WO2021175335A1 - Isopaucifloral F磷酸酯类化合物及其药用用途 - Google Patents
Isopaucifloral F磷酸酯类化合物及其药用用途 Download PDFInfo
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- WO2021175335A1 WO2021175335A1 PCT/CN2021/081571 CN2021081571W WO2021175335A1 WO 2021175335 A1 WO2021175335 A1 WO 2021175335A1 CN 2021081571 W CN2021081571 W CN 2021081571W WO 2021175335 A1 WO2021175335 A1 WO 2021175335A1
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- Prior art keywords
- isopaucifloral
- compound
- pharmaceutically acceptable
- phosphate
- phosphate compound
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Images
Classifications
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- C—CHEMISTRY; METALLURGY
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- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/095—Compounds containing the structure P(=O)-O-acyl, P(=O)-O-heteroatom, P(=O)-O-CN
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
- C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
- C07F9/02—Phosphorus compounds
- C07F9/06—Phosphorus compounds without P—C bonds
- C07F9/08—Esters of oxyacids of phosphorus
- C07F9/09—Esters of phosphoric acids
- C07F9/12—Esters of phosphoric acids with hydroxyaryl compounds
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/66—Phosphorus compounds
- A61K31/661—Phosphorus acids or esters thereof not having P—C bonds, e.g. fosfosal, dichlorvos, malathion or mevinphos
- A61K31/6615—Compounds having two or more esterified phosphorus acid groups, e.g. inositol triphosphate, phytic acid
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C35/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring
- C07C35/22—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system
- C07C35/23—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a ring other than a six-membered aromatic ring polycyclic, at least one hydroxy group bound to a condensed ring system with hydroxy on a condensed ring system having two rings
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
- C07C39/00—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring
- C07C39/02—Compounds having at least one hydroxy or O-metal group bound to a carbon atom of a six-membered aromatic ring monocyclic with no unsaturation outside the aromatic ring
- C07C39/10—Polyhydroxy benzenes; Alkylated derivatives thereof
Definitions
- the technical field of biomedicine of the present invention relates to Isopaucifloral F phosphate compounds and their medicinal uses, and specifically relates to phosphate modified natural molecules Isopaucifloral F compounds and their use in preparing anti-osteoporosis drugs.
- osteoporosis is a multi-cause systemic bone metabolism disease that is easily acquired by the elderly and postmenopausal women. It causes bone strength due to decreased bone mass and degeneration of the fine structure of bone tissue. Decreased, increased bone fragility, and increased fracture risk are the characteristics, and its clinical symptoms are mostly bone pain and fractures. Clinical practice has shown that fractures most often occur in hips, wrists, and spine. The mortality rate within one year after hip fractures can reach 20%-24%, which seriously affects the quality of life of patients, and even their lives. According to statistics, the global incidence of osteoporosis has exceeded 25%, and its incidence has jumped to the seventh place among common and frequently-occurring diseases.
- the World Health Organization believes that osteoporosis is a health problem second only to coronary heart disease in importance. It can occur at any age, any race or ethnic group, and affect millions of people in the world, especially with With the increasing aging of the global population, human health and economic development are facing severe challenges. Therefore, how to treat osteoporosis has become a research hotspot in modern medicine.
- osteoporosis treatment drugs are divided into three categories according to the mechanism of their effects on the bone remodeling process:
- Anti-bone resorption drugs there are four types, namely 1 alendronate sodium diphosphate
- This type of drugs inhibits the key enzyme pyrophosphate synthase in osteoclasts, inhibits the activity of osteoclasts and thereby inhibits osteoclast-mediated bone resorption.
- these drugs strongly inhibit bone resorption and have a long half-life.
- Long-term use has potential bone toxicity (ie, osteonecrosis of the jaw and atypical femoral fracture) and nephrotoxicity.
- the patient's gastrointestinal tract is also intolerant to bisphosphonates.
- denosumab is a human-derived full-length monoclonal antibody that targets to bind nuclear factor-K ⁇ receptor activator ligand (RANKL), thereby Inhibit the formation of osteoclasts and inhibit bone resorption; however, due to the widespread presence of RANKL in most tissues and the immune system, long-term use of denosumab mainly has a variety of side effects.
- RANKL nuclear factor-K ⁇ receptor activator ligand
- Osteogenic drugs the synthetic parathyroid hormone related peptide, teriparatide, is the only effective bone formation stimulant recommended in clinical practice, but teriparatide is expensive and requires subcutaneous injection every day Drug administration severely limits its clinical use, and it is now used as a second-line drug for the treatment of osteoporosis.
- a dual-mode drug that promotes bone formation while inhibiting bone resorption Strontium ranelate (SR) is currently the only dual-mode drug that can simultaneously reduce bone resorption and promote bone formation. It stimulates the formation of new bone tissue while reducing Bone resorption, the drug can reduce the incidence of bone fractures throughout the body, and the therapeutic effect is obvious.
- SR Strontium ranelate
- strontium ranelate can increase the incidence of non-fatal myocardial infarction, prompting regulatory agencies to reconsider its efficacy and risks. Its safety needs to be further improved. improve.
- the research team of the present application isolated a variety of natural polyhydroxy phenolic resveratrol dimers with structural characteristics that are superior to resveratrol in activity and structural characteristics from the medicinal plant genus root, which is used to invigorate the kidney, as a selective estrogen receptor
- the modulator shows an estrogen-like effect on bone, increases bone density, and has no adverse reactions.
- a sample library of four types of natural dominant backbone resveratrol dimer compounds was constructed, with a total of 500 compounds, and they have drug-like properties.
- the seed compound of the dominant skeleton has done the systematic lead compound discovery and drug-making research.
- the natural product Isopaucifloral F can be used as a selective agonist of the estrogen receptor ER ⁇ , but there are some anti-osteoporosis drugs that are not effective defect.
- the present invention intends to provide the use of a class of Isopaucifloral F phosphate compounds in the preparation of anti-osteoporosis drugs.
- This kind of compound has high safety, good metabolic stability, high bioavailability, and exerts anti-osteoporosis drug effect through a dual-mode action mechanism of reducing osteoclast resorption and promoting osteogenesis.
- the purpose of the present invention is to provide a class of Isopaucifloral F phosphate compounds and their use in the preparation of anti-osteoporosis drugs based on the basis and current situation of the prior art.
- the present invention is based on the basis of the prior art, and experiments show that the natural product Isopaucifloral F can be used as a selective agonist of the estrogen receptor ER ⁇ , but there is a defect that the anti-osteoporosis drug effect is not significant; the main reason is that the molecular structure contains Multiple phenolic hydroxyl groups are easily combined with glucuronic acid in the body and excreted from the body.
- the present invention optimizes the molecular structure of Isopaucifloral F to improve the metabolic properties and bioavailability of the lead.
- the anti-osteoporosis drug alendronic acid has a dominant phosphate group in the molecular structure .
- Isopaucifloral F easy to metabolize sites, design and prepare new metabolically stable phosphate target compounds.
- the prepared compounds have high safety, good metabolic stability, high bioavailability, and through reduction
- the dual-mode action mechanism of osteoclast resorption and promotion of osteogenesis exerts anti-osteoporosis drug effect.
- Isopaucifloral F phosphate compound and pharmaceutically acceptable salt thereof having the structure of the following formula (1) of the present invention:
- R 1 is independently a phosphate group or a hydrogen atom, and R 2 is independently a phosphate group;
- R 1 is a hydrogen atom
- R 2 is independently Phosphate group
- R 1 is In the case of a phosphate group
- R 1 R 2 .
- the present invention provides a method for synthesizing the above-mentioned Isopaucifloral F phosphate drug.
- the present invention adopts the previous synthesis method, using 3,5-dimethoxybenzoic acid (2) as the starting material, through the gold coupling reaction, Wittig reaction And Nazarov cyclization to synthesize the key intermediates 6a and 6b, and then introduce the diethyl phosphate group into the molecular structure of the key intermediates 6a and 6b through the nucleophilic substitution reaction under the NaH/THF system, and finally in BBr 3 /CH 2 The methyl group in the molecule is removed in the Cl 2 system to obtain the target products 1a and 1b.
- the base used in the Wittig reaction condition (b) is NaHMDS, KHMDS, LiHMDS, LDA, NaH, tBuONa, tBuOK or n-BuLi.
- the Lewis acid used in the Nazarov cyclization condition (c) is AlCl 3 , BF 3 , TiCl 4 , SnCl 4 , Bi(OTf) 3 or ZnCl 2 .
- the introduction of the phosphate group (d) in the reaction system is NaH, KHMDS, NaHMDS, LiHMDS, t-BuOK or t-BuONa.
- the reagent used in the reaction (e) system for removing the methyl group from the molecule is BBr 3 or AlCl 3 .
- the ratio of Isopaucifloral F to the phosphate group fragment is 1:2.5 (reaction step e).
- the compound has pharmacological research value and can treat osteoporosis.
- the compound has good metabolic stability, high bioavailability and safety, and it can promote bone formation and inhibit bone resorption. Mechanism to play an anti-osteoporosis effect.
- the examples of the present invention provide the lipid-water distribution coefficients of 1a and 1b, the plasma stability test, the mechanism of promoting bone formation and inhibiting bone resorption; and the metabolic stability of human liver microsomes of the candidate drug 1a, in rats Pharmacokinetics, acute toxicity and pharmacodynamic data of zebrafish osteoporosis model.
- the present invention also provides the use of Isopaucifloral F phosphate compounds and their pharmaceutically acceptable salts in the preparation of anti-osteoporosis drugs.
- a pharmaceutical composition for treating osteoporosis is further prepared, which includes the Isopaucifloral F phosphate compound and its pharmaceutically acceptable salts and pharmaceutically acceptable excipients.
- the Isopaucifloral F phosphate racemate mixture or their optically pure isomers and their pharmaceutically acceptable salts of the present invention can be prepared to contain a safe and effective amount of Isopaucifloral F phosphate racemate mixture, optically pure isomers or They are in various formulations of pharmaceutically acceptable salts and pharmaceutical carriers.
- safe and effective amount refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
- the safe and effective amount is determined according to the age, condition, and course of treatment of the subject to be treated.
- pharmaceutical carrier refers to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use, and must have sufficient purity and sufficiently low toxicity.
- composition means that each component of the composition can be blended with the compound of the present invention and between them without significantly reducing the efficacy of the compound.
- examples of pharmaceutically acceptable carriers include sugars (such as glucose, sucrose, lactose, etc.), starch (such as corn starch, potato starch, etc.), cellulose and its derivatives (such as sodium carboxymethyl cellulose, Sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), Polyols (such as propylene glycol, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), coloring agents, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
- sugars such as glucose, sucrose, lactose, etc.
- starch such as corn starch, potato starch, etc
- the most effective anti-osteoporosis drug in clinical practice is a dual-mode drug that promotes bone formation while inhibiting bone resorption. Its only representative drug, strontium ranelate, has the risk of increasing the incidence of non-fatal myocardial infarction. It is safe Needs to be further improved.
- the Isopaucifloral F phosphate compound and its salt of the present invention are highly safe, and play an anti-osteoporotic effect through the dual mechanisms of promoting bone formation and inhibiting bone resorption. This type of compound has good metabolic stability and high bioavailability, and it is expected to meet clinical needs Urgent needs.
- Figure 1 Single crystal formed by alizarin red and calcium nodules observed under a 40x inverted microscope
- FIG. 3 S-1a zebrafish osteoporosis model study (control is the blank control group, Prednisolone is the model group, and Alendronate is the positive drug control group, ns, not significant; ***P ⁇ 0.0001, **P ⁇ 0.001)
- Example 8 Determination of Isopaucifloral F-PP(1a) and Isopaucifloral F-P(1b) Lipid-Water Distribution Coefficient and Stability Test
- 1Determination of fat-water partition coefficient First, formulate a standard curve. Weigh a certain amount of target compounds 1a and 1b to prepare solutions with chromatographic grade acetonitrile. Samples were injected and analyzed by HPLC. The HPLC conditions are: mobile phase: 5%-95% acetonitrile/water, gradient elution, flow rate 0.6mL/min, injection volume 10 ⁇ L, inspection wavelength 254/280nm, run for 20min, record retention time and peak area , Regress the concentration and the peak area to get the standard curve.
- the concentration measured on the upper layer is the drug concentration Co in the oil phase (n-octanol phase), and the concentration measured on the lower layer is the drug concentration Cw in the water phase.
- the logP of Isopaucifloral F-PP(1a) is 2.60; the logP of the compound Isopaucifloral FP(1b) is 2.25, and the logP of the two compounds are both between 1 and 3, which has certain druggability.
- the logP value of the target compound becomes larger, and the fat solubility is significantly improved.
- 2Determination of plasma stability Dissolve an appropriate amount of DMSO stock solution of the target compound in 4mL of plasma to make the concentration 200 ⁇ g/mL, and the DMSO content does not exceed 0.1%. Incubate in a water bath at 37°C for 0, 5, 15, 30 min and 1, 2, 4, 6, 10, 24, 48 h, take 200 ⁇ L, and add 3 times the volume of chromatography grade acetonitrile to precipitate the protein. Vortex for 5 min. After centrifugation, 200 ⁇ L of the supernatant was taken and analyzed by HPLC. The liquid phase conditions were the same as before.
- the concentration of the compound Isopaucifloral F-PP(1a) accounted for 94.17% of the initial concentration; the concentration of the compound Isopaucifloral FP(1b) accounted for 93.74% of the initial concentration. It can be seen that the target compound has a good plasma concentration. The stability.
- 3Determination of cell fluid stability In the intracellular stability test, the MC3T3-E1 subclone 14 cells in the logarithmic growth phase, about 2 million, were digested and collected in a 1mL centrifuge tube, and 200 ⁇ L of cell lysate was added to lyse the cells. . Then add 800 ⁇ L of PBS solution to make the total volume 1 mL. Similarly, incubate in a water bath at 37°C, take 100 ⁇ L at 0, 5, 15, 30, 45, 60 min, and add 3 times the volume of chromatography grade acetonitrile to precipitate the protein. Vortex for 5 min. After centrifugation, 200 ⁇ L of the supernatant was taken and analyzed by HPLC. The liquid phase conditions were the same as before.
- the target compound Isopaucifloral F-PP(1a) and Isopaucifloral FP(1b) basically do not dissociate within 60 minutes in the cell lysate system. It can be judged that the compound should be in the form of the original drug in the cell, and there is no diethyl phosphate. The hydrolysis of the cluster.
- the logP of the compound is between 1 and 3
- the LogP of the compound Isopaucifloral F-PP (1a) is 2.60
- the LogP of the compound Isopaucifloral F-P (1b) is 2.25, which has certain druggability.
- MC3T3-E1 Subclone 14 The proliferation effect of the compound on mouse osteoblast precursor cell MC3T3-E1 Subclone 14 (see Table 1): The MTT method was used to determine the proliferation effect of target compounds 1a and 1b on MC3T3-E1 subclone 14. Genistein and estradiol were used as positive controls.
- MC3T3-E1 subclone14 was cultured in ⁇ -MEM medium containing 10% FBS in a constant temperature incubator at 37°C and 5% CO 2.
- the MC3T3-E1 subclone14 cells in good condition in the logarithmic growth phase were digested with appropriate amount of trypsin (purchased from GIBCO), and the cells were collected.
- the cells were resuspended in ⁇ -MEM medium containing 10% FBS and seeded on a 96-well plate at an amount of 2000 cells/well. After it adheres to the wall, the medium is changed to 10% FBS ⁇ -MEM containing different concentrations of compounds, 200 ⁇ L per well. The cells added with 0.2% DMSO medium were used as the negative control group of the culture medium. The medium was added to the wells not inoculated with cells as a blank control group. Set 6 multiple holes for each concentration. The medium was changed every 2 days, and on the 6th day of administration, 20 ⁇ L of 5 mg/mL MTT/PBS solution was added to the above 96-well plate, and the incubation continued for 4 hours.
- Proliferation rate [(Average OD value of administration group-Average OD value of blank group)/(Average OD value of culture medium control group-Average OD value of blank group)]
- Isopaucifloral F-PP (1a), Isopaucifloral F-P (1b) and Isopaucifloral F have a significantly higher proliferation effect on MC3T3-E1 Subclone 14 than the positive control drug genistein.
- the proliferation rates were 114%, 148% and 124%, respectively.
- MC3T3-E1 subclone 14 (see Table 2): MC3T3-E1 subclone 14 cells were digested with trypsin and collected and then transferred to a 6-well plate at 4 ⁇ 10 4 /well After 12 hours of attachment, the medium was changed to a differentiation medium containing different concentrations of 1a, 1b, Isopaucifloral F and the positive control drug genistein.
- concentrations of the compounds were: 10 -4 to 10 -9 M; the drug concentration of genistein GEN in the positive control group was the concentration with the highest bone formation ability reported in the literature, which was 10 -6 M.
- Isopaucifloral F-PP (1a), Isopaucifloral FP (1b) and Isopaucifloral F have higher ALP enzyme activities than the control group, 2.36 times (enzyme activity increased by 136%), 1.20 times (enzyme activity) Increased by 19.5%) and 1.14 times (enzyme activity increased by 13.5%).
- Isopaucifloral F-PP(1a) has the largest calcium nodule formation at 10 -7 M, and its relative value is 1.446.
- Isopaucifloral FP (1b) has the largest calcium nodule formation at 10 -6 M, and its relative value is 1.182.
- Example 10 Study on the efficacy of Isopaucifloral F-PP (1a) and Isopaucifloral F-P (1b) in inhibiting bone resorption
- mice mononuclear macrophages RAW264.7 were inoculated into 96-well plates. After they adhered, the medium was changed to 1a and 1b with concentrations of 10 -4 M, 10 -6 M, and 10 -8 M.
- the lead compound Isopacifloral F group and the positive control drug Raloxifene (Raloxifene) induction medium, the non-administered group was used as the negative control group (Control group).
- the cells were stained with tartrate-resistant acid phosphatase TRAP incubation solution, mounted with glycerin gelatin, observed under light microscope, and counted the TRAP staining-positive multinucleated cells (as shown in Figure 2), and the inhibitory rate of the drug on the number of TRAP-positive multinucleated cells It is: (n negative control group TRAP-positive multinucleated osteoclasts- n given drug group TRAP-positive multinucleated osteoclasts )/n negative control group TRAP-positive multinucleated osteoclasts ⁇ 100%.
- Their inhibitory activity on osteoclasts is equivalent, significantly higher than Isopacifloral F, which is 62.4% and 65%, respectively, and is 2.2 and 2.3 times that of Isopacifloral F.
- the number of osteoclasts in the Control group is 117; each group has 8 replicate holes.
- S-1a was selected to study the in vivo pharmacokinetics of SD rats.
- the intravenous dose was 5mg/kg and the oral dose was 20mg/kg, at 5min, 8min, 15min, 30min, 1h, 2h, 3h, 4h.
- 6h, 8h, 10h, 24h blood samples were collected through the orbit at each time point, after centrifugation, the concentration of the drug in the plasma at each time point was determined by LC-MS/MS, and the non-compartmental model was used through DAS software (version 2.0)
- the method estimates its preliminary pharmacokinetic parameters in vivo, and the experimental results are as follows:
- Isopaucifloral F-PP (1a) and its precursor Isopaucifloral F for acute toxicity experiments in mice.
- the experimental data was calculated by the SPSS software (IBM SPSS Statistics 19) using the Bliss method.
- S-1a and the clinical first-line drug Alendronate were selected to give zebrafish model juveniles cultured to 10d (10dpf) after fertilization, and used after anesthesia. After fixation with 4% paraformaldehyde, the juveniles were bleached with a bleaching agent containing 30% H 2 O 2 prepared by 1% KOH until the eye pigments were removed. Alizarin red was used to stain the head bones of zebrafish juveniles. A mixed solution of 1% KOH and glycerin with a gradient ratio of 3:1 was used to make the juvenile fish transparent, and finally the juvenile fish were stored in pure glycerin.
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Abstract
Isopaucifloral F磷酸酯类化合物及其药用用途，经实验证实，所述Isopaucifloral F 磷酸酯类化合物及其药学上可接受的盐，对骨重建过程的影响是通过促骨形成和减少破骨吸收双模式作用的机制发挥其抗骨质疏松作用的药效，具有生物利用度高、代谢稳定和安全性好的特点，可用于制备新一代的抗骨质疏松药物。式 I
Description
本发明屈生物医药技术领域，涉及Isopaucifloral F磷酸酯类化合物及其药用用途，具体涉及磷酸酯修饰的天然分子Isopaucifloral F化合物及其在制备抗骨质疏松药物中的用途。
现有技术公开了骨质疏松症(osteoporosis，OP)是一种老人与绝经后妇女易得的多病因的全身性的骨骼代谢疾病，它以骨量降低、骨组织微细结构退变导致骨强度下降、骨脆性增加及骨折危险性增大为特征，其临床症状多为骨骼疼痛和骨折。临床实践显示，骨折最常发生在髋部、手腕和脊柱等部位，其中髋部骨折后一年内的死亡率可达20％-24％，严重影响患者的生活质量，甚至生命。据统计，全球的骨质疏松发病率已超过25％，其发病率已跃居常见病、多发病的第7位。世界卫生组织(WHO)认为骨质疏松症是重要性仅次于冠心病的健康问题，它可以发生在任何年龄阶段，任何种族或民族群体，影响着世界上数百万人，特别是随着全球人口老龄化的加剧，人类的健康和经济发展都面临严峻的挑战。因此，如何治疗骨质疏松成为现代医学的研究热点。
临床实践中根据骨质疏松症治疗药物对骨重建过程影响的作用机制将其分为三大类：(1)抑制骨吸收药物，有四种，分别为①以阿仑膦酸钠二磷酸盐，该类药物抑制破骨细胞内的关键酶焦磷酸合成酶，抑制破骨细胞的活性从而抑制破骨细胞介导的骨吸收。然而这类药物强力抑制骨吸收并且半衰期长，长期使用具有的潜在的骨骼毒性(即颚骨坏死和非典型股骨骨折)以及肾毒性，同时患者胃肠道对二磷酸盐类的不耐受也使其临床应用大打折扣；②雌激素补充疗法，用于改善绝经后妇女的骨质疏松症，雌激素的长期使用时会导致患乳腺癌的危险性增加，故在临床上已不做常规治疗药物；③以雷洛昔芬为代表的选择性雌激素受体调节剂，雷洛昔芬在骨骼表现为激动活性，能提高骨矿物质密度，雷洛昔芬的重要缺陷在于它的绝对生物利用度仅为2％；④以狄诺塞麦为代表的降钙素，是一种人源全长的单克隆抗体，靶向结合核因子-Kβ受体活化因子配体(RANKL)，从而抑制破骨细胞的生成，抑制骨吸收；但是由于大多数组织以及免疫系统中普遍存在RANKL，长期使用狄诺塞麦主要是具有多种毒副作用。(2)促骨形成药物：人工合成的甲状旁腺激素相关肽，特立帕肽，为现今临床推荐的唯一有效的骨形成刺激剂，但是特立帕肽价格昂贵，并且需要每天进行皮下注射给药，严重限制了其临床使用，现作为治疗骨质疏松症的二线药。(3)促骨形成同时抑制骨吸收的双模式药物：雷奈酸锶(SR)是目前唯一能够同时减少骨吸收和促进骨形成的双模式的药物， 刺激新的骨组织的形成的同时减少骨吸收，该药物能够减少全身骨骼的骨折发生率，治疗效果明显，但是有报道，雷奈酸锶能增加非致命性心肌梗死的发生率促使监管机构重新考虑其疗效与风险，其安全有待进一步提高。
药物长期使用的有效性与安全性是目前临床上抗骨质疏松药研究需要解决的主要难题。因此，研究高效低毒同时减少骨吸收和促进骨形成的双模式抗骨质疏松药物具有重大意义。
本申请的研究团队前期从补肾药用植物金雀根中分离得到多种活性优于白藜芦醇、结构特色的天然多羟基酚类白藜芦醇二聚体，作为选择性雌激素受体调节剂，对骨表现出雌激素样作用，增加骨密度，无不良反应；同时构建了四类天然优势骨架白藜芦醇二聚体类化合物样品库共计500个化合物，并对其中具有类药性优势骨架的苗头化合物尽行了系统的先导化合物发现与成药性研究，经实验显示天然产物Isopaucifloral F可作为雌激素受体ERβ的选择性的激动剂，但存在抗骨质疏松药效不显著的缺陷。
基于现有技术的基础与现状，本发明拟提供一类Isopaucifloral F磷酸酯化合物在用于制备抗骨质疏松药物中的用途。该类化合物安全性高、代谢稳定性好、生物利用度高，且通过减少破骨吸收和促进成骨形成的双模式作用机制发挥抗骨质疏松药效。
发明内容
本发明的目的是基于现有技术的基础与现状，提供一类Isopaucifloral F磷酸酯化合物及其在用于制备抗骨质疏松药物中的用途。
本发明基于现有技术的基础，实验显示天然产物Isopaucifloral F可作为雌激素受体ERβ的选择性的激动剂，但存在抗骨质疏松药效不显著的缺陷；其主要原因是分子结构中含有多个酚羟基，易在体内与葡萄糖醛酸结合而排出体外。
本发明对Isopaucifloral F分子结构进行了优化，以改善先导物代谢性质和生物利用度，通过药效团嫁接的药物设计理念，采用抗骨质疏松药物阿仑膦酸分子结构中存在优势磷酸基团，引入到Isopaucifloral F易代谢位点，设计制得新的代谢稳定的磷酸酯目标化合物，经实验证实，制得的该类化合物安全性高、代谢稳定性好、生物利用度高，且通过减少破骨吸收和促进成骨形成的双模式作用机制发挥抗骨质疏松药效。
具体的，本发明如下式(1)结构的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐：
其中：R
1独立地为磷酸酯基团或氢原子，R
2独立地为磷酸酯基团；
当R
1为氢原子时，R
2独立地为
磷酸酯基团；当R
1为
磷酸酯基团时，R
1＝R
2。
本发明提供了上述Isopaucifloral F磷酸酯药物的合成方法，本发明采用前期的合成方法，以3，5-二甲氧基苯甲酸(2)为起始原料，通过金屈偶联反应，Wittig反应以及Nazarov环合合成关键中间体6a与6b，然后在NaH/THF体系下通过亲核取代反应在关键中间体6a与6b的分子结构中引入磷酸二乙酯基团，最后在BBr
3/CH
2Cl
2体系中脱去分子中的甲基，得到目标产物1a和1b。
具体合成路线如反应通式(1)：
Scheme 1：反应通式(1)
作为优选，上述制备方法中，Wittig反应条件(b)所采用的碱为NaHMDS，KHMDS，LiHMDS，LDA，NaH，tBuONa，tBuOK或者n-BuLi。
作为优选，上述制备方法中，Nazarov环合条件(c)所采用的Lewis酸为AlCl
3，BF
3，TiCl
4，SnCl
4，Bi(OTf)
3或者ZnCl
2。
作为优选，上述制备方法中，所述引入磷酸酯基团(d)反应体系中，所采用的碱为NaH，KHMDS，NaHMDS，LiHMDS，t-BuOK或者t-BuONa.
作为优选，上述制备方法中，所述脱去分子中的甲基反应(e)体系中，所采用的试剂为BBr
3或者AlCl
3。
上述反应中，作为优选，Isopaucifloral F与磷酸酯基团片段的比例为1∶2.5(反应步骤e)。
本发明进行了实验，显示所述的化合物具有药理学研究价值，可以治疗骨质疏松，该类化合物代谢稳定性好、生物利用度高以及安全性好，且通过促骨形成与抑制骨吸收双重机制而发挥抗骨质疏松作用。
本发明的实施例中提供了1a和1b的脂水分布系数，血浆稳定性试验，促骨形成能力和抑制骨吸收作用机制数据；以及候选药物1a的人肝微粒体代谢稳定性、大鼠体内药代动力学、急性毒性和斑马鱼骨质疏松模型药效学数据。
本发明还提供了Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐在用于制备抗骨质疏松药物中的用途。进一步制备治疗骨质疏松的药物组合物，其中包括所述的Isopaucifloral F磷酸酯类化合物及其在药学上可接受的盐和药学上可接受的赋形剂。
本发明的Isopaucifloral F磷酸酯消旋体混合物或它们的光学纯异构体及它们在药学上可接受的盐可制成包含安全有效量Isopaucifloral F磷酸酯消旋体混合物，光学纯异构体或它们在药学上可接受的盐及药用载体的各种制剂。
本发明中，“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。安全有效量根据治疗对象的年龄、病情、疗程等来确定。
本发明中，“药用载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。
本发明中，“相容性”指组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。
本发明中，药学上可以接受的载体部分例子有糖(如葡萄糖、蔗糖、乳糖等)，淀粉(如玉米淀粉、马铃薯淀粉等)，纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素 乙酸酯等)，明胶，滑石，固体润滑剂(如硬脂酸、硬脂酸镁)，硫酸钙，植物油(如豆油、芝麻油、花生油、橄榄油等)，多元醇(如丙二醇、甘油、甘露醇、山梨醇等)，乳化剂(如
)、润湿剂(如十二烷基硫酸钠)，着色剂，调味剂，稳定剂，抗氧化剂，防腐剂，无热原水等。
3、与现有技术相比，本发明的有益效果体现在：
目前临床上疗效最好的抗骨质疏松药物是促骨形成同时抑制骨吸收的双模式药物，其仅有的代表药物雷奈酸锶存在增加非致命性心肌梗死的发生率的风险，其安全有待进一步提高。本发明的Isopaucifloral F磷酸酯化合物及其盐安全性高，通过促骨形成与抑制骨吸收双重机制而发挥抗骨质疏松作用，该类化合物代谢稳定性好、生物利用度高，有希望满足临床的迫切需求。
图1： 40倍倒置显微镜下观测到的茜素红与钙结节形成的单晶
图2：各实验组TRAP阳性多核破骨细胞状态(X200)
图3：S-1a斑马鱼骨质疏松模型研究(control为空白对照组，Prednisolone为模型组，Alendronate为阳性药物对照组，ns，not significant；***P＜0.0001，**P＜0.001)
下面结合实施例对本发明作进一步阐述，但这些实施例绝不受本发明的任何限制。
实施例1： 3，3′，5，5′-四甲氧基二苯基乙二酮(3)的合成
在250mL三颈瓶中依次加入23.34g(0.18mol)萘以及1.26g金屈锂带的小碎片。因为金屈锂化学性质的活泼，在空气中其表面易生成氧化物，所以剪切金屈锂带的时候动作要迅速，减少其与空气接触。在氮气保护的条件下，加入150mL的无水THF使萘充分溶解。剧烈搅拌。1-3分钟后，反应液由无色经绿色变至黑色。室温搅拌6～8h，锂萘(LiN)试剂制成待用。另称取15.00g(82.35mmol)3，5-二甲氧基苯甲酸34置于500mL三颈瓶中，氮气保护。加入300mL无水THF使3，5-二甲氧基苯甲酸充分溶解。在冰浴的条件下，用针筒将 事前制备好的LiN试剂吸出，加入到溶有3，5-二甲氧基苯甲酸的THF溶液体系中。反应液经黄色变至深褐。加水终止反应。旋去THF，用溶剂CH
2Cl
2萃取三次，二氯甲烷层经饱和NaCl溶液洗涤，无水Na
2SO
4干燥，浓缩，得到的黄色粗产品。该黄色粗品先经过正相柱的初步分离，用正己烷洗去体系中未反应完的萘后，用溶剂二氯甲烷将剩余黄色固体回收。旋干后，用石油醚∶乙酸乙酯＝2∶1的体系重结晶，得黄绿色固体粉末，即为3，3′，5，5′-四甲氧基二苯基乙二酮(3)的纯品，产率为47.6％。
1H-NMR(CDCl
3，400MHz)δppm：3.83(s，12H)，6.74(s，2H)，7.07(d，J＝1.7Hz，4H)；。
实施例2： 1，2-二-(3，5-二甲氧基苯基)-3-(4-甲氧基苯基)-2-丙烯-1-酮(5a)的合成
于50mL两口瓶中，依次加入500mg(1.51mmol)的3，3′，5，5′-四甲氧基二苯基乙二酮(3)与(4-甲氧基苄基)三苯基氯化膦盐700mg(4a，1.67mmol)通氮气保护，加入20mL无水处理的甲苯使之溶解，匀速滴加KHMDS的THF溶液1.67mL(1.0M，1.67mmol)。TLC显示二酮(3)完全反应，即可加水淬灭反应。乙酸乙酯萃取，有机层经饱和NaCl溶液除水，无水Na
2SO
4干燥，浓缩经硅胶柱层析纯化(正己烷∶丙酮＝10∶1)，得黄色油状物5a。产率为94.4％。
1H-NMR(CDCl
3，400MHz)δppm：3.72-3.77(m，15H)，6.38-6.39(m，1H)，6.56-6.59(m，3H)，6.72-6.75(m，2H)，7.09-7.23(m，5H)。
实施例3： 4，6-二甲氧基-3-(4-羟基苯基)-2-(3，5-二甲氧基苯基)-2，3-二氢茚-1-酮(6a)的合成
称取510.2mg的化合物5a(1.11mmol)置于100mL单口瓶中，用无水甲苯充分溶解后，边搅拌边缓慢加入149mg固体AlCl
3(1.2mmol)，反应体系会渐变为橙红色，室温过夜，TLC监测至反应完全，并且产物点单一。反应体系中加入少量水来终止反应，乙酸乙酯EA萃取，无水Na
2SO
4干燥，浓缩经硅胶柱层析纯化(正己烷∶丙酮＝8∶1)，得泡沫状黄色固体6a。 产率为89.6％。
1H NMR(400MHz，CDCl
3)δppm：6.96(1H，s)，6.72(1H，s)，6.44(4H，s)，6.17(1H，s)，5.91(2H，d，J＝2.30Hz)，4.90(1H，d，J＝2.75Hz)，4.26(1H，d，J＝2.75Hz)，3.88(3H，s)，3.70(3H，s)，3.57(6H，s)。ESI-MS m/z：421.2[M+H]
+；C
25H
24O
6：HRMS calcd.421.1682[M+H]
+，found 421.1335。
实施例4： 4，6-二甲氧基-3-(4-甲氧基苯基)-2-(3，5-二甲氧基苯基)-2，3-二氢茚-1-酮(6b)的合成
称取1.24g的化合物5b(2.85mmol)于100mL单口瓶中，加入40mL二氯甲烷使其充分溶解。室温下慢慢滴加3.8mL的BF
3·Et
2O(14.26mmol)，搅拌过夜，TLC显示原料基本消失，且产物点单一，加少量的水终止反应。用CH
2Cl
2萃取，饱和NaHCO
3水溶液洗，水洗至中性，饱和NaCl溶液除水，无水Na
2SO
4干燥，浓缩溶剂得到的粗品为黄白色固体。正相柱分离，石油醚∶丙酮＝8∶1。产物6b为白色固体，产率：94.4％。
1H NMR(400MHz，CDCl
3)δppm：6.96-6.23(m，9H)，4.52(1H，d，J＝2.60Hz)，3.87(3H，s)，3.78(3H，s)，3.73(6H，s).ESI-MS m/z：435.2[M+H]
+；C
26H
26O
6：HRMS calcd.435.1721[M+H]
+，found 435.1713.。
实施例5：化合物7a的合成
取25mL的两口瓶烘干冷却，称取200mg化合物6a(0.48mmol)以及48mg钠氢(1.19mmol)置于两口瓶中，氮气保护。由于反应体系对无水条件要求严格，无水THF需在使用前再用NaH干燥后加入瓶中，快速搅拌下加入氯磷酸二乙酯(1.90mmol)，回流过夜，反应 体系由浅黄色变为粉红色。TLC监测反应，发现化合物6a反应不完全，但是延长反应时间或补加氯磷酸二乙酯也不能使产率有所提高。终止反应。过滤除去无机盐沉淀，用少量乙酸乙酯洗涤，反应液旋干过正相柱，分离得到化合物7a为无色油状物。产率：21.5％。
1H-NMR(400MHz，CDCl
3)δppm：7.09(2H，d，J＝8.0Hz)，6.98(2H，d，J＝8.0Hz)，6.91(1H，d，J＝2.0Hz)，6.69(2H，d，J＝2.0Hz)，6.28(1H，d，J＝2.0Hz)，6.26(1H，t，J＝2.0Hz，J＝2.0Hz)，4.86(1H，s)，4.16-4.05(8H，m)，3.83(3H，s)，3.70(6H，s)，3.59(3H，s)，1.29-1.20(12H，m)。
实施例6：化合物7b的合成
取25mL的两口瓶烘干冷却，称取127mg化合物6b(0.29mmol)以及24mg钠氢(0.58mmol)置于两口瓶中，氮气保护，无水THF溶解，快速搅拌下加入氯磷酸二乙酯(0.44mmol)，反应体系回流过夜，溶液由浅黄色变为粉红色。TLC监测反应，分离得到纯的化合物7b为无色油状物。产率为：76.0％。
1H-NMR(400MHz，CDCl
3)δppm：7.05(2H，d，J＝8.0Hz)，6.92(1H，d，J＝2.0Hz)，6.73(2H，d，J＝2.0Hz)，6.68(2H，d，J＝8.0Hz)，6.29(1H，d，J＝2.0Hz)，6.27(1H，t，J＝2.0Hz)，4.85(1H，d)，4.25-4.04(4H，m)，3.85(3H，s)，3.71(6H，s)，3.71(3H，s)，3.61(3H，s)，1.37-1.21(6H，m)。
实施例7：目标化合物1a与1b的合成
称取化合物7a或7b100mg，溶于20mL无水二氯甲烷中，在冰浴下加入10～15倍当量 三溴化硼的二氯甲烷溶液(1mol/L)，然后转移至室温，反应8h，TLC检测至反应完全。通过反相色谱柱分离纯化，得到相应的脱甲基保护的目标化合物1a和1b，产率分别为48.0％和54.0％。1a：1H-NMR(400MHz，DMSO-d6)ppm：9.30(1H，s)，9.15(3H，s)，8.99(1H，s)，6.81(2H，d，J＝8.0Hz)，6.48(2H，d，J＝8.0Hz)，6.47(1H，d，J＝2.0Hz)，6.36(2H，d，J＝2.0Hz)，6.06(1H，d，J＝2.0Hz)，6.05(1H，t，J＝2.0Hz)，4.84(1H，s)，4.11-3.93(8H，m)，1.25-1.01(12H，m).ESI-MS m/z：637.15[M+H]
+；C
29H
34O
12P
2：HRMS calcd.637.1596[M+H]
+，Found：637.1622[M+H]
+。1b：
1H-NMR(400MHz，DMSO-d
6)δppm：9.19(1H，s)，9.08(2H，s)，9.02(1H，s)，8.97(1H，s)，6.78(2H，d，J＝8.0Hz)，6.46(2H，d，J＝8.0Hz)，6.45(1H，d，J＝2.0Hz)，6.33(1H，d，J＝2.0Hz)，6.04(1H，d，J＝2.0Hz)，6.02(1H，d，J＝2.0Hz)，4.67(1H，d)，4.13-3.91(4H，m)，1.21(3H，t)，1.11(3H，t).ESI-MS m/z：501.12[M+H]
+；C
25H
25O
9P：HRMS calcd.501.1309[M+H]
+，Found：501.1330[M+H]
+。
实施例8：Isopaucifloral F-PP(1a)和Isopaucifloral F-P(1b)脂水分布系数测定与稳定性试验
①脂水分配系数的测定：首先是制定标准曲线，分别称取一定量的目标化合物1a与1b用色谱级的乙腈配制成溶液。分别用HPLC进样分析。HPLC条件为：流动相：5％～95％的乙腈/水，梯度洗脱，流速为0.6mL/min，进样量为10μL，检验波长为254/280nm，运行20min，记录保留时间与峰面积，以浓度与峰面积回归得到标准曲线。
再分别取10mL水与10mL正辛醇于25mL茄形瓶中，搅拌24h至两相相互饱和。再将混合溶液转移到分液漏斗中，静置，分液。取上层(饱和正辛醇层)1mL，加入约2mg待测化合物，涡旋5min至充分溶解，另取1mL饱和水相与该溶有化合物的饱和正丁醇相混合，涡旋5min，37℃水浴振荡24h后，3500rpm离心15min，分别取上层正辛醇层与下层水层用HPLC条件进样分析。HPLC条件同制定标准曲线时条件相同。记录上层正辛醇与下层水层的峰面积，带入标准曲线中，得出化合物的浓度。上层测得的浓度为油相(正辛醇相)中的药物浓度Co，下层测得的浓度即为水相中的药物浓度Cw，油水分配系数log P的计算方法如下：log P＝logCo/Cw。
Isopaucifloral F-PP(1a)的logP为2.60；化合物Isopaucifloral F-P(1b)的logP为2.25，两个化合物的logP均在1～3之间，具有一定的成药性，与Isopaucifloral F比较(其logP为1.34)，目标化合物的logP值变大，脂溶性显著提高。
②血浆稳定性的测定：取适量目标化合物的DMSO储备液溶于4mL血浆中，使其浓度 为200μg/mL，DMSO含量不超过0.1％。水浴37℃孵育，于0，5，15，30min以及1，2，4，6，10，24，48h，取200μL，加入3倍体积色谱级乙腈沉淀蛋白。漩涡震荡5min，离心后取上层清液200μL，HPLC进样分析，液相条件与之前相同。
在血浆中孵育48h后，化合物Isopaucifloral F-PP(1a)母药浓度占初始浓度的94.17％；化合物Isopaucifloral F-P(1b)母药浓度占初始浓度的93.74％，可见目标化合物在血浆中具有较好的稳定性。
③细胞液稳定性的测定：细胞内稳定性实验是将处于生长对数期的MC3T3-E1 subclone 14细胞，约200万个，消化收集细胞于1mL离心管中，加入200μL细胞裂解液将细胞裂解。然后加入800μL PBS溶液，使得总体积为1mL。同样，在水浴37℃孵育，于0，5，15，30，45，60min取100μL，加入3倍体积色谱级乙腈沉淀蛋白。漩涡震荡5min，离心后取上层清液200μL，HPLC进样分析，液相条件与之前相同。
目标化合物Isopaucifloral F-PP(1a)与Isopaucifloral F-P(1b)在细胞裂解液体系中60min内基本不发生解离，可判断化合物在细胞内应该是以原药形式存在，没有发生磷酸二乙酯基团的水解。
通过对两个目标化合物的稳定性考察发现，发现它们在血浆中稳定，在细胞内不发生解离，以原药形式存在。化合物的logP在1～3之间，化合物Isopaucifloral F-PP(1a)的LogP为2.60；化合物Isopaucifloral F-P(1b)的LogP为2.25，具有一定的成药性。
实施例9.Isopaucifloral F-PP(1a)和Isopaucifloral F-P(1b)在体外的促骨形成能力研究
①化合物对小鼠成骨细胞前体细胞MC3T3-E1 Subclone 14促增殖作用(见表1)：采用MTT法测定目标化合物1a与1b对MC3T3-E1 subclone14的增殖效应。以金雀异黄酮与雌二醇作为阳性对照组。MC3T3-E1 subclone14用含10％FBS的α-MEM培养基培养在37℃，5％CO
2恒温培养箱中。将处于对数生长期，状态良好的MC3T3-E1 subclone14细胞，加入适量胰酶(购自GIBCO)消化，收集细胞。并用含10％的FBS的α-MEM培养基重悬细胞，并以2000个细胞/孔的量接种于96孔板上。待其贴壁后，培养基换成含不同浓度的化合物的10％FBS的α-MEM，每孔200μL。以加入含0.2％DMSO的培养液的细胞中作为培养基的阴性对照组。将培养基加入未接种细胞的孔中作为空白对照组。每个浓度设6个复孔。每2天更换一次培养基，待给药第6天时，在上述96孔板中，加入20μL的5mg/mL的MTT/PBS溶液，继续孵育4h。到时间点后，小心吸去液体，每孔加入150μL的DMSO，使活细胞与MTT形成的紫色晶体甲瓒完全溶解。用酶标仪测定490nm处吸光值(OD)。按以下公式计算增殖 率：
增殖率＝[(给药组平均OD值-空白组平均OD值)/(培养液对照组平均OD值-空白组平均OD值)]
结果显示：Isopaucifloral F-PP(1a)，和Isopaucifloral F-P(1b)以及Isopaucifloral F对MC3T3-E1 Subclone 14促增殖作用明显高于阳性对照药金雀异黄酮。增殖率为分别为114％，148％和124％。
表1：目标化合物对MC3T3-E1 Subclone 14的增殖活性
②化合物对小鼠成骨细胞前体细胞MC3T3-E1 Subclone 14促分化作用研究(见表2)：MC3T3-E1 subclone14细胞用胰酶消化收集后以4×10
4/孔接入6孔板中，12h贴壁后，培养基换成含有不同浓度的1a，1b，Isopaucifloral F以及阳性对照药金雀异黄酮GEN的分化培养基。化合物给药浓度分别为：10
-4～10
-9M；阳性对照药组金雀异黄酮GEN的给药物浓度是根据文献报道的它们具有最高促骨形成能力的浓度，为10
-6M。到给药第6天，每孔经PBS洗2遍后，加入150uL的Western及IP细胞裂解液(不含蛋白抑制剂，购于碧云天)裂解细胞。该操作在冰上进行。取50uL细胞裂解液用PBS稀释至250uL。再取50uL稀释液转移到96孔板，用于ALP酶活性测试。通过酶标仪在405nm处测定96孔板中的每孔吸光度。
结果显示Isopaucifloral F-PP(1a)，和Isopaucifloral F-P(1b)以及Isopaucifloral F的ALP酶活都高于对照组，分别为对照组的2.36倍(酶活力提高了136％)，1.20倍(酶活力提高了19.5％)和1.14倍(酶活力提高了13.5％)。
表2：各实验组ALP酶活性相对值：
③目标化合物成骨细胞钙结节的形成研究(详见附图1和表3)：细胞消化后以4×10
4/孔接入6孔板中，12h贴壁后，培养基换成含有不同浓度的1a，1b，Isopaucifloral F以及阳性对照药金雀异黄酮GEN的分化培养基。化合物给药浓度分别为：10
-4～10
-9M；金雀异黄酮浓度为：10
-6M。到给药第15天，吸出培养基后，每孔经PBS洗2遍后，用95％的乙醇固定细胞10min，再用双蒸水洗2遍。每孔加入0.1％茜素红-Tris.HCl溶液(PH＝8.3)2mL，在37℃下孵育30min后，用双蒸水冲洗数遍，干燥，封片。进一步在40倍镜下拍照观察钙结节的形成。之后每孔加入10％氯化十六烷基吡啶(cetylpyridinium chloride)溶液1mL，共孵育30min，使得茜素红染色释放，将溶液转移至96孔板，每孔200uL，用酶标仪在562nm处测得吸光度进行钙结节的定量分析。
与对照组组相比，目标化合物均对钙结节的形成具有促进作用。Isopaucifloral F-PP(1a)在10
-7M具有最大的钙结节形成的量，其相对值为1.446。Isopaucifloral F-P(1b)在10
-6M具有最大的钙结节形成的量，其相对值为1.182。
表3：各实验组钙结节数量的相对值(Control＝1)
实施例10.Isopaucifloral F-PP(1a)和Isopaucifloral F-P(1b)抑制骨吸收药效研究
将上述小鼠单核巨噬细胞RAW264.7接种于96孔板中，待其贴壁后，培养基更换为浓度 为10
-4M，10
-6M，10
-8M的1a与1b，先导化合物Isopacifloral F组以及阳性对照药雷洛昔芬(Raloxifene)的诱导培养基，以不给药组为阴性对照组(Control组)。培养5天后，细胞经抗酒石酸酸性磷酸酶TRAP孵育液染色，甘油明胶封片，光镜观察，计数TRAP染色阳性多核细胞(如图2所示)，给药物对TRAP阳性多核细胞数的抑制率为：(n
阴性对照组TRAP阳性多核破骨细胞-n
给药物组TRAP阳性多核破骨细胞)/n
阴性对照组TRAP阳性多核破骨细胞×100％。
结果显示(如表4所示)，磷酸酯化合物Isopaucifloral F-PP(1a)与Isopaucifloral F-P(1b)具有优于雷洛昔芬的抑制破骨细胞形成能力，磷酸酯基团的引入使其抑制破骨细胞形成能力大大提高。他们对破骨细胞的抑制活性相当，明显高于Isopacifloral F，分别为62.4％和65％，为Isopacifloral F的2.2倍和2.3倍。
表4：各实验组对破骨细胞的抑制率(Control组的破骨细胞数为117个)
注：Control组的破骨细胞数为117个；每组做8个复孔。
实施例11.Isopaucifloral F-PP(1a)和Isopaucifloral F的人肝微粒体代谢稳定性研究
实验方法：孵育体系包含人肝微粒体、辅助因子、PBS，37℃预孵3min，加入底物启动反应。在反应开始0、1、5、10、15、20、30、60min取样，加入适当终止物终止反应。样品处理(n＝3)：各加合适内标，涡旋后高速离心，取上清液，采用HPLC-MS/MS对底物进行检测。把0min时间点峰面积作为100％。其他时间点的峰面积转换为百分剩余量，各时间点的百分剩余量的自然对数对孵育时间作图，直线回归求算出斜率(-k)，然后按固有清除率(CL
int)＝(k×孵育液体积)/肝微粒体质量，计算出CL
int(mL·min
-1·mg
-1)。T
1/2由Graphpad Prism 5软件计算求得(如表5所示)。
表5：Isopaucifloral F-PP(1a)和Isopaucifloral F在人肝微粒体代谢稳定性数据
从体外人肝微粒体代谢(见表5)结果显示：(1)R-1a和S-1a相较于Isopaucifloral F的代谢稳定性更好，其清除速率CLint(liver)分别为38.4和8.6mL/min/kg；Isopaucifloral F代谢最快，其清除率为123.4mL/min/kg。(2)S-1a和R-1a的代谢稳定性分别是Isopaucifloral F的3.3倍和14.5倍，(3)S-1a的代谢稳定性是R-1a的4.5倍，结果表明S-1a更适于作为候选药物进行研发。
实施例12.Isopaucifloral F-PP(1a)大鼠体内药代动力学研究
选取S-1a进行SD大鼠体内药代动力学研究，分别静脉给药剂量为5mg/kg，口服给药剂量为20mg/kg，于5min，8min，15min，30min，1h，2h，3h，4h，6h，8h，10h，24h各时间点通过眼眶采集血样，离心处理后，采用LC-MS/MS测定各时间点血浆中药物的浓度，并通过DAS软件(2.0版)采用非房室模型的方法估算其体内初步药代动力学参数，实验结果如下：
表6：光学纯异构体S-1a的SD大鼠体内PK数据
表6结果显示：(1)S-1a的口服生物利用度为41.9％适合做口服药物(＞30％)进行药物研发。
实施例13.Isopaucifloral F-PP(1a)和Isopaucifloral F急性毒性研究
为了进一步考察优选化合物的毒性，我们优选Isopaucifloral F-PP(1a)和其前体Isopaucifloral F进行小鼠体内急性毒性实验。将体重为20±2g健康成年的昆明小鼠每组10只，雌雄各半，随机分成5组，通过腹腔注射单次给药，给药容积为0.2mL/10g。对小鼠的行为状态及死亡状况连续观察记录14天。实验数据通过SPSS软件(IBM SPSS Statistics 19)采用Bliss方法计算，结果表明两个化合物的急性毒性LD50＞150mg/kg，治疗指数TI＞有 毒剂量(150mg)/有效剂量(10ug)＝15000，表明该两个化合物具有较好的安全性。
实施例14.Isopaucifloral F-PP(1a)斑马鱼骨质疏松模型药效学研究
在具有良好药代动力学和较好安全性的基础上，选取S-1a以及临床一线用药阿伦磷酸钠(Alendronate)给予斑马鱼模型幼鱼培养至受精后10d(10dpf)，麻醉处死后用4％多聚甲醛固定后，采用1％KOH配制的含30％H
2O
2的漂白剂将幼鱼漂白至眼部色素清除后，采用茜素红对斑马鱼幼鱼头部骨骼染色，再用梯度比分别为3∶1的1％KOH和甘油的混合溶液将幼鱼透明化，最后将幼鱼保存于纯甘油中。使用显微镜拍摄幼鱼头部腹面照，然后采用图像分析软件Image-Pro Plus 6.0将幼鱼头部骨骼染色面积和累积光密度进行分析计算，以反映骨矿化量(如图3所示)；结果表明：(1)S-1a能够浓度依赖(5ug/mL，10ug/mL，25ug/mL)的恢复斑马鱼的骨质疏松；(2)S-1a浓度为(25ug/mL)能够明显恢复骨质疏松程度，和对照药物阿伦磷酸钠(100ug/mL)药效相当，S-1a可以考虑作为临床候选药物。
Claims (9)
- 如式(1)的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐：
其中，R 1独立地为磷酸酯基团或氢原子；R 2独立地为磷酸酯基团。 - 如权利要求1所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，所述的R 1＝H，R 2为磷酸酯基团。
- 如权利要求1所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，所述的R 1＝R 2，为磷酸酯基团。
- 如权利要求1-3任一项所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，所述的磷酸酯基团为
- 如权利要求1所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，所述的化合物其结构选自：
- 如权利要求1-5任一项所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，其为外消旋混合物。
- 如权利要求1-5任一项所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐，其特征在于，其为C3位构型是R或者S构型的光学纯异构体。
- 如权利要求1-7任一项所述的Isopaucifloral F磷酸酯类化合物及其药学上可接受的盐在用于制备抗骨质疏松药物中的用途。
- 一种治疗骨质疏松的药物组合物，其包括权利要求1-7任一项所述的Isopaucifloral F磷酸酯类化合物及其在药学上可接受的盐和药学上可接受的赋形剂。
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