Osteoblast-targeted 11ss-hsd1 inhibitor conjugates, methods of making and using the same

Abstract

The application relates to the field of medicine, and particularly discloses an osteogenic-targeting molecule modified 11beta-HSD1 inhibitor compound, and the structure of the compound is formula (I). The compound can target and inhibit 11beta-HSD1 in osteoblasts, thereby reducing side effects caused by inhibition of 11beta-HSD1, and can effectively treat metabolic diseases such as obesity and diabetes, and can also protect bone formation inhibition, low bone mass and bone structure damage accompanying metabolic diseases.

Description

Technical Field

[0001] This invention relates to the pharmaceutical field, specifically to an osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate, its preparation method, and its application. Background Technology

[0002] 11β-HSD1 is an enzyme that converts biologically inactive glucocorticoids (cortisone in humans and 7-hydroxy11-dehydrocorticosterone in rodents) into active glucocorticoids (cortisol), playing a crucial role in the regulation of endogenous glucocorticoids in the human body. Experiments have shown that animals with the 11β-HSD1 gene knocked out can avoid obesity and metabolic disorders induced by a high-fat diet, while 11β-HSD1 transgenic animals are prone to obesity. Therefore, 11β-HSD1 has long been considered a potential target for treating obesity. Major pharmaceutical companies have developed their own 11β-HSD1 inhibitors for treating obesity; however, none have yet achieved success in clinical trials. There are two main reasons for this: firstly, systemic targeted inhibition of 11β-HSD1 causes severe side effects; and secondly, these 11β-HSD1 inhibitors have not identified the target organs that 11β-HSD1 induces obesity, resulting in poor therapeutic effects after administration.

[0003] AZD8329 is an 11β-HSD1 inhibitor developed by AstraZeneca for the treatment of obesity and diabetes. AZD8329 exhibits significant inhibitory effects on 11β-HSD1 in humans (IC50 = 9 nM), rats (IC50 = 86 nM), dogs (IC50 = 8 nM), and mice (IC50 = 6.1 μM). Therefore, AZD8329 rapidly entered Phase I clinical trials in 2010. However, due to the rapid drug resistance resulting from continuous injection into adipose tissue in humans and rats, and the potential for systemic adverse reactions and reduced therapeutic efficacy with oral administration, the Phase I clinical trial of AZD8329 was discontinued in 2011 (NCT01207089).

[0004] (DSS)6 is a stable osteogenic targeting molecule with a structure of six aspartic-serine-serine polypeptide repeats. It has been shown to form a delivery system with cationic liposomes and specifically deliver drugs to the osteogenic surface. However, this targeting molecule binds to drugs through non-chemical bonds, and the targeting molecule cannot stably bind to drugs.

[0005] CN110669106A discloses a chalcone derivative modified with an osteogenic targeting peptide and its preparation method, wherein the carboxyl group in the chalcone derivative is condensed with the amino group in (DSS)6 to form an amide bond. However, since (DSS)6 itself has multiple carboxyl and hydroxyl groups, this condensation method will generate multiple byproducts, resulting in a low yield of the target conjugate.

[0006] There are currently no publicly reported conjugates of 11β-HSD1 inhibitors that target osteogenic molecules. There is an urgent need to develop new 11β-HSD1 inhibitors that can inhibit the side effects caused by 11β-HSD1 and achieve therapeutic effects for metabolic syndromes such as obesity and diabetes. Summary of the Invention

[0007] In view of the current state of the technology, the present invention provides an osteogenic targeted molecule modified 11β-HSD1 inhibitor conjugate, its preparation method and application. The present invention can reduce the side effects caused by inhibiting 11β-HSD1, while treating metabolic diseases such as obesity and diabetes, and can also protect against bone formation inhibition, low bone mass and bone structure destruction associated with metabolic diseases.

[0008] The structure of the osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate described in this invention is as follows: (I)

[0009]

[0010] This invention also provides a method for preparing the aforementioned osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate, the method comprising:

[0011] (1) In a solvent, BCN-OH and AZD8329 were condensed under the action of a condensing agent to prepare an intermediate containing an alkynyl side chain;

[0012] (2) The intermediate of step (1) reacts with N3(CH2)5OC-(DSS)6 containing azide side chains in a mixed solvent at room temperature;

[0013] (3) After the reaction was completed, the mixture was freeze-dried and the residue was purified by preparative liquid phase to obtain (DSS)6-AZD8329 conjugate.

[0014] In the preparation method of the present invention, as one of the embodiments, the solvent in step (1) is selected from dichloromethane, 1,2-dichloroethane, tetrahydrofuran, ethyl acetate, toluene, acetonitrile or chloroform; preferably, dichloromethane is used as the solvent.

[0015] In the preparation method of the present invention, as one of the embodiments, the condensing agent in step (1) is selected from DCC / DMAP, EDC / DMAP, HATU / DIPEA, TBTU / TEA, or HBTU / DIPEA; preferably, condensation is carried out under the action of DCC / DMAP.

[0016] In the preparation method of the present invention, as one of the embodiments, step (1) further includes: the molar ratio of BCN-OH to AZD8329 is 0.8:1 to 2:1, the amount of DCC is 1.2 equiv to 2 equiv of AZD8329, the amount of DMAP is 0.05 equiv to 0.2 equiv of AZD8329, and the reaction is carried out overnight at room temperature.

[0017] In the preparation method of the present invention, as one of the embodiments, step (2) further includes: the molar ratio of the intermediate to N3(CH2)5OC-(DSS)6 is 1:1 to 2:1, preferably 1:1 to 1.2:1, and the reaction is carried out at room temperature for 24 hours.

[0018] In the preparation method of the present invention, as one embodiment, the mixed solvent in step (2) is selected from tetrahydrofuran / H2O, DMSO / H2O, or acetonitrile / H2O, preferably tetrahydrofuran / H2O. As one embodiment, the volume ratio of tetrahydrofuran to water in the mixed solvent is 0.1:1 to 1:0.1, preferably 0.5:1 to 1:0.5.

[0019] In the preparation method of the present invention, as one of the implementation schemes, step (3) further includes: purifying the conjugate using a preparative chromatographic column, with the following preparation conditions: C-18 reversed-phase chromatographic column, mobile phase of 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile (B), and gradient elution to obtain the conjugate, with the gradient elution program being: gradient of B 20-80%, separation time 60 min.

[0020] In the preparation method of the present invention, N3(CH2)5OC-(DSS)6 can be synthesized by conventional methods in the art or purchased from the market.

[0021] In the preparation method of this invention, AZD8329 can be purchased commercially or prepared using known methods. As one embodiment, the chemical synthesis of AZD8329 includes: using LiHMDS as a base and tetrahydrofuran as a solvent, pinacolone and 1-adamantane isocyanate are reacted at -78°C to construct intermediate 1; using 1,4-dioxane as a solvent, intermediate 1 is reacted with N,N-dimethylformamide dimethyl acetal at 100°C to construct intermediate 2; using ethanol as a solvent, intermediate 2 is reacted with methyl p-hydrazinobenzoate at 80°C under acetic acid catalysis to construct intermediate 3; using 1,4-dioxane as a solvent, intermediate 3 undergoes ester hydrolysis under the action of sodium hydroxide, and finally, after acidification with hydrochloric acid, AZD8329 is obtained as a yellowish-white solid.

[0022] The present invention also provides a pharmaceutical composition comprising the osteogenic-targeting modified 11β-HSD1 inhibitor conjugate described above or prepared by the methods described above.

[0023] The present invention also provides the use of the osteogenic-targeted modified 11β-HSD1 inhibitor prepared by the aforementioned method, or the aforementioned pharmaceutical composition, in the preparation of a medicament for treating metabolic diseases related to 11β-HSD1.

[0024] In one embodiment of the present invention, the metabolic diseases associated with 11β-HSD1 include obesity, diabetes, dyslipidemia, hypertension, or Cushing's syndrome.

[0025] This invention also provides the use of the aforementioned osteogenic-targeted modified 11β-HSD1 inhibitor prepared by the aforementioned method, or the aforementioned pharmaceutical composition, in the preparation of a medicament for treating bone formation inhibition, low bone mass, bone structure destruction, and bone loss. As one embodiment, the bone loss is bone loss caused by a high-fat diet or long-term use of glucocorticoids.

[0026] The present invention also provides the use of the osteogenic-targeted modified 11β-HSD1 inhibitor prepared by the aforementioned method, or the aforementioned pharmaceutical composition, in the preparation of a drug that improves glucose intolerance, inhibits fat accumulation and adipocyte hypertrophy, lowers blood lipids, lowers blood pressure, promotes fat metabolism, and regulates glucocorticoid disorders.

[0027] Current research has shown that mice with systemic 11β-HSD1 knockout do not become obese after high-fat diet induction. However, mice with specific 11β-HSD1 knockout in the adipose tissue, a major energy metabolism organ, still become obese after high-fat diet induction. Similarly, mice with specific overexpression of 11β-HSD2 (which converts activated glucocorticoids to inactive ones, the opposite of 11β-HSD1) in the liver, another major energy metabolism organ, also cannot resist obesity after high-fat diet induction. However, mice with specific overexpression of 11β-HSD2 in bone are resistant to high-fat diet-induced obesity.

[0028] This invention conjugates the osteogenic targeting molecule (DSS)6 with the 11β-HSD1 inhibitor AZD8329, providing an osteoblast-targeting 11β-HSD1 inhibitor conjugate and its preparation method for treating obesity and diabetes. In a high-fat diet-induced mouse model, (DSS)6-AZD8329 effectively inhibited weight gain induced by a high-fat diet, improved glucose intolerance induced by a high-fat diet, and inhibited fat accumulation and adipocyte hypertrophy. Furthermore, (DSS)6-AZD8329 significantly protected against bone formation inhibition, low bone mass, and bone structure destruction induced by a high-fat diet in young mice. In experiments, the therapeutic effect of (DSS)6-AZD8329 was significantly better than that of AZD8329.

[0029] AZD8329, due to its lack of targeting, causes systemic adverse reactions and leads to poor efficacy. The osteogenic-targeting 11β-HSD1 inhibitor (DSS) 6-AZD8329 conjugate of this invention can target and inhibit 11β-HSD1 within osteoblasts, thereby reducing the side effects of 11β-HSD1 inhibition and achieving therapeutic effects for metabolic diseases such as obesity and diabetes. Furthermore, it can protect against bone formation inhibition, low bone mass, and bone structure destruction associated with metabolic diseases. Attached Figure Description

[0030] Figure 1 High-performance liquid chromatography (HPLC) spectrum of the coupling compound (DSS) 6-AZD8329 in Example 2;

[0031] Figure 2 High-resolution mass spectrometry of the conjugate (DSS) 6-AZD8329 in Example 2;

[0032] Figure 3 Distribution and fluorescence intensity statistics of Cy5 fluorescently labeled (DSS)6-AZD8329 in mouse tissues in Experiment Example 1;

[0033] Figure 4 The fluorescence distribution of Cy5-labeled (DSS)6-AZD8329 in bone tissue in Experiment Example 1;

[0034] Figure 5 The dose-response relationship of (DSS)6-AZD8329 in inhibiting 11β-HSD1 activity in bone in Experiment Example 2;

[0035] Figure 6 : Changes in mouse body weight during (left) and after (right) administration in Experiment Example 3;

[0036] Figure 7 Results of oral glucose tolerance test in mice after administration (25 weeks) in Experiment Example 3;

[0037] Figure 8 : The total weight of subcutaneous and gonadal fat in mice after drug administration (25 weeks) in Experiment Example 3 (left) and optical image of fat (right);

[0038] Figure 9 In Experiment 3, after administration of (DSS) 6-AZD8329 for 25 weeks during a high-fat diet induction, the size of mouse adipocytes was observed.

[0039] Figure 10 In Experiment 3, after administration of (DSS) 6-AZD8329 for 25 weeks during a high-fat diet induction, the parameters of cancellous bone in the femur of mice were as follows:

[0040] Figure 11 In Experiment 3, after administration of DSS 6-AZD8329 (25 weeks) during a high-fat diet induction, the cortical bone parameters of mice were as follows:

[0041] Figure 12 Mass spectrum of the structure characterization of the copper-catalyzed conjugate in Example 4. Detailed Implementation

[0042] The following examples and experimental cases are used to further illustrate the present invention, but do not limit the scope of the present invention in any way.

[0043] Example 1: Chemical Synthesis of Compound AZD8329

[0044] The chemical synthesis of compound AZD8329 followed the method reported by Scott's group (J Med Chem, 55:10136-47, as shown below): Using LiHMDS as a base and tetrahydrofuran as a solvent, pinacolone and 1-adamantane isocyanate were reacted at -78 °C to construct intermediate 1. Using 1,4-dioxane as a solvent, intermediate 1 was reacted with N,N-dimethylformamide dimethyl acetal at 100 °C to construct intermediate 2. Using ethanol as a solvent, intermediate 2 was reacted with methyl p-hydrazinobenzoate at 80 °C under acetic acid catalysis to construct intermediate 3. Using 1,4-dioxane as a solvent, intermediate 3 underwent ester hydrolysis in the presence of sodium hydroxide, and finally, after acidification with hydrochloric acid, AZD8329 was obtained as a yellowish-white solid. The final yield was 50%, and the H- and C-spectral data were consistent with those reported in the literature.

[0045]

[0046] Chemical synthetic route of compound AZD8329

[0047] Example 2: Chemical Synthesis of Osteoblast-Targeted (DSS)6-AZD8329 Conjugate

[0048] The chemical synthesis of the (DSS)6-AZD8329 conjugate is shown below. Using dichloromethane as solvent, BCN-OH (CAS: 1263166-90-0, 0.15 g, 1 mmol) and AZD8329 (0.42 g, 1 mmol) were condensed overnight at room temperature in the presence of DCC (0.25 g, 1.2 mmol) and DMAP (5 mg) to prepare an intermediate containing an alkynyl side chain, as a yellowish-white solid, yielding 0.38 g (68%). This intermediate (55 mg, 0.1 mmol) was then reacted with N3(H2C)5OC-(DSS)6 (purchased from Shanghai Qiangyao Biotechnology Co., Ltd., purity >95%) (189 mg, 0.1 mmol) containing an azide side chain in a mixed solvent of tetrahydrofuran and water (1:1 volume ratio) at room temperature for 24 hours. After the reaction was completed, the residue was lyophilized, and the residue was purified by preparative liquid chromatography to obtain (DSS)6-AZD8329 coupling as a white solid of 132 mg, with a yield of 54%.

[0049] High-performance liquid chromatography and high-resolution mass spectra of the couplings, such as Figure 1 and Figure 2 As shown.

[0050]

[0051] Chemical synthesis route of osteoblast-targeted (DSS)6-AZD8329 coupling conjugate

[0052] Purification method: The conjugate was purified using a preparative chromatographic column. The preparation conditions were as follows: C-18 reversed-phase column, mobile phase of 0.1% TFA aqueous solution (A) and 0.1% TFA acetonitrile (B), and gradient elution was used to prepare the conjugate. The gradient elution program was: gradient of B 20-80%, separation time 60 min.

[0053] Example 3 Screening of 11β-HSD1 inhibitors

[0054] 11β-HSD1 inhibitors were primarily screened from those already reported in clinical trials. These include MK0736, BMS823779, AZD4017, AZD8329, BI135585, and HSD016. However, only AZD4017 and AZD8329 are used in the treatment of obesity. Because AZD4017 contains an acylglucuronide metabolite, which can cause severe toxicity in vivo, and AZD8329 is an 11β-HSD1 inhibitor with structural optimization of AZD4017, AZD8329 was ultimately selected as the 11β-HSD1 inhibitor portion of this invention.

[0055] Example 4: Screening of reaction conditions and coupling mechanism

[0056] AZD8329 has poor water solubility, while (DSS)6 has good water solubility; therefore, the coupling of the two requires the selection of a suitable solvent system for the reaction. Initially, we chose a mixed reaction system of dimethyl sulfoxide (DMSO) and water, but due to the high boiling point of DMSO, there was a problem with the post-processing being difficult to distill off. Further optimization led us to use tetrahydrofuran (THF) and water as the reaction solvent, which offers advantages such as high reaction yield and simple post-processing.

[0057] We selected the classic click chemistry reaction to couple AZD8329 and (DSS)6. Initially, we attempted to introduce a straight-chain alkynyl group at the carboxyl terminus of AZD8329 and construct the couple via a copper-catalyzed Click reaction. However, because (DSS)6 contains carboxyl, hydroxyl, and amino groups, it readily chelates copper ions, resulting in a pale blue couple with poor water solubility due to the large amount of copper ions chelated. Therefore, we further opted for a copper-free Click reaction to construct the couple... Linking groups offer advantages such as more environmentally friendly reactions and better water solubility of the resulting coupling products.

[0058] The initial chemical reaction of the conjugate constructed via copper catalysis is shown below, and the structural characterization mass spectrum is shown in [reference needed]. Figure 12 .

[0059]

[0060] Experimental Example 1 (DSS) 6-AZD8329 In vivo distribution experiment

[0061] 1.1 Experimental Samples, Reagents, and Experimental Animals

[0062] Sample: (DSS)6-AZD8329 was prepared according to Example 2 of the present invention; (RKK)6-AZD8329 was synthesized by the inventor.

[0063] Reagents: physiological saline, 4% paraformaldehyde solution, frozen section embedding agent, etc.

[0064] Experimental animals: C57BL / 6J mice.

[0065] 1.2 Experimental Methods

[0066] Wild-type mice (C57BL / 6J mice) were injected with either a cy5-labeled 11β-HSD1 inhibitor conjugate (DSS) 6-AZD8329 or a non-targeting 11β-HSD1 inhibitor conjugate (RKK) 6-AZD8329. Mice were then sacrificed, and bone, brain, heart, lung, liver, spleen, kidney, muscle, and gonadal adipose tissue (GAT) were harvested. The distribution of the inhibitors was detected using biophoton imaging (BPI). Furthermore, frozen sections were prepared from the tibia, and the distribution of the 11β-HSD1 inhibitor within the bone was detected by immunofluorescence.

[0067] (RKK)6 is a non-targeting control peptide of (DSS)6, lacking targeting specificity, and serves as the control group for (DSS)6. In the clinical trials of AZD8329, AZD was administered orally, while in this experiment, (DSS)6-AZD8329 was administered via intraperitoneal injection; furthermore, the molecular weights of (DSS)6-AZD8329 and AZD8329 differ significantly, making them unsuitable as a control. Therefore, (RKK)6-AZD8329, conjugated with a non-targeting control peptide, was selected as the control drug for in vivo distribution experiments. The synthetic pathway of (RKK)6-AZD8329 is as follows:

[0068]

[0069] 1.3 Experimental Results

[0070] Data showed that the fluorescence intensity of CY5-labeled AZD8329 in the bone tissue of mice injected with (DSS)6-AZD8329 was significantly higher than that in mice injected with (RKK)6-AZD8329 (P<0.0001), and the fluorescence intensity in the lung, liver, spleen, kidney, muscle, and gonadal adipose tissue of mice injected with (DSS)6-AZD8329 was significantly lower than that of mice injected with (RKK)6-AZD8329 (P<0.0001). The fluorescence intensity in the brain and heart of both groups of mice was very low. Figure 3 ).

[0071] Furthermore, immunofluorescence assays showed that mice injected with (DSS)6-AZD8329 had significantly more double-positive cells in their bone, denoted by both osteocalcin (green) and AZD8329 (red), compared to mice injected with (RKK)6-AZD8329. Figure 4 ).

[0072] These results indicate that (DSS)6 can specifically deliver conjugated AZD8329 to osteoblasts of wild-type mice, thereby targeting and inhibiting the function of 11β-HSD1 in osteoblasts.

[0073] Experimental Example 2 (DSS) 6-AZD8329 Dose Response Pattern Study

[0074] 2.1 Experimental Samples, Reagents, and Experimental Animals

[0075] Sample: (DSS)6-AZD8329 prepared according to Example 2 of the present invention

[0076] Reagents: physiological saline, 4% paraformaldehyde solution, TRIzol RNA extraction reagent, RNA reverse transcription reagent, SYBR-PCR Mix, etc.

[0077] Experimental animals: C57BL / 6J mice.

[0078] 2.2 Experimental Procedure

[0079] To investigate the dose-response pattern and persistence of (DSS)6-AZD8329, we injected (DSS)6-AZD8329 into 1-month-old mice at doses of 3.0, 5.0, 7.0, and 10.0 mg / kg (n=3). The mRNA expression level of GILZ (a downstream gene of 11β-HSD1) in bone was used to assess the inhibitory efficiency of 11β-HSD1 in bone. Mice were sacrificed on days 1, 2, 3, 5, and 7 after administration, and RNA was extracted from both femurs and tibias. The expression level of GILZ in the extracted bone mRNA was detected.

[0080] 2.3 Experimental Results

[0081] Following a single administration of different doses of (DSS)6-AZD8329, the expression level of GILZ in the bone tissue of mice decreased in a dose-dependent manner, and (DSS)6-AZD8329 had a dose-dependent inhibitory effect on 11β-HSD1 in bone. A single injection of (DSS)6-AZD8329 at a dose of 10.0 mg / kg maintained GILZ expression in mouse bone at a low level (less than 30% of baseline GILZ mRNA level) for 3 days. Figure 5 (Table 1). Therefore, in the subsequent pharmacodynamic study of (DSS)6-AZD8329, administration at a dose of 10 mg / kg every 3 days provided sustained inhibition of 11β-HSD1.

[0082] Table 1

[0083]

[0084] Experimental Example 3 (DSS) 6-AZD8329 Efficacy Study Experiment

[0085] 3.1 Experimental Samples, Reagents, and Experimental Animals

[0086] Sample: (DSS)6-AZD8329 was prepared according to Example 2 of the present invention; AZD8329 was synthesized by the inventors.

[0087] Reagents: high-fat feed, physiological saline, 4% paraformaldehyde solution, glucose injection, insulin, HE staining reagent, etc.

[0088] Experimental animals: C57BL / 6J mice.

[0089] 3.2 Experimental Procedure

[0090] One-month-old normal male wild-type mice were used in the efficacy study and were divided into four groups: a normal diet control group, a high-fat diet control group, a high-fat diet + AZD8329 treatment group, and a high-fat diet + (DSS)6-AZD8329 treatment group, with six mice in each group. Six age-matched normal male mice were sacrificed as the baseline group before high-fat diet induction. During the first week of diet-induced obesity, mice in the high-fat diet + AZD8329 treatment group were given oral AZD8329 (1.72 mg / kg, i.e., 4 μmol / kg), while mice in the high-fat diet + (DSS)6-AZD8329 treatment group were given intravenous (DSS)6-AZD8329 (10 mg / kg, i.e., 4 μmol / kg). Dosing intervals were 3 days. The body weight and feed intake of all mice were checked weekly. After 25 weeks of high-fat diet induction and treatment, oral glucose tolerance tests were performed on mice in each group. After euthanizing the mice, visceral and subcutaneous fat was collected, weighed, and photographed. Paraffin sections were then prepared and H&E staining was performed to determine the fat size in each group. Femurs were fixed and bone mass was analyzed using micro-CT.

[0091] 3.3 Experimental Results

[0092] After 25 weeks of high-fat diet induction, the weight gain of mice in each group was as follows: Figure 6 As shown in Table 2, the weight gain in the high-fat diet control group was significantly higher than that in the normal diet control group (P<0.0001). The weight gain in the high-fat diet + (DSS)6-AZD8329 treatment group was significantly lower than that in the high-fat diet control group (P<0.01), but there was no statistically significant difference in weight gain between the high-fat diet + AZD8329 group and the high-fat diet control group (P=0.76). Furthermore, the weight gain in the high-fat diet + AZD8329 treatment group was significantly higher than that in the normal diet control group (P<0.01), while there was no significant difference in weight gain between the high-fat diet + (DSS)6-AZD8329 treatment group and the normal diet control group. The weight gain in the high-fat diet + (DSS)6-AZD8329 treatment group was lower than that in the normal diet + AZD8329 treatment group (P=0.06), therefore, (DSS)6-AZD8329 is more effective than the original drug AZD8329 in inhibiting weight gain induced by a high-fat diet.

[0093] Table 2

[0094]

[0095] Blood glucose concentrations in each group of the glucose tolerance test are as follows: Figure 7 Table 3 shows the results. The blood glucose levels at all time points after oral glucose administration in the high-fat diet control group were significantly higher than those in the normal diet control group (15 min, 30 min, 90 min, 120 min P<0.05, 60 min P<0.01), and the area under the curve (AUC) in the high-fat diet control group was significantly higher than that in the normal diet control group (P<0.0001). These results indicate that mice in the high-fat diet control group developed glucose intolerance after induction. The AUC in the high-fat diet + AZD8329 treatment group was slightly higher than that in the HFD-vehicle group (P=0.43), while the AUC in the high-fat diet + (DSS)6-AZD8329 treatment group was significantly lower than that in the high-fat diet control group (P<0.01). 120 minutes after oral glucose administration, the blood glucose level in the high-fat diet + (DSS)6-AZD8329 treatment group (7.3 mmol / dL) returned to normal levels compared to the normal diet control group (7.22 mmol / dL). The blood glucose concentrations in the high-fat diet control group (9.4 mmol / dL) and the high-fat diet + AZD8329 treatment group (9.27 mmol / dL) remained abnormal. This result indicates that the targeted inhibitor (DSS) 6-AZD8329 can improve glucose intolerance caused by a high-fat diet, while AZD8329 does not improve glucose intolerance.

[0096] Table 3

[0097]

[0098]

[0099] In addition, the total weight of subcutaneous fat and gonadal fat in each group is as follows: Figure 8 Table 4 shows the results. The total weight of subcutaneous fat and gonadal fat in the high-fat diet control group was significantly higher than that in the normal diet control group (P<0.0001). The total fat weight in the high-fat diet + (DSS)6-AZD8329 treatment group was significantly lower than that in the normal diet control group (P<0.01), while the total fat weight in the high-fat diet + AZD8329 treatment group was slightly lower than that in the high-fat diet control group (P=0.35), but the difference was not statistically significant. The dimensions of adipocytes in each group after H&E staining and IMAGE J analysis of gonadal adipose tissue are shown in the table. Figure 9As shown in Table 4, the mean adipocyte size in the high-fat diet control group was significantly larger than that in the normal diet control group (P<0.01). The mean adipocyte size in the high-fat diet + (DSS)6-AZD8329 treatment group was significantly smaller than that in the high-fat diet control group (P<0.01), while there was no significant difference in adipocyte size between the high-fat diet + AZD8329 treatment group and the high-fat diet control group (P=0.34). Simultaneously, the adipocyte size in the high-fat diet + AZD8329 treatment group was significantly larger than that in the normal diet control group (P<0.05), while there was no significant difference between the high-fat diet + (DSS)6-AZD8329 treatment group and the normal diet control group (P=0.21). These results indicate that after high-fat diet induction, (DSS)6-AZD8329 is more effective than the original drug AZD8329 in inhibiting fat accumulation and adipocyte hypertrophy.

[0100] Table 4

[0101]

[0102] Reports indicate that a high-fat diet can adversely affect bone metabolism, such as inhibiting bone development and bone formation in young mice, ultimately leading to low bone mass and bone structure damage. Therefore, we further investigated whether (DSS)6-AZD8329 protects bone mass and bone structure in young mice fed a high-fat diet.

[0103] Micro-CT analysis of the cancellous bone in the distal femoral metaphysis showed that the Tb.BV / TV (P<0.01), Tb.BMD (P<0.01), Tb.Th (P<0.001), and Tb.N (P<0.01) of the high-fat diet control group were significantly lower than those of the normal diet control group. This indicates that the cancellous bone density and morphology of mice fed a high-fat diet were lower than those of mice fed a normal diet. The high-fat diet + AZD8329 treatment group showed significantly higher levels of Tb.BV (P<0.05), Tb.BMD (P<0.01), and Tb.Th (P<0.05) compared to the high-fat diet control group. The high-fat diet + (DSS)6-AZD8329 treatment group also showed significantly higher levels of Tb.BV / TV (P<0.0001), Tb.BMD (P<0.0001), Tb.Th (P<0.0001), and Tb.N (P<0.0001) compared to the high-fat diet control group. Simultaneously, the high-fat diet + (DSS)6-AZD8329 treatment group also showed significantly higher levels of Tb.BV / TV (P<0.01), Tb.BMD (P<0.01), and Tb.Th compared to the normal diet control group. However, all trabecular bone parameters in the high-fat diet + AZD8329 treatment group showed no significant differences compared to the normal diet control group. Furthermore, the high-fat diet + (DSS)6-AZD8329 treatment group had higher Tb.BV / TV (P<0.01) and Tb.Th (P<0.001) than the high-fat diet + AZD8329 treatment group. Figure 10 (Table 5). These results indicate that, compared to AZD8329, (DSS)6-AZD8329 provides better protection for bone trabeculae in mice induced by a high-fat diet, preventing bone formation inhibition, low bone mass, and bone structure destruction caused by a high-fat diet.

[0104] Table 5

[0105]

[0106] Micro-CT analysis of cortical bone also showed that the Ct.Th (P<0.01) in the high-fat diet control group was significantly lower than that in the normal diet control group. While Ct.BV / TV, Ct.BMD, and Ct.TMD were lower in the high-fat diet control group, the differences were not significant. The high-fat diet + (DSS)6-AZD8329 treatment group had significantly higher Ct.BV / TV (P<0.05) and Ct.Th (P<0.01) compared to the high-fat diet control group, while all cortical bone parameters in the high-fat diet + AZD8329 treatment group were not significantly different from those in the high-fat diet control group. Furthermore, the cortical bone parameters in the high-fat diet + (DSS)6-AZD8329 treatment group were all higher than those in the high-fat diet + AZD8329 treatment group, but again, the differences were not significant. Figure 11(Table 6). These results indicate that, compared to AZD8329, (DSS)6-AZD8329 provides better protection for cortical bone in mice induced by a high-fat diet, preventing bone formation inhibition, low bone mass, and bone structure destruction caused by a high-fat diet.

[0107] Table 6

[0108]

Claims

1. A 11β-HSD1 inhibitor conjugate modified with an osteogenic targeting molecule, characterized in that, The structure of the coupling compound is given by formula (I): Formula (I).

2. The method for preparing the osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate according to claim 1, characterized in that, The method includes: (1) In a solvent, BCN-OH and AZD8329 were condensed under the action of a condensing agent to prepare an intermediate containing an alkynyl side chain; (2) The intermediate of step (1) reacts with N3(CH2)5OC-(DSS)6 in a mixed solvent at room temperature; (3) After the reaction was completed, the residue was freeze-dried and purified by preparative liquid phase to obtain (DSS)6-AZD8329 conjugate.

3. The preparation method according to claim 2, characterized in that, The solvent used in step (1) is selected from dichloromethane, 1,2-dichloroethane, tetrahydrofuran, ethyl acetate, toluene, acetonitrile, or chloroform.

4. The preparation method according to claim 3, characterized in that, The solvent used in step (1) is selected from dichloromethane.

5. The preparation method according to claim 2, characterized in that, The condensing agent in step (1) is selected from DCC / DMAP, EDC / DMAP, HATU / DIPEA, TBTU / TEA, or HBTU / DIPEA.

6. The preparation method according to claim 5, characterized in that, The condensing agent in step (1) is selected from DCC / DMAP.

7. The preparation method according to claim 5, characterized in that, Step (1) further includes: the molar ratio of BCN-OH to AZD8329 is 0.8:1 to 2:1, the amount of DCC is 1.2 equiv to 2 equiv of AZD8329, the amount of DMAP is 0.05 equiv to 0.2 equiv of AZD8329, and the reaction is carried out overnight at room temperature.

8. The preparation method according to claim 2, characterized in that, Step (2) further includes: the molar ratio of the intermediate to N3(CH2)5OC-(DSS)6 is 1:1 to 2:1, and the reaction is carried out at room temperature for 24 hours.

9. The preparation method according to claim 8, characterized in that, Step (2) further includes: the molar ratio of the intermediate to N3(CH2)5OC-(DSS)6 is 1:1 to 1.2:

1.

10. The preparation method according to claim 2, characterized in that, The mixed solvent in step (2) is selected from tetrahydrofuran / H2O, DMSO / H2O, or acetonitrile / H2O.

11. The preparation method according to claim 10, characterized in that, The mixed solvent in step (2) is selected from tetrahydrofuran / H2O.

12. The preparation method according to claim 11, characterized in that, In step (2), the volume ratio of tetrahydrofuran to water is 0.1:1 to 1:0.

1.

13. The preparation method according to claim 12, characterized in that, In step (2), the volume ratio of tetrahydrofuran to water is 0.5:1 to 1:0.

5.

14. The preparation method according to claim 2, characterized in that, Step (3) further includes: purifying the conjugate using a preparative chromatographic column. The purification conditions are: C-18 reversed-phase column, mobile phase: phase A is 0.1% TFA aqueous solution and phase B is 0.1% TFA acetonitrile, gradient elution is used to prepare the conjugate, and the gradient elution program is: gradient of phase B is 20~80%, separation time is 60 min.

15. A pharmaceutical composition comprising an osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate according to claim 1 or an osteogenic-targeting molecule-modified 11β-HSD1 inhibitor conjugate prepared by the method described in claims 2 to 14.

16. The use of the conjugate of claim 1, the conjugate prepared by the method of claims 2-14, or the pharmaceutical composition of claim 15 in the preparation of a medicament for treating obesity or diabetes.

17. The use of the conjugate of claim 1, the conjugate prepared by the method of claims 2-14, or the pharmaceutical composition of claim 15 in the preparation of a medicament for treating bone loss, wherein the bone loss is caused by a high-fat diet or long-term use of glucocorticoids.

18. The use of the conjugate of claim 1, the conjugate prepared by the method of claims 2-14, or the pharmaceutical composition of claim 15 in the preparation of a medicament for lowering blood lipids.

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