Synthesis and Antiosteoporotic Characterization of Diselenyl Maleimides: Discovery of a Potent Agent for the Treatment of Osteoporosis by Targeting RANKL.

To discover new osteoclast-targeting antiosteoporosis agents, we identified forty-six diselenyl maleimides, which were efficiently prepared using a novel, simple, and metal-free method at room temperature in a short reaction time. Among them, 3k showed the most marked inhibition of osteoclast differentiation with an IC50 value of 0.36 ± 0.03 µM. Moreover, 3k significantly suppressed RANKL-induced osteoclast formation, bone resorption, and osteoclast-specific genes expression in vitro. Mechanistic studies revealed that 3k remarkably blocked the RANKL-induced mitogen-activated protein kinase (MAPK) and NF-κB signaling pathways. In ovariectomized mice, intragastric administration of 3k significantly alleviated bone loss, exhibiting an effect similar to that of alendronate. Surface plasmon resonance assay and microscale thermophoresis assay results suggested that RANKL might be a potential molecular target for 3k. Collectively, the findings presented above provided a novel candidate for further development of bone antiresorptive drugs that target RANKL.

Discovery of Novel Antiosteoporosis Leads with Bone Resorption Inhibition and Anabolic Promotion through a Chemotype-Assembly Approach.

Developing a dual-efficiency agent with antiresorptive and anabolic applications is a promising strategy for treating osteoporosis. This study reports the discovery of dual antiosteoporosis agents via a chemotype-assembly approach. Chemotype analysis identified 12 antiresorptive and 12 anabolic chemotypes and 7 dual-function chemotype-assembly rules. Based on these assembly rules, a dual-functional compound S24 was discovered. S24 exhibits osteoclastogenesis inhibition with an IC50 value of 10.28 µM and osteoblast differentiation stimulation at 10 µM. S24 derivatives were designed and synthesized based on the activity relationship of the chemotypes. This yielded a more active compound, S24-14, with an osteoclastogenesis inhibition IC50 value of 0.40 µM and osteoblast differentiation stimulation at 1.0 µM; compound S24-14 also suppressed bone loss in vivo. These results prove that S24-14 can be a potential lead for antiosteoporosis drug development.

Synthesis and biological evaluation of substituted amide derivatives of C4-ageratochromene dimer analog.

Substituted amide derivatives of C4-ageratochromene dimer analog (19) were synthesized through structural modification of precocene-I (4a), isolated from the essential oil of Ageratum conyzoides L. The target compounds (18-20, 23I-VI, 24I-VI, and 25I-VI) were evaluated for their bone-forming effect using osteoblast differentiation assay. Seven compounds (23I, 23II, 23IV, 23VI, 24III, 24VI, and 25VI) presented good activity within 1 pM-1 nM concentration. At 1 pM concentration, the most active compound i.e. 23II showed effective mineralization of osteoblast cells along with expression of osteogenic marker genes viz RUNX 2, BMP-2, and type 1 collagen (Type-1 col) without any toxicity towards osteoblast cells. Single crystal X-ray analysis of 18 and 20 revealed that the core nucleus of these molecules bear phenyl rings in a Trans-stilbenoid system and had a good structural correlation with 17ß-estradiol (1) and diethylstilbestrol (DES, 3). In-silico study about 23II showed its structural complementarities with the LBD of estrogen receptor (ER) which indicated possible ER-mediated activity of compounds.

Coordination Compounds As Multi-Delivery Systems for Osteoporosis.

Osteoporosis therapies leveraging bisphosphonates and mineral components (e.g., magnesium, calcium, and strontium) have been raising attention because of their potential for managing this ever-growing disease. The administration of multicomponent therapeutics (combined therapy) in elderly patients is complex and suffers from low patient adherence. Herein, we report an all-in-one combination of four antiosteoporotic components into a new family of coordination complexes: [M2(H4alen)4(H2O)2]·1.5H2O [where M2+ = Mg2+ (1), (Mg0.535Ca0.465)2+ (2) and (Mg0.505Ca0.450Sr0.045)2+ (3)]. These solid-state complexes were prepared, for the first time, through microwave-assisted synthesis. It is demonstrated that the compounds are capable of releasing their antiosteoporotic components, both in conditions that mimic the path along the gastrointestinal tract and in long periods under physiological conditions (pH ∼7.4). More importantly, when administered in low concentrations, the compounds did not elicit a cytotoxic effect toward liver, kidney, and osteoblast-like cell lines. Besides, it is important to highlight the unique coordination complex with four bone therapeutic components, [(Mg0.505Ca0.450Sr0.045)2(H4alen)4(H2O)2]·1.5H2O (3), which significantly promoted osteoblast metabolic activity up to ca. 1.4-fold versus the control group. These findings bring this type of compounds one-step closer to be considered as an all-in-one and more effective treatment for managing chronic bone diseases, prompting further research on their therapeutic properties.

Tetrahedral Framework Nucleic Acid Promotes the Treatment of Bisphosphonate-Related Osteonecrosis of the Jaws by Promoting Angiogenesis and M2 Polarization.

Bisphosphonates are often used to treat osteoporosis, malignant bone metastases, and hypercalcemia. However, it can cause serious adverse reactions, bisphosphonate-related osteonecrosis of the jaw (BRONJ), which seriously affects the quality of life of patients. At present, the treatment of BRONJ is still difficult to reach an agreement, and there is no effective treatment. Therefore, it is very important to find effective treatments. Many studies have shown that the occurrence of BRONJ may be due to unbalanced bone turnover, anti-angiogenesis, bacterial infection, direct tissue toxicity, and abnormal immune function. The previous research results show that tetrahedral framework nucleic acids (tFNAs), a new type of nanomaterial, can promote various biological activities of cells, such as cell proliferation, migration, anti-inflammation and anti-oxidation, and angiogenesis. Therefore, we intend to explore the potential of tFNAs in the treatment of BRONJ through this study. The results show that tFNAs can promote the treatment of BRONJ by promoting angiogenesis and promoting M2 polarization in macrophages and inhibiting M1 polarization both in vitro and in vivo. These results provide a theoretical basis for the application of tFNAs in the treatment of BRONJ and also provide new ideas and methods for the treatment of other diseases based on ischemia and immune disorders.

Calcitriol tablets with hybrid lipid-based solid dispersions with enhanced stability and content uniformity.

Calcitriol, as the biologically active form of vitamin D3, is essential for patients with renal osteopathy. The solubilization, stabilization, and content uniformity are key issues in its formulation development. In our previous study, the incomplete release of calcitriol was solved by using the hybrid lipid-based solid dispersion (SD) for calcitriol. However, good stability and content uniformity are still urgently needed. In this study, solid lipid with antioxidant properties and liquid lipid compatible with calcitriol were employed as hybrid lipid carrier (HLC) to establish a solid dispersion. Moreover, the content uniformity of tablets with hybrid lipid carrier based SDs (HLCTs) was further guaranteed due to the multi-dispersion of calcitriol in HLC, solidification, and blank granules. Additionally, the compression of the blank granules was adjusted by the water content. The mixing method of calcitriol-containing and blank granules was also optimized. The obtained HLCTs were evaluated for hardness, disintegration time, in vitro drug dissolution, content uniformity, and stability. Satisfactory HLCTs were developed successfully in this study with superior content uniformity and better stability than the commercial soft capsule (Rocaltrol®). It was proved to be a promising formulation for drugs with poor water-solubility, instability to oxygen and heat, and dose-related toxicity.

Self-sacrificial MOFs for ultra-long controlled release of bisphosphonate anti-osteoporotic drugs.

In this paper we report drug delivery systems that are based on phosphonate MOFs. These employ biologically-acceptable metal ions (e.g. Ca2+ and Mg2+) and several anti-osteoporosis bisphosphonate drugs (etidronate, pamidronate, alendronate and neridronate), as the organic linkers. These materials have been synthesized, structurally characterized, and studied for the self-sacrificial release (by pH-driven dissolution) of the bisphosphonate active ingredient. They exhibit variable release rates and final % release, depending on the actual structure of the metal-bisphosphonate material. Their cytotoxicity profiles match those of the active ingredients.

Synthesis and biological activities of drugs for the treatment of osteoporosis.

Osteoporosis is an asymptomatic progressive disease. With the improvement of people's living standard and the aging of population, osteoporosis and its fracture have become one of the main diseases threatening the aging society. The serious medical and social burden caused by this has aroused wide public concern. Osteoporosis is listed as one of the three major diseases of the elderly. At present, the drugs for osteoporosis include bone resorption inhibitors and bone formation promoters. The purpose of these anti-osteoporosis drugs is to balance osteoblast bone formation and osteoclast bone resorption. With the development of anti-osteoporosis drugs, new anti osteoporosis drugs have been designed and synthesized. There are many kinds of new compounds with anti osteoporosis activity, but most of them are concentrated on the original drugs with anti osteoporosis activity, or the natural products with anti-osteoporosis activity are extracted from the natural products for structural modification to obtain the corresponding derivatives or analogues. These target compounds showed good ALP activity in vitro and in vivo, promoted osteoblast differentiation and mineralization, or had anti TRAP activity, inhibited osteoclast absorption. This work attempts to systematically review the studies on the synthesis and bioactivity of anti-osteoporosis drugs in the past 10 years. The structure-activity relationship was discussed, which provided a reasonable idea for the design and development of new anti-osteoporosis drugs.

In-situ forming chitosan implant-loaded with raloxifene hydrochloride and bioactive glass nanoparticles for treatment of bone injuries: Formulation and biological evaluation in animal model.

In-situ forming implants receive great attention for repairing serious bone injuries. The aim of the present study was to prepare novel chitosan in-situ forming implants (CIFI) loaded with bioactive glass nanoparticles and/or raloxifene hydrochloride (RLX). Incorporating raloxifene hydrochloride (RLX) as a selective estrogen receptor modulator was essential to make use of its anti-resorptive properties. The prepared formulae were tested for their in-vitro gelation time, drug release, injectability, rheological properties, erosion rate and morphological properties. Results revealed that the formulation composed of 1% (w/v) chitosan with 2% (w/v) NaHCO3 and 1% (w/v) bioactive glass nanoparticles (CIFI-BG) possessed the most sustained drug release profile which extended over four months with low burst release effect compared to the same formulation lacking bioactive glass nanoparticles (CIFI). Selected formulations were tested for their ability to enhance bone regeneration in induced puncture in rate tibia. Results declared that these formulations were able to enhance bone regeneration after 12 weeks in comparison to the untreated tibial punctures and that containing bioactive glass could be considered as novel approach for treatment of serious bone injuries which require long term treatment and internal mechanical bone support during healing.

Design, Synthesis, and Biological Characterization of Orally Active 17ß-Hydroxysteroid Dehydrogenase Type 2 Inhibitors Targeting the Prevention of Osteoporosis.

Osteoporosis is predominantly treated with drugs that inhibit further bone resorption due to estrogen deficiency. Yet, osteoporosis drugs that not only inhibit bone resorption but also stimulate bone formation, such as potentially inhibitors of 17ß-hydroxysteroid dehydrogenase type 2 (17ß-HSD2), may be more efficacious in the treatment of osteoporosis. Blockade of 17ß-HSD2 is thought to increase intracellular estradiol and testosterone in bone, thereby inhibiting bone resorption by osteoclasts and stimulating bone formation by osteoblasts, respectively. We here describe the design, synthesis, and biological characterization of a novel bicyclic-substituted hydroxyphenylmethanone 17ß-HSD2 inhibitor (compound 24). Compound 24 is a nanomolar potent inhibitor of human 17ß-HSD2 (IC50 of 6.1 nM) and rodent 17ß-HSD2 with low in vitro cellular toxicity, devoid of detectable estrogen receptor α affinity, displays high aqueous solubility and in vitro metabolic stability, and has an excellent oral pharmacokinetic profile for testing in a rat osteoporosis model. Administration of 24 in a rat osteoporosis model demonstrates its bone-sparing efficacy.

Facile Synthesis of Chitosan Capped Mesoporous Silica Nanoparticles: A pH Responsive Smart Delivery Platform for Raloxifene Hydrochloride.

An encapsulation of model drug raloxifene hydrochloride (RAL) inside the chitosan decorated pH responsive mesoporous system has a greater potential for accumulating in the tumor cells. The present study involves synthesis of surface modified mesoporous silica nanoparticles (MSN) with the aim of achieving pH sensitive drug delivery system. A silanol skeleton of MSN has been productively modified to amine intermediate which served as a firm platform to adapt chitosan grafted assembly and systematically evaluated. RAL incorporation inside the featured mesopores was performed employing novel immersion solvent evaporation methodology and evaluated further. The pH responsive behavior of formulated nano framework was studied at three different pH of a phosphate buffer saline individually. The in vitro cell viability assay on MCF-7 breast carcinoma cells was performed in time and concentration dependent manner. Finally, the hemolysis assay of designed nanoparticle was accomplished to envisage the hemocompatibility. The outcome of characterization details unveiled a perfect 2D hexagonal spherical structure gifted with higher surface area and optimum pore size for designed nanoparticles. The higher percentage grafting of amine and chitosan residue, i.e., 4.01 and 28.51% respectively along with 31.89 and 33.57% RAL loading efficiency made MSNs more attractive and applicable. Eventually, in vitro release study exhibited higher RAL release in acidic media for extended time periods confirming successful formation of pH responsive nanoparticle having controlled release property. Conclusively potential of designed nanosystem to serve efficient anti-cancer remedy was confirmed by superior behaviour of chitosan grafted MSN towards MCF-7 cells with supreme hemocompatibility.

Synthesis and evaluation of a novel 99m Tc nitrido radiopharmaceutical with alendronate dithiocarbamate as a potential bone-imaging agent.

Currently, a popular strategy for designing novel radioprobes as bone-imaging agents is based on the concept of bifunctional radiopharmaceuticals. Considering the dithiocarbamate ligand can act as a suitable bifunctional linking agent to attach technetium-99m (99m Tc) to corresponding target molecules, in this study, alendronate dithiocarbamate (ALNDTC) was synthesized and radiolabeled with [99m Tc≡N]2+ core by ligand exchange reaction to produce 99m TcN-ALNDTC complex, for the potential use as a novel probe for bone imaging. The radiochemical purity of the complex was over 90%. The complex was stable in vitro and could bind to hydroxyapatite. The partition coefficient result indicated it was hydrophilic, and an evaluation of biodistribution in mice indicated that the complex exhibited a higher bone uptake than did 99m Tc-labeled methylenediphosphonate (99m Tc-MDP). Further, single photon emission computed tomography imaging study indicated clear accumulation in bone, suggesting that 99m TcN-ALNDTC would be a promising candidate for bone imaging.

A Novel H2S-releasing Amino-Bisphosphonate which combines bone anti-catabolic and anabolic functions.

Bisphosphonates (BPs) are the first-line treatment of bone loss resulting from various pathological conditions. Due to their high affinity to bone they have been used to develop conjugates with pro-anabolic or anti-catabolic drugs. We recently demontrated that hydrogen sulfide (H2S), promotes osteogenesis and inhibits osteoclast differentiation. Here we developed an innovative molecule, named DM-22, obtained from the combination of alendronate (AL) and the H2S-releasing moiety aryl-isothiocyanate. DM-22 and AL were assayed in vitro in the concentration range 1-33 µM for effects on viability and function of human osteoclasts (h-OCs) and mesenchymal stromal cells (h-MSCs) undergoing osteogenic differentiation. Amperometric measures revealed that DM-22 releases H2S at a slow rate with a thiol-dependent mechanism. DM-22 significantly inhibited h-OCs differentiation and function, maintaining a residual h-OCs viability even at the high dose of 33 µM. Contrary to AL, in h-MSCs DM-22 did not induce cytotoxicity as revealed by LDH assay, significantly stimulated mineralization as measured by Alizarin Red staining and increased mRNA expression of Collagen I as compared to control cultures. In conclusion, DM-22 is a new BP which inhibits h-OCs function and stimulate osteogenic differentiation of h-MSCs, without cytotoxicity. DM-22 is an ideal candidate for a novel family of osteoanabolic drugs.

Matrine derivate MASM uncovers a novel function for ribosomal protein S5 in osteoclastogenesis and postmenopausal osteoporosis.

Postmenopausal osteoporosis (POMP) is a public health problem characterized by decreased bone density and increased fracture risk. Over-activated osteoclastogenesis plays a vital role in POMP. Here we developed a novel bioactive compound MASM (M19) based on sophocarpine. Although it showed no significant effects on osteogenesis and adipogenesis for bone marrow-derived mesenchymal stem cells (BMSCs) in vitro, it could significantly inhibit RANKL/M-CSF induced osteoclastogenesis through suppressing NF-κB, MAPKs and PI3K/Akt pathways in vitro and ameliorate bone loss in ovariectomized mice in vivo. Ribosomal protein s5 (RPS5) has been identified as a target of M19 and regulates PI3K/Akt, NF-κB and MAPKs pathways in osteoclastogenesis. Overexpressions of RPS5 synergistically inhibited osteoclastogenesis with M19 while silencing RPS5 compromised M19 inhibitory effects on osteoclastogenesis in vitro. Among the three pathways, Akt plays a major role in M19 effects. The Akt activator SC79 partially reversed the inhibitory effects on osteoclastogenesis by M19 and RPS5-knocking-down. It indicates that RPS5 serves as a potential candidate target for inhibiting osteoclastogenesis and osteoporosis therapy and M19 is a promising agent for POMP treatment.

Smart, programmable and responsive injectable hydrogels for controlled release of cargo osteoporosis drugs.

Easy-to-prepare drug delivery systems, based on smart, silica gels have been synthesized, characterized, and studied as hosts in the controlled release of bisphosphonates. They exhibit variable release rates and final % release, depending on the nature of bisphosphonate (side-chain length, hydro-philicity/-phobicity, water-solubility), cations present, pH and temperature. These gels are robust, injectable, re-loadable and re-usable.

Design, Synthesis, and Pharmacokinetics of a Bone-Targeting Dual-Action Prodrug for the Treatment of Osteoporosis.

A dual-action bone-targeting prodrug has been designed, synthesized, and evaluated for in vitro and in vivo metabolic stability, in vivo tissue distribution, and rates of release of the active constituents after binding to bones through the use of differentially double-labeled derivatives. The conjugate (general structure 7) embodies the merger of a very potent and proven anabolic selective agonist of the prostaglandin EP4 receptor, compound 5, and alendronic acid, a potent inhibitor of bone resorption, optimally linked through a differentially hydrolyzable linker unit, N-4-carboxymethylphenyl-methyloxycarbonyl-leucinyl-argininyl-para-aminophenylmethylalcohol (Leu-Arg-PABA). Optimized conjugate 16 was designed so that esterase activity will liberate 5 and cathepsin K cleavage of the Leu-Arg-PABA element will liberate alendronic acid. Studies with doubly radiolabeled 16 provide a proof-of-concept for the use of a cathepsin K cleavable peptide-linked conjugate for targeting of bisphosphonate prodrugs to bone and slow release liberation of the active constituents in vivo. Such conjugates are potential therapies for the treatment of bone disorders such as osteoporosis.

Rapid, microwave-accelerated synthesis and anti-osteoporosis activities evaluation of Morusin scaffolds and Morusignin L scaffolds.

Described herein is a facile and efficient methodology toward the synthesis of Morusin scaffolds and Morusignin L scaffolds 4-9 and 12via a novel three-step approach (Michael addition or prenylation, cyclization and cyclization) and use a rapid, microwave-accelerated cyclization as the key step. Furthermore, their biological activities have been preliminarily demonstrated by in vitro evaluation for anti-osteoporosis activity. These Morusin, Morusignin L and newly synthesized compounds 5b, 6a, 8e, 8f greatly exhibited the highest potency, especially at the 10-5mol/L (P<0.01), and had good in vitro anti-osteoporosis activities using the commercially available standard drug Ipriflavone as a positive control. The mechanisms associated with anti-osteoporosis effects of these compounds may be through the inhibition of TRAP enzyme activity and bone resorption in osteoclasts, and promotion effect of osteoblast proliferation in vitro. The results indicated that Morusin scaffolds and Morusignin L scaffolds may be useful leads for further anti-osteoporosis activity screenings.

Synthesis of novel 5,6-dehydrokawain analogs as osteogenic inducers and their action mechanisms.

An imbalance between bone resorption by osteoclasts and bone formation by osteoblasts can cause bone loss and bone-related disease. In a previous search for natural products that increase osteogenic activity, we found that 5,6-dehydrokawain (1) from Alpinia zerumbet promotes osteoblastogenesis. In this study, we synthesized and evaluated series of 5,6-dehydrokawain analogs. Our structure-activity relationships revealed that alkylation of para or meta position of aromatic ring of 1 promote osteogenic activity. Among the potential analogs we synthesized, (E)-6-(4-Ethylstyryl)-4-methoxy-2H-pyran-2-one (14) and (E)-6-(4-Butylstyryl)-4-methoxy-2H-pyran-2-one (21) both significantly up-regulated Runx2 and Osterix mRNA expression at 10µM. These osteogenic activities could be mediated by bone morphogenetic protein (BMP) and activation of p38 MAPK signaling pathways. Compounds 14 and 21 also inhibited RANKL-induced osteoclast differentiation of RAW264 cells. These results indicated that novel 5,6-dehydrokawain analogs not only increase osteogenic activity but also inhibit osteoclast differentiation, and could be potential lead compounds for the development of anti-osteoporosis agents.

Reaction of 3-Amino-1,2,4-Triazole with Diethyl Phosphite and Triethyl Orthoformate: Acid-Base Properties and Antiosteoporotic Activities of the Products.

The reaction of diethyl phosphite with triethyl orthoformate and a primary amine followed by hydrolysis is presented, and the reaction was suitable for the preparation of (aminomethylene)bisphosphonates. 3-Amino-1,2,4-triazole was chosen as an interesting substrate for this reaction because it possesses multiple groups that can serve as the amino component in the reaction-namely, the side-chain and triazole amines. This substrate readily forms 1,2,4-triazolyl-3-yl-aminomethylenebisphosphonic acid (compound 1) as a major product, along with N-ethylated bisphosphonates as side products. The in vitro antiproliferative effects of the synthesized aminomethylenebisphosphonic acids against J774E macrophages were determined. These compounds exhibit similar activity to zoledronic acid and higher activity than incadronic acid.

Pyrrolidine-Containing Bisphosphonates as Potential Anti-Resorption Bone Drugs.

Bisphosphonates, particularly those with N-substituted groups, are currently the most popular drugs for the treatment of osteoporosis. However, their chemical structures are still rather simple and new synthetic methods are needed to expand their molecular complexity and also improve their specificity of action towards other targets as anticancer, antibacterial, and antimalarial drugs. Herein, we report a new class of potential antiresorption bisphosphonate drugs that have a pyrrolidine unit with different substituents, obtained through a simple dipolar cycloaddition reaction between azomethine ylides and vinylidenebisphosphonate derivatives as precursors. The methodology led to the efficient preparation of a wide range of (1-methylpyrrolidine-3,3-diyl)bis(phosphonic esters) derivatives with different substituents in position 4.