Telmisartan glucoside derivative, microbial conversion preparation method thereof and application of telmisartan glucoside derivative in preparation of medicine for preventing or treating osteoporosis

Abstract

The invention discloses a telmisartan glucoside derivative, a microbial conversion preparation method thereof and application of the telmisartan glucoside derivative in preparation of a medicine for preventing or treating osteoporosis. The telmisartan glucoside derivative is characterized in that a telmisartan propyl side chain is connected with a glucosyl group. According to the invention, a microbial conversion technology is utilized, telmisartan is taken as a substrate, and glucosyl is introduced into a telmisartan propyl side chain in a regioselective manner, so that the telmisartan glucoside derivative with a novel structure is obtained. Experiments on the osteoblast proliferation promoting activity and the postmenopausal osteoporosis resisting effect of the telmisartan glucoside derivative by using an in-vitro osteoblast model and an ovariectomized induced osteoporosis mouse model prove that the telmisartan glucoside derivative has relatively good osteoblast proliferation promoting activity and postmenopausal osteoporosis resisting activity; the compound can be used as an active component of a medicine for treating osteoporosis, and has wide application.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to a method for preparing telmisartan glycoside derivatives and their applications, particularly to a method for preparing telmisartan glycoside derivatives through microbial transformation and their application in the preparation of drugs for the prevention or treatment of osteoporosis. Background Technology

[0002] Osteoporosis is a systemic metabolic bone disease characterized by decreased bone mass, bone microarchitecture deterioration, and increased bone fragility, significantly increasing the risk of fractures. Its incidence increases with age, becoming a significant threat to the quality of life of middle-aged and elderly individuals. Its pathogenesis involves multiple factors, including genetics, gender, alcoholism, drug abuse, diet, and exercise habits, with declining estrogen levels in women being particularly crucial. Postmenopausal osteoporosis (PO), a specific subtype caused by estrogen deficiency, is characterized by continuous bone loss and bone microarchitecture deterioration. Its global incidence is rising annually, making it one of the major health problems for postmenopausal women. Studies have shown a close correlation between oxidative stress and the occurrence of osteoporosis. Postmenopausal women experience increased oxidative stress due to decreased estrogen levels, manifested as reduced antioxidant enzyme activity and increased lipid oxidation. Currently, commonly used clinical drugs include calcitonin, bisphosphonates, and hormones; however, the need for long-term medication exacerbates the risk of toxic side effects, necessitating the development of novel treatment methods.

[0003] Telmisartan, a representative angiotensin II receptor blocker (ARB), exerts its antihypertensive effect by highly selectively blocking AT1 receptors. Its unique pharmacological mechanism not only effectively controls blood pressure but also protects the heart, kidneys, and vascular endothelial cells, significantly reducing the risk of cardiovascular events in hypertensive patients. It has become a first-line drug for the treatment of hypertension and cardiovascular disease. Regarding safety, common adverse reactions to telmisartan include mild dizziness, gastrointestinal discomfort, and orthostatic hypotension. Notably, this drug may cause rare allergic reactions such as angioedema, requiring close monitoring in clinical use. Regarding drug interactions, caution should be exercised when using telmisartan in combination with diuretics, calcium channel blockers, or other antihypertensive drugs to avoid excessive blood pressure reduction.

[0004] Chemical structural analysis reveals that while the imidazole ring structure of telmisartan enhances its lipophilicity and facilitates tissue penetration, it also limits its water solubility, resulting in low oral bioavailability. Furthermore, the drug's metabolic pathway in vivo is complex, involving multiple cytochrome P450 enzyme systems, and it may competitively inhibit the metabolism of other drugs.

[0005] Significant progress has been made in structural optimization research of telmisartan in recent years. By introducing hydrophilic groups to improve solubility and combining nanotechnology to enhance targeting, novel derivatives have significantly improved bioavailability while maintaining the original antihypertensive effect. Preclinical studies show that these improved formulations have significantly improved antihypertensive efficacy and safety, and some candidate drugs have entered clinical trials. Currently, there are no reports on the anti-osteoporosis effects of telmisartan and its derivatives. Summary of the Invention

[0006] Purpose of the invention: In view of the above, the purpose of this invention is to provide a telmisartan glycoside derivative or a pharmaceutically formable salt thereof and a method for its preparation by microbial transformation. At the same time, the telmisartan glycoside derivative can be used to prepare antitumor and anti-osteoporosis drugs.

[0007] Technical solution: A telmisartan glycoside derivative, wherein the telmisartan glycoside derivative has a glucose group attached to the propyl side chain of telmisartan, and the telmisartan glycoside derivative has the structure of Formula I: The telmisartan glycoside derivative provided by this invention has the structure shown in Formula I: .

[0008] The compound with the structural formula of Formula I is a telmisartan glycoside derivative disclosed for the first time in this invention.

[0009] On the other hand, the present invention also provides a method for preparing telmisartan glycoside derivatives by microbial transformation, comprising the following steps: 1) Fermentation culture of microorganisms: Arbutin was added to the culture medium, followed by transformation culture. After removing the mycelium, the fermentation broth was obtained. The microorganism was Rhizopus stolonifer CGMCC 3.36. 2) The fermentation broth is extracted to obtain the transformed product; 3) The transformed product was purified by reversed-phase silica gel column chromatography. The reversed-phase silica gel column purification used a methanol-water two-phase system for gradient elution, and the methanol-water 60:40 (V / V) elution fractions were collected and combined. 4) The component was purified by reversed-phase high-performance liquid chromatography to obtain telmisartan glycoside derivative.

[0010] In addition, the present invention also provides the use of telmisartan glycoside derivatives or pharmaceutically acceptable salts thereof in the preparation of medicaments for the prevention or treatment of osteoporosis.

[0011] Furthermore, the aforementioned drug for treating osteoporosis is a drug that promotes osteoblast proliferation.

[0012] Furthermore, the aforementioned medication for treating osteoporosis is a medication for treating postmenopausal osteoporosis.

[0013] Furthermore, the drug also contains pharmaceutically acceptable excipients.

[0014] Furthermore, the pharmaceutically acceptable excipients are one or more of the following: diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbents, and lubricants.

[0015] Beneficial effects: Compared with existing technologies, this invention utilizes the catalytic action of microbial enzymes to selectively introduce the glucose domain into the propyl side chain of telmisartan. This yields a novel telmisartan glycoside derivative, Formula I. In vitro cell and in vivo animal experiments have confirmed that Compound I possesses significant anti-osteoporosis activity and can serve as an active ingredient in anti-osteoporosis drugs, with broad applications. Detailed Implementation

[0016] To further illustrate the present invention, the following describes in detail, with reference to embodiments, the preparation method of the telmisartan glycoside derivative provided by the present invention and its application in the preparation of anti-osteoporosis drugs.

[0017] This invention employs a microbial transformation method, using telmisartan as a raw material, to prepare the compound of this invention through fermentation, extraction, and separation. *Rhizopus stolonifer* CGMCC 3.36 was purchased from the Chinese Academy of Sciences Microbial Culture Collection Center (CGMCC), and stored on potato dextrose agar at 4 °C.

[0018] 1) Fermentation, conversion, and extraction: Rhizopus stolonifer CGMCC 3.36 was inoculated into a 250 mL Erlenmeyer flask (containing 100 mL of potato broth) as a seed culture. After shaking culture at 140 rpm and 24 °C for 12 hours, when the mycelial growth was vigorous, 1 mL of the seed culture was added to 10 1000 mL shake flasks (containing 400 mL of potato broth). After shaking culture for 24 hours, 10 mg of telmisartan (0.25 mL, 40 mg / mL anhydrous ethanol-chloroform 1:1 solution) was added to each shake flask, for a total of 200 mg of substrate. Transformation was continued for 7 days under the same conditions. The fermentation broth was filtered to remove mycelia, and the filtrate was extracted three times with an equal volume of ethyl acetate. The extract was concentrated to dryness under reduced pressure to obtain approximately 0.53 g of crude transformant.

[0019] 2) ODS-C18 column chromatography separation: The crude extract was separated by ODS-C18 column chromatography. Methanol:water gradient elution (30:70, 60:40, 100:0). The methanol:water 60:40 fraction was collected.

[0020] 3) Reversed-phase high-performance liquid chromatography purification: The methanol:water 60:40 fraction was purified by reversed-phase high-performance liquid chromatography (RP-HPLC). The preparative conditions were: a YMC ODS A-5 μm column (12.0 × 250 mm), methanol-water (40:60, V / V), flow rate 3.0 mL / min, and detection wavelength 290 nm. The product with the structural formula of Formula I was obtained.

[0021] Compound Formula I: Optical Rotation [α] 22 D: +31.6° ( c = 0.2, MeOH); High-resolution mass spectrometry m / z 715.2751[M+Na] + (calcd. C 39 H 40 N4O8Na, 715.2744); the 1H and 1C NMR spectra are shown below. 1 H NMR (600MHz, CD3OD): d H 8.01 (s, 1H), 7.89 (d, J = 7.5 Hz, 1H), 7.83 (d, J = 7.6 Hz,1H), 7.81 - 7.77 (m, 3H), 7.71 (d, J = 7.5 Hz, 1H), 7.65 - 7.61 (m, 1H), 7.59(ddd, J = 7.5, 6.1, 1.4 Hz, 1H), 7.53 (d, J = 7.3 Hz, 2H), 7.35 - 7.28 (m,2H), 7.25 (d, J = 7.3 Hz, 2H), 5.91 (d, J = 12.5 Hz, 1H), 5.81 (d, J = 12.5Hz, 1H), 4.81 (d, J= 7.2 Hz, 1H), 3.88 (s, 3H), 3.84 - 3.75 (m, 2H), 3.73 -3.62 (m, 2H), 3.59 - 3.51 (m, 2H), 3.30 (dt, J = 12.5, 7.1 Hz, 1H), 3.20 (dt, J = 12.5, 7.1 Hz, 1H), 2.96 - 2.89 (m, 2H), 2.26 - 2.15 (m, 1H), 1.87 - 1.74 (m, 1H). 13 C NMR (150 MHz, CD3OD): d C 169.3, 158.5, 155.6, 143.1, 142.7, 142.3, 137.5, 135.6, 134.7, 133.9, 133.8, 130.3, 129.4, 129.2, 128.6, 127.2, 126.0, 125.8, 122.9, 122.7, 122.3, 120.0, 114.4, 109.5, 103.6, 75.8, 73.6, 72.7, 70.9, 68.5, 62.7, 46.2, 31.6, 29.9, 27.9.

[0022] Example 2: Effect of Telmisartan glycoside derivative formula I on the survival rate of MC3T3-E1 cells To further illustrate the present invention, the following detailed description, in conjunction with embodiments, describes the application of the telmisartan glycoside derivatives or pharmaceutically formable salts thereof provided by the present invention in the preparation of medicaments for the prevention or treatment of osteoporosis.

[0023] This invention investigated the effect of telmisartan glycoside derivative Formula I on osteoblast viability. Pre-osteoblast MC3T3-E1 cells were resuscitated and cultured in medium containing 10% fetal bovine serum at 37°C in a 5% CO2 incubator for induced differentiation, followed by passage. Osteoblasts in logarithmic growth phase were seeded at a concentration of 1×10⁵ / ml in 96-well plates. Cells were divided into a blank control group, an H₂O₂ group (0.5 mM), a low-dose H₂O₂ + Formula I group (1 mM Formula I), a medium-dose H₂O₂ + Formula I group (10 mM Formula I), and a high-dose H₂O₂ + Formula I group (20 mM Formula I). ​​After 72 h of culture, the absorbance (A) at 490 nm was measured using the MTT assay, and cell viability was calculated [viability = experimental group A value / control group A value × 100%]. The experiment was repeated three times. Analysis of variance and LSD-t test were performed using SPSS 22.0 software, and the results are shown in Table 1.

[0024]

[0025] Compared with the control group * P < 0.05; compared with the H2O2 group # P < 0.05; ## P < 0.01.

[0026] The results show that compound I of the present invention can promote osteoblast proliferation in a dose-dependent manner and inhibit H2O2-induced cytotoxicity, and can be used as an active ingredient in anti-osteoporosis drugs.

[0027] Example 3: Effect of Telmisartan glycoside derivative Formula I on BMD in OVX mice A postmenopausal osteoporosis model in mice was established using bilateral ovariectomy (OVX). Twelve-week-old female C57BL / 6J mice were randomly divided into a sham-operated group, a model group, a positive control group, and two treatment groups (low-dose (2 mg / kg) and high-dose (10 mg / kg) of compound I, with eight mice in each group. The sham-operated and model groups were administered 0.9% sodium chloride solution by gavage post-surgery, the positive control group was administered 0.2 mg / kg estradiol valerate solution by gavage, and the treatment groups were administered compound I by gavage once daily for eight weeks. Bone mineral density (BMD) values ​​of the fourth lumbar vertebra and right femur were measured using a bone mineral density analyzer after anesthesia. Analysis of variance and LSD-t test were performed using SPSS 22.0 software. The results are shown in Table 2.

[0028]

[0029] Compared with the blank group * P < 0.05; compared with the model group # P < 0.05.

[0030] Compared with the blank control group, the BMD values ​​of the fourth lumbar vertebra and right femur in the model group were significantly reduced. P < 0.05). Compared with the model control group, the BMD values ​​of the fourth lumbar vertebra and right femur were significantly increased in the positive control group and the low- and high-dose groups. P < 0.05). Compared with the low-dose group, the high-dose group showed a significant increase in BMD values ​​of the fourth lumbar vertebra and right femur. The experimental results indicate that compound I can significantly increase bone mineral density in postmenopausal osteoporotic mice in a dose-dependent manner, and can be used as an active ingredient in anti-osteoporosis drugs.

[0031] The foregoing description clearly and completely illustrates the technical solutions in the embodiments of the present invention, enabling those skilled in the art to better understand the advantages and features of the present invention, thereby providing a clearer definition of the scope of protection of the present invention. The embodiments described in this invention are merely some embodiments of the present invention, not all embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

Claims

1. A glycoside derivative of telmisartan, characterized by: The telmisartan glycoside derivative is a telmisartan propyl side chain with a glucose group, and the telmisartan glycoside derivative has the structure of formula I: 。 2. A process for the microbial conversion of a glycoside derivative of telmisartan according to claim 1, characterized in that: The method comprises the following steps: 1) Pre-culturing a microorganism, adding telmisartan to the culture medium, then carrying out transformation culture, removing mycelium to obtain a fermentation liquor, the microorganism being Rhizopus stolonifer Rhizopus stolonifer CGMCC 3.36; 2) The fermentation broth is extracted to obtain a conversion product; 3) The conversion product is purified by a reversed-phase silica gel column chromatography, the reversed-phase silica gel column purification adopts a methanol-water two-phase system gradient elution, and the elution components of methanol-water 60:40 (V / V) are collected and combined; 4) The components are purified by a reversed-phase high performance liquid chromatography to obtain the telmisartan glycoside derivative with the structure of formula I.

3. Use of the telmisartan glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing or treating osteoporosis.

4. Use of the telmisartan glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 3 for the manufacture of a medicament for preventing or treating osteoporosis, characterized by: The drug for treating osteoporosis is a drug for promoting the proliferation of osteoblasts.

5. Use of the telmisartan glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 3 for the manufacture of a medicament for preventing or treating osteoporosis, characterized in that: The drug for treating osteoporosis is a drug for treating postmenopausal osteoporosis.

6. Use of the telmisartan glycoside derivative or a pharmaceutically acceptable salt thereof according to claim 3 for the manufacture of a medicament for preventing or treating osteoporosis, characterized by: The drug further comprises a pharmaceutically acceptable excipient.

7. Use of the osiglycoside derivative of telmisartan or a pharmaceutically acceptable salt thereof according to claim 6 for the manufacture of a medicament for the prevention or treatment of osteoporosis. The pharmaceutically acceptable excipient is one or more of a diluent, an excipient, a filler, a binder, a wetting agent, a disintegrant, an absorption promoter, a surfactant, an adsorption carrier and a lubricant.

Images