NO-donating Genistein Prodrug: Design, Synthesis And It's Bioactivities On Osteoblast

BackgroundOsteoporosis (OP) is a kind of systematic skeleton disease. The characteristics of which are reduced bone content, degenerated microstructure of bone tissues, increased fragility of bone and chanciness of bone fracture. OP occurs mainly among postmenopausal women and aged men. Epidemiological statistics shows that the incidence of OP ranked second in metabolic bone diseases. Along with the developing aging tendency of human, the incidence of bone fracture which accompanied with OP is increasing.Postmenopausal osteoporosis (PMO) accounted for a large portion of OP. For a long time, hormone replacement therapy (HRT) has been the main treatment for prevention and therapy. HRT in the prevention and treatment of PMO has good results, however, the serious side effects and contraindications of HRT hamper its clinical use. Recently, the American National Institute of Health stopped a clinical trial with HRT in healthy postmenopausal women after 5.2 years more than 3 years earlier than planned due to an increased risk of invasive breast cancer and of cardiovascular risks. It was concluded that these risks outweighed any benefits of HRT. As a consequence of reports of these results in scientific publications and in mass media, people are looking for safe, effective alternatives which may also relieve climacteric symptoms. Phytoestrogens are plant origin isoflavones. They have similar structures to that of estrogen, with weaker estrogen-like activity, and may bond to estrogen receptor (ER) selectively. Compared with estrogen, they have less side effects, espscially estrogen-related cancers. As for their weaker bioactivity, their effects are not very stable and can not improve climacteric symptom effectively. Genistein (Gen), daidzein and glycitein are the most studied ones.1994, the study of Kasten TP found that inhibition of nitric oxide synthase (NOS) may result in the increasing of bone resorption in ovariectomized rats (OVX), the inhibition of NOS may inhibited the proliferation of osteoblast (OB) dose-dependently. Many studies show that serum NO was decreased significantly when bone mineral density (BMD) and estrogen were all droped in OVX rats. The descendent level of estrogen cause reduced NO level, which in turn enhances bone resorption. Then bone content loses and BMD reduces. This may be one of the important mechanisms of PMO. Because PMO patients could not generate necessary NO to maintain normal physiologic function, it provides a reasonable biologic foundation for NO replacement therapy. Some studies also indicated that drugs regulated NO are likely to become new candidates for OP treatment with broad prospects.AimsBased on former studies, consulting the structures of NO-donating non-steroidal antiinflammatory drugs (NO-NSAIDs) and analyzing the structure characteristics of Gen, we design, synthesize a structure by conjugate Gen and NO-donor moiety together. After biotransformation, this structure may release NO slowly and its parent drug Gen, and may synergism the treatment effect of OP.Methods1. Design, synthesis, and structure determination of NO-G. By analyzing the structure of Gen, refering to the strcture of NO-NSAIDs which has the function to release NO slowly, we got a target compound by esterification and nitration reaction. The structure of this new compound was confirmed by nuclear magnetic resonance (NMR), infrared spectrum (IR) and mass spectrometry (MS).2. HPLC method was used to determine the new compound, and its stability in vitro was also studied.2.1 Refer to the determination method of Gen and experimental exploration, we established the HPLC method to determine NO-G.2.2 Studies on the stability of NO-G in vitro.2.2.1 Stability under simulated physiological environment. The solution of NO-G was shaking under 37℃at pH1.0 (simulated gastric environment) and pH6.8 (simulated small intestinal environment) respectively. Samples were collected at 0,0.5,1,1.5,2,3h and were determined by HPLC;2.2.2 Stability on osteoblast cells in vitro. MC3T3-E1 cells were used in this study to observe the stability of NO-G by HPLC determination.3. Studies on the bioactivity of NO-G. These were all performed on MC3T3-E1 cells.3.1 NO releasing capacity of NO-G in MC3T3-E1 cells. Griess reagent was used to determine NO releasing capacity by determine the content of nitrite in cultured media. SNAP was used as control to compare their NO releasing characters.3.2 Bioactivity of NO-G in vitro. Studies were performed on proliferation, differentiation and mineralization of NO-G on MC3T3-E1 cells compared with Gen, SNAP and estrodial(E2).3.1 Effects on cell proliferation a. MTT assay was used to study the dose- and time- dependent effect of NO-G on the proliferation of MC3T3-E1 cells, and compared the effect with other drugs . b. Cell cycle analysis was performed by flow cytometry to further determine the effect of NO-G on cell proliferation.3.2.2 Effects on cell differentiation a. Alkaline phosphatase (ALP) kit was used to study the dose- and time- dependent effects of NO-G on ALP activity, and compared the effect with other drugs.b. OCN ELISA kit was used to study the dose- and time- dependent effects of NO-G on OCN secretion, and compared the effect with other drugs.3.2.3 Mineralization assay Mineralization in OB cultures was determined by staining of Alizarin Red-S, and quantitive analysis was performed. Results1. A new compound (NO-G) was obtained by chemical synthesis. It was a light beige powder, which melting point was ranged from 125.5 to 127.5oC, and the yield was 87.6%. The mass spectrometric analysis showed the relative molecular mass was 532.04, and the data of 1H NMR spectra and infrared spectrum of the product were in agreement with the designed structure.2. Established HPLC determination method of NO-G. The chromatographic condition is: mobile phase: CH3 CN: 0.1%CH3 COOH solution (60:40); flow rate: 254nm; chromatographic column: C18(250×4.6mm, 5μm); column temperature: 25 oC; sample injiection: 20μl.3. In simulated stomach (pH1.0) condition, Gen can be determined half an hour shaking in 37 oC waterbath; and NO-G almost wholly decomposed after 3h. In simulated small intestinal (pH6.8) condition NO-G was stsble for about 3h. NO-G and Gen may be detected in MC3T3-E1 cells 30min after drug donation.4. NO-G may liberate NO in MC3T3-E1 cells. Compared with SNAP, its manner was slow and moderate, which may keep up to 5h.5. NO-G may promote proliferation, differentiation of MC3T3-E1 cells, the effects was both time- and dose- dependent. All the effects were better than its parent drugs (P<0.05 or 0.01).6. NO-G may promote the form of mineralization. Mineralization may be formed after cultured for 18d. Quantitative analysis showed that NO-G may promote mineralization effectively compared with its parent drugs (P<0.05 vs Gen, SNAP, and Gen+SNAP).Conclusions1. We designed and synthesized the structure of NO-G, which may release NO slowly. The desighed synthetic routs are simple and easy to perform. The operative procedures could be controlled easily, and the product had better purity and proper yield. The structure of NO-G didn't had been reported.2. The estabilished HPLC method was stable and repeatable. It may determine NO-G and Gen at the same time. Under this condition, the peak of Gen, NO-G and dopant may be well separated. The retention time is appropriate and blank solution has no special absorption peak.3. NO-G was not stable under simulated stomach condition, but it was stable under simulated small intestinal condition. NO-G could enter OB mostly, then decomposed and liberated Gen.4. Compared with SNAP, NO-G may releasing low dose NO slowly in OB. This may be the basis of enhanced effect of NO-G.5. NO-G showed time- and dose- dependent effects on proliferation and differentiation of OB, and effectively promoted mineralization. All the effects were better than its parent drugs.We believe NO-G may be a new alternative for PMO.