Discovery of a novel benzopyranyl compound as a potent in vitro and in vivo osteogenic agent

Sangmi Oh; Sun Wook Cho; Jae-Yeon Yang; Hyun Jin Sun; Young Sun Chung; Chan Soo Shin; Seung Bum Park

doi:10.1039/C0MD00149J

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DOI: 10.1039/C0MD00149J (Concise Article) Med. Chem. Commun., 2011, 2, 76-80

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Discovery of a novel benzopyranyl compound as a potent in vitro and in vivo osteogenic agent†

Sangmi Oh ^a, Sun Wook Cho ^b, Jae-Yeon Yang ^b, Hyun Jin Sun ^b, Young Sun Chung ^d, Chan Soo Shin *^b and Seung Bum Park *^ac
^aDepartment of Chemistry, Seoul National University, Seoul, Korea. E-mail: sbpark@snu.ac.kr; Fax: (+)82 2 884 4025; Tel: (+)82 2 880 9090
^bDepartment of Internal Medicine, Seoul National University College of Medicine, Seoul, Korea. E-mail: csshin@snu.ac.kr; Fax: (+)82 2 765 3734; Tel: (+)82 2 2072 3734
^cDepartment of Biophysics and Chemical Biology, Seoul National University, Seoul, Korea
^dDepartment of Counselling, Korea Cyber University, Seoul, Korea

Received 2nd September 2010 , Accepted 8th October 2010

First published on 1st November 2010

Abstract

A novel osteogenic agent containing a benzopyranyl core skeleton was discovered from cell-based alkaline phosphatase (ALP) assay of a small-molecule library constructed by diversity-oriented synthetic approach. The subsequent structure–activity relationship study using ALP and TOPflash reporter gene assay confirmed 6b as an osteogenesis activator. In in vivo study, we successfully demonstrated that the oral treatment of ovariectomized mice with 6b resulted in increased bone mass and bone volume as well as improved bone quality.

Introduction

Since the completion of the human genome project in 2003, biomedical communities have been focusing on the elucidation of gene functions and the associated control of gene products with small-molecule modulators.¹ The core research areas in chemical biology are the systematic identification of small-molecule modulators that act as perturbing agents in biological systems and the use of these agents for the control of specific gene products.² Therefore, there is great demand for the development of drug-like compound libraries that cover a wide range of molecular diversity for the identification of specific small-molecule modulators. Diversity-oriented synthesis (DOS) has been developed a efficient synthetic strategy for populating the chemical space with skeletally and stereochemically diverse small molecules synthesized by complexity-generating reactions.³ The use of the DOS strategy for constructing natural product-like and drug-like small molecules has been proved to be effective for the discovery of bioactive small-molecule modulators and therapeutic agents.⁴

Skeletal homeostasis is a dynamic state, which is the balance regulated mainly by two cell types, osteoblast for bone-formation and osteoclast for bone-resorption. Imbalance of this regulation can lead to skeletal diseases such as osteoporosis which characterized by the loss of bone mass and lead to an increased risk of fracture.⁵ Osteoporosis is a common disease that affects many people, both men and women. Further, it is expected to become more prevalent in the future as a result of rising average life expectancy; this has significant implications for public health care. Most of the current therapeutic approaches to osteoporosis are principally based on antiresorptive agents such as estrogen, bisphosphonates, raloxifen, and calcitonin.⁶ Although these conventional drugs have been shown to effectively increase bone mass and reduce risk of subsequent fracture,⁷ there have been concerns with respect to their long-term safety. The only available bone anabolic medication for the treatment of osteoporosis is human parathyroid hormone (PTH);⁸ however, this treatment still involves many unsolved problems such as daily injections, high cost, and unknown long-term effects.^8c Therefore, there is a pressing need for the development of a new anabolic drug that can be used in combination with conventional antiresorptive drugs to treat osteoporosis.

In this paper, we report a novel, small-molecule bone anabolic agent (6b) that was identified by performing a high-throughput alkaline phosphatase (ALP) assay in C3H10T1/2 mesenchymal cells. This benzopyranyl compound was derived from an in-house library containing divergent chemical scaffolds constructed by diversity-oriented synthetic pathways. Our data clearly shows that this novel small molecule has osteogenic effects, and its efficacy was confirmed by in vitro and in vivo experiments

Results and discussion

ALP is a typical marker of osteoblastic differentiation and its activity has been shown to be highly correlated with the early phase of osteoblast differentiation.⁹ Using this cell-based ALP assay, we screened the in-house drug-like small-molecule library, constructed by DOS, which has been proven to be essential for the rapid discovery of biologically active small molecules. We have been developing a series of efficient DOS routes for the construction of a natural product-like, small-molecule library with diverse heterocyclic core skeletons embedded with privileged substructural motifs, such as benzopyran, pyridine, pyrazole, pyrazolopyrimidine, and pyrimidine.¹⁰ In particular, we constructed a benzopyran-containing small-molecule library with five discrete core skeletons using DOS approach (Fig. 1).^10f With the molecular diversity of small-molecule libraries in our group, we pursued the discovery of novel small-molecule modulators to boost the osteoblastic differentiation. After the initial cell-based ALP assay with benzopyranyl compound library, we identified several compounds having core skeleton A-1 in Fig. 1 with reasonable efficacy and prioritized these initial hits through secondary ALP assay with a dose-dependent treatment.


	Fig. 1 Diversity-oriented synthetic pathway to construct a novel collection of small molecules containing benzopyranyl substructure.

The initial structure–activity relationship of confirmed hit compound 6a revealed that the hydroxyl moiety at the R¹ position is essential for the activity through the comparison with other structural analogs in our library. In addition, the replacement of R³-substituted aryl groups with heteroaryl moieties, such as indole, pyrrole, thiophene, or pyridine, eliminates the ALP activity in our preliminary SAR study (Fig. S1 in ESI†). Therefore, we synthesized a focused library, containing 13 analogs of 6a based on the benzopyranyl scaffold A-1, to explore the structure–activity relationships. As shown in Table 1, the synthesis of desired analogs (6a–6m) was accomplished through a series of chemical transformations; [1] the cyclization of hydroxyacetophenones (1) with different ketones and the subsequent silyl protection of phenolic OH to produce 3; [2] the triflation of carbonyl group on chromenone moiety to produce benzopyranyl intermediates containing vinyl triflate (4); [3] palladium-mediated Suzuki coupling of 4 with boronic acid derivatives and subsequent desilylation to obtain the desired molecular framework 6 in 60∼70% overall yields (see ESI†). After the construction of a 13-member focused library, we evaluated their bone-anabolic activity viacell-based ALP activity. First of all, the significant activity enhancement was observed through the substituent pattern at the R³ position; 3-nitro group at the R³ position of 6b allows 6.8-fold enhancement of ALP activity, in comparison with 3.1-fold enhancement in the case of 4-nitro group of 6a. In contrast, the ortho-positional alteration of the nitro group at the R³ position in 6c diminished the functional activation of ALP. We also identified significant reduction in cellular ALP activity based on substituent differences at the R¹ and R² positions in 6d, as compared to 6b, which is consistent with our previous observation in ALP assay using original library members. Therefore, we hypothesized that there is a specific hydrogen bonding interaction of hydroxyl moiety at the R¹ position in hit compounds with cellular target proteins, and kept the hydroxy moiety at the R¹ position for the construction of the focused library. Cyclopentyl and alkyl ester groups at the R position of benzopyran substructure in 6e and 6f significantly attenuated their cellular ALP activity. The introduction of various substituents with various electrostatic and steric properties at the R³ position (6g–6m) confirmed that 6b, with an electronegative nitro group in meta position, is the best candidate as a bone anabolic small-molecule agent for the further biological investigation. As shown in Fig. 2A, 6b activates the osteogenic ALP activity of C3H10T1/2 cells in a dose-dependent manner. Consistent with this enzyme activity, 6b also induces osteoblastic activity through the functional activation of alkaline phosphatase which was monitored by cellular ALP staining with nitroblue tetrazolium chloride (NBT) and 5-bromo-4-chloro-3-indolyl phosphate p-toluidine salt (BCIP) as a chromogenic phosphatase substrate. We also observed the enhanced calcium deposition in C3H10T1/2 cells upon treatment with 6b, monitored by Alizarin Red S (an anthraquinone derivative) staining (Fig. S2†).

Table 1 Representative compounds in focused library and their osteogenic activities on specific markers


Cpd.	R	R′	R¹	R²	R³	ALP activity (Fold)^a
a Relative ALP activity refers the fold enhancement compared to control upon the treatment of individual compounds at 2 μM. The maximum ALP activity was shown under the treatment of Wnt-3a. All experiments were performed at least six times, in triplicate within each individual experiment, and the mean value of all experiments was used.
6a	Methyl	Methyl	Hydroxy	Hydro	4-Nitro	3.1
6b	Methyl	Methyl	Hydroxy	Hydro	3-Nitro	6.8
6c	Methyl	Methyl	Hydroxy	Hydro	2-Nitro	1.2
6d	Methyl	Methyl	Methoxy	Hydroxy	3-Nitro	1.5
6e	R–CH₂CH₂CH₂CH₂–R′		Hydroxy	Hydro	3-Nitro	0.6
6f	Methyl	3-Ethoxy-3-oxopropyl	Hydroxy	Hydro	3-Nitro	1.4
6g	Methyl	Methyl	Hydroxy	Hydro	3-(Dimethylamino)	1.6
6h	Methyl	Methyl	Hydroxy	Hydro	3-Amino	2.5
6i	Methyl	Methyl	Hydroxy	Hydro	3-Hydroxy	1.4
6j	Methyl	Methyl	Hydroxy	Hydro	3-Acetyl	1.9
6k	Methyl	Methyl	Hydroxy	Hydro	3,5-Dichloro	1.5
6l	Methyl	Methyl	Hydroxy	Hydro	4-(Dimethylamino)	0.7
6m	Methyl	Methyl	Hydroxy	Hydro	4-Amino	3.9
Wnt-3a	—	—	—	—	—	11.8


	Fig. 2 Osteogenic activity of 6b: (A) Cellular alkaline phosphatase (ALP) staining and functional ALP assay upon treatment with 6b in dose-dependent pattern for 72 h. The results are expressed as mean ± SD; (B) TOPflash reporter gene assay in C3H10T1/2 cell lines treated with 6b; (C) Chemical structure of 6b.

The dose-dependent activation of cellular ALP function can strongly support its potential activity toward osteogenic induction without cellular cytotoxicity (Fig. S3†). To gain more mechanistic insight of the osteogenic effect upon treatment with 6b, we screened the downstream target genes of Wnt/β-catenin signaling pathway, one of the major osteogenic pathways,¹¹ using a cell-based reporter gene assay system: the TCF-reporter plasmid contains eight-copies of β-catenin DNA-binding site in TCF upstream of a luciferase reporter gene and stably transfected into C3H10T1/2 cells—TOPflash assay. The treatment of 6b at 2 μM could increase the transcriptional activities of Wnt/β-catenin signaling pathways, which is comparable to Wnt-3a, a gold standard bone anabolic molecule (Fig. 2B). This in vitro study strongly suggested that the pharmacological regulation of 6b with various biopolymers in the Wnt/β-catenin signaling pathway can lead to an increase in bone mass through the stimulation of the osteoblast function. However, the detailed mechanistic understanding on the osteogenic effect of 6b and the identification of its protein targets remain to be determined.

Finally, we applied an oral treatment of 6b for 4 weeks to ovariectomized C57BL/6 mice to evaluate in vivo efficacy of 6b as an osteogenic agent. We classified 12-week-old female mice into five groups as follows: sham-operated mice were treated with a vehicle [Sham op + vehicle], four groups of mice that were ovariectomized and treated with a vehicle [OVX + vehicle], 17β-estradiol [OVX + estradiol (60 mg kg⁻¹/week)], 5 mg kg⁻¹ of 6b [OVX + 6b (5 mg kg⁻¹ day⁻¹)] and 10 mg kg⁻¹ of 6b [OVX + 6b (10 mg kg⁻¹ day⁻¹)]. To avoid periods of acute bone loss, caused by the activation of osteoclast immediately following ovariectomy, as well as to discern the anabolic effect from the antiresorptive effect, the mice were allowed to drop bone mass for 4 weeks and the compounds or vehicle were applied orally for the subsequent 4 weeks. Among the five study groups, all OVX mice (other than the Sham-op group) showed a marked increase in fat percent and body weight, which indicated that the successful induction of estrogen deficiency had taken place (data not shown). Measurement of whole body bone mineral density (BMD, g cm⁻²), assessed by bone mineral density using DEXA, also revealed that all OVX mice showed a significant decrease in BMD over the 4 weeks following ovariectomy, as expected. Four weeks of oral treatment with 6b (5 and 10 mg kg⁻¹ day⁻¹) resulted in significantly increased BMD (∼3.7%), as compared with the OVX + vehicle treatment group (Fig. 3). Micro-CT analysis also revealed that bone volume and trabecular number were significantly increased, and that trabecular space was decreased in the OVX + 6b (10 mg kg⁻¹) treatment group, as compared to the OVX + vehicle treatment group (Fig. 4), which confirms that the oral treatment of 6b resulted in increased bone mass and bone volume as well as improved bone quality.


	Fig. 3 Bone mineral density induced by 6b in ovariectomized C57BL/6 mice: 4 weeks after the ovariectomy, the mice received 6b (orally 5 or 10 mg kg⁻¹ day⁻¹), 17β-estradiol (intramuscular injection, 60 mg kg⁻¹/week), or vehicle for 4 weeks.


	Fig. 4 In vivo confirmation of osteogenic activity of 6b: (A) Three-dimensional Micro-CT reconstruction of proximal femur obtained after 4 weeks of treatment; (B) Quantitative analysis of bone volume (% bone volume/tissue volume), trabecular number, trabecular thickness, and trabecular separation. * p < 0.05 vs.OVX + vehicle.

Conclusions

We identified a novel, small-molecule bone anabolic agent containing privileged benzopyranyl substructure. Initial core skeleton A-1 was discovered from in-house benzopyranyl compound library, constructed by diversity-oriented synthetic pathways, viacell-based ALP assay which provide functional assessments of osteogenic activity. The subsequent lead optimization and SAR study using a 13-member focused library revealed a final candidate, 6b, for further biological evaluation. The osteogenic activity of 6b was confirmed by in vitro TOPflash reporter gene assay, functional ALP assay, cellular ALP staining, and Alizarin Red S staining. Finally, the in vivo efficacy of 6b as a bone anabolic agent was evaluated by the oral application to ovariectomized C57BL/6 mice for a period of 4 weeks, which effectively alleviated OVX-induced bone loss and improved bone quality analyzed by micro-CT. These in vitro and in vivo results clearly demonstrate that 6b is a novel osteogenic agent and may constitute a promising anabolic agent for bone disease.

Acknowledgements

This work was supported by (1) the National Research Foundation of Korea (NRF), (2) the WCU program funded by NRF and the Korean Ministry of Education, Science and Technology (MEST) and (3) the MarineBio Technology Program funded by the Ministry of Land, Transport, and Maritime Affairs (MLTM), Korea. S.O. is grateful for the fellowship award of the BK21 Program.

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Footnote

† Electronic supplementary information (ESI) available: Detailed synthetic procedures, biological procedures, characterization data and copies of NMR spectra for all compounds. See DOI: 10.1039/c0md00149j.

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