Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents

Article abstract of DOI:10.1016/j.bmcl.2017.02.004

The structural modifications of pregnenolone have been described via the introduction of heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized derivatives, four compounds showed significantly increased ALP activity. Among all four active compounds, the novel compound 3a has shown significant bone matrix mineralization and mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1 pM concentration.

Full text of DOI:10.1016/j.bmcl.2017.02.004

Accepted Manuscript
Synthesis and biological evaluation of heterocyclic analogues of pregnenolone
as novel anti-osteoporotic agents
Saransh Wales Maurya, Kapil Dev, Krishna Bhan Singh, Reena Rai, Ibadur
Rahman Siddiqui, Divya Singh, Rakesh Maurya
PII:
S0960-894X(17)30125-7
DOI:
http://dx.doi.org/10.1016/j.bmcl.2017.02.004
BMCL 24676
Reference:
Bioorganic & Medicinal Chemistry Letters
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Revised Date:
Accepted Date:
3 January 2017
25 January 2017
2 February 2017
Please cite this article as: Maurya, S.W., Dev, K., Singh, K.B., Rai, R., Siddiqui, I.R., Singh, D., Maurya, R.,
Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as novel anti-osteoporotic agents,
Bioorganic & Medicinal Chemistry Letters (2017), doi: http://dx.doi.org/10.1016/j.bmcl.2017.02.004
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Saransh Wales Maurya, Kapil Dev, Krishna Bhan Singh, Reena Rai, Ibadur Rahman Siddiqui, Divya Singh,
Rakesh Maurya

Bioorganic & Medicinal Chemistry Letters
Synthesis and biological evaluation of heterocyclic analogues of pregnenolone as
novel anti-osteoporotic agents
Saransh Wales Mauryaa, b, Kapil Deva, Krishna Bhan Singhc, Reena Raic, Ibadur Rahman Siddiquib, Divya
Singhc, Rakesh Mauryaa,
aMedicinal and Process Chemistry Division, CSIR-Central Drug Research Institute, Lucknow-226031, India
bDepartment of Chemistry, University of Allahabad, Allahabad-211001, India
cEndocrinology Division, CSIR-Central Drug Research Institute, Lucknow-226031, India
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
The structural modifications of pregnenolone have been described via the introduction of
heterocyclic moieties at C-17 position by limiting the acyl group. Novel heterocyclic analogues
of pregnenolone have been synthesized by using Friedlander and Claisen-Schmidt reactions, and
the synthesized compounds were evaluated for their osteogenic activity. Among the synthesized
derivatives, four compounds showed significantly increased ALP activity. Among all four active
compounds, the novel compound 3a has shown significant bone matrix mineralization and
mRNA expressions of osteogenic marker genes, BMP2, RUNX-2 and OCN at 1 pM
concentration.
Keywords:
Pregnenolone
Friedlander reaction
Chalcone derivatives
Osteoporosis
2009 Elsevier Ltd. All rights reserved.
ALP activity
The skeleton is extremely dynamic tissue where bone
remodeling is ongoing and activated osteoclasts resorb bone
while osteoblasts generate bone matrix.1 This process serves to
repair microdamage and ensure skeletal strength.2 Numerous
systemic mediators regulate bone cell activity, including
because of multiple factors, including cost and the inconvenience
of daily injections.10 Besides there are reports of osteosarcoma
risk in patients taking PTH due to which its therapy is restricted
to two years.11 Other treatments include Denosumab,
a
monoclonal antibody that potently blocks the binding of
osteoblast-derived RANKL to its osteoclast-derived receptor
(RANK), an interaction that is required for osteoclast formation,
activation, and survival. By blocking this receptor binding,
denosumab potently inhibits osteoclast mediated bone
resorption.12 Romosozumab is another monoclonal antibody
targeted against sclerostin, inhibitor of Wnt signaling pathway
that is developed for osteoporosis treatment.13
endogenous parathyroid hormone, vitamin
D metabolites,
prostaglandins, cortisol, and hormones. Disruption in the growth
and maintenance of skeleton can result in bone disorders like
osteoporosis.3 Osteoporosis is characterized by low bone mass
and loss of structural integrity resulting in increased bone
fragility and susceptibility to fracture.4 Osteoporosis affects
nearly 75 million people in Europe, USA and Japan.5 In India,
osteoporosis has emerged as a serious problem. One out of eight
males and one out of three females in India suffers from
osteoporosis, making India one of the largest affected countries
in the world.6 Approximately 80% of the urban Indian population
is vitamin D deficient and hip fractures occur about a decade
earlier than in Western nations.7
Despite these advances, there is need for newer therapies as
the above mentioned therapeutic options are associated with
some or the other side effects. For instance, anti-resorptive
therapies though prevent bone loss but do not increases bone
mass. On the other hand, anabolic therapies like PTH are
associated with osteosarcoma risks and also do not inhibit bone
resorption. Thus, our efforts are to discover new potential agents
that promote osteoblast differentiation and may have a role in
management of bone related disorders.
Therapeutic options for osteoporosis have greatly expanded
over the past few decades. These include agents like nitrogen-
containing bisphosphonates, which act by suppressing bone
resorption.8,9 Parathyroid hormone (PTH) and teriparatide, the
only FDA-approved anabolic drug, also help to prevent fractures,
but are generally reserved for patients with severe disease
———
The core structure of the steroids is a seventeen carbon
arrangement of four fused rings, in which three rings are six
membered as cyclohexane and the fourth ring as five membered
*Corresponding author. Tel +91-(522)-2772450; fax: +91-(522)-2771941; e-mail: mauryarakesh@rediffmail.com

skeleton of cyclopropane.14 Steroids are well known for their
chemical and therapeutical importance as they have played major
role in drug discovery and development for different diseases
such as cancer, inflammatory diseases, and asthma etc.15-21
2-amino pyridine carboxaldehyde derivatives in ethanol and
KOH (aq.) at room temperature for 24h in good yields. Similarly,
the heterocyclic chalcone analogues (4a-4e) of pregnenolone
were synthesized by employing the Claisen-Schmidt reaction, in
which compound 1 was stirred with heteroaromatic aldehydes in
ethanol and KOH (aq.) at room temperature for 24-48h [Scheme
1].
Pregnenolone (1), a naturally occurring endogenous steroid
and is a well-known precursor for the biosynthesis of various
steroidal hormones like estrogen, progesterone, testosterone,
glucocorticoids and mineralocorticoids.22,23 Chemically, it
consists of four interconnected cyclic hydrocarbon rings with a
hydroxyl group at C-3 position, a double bond between C-5 & C-
6 and an acyl group at C-17 position.
As our approach relies on the modification of acyl group
present at C-17 of pregnenolone, hence we further synthesized
the imine and hydrazone analogues (5a-5c), by using hydroxyl
amine hydrochloride, hydrazine hydrate and p-toluene
sulfonylhydrazide [Scheme 2]. The compound 2 (1.0 eq.) was
stirred with hydroxylamine hydrochloride (1.1 eq.), 30% KOH
(aq.) in ethanol at room temperature for 4h, afforded compound
5a in 91% yield. Similarly, the compound 5b and 5c were
synthesized by heating the mixture of compound 2 (1.0 eq),
hydrazine hydrate or p-toluene sulfonylhydrazide (1.1 eq.), acetic
acid (cat.) in methanol (15 mL) at 50 C for 4-5h, respectively.
Furthermore, the existence of a double bond between C-5 & C-6
position of pregnenolone skeleton provided us an opportunity for
the synthesis of epoxide derivative [Scheme 3]. The epoxide
derivatives (6a & 6b) of pregnenolone 1 and pregnenolone
acetate 2 were synthesized by the reaction of mCPBA (1.5 eq.) in
chloroform for 24 h. Surprisingly, it was observed that the
epoxidation of pregnenolone yielded 5α,6α-epoxy pregnenolone,
whereas pregnenolone acetate provided a diastereomeric mixture
(3:1) of epoxy pregnenolone acetate (6b). The same observations
were also reported by other research groups.30,31 All the novel and
known synthesized compounds were characterized with the help
of extensive analysis of spectroscopic data and the purity was
analyzed by HPLC analysis before biological evaluation.
Figure 1: Structure of pregnenolone with possible active centers for
chemical modifications.
The unique structural features and the characteristic biological
properties of the compound 1, attracted several research groups
and several possible chemical modifications have been
reported.24,25 The literature surveys have demonstrated that the
synthesis of chalcone derivatives with aromatic aldehydes by
Claisen-Schmidt reaction is very common whereas very few
reports are available on the introduction of heterocyclic groups
by modifications of the acyl group in pregnenolone. The C-20
hydroxylated derivatives of pregnenolone have been found
effective in calcium-dependent processes and hence affect the
degree of depolarization of smooth muscle.26 The previously
pyrazoline,27 pyrazole,27,28 triazole,29 epoxide30,31 and chalcone28
analogues of pregnenolone have shown significant effect against
cancer cells. Similarly, Guo et al. have synthesized a series of 21-
aylidenepregnenolone and their epoxide analogues of
pregnenolone and evaluated their neuroprotective effect against
To determine the osteogenic potential of the synthesized
compounds, osteoblast differentiation assay were performed in
vitro because the osteoblast differentiation is one of the important
aspects in osteoblast development. The alkaline phosphatase
(ALP), osteocalcin (OCN) and type-1 collagen are some
important bone markers which are frequently used to follow the
differentiation process of osteoblasts. Thus, we decided to
determine the osteogenic efficacy of compounds by measuring
their ALP activity in mice calvarial osteoblasts. For the
measurement of Alkaline phosphatase (ALP) activity, 2x103 cells
were seeded per well of 96 well plates. Cells were treated with
different concentrations of compounds for 48 hr concentration in
α-MEM media containing 5% FCS, 10-mM β- glycerophosphate,
50µg/ml ascorbic acid and 1% penicillin/streptomycin (osteoblast
differentiation medium). After 48 hr incubation total ALP
activity was measured using p-nitrophenylphosphate (PNPP) as a
substrate and quantified colorimetrically at 405 nm.
beta-amyloid
and
hydrogen
peroxide
(H2O2)-induced
neurotoxicity in PC12 cells, and oxygen-glucose deprivation
(OGD)-induced neurotoxicity in SH-SY5Y cells.31
As per our aim of drug discovery from natural products and
their semi-synthetic analogues for anti-osteoporotic activity, we
have isolated and/or synthesized several natural product
analogues for the management of osteoporosis.32 Three active
centers are present in pregnenolone 1 which could facilitate and
attracted us for the possibility of new drug discovery and
development from this steroidal molecule.
All the compounds were treated in mouse calvarial osteoblast
cells at various concentrations ranging from 1 pM to 1 µM and
ALP activity was measured spectrophotometrically [Figure 2].
Among all the tested compounds, only four compounds 2, 3a, 3d,
and 5a showed significant increase in ALP activity compared to
the control untreated cells [Figure 2]. Among the compounds
under study, compound 3a showed maximum osteogenic efficacy
as assessed by ALP activity. It led to 100% and 85%
enhancement in ALP activity at concentrations of 1pM and
100pM, respectively which was the best amongst all other active
compounds. Hence, compound 3a was further chosen for
mineralization and qPCR experiments which determine the
expression of osteogenic mRNA levels.
In order to achieve our aim, we designed and synthesized the
heterocyclic analogues of pregnenolone by using Friedlander and
Claisen-Schmidt reactions to introduce the heterocyclic moiety in
pregnenolone skeleton. The details of syntheses of these
compounds are shown in schemes 1-3. Initially, the compound 1
was acetylated with Ac2O/pyridine to afford pregnenolone
acetate 2. The presence of an acyl group at C-17 provided us an
opportunity to employ the Friedlander reaction to generate
heterocyclic core from the application of aromatic amino
aldehydes. To achieve the synthesis of novel heterocyclic
analogues (3a-3d) of pregnenolone by Friedlander reaction, we
stirred the solution of compound 1 and 2-amino benzaldehyde or
The process of osteoblastogenesis involves differentiation of
preosteoblastic cells into terminally differentiated osteoblast cells
mainly in three stages. These are proliferation, matrix

development and maturation and mineralization. The most
reliable indicator of differentiation of osteoblastic precursor cells
to the terminal osteoblastic phenotype is the mineralization.
Therefore, compound 3a was further evaluated for its osteogenic
activity in osteoblast mineralization assay. To assess the
osteoblast mineralization efficacy of the compound, calvarial
osteoblast cells were cultured for 18 days in differentiation media
containing 10mM β-glycerophosphate and 50 μg/ml ascorbic acid
in presence or absence of compound at 1pM and 100pM
concentrations. Cells were then stained with 40 mM alizarin red-
S and dye was extracted to quantify the extent of osteoblast
mineralization. The alizarin dye stains newly formed mineralized
nodes which accumulate calcium. After treatment, optical
densitometry was used to quantitate mineralization of osteoblast
cells by measuring alizarin extracted from stained cultures.
Compound 3a exhibited significant increase in mineral nodule
formation at concentrations as low as 100pM and 1pM compared
to the control untreated cells with best effect at 1pM
concentration [Figure 3(a) & 3(b)].
Scheme 1: Reaction Conditions: (a). Ac2O (1.1 eq.), pyridine, dry CH2Cl2, 0C-rt, 2 h (b) 2-amino benzaldehyde or 2-amino pyridine carboxaldehyde
derivatives (1.0 eq.), 50% KOH (aq.), ethanol, rt, 24-30 h (c) R1CHO (1.0 eq.), 50% KOH (aq.), ethanol, rt, 24-48 h.

Scheme 2: Reaction Conditions: (a) NH2OH.HCl (1.1 eq.), 30% KOH, ethanol, rt, 4 h (b) NH2NH2.H2O (1.1 eq.), acetic acid (cat.), methanol, 50C, 5 h (c)
p-toluenesulfonyl hydrazide (1.1 eq.), acetic acid (cat.), methanol, 50C, 4 h.
Scheme 3: Reaction Conditions: (a) mCPBA (1.5 eq.), CHCl3, rt, 24 h.

Figure 2: Effect of compounds on osteoblast differentiation. Mice calvarial osteoblasts (2 X 103 cells) were seeded in 96-well plate and treated
with different concentrations of compounds for 48 h. ALP activity was quantified spectrophotometrically at 405nm. Data shown as mean ±
SEM; n=4; *P < 0.05, **P < 0.01 and ***P < 0.001 compared with vehicle treated cells.
Figure 3: Effect of compound 3a on mineralization of mice calvarial osteoblasts. 2x103 osteoblast cells were seeded in 12-well plates and
exposed to different concentrations of compounds for 21 days. At the end of the incubation, cells were fixed and stained with alizarin red-S.
The stain was extracted and O.D. measured colorimetrically as described in material and method. (a) Representative images of bone mineral
nodule formation (b) Quantitative estimation of alizarin Red-S staining of mineralized nodules. Values are obtained from three independent
experiments in the replicate of six/treatment point and expressed as mean ± SEM; ***P<0.01compared with vehicle.

Figure 4: Effect of compound 3a on mRNA expression of osteogenic markers. Osteoblast cells were cultured with or without compound 3a
and RNA was collected at 24 h time point. qPCR for BMP-2, RUNX-2 and OCN was performed as described in materials and methods. Data
shown as mean ± SEM; n=4; *P < 0.05 and ***P < 0.001 compared with vehicle treated cells.
Table 1: Primer sequences used for qPCR
GENE NAME
PRIMER SEQUENCE
RUNX-2
F-TTGACCTTTGTCCCAATGC
R-AGGTTGGAGGCACACATAGG
F-GCCCTGAGTCTGACAAAGGTA
R-GGTGATGGCCAAGACTAAGG
F-AGATCTGTACCGCAGGCACT
R-GTTCCTCCACGGCTTCTTC
F-AGCTTGTCATCAACGGGAAG
R-TTTGATGTTAGTGGGGTCTCG
Osteocalcin
BMP2
GAPDH
Bone morphogenetic proteins (BMPs) are members of
transforming growth factor-β (TGF-β) superfamily which plays
an important role in osteoblast differentiation.33 Among other
BMPs, BMP-2 stimulates osteoblast phenotype expression such
as increase in ALP activity and collagen synthesis. Additionally,
Runt-related transcription factor-2 (RUNX-2) is a protein which
is a transcription factor associated with osteoblast differentiation
and skeletal morphogenesis.34 Further, osteocalcin (OCN) is an
established differentiation marker for bone formation.34
Considering these markers as important indicators of bone
formation, the mRNA expression levels of these markers were
evaluated in presence of compound 3a for further confirmation of
its bone forming potential. To determine the effect of compound
3a on osteogenic markers BMP-2, RUNX-2 and OCN, osteoblast
cells were treated with compounds at 1 pM concentration for 24
h, total RNA was isolated and cDNA was synthesized. cDNA
was used as a template in real-time quantitative PCR. The
housekeeping gene GAPDH was used as the internal control in
our experiments [Table 1]. Results showed that treatment of
osteoblast cells with compound 3a at 1 pM concentration
increased the transcript levels of Runx-2 (~ 1.5 fold at 24 h),
OCN (~3 fold at 24 h) and BMP-2 (~2 fold at 24 h) [Figure 4].
Thus, compound 3a up-regulates the expression of osteogenic
expression markers.
of additional heteroatom away from the periphery of acyl group,
decreases the osteogenic effect. Similarly, the presence of the
chalcone functionality with either heteroaryl or aryl group
showed no osteogenic efficacy. The presence of acetate at C-3
and the double bond at C-5 & C-6 position could be also
important for the osteogenic activity up to some extent.
In conclusion, our study has shown that the role of steroidal
molecule pregnenolone and its heterocyclic analogues for anti-
osteoporotic activity. We have synthesized these heterocyclic
analogues by the application of Friedlander and Claisen-Schmidt
reactions. Our preliminary results could be useful for the design
of novel steroidal osteogenic lead molecules. Further, studies in
search of more potent analogue from this class of compounds and
understanding of mechanism of action are in progress.
Acknowledgments
SWM is thankful University of Allahabad, Allahabad, India
for providing scholarship and Director CSIR-CDRI for providing
opportunity of academic training in CDRI, Lucknow, India. KD
is grateful to CSIR, New Delhi, India for providing senior
research fellowship. Authors are thankful to SAIF division of
CSIR-CDRI for providing spectral data. CSIR-CDRI
communication number for this manuscript is
The structure activity relationship revealed that the presence
of a heterocyclic group in the periphery of acyl group at C-17
position showed significant enhancement in the osteogenic
activity e.g. compounds 3a, 3d and 5a, whereas, the introduction
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