CN114933623B - Estradiol derivative and preparation method and application thereof - Google Patents
Estradiol derivative and preparation method and application thereof Download PDFInfo
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- 238000002360 preparation method Methods 0.000 title claims abstract description 24
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07J—STEROIDS
- C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
- C07J1/0051—Estrane derivatives
- C07J1/0066—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
- A61P15/08—Drugs for genital or sexual disorders; Contraceptives for gonadal disorders or for enhancing fertility, e.g. inducers of ovulation or of spermatogenesis
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Reproductive Health (AREA)
- Medicinal Chemistry (AREA)
- Pregnancy & Childbirth (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Endocrinology (AREA)
- Pharmacology & Pharmacy (AREA)
- Life Sciences & Earth Sciences (AREA)
- Animal Behavior & Ethology (AREA)
- Gynecology & Obstetrics (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Steroid Compounds (AREA)
Abstract
The application discloses an estradiol derivative, a preparation method and application thereof. The estradiol derivative of the application prepares the compound with the NO donor performance and E of L-arginine (L-Arg) through the esterification reaction between 17 beta-estradiol and L-arginine 2 The ER targeting compound is expected to be applied to medicines for promoting reproductive function, can be used as estrogen receptor agonist medicines and/or NO donor medicines, can enhance normal endometrium and ovarian function, and plays a role in promoting reproduction.
Description
Technical Field
The application belongs to the technical field of estrogen-related medicines, and particularly relates to an estradiol derivative, and a preparation method and application thereof.
Background
In the current culture systems of the In Vitro Fertilization (IVF) cycle, the gaseous environment is a very important influencing factor. Existing CO 2 Both culture systems and triple-gas culture systems were developed by studying the microenvironment in vivo and then performing simulations in vitro. In fact, there are a variety of gaseous molecules in mammals that regulate life processes, such as Nitric Oxide (NO), carbon monoxide, hydrogen sulfide, reactive Oxygen Species (ROS), etc. Their biological function in the reproductive systemAnd the possible medical applications still require detailed investigation.
Nitric oxide is the first gas signaling molecule found to be involved in cell signaling. In mammals, NO is produced by the oxidation of L-arginine (L-Arg) catalyzed by three Nitric Oxide Synthases (NOs). Since NO is a water-soluble molecule, it can be rapidly combined with oxygen to generate various Reactive Nitrogen (RNS) radicals and develop biological functions. The first biological function of NO is its role in the cardiovascular system, a smooth muscle relaxing factor. In addition, it plays a role in the immune system, nervous system and anti-tumor response. NO plays a very wide range of roles in mammalian reproduction, for example regulating the development and function of the reproductive system, and the process of embryonic development. Because of the broad role of NO on the reproductive system, the use of NO donors and NOs inhibitors to regulate target reproductive organs is considered to be a clinically significant intervention. For example, existing clinical studies provide a reference for guiding embryo screening and transplantation by detecting NO metabolism in the medium during the IVF cycle.
Estradiol (E) 2 ) Also known as 17β -estradiol, is secreted mainly by the ovaries. E (E) 2 Is the major female sex hormone responsible for regulating female characteristics, maturation of accessory organs and the menstrual-ovulation cycle. E (E) 2 Mainly by binding to the Estrogen Receptor (ER). 17 beta-estradiol has a high binding affinity for ER. After binding to ER, estradiol exerts a variety of functions on the ovary, uterus and other tissues.
If it is possible to develop a novel L-arginine (L-Arg) donor having both the NO donor property and E 2 The ER targeting ability of the drugs of (C) will have great significance in promoting reproductive system function.
Disclosure of Invention
The application aims to overcome the defects in the prior art and provides an estradiol derivative, a preparation method and application thereof, so as to solve the problem that the traditional auxiliary reproductive medicine cannot have both NO donor performance and E 2 Technical problem of ER targeting ability.
In order to achieve the object of the above application, a first aspect of the present application provides an estradiol derivative having a structure represented by the following formula (1):
in a second aspect, the application provides the use of an estradiol derivative of the application in a medicament for promoting reproductive function.
Further, the reproductive function promoting drug includes at least one of estrogen receptor agonist drugs and NO donor drugs.
In a third aspect of the present application, there is provided a process for the preparation of an estradiol derivative of the application, the estradiol derivative being prepared by an esterification reaction comprising 17 beta-estradiol and L-arginine.
Further, the preparation method of the estradiol derivative comprises the following steps:
preparing an organic solvent mixed solution containing Boc-Arg (Pbf) -OH, 17 beta-estradiol and 4-dimethylaminopyridine under an inert atmosphere;
EDC & HCl, namely 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, is added into the mixed solution to perform a first reaction to obtain an intermediate product;
a TFA/DCM mixed solvent is added to the intermediate product under an inert atmosphere for a second reaction to obtain a solution containing the product.
Further, the preparation method of the application also comprises a product purification treatment step, wherein the purification treatment step comprises the following steps: the product-containing solution was concentrated and added dropwise to methyl tert-butyl ether to obtain a precipitated product.
Further, the molar ratio of Boc-Arg (Pbf) -OH, 17 beta-estradiol, 4-dimethylaminopyridine and EDC & HCl is 1: (1.1-1.5): (1.0-1.5): (1.1-1.5).
Further, the organic solvent is dichloromethane or chloroform.
Further, the volume ratio of TFA and DCM in the TFA/DCM mixed solvent is 9:1.
further, the temperature of the first reaction is 0-10 ℃ and the time is 8-12 h.
Compared with the prior art, the application has the following technical effects:
the estradiol derivative of the application has both the NO donor property of L-arginine (L-Arg) and E 2 Is described herein.
The estradiol derivative can be applied to medicines for promoting reproductive function, can be used as estrogen receptor agonist medicines and/or NO donor medicines, can enhance normal endometrium and ovarian function, and plays a role in promoting reproduction.
The preparation method of the estradiol derivative can prepare the compound with the NO donor performance and E of L-arginine (L-Arg) through the esterification reaction between 17 beta-estradiol and L-arginine 2 The ER targeting ability of the compound is expected to be applied to medicaments for promoting reproductive function, and can be used as estrogen receptor agonist medicaments and/or NO donor medicaments, and the medicament compound can enhance normal endometrium and ovarian function and plays a role in promoting reproduction. In addition, the preparation method of the estradiol derivative has the advantages of simple synthetic route, easy control of preparation conditions, high target yield and few byproducts.
Drawings
In order to more clearly illustrate the embodiments of the present application or the technical solutions in the prior art, the drawings that are needed in the description of the embodiments or the prior art will be briefly described, and it is obvious that the drawings in the description below are some embodiments of the present application, and other drawings can be obtained according to the drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram showing the molecular structure of an estradiol derivative (GDT) prepared in example 1 of the present application;
FIG. 2 shows the estradiol derivative (GDT) prepared in example 1 of the present application 1 H NMR spectrum;
FIG. 3 is a MS spectrum of an estradiol derivative (GDT) prepared in example 1 of the present application;
FIG. 4 is a graph showing the comparison of the Griess assay for evaluating NO production of drugs in mouse blood provided by the application example of the present application;
FIG. 5 is a graph showing comparison of IHC staining results of CD34, CD105 and iNOS of mouse ovarian tissue provided in the application example of the present application;
FIG. 6 is a graph showing the average optical density of CD 34-organized sections of mouse ovarian tissue provided in the application example of the present application;
FIG. 7 is a graph showing the average optical density of CD105 organized sections of mouse ovarian tissue provided in the application example of the present application;
FIG. 8 is a graph showing the average optical density of iNOS histochemical sections of mouse ovarian tissue in accordance with an embodiment of the present application;
FIG. 9 is a graph showing comparison of IHC staining results of CD34, CD105 and iNOS of mouse uterine tissue provided in the application example of the present application;
FIG. 10 is a graph showing comparison of endometrium thickness of mice according to an application example of the present application;
FIG. 11 is a graph showing the average optical density of CD 34-organized sections of mouse uterine tissue according to an application example of the present application;
FIG. 12 is a graph showing the average optical density of CD105 organized sections of mouse uterine tissue provided in the application example of the present application;
FIG. 13 is a graph showing the comparison of the average optical densities of iNOS histochemical sections of mouse uterine tissue provided in the application example of the present application.
The different letter designations (a, b, c, d) in FIGS. 4, 6-8 and 10-13 represent significant differences (p.ltoreq.0.05).
Detailed Description
In order to make the technical problems, technical schemes and beneficial effects to be solved more clear, the application is further described in detail below with reference to the embodiments. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the application.
In the present application, the term "and/or" describes an association relationship of an association object, which means that three relationships may exist, for example, a and/or B may mean: a alone, a and B together, and B alone. Wherein A, B may be singular or plural. The character "/" generally indicates that the context-dependent object is an "or" relationship.
In the present application, "at least one" means one or more, and "a plurality" means two or more. "at least one of" or the like means any combination of these items, including any combination of single item(s) or plural items(s). For example, "at least one (individual) of a, b, or c," or "at least one (individual) of a, b, and c," may each represent: a, b, c, a-b (i.e., a and b), a-c, b-c, or a-b-c, wherein a, b, c may be single or multiple, respectively.
It should be understood that, in various embodiments of the present application, the sequence number of each process described above does not mean that the execution sequence of some or all of the steps may be executed in parallel or executed sequentially, and the execution sequence of each process should be determined by its functions and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.
The terminology used in the embodiments of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application. As used in this application and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.
The weights of the relevant components mentioned in the description of the embodiments of the present application may refer not only to the specific contents of the components, but also to the proportional relationship between the weights of the components, so long as the contents of the relevant components in the description of the embodiments of the present application are scaled up or down within the scope of the disclosure of the embodiments of the present application. Specifically, the mass described in the specification of the embodiment of the application can be mass units known in the chemical industry field such as mu g, mg, g, kg.
The terms "first" and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implicitly indicating the number of technical features indicated for distinguishing between objects such as substances from each other. For example, a first XX may also be referred to as a second XX, and similarly, a second XX may also be referred to as a first XX, without departing from the scope of embodiments of the application. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature.
In a first aspect, embodiments of the present application provide an estradiol derivative (GDT) having a structure represented by the following formula (1):
the estradiol derivative (GDT) of the embodiment of the application introduces the guanidyl and the amino on the basis of the molecular structure of 17 beta-estradiol, and the introduction of the guanidyl and the amino is beneficial to improving the water solubility of the estradiol derivative, thereby improving the bioavailability of the estradiol derivative and better exerting the pharmaceutical activity of the estradiol derivative.
The estradiol derivative (GDT) of the embodiment of the application can be obtained from 17 beta-estradiol and L-arginine (L-Arg) through esterification reaction, namely the chemical structure of the estradiol derivative (GDT) of the embodiment of the application mainly comes from 17 beta-estradiol and L-arginine (L-Arg). Because 17β -estradiol is a natural steroid hormone that can bind to the Estrogen Receptor (ER) and perform a targeted delivery function, while L-arginine (L-Arg) is a key substrate for NO synthesis in mammals, the estradiol derivatives (GDT) of the present embodiments bind to the dual advantages of 17β -estradiol and L-arginine (L-Arg) molecular structures, making it desirable to achieve targeted delivery of NO to tissues rich in estrogen receptors, such as the reproductive organs.
In a second aspect of the embodiment of the application, the application of the estradiol derivative in medicines for promoting reproductive function is provided. Further, the application of the estradiol derivative in the estrogen receptor agonist medicines and/or NO donor medicines is provided. Namely, the estradiol derivative of the embodiment of the application can be used as an estrogen receptor agonist drug, can be used as an NO donor drug, and can also be used as an estrogen receptor agonist drug and an NO donor drug.
Through the application study of the estradiol derivative (GDT) on female mice, the application can enhance the normal endometrium and ovary functions of the mice and play a role in promoting reproduction when the daily administration amount of the GDT is 15 mug under the condition of continuous administration for 4 days.
In a third aspect of the embodiment of the application, a preparation method of the estradiol derivative is provided, wherein the estradiol derivative is prepared from 17 beta-estradiol and L-arginine through esterification reaction.
In a specific embodiment, the preparation method of the estradiol derivative according to an embodiment of the application may include the following steps:
s01: preparing an organic solvent mixed solution containing Boc-Arg (Pbf) -OH, 17 beta-estradiol and 4-dimethylaminopyridine under the protection of inert atmosphere, such as nitrogen;
s02: then EDC and HCl are added into the mixed solution to carry out a first reaction to obtain an intermediate product; s03: a TFA/DCM mixed solvent is added to the intermediate product under an inert atmosphere for a second reaction to obtain a solution containing the product.
Further, the preparation method of the embodiment of the application further comprises a product purification treatment step, namely S04: the product-containing solution was concentrated and added dropwise to methyl tert-butyl ether to obtain a precipitated product.
Wherein, the mixing treatment of the step S01 is to fully mix the reactants in the organic solvent to form a uniform reaction system. In the examples, the molar ratio of Boc-Arg (Pbf) -OH, 17β -estradiol, 4-dimethylaminopyridine in the mixed solution was 1: (1.1-1.5): (1.0-1.5). By controlling the reaction system of the content ratio, the target product, namely the estradiol derivative (GDT), can be obtained with high efficiency under the condition of reasonable raw material proportion.
In an embodiment, the organic solvent in step S01 may be dichloromethane or chloroform-based organic solvent. The methylene dichloride or chloroform organic solvent is selected, so that the reaction raw materials have good solubility in a mixed system, and a uniform solution system is formed.
The inert atmosphere in step S01 is performed under an oxygen-free condition to protect the reaction system, and in the embodiment, the inert atmosphere may be an atmosphere formed by a chemical inert gas such as nitrogen, argon, or the like, and may be a vacuum environment.
The first reaction of step S02 is a reaction of reactants Boc-Arg (Pbf) -OH, 17β -estradiol, 4-dimethylaminopyridine, edc·hcl in organic solvent dichloromethane, specifically a reaction in the following chemical reaction formula (1):
in the examples, EDC & HCl was used in a molar ratio of Boc-Arg (Pbf) -OH to EDC & HCl of 1: the ratio of (1.1 to 1.5) is added to the reaction system, specifically to the mixed solution in step S01. By controlling the reaction system with the content ratio, the intermediate product can be efficiently obtained under the condition of reasonable raw material proportion, and the target product can be obtained with high yield.
In the examples, the temperature of the first reaction in step S02 is preferably 0 to 10℃and the time of the first reaction is preferably 8 to 12 hours, under which conditions the intermediate product can be obtained in high yield. In the first reaction process, protective nitrogen is introduced into the reaction system, the reaction progress is monitored by Thin Layer Chromatography (TLC), and after the reaction is finished, 50-80 mL of water is added into the reaction system to quench the reaction. The mixture was extracted with Dichloromethane (DCM) or chloroform, after which the organic phases were combined and then rotary evaporated in vacuo to give the crude product. The crude product was purified by silica gel column (elution conditions, petroleum ether/ethyl acetate, 3/1 to 1/1) to give an intermediate.
The reaction of step S03 is a reaction of removing Boc group in a TFA/DCM mixed solvent to obtain the final objective estradiol derivative (GDT), specifically, a reaction in the following chemical reaction formula (2):
for more efficient removal of the Boc group on the intermediate, it is preferred that the TFA/DCM mixed solvent in step S03 has a volume ratio of TFA to DCM of 9:1. the progress of the reaction was monitored by Thin Layer Chromatography (TLC), and the reaction was terminated after observing the change of the reaction solution from a colorless transparent solution to a reddish brown solution.
In the example, in step S04, after the completion of the reaction, the solution was concentrated to 3 to 5mL by vacuum rotary evaporation. Then, the mixture was slowly added dropwise to methyl tert-butyl ether, and a large amount of white solid precipitate was observed. The solid precipitate was sonicated. It is then centrifuged for 3-5 min and the supernatant removed. And then washing the solid with methyl tertiary butyl ether for 2-3 times, centrifuging to recover a solid part, washing with methanol to remove impurities, and filtering to obtain the product estradiol derivative.
The preparation method provided by the embodiment of the application has the advantages of simple synthetic route, easy control of preparation conditions, high target yield and few byproducts.
The chemical structure of the estradiol derivative GDT mainly comes from 17 beta-estradiol and L-Arg.17 beta-estradiol is a natural steroid hormone that can bind to the ER and achieve targeted delivery. L-arginine is a key substrate for NO synthesis in mammals. The application prepares the compound with the NO donor performance and E of L-arginine (L-Arg) through the esterification reaction between 17 beta-estradiol and L-arginine 2 The ER targeting compound of (2) introduces guanidine and amino on the basis of the structure of estradiol, improves the water solubility and bioavailability of estradiol derivatives, makes the compound be expected to be applied to medicines for promoting reproductive function, can be used as estrogen receptor agonist medicines and/or NO donor medicines, can enhance normal endometrium and ovarian function, and plays a role in promoting reproduction.
The following examples illustrate the estradiol derivatives (GDT) and methods for preparing and using the same, and the like, according to embodiments of the present application.
1. Estradiol derivatives (GDT) and examples of preparation methods thereof:
example 1
This example provides a estradiol derivative (GDT) and a method for preparing the same.
The preparation method of the estradiol derivative (GDT) comprises the following steps:
s01: boc-Arg (Pbf) -OH (2.63 g,5mmol,1.0equiv, CAS #200124-22-7, shanghai Aba Ding Shenghua technologies Co., ltd.), 17 beta-estradiol (1.1 equiv, CAS #50-28-2, shanghai Aba Ding Shenghua technologies Co., ltd.) and 4-dimethylaminopyridine (DMAP, 1.0 equiv) were added to the reaction flask at room temperature. The reaction flask was then evacuated and filled with nitrogen. 20mL of methylene chloride was added under nitrogen.
S02: EDC. HCl (1.1 equiv) was then added to the ice bath and the reaction was allowed to warm naturally for 10h. The progress of the reaction was monitored by Thin Layer Chromatography (TLC) and after completion of the reaction, 50mL of water was added to quench the reaction. The mixture was extracted twice with Dichloromethane (DCM) and the combined organic phases were dried and then rotary evaporated in vacuo to give the crude product. Purifying with silica gel column (eluting condition, petroleum ether/ethyl acetate, 3/1-1/1) to obtain intermediate product.
S03: the intermediate was placed in a 50mL round bottom flask. The flask was evacuated and filled with nitrogen. Then, 20mL of TFA/DCM (9/1;V/V) was added and the solvents were mixed under nitrogen. The reaction solution was observed to change from a colorless transparent solution to a reddish brown solution. The reaction was monitored by TLC. The solution was concentrated to 3mL by rotary evaporation under vacuum.
S04: then, the mixture was slowly added dropwise to methyl tert-butyl ether, and a large amount of white solid precipitate was observed. The solid precipitate was sonicated. It was then centrifuged for 3min and the supernatant removed. And then washing the solid with methyl tertiary butyl ether for 3 times, centrifuging to recover a solid part, washing with methanol to remove impurities, and filtering to obtain a pure product. The dried white solid product powder was purified by hydrogen nuclear magnetic resonance 1 H NMR, bruker Magnet System, karlsruhe, germany,400MHz/54 mm) and mass spectrometry (MS, waters LC-MS System, milford, mass., USA).
The molecular structure of the product estradiol derivative is shown in figure 1, which 1 The H NMR spectrum is shown in FIG. 2, and the MS spectrum is shown in FIG. 3. 1 H NMR and MS characterization indicated that this example successfully synthesized the estradiol derivative GDT. At the position of 1 Each peak of GDT was found in H NMR (FIG. 2, at 500. Mu.L DMSO-d 6 And 5 μl formic acid), and 429.28Da following GDT in sizeMolecular structure (fig. 3). 1 H NMR(400MHz,DMSO-d 6 ):δ8.99(br s,2H),7.36(d,J＝8.6Hz,1H),7.24(s,1H),7.04(d,J＝8.5Hz,1H),6.97–6.78(m,1H),6.51(dt,J＝7.1,3.2Hz,1H),6.44(d,J＝2.6Hz,1H),4.92(q,J＝8.2Hz,1H),4.29–3.55(m,3H),3.17(s,1H),2.90–2.61(m,2H),2.41–0.98(m,18H),0.83(d,J＝7.0Hz,2H),0.70(d,J＝7.8Hz,1H).MS(ESI + ):calculated for C 24 H 36 N 4 O 3 ,428.28；found 429.28[M] + .
Example 2
This example provides a estradiol derivative (GDT) and a method for preparing the same.
The preparation method of the estradiol derivative (GDT) comprises the following steps:
s01: boc-Arg (Pbf) -OH (2.63 g,5mmol,1.0equiv, CAS #200124-22-7, shanghai Aba Ding Shenghua technologies Co., ltd.), 17 beta-estradiol (1.3 equiv, CAS #50-28-2, shanghai Aba Ding Shenghua technologies Co., ltd.) and 4-dimethylaminopyridine (DMAP, 1.2 equiv) were added to the reaction flask at room temperature. The reaction flask was then evacuated and filled with nitrogen. 20mL of methylene chloride was added under nitrogen.
S02: EDC. HCl (1.3 equiv) was then added to the ice bath and the reaction was allowed to warm for 8h. The progress of the reaction was monitored by Thin Layer Chromatography (TLC) and after completion of the reaction, 60mL of water was added to quench the reaction. The mixture was extracted twice with Dichloromethane (DCM) and the combined organic phases were dried and then rotary evaporated in vacuo to give the crude product. Purifying with silica gel column (eluting condition, petroleum ether/ethyl acetate, 3/1-1/1) to obtain intermediate product.
S03: the intermediate was placed in a 50mL round bottom flask. The flask was evacuated and filled with nitrogen. Then, 20mL of TFA/DCM (9/1;V/V) was added and the solvents were mixed under nitrogen. The reaction solution was observed to change from a colorless transparent solution to a reddish brown solution. The reaction was monitored by TLC. The solution was concentrated to 4mL by rotary evaporation under vacuum.
S04: then, the mixture was slowly added dropwise to methyl tert-butyl ether, and a large amount of white solid precipitate was observed. The solid precipitate was sonicated. It was then centrifuged for 4min and the supernatant removed. And then washing the solid with methyl tertiary butyl ether for 3 times, centrifuging to recover a solid part, washing with methanol to remove impurities, and filtering to obtain a pure product.
Example 3
This example provides a estradiol derivative (GDT) and a method for preparing the same.
The preparation method of the estradiol derivative (GDT) comprises the following steps:
s01: boc-Arg (Pbf) -OH (2.63 g,5mmol,1.0equiv, CAS #200124-22-7, shanghai Aba Ding Shenghua technologies Co., ltd.), 17 beta-estradiol (1.5 equiv, CAS #50-28-2, shanghai Aba Ding Shenghua technologies Co., ltd.) and 4-dimethylaminopyridine (DMAP, 1.5 equiv) were added to the reaction flask at room temperature. The reaction flask was then evacuated and filled with nitrogen. 20mL of chloroform was added under nitrogen.
S02: EDC. HCl (1.5 equiv) was then added to the ice bath and the reaction was allowed to warm for 12h. The progress of the reaction was monitored by Thin Layer Chromatography (TLC) and after completion of the reaction, 80mL of water was added to quench the reaction. The mixture was extracted twice with chloroform and the combined organic phases were dried and then rotary evaporated in vacuo to give the crude product. Purifying with silica gel column (eluting condition, petroleum ether/ethyl acetate, 3/1-1/1) to obtain intermediate product.
S03: the intermediate was placed in a 50mL round bottom flask. The flask was evacuated and filled with nitrogen. Then, 20mL of TFA/DCM (9/1;V/V) was added and the solvents were mixed under nitrogen. The reaction solution was observed to change from a colorless transparent solution to a reddish brown solution. The reaction was monitored by TLC. The solution was concentrated to 5mL by rotary evaporation under vacuum.
S04: then, the mixture was slowly added dropwise to methyl tert-butyl ether, and a large amount of white solid precipitate was observed. The solid precipitate was sonicated. It was then centrifuged for 5min and the supernatant removed. And then washing the solid with methyl tertiary butyl ether for 3 times, centrifuging to recover a solid part, washing with methanol to remove impurities, and filtering to obtain a pure product.
2. Application example
In vitro cytotoxicity experiments, the application does not find that GDT drug saturated solution has obvious effect on HELA cellsIs a toxic property. Subsequently, 40C 57/BL6 female mice of 8 weeks of age were taken for in vivo studies. All animals were kept at 25 ℃ for a cycle of 12 hours light and 12 hours dark, with water and food ad libitum. All animal experiments were performed according to the protocol approved by the SPF laboratory animal center ethics Committee for animal experiments (IACUC-2021-0102) of Shenzhen City Tuo Pu Biotechnology Co. Based on the duration of the normal oestrus cycle of the mice (2-8 days) and the pair of mouse reproductive systems E 2 The experimental design of the drug is as follows: all mice were fed adaptively for 4 days. Then, the administration was performed by intraperitoneal injection twice daily. After 4 days of continuous administration, mice were anesthetized with isoflurane and blood was collected from the eyeballs. After cervical dislocation sacrifice, the mouse ovary and uterus tissues were taken for HE staining and immunohistochemical analysis. In addition, mouse serum was collected, and NO and its end products (NO 2 - ) The formation in blood. All mice were divided into 5 groups (8 per group) according to the structural features of the estradiol derivative (GDT) and the purpose of the application. Group 1 was dosed with 15 μg/mouse/day GDT, group 2 was dosed with 10 μg/mouse/day E 2 Administration, group 3 was administered with 6. Mu.g/mouse/day L-Arg, group 4 was administered with 10. Mu.g/mouse/day E 2 And 6. Mu.g/mouse/day L-Arg were mixed and administered, and group 5 was treated with injection solvent (200. Mu.L) as a control. The drug was dissolved in aqueous HCl (ph=4.0) at the time of administration.
Detection of NO in mouse serum
The collected mouse blood was left at room temperature for 2 hours. Then, it was centrifuged at 3500rpm for 15 minutes, and the supernatant (serum) was stored at-20 ℃. The experiment used classical Griess methods (Shanghai Biyun biotechnology Co., ltd.) to measure NO production of GDT and other drugs. After each set of experimental treatments, mouse serum was collected to detect NO metabolite concentrations. Mouse serum was collected from heart blood. The specific method comprises the following steps: 50 microliters of serum samples were mixed with 50 microliters of Griess reagent I in 96-well plates and shaken at room temperature in the dark for 10 minutes. To the mixture was added 50 μ L Griess Reagent II and the solution mixture was incubated at room temperature for 10 minutes in the dark. Finally, the mixture was measured using an ultraviolet-visible spectrophotometer (540 nm). Using NaNO 2 Standard sample (0.05-2)5 μm) was established to calculate NO concentration.
The NO detection in the mouse serum is shown in FIG. 4. The results showed that the test piece was similar to group 4 (E 2 ) Group 1 (GDT), group 2 (L-Arg) and group 3 (E) as compared to group 5 (control) 2 +L-Arg) serum concentration of NO. In addition, the difference in NO concentration was not significant in these three groups (groups 1, 2, 3) compared with groups 4, 5.
Histological analysis
The collected mouse organs were fixed with paraformaldehyde and paraffin sections were prepared. The sections were then dewaxed for HE and Immunohistochemical (IHC) staining. For immunohistochemical evaluation, the present application performed antigen retrieval, endogenous peroxidase blocking, serum blocking, primary antibody incubation, secondary antibody incubation, DAB chromogenic and nuclear counterstain on these tissue sections. We used CD34, CD105 and iNOS (inducible nitric oxide synthase) as biomarker targets. The primary antibody (rabbit anti-mouse, abcam) concentration was set as: CD34 is 1:300, CD105 is 1:100, iNOS is 1:100. All HE and IHC slides were dehydrated and mounted for microscopy and image analysis. The processed image data of IHC slices were calculated as Average Optical Density (AOD) for quantitative evaluation of ImageJ.
Statistical analysis
Data were analyzed by Graphpad Prism statistical analysis program using paired t-test. Results are expressed as standard error of the mean (SEM). A value of p.ltoreq.0.05 is considered statistically significant.
All HE and IHC slides were dehydrated and mounted for microscopy and image analysis (fig. 5-13). To observe proliferation of tissues and vessels, the present application incorporates CD34 and CD105 markers for immunohistochemical analysis. Further, iNOS is set as an index that NO signal pathway is affected and NOs is consumed. The CD34, CD105 positive regions are mainly concentrated in sites rich in new blood vessels, such as the corpus luteum and neointimal tissue, while iNOS positive regions are mainly concentrated in sites rich in epithelial and endothelial cells, such as follicular granulosa cells (fig. 5) and uterine villi (fig. 9). In ovarian sections, mice from different treatment groups showed different results. L-ArgGroup (group 2) CD34 was significantly more expressed than the other groups (groups 1, 3, 4, 5) (FIG. 6), whereas GDT group (group 1), L-Arg group (group 2) and E 2 CD105 expression was significantly higher in the +L-Arg group (group 3) than in the other groups (groups 4, 5). GDT group, L-Arg group and E 2 In the +L-Arg group, mice showed a trend of increased expression of CD34 and CD105 due to the introduction of guanidyl NO donor (FIG. 7). In contrast, their iNOS expression (groups 1, 2, 3) exhibited inhibitory characteristics (fig. 8). In endometrium sections (FIG. 9), GDT groups, E 2 +L-Arg group and E 2 The endometrium of the group was significantly thickened (fig. 10). Accordingly, the expression of CD34 and CD105 was also higher in the three groups (groups 1, 3, 4), and the expression of CD34 was significantly higher than in the control group and the L-Arg group (FIGS. 11 and 12). In endometrial tissue, E 2 Group E 2 The expression of +L-Arg group iNOS was significantly higher than that of the other three groups (groups 1, 2, 5) (FIG. 13).
The mouse experiment shows that the synthesized estradiol derivative GDT has E 2 Is a part of the biological functions of (a). And in use with GDT or E 2 After treatment, the endometrium of the mice was significantly thickened compared to the L-arginine treatment and the blank. In humans, at least 5 days of estrogen exposure are required to build up a sufficiently thick endometrium to allow embryo implantation. Similar to human observations, the proliferative response of the mouse epithelial cells was visible after 1 day of treatment. In GDT-dosed mice, there were some beneficial changes in endometrial preparation, including a significant increase in expression of biomarkers associated with vascular and endometrial proliferation. Furthermore, the results also indicate that NO donors (GDT or L-Arg) reduce the abundance of iNOS expression in tissues, which further demonstrates that NO exerts its biological effect. Vasodilation and blood flow increasing effects of NO with E 2 In combination with the promotion of endometrial growth, the present application has found that GDT is a promising small molecule drug that enhances normal endometrial and ovarian function in mammals and may play a role in promoting reproductive system function.
The foregoing examples illustrate only a few embodiments of the application, which are described in detail and are not to be construed as limiting the scope of the application. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the application, which are all within the scope of the application. Accordingly, the scope of protection of the present application is to be determined by the appended claims.
Claims (6)
1. An estradiol derivative characterized by having a structure represented by the following formula (1):
。
2. a process for the preparation of an estradiol derivative as defined in claim 1, characterized in that said process comprises the steps of:
preparing an organic solvent mixed solution containing Boc-Arg (Pbf) -OH, 17 beta-estradiol and 4-dimethylaminopyridine under an inert atmosphere;
EDC and HCl are added into the mixed solution to perform a first reaction to obtain an intermediate product; the temperature of the first reaction is 0-10 ℃;
adding a TFA/DCM mixed solvent into the intermediate product under an inert atmosphere for a second reaction to obtain a solution containing the product;
the volume ratio of TFA and DCM in the TFA/DCM mixed solvent is 9:1, a step of;
wherein the estradiol derivative has a structure represented by the following formula (1):
。
3. the method of claim 2, further comprising a product purification treatment step of: the product-containing solution was concentrated and then added dropwise to methyl tert-butyl ether to obtain a precipitated product.
4. The preparation method according to claim 2, wherein the molar ratio of Boc-Arg (Pbf) -OH, 17 β -estradiol, 4-dimethylaminopyridine and edc.hcl is 1: (1.1 to 1.5): (1.0 to 1.5): (1.1 to 1.5).
5. The preparation method according to claim 2, wherein the organic solvent is dichloromethane or chloroform.
6. The method of claim 2, wherein the first reaction time is 8 to 12 hours.
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| CN110563598A (en) * | 2019-08-26 | 2019-12-13 | 沈阳药科大学 | Amino acid derivative and salt thereof, preparation and application |
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| CN110563598A (en) * | 2019-08-26 | 2019-12-13 | 沈阳药科大学 | Amino acid derivative and salt thereof, preparation and application |
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| Title |
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| Jing Fan et al..The effect of a novel small molecule estradiol guanidine derivative on the ovary and uterus of female mice.《Science of Advanced Materials》.2022,第14卷(第2期),263-271. * |
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