Differentiation of skeletal osteogenic progenitor cells

European Journal of Medicinal Chemistry 121 (2016) 82e99

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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech

Research paper

Differentiation of skeletal osteogenic progenitor cells to osteoblasts with 3,4-diarylbenzopyran based amide derivatives: Novel osteogenic agents Atul Gupta a, *, Imran Ahmad a, Jyoti Kureel b, Aijaz A. John b, Eram Sultan c, Debabrata Chanda c, Naresh Kumar Agarwal d, f, Alauddin d, f, Wahajuddin d, S. Prabhaker e, Amita Verma a, Divya Singh b a

Medicinal Chemistry Department, CSIR-Central Institute of Medicinal and Aromatic Plants, PO. CIMAP, Kukrail Road, Lucknow, 226015, India Division of Endocrinology, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, India c Molecular Bioprospection Department, CSIR-Central Institute of Medicinal and Aromatic Plants, PO. CIMAP, Kukrail Road, Lucknow, 226015, India d Division of Pharmacokinetics and Metabolism, CSIR-Central Drug Research Institute, Sector-10, Jankipuram Extension, Lucknow, 226031, India e National Centre for Mass Spectrometry, CSIR-Indian Institute of Chemical Technology, Tarnaka, Hyderabad, 500 007, India f Department of Zoology, HNB Garhwal Central University, Badshahi Thaul Campus, Tehri Garhwal 249 199, Uttarakhand, India b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 9 February 2016 Received in revised form 8 April 2016 Accepted 7 May 2016 Available online 9 May 2016

A series of 3,4-diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity in in vitro and in vivo models of osteoporosis. Compounds 17a, 21bec and 22aeb showed significant osteogenic activity in osteoblast differentiation assay. Among the synthesized compounds, 22b was identified as lead molecule which showed significant osteogenic activity at 1 pM concentration in osteoblast differentiation assay and at 1 mg kg1 body weight dose in estrogen deficient balb/c mice model. In vitro bone mineralization and expression of osteogenic marker genes viz BMP-2, RUNX-2, OCN, and collagen type 1 further confirmed the osteogenic potential of 22b. Gene expression study for estrogen receptor a and b (ER-a and ER-b) in mouse calvarial osteoblasts (MCOs) unveiled that possibly 22b exerted osteogenic efficacy via activation of Estrogen receptor-b preferentially. In vivo pharmacokinetic, estrogenicity and acute toxicity studies of 22b showed that it had good bioavailability and was devoid of uterine estrogenicity at 1 mg kg1 and inherent toxicity up to 1000 mg kg1 body weight dose respectively. © 2016 Elsevier Masson SAS. All rights reserved.

Keywords: Osteogenic Isoflavone Benzopyran Estrogen Antiosteoporotic

1. Introduction Osteoporosis is the most prevalent metabolic bone disease among men and women with an average age above 50 years [1,2]. It is defined as a systemic skeletal disease characterized by low bone mass and micro-architectural deterioration of bone tissue with a consequent increase in bone fragility and susceptibility to nontraumatic fractures [3e7]. The incidence of osteoporotic fractures increases with age. Osteoporosis is now considered as one of the major and growing health care problem around the world [8]. This disease occurs due to an imbalance in the process of bone remodeling which leads to exaggerated bone resorption [9e11]. Any

* Corresponding author. E-mail address: [email protected] (A. Gupta). http://dx.doi.org/10.1016/j.ejmech.2016.05.023 0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

defect in osteoprotegerin (OPG)/receptor activator NFkB ligand (RANKL)/receptor activator NFkB (RANK) system, a dominant mediator of osteoclastogenesis, leads to imbalance in bone remodeling [12,13]. Osteoprotegerin acts as a decoy receptor for the receptor activator NFkB ligand (RANKL) in osteoblast cells and prevents the RANK-RANKL interaction between osteoblast and osteoclast precursor cells which restrain maturation of premature osteoclast cells [14]. The production of osteoprotegerin is stimulated by estrogen in a dose and time dependent manner [15]. Saika et al. reported that estrogen induced increase in OPG is likely to involve its genomic action through estrogen receptor-a (ER-a) [16]. Many cytokines such as interleukins (IL-1, IL-6), TNF-a and monocyte/macrophage colony stimulating factor (M-CSF) involved in bone resorption are also known to be down regulated by estrogen [17]. Furthermore, estrogen is also implicated in stimulation, differentiation and activity of osteoblast cells in cultures [18,19].

A. Gupta et al. / European Journal of Medicinal Chemistry 121 (2016) 82e99

In postmenopausal women, the level of reproductive hormones fluctuates causing a series of immunological and metabolic alterations which leads to lack of adequate bone formation and increases bone resorption [20,21]. The use of estrogen replacement or hormone replacement therapy (ERT or HRT) improves the situation. However, these options have their limitations due to associated side effects especially in reproductive tissues [22,23]. The approved therapies for osteoporosis treatment are mainly focused on bone resorption inhibitors [24]. The recent observations reveal that bone resorption is accompanied by inhibited bone formation due to coupling of these two processes which represents the main disadvantage associated with bone resorption inhibitors [25e27]. The attention has now been focused on osteogenic agents for osteoporosis treatment [28e30]. In order to develop osteogenic drugs, 3-arylbenzopyran core which structurally simulates with tetracyclic nucleus of estran (e.g. 17b-estradiol) and stilbene core of diethylstilbestrol (DES) as well as resveratrol, has been realized as an important nucleus. This core is present in many naturally occurring osteogenic compounds such as geinstein (1), daidgein (2), formonentin (3), isoformonenetin (4), equol and medicarpin etc [31e37]. 3-arylbenzopyran core is also present in various synthetic compounds such as 5e10 (Fig. 1) [33e35,38e43]. These compounds possess potential osteogenic activity with minimal or no toxicity. In our approach to investigate novel osteogenic agents, 3,4diarylbenzopyran based amide derivatives 15aec, 16aec, 21aec and 22aec were designed and synthesized. To achieve the targeted osteogenic activity, the proposed molecules were designed to have 3-arylbenzopyran core for their binding with estrogen receptors (ER) and a long chain alkylamide bearing phenyl group at position 4 of 3-arylbenzopyran core for induction of mixed estrogen agonistic and antagonistic activity. The synthesized compounds were evaluated for osteogenic activity in osteoblast differentiation assay using mouse calvarial osteoblast cells (MCOs). The osteogenic potential of most active compound 22b was further confirmed by bone mineralization activity and osteogenic gene expression analysis by qPCR. Following in vitro osteogenic activity data, 22b was

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further evaluated for osteogenic activity in in vivo estrogen deficient balb/c mice model for osteoporosis. The effect of 22b on trabecular bone volume (BV/TV; %), trabecular number (Tb.N), and trabecular separation (Tb.Sp), trabecular thickness (Tb.Th) was evaluated. Furthermore, 22b was evaluated for its pharmacokinetic profile, estrogenicity and acute toxicity studies in animal models. 1.1. Chemistry The synthesis of designed compounds was started with the synthesis of 2,2-dimethyl-4-(4-hydroxyphenyl)-7-methxoy-3phenyl-2H-benzopyran (13) and 2,2-dimethyl-4-(4hydroxyphenyl)-7-methxoy-3-phenyl-3H-benzopyran (18) from 3-methoxyphenol (11) and 4-hydroxybenzoic acid (12) following reported methodology [44]. For synthesis of 16aec and 17aec, 13 was alkylated with different bromo alkyl esters in presence of anhydrous K2CO3 in dry acetone which gave corresponding ester derivatives (14) in 76e80% yields (Scheme 1). On basic hydrolysis with 10% NaOH in methanol at reflux, compound 14 yielded 15 in quantitative yield. Subsequently, 15 was treated with 2methylaminopyridine or N-methyl-N-butylamine using 1-ethyl-3(3-dimethylaminopropyl)carbodiimide (EDC) and 1hydroxybenzotrizole (HOBt) in dry dimethylformamide (DMF) at room temperature to yield target amides, 16 and 17, in 69e75% yields. Similarly, 21aec and 22aec were synthesized through alkylation of 19 with different alkyl esters followed by basic hydrolysis to synthesize corresponding acid derivatives (20) in 82e88% yield following above protocol used for synthesis of 16 and 17 (Scheme 2). The target amides, 21 and 22, were achieved in 68e74% yields, through reaction of 20 with 2-methylaminopyridine or N-methylN-butylamine using EDC and HOBt in dry DMF at room temperature. Synthesized compounds were characterized using NMR, IR and mass spectrometry. The trans orientation of two aryl groups present at C-3 and C-4 of 3,4-diaryl-3,4-dihydrobenzopyran core was ascertain with the help of coupling constants (J values). The

Fig. 1. Some 3-arylbenzopyran based osteoprotective agents.

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Scheme 1. Reagents and conditions: (a) Ref. [44] (b) K2CO3, anhy. acetone, reflux (c) 10% NaOH, methanol, reflux (d) 2-methylaminopyridine or N-methyl-N-butylamine, EDC, HOBt, Et3N, DMF, RT.

Scheme 2. Reagents and conditions: (a) Ref. [44] (b) K2CO3, anhy. acetone, reflux (c) 10% NaOH, methanol, reflux (d) 2-methylaminopyridine or N-methyl-N-butylamine, EDC, HOBt, Et3N, DMF, RT.

chemical shift (ppm) values for different protons and carbons were in agreement with the values reported in literature. Interestingly, in

the case of 17aec and 22aec having N-methyl-N-butyl amino group, there were two triplets for eNCH2 group (each one


corresponds to one proton with total integration for two protons) and a doublet for eNCH3 instead of a singlet which theoretically should appear as one singlet and one triplet respectively. Additionally, in 13C spectra, these compounds showed double number of the signals in upfield region (mainly in aliphatic part, below 100 ppm). The initial consideration for such abnormalities was given to racemic nature of 3,4-diaryl-3,4-dihydrobenzopyran which was ruled out because similar trend was observed in case of 3,4-diaryl-2H-benzopyran based compounds. To clarify the impact of basic skeleton of compounds on such abnormalities, we synthesized structurally simple amide derivatives of 4-(3methoxyphenoxy)-butanoic acid (24) using n-butyl amine and Nmethyl-N-butylamine (25a & 25b) (Scheme 3). Compound 25b showed all spectral abnormalities as presented by compounds 17aec and 22aec whereas compound 25a showed a singlet for NeCH3 and a triplet for eNeCH2 group respectively with correct number of signal in 13C spectra as anticipated theoretically. These observations indicated that the spectral abnormalities were possibly due to near atropisomerism of amide function and core structure of the molecules had no impact on such phenomenon. Further, temperature variant (at 300e360 K) NMR analysis of 22b and 25b in deuterated pyridine confirmed our assumption (Figs. S1 and S2, supporting information). With the increase in temperature, the two triplets and a doublet appeared for eNCH2 and NCH3 gradually merged. At 360 K, both compound showed singlet for NeCH3 and broad singlet for eNCH2. For synthesis of N-butyl-4-(3-methoxyphenoxy)butanamide (25a) and N-butyl-4-(3-methoxyphenoxy)-N-methylbutanamide (25b), 11 was alkylated with ethyl 4-bromobutyrate in the presence of anhydrous K2CO3 in dry acetone which yielded corresponding ester derivatives (23, Scheme 1) in 84% yield. On basic hydrolysis with 10% NaOH in methanol at reflux, compound 23 yielded 24 in quantitative yield. Subsequently, 24 was reacted with N-methyl-Nbutyl amine or N-butyl amine using EDC and HOBt in dry DMF at room temperature to yield target amides, 25aeb, in 75e88% yields. 1.2. Biology 1.2.1. Assessment of bone forming potential of compounds using osteoblast differentiation assay The process of osteogenesis is facilitated by differentiation of preosteoblasts to mature osteoblasts. Osteoblast differentiation involves modulation/activation of some important osteogenic factors viz alkaline phosphatase (ALP), osteocalcin (OCN) and collagen type 1, etc [3,5]. The extent of osteogenesis can be easily quantified by measuring activity of ALP, OCN and collagen type 1. Since, ALP and OCN are early markers of osteoblastogenesis, we initially measured the ALP activity in MCOs as an indicator of osteoblast differentiation. For this purpose, MCOs were treated with test compounds for 48 h and ALP activity was measured

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spectrophotometrically. Compounds 16aec, 17aec, 21aec and 22aec were evaluated for their osteogenic activity at different concentrations in this assay. 17a, 21bec and 22aeb showed significant increase in ALP activity compared to control untreated cells (Fig. 2). In this experiment, 17b-estradiol (17b-E2) was used as positive control. Among others, 22b exhibited the maximum increase in ALP activity at 1 pM concentration. Therefore, 22b was selected for further confirmation of osteogenic activity through assessment of its bone mineralization and osteogenic gene expression capabilities. 1.2.2. Mineralization efficacy of 22b in mouse calvarial osteoblast cells The mineralization of osteoblast cells is an imperative indicator of its differentiation. Following significant increase in ALP activity by 22b at 1 pM, its effect on bone mineralization was evaluated. For this purpose, MCOs were cultured for 15 days with or without 22b at 1 pM in differentiation media containing 10 mM b-glycerophosphate and 50 mg/mL ascorbic acid. The cells were then stained with alizarin red S dye and the excess dye was extracted to quantify the extent of osteoblast mineralization. In this assay, 22b significantly induced the formation of mineralized nodules compared to the control untreated cells (Fig. 3aeb). 1.2.3. Effect of 22b on osteogenic gene expression Certain biomarkers of bone formation such as bone morphogenetic proteins (BMPs), runt-related transcription factor-2 (RUNX2) and osteocalcin (OCN) are important indicators of osteoblast activity [45,46]. While BMP-2 stimulates an increase in ALP activity and collagen synthesis, RUNX-2 and OCN are associated with osteoblast differentiation and skeletal morphogenesis [47e50]. Considering the importance of these biomarkers in bone formation (osteogenic activity), the effect of 22b on mRNA expression levels of BMP-2, RUNX-2, OCN, and collagen type 1 was evaluated by qPCR to substantiate its osteogenic efficacy. To determine the effect of 22b on osteogenic markers viz BMP-2, RUNX-2, OCN and collagen type 1, osteoblast cells were treated with 22b at 1 pM for 24 and 48 h. Total RNA was then isolated followed by cDNA synthesis which was used as a template in qPCR. GAPDH was used as an internal control for normalization. The results showed that treatment of osteoblast cells with 22b at 1 pM increased the transcript level of RUNX-2 at 24 h significantly compared to control (Fig. 4). BMP-2 and OCN expression levels were significantly enhanced at 48 h while collagen type 1 level was increased at 24 h and 48 h time points over the control (Fig. 4). Based on the mineralization and qPCR data, 22b was further taken up for in vivo studies in estrogen deficient balb/c mice model. 1.2.4. In vivo osteogenic activity of 22b The osteoprotective effect of 22b was evaluated against

Scheme 3. Reagents and reaction conditions: (a) ethyl 4-bromobutyrate, anhy. K2CO3, dry acetone, reflux (b) 10% NaOH, methanol, 40 C (c) N-methyl-N-butylamine or Nbutylamine, EDC, HOBt, Et3N, DMF, RT.

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Fig. 2. Effect of test compounds on osteoblast differentiation. MCOs were seeded in 96 well plates and exposed to various concentrations of compounds ranging from 1 pM to 1 mM for 48 h and ALP activity was determined spectrophotometrically at 405 nm. Estradiol at 100 pM was used as a positive control. Data shown as mean ± SEM; n ¼ 8; (*) P < 0.05, (**) P < 0.01, (***)P < 0.001 compared with untreated cells taken as control.

Fig. 3. aeb. MCOs were seeded in 12 well plates at a density of 2 103 cells per well in differentiation medium and treated with and without 22b for 15 days. At the end of the incubation, cells were fixed and stained with alizarin red-S. Stain was extracted, and optical density was measured calorimetrically. Data shown as mean ± S.E.M; n ¼ 3; ***p < 0.001.

deterioration of trabecular microarchitecture under ovariectomized condition of test animals. Ovariectomized balb/c mice (Ovx mice) were treated with vehicle or 22b by oral gavage for a period of one month. 17b-Estradiol was taken as a positive control. At the completion of experiment, femur bones were dissected out and the changes in trabecular microarchitecture were quantified by mCT. As anticipated, deterioration in trabecular structures was clearly visible in the Ovx þ vehicle group compared with the sham þ vehicle group. Upon quantitation, Ovx þ vehicle group was found to have reduced bone volume density (BV/TV), trabecular number (Tb.N) and increased trabecular spacing (Tb.Sp) values compared to sham þ vehicle group (Fig. 5). Ovx mice treated with 22b exhibited higher values for bone volume density (BV/TV), trabecular number (Tb.N) and lower values of trabecular spacing (Tb.Sp) at 1 and 10 mg kg1 doses compared with Ovx þ vehicle group and these values were comparable with sham þ vehicle group (Fig. 5). However, in 17b-E2 treated group, these effects were more robust possibly due to its potent estrogen agonist action. The above mentioned parameters are key measures to characterize the 3D-structure of trabecular bone. Thus, our data suggested that

compared to vehicle treated estrogen deficient ovariectomized mice, 22b treated Ovx mice showed improved trabecular microarchitecture in femoral bones and this effect was almost equal to sham animals. Thus, 22b had bone conserving effect. 1.2.5. Effect of 22b on bone biomechanical properties It is desirable for a compound to confer better bone quality. Therefore, bone biomechanical properties of 22b were determined by bone strength testing using TKC Muromachi bone strength tester (Fig. 6). It was observed that compared to vehicle treated Ovx group, 22b treatment led to increased energy, stiffness and force required to break. 1.2.6. Effect of 22b on bone turnover markers The rate of bone turnover increases significantly under ovariectomized (Ovx) condition. As anticipated, the levels of bone turnover markers viz serum osteocalcin and carboxy-terminal collagen crosslinks (CTx) were elevated in the Ovx animals compared with sham group (treated with vehicle only). It was observed that 22b treated groups at 1 and 10 mg kg1 body weight


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Fig. 4. qPCR analysis showing increase in mRNA expression of osteogenic marker genes. MCOs were treated with 22b (1 pM) for 24 h and 48 h. Expression of mRNA level of BMP-2, RUNX -2, OCN and Collagen type 1 were determined. qPCR data of the indicated genes represent the mean ± SEM from three independent experiments; (**)P < 0.01, (***)P < 0.001 compared to control.

Fig. 5. Micro architectural Parameters of Tibia; treatment of 22b increased significantly trabecular bone volume (BV/TV), trabecular number (Tb.N) and trabecular spacing (Tb.Sp) which decreases in OVx animals. Each parameter represents pooled data from 10 mice/group, and values are expressed as the mean ± SE: (***) p < 0.001, (**) p < 0.01, and (*) p < 0.05 compared with Ovx þ vehicle group. Inter dose comparison of compound 18 shows that for 1 (mg/kg)/day dose P < 0.01, P < 0.05 when compared with Ovx þ 5 (mg kg1)/ day group and p < 0.01, p < 0.05 when compared with Ovx þ 10 ( mg kg1)/day group. p < 0.01 when Ovx þ5 (mg kg1)/day compared with Ovx þ 10 (mg kg1)/day.

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Fig. 6. At 1 mg kg1 and 10 mg kg1 body weight doses, 22b led to increase in bone biomechanical parameters such as energy, stiffness and force required to breakage of bone. Data represent the mean ± SEM: (***) P < 0.001, (**) P < 0.01 compared with Ovx þ vehicle group.

Fig. 7. 22b is effective in reducing bone turnover markers in OVx mice. (A) serum carboxy-terminal collagen crosslinks (CTx) and (B) Serum osteocalcin levels in Ovx mice treated with dose (1 and 10 mg kg1 body weight dose compared with Ovx þ vehicle group. n ¼ 10 mice/group were used for serum samples assayed in duplicate. Data represent the mean ± SEM: (***) P < 0.001, (**) P < 0.01 compared with Ovx þ vehicle group.

doses for 30 days, significantly reduced the concentration of serum CTx and serum OCN (Fig. 7). These results indicated that 22b could potentially inhibit the aggravated bone turnover caused due to estrogen deficiency which is evidently seen in aging women. 1.2.7. Assessment of uterine estrogenicity of 22b The lack of estrogenic activity in reproductive and adipose tissues is a desirable pre-requisite from an osteoporotic drug to avoid the risk of estrogen induced cancers such as ovarian, uterine and breast cancers. To assess the estrogenicity of 22b, six to eight week old balb/c mice (n ¼ 10) were ovariectomized and oral administration of 22b was given at 1 mg kg1 day1 and 10 mg kg1 day1 doses for 30 consecutive days. 17b-E2 was administered as positive control at 0.01 mg kg1 day1. Uterine weights were recorded in 22b and 17b-E2 treated groups. It was observed that uterine

weights were not different than Ovx group at 1 mg kg1 dose (Fig. 8). However, a slight increase was observed at 10 mg kg1 dose which was significantly lower than the 17b-E2 treated group (Fig. 8). These results showed that 22b was devoid of any uterine estrogenicity. 1.2.8. Effect of 22b in ER-regulated osteoblast functions The osteobalst cells are reported to express both ER-a and ER-b and their differential level of expression during human osteoblast differentiation have revealed a gradual increase in ER-b mRNA expression [51]. The observed differential regulation of ER is suggestive for an additional functional role of ER-b to ER-a in bone maintenance. Keeping these observations in our consideration, we studied the effect of 22b and 17b-E2 in the expression of ER-a and ER-b genes in MCOs. Consistent with previous reports [37], in


Fig. 8. 22b is devoid of statistically non significant estrogenic activity at 1 and 10 mg kg1 body weight dose compared with OVx þ vehicle group. n ¼ 10 mice/group were used. Data represent the mean ± SEM: (***) P < 0.001, (**) P < 0.01 compared with Ovx þ vehicle group.

MCOs, ER-a protein was readily detectable whereas the abundance of ER-b protein appeared in low levels (Fig. 9). Although basal (unstimulated) levels of ER-b protein were low in MCOs, both 22b (at 1 pM concentration) and 17b-E2 (at 10 nM concentration) substantially increased the protein levels of ER-b in MCOs but not ER-a (Fig. 9). From these results, it appeared that ER-b could be involved in mediating osteogenic effect of 22b. 1.2.9. Plasma pharmacokinetic study of lead compound 22b The pharmacokinetic profile of 22b was assessed in order to evaluate its oral bioavailability and metabolic stability. For this purpose female Sprague-Dawley rats (n ¼ 4) were fasted overnight (12e14 h) before dosing and had free access to water throughout the experimental period. 22b was administered orally at 1 mg kg1

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in 0.25% CMC suspension and via a lateral tail vein as a bolus dose at 1 mg kg1. Blood samples were collected, processed and analyzed for maximum plasma concentration (Cmax) and the time to reach the maximum plasma concentration (Tmax). The mean plasma concentration-time profile of 22b and its estimated pharmacokinetic parameters are presented in the (Fig. 10 and Table 1). Upon intravenous administration, the apparent volume of distribution was found to be 5.6 ± 0.05 L/kg and clearance was found to be 1.10 ± 0.04 L/h/kg. The time taken for the systemic levels to reduce to half (half life, T1/2) was 3.52 ± 0.12 h and the elimination rate constant (Ke) was found to be 0.197 ± 0.006 h1. The area under the concentration-time profile (AUC0-∞) representing the total systemic drug exposure was 906.01 ± 38.12 and 119.40 ± 15.26 h*ng/ mL after intravenous and oral drug administration respectively. The results showed that 22b had 13.18% oral bioavailability (F). The maximum plasma concentration (Cmax) of 22b reached after oral administration was 38.90 ± 12.19 ng/mL and time required to reach this maximum concentration was 1.50 ± 0.50 h. 1.2.10. In vivo acute toxicity studies of 22b Following in vivo osteogenic, estrogenicity and pharmacokinetic studies, 22b was evaluated for possible adverse effects on the other major organ systems due to its inherent toxicity in swiss albino mice following the OECD Guideline no. 420, 423 and 425. In acute oral toxicity study, mice treated with 22b at 5, 50, 300 and 1000 mg kg1 as a single acute oral dose did not show any morbidity, mortality or observational changes during the entire period of experimentation of 7 days. At the end of experiment, blood and tissue samples were collected from animals of all the experimental groups including control and used for analysis of hematological and biochemical parameters (Tables 2 and 3). Hematological parameters like total RBC count, WBC count, DLC and hemoglobin did not show any change in any experimental group compared to control. Similarly, biochemical parameters like SGOT, SGPT, ALP, creatinine, triglycerides, serum proteins, tissue protein etc. and tissue biochemical parameters like MDA, reduced GSH etc.

Fig. 9. Effect of 22b in ER-regulated osteoblast function. Mouse osteoblast cells were treated with or without compound 22b and positive control E2 (100 pM) for 48 h time point. Protein lysates were collected and transblotted onto a PVDF membrane and probed with primary antibodies followed by the corresponding HRP linked secondary antibodies and these were normalized with b-actin. Graph shows the densitometric analysis (fold change) of the observed change in expression of the proteins.

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kidneys, spleen, uterus, ovary and testes were collected after sacrifice of the animals and were examined for gross pathological changes and weighed. The organ weight in both absolute and relative terms did not produce any change in any experimental group compared to control. These results are presented in Tables 2e5. 1.3. Structure activity relationship (SAR)

Fig. 10. Mean plasma concentration-time profile of 22b upon oral and intravenous administration. Data represented as mean ± S.D. (n ¼ 4).

did not show significant changes in any experimental group compared to control. Vital organs like brain, lungs, heart, liver,

Table 1 Pharmacokinetic parameters of 22b upon intravenous (i.v.) and oral administration. Data represented as mean ± S.D. (n ¼ 4).

3-aryl benzopyran core is a well established pharmacophore for many biological targets including estrogen receptor-a and b (ER-a & ER-b). This core is widely present in isoflavone based phytoestrogens such as geinstein (1) and daidgein (2) etc. Simple structural modification in this core often brings receptor subtype selectivity which can be exemplified by enantiomers of equol molecule [(R) equol is ER-a selective while (S) equol is for ER-b)] [52]. Further, structural modifications in this core led to the 4-benzyl-3-arylbenzopyran, 2,3-diarylbenzopyran and 3,4-diarylbenzopyan pharmacophores with established ER modulating ability [44,53]. More recently, we could identify some novel osteogenic agents through modification of 3-arylbenzopyran core [38a].

Table 4 Effect of 22b at 5, 50, 300 and 1000 mg/kg body weight as a single acute oral dose on absolute organ weight (g) (Mean ± SE, n ¼ 6).

Parameter

I.V.

Oral

Organ

Control

Ke (1/h) T1/2 (h) Tmax (h) C0/Cmax (ng/mL) AUC0e24 (h*ng/mL) AUC0-∞ (h*ng/mL) Vd (L/kg) CL (L/h/kg) F (%)

0.197 ± 0.006 3.524 ± 0.115 e 610.313 ± 45.427 851.646 ± 35.037 906.019 ± 38.128 5.612 ± 0.052 1.105 ± 0.046 e

0.194 ± 0.029 3.621 ± 0.556 1.500 ± 0.500 38.900 ± 12.194 113.722 ± 9.473 119.404 ± 15.269 5.822 ± 1.607 1.100 ± 0.134 13.179

Liver Lungs Brain Spleen Kidneys Heart Uterus Ovary Testes

1.11 0.16 0.37 0.17 0.29 0.12 0.27 0.04 0.15

± ± ± ± ± ± ± ± ±

0.04 0.01 0.02 0.03 0.02 0.00 0.07 0.02 0.01

5 mg/kg 1.18 0.15 0.35 0.11 0.28 0.12 0.30 0.07 0.15

± ± ± ± ± ± ± ± ±

50 mg/kg

0.05 0.01 0.01 0.01 0.01 0.01 0.02 0.01 0.01

1.23 0.20 0.36 0.13 0.26 0.12 0.24 0.07 0.17

± ± ± ± ± ± ± ± ±

0.08 0.03 0.02 0.02 0.05 0.01 0.04 0.01 0.01

300 mg/kg 1.13 0.17 0.35 0.13 0.27 0.12 0.33 0.06 0.14

± ± ± ± ± ± ± ± ±

1000 mg/kg

0.08 0.02 0.01 0.01 0.02 0.01 0.04 0.01 0.01

1.07 0.15 0.33 0.10 0.29 0.12 0.38 0.05 0.18

± ± ± ± ± ± ± ± ±

0.07 0.02 0.04 0.02 0.04 0.01 0.09 0.01 0.03

Table 2 Effect of 22b at 5, 50, 300 and 1000 mg/kg body weight as a single acute oral dose on body weight, hematological and biochemical parameters. Parameters studied

Treated groups Control

5 mg/kg

50 mg/kg

300 mg/kg

1000 mg/kg

Body weight0 day (g) Body weight7th day (g) Haemoglobin (gm/dL) RBC (million/mm3) WBC (thousands/mm3) ALKP (U/L) SGOT (U/L) SGPT (U/L) Albumin (g/dL) Creatinine (mg/dL) Triglycerides (mg/dL) Cholesterol (mg/dL) Serum protein (mg/ml) Tissue protein (mg/ml) Reduced GSH (mg/mg protein) Malondialdehyde (nM/mg protein)

20.38 ± 0.24 22.75 ± 0.38 10.86 ± 0.87 6.40 ± 0.28 6.84 ± 0.83 379.25 ± 27.08 31.99 ± 3.37 14.04 ± 1.45 3.24 ± 0.30 0.97 ± 0.07 58.51 ± 10.82 265.76 ± 24.78 0.66 ± 0.01 0.029 ± 0.004 18.20 ± 1.56 0.14 ± 0.01

20.65 ± 0.48 24.25 ± 0.59 12.94 ± 0.46 7.14 ± 1.59 6.10 ± 0.86 278.70 ± 48.44 33.05 ± 4.24 17.44 ± 0.99 3.97 ± 0.26 0.78 ± 0.05 67.53 ± 8.22 288.67 ± 20.54 0.74 ± 0.01 0.035 ± 0.002 19.35 ± 0.42 0.14 ± 0.01

20.38 ± 1.43 23.52 ± 2.06 13.14 ± 0.82 8.16 ± 0.63 5.69 ± 0.16 306.33 ± 20.85 40.13 ± 4.07 17.18 ± 2.13 2.77 ± 0.21 0.80 ± 0.05 83.78 ± 14.48 219.09 ± 8.78 0.74 ± 0.01 0.036 ± 0.002 15.00 ± 1.85 0.14 ± 0.01

19.61 ± 0.77 23.31 ± 1.35 12.92 ± 1.36 7.31 ± 0.75 6.59 ± 0.52 329.28 ± 18.78 42.66 ± 5.02 17.60 ± 1.24 3.73 ± 0.44 0.97 ± 0.09 57.55 ± 10.74 257.26 ± 44.40 0.67 ± 0.02 0.034 ± 0.001 14.31 ± 2.13 0.13 ± 0.01

18.42 ± 0.95 20.30 ± 0.64 11.35 ± 0.94 6.27 ± 1.01 5.10 ± 0.26 378.12 ± 47.94 38.60 ± 5.72 17.21 ± 0.49 3.98 ± 0.58 0.69 ± 0.06 92.31 ± 25.05 267.94 ± 41.48 0.75 ± 0.03 0.032 ± 0.003 13.98 ± 1.63 0.16 ± 0.02

Table 3 Effect of 22b at 5, 50, 300 and 1000 mg/kg body weight as a single acute oral dose on DLC (%) (Mean ± SE, n ¼ 6). Groups

Control

5 mg/kg

50 mg/kg

300 mg/kg

1000 mg/kg

Neutrophil Lymphocyte Monocyte Eosinophill Basophill

41.50 ± 4.08 37.97 ± 1.80 12.58 ± 2.86 6.34 ± 1.04 1.50 ± 0.29

42.94 ± 5.01 39.08 ± 3.12 8.94 ± 1.95 8.35 ± 2.28 0.67 ± 0.67

44.27 ± 3.10 35.79 ± 3.50 12.35 ± 1.19 6.99 ± 0.58 0.50 ± 0.29

41.54 ± 3.82 42.90 ± 2.54 8.99 ± 0.35 5.27 ± 1.65 1.25 ± 0.75

46.03 ± 2.68 34.49 ± 2.08 12.27 ± 1.57 5.35 ± 0.61 1.75 ± 0.48

A. Gupta et al. / European Journal of Medicinal Chemistry 121 (2016) 82e99 Table 5 Effect of 22b at 5, 50, 300 and 1000 mg/kg body weight as a single acute oral dose on relative organ weight (%) (Mean ± SE). Groups

Control

Liver Lung Brain Spleen Kidney Heart Uterus Ovary Testes

6.81 0.70 1.63 0.73 1.25 0.51 1.63 0.07 0.62

± ± ± ± ± ± ± ± ±

2.14 0.08 0.13 0.14 0.08 0.02 0.71 0.02 0.03

5 mg/kg 4.84 0.61 1.46 0.46 1.13 0.48 1.77 0.20 0.60

± ± ± ± ± ± ± ± ±

0.10 0.03 0.07 0.05 0.03 0.04 0.36 0.16 0.04

50 mg/kg 5.17 0.84 1.54 0.55 1.09 0.50 1.17 0.33 0.63

± ± ± ± ± ± ± ± ±

0.26 0.14 0.13 0.03 0.10 0.03 0.11 0.04 0.03

300 mg/kg 4.84 0.73 1.54 0.57 1.14 0.49 1.23 0.29 0.54

± ± ± ± ± ± ± ± ±

0.08 0.15 0.15 0.01 0.06 0.02 0.67 0.07 0.03

91

adequate insight for further development as osteogenic therapeutic for the treatment of osteoporosis following preclinical studies.

1000 mg/kg 5.28 0.75 1.63 0.50 1.44 0.57 1.82 0.05 0.88

± ± ± ± ± ± ± ± ±

0.28 0.08 0.16 0.09 0.23 0.03 0.33 0.00 0.18

In the present study, the target compounds were synthesized based on 3-arylbenzopyran core. In general, designed compounds belong to two types of subsets namely 3,4-diaryl-2H-benzopyran (16aec and 17aec) and 3,4-diaryl-3H-benzopyran (21aec and 22aec) derivatives. Structurally, these molecules possess long chain amide group tethered with 4-phenyl group of 3,4-diaryl-2Hbenzopyran and 3,4-diaryl-3H-benzopyran nucleus (compounds 16aec, 17aec, 21aec and 22aec). For the preparation of long chain amide group, N-butyl-N-methyl amine or 2-methylaminopyridine was used with carbon linker of variable chain lengths (C4 to C6). Among these two types of subsets total five compounds (17a, 21bec and 22aeb) were found active. The biological activity data raveled that 3,4-diaryl-3H-benzopyran derivatives (21bec and 22aeb) were more active than 3,4-diaryl-2H-benzopyran derivative (17a) which might be due to non planarity and greater molecular thickness of 3,4-diaryl-3H-benzopyran nucleus. As far as the length of linker is concern, a linker with five carbon (C5) atoms is optimal for induction of better biological activity. Interestingly, it was noticed that amide derivatives with N-butyl-N-methyl amine were better than 2-methylaminopyridine based derivatives in both the subtypes of compounds. The better activity of N-butyl-Nmethyl amine based amide derivatives could be ascribed due to its greater accessibility within the target site for more favorable electrostatic contacts with target protein as described for ICI 164384 molecule [54]. 2. Conclusion In anticipation of osteogenic activity, a series of 3,4diarylbenzopyran based amide derivatives was synthesized and evaluated for osteogenic activity using osteoblast differentiation assay and estrogen deficient balb/c mice model of osteoporosis. Among other active compounds, 22b was characterized as lead molecule which showed potent osteogenic activity at 1 pM concentration as measured by ALP activity in osteoblast differentiation assay using MCOs. The osteogenic potential of 22b was further confirmed by increased bone mineralization activity and expression of osteogenic genes viz BMP-2, RUNX-2, osteocalcin and collagen type 1. In vivo osteogenic study of 22b in balb/c mice model showed potential osteogenic activity at 1 mg kg1 body weight dose in terms of increased bone volume density (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N) and low values of trabecular spacing (Tb.S). The in vivo pharmacokinetic study of 22b in Sprague-Dawley rats revealed good bioavailability of 22b. Furthermore, 22b showed no apparent toxicity upto 1000 mg kg1 body weight dose in in vivo model. Interestingly, from in-vivo osteogenic, toxicity and pharmacokinetic studies, it appeared that 22b had no uterotropic activity in these three animal models. Overall, based on biological activity data, 22b has been identified as a potential osteogenic lead molecule which can give

3. Experimental section 3.1. Chemical methods: reagents and chemicals All reagents and solvents used were of analytical or laboratory grade and used without further purification. The reactions were monitored on Merck aluminium thin layer chromatography (TLC, UV254nm) plates. Column chromatography was carried out on silica gel (100e200 mesh). The melting points were determined on Buchi melting point M560 apparatus in open capillaries and are uncorrected. 1H and 13C NMR spectra were recorded on a Bruker WM-300 (300 MHz) using CDCl3, acetone-d6, pyridine-d5 and DMSO-d6 as the solvent. Chemical shift are reported in parts per million shift (dvalue). Signal patterns are indicated as s, singlet; bs, broad singlet; d, doublet; dd, double doublet; t, triplet; m, multiplet; brm, broad multiplet. Coupling constants (J) are given in Hertz. Infrared (IR) spectra were recorded on a Perkin-Elmer AX-1 spectrophotometer in KBr disc and reported in wave number (cm1). ESI mass spectra were recorded on Shimadzu LC-MS after dissolving the compounds in acetonitrile and methanol. Elemental analyses were performed on a Carlo-Erba-1108 C, H, N elemental analyzer (Italy). 3.1.1. 4-(4-hydroxyphenyl)-7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran (13) Yield: 69%; Mp. 170e172 C; Rf: 0.30 (20% ethyl acetate-hexane); 1 H NMR (CDCl3, 300 MHz, d ppm): 1.46 (bs, 6H, 2xCH3), 3.787 (s, 3H, OCH3), 4.87 (bs, 1H, OH), 6.38e6.34 (dd, J ¼ 3.6 and 2.7 Hz, 1H, ArH), 6.510e6.02 (d, J ¼ 2.1 Hz, 1H, ArH), 6.67e6.53 (m, 3H, ArH), 6.87e6.84 (d, J ¼ 8.4 Hz, 2H, ArH), 7.05e7.00 (m, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 27.35 (2xC), 55.76, 79.27, 102.47, 107.17, 115.03 (2xC), 118.73, 126.78, 127.67, 128.06 (2xC), 130.31 (2xC), 130.55, 132.25 (3xC), 136.60, 139.12, 154.30, 154.44, 160.90; MS (C24H22O3): m/z ¼ 357 [MH]þ, 381 [MþNa]þ. 3.1.2. Synthesis of ethyl 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl2H-benzopyran-4-yl)phenoxy)butanoate (14a) In a round bottom flask, compound 13 (1.0 g, 2.78 mmol) was dissolved in dry acetone (25 mL). To this solution anhydrous potassium carbonate (1.10 g, 8.34 mmol) and ethyl 4-bromobutyrate (0.63 mL, 4.17 mmol) were added. The solution was heated under reflux for 6 h. The reaction mixture was filtered and filtrate was evaporated to dryness. The residue was dissolved into ethylacetate and washed with water. The organic layer was separated, dried over anhydrous sodium sulfate and concentrated. The crude material was then chromatographed over a column of silicagel eluting with the ethylacetate-hexane (5:95) to afford compound 14a as white solid. Yield: 76%; m.p. 100e101 C; Rf: 0.60 (20% ethyl acetatehexane); IR (KBr, nmax/cm1): 2970, 1732, 1608, 1508, 1283, 1244, 1202, 1034, 842, 705; 1H NMR (CDCl3, 300 MHz, d ppm): 1.26 (bs, 3H, CH3), 1.49 (bs, 6H, 2xCH3), 2.08 (bs, 2H, CH2), 2.50 (bs, 2H, CH2), 3.93 (s, 3H. OCH3). 4.06 (bs, 2H, CH2), 4.14e4.17 (m, 2H, CH2), 6.37e6.40 (m, 1H, ArH), 6.53 (bs, 1H, ArH), 6.66e6.69 (d, J ¼ 7.2 Hz, 3H, ArH), 6.92 (d, J ¼ 7.8 Hz, 2H, ArH), 7.05 (bs, 2H, ArH), 7.14 (bs, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.63, 25.08, 27.36 (2xC), 31.25, 55.75, 60.81, 66.90, 102.42, 107.12, 114.01 (3xC), 118.77, 126.76, 127.70, 128.07 (3xC), 130.31 (2xC), 132.06 (2xC), 132.15, 136.49, 139.15, 154.33, 157.74, 160.90, 173.65; MS (C30H32O5): m/ z ¼ 495 [MþNa]þ; Anal. Cacld (C30H32O5): C, 76.25; H, 6.83 Found C, 76.20; H, 6.91.

92


3.1.3. Ethyl 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran-4-yl)phenoxy)pentanoate (14b) Yield: 80%, m.p. 90e91 C, Rf: 0.50 (20% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2983, 1734, 1612, 1507, 1289, 1240, 1165, 1128, 1035, 852, 701; 1H NMR (CDCl3, 300 MHz, d ppm): 1.25 (t, J ¼ 7.0 Hz, 3H, CH3), 1.46 (bs, 6H, 2xCH3), 1.77 (bs, 4H, 2xCH2), 2.34 (bs, 2H, CH2), 3.79 (s, 3H, OCH3), 3.86 (bs, 2H, CH2), 4.12 (q, 2H, CH2), 6.36 (dd, J ¼ 8.7 and 8.4 Hz, 1H, ArH), 6.51 (d, J ¼ 2.4 Hz, 1H, ArH), 6.63e6.72 (m, 3H, ArH), 6.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.01e7.03 (m, 2H, ArH) 7.08e7.13 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.21, 21.62, 26.72 (2xC), 28.67, 33.94, 55.30, 60.25, 67.09, 78.80, 101.98, 106.67, 113.56 (2xC), 118.35, 126.31, 127.27, 127.62 (2xC), 129.87 (3xC), 131.60 (2xC), 131.74, 136.03, 138.72, 153.89, 157.42, 160.45, 173.42; MS (C31H34O5): m/z ¼ 487 [MþH]þ, 509 [MþNa]þ; Anal. Cacld (C31H34O5): C, 76.52; H, 7.04 Found C, 76.42; H, 6.93. 3.1.4. Ethyl 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran-4-yl)phenoxy)hexanoate (14c) Yield: 78%; m.p. 96e97 C; Rf, 0.50 (20% ethyl acetate-hexane); IR (KBr, nmax/cm1): 1728, 1610, 1508, 1241, 1168, 1042, 811, 704; 1H NMR (CDCl3, 300 MHz, d ppm): 1.25 (t, J ¼ 7.2 Hz, 3H, CH3), 1.47 (bs, 8H, CH2 and 2xCH3), 1.62e1.79 (m, 4H, 2xCH2), 2.32 (t, J ¼ 7.5 Hz, 2H, CH2), 3.79 (s, 3H, OCH3), 3.86 (t, J ¼ 6.3 Hz, 2H, CH2), 4.13 (q, 2H, OCH2), 6.36 (dd, J ¼ 2.4 and 2.4 Hz, 1H, ArH), 6.50 (d, J ¼ 6.3 Hz, 1H, ArH), 6.65 (dd, J ¼ 3.0 and 3.0 Hz, 3H, ArH), 6.90 (d, J ¼ 8.4 Hz, 2H, ArH), 7.02 (d, J ¼ 6.3 Hz, 2H, ArH), 7.08e7.16 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.65, 25.12, 26.06, 27.36 (2xC), 29.36, 34.65, 55.74, 60.64, 67.80, 79.24, 102.41, 107.11, 114.01 (2xC), 118.80, 126.74, 127.72, 128.05 (2xC), 130.31 (2xC), 132.03 (3xC), 132.19, 136.45, 139.17, 154.33, 157.93, 160.88, 174.05; MS (C32H36O5): m/ z ¼ 525 [MþNaþ2H]þ; Anal. Cacld (C32H36O5): C, 76.77; H, 7.25 Found C, 76.86; H, 7.41. 3.1.5. Synthesis of 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran-4-yl)phenoxy)butanoic acid (15a) In a round bottomed flask, 14a (0.58 g, 1.22 mmol) was dissolved in methanol and 10% aq. NaOH (10 mL) was added to this mixture and reaction was allowed to reflux for 1.5e2 h. After completion of the reaction, excess solvent was evaporated and the reaction mixture was acidified with conc. HCl. Subsequently, the content was extracted using ethyl acetate. Organic layer was separated, dried over anhydrous sodium sulfate and concentrated. The crude product was recrystallized by using hexane-ethylacetate mixture to yield pure 15a as white solid. Yield: 84%; Rf:0.12 (30% ethyl acetate-hexane); IR (KBr, nmax/ cm1): 3449, 1704, 1615, 1509, 1239, 1164, 857, 703; 1H NMR (CDCl3, 300 MHz, d ppm): 1.49 (s, 6H, 2xCH3), 2.08 (t, J ¼ 6.3 Hz, 2H, CH2), 2.56 (t, J ¼ 7.1 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.94 (t, J ¼ 5.8 Hz, 2H, OCH2), 6.37e6.39 (m, 1H, ArH), 6.53 (s, 1H, ArH), 6.67 (d, J ¼ 8.4 Hz, 3H, ArH), 6.93 (d, J ¼ 8.4 Hz, 2H, ArH), 7.03e7.05 (m, 2H, ArH), 7.11e7.16 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 24.82, 27.36 (2xC), 30.96, 55.74, 66.70, 79.24, 102.43, 107.14, 114.03 (2xC), 118.77, 126.77, 127.69, 128.07 (3xC), 130.30 (3xC), 130.55, 132.07 (2xC), 136.52, 139.14, 154.33, 157.65, 160.90; MS (C28H28O5): m/ z ¼ 443 [MH]þ, 467 [MþNa]þ; Anal. Cacld (C28H28O5): C, 75.65; H, 6.35 Found C, 75.89; H, 6.51. 3.1.6. 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2H-benzopyran-4yl)phenoxy)pentanoic acid (15b) Yield: 82%; m.p. 123e124 C; Rf: 0.11 (30% ethyl acetatehexane); IR (KBr, nmax/cm1): 3328, 2928, 1706, 1606, 1508, 1282, 1241, 1114, 1031, 840, 703; 1H NMR (CDCl3, 300 MHz, d ppm): 1.49 (bs, 6H, 2xCH3), 1.81 (bs, 4H, 2xCH2), 2.48 (bs, 2H, CH2), 3.81 (s, 3H, OCH3), 3.90 (bs, 2H, OCH2), 6.27e6.40 (m, 1H, ArH), 6.53 (d, J ¼ 2.1 Hz, 1H, ArH), 6.68 (dd, J¼ 2.7 and 2.4 Hz, 3H, ArH), 6.92 (d,

J ¼ 8.4 Hz, 2H, ArH), 7.16e7.05 (m, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 21.80, 27.37 (2xC), 28.98, 34.00, 55.75, 67.47, 79.25, 102.43, 107.14, 114.01 (2xC), 118.80, 126.76, 127.72, 128.07 (2xC), 130.32 (3xC), 132.06 (2xC), 132.18, 136.49, 139.16, 154.33, 157.82, 160.89, 179.82; MS (C29H30O5): m/z ¼ 457 [MH]þ, 481 [MþNa]þ; Anal. Cacld (C29H30O5): C, 75.96; H, 6.59; O, 17.45Found C, 75.75; H, 6.65. 3.1.7. 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2H-benzopyran-4yl)phenoxy)hexanoic acid (15c) Yield: 80%; m.p. 103e104 C: Rf: 0.12 (30% ethyl acetatehexane); IR (KBr, nmax/cm1): 3349, 2941, 1714, 1610, 1508, 1243, 1165, 851, 700; 1H NMR (CDCl3, 300 MHz, d ppm): 1.49 (bs, 8H, CH2 and 2xCH3), 1.67e1.80 (m, 4H, 2xCH2), 2.40 (t, J ¼ 7.2 Hz, 2H, CH2), 3.81 (s, 3H, OCH3), 3.88 (t, J ¼ 6.3 Hz, 2H, OCH2), 6.39 (dd, J ¼ 2.4 and 2.1 Hz, 1H, ArH), 6.53 (d, J ¼ 2.4 Hz, 1H, ArH), 6.68 (dd, J ¼ 3.0 and 2.7 Hz, 3H, ArH), 6.92 (d, J ¼ 8.4 Hz, 2H, ArH), 7.05 (d, J ¼ 7.8 Hz, 2H, ArH), 7.10e7.16 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 27.36, 29.42 (2xC), 36.94, 44.80, 55.74, 67.83, 79.23, 102.41, 107.10, 114.01 (2xC), 118.0, 122.57, 122.77, 126.74, 127.72 (2xC), 128.05 (2xC), 130.22 (2xC), 130.30, 132.02, 132.18, 136.44 (2xC), 137.18, 139.16 (2xC), 149.37, 154.33, 156.80, 157.93, 160.87, 173.34; MS (C29H32O5): m/z ¼ 471 [MH]þ, 495 [MþNa]þ; Anal. Cacld (C30H32O5): C, C, 76.25; H, 6.83 Found C, 76.34; H, 7.01. 3.1.8. Synthesis of 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopran-4-yl)phenoxy)-N-(pyridin-2-ylmethyl)butanamide (16a) In a round bottom flask, 15a (0.20 g, 0.42 mmol) was dissolved in dry dimethylformamide (DMF) and DCC (0.13 g, 0.63 mmol), HOBt (0.08 g, .63 mmol) and 3e4 drops of triethylamine were added to this solution at 40 C. The reaction was allowed to stir at 40 C for 30 min. Addition of N-methyl-N-butylamine (0.04 mL, 0.46 mmol) was done and stirring was continued at this temperature for 4 h. After completion of the reaction, water was added to reaction mixture which was extracted with ethyl acetate. The organic layer was separated, dried over anhydrous sodium sulfate and concentrated. The crude product was purified by column chromatography using silicagel (100e200) and ethylacetate-hexane (40:60) which gave pure 16a. Yield: 75%; m.p. 142e143 C; Rf: 0.40 (40% ethyl acetatehexane); IR (KBr, nmax/cm1): 3353, 2924, 1643, 850; 1H NMR (CDCl3, 300 MHz, d ppm): 1.48 (m, 6H, 2xCH3), 2.11e2.19 (m, 2H, CH2), 2.46e2.50 (m, 2H, CH2), 3.81 (s, 3H, OCH3), 3.92e3.93 (m, 2H, CH2), 4.57 (d, J ¼ 4.2 Hz, 2H, CH2), 6.37 (d, J ¼ 8.4 Hz, 1H, ArH), 6.53 (d, J ¼ 1.8 Hz, 1H, ArH), 6.67 (d, J ¼ 8.1 Hz, 3H, ArH), 6.87e6.92 (m, 3H, ArH), 7.03e7.27 (m, 7H, ArH), 7.63e7.68 (m, 1H, ArH), 8.50 (bs, 1H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 25.64, 27.35 (2xC), 33.29, 44.85, 55.75, 67.10, 79.24, 102.40, 107.13, 114.05 (2xC), 118.77, 122.50, 122.77, 126.77, 127.70, 128.07 (2xC), 130.30 (2xC), 130.40 (2xC), 132.05 (2xC), 136.48, 137.19, 139.14, 149.41, 154.33, 156.77, 157.76, 160.88, 172.82; HRMS (ESI): calc. for C34H34N2O4: 535.2591 (MþþH); found: 535.2591. The synthesis of 16bec was accomplished from 15bec following same synthetic methodology. 3.1.9. 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2H-benzopyran-4yl)phenoxy)-N-(pyridin-2-ylmethyl)pentanamide (16b) Yield: 69%, m.p. 111e112 C, Rf: 0.32 (40% ethyl acetate-hexane); IR (KBr, nmax/Cm1): 3283, 2936, 1654, 1285, 1240, 1040, 984, 760, 702; 1H NMR (CDCl3, 300 MHz, d ppm): 1.24 (bs, 6H, 2xCH3), 1.79 (bs, 4H, 2xCH2), 2.32 (bs, 2H, CH2), 3.78 (s, 3H, OCH3), 3.86 (bs, 2H, OCH2), 4.55 (d, J ¼ 3.4 Hz, 2H, CH2), 6.36 (d, J ¼ 8.1 Hz, 1H, ArH), 6.49 (s, 1H, ArH), 6.64 (d, J ¼ 5.1 Hz, 3H, ArH), 6.81 (bs, 1H, NH), 6.88 (d, J ¼ 7.8 Hz, 2H, ArH), 7.00e7.25 (m, 7H, ArHþpyrdinyl-H), 7.61e7.66


(m, 1H, Pridinyl-H), 8.50 (bs, 1H, Pridinyl-H); 13C NMR (CDCl3, 75 MHz, d ppm): 22.84, 27.36 (2xC), 29.20, 36.60, 44.81, 55.74, 67.69, 79.24, 102.41, 107.11, 114.00 (2xC), 118.79, 122.56, 122.78, 126.76, 127.72, 128.06 (2xC), 130.30 (3xC), 132.03 (2xC), 132.17, 136.46, 137.19, 139.15, 149.38, 154.32, 156.77, 157.86, 160.88, 173.14; MS (C35H36N2O4): m/z ¼ 547[MH], 549[MþH], 571[MþNa]. 3.1.10. 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2H-benzopyran-4yl)phenoxy)-N-(pyridin-2-ylmethyl)hexanamide (16c) Yield: 72%; m.p. 105e106 C; Rf: 0.40 (40% ethyl acetatehexane): IR (KBr, nmax/Cm1): 3296, 2943, 1640, 1285, 1242, 1199, 1110, 1041, 821, 758, 704; 1H NMR (CDCl3, 300 MHz, d ppm): 1.46 (s, 8H, CH2 and 2xCH3), 1.68e1.74 (m, 4H, 2xCH2), 2.29 (t, J ¼ 7.5 Hz, 2H, CH2), 3.78 (s, 3H, OCH3), 3.84 (t, J ¼ 6.3 Hz, 2H, OCH2), 4.55 (d, J ¼ 4.8 Hz, 2H, CH2), 6.36 (dd, J ¼ 2.4 and 2.4 Hz, 1H, ArH), 6.50 (d, J ¼ 2.1 Hz, 1H, ArH), 6.50e6.67 (m, 3H, ArH), 6.78 (bs, 1H, NH), 6.89 (d, J ¼ 8.4 Hz, 2H, ArH), 7.02 (d, J ¼ 7.5 Hz, 2H, ArH), 7.07e7.26 (m, 4H, ArH þ Pridinyl-H), 7.24 (d, J ¼ 7.2 Hz, 1H, Pridinyl-H), 7.61e7.67 (m, 1H, Pridinyl-H), 8.50 (bs, 1H, Pridinyl-H); 13C NMR (CDCl3, 75 MHz, d ppm): 20.57, 22.85, 29.21, 36.60, 44.33, 44,80, 55.63, 55.89, 67.64, 78.61, 101.78, 107.87, 114.34 (2xC), 119.03, 122.56, 122.78, 127.08, 128.35 (3xC), 130.39 (3xC), 131.34, 136.32, 137.20, 139.83, 149.38, 154.55, 156.76, 157.58, 159.59, 173.16; HRMS (ESI): calc. for C36H38N2O4: 563.2904 (MþþH); found: 563.2886. The synthesis of 17aec was accomplished from 15aec and 2aminnomethylpyridine, following the synthetic methodology described for synthesis of 15aec. 3.1.11. Synthesis of N-butyl-4-(4-(7-methoxy-2,2-dimethyl-3phenyl-2H-benzopyran-4-yl)phenoxy)-N-methylbutanamide (17a) Yield: 72%; m.p. oil; Rf: 0.35 (30% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2924, 1689, 1606, 1507, 1285, 1243, 1161, 787; 1H NMR (CDCl3, 300 MHz, d ppm): 0.85e0.94 (m, 3H, CH3), 1.25e1.35 (m, 3H, CH3), 1.46 (bs, 7H, 2xCH2 and CH3), 2.07 (bs, 2H, CH2), 2.43e2.51 (m, 2H, CH2), 2.92 (d, J ¼ 10.2 Hz, 3H, NCH3), 3.23 (t, J ¼ 7.5 Hz, 1H, CH of CH2), 3.34 (t, J ¼ 7.3 Hz, 1H, CH of CH2), 3.78 (s, 3H, OCH3), 3.93 (bs, 2H, OCH2), 6.35 (dd, J ¼ 2.4 and 2.4 Hz, 1H, ArH), 6.50 (d, J ¼ 2.4 Hz, 1H, ArH), 6.63e6.66 (m, 3H, ArH), 6.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.00 (d, J ¼ 6.3 Hz, 2H, ArH), 7.08e7.15 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.22, 20.35, 20.48, 25.25, 25.36, 25.52, 27.35 (2xC), 29.46, 29.83, 30.08, 30.19, 30.95, 33.80, 34.35, 35.60, 47.92, 50.07, 55.73, 67.21, 67.29, 79.22, 102.43, 107.11, 114.02 (2xC), 118.78, 126.73, 127.68, 128.04 (2xC), 130.32 (2xC), 132.04 (2xC), 132.18, 136.48, 139.18, 154.34, 157.83, 160.91, 172.39, 172.50; HRMS (ESI): calc. for C33H40NO4: 514.2947 (MþþH); found: 514.2932. 3.1.12. N-butyl-5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran-4-yl)phenoxy)-N-methylpentanamide (17b) Yield: 73%; m.p. oil; Rf: 0.03 (30% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2924, 1645, 1611, 1505, 1243, 1036, 790; 1H NMR (CDCl3, 300 MHz, d ppm): 0.89e0.95 (m, 3H, CH3), 1.25e1.33 (m, 3H, CH3), 1.46 (bs, 7H, 2xCH2 and CH3), 1.78 (bs, 4H, 2xCH2), 2.35 (bs, 2H, CH2), 2.90 (d, J ¼ 15.6 Hz, 3H, NCH3), 3.23 (t, J ¼ 7.5 Hz, 1H, CH of CH2), 3.34 (t, J ¼ 7.3 Hz, 1H, CH of CH2), 3.78 (s, 3H, OCH3), 3.88 (bs, 2H, OCH2), 6.35 (dd, J ¼ 2.4 and 2.4 Hz, 1H, ArH), 6.50 (d, J ¼ 2.7 Hz, 1H, ArH), 6.62e6.67 (m, 3H, ArH), 6.89 (d, J ¼ 8.7 Hz, 2H, ArH), 7.00e7.03 (m, 2H, ArH), 7.07e7.15 (m, 3H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.28, 20.37, 20.49, 22.14, 22.51, 27.36 (2xC), 29.37, 29.84, 31.03, 32.95, 33.59, 33.77, 35.67, 47.87, 50.14, 55.73, 67.82, 79.23, 102.42, 107.09, 113.99 (2xC), 118.79, 126.74, 127.71, 128.05 (2xC), 130.23 (2xC), 130.30, 132.02 (2xC), 132.19, 136.45, 139.16, 154.33, 157.92, 160.88, 172.73, 172.86; HRMS (ESI): calc. for C34H41NO4: 528.3114 (MþþH); found: 528.3081.

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3.1.13. N-butyl-6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-2Hbenzopyran-4-yl)phenoxy)-N-methylhexanamide (17c) Yield: 74%; m.p. oil; Rf: 0.30 (30% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2933, 1645, 1506, 1241, 837, 705; 1H NMR (CDCl3, 300 MHz, d ppm): 0.91e0.98 (bs, 3H, CH3), 1.27e1.37 (m, 3H, CH3), 1.43 (bs, 7H, 2xCH2 and CH3), 1.55e1.80 (m, 8H, 4xCH2), 2.30e2.36 (m, 2H, CH2), 2.94 (d, J ¼ 14.4 Hz, 3H, NCH3), 3.28 (t, J ¼ 6.9 Hz, 1H, CH of CH2), 3.38 (t, J ¼ 6.9 Hz, 1H, CH of CH2), 3.81 (s, 3H, OCH3), 3.88 (t, J ¼ 6.3 Hz, 2H, OCH2), 6.37e6.39 (m, 1H, ArH), 6.51 (d, J ¼ 2.4 Hz, 1H, ArH), 6.65e6.69 (bs, 3H, ArH), 6.90 (d, J ¼ 6.4 Hz, 2H, ArH), 7.02e7.15 (m, 5H, ArH); 13C NMR (acetone-d6, 75 MHz, d ppm): 13.71, 20.09, 20.17, 25.43, 25.72, 26.47, 27.12 (2xC), 31.27, 32.95, 33.56, 35.16, 47.40, 49.79, 55.51, 68.14, 79.15, 102.72, 107.20, 114.37 (2xC), 118.94, 127.15, 127.81, 128.39 (2xC), 130.44, 130.69 (2xC), 132.26 (2xC), 132.56, 136.81, 139.57, 154.77, 158.61, 161.53, 172.19; HRMS (ESI): calc. for C35H43NO4: 542.3270 (MþþH); found: 542.3244. 3.1.14. 4-(4-hydroxyphenyl)-7-methoxy-2,2-dimethyl-3-phenyl3,4-dihydrobenzopyran (18) Yield: 72%; Mp. 264e265 C; Rf: 0.30 (20% ethyl acetatehexane); 1H NMR (acetone-d6, and 2d DMSO-d6, 300 MHz, d ppm): 1.18 (s, 3H, CH3), 1.31 (s, 3H, CH3), 2.05e2.09 (d, J ¼ 10.8 Hz, 1H, CH), 3.77 (s, 3H. OCH3), 4.40e4.44 (d, J ¼ 12.3 Hz, 1H, CH), 6.37e6.34 (d, J ¼ 8.7 Hz, 2H, ArH). 6.55e6.53 (d, J ¼ 7.87 Hz, 3H, ArH), 6.88e6.85 (d, J ¼ 7.8 Hz, 2H, ArH), 7.12e7.29 (m, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): (acetone-d6, and 2d DMSO-d6, 300 MHz, d ppm): 20.26, 28.97, 43.46, 55.26, 59.95, 78.38, 101.80, 107.42, 115.50 (2xC), 119.60, 127.06, 128.31 (3xC), 131.23, 134.57, 140.24, 154.47, 156.45, 159.61; MS (C24H24O3): m/z ¼ 359 [MH]þ, 383 [MþNa]þ. 3.1.15. Synthesis of ethyl 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl3,4-dihydrobenzopyran-4-yl)phenoxy)butanoate (19a) The synthesis of 19aec was accomplished from 18 following the synthetic methodology described for synthesis of 14aec. Yield: 78%; m.p. 94e95 C Rf: 0.5 (20% ethyl acetate-hexane): IR (KBr, nmax/cm1): 1738, 1505, 1248, 1104, 834; 1H NMR (CDCl3, 300 MHz, d ppm): 1.20e1.24 (m, 9H, 3xCH3), 2.01 (t, J ¼ 6.0 Hz, 2H, CH2), 2.44 (t, J ¼ 6.0 Hz, 2H, CH2), 3.16 (d, J ¼ 12.1 Hz, 1H, CH), 3.76 (s, 3H, OCH3), 3.85e3.99 (m, 2H, OCH2), 4.10 (q, 2H, OCH2), 4.30 (d, J ¼ 12.3 Hz, 1H, CH), 6.34e6.42 (m, 1H, ArH), 6.49 (bs, 1H, ArH), 6.59 (d, J ¼ 8. 1 Hz, 3H, ArH), 6.85 (d, J ¼ 8.1 Hz, 2H, ArH), 7.16 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.62, 20.57, 25.09, 29.22, 31.24, 44.34, 55.63, 57.89, 60.79, 66.85, 78.60, 101.79, 107.88, 114.36 (2xC), 119.00, 127.09, 128.35 (3xC), 130.41 (3xC), 131.32, 136.82, 139.82, 154.56, 157.47, 159.60, 173.66; MS (C30H34O5): m/z ¼ 497 [MþNa]þ; Anal. Cacld (C30H34O5): C, 75.92; H, 7.22 Found C, 76.20; H, 7.61. 3.1.16. Ethyl 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)pentanoate (19b) Yield: 80%; Rf: 0.50 (20% ethyl acerare-hexane); IR (KBr, nmax/ cm1): 2932, 1736, 850; 1H NMR (CDCl3, 300 MHz, d ppm): 1.20e1.25 (m, 6H, 2xCH3), 1.37 (s, 3H, CH3), 1.68e1.74 (m, 4H, 2xCH2), 2.31e2.33 (m, 2H, CH2), 3.17 (d, J ¼ 12.3 Hz, 1H, CH), 3.76 (s, 3H, OCH3), 3.82 (bs, 2H, OCH2), 4.10 (q, 2H, OCH2), 4.31 (d, J ¼ 12.0 Hz, 1H, CH), 6.35 (dd, J ¼ 2.4 and 2.4 Hz, 1H, ArH), 6.43 (d, J ¼ 2.4 Hz, 1H, ArH), 6.59 (d, J ¼ 8.4 Hz, 3H, ArH), 6.86 (d, J ¼ 8.4 Hz, 2H, ArH), 7.18 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.63, 20.57, 22.06, 29.11, 31.32, 34.37, 44.33, 55.62, 57.89, 60.68, 67.47, 78.60, 101.78, 107,86, 114.33 (2xC), 119.02, 127.07, 128.34 (3xC), 130.39 (3xC), 131.33, 136.32, 139.83, 154.55, 157.57, 159.58, 173.88; MS (C31H36O5): m/z ¼ 488 [MþH]þ; Anal. Cacld (C31H36O5): C, 76.20; H, 7.43 Found C, 76.35; H, 7.65.

94


3.1.17. Ethyl 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)hexanoate (19c) Yield: 80%; m.p. 79-80 C; Rf: 0.40 (20% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2941, 1730, 1504, 1241, 1103, 1029, 836, 706; 1H NMR (CDCl3, 300 MHz, d ppm): 1.21e1.26 (m, 6H, 2xCH3), 1.37 (s, 3H, CH3), 1.37e1.48 (m, 2H, CH2), 1.63e1.76 (m, 4H, 2xCH2), 2.29 (t, J ¼ 7.5 Hz, 2H, CH2), 3.17 (d, J ¼ 12.0 Hz, 1H, CH), 3.76 (s, 3H, OCH3), 3.81 (t, J ¼ 6.0 Hz, 2H, OCH2), 4.12 (q, 2H, OCH2), 4.31 (d, J ¼ 12.0 Hz, 1H, CH), 6.36 (dd, J ¼ 2.7 and 2.4 Hz, 1H, ArH), 6.43 (d, J ¼ 2.4 Hz, 1H, ArH), 6.60 (d, J ¼ 8.4 Hz, 3H, ArH), 6.84e6.87 (d, J ¼ 8.7 Hz, 2H, ArH), 7.16 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.63, 20.58, 25.11, 26.06, 29.20, 34.64, 44.36, 55.61, 57.91, 60.61, 67.77, 78.59, 101.81, 107.86, 114.36 (2xC), 119.04, 127.06, 128.32 (3xC), 130.38 (2xC), 131.32, 136.27, 139.86, 154.57, 157.67, 159.60, 174.03; MS (C32H38O5): m/z ¼ 525 [MþNaþ2]þ; Anal. Cacld (C32H38O5): C, 76.46; H, 7.62 Found C, 76.48; H, 7.70. 3.1.18. Synthesis of 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)butanoic acid (20a) The synthesis of 20aec was accomplished from 19aec following the synthetic methodology described for synthesis of 15aec. Yield: 88%, m.p, 127e128 C; Rf: 0.12 (20% ethyl acetate-hexane); 1 H NMR (CDCl3, 300 MHz, d ppm): 1.25 (s, 3H, CH3), 1.39 (s, 3H, CH3), 2.046 (t, J ¼ 6.0 Hz, 2H, CH2), 2.53 (t, J ¼ 6.0 Hz, 2H, CH2), 3.19 (d, J ¼ 11.7 Hz, 1H, CH), 3.78 (s, 3H, OCH3), 3.88 (s, 2H, OCH2), 4.33 (d, J ¼ 12.0 Hz, IH, CH), 6.37 (d, J ¼ 8.7 Hz, 1H, ArH), 6.50 (s, 1H, ArH), 6.61 (d, J ¼ 8.1 Hz, 3H, ArH), 6.88 (d, J ¼ 7.2 Hz, 2H, ArH), 7.18 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 20.60, 24.81, 29.21, 30.91, 44.39, 55.62, 57.94, 66.67, 78.60, 101.86, 107.90, 114.39 (2xC), 119.01, 127.09, 128.35 (3xC), 130.43 (3xC), 131.31, 136.60, 139.84, 154.59, 157.40, 159.63, 179.36; MS (C28H30O5): m/z ¼ 445 [MH]þ, 469 [MþNa]þ; Anal. Cacld (C28H30O5): C, 75.31; H, 6.77 Found C, 75.28; H, 6.67. 3.1.19. 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)pentanoic acid (20b) Yield: 85%; m.p. 140e141 C; Rf: 0.12 (20% ethyl acetatehexane); IR (KBr, nmax/cm1): 2928, 1706, 1606, 1508, 1241, 1031, 840, 703; 1H NMR (CDCl3, 300 MHz, d ppm): 1.22e1.25 (m, 6H, 2xCH3), 1.75 (bs, 4H, 2xCH2), 2.38 (bs, 2H, CH2), 3.16 (d, J ¼ 12.0 Hz, 1H, CH), 3.78 (s, 3H, OCH3), 3.82 (bs, 2H, OCH2), 4.30 (d, J ¼ 12.0 Hz, 1H, CH), 6.33e6.37 (m, 1H, ArH), 6.43 (d, J ¼ 2.4 Hz, 1H, ArH), 6.59 (dd, J ¼ 2.1 and 2.1 Hz, 3H, ArH), 6.86 (d, J ¼ 8.4 Hz, 2H, ArH), 7.16 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 20.58, 21.80, 28.99, 29.22, 30.11, 33.97, 44.35, 55.63, 57.91, 67.42, 78.63, 101.80, 107.89, 114.35 (2xC), 119.03, 127.09, 128.35 (3xC), 130.41(2xC), 131.34, 136.39, 139.83, 154.56, 157.54, 159.59, 179.64; MS (C29H32O5): m/ z ¼ 461[MþH], 459 [MH], 483 [MþNa]þ ]þ; Anal. Cacld (C29H32O5): C, 75.63; H, 7.00; Found C, 75.41; H, 6.89. 3.1.20. 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)hexanoic acid (20c) Yield: 82%; m.p. 122e123 C; Rf: 0.12 (20% ethyl acetatehexane); IR (KBr, nmax/cm1): 2943, 1705, 1507, 1245, 1161, 837, 702; 1H NMR (CDCl3, 300 MHz, d ppm): 1.24 (s, 3H, CH3), 1.38 (s, 3H, CH3), 1.43e1.49 (m, 2H, CH2), 1.62e1.74 (m, 4H, 2xCH2), 2.37 (t, J ¼ 6.0 Hz, 2H, CH2), 3.18 (d, J ¼ 12.0 Hz, 1H, CH), 3.77 (s, 3H, OCH3), 3.82 (t, J ¼ 4.5 Hz, 2H, OCH2), 4.30e4.34 (d, J ¼ 12.0 Hz, 1H, CH), 6.35e6.38 (dd, J ¼ 2.4 and 2.7 Hz, 1H, ArH), 6.44 (bs, 1H, ArH), 6.59e6.62 (d, J ¼ 8.7 Hz, 3H, ArH), 6.85e6.88 (d, J ¼ 8.1 Hz, 2H, ArH), 7.16 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 20.58, 24.83, 26.01, 29.21, 29.36, 34.27, 44.36, 55.63, 57.91, 67.74, 78.62, 101.82, 107.88, 114.38 (3xC), 119.05, 127.07, 128.34 (3xC), 130.40 (3xC), 131.34, 136.31, 139.85, 154.56, 157.64, 159.59; MS (C30H34O5): m/ z ¼ 473[MH]þ, 497 [MþNa]þ; Anal. Cacld (C30H34O5): C, 75.92; H,

7.22 Found C, 75.84; H, 7.31. The synthesis of 21aec was accomplished from 20aec and 2aminnomethylpyridine, following the synthetic methodology described for synthesis of 16aec.

3.1.21. 4-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-(pyridin-2-ylmethyl) butanamide (21a) Yield: 72%; m.p. 130e131 C; Rf: 0.40 (40% ethyl acetatehexane); IR (KBr, nmax/cm1): 3261, 3068, 2973, 1648, 1162, 1117, 841, 756, 703; 1H NMR (CDCl3, 300 MHz, d ppm): 1.20e124 (m, 9H, 3xCH3), 1.99e2.03 (t, 2H, CH2), 2.42e2.46 (t, 2H, CH2), 3.76 (s, 3H, CH3), 3.85e3.99 (m, 2H, CH2), 4.07e4.11 (q, 2H, CH2), 4.28e4.32 (d, J ¼ 12.3 Hz, 1H, CH), 6.34e6.42 (m, 1H, ArH), 6.48e6.51 (bs, lH, ArH), 6.58e6.61 (d, J ¼ 8.1 Hz, 3H, ArH), 6.84e6.87 (d, J ¼ 8.1 Hz, 3H, ArH), 7.16 (bs, 6H, pyridyl-H þ ArH), 7.58e7.64 (m, 1H, pyridyl-H), 8.40 (d, J ¼ 3.6 Hz, 1H, pyridyl-H): 13C NMR (CDCl3, 75 MHz, d ppm): 20.57, 25.64, 29.21, 33.27, 44.34, 44.78, 55.62, 57.89, 67.05, 78.58, 101.82, 107.88, 114.42 (2xC), 119.00, 122.64, 122.81, 127.09, 128.34 (2xC), 130.39 (2xC), 131.31 (2xC), 136.44 (2xC), 137.35, 139.84, 149.23, 154.56, 156.84, 157.50, 159.61, 172.90; HRMS (ESI): calc. for C34H37N2O4: 537.2747 (MþþH); found: 537.2728.

3.1.22. 5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-(pyridin-2-ylmethyl) pentanamide (21b) Yield: 70%; m.p. 135-136 C; Rf: 0.12 (20% ethyl acetate-hexane); IR (KBr, nmax/cm1): 3348, 2932, 1657, 1243, 1163,1104, 1031, 838, 753; 1H NMR (CDCl3, 300 MHz, d ppm): 1.22 (s, 6H, CH3), 1.36 (s, 3H, CH3), 1.78 (bs, 4H, 2xCH2), 2.31 (t, J ¼ 6.6 Hz, 2H, CH2), 3.16 (d, J ¼ 12.0 Hz, 1H, CH), 3.75 (s, 3H, OCH3), 3.83 (bs, 2H, OCH2), 4.30 (d, J ¼ 12.0 Hz, 1H, CH), 6.34e6.37 (m, 1H, ArH), 6.41 (d, J ¼ 2.1 Hz, 1H, ArH), 6.58e6.60 (m, 3H, ArH), 6.79 (bs, 1H, NH), 6.85 (d, J ¼ 8.4 Hz, 2H, ArH), 7.15 (bs, 5H, ArH), 7.23 (d, J ¼ 8.4 Hz, 2H, Pyridyl-H), 7.63 (dd, J ¼ 6.0 and 6.3 Hz, 1H, Pyridyl-H), 8.50 (d, J ¼ 3.3 Hz, 1H, Pyridyl-H)); 13C NMR (CDCl3, 75 MHz, d ppm): 20.57, 22.85, 29.21 (2xC), 36.60, 44.33, 44.80, 55.63, 55.89, 67.64, 78.61, 101.78, 107.87, 114.34 (2xC), 119.03, 122.56, 122.78, 127.08, 128.35 (3xC), 130.39 (3xC), 131.34, 136.32, 137.20, 139.83, 149.38, 154.55, 156.76, 157.58, 159.59, 173.16; MS (C35H38N2O4): m/z ¼ 551[MþH]þ.

3.1.23. 6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-(pyridin-2-ylmethyl) hexanamide (21c) Yield: 73%; m.p. 132e133 C; Rf: 0.40 (30% ethyl acetatehexane); 1H NMR (CDCl3, 300 MHz, d ppm): 1.24 (bs, 3H, CH3), 1.42e1.50 (m, 5H, CH2 and CH3), 1.66e1.76 (m, 4H, 2xCH2), 2.27 (t, J ¼ 7.5 Hz, 2H, CH2), 3.17 (d, J ¼ 12.0 Hz, 1H, CH), 3.76e3.82 (m, 5H, OCH2 and OCH3), 4.31 (d, J ¼ 12.0 Hz, 1H, CH), 4.54 (d, J ¼ 4.8 Hz, 2H, CH2), 6.35 (dd, J ¼ 2.4 and 2.7 Hz, 1H, ArH), 6.43 (d, J ¼ 2.7 Hz, 1H, ArH), 6.57e6.62 (m, 3H, ArH), 6.76 (bs, 1H, NH), 6.85 (d, J ¼ 8.7 Hz, 2H, ArH), 7.16e7.26 (m, 7H, ArH and Pyridyl-H), 7.61e7.66 (m, 1H, Pyridyl-H), 8.50 (d, J ¼ 4.2 Hz, 1H, Pyridyl-H); 13C NMR (CDCl3, 75 MHz, d ppm): 20.57, 25.80, 26.20, 29.44, 36.91, 44.35, 44.76, 55.60, 57.60, 57.89, 67.80, 78.58, 101.81, 107.84, 114.37 (3xC), 119.03, 122.61, 122.76, 127.05, 128.31 (2xC), 130.37 (2xC), 131.31, 136.24, 137.20, 139.85, 149.37 (2xC), 154.56, 156.82, 157.66, 159.58, 173.34; HRMS (ESI): calc. for C36H41N2O4: 565.3066 (MþþH); found: 565.3037. The synthesis of 22aec was accomplished from 20aec, following the synthetic methodology described for synthesis of 17aec.


3.1.24. N-butyl-4-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-methylbutanamide (22a) Yield: 73%; m.p. oil; Rf: 0.04 (30% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2918, 1644, 1618, 1505, 1242, 1161, 1104, 787, 763, 702; 1 H NMR (CDCl3, 300 MHz, d ppm): 0.88e0.93 (m, 3H, CH3), 1.20e1.32 (m, 6H, 3xCH3), 1.47e1.74 (m, 2H, CH2), 1.75 (bs, 4H, 2xCH2), 2.32e2.34 (bs, 2H, CH2), 2.91 (d, J ¼ 15.0 Hz, 3H, NCH3), 3.17 (d, J ¼ 12.0 Hz, 1H, CH), 3.22 (t, J ¼ 7.3 Hz, 1H, CH of CH2), 3.33 (t, J ¼ 7.3 Hz, 1H, CH of CH2), 3.76 (s, 3H, OCH3), 3.83 (bs, 2H, OCH2), 4.30 (d, J ¼ 12.3 Hz, 1H, CH), 6.35 (dd, J ¼ 2.7 and 2.7 Hz, 1H, ArH), 6.43 (d, J ¼ 2.7 Hz, 1H, ArH), 6.58e6.62 (m, 3H, ArH), 6.85 (d, J ¼ 8.7 Hz, 2H, ArH), 7.15 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.24, 14.29, 20.35, 20.48, 20.58, 22.13, 22.50, 29.21, 29.38, 29.83, 31.01, 32.94, 33.58, 33.76, 35.67, 44.34, 47.86, 50.14, 55.62, 57.90, 67.77, 78.60, 101.78, 107.86, 114.34 (2xC), 119.04, 127.07, 128.33 (2xC), 130.38 (2xC), 131.33, 136.25, 139.84, 154.56, 157.64, 159.59, 172.77, 172.89; MS (C33H41NO4): m/z ¼ 514[MH], 537 [MHþNa]; HRMS (ESI): calc. for C33H42NO4: 516.3114 (MþþH); found: 516.3092. 3.1.25. N-butyl-5-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-methylpentanamide (22b) Yield: 74%; m.p. oil; Rf: 0.32 (30% ethyl acetate-hexane); IR (KBr, nmax/cm1): 2918, 1644, 1508, 1242, 837, 705; 1H NMR (pyridine-d5, 400 MHz, d ppm): 0.84e0.89 (bs, 3H, CH3), 1.18e1.49 (m, 10H, 2xCH2 and 2xCH3), 1.80e1.89 (m, 4H, 2xCH2), 2.30 (t, J ¼ 9.2 Hz, 1H, CH of CH2), 2.37 (t, J ¼ 9.2 Hz, 1H, CH of CH2), 2.87 (d, J ¼ 55.2 Hz, 3H, NCH3), 3.13 (t, J ¼ 9.8 Hz, 1H, CH of CH2), 3.42 (t, J ¼ 9.2 Hz, 1H, CH of CH2), 3.48 (d, J ¼ 16.4 Hz, 1H, CH), 3.73e3.79 (m, 5H, OCH2 and OCH3), 4.60 (d, J ¼ 16.4 Hz, 1H, CH), 6.61 (dd, J ¼ 3.2 and 2.8 Hz, 1H, ArH), 6.80e6.87 (m, 4H, ArH), 7.12e7.30 (m, 5H, ArH þ pyridine), 7.37 (d, J ¼ 9.6 Hz, 2H, ArH); 13C NMR (pyridine-d5, 400 MHz, d ppm): 14.54, 14.59, 20.64, 20.82, 20.89, 22.68, 23.02, 29.58, 29.81, 30.16, 30.27, 30.53, 31.33, 33.10, 33.73, 35.50, 44.48, 47.85, 50.02, 55.82, 58.00, 68.26, 79.03, 102.81, 108.28, 115.11 (2xC), 120.03, 127.68, 128.98 (2xC), 131.18 (2xC), 132.06, 136.99, 140.67, 155.39, 158.46, 160.44, 172.74; MS (C34H43NO4): m/z ¼ 552 [MþNa]þ; HRMS (ESI): calc. for C34H44NO4: 530.3270 (MþþH); found: 530.3240. NMR @ 360 K temperature. 1 H NMR (pyridine-d5, 400 MHz, d ppm): 0.92e0.97 (t, J ¼ 9.8 Hz, 3H, CH3), 1.29e1.57 (m, 10H, CH2 and 2xCH3), 1.83e1.95 (m, 4H, 2xCH2), 2.41 (t, J ¼ 9.2 Hz, 2H, CH2), 2.93 (s, 3H, NCH3), 3.376 (bs, 2H, CH2), 3.49 (d, J ¼ 12.0 Hz, 1H, CH), 3.79 (s, 3H, OCH3), 3.89e4.02 (m, 2H, OCH2), 4.61 (d, J ¼ 12.0 Hz, 1H, CH), 6.60 (dd, J ¼ 3.2 and 3.2 Hz, 1H, ArH), 6.78e6.91 (m, 4H, ArH), 7.12e7.39 (m, 7H, ArH þ pyridine); 13C NMR (pyridine-d5, 400 MHz, d ppm): 14.23, 20.69, 21.20, 22.83, 29.55, 29.93, 30.35, 33.49, 45.04, 55.90, 58.54, 68.79, 78.98, 103.31, 108.38, 115.49 (2xC), 120.10, 127.54, 128.81 (2xC), 130.61 (2xC), 131.08 (2xC), 131.80, 137.16, 140.85, 155.55, 158.70, 160.69, 172.70. 3.1.26. N-butyl-6-(4-(7-methoxy-2,2-dimethyl-3-phenyl-3,4dihydrobenzopyran-4-yl)phenoxy)-N-methylhexanamide (22c) Yield: 68%; m.p. oil; Rf: 0.40 (30% ethyl acetate-hexane); 1H NMR (CDCl3, 300 MHz, d ppm): 0.91e0.96 (bs, 3H, CH3), 1.23e1.37 (m, 8H, CH2 and 3xCH3), 1.43e1.47 (m, 4H, 2xCH2), 1.70e1.75 (m, 4H, 2xCH2), 2.26e2.31 (m, 2H, CH2), 2.92 (d, J ¼ 15.3 Hz, 3H, NCH3), 3.17 (d, J ¼ 12.3 Hz, 1H, CH), 3.22 (t, J ¼ 9.3 Hz, 1H, CH of CH2), 3.35 (t, J ¼ 7.3 Hz, 1H, CH of CH2), 3.76e3.83 (m, 5H, OCH2 and OCH3), 4.31 (d, J ¼ 12.0 Hz, 1H, CH), 6.36 (d, J ¼ 8.4 Hz, 1H, ArH), 6.43 (s, 1H, ArH), 6.60 (d, J ¼ 6.9 Hz, 3H, ArH), 6.85 (d, J ¼ 7.5 Hz, 2H, ArH), 7.26 (bs, 5H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 13.81, 19.93, 20.05, 20.14, 24.79, 25.15, 25.91, 26.00, 28.78, 19.15, 29.40, 30.60, 32.80, 33.34, 33.43, 35.26, 43.91, 47.42, 49.73, 55.19, 57.46, 67.43, 78.17,

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101.35, 107.43, 113.92 (2xC), 118.62, 126.63, 127.90 (2xC), 129.95 (2xC) 130.91, 135.79, 139.42, 154.13, 157.27, 159.16, 172.66; HRMS (ESI): calc. for C35H46NO4: 544.3421 (MþþH); found: 544.3400. 3.1.27. N-butyl-4-(3-methoxyphenoxy)butanamide (25a) Yield: 75%; m.p. oil; Rf: 0.45 (20% ethyl acetate-hexane); 1H NMR (CDCl3, 300 MHz, d ppm): 0.89 (t, J ¼ 7.2 Hz, 3H, CH3), 1.24e1.36 (m, 2H, CH2), 1.39e1.48 (m, 2H, CH2), 2.05e2.16 (m, 2H, CH2), 2.35 (t, J ¼ 7.2 Hz, 2H, CH2), 3.23 (q, 2H, CH2), 3.77 (s, 3H, OCH3), 3.98 (q, 2H, CH2),5.63 (bs, 1H, NH), 6.439e6.50 (m, 3H, ArH),7.15(dd, J ¼ 8.1 and 8.4 Hz, 1H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.10, 20.45, 25.69, 32.10, 33.46, 39.67, 55.64, 67.31, 101.39, 106.72, 107.10, 130.29, 106.52, 161.26, 172.61; MS (C15H23NO3): m/z ¼ 266 [MþH]þ; Anal. Cacld (C30H34O5): C, 67.90; H, 8.74; N, 5.28 Found C, 68.10; H, 8.86; N, 5.43. 3.1.28. N-butyl-4-(3-methoxyphenoxy)-N-methylbutanamide (25b) Yield: 88%; m.p. oil; Rf: 0.45 (20% ethyl acetate-hexane); 1H NMR (CDCl3, 300 MHz, d ppm): 0.92 (bs, 3H, CH3), 1.24e1.30 (m, 2H, CH2), 1.42e1.56 (m, 2H, CH2), 2.11 (bs, 2H, CH2), 2.48 (q, 2H, CH2), 2.92 (d, J ¼ 10.6 Hz, 3H, NCH3), 3.25 (t, J ¼ 7.5 Hz, 1H, CH of CH2), 3.34 (t, J ¼ 7.2 Hz, 1H, CH of CH2), 3.37 (s, 3H, OCH3), 3.90 (t, J ¼ 5.8 Hz, 2H, OCH2)6.44e6.49 (m, 3H, ArH), 7.13 (t, J ¼ 8.1 Hz, 1H, ArH); 13C NMR (CDCl3, 75 MHz, d ppm): 14.21, 14.26, 20.35, 20.48, 25.20, 25.45, 29.42, 29.82, 30.14, 30.94, 33.81, 35.62, 4792, 50.09, 55.62, 67.39, 67.48, 101.29, 106.62, 107.08 (2xC), 130.25, 160.57, 161.23 (2xC), 172.39, 172.49; MS (C16H25NO3): m/z ¼ 280 [MþH]þ; Anal. Cacld (C16H25NO3): C, 68.79; H, 9.02; N, 5.01 Found C, 68.92; H, 9.22; N, 5.32. 3.2. Biological methods: reagents and chemicals Cell culture media and supplements were purchased from Invitrogen (Carlsbad, CA) and all fine chemicals from Sigma Aldrich (St. Louis, MO). ECL kit was obtained from Amersham Pharmacia, USA. All antibodies for Western blot analysis were obtained from Cell Signaling Technologies (Danvers, MA). 3.3. Culture of mice calvarial osteoblasts Mouse calvarial osteoblasts were obtained following our previously published protocol of sequential digestion. In brief, calvaria from 1- to 2-day old mice (both sexes) were pooled. After surgical isolation from the skull and removal of sutures and adherent mesenchymal tissues, calvaria were subjected to five sequential (10e15 min) digestions at 37 C in a solution containing 0.1% dispase and 0.1% collagenase P. Cells released from the second to fifth digestions were pooled, centrifuged, resuspended, and plated in T25 cm2 flasks in a-MEM containing 10% FCS and 1% penicillin/ streptomycin (complete growth medium). 3.4. Osteoblast differentiation For the measurement of alkaline phosphatase (ALP) activity, osteoblasts at ~80% confluence were trypsinized and 2 103 cells per well were seeded in 96-well plates. Cells were treated with different concentrations of the compounds for 48 h in a-MEM supplemented with 5% Fetal bovine serum, 10 mM b glycerophosphate, 50 mg/mL ascorbic acid and 1% penicillin/streptomycin (osteoblast differentiation medium). At the end of the incubation period, the total ALP activity was measured using p-nitrophenylphosphate (PNPP) as a substrate and quantified colorimetrically at 405 nm.

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3.5. Mineralization of calvarial osteoblast cells For mineralization studies, calvarial cells were cultured in aMEM medium, supplemented with 10% fetal bovine serum, 50 mg/ mL ascorbic acid, and 10 mM b glycerophosphate. Cells were cultured with and without compounds for 21 day at 37 C in a humidified atmosphere of 5% CO2 and 95% air, and the medium was changed every 48 h. After 21 days, the attached cells were fixed in 4% formaldehyde for 20 min at room temperature and rinsed once in PBS. After fixation, the specimens were processed for staining with 40 mM alizarin red S, which stains areas rich in nascent calcium. 17b-E2 was used as a positive control. For quantification of alizarin red-S staining, 800 mL of 10% (v/v) acetic acid was added to each well, and plates were incubated at room temperature for 30 min with gentle shaking. Monolayer, loosely attached to the plate, was then scraped with a cell scraper and transferred with 10% (v/v) acetic acid to a 1.5-mL tube. After vortexing for 30 s, the slurry was over laid with 500 mL mineral oil (SigmaeAldrich), heated to exactly 85 C for 10 min, and transferred to ice for 5 min. The slurry was then centrifuged at 20,000g for 15 min and 500 mL of the supernatant was removed to a new tube. Then 200 mL 10% (v/v) ammonium hydroxide was added to neutralize the acid. OD (405 nm) of 150 mL aliquots of the supernatant were measured in 96-well plates. 3.6. qPCR assay Total RNA was extracted from the cultured cells using TRIzol (Invitrogen, Carlsbad, CA). cDNA was synthesized from 2 mg total RNA with the Revert Aid™ H Minus first strand cDNA synthesis kit (Fermentas, Vilnius, Lithuania). SYBR green chemistry was used for quantitative determination of the mRNAs for BMP-2, Runx-2, osteocalcin and a housekeeping gene, GAPDH, following an optimized protocol. The design of sense and antisense oligonucleotide primers was done using the Universal Probe Library (Roche Diagnostics, Indianapolis, IN). For real-time PCR, the cDNA was amplified with Light Cycler 480 (Roche Diagnostics Pvt Ltd, Indianapolis, IN). The double-stranded DNA-specific dye SYBR Green I was incorporated into the PCR buffer provided in the Light Cycler 480 SYBER Green I Master (Roche Diagnostics Pvt. Ltd, Indianapolis, IN) to allow for quantitative detection of the PCR product in a 20 mL reaction volume. The temperature profile of the reaction was 95 C for 5 min, 40 cycles of denaturation at 94 C for 2 min, and annealing and extension at 62 C for 30 s extension at 72 C for 30 s. GAPDH was used to normalize differences in RNA expression. 3.7. In vivo activity The study was conducted in accordance with current legislation on animal experiments [Institutional Animal Ethical Committee (IAEC) at C.D.R.I.]. Eight week old adult female balb/c mice were used for the study (Fujioka et al., 2007). Animals were housed at 21 C. in 12-h light: 12-h dark cycles. Normal chow diet and water were provided ad libitum. Ten mice in each group were taken for the study and the groups were as follows: sham (ovary intact) þ vehicle (gum acacia in distilled water), Ovx þ vehicle, Ovx þ 1.0 or 10.0 mg kg1 body weight dose of 22b. Mice were treated with 1.0 and 10.0 mg kg1 body weight dose of 22b or vehicle once daily for 4 weeks by oral gavage. After a period of four weeks, animals were sacrificed and femur bones were collected for analysis of trabecular microarchitecture. mCT experiments were carried out using Sky Scan 1076 microCT scanner (Aartselaar, Belgium) as previously reported (Trivedi et al., 2009). Femora were dissected from the animals after sacrifice, cleaned of soft tissue and fixed prior to storage in alcohol. The

samples were scanned in batches of three, at a nominal resolution (pixel) of ISum. Reconstruction was carried out employing a modified Feldkamp algorithm using the Sky Scan Nrecon software which facilitates network distributed reconstruction carried out on four PC’s running simultaneously. The X-ray source was set at 70 kV and 100 mA, with a pixel size at 181lm. Hundred projections were acquired over an angular range of 180’. The image slices were reconstructed using the cone-beam reconstruction software version 2.6 based on the Feldkamp algorithm (Skyscan). Trabecular bone volume (BV/TV; %), trabecular number (Tb.N), and trabecular separation (Tb.Sp) were calculated by the mean intercept length method. Trabecular thickness (Tb.Th) was calculated according to the method of Hilderbrand et al. (Hildebrand & Ruegsegger 1997). 3D parameters were based on analysis of a Marching cubes type model with a rendered surface. 3.8. Western blotting Cells were grown to 60e70% confluence following which they were treated with and without compound for 48 h. Cells were lysed with cell lysis buffer (Sigma-Aldrich) with protease inhibitor cocktail (Sigma-Aldrich). Cell lysate was centrifuged at 12,000 g for 15 min and supernatant was collected. Estimation of protein concentration was determined by Bradford assay. Thirty micrograms of total protein was then resolved by 10% SDS-PAGE gel. After electrophoresis, proteins were transferred onto PVDF membranes (Immobilon-P, Millipore, Billerica, MA, USA). The membranes were probed with ER-a, ER-b, PARP and b-Actin antibodies (Cell Signaling Technology, Danvers, MA, USA) and then incubated with secondary antibodies conjugated with HRP (Cell Signaling Technology). Immunodetection was done using an enhanced chemiluminescence kit (GE Healthcare, Little Chalfont, UK) using Image Quant LAS 4000 (GE Healthcare). Densitometry of blots was done using quantity 1D analysis software and gel doc imaging system. 3.8.1. Pharmacokinetic study Female Sprague-Dawley rats (n ¼ 4) were fasted overnight (12e14 h) before dosing and had free access to water throughout the experimental period. 22b was administered orally at a dose of 1 mg kg1 in 0.25% CMC suspension. Animals were provided with standard diet after 3 h of dosing. The rats were anaesthetized using ether and blood samples were collected from the retro-orbital plexus into heparinized microfuge tubes at 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h post-dosing. In another group of female SpragueDawley rats (n ¼ 4), solution of 22b was administered to rats via a lateral tail vein as a bolus dose of 1 mg kg1. The intravenous formulation was prepared in DMSOePEG400eEthanolewater (4: 3: 1: 2 v/v) and finally filtered through 0.22 mM filter before administration. Animals had free access to food and water throughout the experimentation period. Blood samples were collected from the retro-orbital plexus into heparinized microfuge tubes at 0.08, 0.25, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10 and 24 h post-dosing. Plasma was harvested by centrifugation of blood at 13,000 rpm for 10 min on Sigma 1e15K (Frankfurt, Germany) and stored frozen at 70 ± 10 C until bioanalysis. The observed maximum plasma concentration (Cmax) and time to reach the maximum plasma concentration (Tmax) were obtained by visual inspection of the experimental data. The data was subjected to non-compartmental pharmacokinetics analysis using WinNonlin (version 5.1, Pharsight Corporation, Mountain View, USA). Area under curve (AUC) from 0 to 24 h (AUC0e24) was calculated using linear trapezoidal rule. AUC from 0 to infinity (AUC0-∞) was calculated as the sum of AUC0-t and Clast/kel, where, Clast represents the last quantifiable concentration and kel represents the elimination rate constant. The absolute bioavailability (%


F) was calculated using the relationship. %F ¼ [AUC(0∞)oral Dose(i.v.)/AUC(0∞) i.v. Dose(oral)] 100.

3.9. Animals and legal prerequisite

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tail vein of the animals after the 7th day of experiment for serum separation and hematogram. Biochemical parameters like serum SGPT, SGOT, ALKP, triglycerides, creatinine, albumin, serum protein, tissue protein, reduced GSH and MDA activity were performed. At the end of experiment, animals were sacrificed and significant changes (if any) were observed in organs like brain, lungs, heart, liver, kidneys, spleen, uterus, ovary and testes etc. A slice of liver was collected for homogenate preparation from each experimental animal. Homogenate was analyzed for tissue protein, MDA and reduced GSH.

Young, adult female Sprague-Dawley (SD) rats, weighing 200 ± 20 g, were procured from the National Laboratory Animal Center, C.S.I.R. e C.D.R.I. (Lucknow, India). Rats were housed in well ventilated cages at room temperature (24 ± 2 C) and 40e60% relative humidity while on a regular 12 h light-dark cycle. The animals were acclimatized for a minimum period of 3 days prior to the experiment. Approval from the Local Animal Ethics Committee was sought and the study protocols were approved before the commencement of the studies.

Data are expressed as mean ± S.E.M. Cell cycle data was analyzed by student’s t-test. The data obtained from remaining experiments with multiple treatments were subjected to one-way ANOVA followed by NewmaneKeuls test of significance using Prism version 3.0 software.

3.10. Sample preparation

Acknowledgements

The study samples generated were analyzed by the validated LCMS/MS method. To 100 mL of plasma, 10 mL of IS (Halofantrine, equivalent to 10 ng) was added and mixed for 15 s on a cyclomixer (Spinix Tarsons, Kolkata, India), followed by extraction with 2.0 mL of n-hexane. The mixture was vortexed for 3 min, followed by centrifugation for 5 min at 2000g on Sigma 3e16K (Frankfurt, Germany). The organic phase was separated and evaporated. The residue was reconstituted with 150 mL of mobile phase and 10 mL of this solution was subjected to LC-MS/MS analysis.

A.G. acknowledges DST India for financial support in the form of DST fast track project (SR/FT/CS-008/2009). I.A. acknowledges CSIR, New Delhi, India for senior research fellowship. Support received from Director, CSIR-CIMAP, CSIR-CDRI and CSIR-IICT India is dully acknowledged.

3.11. LC-MS/MS based bioanalytical method for analysis of plasma samples LC-MS/MS analyses were carried out using a HPLC system consisting of shimadzu SCL 20Avp and Shimadzu SIL-HTC autosampler on a Waters X-bridge C18-column (4.6 50 mm, 5.0 mm). The system was run in isocratic mode with a mobile phase consisting of 0.01 M ammonium acetate (pH ¼ 4.5) and acetonitrile in a ratio of 10:90 v/v. The mass spectrometer was operated in positive ion mode and detection of the ions was performed in the multiple reaction monitoring (MRM) mode: monitoring transition of m/z 531.1 precursor ion [MþH]þ to the m/z 170.0 product ion for 22b and m/z 502.0 precursor ion [MþH]þ to the m/z 142.2 product ion for I.S. Linearity in the range of 0.5e200 ng/mL was prepared for 22b in plasma. Prior to the analysis of samples, three concentrations (nominal concentrations of 1.2, 80 and 160 ng/mL) of quality control (QC) samples were prepared in rat plasma. Along with the study samples, QC samples (N ¼ 3, at each concentration level) were distributed among the unknown samples in the analytical run. 3.12. In vivo acute toxicity In order to generate preclinical safety data of 22b before its preclinical studies in in vivo models in the field of osteoporosis, acute toxicity studies were carried out in Swiss albino mice following OECO guidelines [55e56]. In acute oral toxicity study, mice were treated with 22b and vehicle at 5, 50, 300, 1000 mg kg1 body weight dose as a single acute oral dose. Animals were carefully observed in whole period of experiment. A daily cage side examinations were carried out during the entire period of observation i.e 7 days in acute oral toxicity study. The animals were studied for different observational, hematological, biochemical and gross pathological indices. Blood samples were collected from the

3.13. Statistical analysis

List of abbreviations ALP BMPs cDNA Col 1 CTx mCT ER MCOs NFkB OD OCN OPG Ovx qPCR RANKL RANK RUNX-2

alkaline phosphatase bone morphogenetic proteins complementary DNA type-1 collagen carboxy-terminal collagen crosslinks micro computed tomography estrogen receptor mouse calvarial osteoblasts nuclear factor-kappaB optical density osteocalcin osteoprotegerin ovariectomized quantitative polymerase chain reaction receptor activator NFkB ligand Receptor activator NFkB runt-related transcription factor-2

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Differentiation of skeletal osteogenic progenitor cells

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