Synthesis and Evaluation of Galloyl Conjugates of Flavanones as BMP-2 Upregulators with Promising Bone Anabolic and Fracture Healing Properties

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Synthesis and Evaluation of Galloyl Conjugates of Flavanones as

BMP‑2 Upregulators with Promising Bone Anabolic and Fracture

Healing Properties

Alka Raj Pandey,^∇Divya Rai,^∇Suriya Pratap Singh, Ashish Kumar Tripathi, Anirban Sardar,

Alisha Ansari, Anjali Mishra, Sudha Bhagwati, Rabi Sankar Bhatta, Mohammad Imran Siddiqi,

Naibedya Chattopadhyay, Ritu Trivedi,* and Koneni V. Sashidhara *

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ABSTRACT: The molecular hybridization concept led us to design a

series of galloyl conjugates of ﬂavanones that have potent osteoblast

diﬀerentiation ability in vitro and promote bone formation in vivo. An

array of in vitro studies, especially gene expression of osteogenic

markers, evinced compound 5e as the most potent bone anabolic agent,

found to be active at 1 pM, which was then further assessed for its

osteogenic potential in vivo. From in vivo studies on rat calvaria and a

fracture defect model, we inferred that compound 5e, at an oral dose of

5 mg/(kg day), increased the expression of osteogenic genes (RUNX2,

BMP-2, Col1, and OCN) and the bone formation rate and signiﬁcantly

promoted bone regeneration at the fracture site, as evidenced by the

increased bone volume/tissue fraction compared with vehicle-treated

rats. Furthermore, structure−activity relationship studies and pharma-

cokinetic studies suggest 5e as a potential bone anabolic lead for future osteoporosis drug development.

monoclonal antibody against RANKL (denosumab), have an

INTRODUCTION

■

ameliorative eﬀect in halting further bone resorption in

osteoporosis patients. The chief pitfall with the use of

antiresorptive agents is that the amount of bone that is already

lost cannot be restored.⁴Bone morphogenetic proteins (BMPs),

which belong to the transforming growth factor β (TGF-β)

superfamily, have been reported to promote fracture healing in

various animal models and in humans. At the fracture sites,

BMPs are generally delivered as recombinant proteins (BMP-2/

BMP-7). BMP-2 is expressed by the bone-forming osteoblast

cells and promotes osteoblast diﬀerentiation and bone

regeneration in various animal models.⁵Local application of

BMP-2 at the fracture site has received FDA approval for

orthopedic use. However, poor bioavailability, expensive

synthesis, and puriﬁcation process of recombinant BMP-2

protein limit its systemic use. Hence, in this direction, synthesis

of small, orally eﬃcacious, and inexpensive BMP-2 upregula-

tors/secretagogues may provide new bone anabolic agents that

Osteoporosis, a silent metabolic disorder, is depicted by an

aberrant bone remodeling process. Remodeling of a living and

dynamic tissue like bone is incessantly balanced by two opposite

events, i.e., bone resorption and bone formation. Osteoclasts

and osteoblasts are the two active participants of bone

resorption and bone formation processes, respectively, and in

bone remodeling, these two processes are tightly coupled.¹An

imbalance in the homeostasis of bone remodeling that causes

greater bone resorption over bone formation results in

osteoporosis, which is characterized by low bone mass and

structurally compromised bone tissue, thereby predisposing

bones to a high risk of fractures.²Osteoporosis is not only

associated with compromised bone mechanical strength but also

a decreased quality of life. According to recent studies,

osteoporosis aﬄicts about 200 million globally and this number

is expected to surge 2-fold by 2040.³

As of now, suppression of osteoclast functions and stimulation

of osteoblast activity are the key therapeutic tools for rectifying

the aberrant bone remodeling process in osteoporosis.

Currently, drugs of two diﬀerent categories are in clinical use,

which include antiresorptive and bone anabolic agents.

Antiresorptive agents, e.g., bisphosphonates (alendronic acid,

ibandronic acid, risedronic acid, and zoledronic acid), selective

estrogen receptor modulators (SERMs) like raloxifene, and

Received: January 21, 2021

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A

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Figure 1. Molecular hybridization approach for designing galloyl conjugates of ﬂavanones.

a

Scheme 1. Synthesis of Galloyl Conjugates of Flavanones

a

Reagents and conditions: (i) NaOH (4 M), ethanol, r.t. (overnight); (ii) conc. H₂SO₄, ethanol, reﬂux (3−4 h); (iii) Fe, CaCl₂, H₂O, ethanol,

reﬂux (3−4 h); (iv) Et₃N, dichloromethane (DCM), r.t.; (v) NaBH₄, r.t. 1 h.

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Figure 2. (A) Compounds stimulating osteoblast diﬀerentiation in an ALP activity assay. 6p, 5e, 5n, and 5l treatments showed elevated ALP

production compared to the vehicle-treated cells. (B) Compounds stimulating cell viability. 6p, 5e, 5n, and 5l treatments enhanced the viability of

osteoblasts compared to the vehicle-treated cells. (C) Compounds promoting mineralized nodule formation as assessed by Alizarin red-S staining

shown by the representative microscopic images and (D) quantiﬁcation of the extracted stain. Data are represented as mean standard error of the

mean (SEM) of three independent experiments compared to the vehicle, *p < 0.05, **p < 0.01, and ***p < 0.001 vs vehicle.

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not only induce new bone formation but also show lesser

adverse eﬀects.⁶

the diastereotopic H-3a and H-3b protons as two doublet of

doublets at δ2.89 and 3.07, respectively, in ¹H NMR spectra of

the most active compound 5e conﬁrmed the presence of the

chromane ring of the ﬂavanone structure. H-3a and H-3b of the

ﬂavanone ring showed geminal coupling with a coupling

constant of 17.0 Hz. On the other hand, a coupling constant

value of 13.2 Hz for H-2 and H-3a indicated the axial position for

H-3a, whereas J_2,3b= 2.8 Hz further conﬁrmed the equatorial

position of H-3b.¹⁸In this way, the half chair conformation of

the chromane ring in ﬂavanone was conﬁrmed. In addition, one

singlet at δ 3.85 (s, 3H) corresponded to the methoxy group of

ﬂavanone, while two singlets at 3.90 (s, 3H) and 3.93 (s, 6H)

corresponded to the three methoxy groups of gallic acid. Peaks

ranging from δ 6.91 to 8.15 correspond to the remaining nine

aromatic protons. ¹³C NMR spectra also showed all of the

relevant number of resonances corresponding to 26 carbon

atoms. The characteristic peaks at δ 165.7 and 192.0 conﬁrmed

the presence of carbonyl of the amide bond through which gallic

acid and ﬂavanone were coupled and carbonyl of the ﬂavanone

structure, respectively. Furthermore, the molecular ion peak [M

+ H]⁺at m/z = 464.1705 in the high-resolution mass spectra

(HRMS) established its molecular formula as C₂₆H₂₆NO₇.

Based on the above spectral data evidence and reported

literature values, compound 5e was conﬁrmed to be 3,4,5-

trimethoxy-N-(2-(3-methoxyphenyl)-4-oxochroman-6-yl) ben-

zamide. Further, it was observed that the stereocenter at the C-2

position imparts chirality to compound 5e and thus was

obtained as a racemic mixture; it was resolved into its

component isomers with the use of chiral separation over

normal-phase high-performance liquid chromatography

(HPLC). Assignment of the absolute conﬁguration R/S was

achieved utilizing circular dichroism (CD) spectroscopy

analysis.²²

Biological Activity. Primary Screening of Flavanones

Using Osteoblast Diﬀerentiation Assay. In calvarial osteoblast

cells, alkaline phosphatase (ALP) activity, a surrogate of

osteoblast diﬀerentiation, was used to screen the synthesized

ﬂavanones.²³The ALP activity was read with a spectropho-

tometer, and the optical density was measured at 405 nm in

calvarial osteoblast cells by treating them with test compounds at

concentrations ranging from 1 pM to 1 μM. Four compounds,

namely, 6p, 5e, 5n, and 5l, among the total of 20 exhibited

increased ALP activity in comparison to the vehicle (with no

treatment). Compound 6p increased the ALP activity

signiﬁcantly at concentrations ranging from 1 pM to 1 nM,

compound 5e increased the activity from 1 pM to 10 nM,

compound 5n increased it at concentrations from 1 pM to 1 μM,

and compound 5l increased the activity at 1, 10, and 100 pM, as

shown in Figure 2A. Table 1 shows the E_maxvalues (maximum

Currently available bone anabolic agents including teripara-

tide, abaloparatide, and romosozumab are biologics and are

administered through the parenteral route.^7,8Treatment

adherence is greater with oral medication over the parenteral

drugs, and hence, oral bone anabolic drug is an unmet clinical

need.

Gallic acid or 3,4,5-trihydroxy benzoic acid and its derivatives

embrace a group of polyphenolic compounds belonging to the

family of natural antioxidants. In nature, they are present in

diﬀerent forms such as methylated gallic acids (e.g., syringic

acid), galloyl conjugates of catechin derivatives (e.g., epigallo-

catechin), and polygalloyl esters of glucose, quinnic acid, or

glycerol. These polyphenolic compounds are known to possess

strong antioxidant and anti-inﬂammatory activities.⁹Besides,

gallic acid has been known for its osteogenic activity since

long.¹⁰However, despite having substantial pharmacological

responses, the hydrophilicity of gallic acid has hampered its

implementation as a potent antioxidant clinically by lowering its

absorption, increasing the rate of elimination, and decreasing its

bioavailability. Also, a structure−activity relationship study

carried out on gallic acid and its derivatives for evaluating their

antioxidant ability revealed that derivatives with hydrophobicity

higher than gallic acid such as epigallocatechin gallate and

epicatechingallate have better scavenging eﬀects in comparison

to gallic acid.¹¹Thus, to improve the bone anabolic potential of

gallic acid, sulfonamide-based gallates such as JEZ-C and

ZXHA-TC were synthesized, and indeed, both displayed

promising bone anabolic agents.^12,13

From the above description, it was reasonable to consider the

synthesis of gallic acid to harness its salutary biological eﬀects

and improve on its major limitation such as poor bioavail-

ability.¹⁴Moreover, several studies have been reported, in which

ﬂavanones demonstrated a wide spectrum of biological

activities. Interestingly, ﬂavanones such as Naringin,¹⁵Hesper-

etin,¹⁶and Poncirin¹⁷have been reported to possess an

osteoprotective eﬀect, and these reports prompted us to design,

synthesize, and evaluate amide-based galloyl conjugates of

ﬂavanones, which have not been explored much to date for their

skeletal action. Figure 1 shows the strategy for designing galloyl

conjugates of ﬂavanones.

RESULTS AND DISCUSSION

■

Chemistry. A general synthesis of galloyl conjugates of

ﬂavanones 5a−n is outlined in Scheme 1. Their synthesis started

with the condensation of 2′-hydroxy-5′-nitroacetophenone 1a−

n with the corresponding aromatic aldehyde in a basic medium,

which aﬀorded 2′-hydroxy-5′-nitro substituted chalcones 2a−

n.¹⁸These chalcones were then reﬂuxed in ethanol in the

presence of conc. H₂SO₄to give 5′-nitro substituted ﬂavanones

3a−n.¹⁹Thus-obtained nitro ﬂavanones were reduced with Fe/

CaCl₂in aqueous ethanol to corresponding amines 4a−n and

subsequently coupled with 3,4,5-trimethoxybenzoyl chloride to

give the ﬁnal galloyl conjugates of ﬂavanones 5a−n in good

yields.²⁰To study their structure−activity relationship (SAR),

six ﬂavanones were further reduced with NaBH₄to obtain

galloyl conjugates of ﬂavan-4-ols 6o−t.²¹All of the newly

synthesized galloyl conjugates were characterized by ¹H NMR,

¹³C NMR, two-dimensional (2D) NMR, and high-resolution

mass spectrometry.

Table 1. EC_maxof the Four Bioactive Compounds

Determined by the ALP Assay

compound

EC_max

6p

100 pM

5e

1 nM

5n

10 pM

5l

10 pM

eﬀective concentration) for all four bioactive compounds. This

experiment evidently demonstrated that these four compounds

act as promising osteoblast diﬀerentiating agents.

Enhanced Proliferation of Osteoblasts within Eﬀective

Concentrations. Four bioactive compounds were tested for

toxicity on calvarial osteoblast cells by the cell viability assay

using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bro-

Characterization of Compound 5e. The presence of the

characteristic peak of H-2 as a doublet of doublets at δ5.46 and

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Figure 3. Assessment of osteogenic gene expression by qPCR. (A) 6p, (B) 5e, (C) 5n, and (D) 5l upregulate ALP, BMP-2, Col1, and RUNX2

expressions compared to the vehicle. Data are represented as mean SEM of three independent experiments relative to the vehicle, *p < 0.05, **p <

0.01, and ***p < 0.001 vs the vehicle.

mide (MTT). Compounds 6p, 5e, 5n, and 5l showed no toxicity

to osteoblast cells when compared with the vehicle group at

concentrations ranging from 1 pM to 1 μM. Furthermore,

compound 5e increased proliferation (Figure 2B) at concen-

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Figure 4. (A) 5e showing elevated ALP activity at the indicated concentrations in rat primary osteoblast cells compared with its other structural

analogues. (B) 5e (2R) and 5e (2S) exhibited elevated ALP activity at the indicated concentrations and compared with the vehicle-treated control. (C)

5e diﬀerentiated osteoblast cells through the BMP-2/Smad pathway. Immunoblot showing elevated expressions of RUNX2, BMP-2, p-Smad 1, and

Smad 1 at 1 pM. (D) 5e increased the ALP activity in osteoblasts, and the eﬀect was completely abrogated by noggin, a BMP-2 antagonist. (E) Presence

of 5e had no eﬀect on the ALP activity of BMP-2 (100 ng/mL). Data are represented as mean SEM compared to the vehicle, *p < 0.05, **p < 0.01,

and ***p < 0.001 vs vehicle and ^#5e vs Noggin.

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trations ranging from 1 pM to 1 μM, whereas compounds 6p and

5n showed a signiﬁcant increase in proliferation at 1 pM (Figure

2B).

treated with BMP-2 were taken as a positive control. 5e

signiﬁcantly increased the ALP activity at 1 pM, whereas the

other three required concentrations higher than this (Figure

4A). 5e increased the ALP activity compared to 3,4,5-

trimethoxybenzoic acid (p < 0.05), gallic acid (p < 0.05), and

ﬂavanone 3e (p < 0.01). At 10 pM, 5e showed a greater activity

compared with 3,4,5-trimethoxybenzoic acid (p < 0.05), gallic

acid (p < 0.01), and ﬂavanone 3e (p < 0.001). At 100 pM, the

same pattern was also observed although not signiﬁcant. For the

statistical examination, a two-tailed Student’s t test was

employed.

To conﬁrm the activity of the racemic mixture of the lead

compound 5e, isomers were separated and both (2R and 2S)

were found active compared to the vehicle, as indicated by the

enhanced ALP activity (Figure 4B). This data suggested that the

activity of 5e as a racemic mixture was not dependent on any one

isomer. Changes in the protein level of osteogenic target genes

were quantiﬁed with reference to β-actin. Calvarial osteoblasts

were treated with 5e (1, 10, and 100 pM) for 48 h, which

resulted in an increase in RUNX2 and BMP-2 expressions

maximally at 1 pM as assessed by immunoblotting (Figure 4C).

Moreover, phosphorylation of Smad 1 (p-Smad 1), the

downstream of BMP receptor activation, was strongly increased

by 5e, which suggested that BMP-2 upregulation by the

compound likely elicited the BMP receptor response triggering

osteogenesis (Figure 4C).³⁰

Next, we studied whether or not the osteogenic eﬀect of 5e

was BMP-2 dependent. Noggin is an endogenous BMP

antagonist and is known to diminish osteoblast diﬀerentiation,²⁸

thereby regulating the amount of bone deposition at the

physiologic level. The presence of noggin completely abrogated

the osteogenic eﬀect of 5e, as assessed by ALP production from

osteoblasts (Figure 4D), thus suggesting that 5e required BMP-

2 to achieve an osteogenic impact. Furthermore, the osteogenic

eﬀect of BMP-2 was not altered by 5e, thus suggesting that the

compound signaled via the BMP-2 pathway (Figure 4E). Taken

together, it appears that in osteoblasts, 5e is a BMP-2

“secretagogue”.

Because BMP-2 is endogenously produced by osteoblasts and

acts locally to promote bone formation, we considered it

important to assess whether 5e interfered with the action of

BMP-2. At the functional level, two BMP-2 molecules form an

intermolecular disulﬁde bond (Cys78 from both ligand

molecules).³⁰We performed molecular docking to predict the

binding of 5e with the BMP-2 dimer and to test whether it

aﬀected the dimer (Supporting Figures S3 and S4). Indeed, the

prediction supports the preservation of the BMP-2 dimer and

further received credence from Figure 4E showing that BMP-2

function is completely retained in the presence of 5e besides

proving that 5e exclusively signals through the BMP-2-

dependent mechanism involving its upregulation.

Mineralization Ability in Calvarial Osteoblast Cells.

Compounds selected from the series of 20 compounds after

the assessment of bioactivity in the ALP assay were further

analyzed for their mineralizing ability as it is a physiological

process in the bone due to the inherent potential of osteoblasts

to diﬀerentiate and form mineralized nodules in vitro.²⁴Calvarial

osteoblast cells were treated with diﬀerent concentrations of

compounds, and cultures were maintained for 14 days. Based on

our ALP assay, we selected the lowest active dose of each

compound and the mineralization assay was performed at the

lowest active dose. Cells were ﬁxed and stained with Alizarin red-

S, a calcium chelator (Figure 2C).²⁵Compounds 6p, 5e, 5n, and

5l showed signiﬁcantly increased mineralized nodules compared

to the untreated cells following the quantiﬁcation of optical

density of Alizarin red-S extraction from stained cultures (Figure

2D). These data suggested that 6p and 5n were the most potent

at 10 nM, 5e at 1 pM, and 5l at 100 pM in inducing osteoblast

mineralization in vitro (Figure 2D).

Active Compounds Increased the Gene Expression of

Osteogenic Markers. After examining the mineralizing ability of

active compounds, we studied their response on the expression

of various osteogenic genes including RUNX2 (Runt-related

transcription factor 2), BMP-2, ALP, and Col1 (Type 1

collagen) in calvarial osteoblast cells by the quantitative real-

time polymerase chain reaction (qPCR). Housekeeping gene

glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was

used as an internal control (to normalize input RNA).

RUNX2, a bone-speciﬁc transcription factor, is a master

transcription factor required for determination of osteoblast

cell fate.²⁶A number of transcriptional regulators interact with

RUNX2 to control osteoblast diﬀerentiation. RUNX2 upregu-

lates the expression of ALP and Col1 to drive osteoblast

diﬀerentiation.²⁷

BMP-2 is a strong inducer of bone formation and is under the

regulation of the parathyroid hormone²⁸and estrogen.²⁹The

mRNA levels of BMP-2, RUNX2, ALP, and Col1 were

quantiﬁed by qPCR after the treatment of calvarial osteoblasts

with bioactive compounds at concentrations ranging from 1 pM

to 10 nM. 6p increased the expressions of ALP, BMP-2, Col1,

and RUNX2 by 2.3-, 1.83-, 1.55-, and 1.80-fold, respectively, at

100 pM for ALP and others at 10 nM concentrations (Figure

3A).

5e increased the expression of these osteogenic genes at a

wide range of concentrations, but at its minimum active

concentration, i.e., 1 pM, the expressions of ALP, BMP-2, Col1,

and RUNX2 were increased by 5.5-, 4.4-, 2.9-, and 3.7-fold,

respectively (Figure 3B). 5n increased ALP, BMP-2, and

RUNX2 expressions by 2.8-, 1.7-, and 1.9-fold, respectively;

however, it did not signiﬁcantly increase the expression of Col1

(Figure 3C). 5l increased the expressions of ALP, BMP-2, and

Col1 by 1.3-, 1.9-, and 1.8-fold at 100 pM and RUNX2 by 2-fold

at 1 pM (Figure 3D). From this experiment, we conﬁrmed the

osteogenic eﬀect of four compounds. To determine the lead

compound, we considered the lowest active in vitro osteogenic

concentration and the highest fold induction of the expression of

osteogenic genes by these four compounds. Compound 5e best

met these two criteria.

In Vivo Evaluation of 5e as an Osteoanabolic Agent. To

provide in vivo evidence of the osteogenic eﬀect of 5e, we

administered it subcutaneously in 7 day old rat pups for 6 days at

1 mg/kg, 5 mg/kg, and 10 mg/kg doses. Once the treatment was

over, pups were sacriﬁced and the calvarial tissue was isolated for

each pup to measure the mRNA expression of osteogenic genes

including BMP-2, RUNX2, Col1, and osteocalcin (OCN). We

also analyzed the quantitative histomorphometry and histology

in response to 5e treatment. Neonatal calvaria is rich in

preosteoblasts, and we used newborn rat pups in a subchronic

dosing model to assess rapid osteogenic response if any achieved

by the stimulation of intramembranous ossiﬁcation of a test

Comparison of 5e with Other Structural Analogues. The

osteoblastic ALP activity of 5e was compared with 3,4,5-

trimethoxybenzoic acid, gallic acid, and ﬂavanone 3e. Cells

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Figure 5. (A) 5e upregulated mRNA expressions of RUNX2, BMP-2, OCN, and Col1 in the calvarial tissue. (B) 5e induced in vivo bone formation

measured by double calcein labeling and shown are the representative images. (C) Quantiﬁcation of various parameters derived from the double

labeling experiment at the indicated doses; MS/BS, mineralizing surface normalized with bone surface; MAR, mineral apposition rate; and BFR, bone

formation rate. Box indicates continuous double labeling at 5 mg/kg dose and discontinuous double labeling at 1 mg/kg dose. (D) Representative

histological sections of the calvarial tissue showing increased width at the indicated doses compared with the vehicle and as indicated by the

bidirectional arrow. (E) Quantiﬁcation of the data derived from panel D. Data are represented as mean SEM compared to the vehicle, *p < 0.05 and

**p < 0.01 vs the vehicle.

compound in vivo^31,32before undertaking conﬁrmatory tests in

models that require a prolonged dosing regimen.

All osteogenic genes in the calvaria were maximally stimulated

by 5e at 5 mg/kg dose (Figure 5A). RUNX2 was upregulated by

∼2-fold (*p < 0.01) at 5 mg/kg dose compared with the vehicle.

BMP-2 expression showed a robust dose-dependent increases in

response to 5e between 1 and 5 mg/kg doses, and the induction

ranged from 30- to 38-fold over the vehicle-treated cells.

Expression of the most abundant bone matrix protein, Col1, was

increased by ∼138- and ∼92-fold by 5- and 10 mg/kg doses,

respectively. Expression of OCN, the most abundant non-

collagenous protein of the matrix, was upregulated by ∼18-fold

and ∼10-fold, respectively, by 5 and 10 mg/kg doses and notably

displayed a dose-dependent decrease. OCN upon carboxylation

favors calcium nucleation, a crucial step in mineralization.³³

Therefore, our in vitro and in vivo data suggest that 5e exerts its

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Figure 6. 5e promotes fracture healing. (A) Representative 2D structure of the fracture site from microCT. (B) 3D representations of the same. (C) 5e

at 5 mg/kg dose shows an increased bone volume/tissue volume fraction (BV/TV %), trabecular thickness (Tb.Th.), and trabecular number (Tb.N) of

callus compared to the vehicle. (D) Representative microscopic images of calcein labeled callus (deposition of nascent bone) at the fracture site. (E)

Mean intensity of calcein binding quantiﬁed from the images shown in panel (D). Data are represented as mean SEM compared to the vehicle, *p <

0.05, **p < 0.01, and ***p < 0.001 compared with the vehicle.

osteogenic eﬀect by increasing the synthesis of the matrix

protein Col1 and mineralization by increasing OCN.

kg dose of 5e but did not reach signiﬁcance. The bone formation

rate (BFR), a cumulative measure of mineralized bone

formation per unit time, however, was signiﬁcantly increased

at 5 mg/kg dose over the vehicle (*p < 0.01) (Figure 5B). The

bone formation rate in 10 mg/kg is greater than or equal to the

vehicle group.³⁵

5e Promotes New Bone Formation in Calvaria. We

analyzed surface referent bone formation that serves as the

“litmus” test for osteoanabolic eﬀect.³⁴Time-spaced calcein

labeling (Figure 5B) was used to calculate various parameters

shown in Figure 5C. The mineral apposition rate (MAR), a

measure of osteoblast vigor, showed an increasing trend at 5 mg/

5e Increases Calvarial Thickness In Vivo. We next assessed

whether or not an increase in BFR translated to calvarial

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Figure 7. (A) Gene expressions of OCN and Col1 at the newly formed callus. (B) Histological representation of the fracture site and associated callus

as seen in H&E staining and toluidine blue staining. The ﬁrst panel indicates the lower magniﬁcation of 4×, whereas the other two panels indicate the

higher magniﬁcation (10×) of the callus region. Data are represented as mean SEM compared to the vehicle, *p < 0.05 and ***p < 0.001 vs the

vehicle.

thickness by quantitative histology. Hematoxylin and eosin

staining of the sections showed an increase in calvarial thickness

induced by 5e at the 5 mg/kg dose over the vehicle (*p < 0.01)

(Figure 5D,E).³⁵

(Figure 6C). Increased BV/TV, Tb.Th, and Tb.N at the fracture

site indicated greater callus formation through new bone

regeneration (woven bone), which would facilitate its trans-

formation into the lamellar bone and improved callus

biomechanical property.³⁷From the calcein labeling study, we

observed that 5e (5 mg/kg) was comparable to PTH (130%

increase with 5e vs 184% with PTH) (Figure 6D,E). These data

support drawing the conclusion that 5e promotes fracture

healing maximally at 5 mg/kg dose and the eﬀect is comparable

to the clinically used osteoanabolic drug PTH. PTH, due to its

osteoanabolic properties, is suitable for fracture healing and has

been reported in various studies and thus taken as a positive

control. Its eﬀectiveness and safety are also well established.^38,39

5e Upregulated the Expressions of Col1 and OCN in

Fracture Calluses. Consistent with our observation with calvaria

(Figure 5A), the expressions of OCN and Col1 too were

upregulated by 5e at the callus compared to the vehicle-treated

animals. The fold increases of Col1 and OCN by 5e were ∼2.5-

and ∼2.2-fold, respectively (Figure 7A). Col1 is the most

abundant matrix protein, and OCN is the most abundant

noncollagenous protein, and together they provide the substrate

and catalyze mineralization of the matrix. Upregulation of Col1

and OCN thus provides a mechanistic basis for increased calcein

deposition (a surrogate of nascent bone formation) at the callus

by 5e.^40,41

Evaluation of 5e in Fracture Healing through Micro-

computed Tomography (μCT) and Bone Histomorphometry.

Having shown that 5e has an osteogenic eﬀect in neonatal

animals characterized by heightened bone formation, we next

assessed whether or not it has an osteogenic eﬀect in adult

animals. To this aim, we tested its bone regenerative ability in a

femur osteotomy (fracture) model, which represents a stable

fracture that heals by primary ossiﬁcation.³⁶The treatment of

compound 5e was started the very next day of the injury and was

given once daily for 14 days at doses of 1, 5, and 10 mg/kg body

weight, orally. Human parathyroid hormone (1−34) (PTH)

was used as the comparator as it is the most widely used

standard-of-care osteogenic drug and is reported to mediate its

eﬀect through BMP-2.²⁸Moreover, BMP-2 has no permissible

systemic use. In comparison to the vehicle, 5e healed the fracture

and promoted callus formation at the injury site as shown by 2D

and three-dimensional (3D) representations of μCT (Figure

6A,B). μCT analysis showed that at 5 mg/kg dose, 5e increased

BV/TV by ∼44.3% at 5 mg/kg, which was comparable to PTH,

which increased it by ∼46.0% when compared to the vehicle

group. Tb.N was signiﬁcantly increased, while Tb.Th showed an

increasing trend in 5e-treated rats compared with the vehicle

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Figure 8. In vitro stability of compound 5e in (A) simulated gastric ﬂuid, (B) intestinal ﬂuid, (C) plasma, and (D) rat liver microsomes (RLMs).

Eﬀect of 5e on the Restoration of Bone Morphology.

Sections made through the calluses were stained with H&E and

toluidine blue to determine the morphology and cartilage

formation after 5e and PTH treatments in adult rats. The

connective tissue at the drill-hole site was replaced by the mature

osteoblasts and ﬁbrous callus in the 5e (1, 5, and 10 mg/(kg

day)) and PTH groups but not in the vehicle group. In the

regions of the drill hole that are away from the periosteal and

endosteal surfaces, the progenitor cells diﬀerentiate into

chondrocytes and healing begins through endochondral

ossiﬁcation. Toluidine blue stains proteoglycan, a major

cartilage protein, and accordingly, we observed extensive blue-

stained areas in the sections from animals treated with 5e (5 and

10 mg/kg) and PTH in contrast to sparse staining in the vehicle

group (Figure 7B). Together with Figure 6D showing increased

mineralization, enhanced proteoglycan formation by 5e

indicates stimulation of chondrogenesis besides the induction

of osteogenesis.⁴²

CYP enzymes, as 53% was metabolized after 1 h of incubation by

RLM. The in vitro half-life (t_1/2) and Cl_intof the compound were

calculated by plotting the normal logarithmic of the compound

remaining over the incubation time up to 60 min. The half-life

and Cl_intwere found to be 62.02 h and 0.04 mL/min/mg,

respectively. The signiﬁcant RLM stability of 5e could be

attributed to the presence of the methoxy group that likely

resisted hepatic glucuronidation observed in unmethylated

ﬂavonoids, resulting in the poor metabolic stability of the

latter.⁴³

In Vivo Pharmacokinetic Analysis. Gallic acid and ﬂavonoids

including ﬂavanones suﬀer from poor oral bioavailability, which

dampens their development as drugs despite displaying salutary

eﬀects in vitro. We therefore assessed the oral bioavailability of

5e in adult rats. Figure 9 shows the mean plasma concentration

Overall, the eﬀect of 5e (5 mg/kg) on fracture healing and

bone regeneration was comparable to that of PTH. As there is

neither an oral osteoanabolic drug nor a small molecule (all

three clinically used osteoanabolic drugs are biologics), the oral

eﬃcacy of 5e on par with PTH is suﬃciently tantalizing to

ascribe it as a potential ﬁrst-in-class oral osteoanabolic lead.

Metabolic Stability and Pharmacokinetic Studies. In

Vitro Stability Study in Gastric Fluid and Intestinal Fluid. The

stability of 5e in simulated gastric ﬂuid (SGF, pH 1.2) and

simulated intestinal ﬂuid (SIF, pH 6.5) is a prerequisite criterion

for oral bioavailability. The result shows that the compound was

stable in SIF (69.0 5.01% remaining) and completely stable in

SGF (88.0 1.72% remaining) after 1 h of incubation at 37 °C,

as shown in Figure 8A,B.

In Vitro Plasma and Microsomal Stability. Plasma stability

of the compound is shown in Figure 8C, from which it can be

concluded that 5e was stable in rat plasma as 89.01 3.74%

remained intact in the plasma after 1 h of incubation. The in vitro

microsomal stability assay was useful in measuring the intrinsic

clearance Cl_intor in identifying the metabolites of compounds

formed by liver enzymes. The compound was found to be stable

without NADPH in rat liver microsomes (RLMs), as shown in

Figure 8D. This revealed that 5e was mainly metabolized by

Figure 9. Mean plasma concentration vs time of 5e at the indicated

doses in adult male SD rats.

versus time proﬁle in male Sprague-Dawley (SD) rats, and Table

2 shows the pharmacokinetic parameters that were estimated by

Phoenix WinNonlin (version 8.1, CertaraInc). After the oral

administration of 5e (5 and 10 mg/kg), the maximum plasma

concentration (C_max) and the overall systemic exposure (AUC)

were found to be 736 (ng/mL) and 4824.5 (h*ng/mL),

respectively, which shows the rapid absorption of the compound

from the gut. Volume of distribution (V_d) and clearance (Cl)

were found to be 8.42 (L/kg) and 1.04 (L/(h kg)), respectively.

At 5 mg/kg (the most active dose), C_maxof 5e (736 ng/mL or 1.6

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m-methoxy substituents in the “C” ring of ﬂavanone imparting

the optimum levels of lipophilicity. A pictorial representation of

the preliminary structure−activity relationships (SARs) is

depicted in Figure 10.

Table 2. Oral Pharmacokinetic Parameters of 5e

PK parameters

unit

dose 5 mg/kg

dose 10 mg/kg

C_max

AUC

T_1/2

Cl

(ng/mL)

(H*ng/mL)

H

L/(h kg)

L/kg

736.00

4824.50

5.60

857.50

6531.55

5.85

1.54

12.99

9.17

CONCLUSIONS

■

1.04

8.42

A series of 20 new galloyl conjugates of ﬂavanones were

synthesized and studied in rat primary osteoblasts in vitro and in

a drill-hole fracture model in rats for assessing their osteogenic

potential. Through a series of in vitro and in vivo studies, we

identiﬁed a lead compound 5e that was orally bioavailable and

induced osteoanabolic eﬀect that was on par with the standard-

of-care, PTH. At the mechanistic levels, 5e induced osteogenic

eﬀect by acting as a BMP-2 secretagogue, i.e., by stimulating the

synthesis of BMP-2 in osteoblasts. Given that an oral

osteoanabolic drug is an unmet clinical need, 5e appears suitable

for further development as a novel class of osteoanabolic drug.

V_d

MRT

H

7.68

μM) was a log scale higher than its in vitro osteogenic EC_maxof 1

nM, which suggested that the compound has substantial oral

bioavailability.

The times taken for the systemic level to reduce to half (half-

life, t_1/2) mean resident time (MRT) were found to be 5.6 (h)

and 7.18 (h). It could be inferred from the above

pharmacokinetic parameters that 5e has an adaptable oral

exposure and can therefore be considered a lead molecule for

potential optimizations.

EXPERIMENTAL SECTION

■

Structure−Activity Relationship (SAR). Primarily, all of the

synthesized hybrids and their analogues were evaluated in vitro

for their antiosteoporotic potential using the ALP activity, which

yielded four active compounds 6p, 5e, 5n, and 5l. SAR deduced

from the in vitro activity revealed that the phenyl ring of

ﬂavanone whether unsubstituted or with the substituent p-tert-

butyl or m-methoxy group retained good activity. Interestingly,

replacement of the phenyl ring with the heteroaromatic ring

(5m) did not improve the activity, nor did the reduction of the

carbonyl group (as in compounds 6o−t). To show the

indispensable role of pharmacophoric subunits in the overall

activity of our hybrid molecule, a comparison of the osteoblastic

ALP activity of 5e with 3,4,5-trimethoxybenzoic acid, gallic acid,

and ﬂavanone (3e) was done, which revealed that although gallic

acid and ﬂavanones were active individually, their hybrid with a

suitable substitution on the phenyl ring possessed better activity

(Figure 4A). In addition, as 5e turned out to be the most active

in the series, further probing of its two isomers (2R and 2S) was

done. Interestingly, both were found active and comparable to

their racemic form 5e (Figure 4B). Thus, chirality did not play

any signiﬁcant role in the bioactivity. Overall, it was inferred that

both gallic acid and ﬂavanone subunits constitute the core

pharmacophore of the synthesized hybrids, with p-tert-butyl or

General Chemistry. Reagents obtained from commercial sources

were used without further puriﬁcation. A 100−200-mesh silica gel was

used in column chromatography for puriﬁcation of compounds. The

pace of chemical reactions was monitored by thin-layer chromatog-

raphy (TLC) (silica gel plates with ﬂuorescence F254). Melting points

were uncorrected. The ¹H and ¹³C NMR spectra were recorded at 400

and 100 MHz, respectively, using CDCl₃and DMSO-d₆as solvents. All

chemical shift values were described in ppm and their multiplicities

expressed as follows: m, multiplet; q, quartet; t, triplet; dd, double

doublet; brd, broad doublet; d, doublet; brs, broad singlet; and s,

singlet. The ESI-MS spectra were recorded on an ion trap LCQ

Advantage Max mass spectrometer (Thermo Electron Corporation),

and HRMS spectra were recorded by Q-TOF (Agilent 6520). All of the

ﬁnal compounds were found to be >95% pure as determined by an

Agilent HPLC equipped with an Eclipse Plus C18 column (4.6 mm ×

250 mm, 5 μm mesh size), with acetonitrile (solvent A) and water

(solvent B) as an eluent in the ratio 9:1, at a ﬂow rate of 0.80 mL/min

and run time of 10 min. Detection was done by UV at 220 and 254 nm.

General Synthesis of 2′-Hydroxy-5′-nitro Substituted Chalcones

(2a−n). To a well-stirred solution of 2′-hydroxy-5′-nitroacetophenone

(10 mmol) and the respective aldehyde (10 mmol) in ethanol, sodium

hydroxide (4 M) was added, and the reaction mixture was allowed to

stir overnight at room temperature. On completion of the reaction, the

reaction mixture was neutralized with dil. HCl (1 N) to precipitate

chalcones, followed by ﬁltration and drying in a vacuum. In any case,

Figure 10. Structure−activity relationship (SAR) of synthesized compounds.

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chalcone was not precipitated; it was extracted with ethylacetate, dried

over anhyd. Na₂SO₄, and evaporated in a high vacuum.

2.7 Hz, 1H), 10.20 (s, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ43.4, 56.1,

60.1, 78.1, 105.3, 113.3, 113.5, 115.2, 115.3, 117.6, 118.2, 120.3, 122.6,

129.3, 129.6, 130.6, 130.7, 133.1, 140.4, 141.7, 152.6, 157.1, 161.2,

163.1, 164.6, 191.2; ESI-MS (m/z): 452 [M + H]⁺; HRMS m/z calcd

for C₂₅H₂₃FNO₆[M + H]⁺452.1509, found 452.1503.

3,4,5-Trimethoxy-N-(4-oxo-2-(m-tolyl)chroman-6-yl)benzamide

(5c). Yellow solid; yield: 80%; mp: 191−193 °C; ¹H NMR (CDCl₃, 400

MHz): δ2.40 (s, 3H), 2.86 (dd, J = 3.0 Hz, 17.0 Hz, 1H), 3.08 (dd, J =

13.4 Hz, 17.0 Hz, 1H), 3.89 (s, 3H), 3.92 (s, 6H), 5.44 (dd, J = 2.8 Hz,

13.3 Hz, 1H), 7.09 (d, J = 9.0 Hz, 1H), 7.11 (s, 2H), 7.21 (d, J = 7.5 Hz,

1H), 7.27−7.34 (m, 3H), 7.86 (d, J = 2.7 Hz, 1H), 8.07 (s, 1H), 8.13

(dd, J = 2.8 Hz, 9.3 Hz, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ21.6, 44.6,

56.5, 60.5, 61.0, 79.9, 104.9, 118.2, 119.0, 120.8, 123.4, 127.0, 128.9,

129.7, 129.9, 130.0, 132.4, 138.6, 138.8, 141.5, 153.4, 158.6, 165.9,

192.2; ESI-MS (m/z): 487 [M + K]⁺; HRMS m/z calcd for C₂₆H₂₆NO₆

[M + H]⁺448.1760, found 448.1753.

N-(2-(3-Hydroxyphenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxy-

benzamide (5d). Yellow solid; yield: 79%; mp: 111−113 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ2.83 (dd, J = 2.9 Hz, 16.8 Hz, 1H), 3.19 (dd, J

= 12.6 Hz, 16.8 Hz, 1H), 3.74 (s, 3H), 3.88 (s, 6H), 5.59 (dd, J = 2.9 Hz,

12.6 Hz, 1H), 6.76−6.78 (m, 1H), 6.93−6.95 (m, 2H), 7.13 (d, J = 9.0

Hz, 1H), 7.22 (t, J = 8.2 Hz, 1H), 7.31 (s, 2H), 7.99 (dd, J = 2.8 Hz, 9.0

Hz, 1H), 8.17 (d, J = 2.7 Hz, 1H), 9.67 (brs, 1H), 10.20 (s, 1H); ¹³C

NMR (DMSO-d₆, 400 MHz): δ43.6, 56.1, 60.1, 78.8, 105.3, 113.4,

115.4, 117.0, 117.6, 118.1, 120.4, 129.3, 129.6, 129.7, 133.0, 140.4,

152.7, 157.3, 157.6, 164.7, 191.6; ESI-MS (m/z): 450 [M + H]⁺;

HRMS m/z calcd for C₂₅H₂₄NO₇[M + H]⁺450.1553, found 450.1548.

3,4,5-Trimethoxy-N-(2-(3-methoxyphenyl)-4-oxochroman-6-yl)-

benzamide (5e). Yellow solid; yield: 84%; mp: 171−173 °C; ¹H NMR

(CDCl₃, 400 MHz): δ2.89 (dd, J = 3.0 Hz, 17.0 Hz, 1H), 3.07 (dd, J =

13.2 Hz, 17.0 Hz, 1H), 3.85 (s, 3H), 3.90 (s, 3H), 3.93 (s, 6H), 5.46

(dd, J = 2.8 Hz, 13.2 Hz, 1H), 6.91−6.94 (m, 1H), 7.04−7.05 (m, 2H),

7.10 (fused d, 1H), 7.11 (s, 2H), 7.35 (t, J = 8.1 Hz, 1H), 7.85 (d, J = 2.7

Hz, 1H), 8.00 (s, 1H), 8.15 (dd, J = 2.8 Hz, 9.0 Hz, 1H); ¹³C NMR

(CDCl₃, 400 MHz): δ44.7, 55.5, 56.5, 61.1, 79.7, 104.8, 112.0, 114.3,

118.2, 118.4, 119.0, 120.8,129.9, 130.0, 130.1, 132.4, 140.2, 141.5,

153.4, 158.5, 160.1, 165.7, 192.0; ESI-MS (m/z): 464 [M + H]⁺;

HRMS m/z calcd for C₂₆H₂₆NO₇[M + H]⁺464.1709, found 464.1705.

N-(2-(4-Chlorophenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxy-

benzamide (5f). Yellow solid; yield: 78%; mp: 218−220 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ2.87 (dd, J = 2.9 Hz, 16.8 Hz, 1H), 3.26 (dd, J

= 12.9 Hz, 16.8 Hz, 1H),3.74 (s, 3H), 3.88 (s, 6H), 5.71 (dd, J = 2.8 Hz,

13.0 Hz, 1H), 7.15 (d, J = 8.9 Hz, 1H), 7.31 (s, 2H), 7.51 (d, J = 8.5 Hz,

2H), 7.60 (d, J = 8.5 Hz, 2H), 7.99 (dd, J = 2.6 Hz, 8.9 Hz, 1H), 8.18 (d,

J = 2.6 Hz, 1H), 10.19 (s, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ43.4,

56.1, 60.1, 78.1, 105.3, 117.5, 118.1, 120.3, 128.5, 128.5, 129.2, 129.6,

133.1, 138.0, 140.4, 152.6, 157.2, 164.6, 191.3; ESI-MS (m/z): 468 [M

+ H]⁺; HRMS m/z calcd for C₂₅H₂₃ClNO₆[M + H]⁺468.1214, found

468.1208.

(E)-1-(2-Hydroxy-5-nitrophenyl)-3-(3-methoxyphenyl)prop-2-en-

1

1-one (2e). Yellow solid; yield: 80%; mp: 119−121 °C; H NMR

(DMSO-d₆, 400 MHz): δ3.81 (s, 3H), 7.04−7.07 (m, 1H), 7.19 (d, J =

9.1 Hz, 1H), 7.38 (t, J = 8.0 Hz, 1H), 7.43−7.45 (m, 2H), 7.74 (d, J =

15.8 Hz, 1H), 7.85 (d, J = 15.8 Hz, 1H), 8.34 (dd, J = 2.9 Hz, 9.2 Hz,

1H), 8.70 (d, J = 2.8 Hz, 1H), 12.67 (s, 1H); ¹³C NMR (DMSO-d₆, 400

MHz): δ55.3, 114.0, 117.0, 118.4, 121.6, 123.3, 123.9, 126.6, 129.6

130.0, 135.7, 139.6, 145.1, 159.6, 164.6, 191.5.

General Synthesis of 5′-Nitro Flavanones (3a−n). The respective

chalcones (0.01 mol) were suspended in ethanol followed by slow

addition of concentrated H₂SO₄. The resultant reaction mixture was

allowed to reﬂux for 4−6 h. On completion, the reaction mixture was

allowed to cool down to room temperature followed by neutralization

with saturated sodium bicarbonate solution and then extraction with

ethylacetate. The combined organic layer was dried over anhyd.

Na₂SO₄and evaporated in a high vacuum. Thus-obtained 5′-nitro

ﬂavanones were pure enough to be directly used in the next step for

reduction without any further puriﬁcation by column chromatography.

2-(3-Methoxyphenyl)-6-nitro-4H-chromen-4-one (3e). Light-

1

brown solid; yield: 74%; mp: 142−144 °C; H NMR (DMSO-d₆,

400 MHz): δ2.99 (dd, J = 3.0 Hz, 17.0 Hz, 1H), 3.43 (dd, J = 12.8 Hz,

16.9 Hz, 1H), 3.78 (s, 3H), 5.86 (dd, J = 3.0 Hz, 12.8 Hz, 1H), 6.97−

7.00 (m, 1H), 7.11−7.14 (m, 2H), 7.35−7.39 (m, 2H), 8.41 (dd, J = 3.0

Hz, 9.2 Hz, 1H), 8.52 (d, J = 2.8 Hz, 1H); ¹³C NMR (DMSO-d₆, 400

MHz): δ42.7, 55.2, 79.6, 112.4, 114.3, 118.8, 119.9, 120.2, 122.2, 129.8,

130.5, 139.4, 141.5, 159.4, 165.1, 190.2.

General Synthesis of 5′-Amino Flavanones (4a−n). The above-

obtained respective ﬂavanones (0.01 mol) were subjected to reduction

by suspending them in a mixture of ethanol and water, followed by

addition of Fe powder (0.03 mol) and calcium chloride (0.01 mol). The

resulting suspension was then reﬂuxed for 4−5 h. On completion of the

reaction, the reaction mixture was then ﬁltered over celite for removal of

iron residues followed by extraction with ethylacetate to obtain the

respective amines.

6-Amino-2-(3-methoxyphenyl)-4H-chromen-4-one (4e). Reddish

oil; yield: 89%; ¹H NMR (DMSO-d₆, 400 MHz): 2.72 (dd, J = 2.9 Hz,

16.9 Hz, 1H), 3.12 (dd, J = 12.9 Hz, 16.9 Hz, 1H), 2.78 (s, 3H), 4.98 (s,

2H), 5.46 (dd, J = 2.8 Hz, 13.0 Hz, 1H), 6.84 (d, J = 8.7 Hz, 1H), 6.88

(dd, J = 2.8 Hz, 8.7 Hz, 1H), 6.92−6.95 (m, 1H), 6.97 (d, J = 2.6 Hz,

1H), 7.08−7.10 (m, 2H), 7.31−7.35 (m, 1H); ¹³C NMR (DMSO-d₆,

400 MHz): δ44.1, 55.1, 78.5, 79.2, 108.5, 112.1, 113.7, 118.2, 118.6,

120.8, 123.5, 129.6, 141.0, 143.1, 152.7, 159.3, 191.9.

General Synthesis of Galloyl Conjugates of Flavanones (5a−n).

The respective amines (0.01 mol) thus obtained were dissolved in

dichloromethane at 0 °C followed by slow addition of triethylamine

(0.15 mol). This reaction mixture was then allowed to stir for 15 min,

and a solution of 3,4,5-trimethoxybenzoyl chloride (0.01 mol) in

dichloromethane was added slowly. The resulting reaction mixture was

then stirred at room temperature for 2 h. On completion, the usual

workup was done followed by puriﬁcation of the crude residue obtained

by column chromatography using ethylacetate/hexane as an eluent.

N-(2-(2,4-Dichlorophenyl)-4-oxochroman-6-yl)-3,4,5-trimethox-

Ethyl 4-(4-Oxo-6-(3,4,5-trimethoxybenzamido)chroman-2-yl)-

1

benzoate (5g). Yellow solid; yield: 76%; mp 177−179 °C; H NMR

(CDCl₃, 400 MHz): δ1.41 (t, J = 7.2 Hz, 3H), 2.92 (dd, J = 3.2 Hz, 17.0

Hz, 1H), 3.05 (dd, J = 12.9 Hz, 16.9 Hz, 1H), 3.90 (s, 3H), 3.93 (s, 6H),

4.40 (q, J = 7.2 Hz, 2H), 5.56 (dd, J = 3.3 Hz, 13.0 Hz, 1H), 7.11 (s,

2H), 7.12 (fused d, 1H), 7.56 (d, J = 2.8 Hz, 2H), 7.86 (d, J = 2.7 Hz,

1H), 8.10−8.16 (m, 3H); ¹³C NMR (CDCl₃, 400 MHz): δ14.4, 44.6,

56.5, 61.0, 61.3, 79.2, 104.9, 118.2, 119.0, 120.8, 126.0, 130.0, 130.2,

131.0, 132.7, 141.5, 143.4, 153.4, 158.2, 165.8, 166.2, 191.4; ESI-MS

(m/z): 506 [M + H]⁺; HRMS m/z calcd for C₂₈H₂₈NO₈[M + H]⁺

506.1815, found 506.1802.

1

ybenzamide (5a). Yellow solid; yield: 74%; mp: 203−205 °C; H

NMR (CDCl₃, 400 MHz): δ 2.85 (dd, J = 13.5 Hz, 17.0 Hz, 1H), 3.02

(dd, J = 2.8 Hz, 17.0 Hz, 1H), 3.91 (s, 3H), 3.94 (s, 6H), 5.82 (dd, J =

2.8 Hz, 13.5 Hz, 1H), 7.11 (s, 2H), 7.13 (s, 1H), 7.39 (dd, J = 2.1 Hz,

8.4 Hz, 1H), 7.45 (d, J = 2.0 Hz, 1H), 7.70 (d, J = 8.4 Hz, 1H), 7.87 (d, J

= 2.7 Hz, 1H), 7.91 (s, 1H), 8.16 (dd, J = 2.8 Hz, 9.0 Hz, 1H); ¹³C NMR

(CDCl₃, 400 MHz): δ43.4, 56.5, 61.1, 76.4, 104.8, 118.2, 119.0, 120.8,

128.0, 128.3, 129.7, 129.9, 130.0, 132.4,132.8, 135.1, 135.4, 141.6,

153.5, 158.3, 165.8, 191.2; ESI-MS (m/z): 502 [M + H]⁺; HRMS m/z

calcd for C₂₅H₂₂Cl₂NO₆[M + H]⁺502.0824, found 502.0820.

N-(2-(4-Ethylphenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxyben-

1

zamide (5h). Yellow solid; yield: 81%; mp: 178−180 °C; H NMR

(CDCl₃, 400 MHz): δ1.26 (t, J = 7.4 Hz, 3H), 2.68 (q, J = 7.5 Hz, 2H),

2.84 (dd, J = 2.0 Hz, 16.8 Hz, 1H), 3.07 (dd, J= 13.1 Hz, 16.8 Hz, 1H),

3.87 (s, 3H), 3.90 (s, 6H), 5.43 (d, J = 11.7 Hz, 1H), 7.05 (d, J = 9 Hz,

1H), 7.13 (s, 2H), 7.25−7.27 (m, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.87 (s,

1H), 8.13 (d, J = 7 Hz, 1H), 8.28 (s, 1H); ¹³C NMR (CDCl₃, 400

MHz): δ15.6, 28.7, 44.5, 56.5, 61.0, 79.8, 104.9, 118.2, 119.0, 120.8,

126.4, 128.5, 130.0, 130.0, 132.4, 135.8, 141.4, 145.3, 153.4, 158.7,

N-(2-(3-Fluorophenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxy-

benzamide (5b). Yellow solid; yield: 72%; mp: 217−219 °C; ¹H NMR

(CDCl₃, 400 MHz): δ2.90 (dd, J = 3.0 Hz, 16.9 Hz, 1H), 3.24−3.28 (m,

1H), 3.74 (s, 3H), 3.88 (s, 6H), 5.72 (dd, J = 2.8 Hz, 12.8 Hz, 1H), 7.17

(d, J = 9.0 Hz, 1H), 7.21−7.26 (m, 1H), 7.31 (s, 2H), 7.40−7.44 (m,

2H), 7.47−7.52 (m, 1H), 7.99 (dd, J = 2.7 Hz, 9.0 Hz, 1H), 8.18 (d, J =

M

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165.8, 192.3; ESI-MS (m/z): 462 [M + H]⁺; HRMS m/z calcd for [M +

H]⁺C₂₇H₂₈NO₆462.1917, found 462.1909.

General Synthesis of Galloyl Conjugates of Flavan-4-ols (6o−t).

The respective galloyl conjugate of ﬂavanone 5 (1.0 mmol) was taken in

methanol (10 mL) followed by treatment with an excess of NaBH₄(1.5

mmol) at 0 °C. The resulting reaction mixture was allowed to stir at

room temperature for 30 min. On completion of the reaction as

monitored by TLC, the reaction mixture was then concentrated in a

high vacuum. The remaining residue was dispersed in water and

extracted with dichloromethane. Thus-obtained organic layers were

combined, dried over anhydrous sodium sulfate, and ﬁnally

concentrated in a vacuum to yield the desired compound (6o−t) in

the pure form.

N-(2-(3-Fluorophenyl)-4-hydroxychroman-6-yl)-3,4,5-trimethox-

ybenzamide (6o). White solid; yield: 88%; mp: 159−161 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ1.93 (q, J = 12.0 Hz, 1H), 2.32−2.38 (m, 1H),

3.73 (s, 3H), 3.87 (s, 6H), 4.95−4.99 (m, 1H), 5.29 (d, J = 10.8 Hz,

1H), 5.63 (s, 1H), 6.81 (d, J = 8.7 Hz, 1H), 7.16−7.21 (m, 1H), 7.29−

7.31 (m, 1H), 7.31 (s, 2H), 7.32−7.34 (m, 2H), 7.44−7.50 (m, 1H),

7.57 (dd, J = 2.5 Hz, 8.8 Hz, 1H), 7.81 (d, J = 2.1 Hz, 1H), 10.04 (s,

1H); ¹³C NMR (DMSO-d₆, 400 MHz): δ56.1, 60.1, 63.9, 75.7, 79.2,

105.2, 112.8, 112.9, 114.7, 115.6, 120.1, 121.4, 122.1, 127.3, 130.0,

130.4, 130.5, 132.0, 140.2, 144.0, 150.2, 152.6, 164.3; ESI-MS (m/z):

440 [M + H]⁺; HRMS m/z calcd for C₂₅H₂₅FNO₆[M + H]⁺454.1666,

found 454.1654.

N-(4-Hydroxy-2-(m-tolyl)chroman-6-yl)-3,4,5-trimethoxybenza-

mide (6p). White solid; yield: 86%; mp: 156−158 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ1.96 (q, J = 12.1 Hz, 1H), 2.28−2.32 (m, 1H),

2.35 (s, 1H), 3.73 (s, 3H), 3.87 (s, 6H), 4.96−5.00 (m, 1H), 5.20 (d, J =

11.0 Hz, 1H), 5.55 (d, J = 7.0 Hz, 1H), 6.51 (s, 1H), 6.78 (d, J = 8.7 Hz,

1H), 7.16 (d, J = 7.2 Hz, 1H), 7.24−7.28 (m, 2H), 7.30 (s, 2H), 7.55

(dd, J = 2.5 Hz, 8.7 Hz, 1H), 7.79 (d, J = 2.1 Hz, 1H), 10.00 (s, 1H); ¹³C

NMR (DMSO-d₆, 400 MHz): δ21.1, 56.1, 60.1, 64.1, 76.5, 105.2,

115.6, 120.1, 121.4, 123.3, 126.7, 127.3, 128.3, 128.5, 130.1, 131.8,

137.6, 140.1, 141.0, 150.6, 152.6, 164.3; ESI-MS (m/z): 450 [M + H]⁺;

HRMS m/z calcd for C₂₆H₂₈NO₆[M + H]⁺450.1917, found 450.1909.

N-(4-Hydroxy-2-(3-methoxyphenyl)chroman-6-yl)-3,4,5-trime-

thoxybenzamide (6q). White solid; yield: 87%; mp: 167−169 °C; ¹H

NMR (DMSO-d₆, 400 MHz): δ1.96 (q, J = 12.0 Hz, 1H), 2.30−2.35

(m, 1H), 3.73 (s, 3H), 3.78 (s, 1H), 3.87 (s, 6H), 4.94−5.00 (m, 1H),

5.22 (d, J = 11.3 Hz, 1H), 5.56 (d, J = 7 Hz, 1H), 6.79 (d, J = 8.7 Hz,

1H), 7.92 (dd, J = 2.1, 8.2 Hz, 1H), 7.03−7.06 (m, 2H), 7.31 (s, 2H),

7.31−7.35 (m, 1H), 7.55 (dd, J = 2.6 Hz, 8.7 Hz, 1H), 7.80 (d, J = 2.3

Hz, 1H), 10.00 (s, 1H); ¹³C NMR (DMSO-d₆, 400 MHz): δ55.1, 56.1,

60.1, 64.0, 76.4, 105.2, 111.7, 113.3, 115.6, 118.2, 120.1, 121.4, 127.3,

129.5, 130.0, 131.9, 140.2, 142.6, 150.5, 152.6, 159.3, 164.3; ESI-MS

(m/z): 466 [M + H]⁺; HRMS m/z calcd for C₂₆H₂₈NO₇[M + H]⁺

466.1866, found 466.1860.

N-(2-(4-Chlorophenyl)-4-hydroxychroman-6-yl)-3,4,5-trimethox-

ybenzamide (6r). White solid; yield: 88%; mp: 117−119 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ1.93 (q, J = 12.0 Hz, 1H), 2.31−2.35 (m, 1H),

3.73 (s, 3H), 3.87 (s, 6H), 5.00 (m, 1H), 5.26−5.29 (m, 1H), 5.58−

5.60 (m, 1H), 6.51 (s, 1H), 6.80 (d, J = 8.7 Hz, 1H), 7.30 (s, 2H), 7.47−

7.57 (m, 5H), 7.80 (d, J = 2.1 Hz, 1H), 10.00 (s, 1H); ¹³C NMR

(DMSO-d₆, 400 MHz): δ56.1, 60.1, 63.9, 75.7, 105.2, 115.6, 120.1,

121.4, 127.3, 128.0, 128.4, 130.0, 132.0, 140.0, 140.2, 150.3, 152.6,

164.3; ESI-MS (m/z): 470 [M + H]⁺; HRMS m/z calcd for

C₂₅H₂₅ClNO₆[M + H]⁺470.1370, found 470.1360.

N-(2-(4-Fluorophenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxy-

benzamide (5i). Yellow solid; yield: 74%; mp: 215−217 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ2.83−2.87 (m, 1H), 3.26−3.33 (m, 1H), 3.74

(s, 3H), 3.88 (s, 6H), 5.68−5.70 (m, 1H), 7.13−7.15 (m, 3H), 7.31 (s,

2H), 7.63 (s, 2H), 7.98−8.00 (m, 1H), 8.19 (s, 1H), 10.20 (s, 1H); ¹³C

NMR (DMSO-d₆, 400 MHz): δ43.6, 56.2, 60.2, 78.3, 105.3, 115.3,

115.5, 117.7, 118.2, 120.3, 129.0, 129.0, 129.3, 129.7, 133.1, 135.3,

140.4, 152.7, 157.4, 160.9, 163.3, 164.8, 191.6; ESI-MS (m/z): 452 [M

+ H]⁺; HRMS m/z calcd for C₂₅H₂₃FNO₆[M + H]⁺452.1509, found

452.1506.

N-(2-(4-Isopropylphenyl)-4-oxochroman-6-yl)-3,4,5-trimethoxy-

benzamide (5j). Yellow solid; yield: 79%; mp: 186−189 °C; ¹H NMR

(CDCl₃, 400 MHz): δ1.27 (d, J = 6.9 Hz, 6H), 2.89 (dd, J = 3.0 Hz, 17.0

Hz, 1H), 2.96 (q, J = 6.9 Hz, 1H), 3.12 (dd, J = 13.3 Hz, 16.9 Hz, 1H),

3.90 (s, 3H), 3.93 (s, 6H), 5.46 (dd, J = 2.7 Hz, 13.3 Hz, 1H), 7.09

(fused d, 1H), 7.10 (s, 2H), 7.30 (d, J = 8.2 Hz, 2H), 7.41 (d, J = 8.1 Hz,

2H), 7.85 (d, J = 2.7 Hz, 1H), 7.90 (s, 1H), 8.12 (dd, J = 2.7 Hz, 9.0 Hz,

1H); ¹³C NMR (CDCl₃, 400 MHz): δ24.1, 34.1, 44.5, 56.6, 61.1, 79.8,

104.9, 118.2, 119.0, 120.9, 126.4, 127.1, 129.9, 130.1, 132.3, 136.0,

141.6, 150.0, 153.5, 158.7, 165.8, 192.3; ESI-MS (m/z): 476 [M + H]⁺;

HRMS m/z calcd for C₂₈H₃₀NO₆[M + H]⁺476.2073, found 476.2063.

3,4,5-Trimethoxy-N-(4-oxo-2-(p-tolyl)chroman-6-yl)benzamide

(5k). Yellow solid; yield: 81%; mp: 195−198 °C; ¹H NMR (CDCl₃, 400

MHz): δ2.39 (s, 3H), 2.87 (dd, J = 2.9 Hz, 17.0 Hz, 1H), 3.09 (dd, J =

13.1 Hz, 16.9 Hz, 1H), 3.90 (s, 3H), 3.93 (s, 6H), 5.45 (dd, J = 2.7 Hz,

13.2 Hz, 1H), 7.09 (d, J = 9.1 Hz, 1H), 7.10 (s, 2H), 7.25 (d, J = 8.2 Hz,

1H), 7.37 (d, J = 8.0 Hz, 2H), 7.85 (d, J = 2.5 Hz, 1H), 7.93 (s, 1H),

8.13 (dd, J = 2.5 Hz, 8.9 Hz, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ21.3,

44.5, 56.5, 61.1, 79.8, 104.9, 118.2, 119.0, 120.8, 126.3, 129.6, 129.9,

130.0, 132.4, 135.6, 139.0, 141.5, 153.4, 158.7, 165.8, 192.3; ESI-MS

(m/z): 448 [M + H]⁺; HRMS m/z calcd for C₂₆H₂₆NO₆[M + H]⁺

448.1760, found 448.1749.

3,4,5-Trimethoxy-N-(4-oxo-2-phenylchroman-6-yl)benzamide

(5l). Yellow solid; yield: 80%; mp: 242−246 °C; ¹H NMR (DMSO-d₆,

400 MHz): δ2.87 (dd, J = 2.8 Hz, 16.9 Hz, 1H), 3.25−3.29 (m, 1H),

3.74 (s, 3H), 3.88 (s, 6H), 5.69 (dd, J = 2.9 Hz, 13.0 Hz, 1H), 7.15 (d, J

= 8.9 Hz, 1H), 7.31 (s, 2H), 7.38−7.47 (m, 3H), 7.57 (d, J = 7.0 Hz,

2H), 7.99 (dd, J = 2.6 Hz, 8.9 Hz, 1H), 8.19 (d, J = 2.7 Hz, 1H), 10.20

(s, 1H); ¹³C NMR (DMSO-d₆, 400 MHz): δ43.6, 56.1, 60.2, 78.9,

105.3, 117.6, 118.2, 120.3, 126.6, 128.6, 129.3, 129.6, 133.0, 139.0,

140.4, 152.7, 157.4, 164.7, 191.6; ESI-MS (m/z): 434 [M + H]⁺;

HRMS m/z calcd for C₂₅H₂₄NO₆[M + H]⁺434.1604, found 434.1590.

3,4,5-Trimethoxy-N-(4-oxo-2-(thiophen-3-yl)chroman-6-yl)-

benzamide (5m). Yellow solid; yield: 78%; mp: 230−235 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ2.97 (dd, J = 3.2 Hz, 16.9 Hz, 1H), 3.25−3.30

(m, 1H), 3.74 (s, 3H), 3.88 (s, 6H), 5.74 (dd, J = 3.1 Hz, 13.9 Hz, 1H),

7.12 (d, J = 9.0 Hz, 1H), 7.29−7.31 (m, 1H), 7.32 (s, 2H), 7.59−7.61

(m, 1H), 7.64−7.65 (m, 1H), 7.99 (dd, J = 2.7 Hz, 9.0 Hz, 1H), 8.18 (d,

J = 2.6 Hz, 1H), 10.24 (s, 1H); ¹³C NMR (DMSO, 400 MHz): δ42.9,

56.1, 60.1, 75.0, 105.3, 117.5, 118.1, 120.3, 123.6, 126.4, 127.0, 129.2,

129.6, 133.0, 140.1, 140.4, 152.6, 157.1, 164.6, 191.4; ESI-MS (m/z):

440 [M + H]⁺; HRMS m/z calcd for C₂₃H₂₂NO₆S [M + H]⁺440.1168,

found 440.1165.

N-(2-(4-(tert-Butyl)phenyl)-4-oxochroman-6-yl)-3,4,5-trimethox-

1

ybenzamide (5n). Yellow solid; yield: 78%; mp: 169−172 °C; H

N-(4-Hydroxy-2-phenylchroman-6-yl)-3,4,5-trimethoxybenza-

mide (6s). White solid; yield: 85%; mp: 152−154 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ1.92−2.01 (m, 1H), 2.30−2.35 (m, 1H), 3.73

(s, 3H), 3.87 (s, 6H), 4.95−5.04 (m, 1H), 5.25 (d, J = 10.0 Hz, 7.0 Hz,

1H), 5.57 (d, J = 6.8 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 7.31 (s, 2H),

7.33−7.38 (m, 1H), 7.40−7.44 (m, 2H), 7.7−7.50 (m, 2H), 7.56 (dd, J

= 2.5 Hz, 8.7 Hz, 1H), 7.80 (d, J = 2.1 Hz, 1H), 10.01 (s, 1H); ¹³C NMR

(DMSO-d₆, 400 MHz): δ56.1, 60.1, 64.0, 76.5, 79.2, 105.2, 115.5,

120.1, 121.4, 126.2, 127.3, 127.9, 128.4, 130.0, 131.9, 140.1, 141.0,

150.5, 152.6, 164.3; ESI-MS (m/z): 475 [M + K + H]⁺; HRMS m/z

calcd for C₂₅H₂₆NO₆[M + H]⁺436.1760, found 436.1747.

NMR (CDCl₃, 400 MHz): δ1.34 (s, 9H), 2.82−2.90 (m, 2H), 2.98−

3.13 (m, 2H), 3.87 (s, 3H), 3.88 (s, 1H), 3.90 (s, 6H), 3.91 (s, 6H), 5.44

(dd, J = 2.8 Hz, 13.3 Hz, 1H), 5.86 (dd, J = 2.9 Hz, 13.4 Hz, 1H), 7.06

(d, J = 8.9 Hz, 1H), 7.09 (d, J = 8.9 Hz, 1H), 7.13 (s, 2H), 7.14 (s, 2H),

7.30−7.38 (m, 2H), 7.40−7.42 (m, 4H), 7.45−7.47 (m, 2H), 7.73−

7.76 (m, 1H), 7.87 (d, J = 2.7 Hz, 1H), 7.89 (d, J = 2.7 Hz, 1H), 8.13

(dd, J = 2.7 Hz, 9.0 Hz, 1H), 8.17 (dd, J = 2.7 Hz, 9.0 Hz, 1H), 8.25 (s,

1H), 8.28 (s, 1H); ¹³C NMR (CDCl₃, 400 MHz): δ29.8, 31.4, 34.8,

43.5, 44.4, 56.5, 61.0, 79.7, 104.9, 118.2, 118.3, 118.9, 120.8, 120.8,

125.9, 126.2, 127.4, 129.8, 129.9, 129.9, 130.0, 130.0, 131.8, 132.4,

132.7, 135.5, 136.7, 141.5, 141.5, 152.2, 153.4, 158.5, 158.7, 165.8,

191.7, 192.3; ESI-MS (m/z): 490 [M + H]⁺; HRMS m/z calcd for

C₂₉H₃₂NO₆[M + H]⁺490.2230, found 490.2221.

N-(2-(4-(tert-Butyl)phenyl)-4-oxochroman-6-yl)-3,4,5-trimethox-

ybenzamide (6t). White solid; yield: 85%; mp: 147−149 °C; ¹H NMR

(DMSO-d₆, 400 MHz): δ1.30 (s, 9H), 1.86−2.03 (m, 2H), 2.28−2.33

N

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(m, 1H), 2.38−2.43 (m, 1H), 3.73 (s, 6H), 3.87 (s, 12H), 4.96−5.04

(m, 2H), 5.20 (d, J = 10.7 Hz, 1H), 5.52−5.56 (m, 2H), 5.64 (d,J = 6.6

Hz, 1H), 6.51 (s, 1H), 6.76 (d, J = 8.7 Hz, 1H), 6.83 (d, J = 8.8 Hz, 1H),

7.29−7.31 (m, 4H), 7.38−7.47 (m, 6H), 7.51 (dd, J = 1.4 Hz, 7.7 Hz,

1H), 7.56 (dd, J = 2.5 Hz, 8.7 Hz, 1H), 7.59 (dd, J = 2.5 Hz, 8.8 Hz, 1H),

7.67 (dd, J = 1.7 Hz, 7.6 Hz, 1H), 7.81 (dd, J = 2.2 Hz, 7.1 Hz, 2H), 9.10

(s, 1H), 10.24 (s, 1H); ¹³C NMR (DMSO-d₆, 400 MHz): δ 31.1, 34.3,

37.8, 56.1, 60.1, 63.9, 64.1, 73.5, 76.4, 105.2, 115.5, 115.6, 120.1, 120.2,

121.4, 121.5, 125.1, 126.0, 127.2, 127.3, 127.5, 127.7, 129.5, 129.6,

130.0, 130.0, 131.0, 131.8, 132.1, 138.0, 138.1, 140.2, 140.2, 150.2,

150.4, 150.6, 152.6, 164.3, 164.3; ESI-MS (m/z): 492 [M + H]⁺;

HRMS m/z calcd for C₂₉H₃₄NO₆[M + H]⁺492.2386, found 492.2373.

Chiral Separation of 5e. Racemic 5e was subjected to chiral

separation over normal-phase Shimadzu HPLC with a PDA detector

using an analytical Chiralpak IA column (4.6 mm × 250 mm, 5 μm

mesh size) and eluted with isocratic solvent system isopropyl alcohol

and n-hexane in the ratio 2:8 at the ﬂow rate of 1 mL/min; two peaks

were resolved with retention times at 15.17 and 32.54 min. With the

optimized method in hand, pure R and S isomers were obtained with

preparative HPLC using a Chiralpak IA column (20 mm × 250 mm, 5

μm mesh size).

experiment, cells were washed with phosphate-buﬀered saline (PBS)

and ﬁxed with 4% paraformaldehyde in PBS for 15 min. The ﬁxed cells

were stained with Alizarin red-S (40 mM, pH 4.5) for 30 min followed

by washing with tap water. Stained cells that appear as mineralized

nodules were photographed by a camera (Canon SX410). For

quantiﬁcation of mineralized nodules, Cetylpyridinium chloride

method was taken into account. Brieﬂy, 500 μL of 10% (w/v) CPC

was added in a plate and incubated for 1 h. After the end of the

incubation period, the dye solution was transferred in a 96-well plate

and absorbance was taken at 595 nm.

Quantitative Real-Time Polymerase Chain Reaction (qPCR) for

RCO In Vitro Study. Cells were treated with diﬀerent concentrations of

test compounds 6p, 5e, 5n, and 5l for 48 h. At the end of the treatment,

cells were collected in QIAazol (Qiagen) and the total RNA was

isolated according to the manufacturer’s protocol. The qPCR reaction

was performed for quantitative comparative measurement of the

expression of osteoblast-speciﬁc genes ALP, ColI, BMP-2, and RUNX2

following an optimized protocol using a high-capacity cDNA reverse

transcription kit (Thermo Fisher) using 2.0 μg of total RNA. The

housekeeping gene GAPDH was used as an internal control. Primers

were designed using the Universal Probe Library (Roche Applied

Sciences) for the selected genes (Table 1). SYBR Green chemistry was

used to perform quantitative determination of the relative expression of

transcripts for all genes. All genes were analyzed using the Quant Studio

3 (Applied Biosystem Foster City, California) real-time PCR

machine.⁴⁴

CD Spectroscopy. CD spectra for 5e isomers were recorded at 25

°C using a J-1500, JASCO CD spectrophotometer. Samples were

prepared in methanol with 2 μM concentration, and the ﬁnal values

were obtained from the average of three consecutive scans with solvent

subtraction. CD values were obtained as degrees of ellipticity “θ” in

mdeg against wavelength in nm.

Western Blot Analysis. Cells were seeded in a T-25 ﬂask for 24 h to

reach approximately 70−80% conﬂuency. Treatment of our lead

compound (5e) was performed at three diﬀerent concentrations (1, 10,

and 100 pM) for 48 h in a 10% osteoblast diﬀerentiating medium. Cells

were washed with cold PBS, and lysates were prepared using a RIPA

buﬀer containing a protease and phosphatase inhibitor mixture. A total

of 30 μg of cell lysates was separated on sodium dodecyl sulfate

polyacrylamide gel electrophoresis (SDS-PAGE) under reducing

conditions (Bio-Rad, Hercules, CA) and then transferred to poly-

(vinylidene diﬂuoride) (PVDF) membranes (Millipore, Watford,

U.K.). Membranes were blocked by 5% BSA and then kept at 4 °C

overnight with the primary antibody BMP-2 (ab-14933, dilution

1:1000), RUNX2 (12 556s, dilution 1:1000), p-Smad 1 (ab-63439,

dilution 1:1000), Smad 1 (SC-7965, dilution 1:1000), and β-actin

(A3854, dilution 1:50 000). After overnight incubation, membranes

were washed with a wash buﬀer (PBST) thrice with gentle shaking and

probed with an HRP conjugated secondary antibody at room

temperature for 2 h, and then, western blot signals were visualized by

a chemiluminescence system (GE Healthcare Life Sciences, Bangalore,

India) according to the manufacturer’s instructions.⁴⁵

In Vivo Calvarial Injection and qPCR. To establish the potency of

compound 5e in vivo, 7 day old rat pups with their mother were taken.

Pups were divided into four groups (n = 5/group), namely, Vehicle, 1

mg/kg, 5 mg/kg, and 10 mg/(kg day) body weight. Lead compound 5e

was injected subcutaneously in the pup’s calvaria for 6 days. After the

end of the experimental time point, pups were euthanized and calvariae

were separated by making a straight cut across the sagittal suture and

collected in chilled PBS. Calvariae were homogenized with QIAazol

(Qiagen), and RNA was isolated according to the manufacturer’s

protocol. Further, cDNA was synthesized by a high-capacity cDNA

reverse transcription kit with random primers. Gene expressions of

BMP-2, RUNX2, OCN, and Col1 were assessed using Power up SYBR

Green in Applied Biosystem Quantstudio3 RT-PCR. GAPDH was

taken as an endogenous control.³⁵

Biological Assays. Rat Calvaria Osteoblast Culture (RCO). The

primary cell population of osteoblast was obtained by sequential

enzymatic digestion of the calvarial bone matrix. Rat pups (1 or 2 days

old) were used for the isolation of cells. Institutional Animal Ethics

Committee approval was obtained (IAEC/2020/65). Calvariae of pups

were isolated, sutures were segregated, and adherent tissues were

discarded by scrapping, and cleaned calvariae were crumbled. An

enzyme cocktail of Dispase II (1 mg/mL) and Collagenase type-P (1

mg/mL) was prepared. Crumbled calvaria was incubated at 37 °C in the

enzyme cocktail for ﬁve consecutive digestions (15 min/digestion) to

release the cells. The supernatant from the ﬁrst digestion was discarded,

and cells were collected from the 2nd to 5th digestions, in a complete α-

MEM medium containing 10% fetal bovine serum (FBS) and 1%

antibiotic-antimycotic solution.

Alkaline Phosphatase Assay. Rat calvarial osteoblasts were

trypsinized after attainment of 70−80% conﬂuency, and 2 × 10³

cells/well were seeded in 96-well plates to assess the ALP activity.

Once the desired conﬂuency was obtained, cells were treated with

diﬀerent concentrations of 20 test compounds for 48 h in α-MEM

supplemented with 10% FBS, 10 mM β-glycerophosphate, 50 μg/mL

ascorbic acid, and 1% penicillin/streptomycin (osteoblast diﬀer-

entiation medium). At the end of the incubation period, media were

evacuated and PBS was added to wash out. Plates were kept at −80 °C

overnight and then incubated at 37 °C to induce rupturing of the cells.

The total ALP activity was measured using p-nitrophenyl phosphate

(PNPP) as the substrate, and absorbance was taken at 405 nm.

Cell Viability Assay. The toxicity of compounds was tested on

calvarial osteoblast cells. Cells were seeded in a 96-well plate (2 × 10³

cells/well) for 24 h at 37 °C and 5% CO₂. Once the conﬂuency reaches

70−80%, cells were treated with various concentrations of 6p, 5e, 5n,

and 5l compounds ranging from 1 pM to 1 μM in 10% proliferating (α-

MEM with 10% FBS, 1% antibiotic and antimycotic). After 48 h, 10 μL

of MTT solution (5 mg/mL) was added to each well for 4 h at 37 °C.

Formazan crystals were dissolved by adding 100 μL of dimethyl

sulfoxide (DMSO) in each well after the removal of media, and the OD

was taken at 570 nm.

Calvarial Histomorphometry. Dynamic bone indices were

evaluated from ﬂuorescein (5 mg/kg)³⁵given at an interval of 5 day

double-labeled calvaria sectioned at 50 μm using an Isomet bone cutter

and photographed under a ﬂuorescent microscope. Brieﬂy, the calvariae

were embedded in an acrylic material for the determination and

calculation of the bone formation rate/bone surface (BFR/BS) and the

mineral appositional rate (MAR) using Leica IM50 Image Acquisition

software following a previously described protocol, and analysis was

done using Image pro software.

Mineralized Nodule Formation Assay. Upon diﬀerentiation

induction calvarial osteoblast display eﬃcient mineralizing ability. To

analyze this activity, cells were seeded onto 12-well plates (10 000 cells/

well). In a 10% osteoblast diﬀerentiation medium, osteoblasts were

treated with or without test compounds for 14 days at diﬀerent

concentrations and in triplicates; after every 48 h, fresh media along

with the compound were supplied to the cells. At the end of the

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Histology of Calvaria. Calvariae were ﬁxed in 4% paraformaldehyde

for 2 days and then kept in a decalcifying solution overnight (D0818-1L

Sigma-Aldrich) for decalciﬁcation. Calvariae were then embedded in

paraﬃn wax, and sections of 5 μm thickness were obtained (Leica

Microsystems, Wetzlar, Germany). Tissue sections were deparaﬃnized

in xylene and in graded ethanol series (100, 90, 70, and 50%) and

double-stained with hematoxylin and eosin. The hematoxylin and

eosin-stained preparations were visualized with a bright-ﬁeld optical

microscope (Evosxl bright-ﬁeld microscope) at 20× magniﬁcation and

captured for analysis of calvarial width using Image J software.⁴⁶

Fracture Study. All animal experimental procedures were approved

by the Institutional Animal Ethics Committee (IAEC/2020/65).

Female SD rats of weight 180 20 g were obtained from the National

Laboratory Animal Centre. Animals were treated with good care and

were subjected to a 12 h light−dark cycle, appropriate temperature

(22−24 °C), humidity (50−60%), and easy access to rodent food and

water.

Drill-Hole Defect at the Mid-Diaphysis of the Femur. Fifty adult

Sprague-Dawley rats were taken and randomly divided into ﬁve groups

[Vehicle, PTH (30 μg/kg subcutaneously), and 5e at 1, 5, and 10 mg/

kg] for drill-hole injury. For this, a 1 cm incision in the front skin of the

mid-diaphysis of the femur was made to expose the region to be

inﬂicted with injury. Nearby muscles were removed, and the periosteum

was stripped oﬀ to expose the femoral bone surface. A drill-hole injury

was made by inserting a drill bit of diameter 0.8 mm in the anterior

portion of the diaphysis, 2 cm above the knee joint. Injury was made in

all of the aforementioned groups, and treatment was started from the

very next day and continued for 14 days. After 2 weeks, animals were

euthanized and femurs were collected for measurement of bone

microarchitectural parameters and dynamic histomorphometric study

at the drill-hole site.⁴⁷

Fracture Site Bone Regeneration. Each animal was given calcein

(20 mg/kg body weight) intraperitonially, 24 h before autopsy. Bones

were embedded in an acrylic material, and 50 μm sections were cut

using an Isomet bone cutter and photographs were taken under a

confocal microscope aided with appropriate ﬁlters. The intensity of

calcein binding at the fracture site, which is indicative of new mineral

deposition, was calculated using Image J software.⁴⁸

To quantify the microarchitecture of callus formed in the drill hole,

μCT analysis was performed, using a Sky Scan 1276 CT scanner

(Aartselaar, Belgium). Soft tissues were removed carefully without

disturbing the callus of the bone and scanned using an X-ray source of

70 kV, 200 mA with a pixel size of 18 mm. The images were

reconstructed using Sky Scan Nrecon software. The region of interest

(ROI) was drawn around the callus area, and analysis was performed

using CTAn software. Microarchitectural parameters including bone

volume fraction (BV/TV), trabecular thickness (Tb.Th), and

trabecular number (Tb.N) were quantiﬁed.⁴⁹

Quantitative Real-Time PCR (qPCR). Three femoral specimens from

each group were used to evaluate the mRNA expressions in the callus

region by quantitative real-time polymerase chain reactions (RT-

PCRs). The drilled regions, exhibiting newly formed callus, were

excised as much free from the bone part by a surgical sterilized scalpel.

The piece of bone was frozen and powdered in liquid nitrogen using a

mortar and pestle. Total RNA was isolated using QIAazol (Qiagen)

according to the manufacturer’s protocol. The concentration and the

purity of RNA were determined by measuring the absorbance at 260/

280 nm using a spectrophotometer (NANO-Drop). Further, cDNA

was synthesized by a high-capacity cDNA reverse transcription kit with

random primers. Gene expressions of OCN and Col1 were assessed

using Power up SYBR Green in Applied BiosystemQuantstudio 3 RT-

PCR. GAPDH was taken as an internal control.⁵⁰

Histology at the Fracture Site. Femur bones with drill-hole injury

were dissected out and cleaned. Bones were ﬁxed in 4%

paraformaldehyde for 48 h and then transferred to 70% isopropanol

for another 48 h. Bones were then decalciﬁed in a decalcifying solution

(Sigma-Aldrich) for 4 days and subsequently embedded in paraﬃn. For

histological analysis of the bone (hard callus) at the fracture site,

sections of 5 μm size were cut through transversely. Paraﬃn sections

were dewaxed, rehydrated, stained with hematoxylin and eosin,

dehydrated, and mounted. For the analysis of soft callus at the fracture

site, sections of 5 μm were cut and double-stained with toluidine blue

and fast green. Representative images of the stained sections were

selected for observation.

Statistical Analysis. All experiments were performed three times,

independently. Data are expressed as mean SE. One-way analysis of

variance (ANOVA) was used for experiments with multiple

comparisons followed by Newman−Keul’s test of signiﬁcance using

Graph Pad Prism v.5. Data were presented as the mean SE, *p < 0.05,

**p < 0.01, ***p < 0.001 versus the vehicle group.

LCMS/MS Conditions for In Vivo and In Vitro PK Sample Analysis.

The method of compound 5e was developed using API3200 (Applied

Biosystem, MDS Sciex, Toronto, Canada) mass spectroscopy

connected with a Waters HPLC system consisting of a Waters 2707

autosampler with Waters Milford USA (quaternary pump-600) for the

analysis of in vitro and in vivo study samples. The processed samples

were injected into HPLC (injection volume, 20 μL), and a

chromatographic resolution was obtained by the Water symmetry

shield C18 column (150 mm × 4.6 mm × 5 μm) attached with a C18

guard column. The isocratic mode was applied for sample elution, and

the mobile phase ratio was 90:10 v/v (0.1% formic acid in total distilled

water (TDW)/acetonitrile (ACN)) at a ﬂow rate of 0.7 mL/min.

Analyte detection was performed using an electrospray ionization

source (ESI) in the positive mode, and 4000 ion spray voltage was

applied. Compound-dependent parameters such as declustering

potential, collision energy, and cell exit potential were set to 92.1,

41.63, and 2, respectively. Multiple reaction monitoring (MRM) modes

were used to detect and quantify the analyte as well as IS

(carbamezapine) using a precursor → product ion combination of

464.1/195.0 and 237.2/194, respectively.

In Vitro SGF, SIF, and Plasma Stability. The SIF, SGF, and rat

plasma stability studies of compound 5e were performed at 1 μm/mL in

triplicate. SGF and SIF were prepared as per the USP guideline.⁵¹In

brief, SGF, SIF, and plasma were taken in glass vials separately and

preincubated in a shaking water bath at 37 °C for 10 min. Further, the

aliquots of the analyte were spiked separately in both the ﬂuids and 100

μL of the sample was withdrawn at each time point (0, 5, 10, 20, 30, and

60 min). Further, each sample was quenched with acetonitrile (1:3) v/v

followed by vortex and centrifugation at 5 min and 12 000 rpm for 10

min, respectively. Finally, 100 μL of the supernatant was collected and

loaded in LCMS/MS for analysis.

In Vitro Microsomal Stability. Rat liver microsomes were used for

microsomal stability of the analyte, and the study was conducted in

triplicate. In brief, 886.5 μL of tris buﬀer (50 mM, pH 7.4), magnesium

chloride (500 mM), RLM protein (0.25 mg/mL), and drug (1 μm/mL)

was spiked in each glass test tube separately and preincubated at 37 °C

for 10 min. Positive and negative controls were also done for validation

and microsomal activity. After preincubation, NADPH (40 mM) was

added to each vial to initiate the reaction. The reaction was terminated

by adding acetonitrile (200 μL) in each sample (100 μL collection

volume) at each time point (0, 5, 10, 20, 30, 40, and 60 min). Finally,

the quenched sample was vortexed and centrifuged for 5 min and at

12 000 rpm for 10 min, respectively, and load into LCMS/MS for

further analysis.

In Vivo Pharmacokinetic Study. In vivo oral pharmacokinetic study

of the compound was performed in male SD rats (n = 3). The

compound was administered at 5 mg/kg dose through oral gavage to

each rat. The formulation was formulated in 0.5 per cent sodium CMC

solution in the form of an oral suspension. The blood was collected in

microcentrifuge tubes containing heparin at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8,

10, 12, 16, and 24 h after dosing. Before blood collection, each animal

was anesthetized using light ether anesthesia. Plasma was collected from

blood by centrifugation at 5000 rpm for 20 min and stored at −80 °C

until bioanalysis. Protein precipitation technique was used to extract the

compound from plasma using acetonitrile (300 μL/100 μL plasma)

containing carbamezapine as the internal standard. Finally, the

supernatant was collected and injected into LCMS/MS.

Data Analysis. The percentage of the analyte remaining intact at

diﬀerent time intervals after incubation was determined by eq 1, and the

graph was plotted between the normal logarithmic of the compound

P

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remaining over time.^52,53The curve slope was deﬁned as a depletion

rate constant K (min⁻¹). The half-life (t_1/2) and in vitro intrinsic

clearance (CL_ins) were determined by eqs 2 and 3, respectively.

Anjali Mishra − Pharmacokinetics and Metabolism Division,

CSIR-Central Drug Research Institute, Lucknow 226031,

India; Academy of Scientiﬁc and Innovative Research (AcSIR),

Ghaziabad 201002 U.P., India

Sudha Bhagwati − Molecular and Structural Biology Division,

CSIR-Central Drug Research Institute, Lucknow 226031,

India

% drug remaining = (concentration of drug after incubation

/concentration of drug before incubation)

(1)

× 100

Rabi Sankar Bhatta − Pharmacokinetics and Metabolism

Division, CSIR-Central Drug Research Institute, Lucknow

226031, India; orcid.org/0000-0003-4844-2388

Mohammad Imran Siddiqi − Molecular and Structural Biology

Division, CSIR-Central Drug Research Institute, Lucknow

226031, India; orcid.org/0000-0001-5655-5484

Naibedya Chattopadhyay − Division of Endocrinology and

Centre for Research on ASTHI, CSIR-Central Drug Research

Institute, Lucknow 226031, India; Academy of Scientiﬁc and

Innovative Research (AcSIR), Ghaziabad 201002 U.P.,

India; orcid.org/0000-0003-2473-0246

half life (t_1/2) = 0.693/K

(2)

intrinsic clearance (CL_ins

)

= K × volume of incubation (μL)

/amount of protein incubated (mg)

(3)

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00112.

■

sı

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00112

¹H NMR and ¹³C NMR spectra of all compounds (PDF)

Molecular formula strings (CSV)

Author Contributions

^∇A.R.P. and D.R. contributed equally.

AUTHOR INFORMATION

Corresponding Authors

■

Notes

The authors declare no competing ﬁnancial interest.

Ritu Trivedi − Division of Endocrinology and Centre for

Research on ASTHI, CSIR-Central Drug Research Institute,

Lucknow 226031, India; Academy of Scientiﬁc and Innovative

Research (AcSIR), Ghaziabad 201002 U.P., India;

Phone: +91-0522-2772450; Email: ritu_trivedi@cdri.res.in

Koneni V. Sashidhara − Medicinal and Process Chemistry

Division, CSIR-Central Drug Research Institute, Lucknow

226031, India; Sophisticated Analytical Instrument Facility &

Research, CSIR-Central Drug Research Institute, Lucknow

226031 U.P., India; Academy of Scientiﬁc and Innovative

Research (AcSIR), Ghaziabad 201002 U.P., India;

orcid.org/0000-0002-8339-568X; Phone: +91-522-

2772450; Email: kv_sashidhara@cdri.res.in; Fax: +91-522-

2771942/2771970

ACKNOWLEDGMENTS

■

The authors are thankful to Director, CSIR-CDRI, for providing

necessary research facilities. A.R.P. and A.M. are thankful to the

Department of Science and Technology for the Inspire

fellowship. D.R. and A.K.T. acknowledge AcSIR, New Delhi,

and A.A., D.R., and S.B. acknowledge UGC, New Delhi, for their

research fellowships. The authors acknowledge Dr. Chandra

Bhushan Tripathi for helping in chiral separation of the

compound. The authors are also thankful to the in-house

Sophisticated Analytical Instrumentation Facility & Research

(SAIF & R) for providing the required spectral data. This

manuscript has CDRI Communication no. 10279.

Authors

ABBREVIATIONS USED

■

Alka Raj Pandey − Medicinal and Process Chemistry Division,

CSIR-Central Drug Research Institute, Lucknow 226031,

India; Academy of Scientiﬁc and Innovative Research (AcSIR),

Ghaziabad 201002 U.P., India

Divya Rai − Division of Endocrinology and Centre for Research

on ASTHI, CSIR-Central Drug Research Institute, Lucknow

226031, India; Academy of Scientiﬁc and Innovative Research

(AcSIR), Ghaziabad 201002 U.P., India

Suriya Pratap Singh − Medicinal and Process Chemistry

Division, CSIR-Central Drug Research Institute, Lucknow

226031, India

Ashish Kumar Tripathi − Division of Endocrinology and Centre

for Research on ASTHI, CSIR-Central Drug Research

Institute, Lucknow 226031, India

Anirban Sardar − Division of Endocrinology and Centre for

Research on ASTHI, CSIR-Central Drug Research Institute,

Lucknow 226031, India; Academy of Scientiﬁc and Innovative

Research (AcSIR), Ghaziabad 201002 U.P., India

Alisha Ansari − Medicinal and Process Chemistry Division,

CSIR-Central Drug Research Institute, Lucknow 226031,

India; Academy of Scientiﬁc and Innovative Research (AcSIR),

Ghaziabad 201002 U.P., India

ALP, alkaline phosphatase; BFR, bone formation rate; BMP-2,

bone morphogenic protein 2; GAPDH, glyceraldehyde 3-

phosphate dehydrogenase; IAEC, Institutional Animal Ethics

Committee; MAR, mineral appositional rate; PTH, parathyroid

hormone; qPCR, quantitative real-time polymerase chain

reaction; RCO, rat calvarial osteoblast; RUNX2, runt-related

transcription factor 2; Col1, collagen 1; OCN, osteocalcin; BV/

TV, bone volume/tissue fraction; Tb.Th, trabecular thickness;

Tb.N, trabecular number; SAR, structure−activity relationship;

SERMs, selective estrogen receptor modulators; NMR, nuclear

magnetic resonance; HPLC, high-performance liquid chroma-

tography; EC₅₀, half-maximal eﬀective concentration; E_max

,

maximum eﬃcacy; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyltetrazolium bromide; SIF, simulated intestinal ﬂuid;

SGF, simulated gastric ﬂuid; NADPH, nicotinamide adenine

dinucleotide phosphate; RLM, rat liver microsomes; CL_int,

intrinsic clearance; AUC, area under the curve; t_1/2, half-time;

C

_max, maximum concentration; V_d, volume of distribution; Cl,

clearance; MRT, mean resident time; MRM, multiple reaction

monitoring; CPC, cetylpyridinium chloride; PNPP, p-nitro-

phenylphosphate; ESI, electrospray ionization source

Q

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potential apoptosis-inducing agents: synthesis and biological activity

evaluation. Eur. J. Med. Chem. 2012, 58, 573−580.

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https://doi.org/10.1021/acs.jmedchem.1c00112

J. Med. Chem. XXXX, XXX, XXX−XXX

Article Doi

Synthesis and Evaluation of Galloyl Conjugates of Flavanones as BMP-2 Upregulators with Promising Bone Anabolic and Fracture Healing Properties

DOI: 10.1021/acs.jmedchem.1c00112

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Full text of DOI:10.1021/acs.jmedchem.1c00112

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