
To determine the effect of the compound 4d on the
ability of bone marrow cell differentiation towards osteogenic
lineage, ex-vivo culture of BMC’s was performed. BMC’s from
long bone were isolated and cultured for 21 days in osteogenic
differentiation media. Fig. 6(A) showed that BMC’s harvested
after treatment of compound 4d show significantly enhanced
alkaline phosphatase (ALP-Early osteoblast differentiation)
activity (about ~2 fold) as compared to OVx group. Further, we
analyzed the mineralizing ability of the BMC’s that started to
form mineralizing nodules at 12th day of culture and maximum
calcium nodules were observed at day 21 of culture. At day 21,
the number of Alizarin Red-stained calcium nodules [Fig. 6(B)]
was increased in compound 4d at 1.0 mg/kg dose treated group.
Further, on the assessment of compound 4d in fracture
healing model of mice by administrating the 1.0 mg/kg dose for
21 days in mice model, we analyzed the µ-CT parameters at drill
hole site of the various group (representative images in fig. 7
A).Quantification of various µ-CT parameters showed that active
dose of 1.0 mg/kg improved the bone micro-architecture
parameters. Compound 4d at active dose increased bone volume
(BV/TV, p<0.05), trabecular thickness (Tb.Th, p<0.001),
trabecular number (Tb.N, p<0.01), and decreased trabecular
separation (Tb.Sp, P<0.01) and structure model index (SMI,
P<0.001) (Table 3) as compared to control.

Fig. 6: Compound 4d promotes osteoblast mineralization in BMCs through inhibiting osteoclast gene expression. (A) Ex vivo experiments showed that
BMCs (Bone marrow cells) were differentiation toward osteoblast lineage measured by ALP activity compared with OVx group (B) Oral administration of
Compound 4d has the ability to form mineralized nodule in BMCs compared to OVx group assessed by mineralization assay. The upper panel showed the
representative photomicrographs of mineralized nodules formation in various experimental groups (C) lower panel showed quantification of alizarin dye. O.D.
has taken at 405nM. Both data are represented as the means SE. ∗∗∗p<0.001, ∗∗p<0.01 and ∗p<0.05 compared with OVx group (D) Relative m-RNA
expression of osteogenic genes in treated and control groups. Compound 4d induced the m-RNA expression of osteogenic genes Bone morphogenic protein-2
(BMP-2), RunX-2, Osterix (Osx), alkaline phosphatase (ALP), Osteoprotegerin (OPG), Osteocalcin (OCN) and Collagen I (Col-I) in femoral bone. All values
were normalized with GAPDH. (E) Relative m-RNA expression of osteoclast genes including Receptor Activator of Nuclear Factor k (RANK), Receptor
Activator of Nuclear Factor-k legend (RANKL), and Tartrate-Resistant Acid Phosphatase (TRAP) and normalizing with housekeeping gene glyceraldehydes-3-
phosphate dehydrogenase (GAPDH). Data was represented as the means SEM. ∗∗∗p<0.001, ∗∗p<0.01 and *p<0.05 compared with OVx group. (F)
Measurement of serum osteocalcin levels (bone turnover marker). Administration of Compound 4d at 1mg/kg dose modulated the bone turnover in favor of bone
formation. Results represent pooled data as means SE. ∗∗p<0.01, compared with OVx group.

Table 3: Micro-CT analysis of bone at drill-hole site
Parameters
Acknowledgement
Authors are thankful to CSIR and UGC for providing
Control
1 mg/kg
fellowships. Saransh W. Maurya is thankful to University of
Allahabad, Allahabad for providing fellowship and Director
CSIR-CDRI for providing opportunity of academic training in
CDRI, Lucknow, India. Rakesh Maurya is grateful to CSIR, New
Delhi, India, for providing emeritus scientist scheme with
reference no. 21(1019)/16/EMR-II. The grant from DBT
(Department of Biotechnology GAP0127), CSIR sponsored
network project CSC0130, BSC103, AcSIR and CSIR,
Government of India are acknowledgment. Authors are also
grateful to SAIF division of CDRI for providing spectral data.
BV/TV (%)
Tb.Th (mm)
Tb.No (mm-1)
Tb.Sp (mm)
SMI
32.924 3.806
0.111 0.006
2.194 0.265
0.219 0.015
2.585 0.387
44.359 1.912*
0.166 0.010***
3.442 0.262**
0.164 0.007**
0.496 0.081***
Compound 4d (1mg/kg) improves micro-architectural parameters at the
drill-hole site. Bone volume/tissue volume (BV/TV), Trabecular thickness.
(Tb.Th), Trabecular number (Tb.No), Trabecular separation (Tb.Sp),
Structure model index (SMI). All values are expressed as mean
SE;
*P< 0.05; **P< 0.01 and ***P< 0.001.
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Graphical Abstract
Design and synthesis of dalbergin analogues and evaluation
of anti-osteoporotic activity
Leave this area blank for abstract info.
Padam Kumar, Priyanka Kushwaha, Naseer Ahmad, Saransh Wales Maurya, Kapil Dev, Vikram Khedgikar,
Ibadur Rahman Siddiqui, Ritu Trivedi and Rakesh Maurya
A series of dalbergin analogues have been synthesized. Among them, compound 4d showed singnificant increased
proliferation as assessed by alkaline phosphatase activity and mineralization in calvarial osteoblast cells in in vitro
as well as in vivo.