WO2023165381A1

WO2023165381A1 - Acyl-based statin compound with lipase inhibitory activity, preparation method therefor, and use thereof

Info

Publication number: WO2023165381A1
Application number: PCT/CN2023/077498
Authority: WO
Inventors: 王勇; 刘海利; 许建林
Original assignee: 上海临贤生物科技有限公司
Priority date: 2022-03-01
Filing date: 2023-02-21
Publication date: 2023-09-07
Also published as: CN116731091A

Abstract

Provided are an acyl-based statin compound with lipase inhibitory activity, a preparation method therefor, and use thereof in controlling body weight and treating obesity.

Description

Acyltadine compound with lipase inhibitory activity, preparation method and application thereof

technical field

The invention belongs to the field of biomedicine, and more specifically, the invention relates to acyltadine compounds having lipase inhibitory activity and a preparation method thereof, and the role of such lipase inhibitors in preventing and treating obesity, type 2 diabetes and cancer induced by obesity applications in diseases.

Background technique

Body mass index (BMI) greater than 30kg/m ² or more than 20% of the standard body weight is called obesity. It is a chronic disease of rapid accumulation of body fat due to energy intake exceeding metabolic expenditure. Studies have shown that obesity can damage the organs and systems of the human body, and is a risk factor for various diseases such as type 2 diabetes, hypertension, cardiovascular disease and cancer. In recent years, due to excessive intake of high-calorie substances and lack of physical exercise, the number of overweight people has increased dramatically. According to statistics, in 2019, the number of obese people in China exceeded 250 million, ranking first in the world. Diseases caused by obesity consume a large amount of medical expenses, making it a public health problem that has to be ignored. Therefore, it is of great significance to carry out basic research related to obesity.

Currently, commonly used drugs for treating obesity include orlistat, liraglutide, phentermine hydrochloride/topiramate, lorcaserin, and naltrexone/bupropion combination, etc. Although these drugs can reduce the weight of obese people and reduce the risk of other diseases to a certain extent, their adverse reactions restrict their clinical application. For example, both orlistat and liraglutide can cause unpleasant gastrointestinal effects. Moreover, long-term use of orlistat can damage the liver and increase the incidence of colon cancer. The most serious adverse reaction of phentermine hydrochloride is coronary artery spasm, which can cause hemorrhagic and ischemic stroke. The most common side effect of lorcaserin is headache, while the naltrexone/bupropion combination mainly has adverse reactions such as headache, dizziness, nausea and vomiting, diarrhea, constipation and insomnia. In addition, the diet drug rimonabant, which caused severe mental illness, and sibutramine, which increased the risk of cardiovascular disease, were discontinued in 2009 and 2010, respectively.

Therefore, new candidate compounds with high efficiency and low toxicity are still needed in this field to provide a scientific basis for the development of weight loss drugs, meet the needs of overweight people for healthy weight loss, and reduce the risk of other diseases caused by obesity.

Contents of the invention

The object of the present invention is to provide acyltadine compounds with lipase inhibitory activity and a preparation method, as well as the application of the lipase inhibitors in weight control and obesity treatment.

In the first aspect of the present invention, there is provided a compound represented by formula (I) or a pharmaceutically or food-acceptable salt or ester thereof, isomers, racemates, solvates, hydrates or precursors,

Wherein, n is 2, 3 or 4;

m is 2, 3 or 4;

Each R ₁ , R ₂ , R ₃ , R ₄ , R ₅ is each independently selected from the following group: hydrogen, C ₁ -C ₄ alkyl or -OZ; wherein each Z is independently selected from the following group: hydrogen, C ₃ -C ₆ acyl; and, at least one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ is -OZ;

Each R ₁ ′, R _{2 ′} , R ₃ ′, R ₄ ′, R ₁ ″, R ₂ ″, R ₃ ″, R ₄ ″, Ra are each independently selected from the following group: hydrogen, hydroxyl, C ₁ -C ₄ alkyl, halogen;

Each Y ₂ and Y ₃ are independently selected from the following group: -O-, -NH-;

When m is 3 and n is 2, R is _not hydroxyl or

In one or more embodiments, each R ₁ , R ₂ , R ₄ , R 1 ′, R ₂ _′ , R ₄ ′, R _{1 ″, R 2} _″ , R ₄ ″, Ra are each independently selected from the following Group: hydrogen, C ₁ -C ₂ alkyl or hydroxy.

In another preferred example, each R ₂ is independently C ₁ -C ₂ alkyl. Place

In another preferred example, each R ₂ is independently C ₁ -C ₂ alkyl.

In one or more embodiments, the compound of formula (I) has the structure shown in formula (I-1) or formula (I-2),

Wherein, R ₃ is as defined above.

In one or more embodiments, the compound of formula (I) is selected from:

In the second aspect of the present invention, there is provided a method for preparing the compound of formula (I), comprising: cultivating Streptomyces sp. HO1518, and isolating the compound of formula (I) from its culture product. Preferably, the culture product is a culture supernatant.

In one or more embodiments, said isolating comprises purifying the culture product and isolating different compounds of formula (I) from different eluents. The purification is preferably gradient elution.

In one or more embodiments, the Streptomyces HO1518 Streptomyces sp. HO1518 is Streptomyces HO 1518 Streptomyces sp. HO1518 with a preservation number of CCTCC NO: M 2018176.

In a third aspect of the present invention, a composition is provided, comprising:

(1) The compound of formula (I) as described in the first aspect of the present invention or its pharmaceutically or food acceptable salt or ester, isomer, racemate, solvate, hydrate or precursors; and

Optional (2) pharmaceutically or food-acceptable excipients.

In one or more embodiments, the composition may contain other ingredients required as pharmaceuticals or foods. For example, proteins, lipids, carbohydrates, dietary fibers, vitamins, and the like may be contained.

In one or more embodiments, the composition is selected from daily chemical products, foods, health products, and pharmaceutical compositions.

In a fourth aspect of the present invention, a kit is provided, characterized in that, comprising:

(1) The compound of formula (I) as described in the first aspect of the present invention or its pharmaceutically or food-acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor, Or as described by the third party of the present invention composition; and

Optionally (2) using or administering other agents as needed; and

Optional (3) instructions.

In a fifth aspect of the present invention, there is provided the compound of formula (I) described herein or its pharmaceutically or food-acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor Use in the manufacture of a product for inhibiting lipase activity, controlling body weight, improving fat utilization, or preventing, alleviating or treating a disease or condition that would benefit from reduced fat absorption.

In one or more embodiments, diseases or conditions that would benefit from reduced fat absorption include hyperlipidemia, obesity, or fat storage lipid metabolism disorders.

In one or more embodiments, the lipase is selected from animal lipase, plant lipase, microbial lipase. In one or more embodiments, the lipase is animal lipase, preferably pancreatic lipase.

In one or more embodiments, the product includes daily chemical products, food, health products, pharmaceutical compositions or kits.

In a sixth aspect of the present invention, a non-diagnostic, non-therapeutic method for inhibiting lipase activity is provided, comprising: making the compound of formula (I) described herein or its pharmaceutically or food-acceptable salt or ester, iso Constructs, racemates, solvates, hydrates or precursors are in contact with lipase-containing samples, thereby inhibiting the activity of lipase.

In one or more embodiments, the method of inhibiting lipase activity is a non-diagnostic or therapeutic method.

In a seventh aspect of the present invention, there is provided a method of controlling body weight, improving fat utilization, or preventing, alleviating or treating a disease or condition benefiting from reduced fat absorption, comprising: administering to a subject in need thereof an effective amount of The compound represented by formula (I) or its pharmaceutically or food acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor.

In the eighth aspect of the present invention, the Streptomyces HO 1518 Streptomyces sp. HO1518 or its culture with the preservation number CCTCC NO: M 2018176 is provided for producing the compound represented by the formula (I) described herein.

In the ninth aspect of the present invention, there is provided a method for improving the inhibitory activity of amino oligosaccharides on lipase The method comprises: adding at least one acyl group to the amino oligosaccharide compound.

Description of drawings

Figure 1A, a diagram of the cleavage pattern of acyltadine compounds 2-5.

Figure 1B, HRESIMS/MS spectrum of compound 2.

Figure 1C, HRESIMS/MS spectrum of compound 3.

Figure 1D, HRESIMS/MS spectrum of compound 4.

Figure 1E, HRESIMS/MS spectrum of compound 5.

Fig. 2A, a schematic diagram of the cleavage mode of acyltadine compounds 6-8.

Figure 2B, HRESIMS/MS spectrum of compound 6.

Figure 2C, HRESIMS/MS spectrum of compound 7.

Figure 2D, HRESIMS/MS spectrum of compound 8.

Figure 3, the inhibitory effect of acyltadine compounds on pancreatic lipase.

Figure 4. The general structural formula of the alcatrazine family compounds in Streptomyces sp.HO1518. (A) Aca-glu type; (B) glu-Aca-glu type; (C) incAca-glu type.

Detailed ways

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (such as the embodiments) can be combined with each other to form a preferred technical solution.

The present invention discloses a new class of acyl hepatidine compounds for the first time, and finds that the acyl hepatidine compounds have high-efficiency lipase inhibitory activity.

The above-mentioned acyl hepatidine compound has a compound represented by formula (I),

Wherein, n is 2, 3 or 4;

m is 2, 3 or 4;

Each R ₁ , R ₂ , R ₃ , R ₄ , R ₅ is independently selected from the group consisting of hydrogen, C ₁ -C ₄ alkyl or -OZ; In, each Z is independently selected from the following group: hydrogen, C ₃ -C ₆ acyl; and, at least one of R ₁ , R ₂ , R ₃ , R ₄ , R ₅ is -OZ;

Each of Y ₂ and Y ₃ is independently selected from the following group: -O-, -NH-.

In the study of the compounds in the Streptomyces strain HO 1518 Streptomyces sp. HO1518, the inventors predicted and discovered a series of Aka The structure of the tadine family compound (compound shown in formula (I)) has been deeply studied, and it has been unexpectedly found that it has a high-efficiency lipase inhibitory activity. On this basis, the present invention has been completed.

The present invention also includes its pharmaceutically or food acceptable salts, isomers, racemates, solvates, hydrates or precursors of the compounds of the above formula (I), as long as they also have the same properties as those of the formula (I) The compounds have the same or substantially the same function.

As used herein, the term "alkyl" alone or in combination with other terms refers to a saturated aliphatic alkyl group, including straight or branched chain alkyl groups of 1-20 carbon atoms. Preferably, the alkyl group refers to a medium alkyl group containing 1-10 carbon atoms, such as methyl, ethyl, propyl, 2-isopropyl, n-butyl, isobutyl, tert-butyl, pentyl and Similar to alkyl. More preferably, it refers to a lower alkyl group having 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-isopropyl, n-butyl, isobutyl, t-butyl and the like. Alkyl groups may be substituted or unsubstituted. When substituted, the number of substituents is 1 or more, preferably 1-3, more preferably 1 or 2, and the substituents are independently selected from halogen, hydroxyl, lower alkoxy and aryl.

The term "alkenyl" as used herein includes straight and branched chain hydrocarbon groups containing at least one carbon-carbon double bond and 2-4 carbon atoms, preferably 2-3 carbon atoms.

The term "alkynyl" as used herein includes straight and branched chain hydrocarbon groups containing at least one carbon-carbon triple bond and 2-4 carbon atoms, preferably 2-3 carbon atoms.

The term "halogen" as used herein refers to F, Cl, Br, or I.

As used herein, the term "substituted" refers to a compound having a substituent comprising at least one carbon atom, nitrogen atom, oxygen atom or sulfur atom with one or more hydrogen atoms. If a substituent is described as being "substituted," it means that a non-hydrogen substituent occupies the position of a hydrogen on a carbon, nitrogen, oxygen, or sulfur. In the present invention, the alkyl, alkenyl and alkynyl groups may be substituted; for example, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl. Unless otherwise defined, a substituted group has a substituent at one or more appropriate positions, and when more than one position is substituted, the substituents at each substituted position may be the same or different. The substituents of the substituted acyl include: hydroxyl, C1-C4 alkyl, C2-C4 Alkenyl, C2-C4alkynyl, halogen.

The term "isomer" as used herein includes: geometric isomers, enantiomers, diastereomers (such as cis-trans isomers, conformational isomers). The compounds disclosed in the present invention or their salts may contain one or more asymmetric centers, so there will be enantiomers, diastereoisomers and other stereoisomeric forms that can be defined, and can be divided according to stereochemistry. is (R)- or (S)-, (D)- or (L)- for amino acids. The present invention is meant to include all such possible isomers, as well as racemic and optically pure forms. Optically active (+) and (-), (R)- and (S)- or (D)- and (L)-isomers can be prepared by chiral synthons or chiral reagents, or by conventional techniques Such as the use of high performance liquid phase chiral column to separate the preparation. When the compounds described herein contain olefinic double bonds or other centers of geometric asymmetry, unless otherwise stated, it is meant that the compounds include both E and Z geometric isomers. Likewise, all tautomers are included.

As used herein, the expression method of the ellipsis "..." in the general formula is well known to those skilled in the art, which means that there is any, one or more omitted units. The unit is structurally the same as the unit before "..." or corresponding.

In the present invention, "food or pharmaceutically acceptable" ingredients are substances that are suitable for humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergic reactions), that is, substances with a reasonable benefit/risk ratio.

The "food or pharmaceutically acceptable salts" mentioned herein include acid salts and basic salts.

"Pharmaceutically acceptable acid salt" refers to a salt that can maintain the biological activity and properties of the free base, and such salts will not have undesired biological activity or other changes. Such salts may be formed from inorganic acids such as, but not limited to, hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, and the like. Such salts may also be formed from organic acids such as, but not limited to, acetic acid, dichloroacetic acid, adipic acid, alginic acid, ascorbic acid, aspartic acid, benzenesulfonic acid, benzoic acid, 4-acetamidobenzoic acid, camphoric acid, Camphorsulfonic acid, capric acid, caproic acid, caprylic acid, carbonic acid, cinnamic acid, citric acid, cyclamic acid, dodecylsulfonic acid, 1,2-ethanedisulfonic acid, ethanesulfonic acid, isethionic acid , formic acid, fumaric acid, galactaric acid, gentisic acid, glucoheptonic acid, gluconic acid, glucuronic acid, glutamic acid, glutaric acid, 2-oxoglutaric acid, glycerophosphate , glycolic acid, hippuric acid, isobutyric acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, malonic acid, mandelic acid, methanesulfonic acid, mucic acid, naphthalene-1,5-di Sulfonic acid, 2-naphthalenesulfonic acid, 1-naphthol-2-carboxylic acid, niacin, oleic acid, orotic acid, oxalic acid, palmitic acid, pamoic acid, propionic acid, pyroglutamic acid, pyruvic acid, water Cylic acid, 4-aminosalicylic acid, sebacic acid, stearic acid, succinic acid, tartaric acid, thiocyanic acid, p-toluenesulfonic acid, trifluoroacetic acid, undecylenic acid and similar acids.

"Pharmaceutically acceptable basic salt" refers to a salt that can maintain the biological activity and properties of the free acid, and such salts will not have undesired biological activity or other changes. These salts are prepared by adding an inorganic or organic base to the free acid. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum and the like. Preferred inorganic salts are ammonium salts, sodium salts, potassium salts, calcium salts and magnesium salts. Salts derived from organic bases include, but are not limited to, primary, secondary, and tertiary ammonium salts, substituted amines include naturally substituted amines, cyclic amines, and basic ion exchange resins such as ammonia, isopropylamine, trimethylamine, di Ethylamine, triethylamine, tripropylamine, diethanolamine, ethanolamine, tannol, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, coffee Inine, procaine, halamine, choline, betaine, phenethylbenzylamine, N,N'-bisbenzylethylenediamine, ethylenediamine, glucosamine, methylglucosamine, theobromine, three Ethanolamine, tromethamine, purine, piperazine, piperidine, N-ethylpiperidine, polyamide resin, and similar structures. Preferred organic bases are isopropylamine, diethylamine, ethanolamine, trimethylamine, dicyclohexylamine, choline and caffeine.

Often crystallization will result in solvated products of the disclosed compounds. As used herein, the term "solvate" refers to a polymer comprising one or more molecules of a compound disclosed herein and one or more solvent molecules. The solvent may be water, in which case the solvate may be a hydrate. Alternatively, the solvent may also be an organic solvent. Therefore, the compounds disclosed in this patent can exist as hydrates, including monohydrates, dihydrates, hemihydrates, sesquihydrates, trihydrates, tetrahydrates and similar structures, and can also be used as corresponding solvated products exist. The compounds disclosed herein may be true solvates, while in other cases, the compounds disclosed herein may retain some water only, or a mixture of water and some solvent.

The "precursor of the compound" refers to that when taken in an appropriate way, the precursor of the compound undergoes metabolism or chemical reaction in the patient's body and converts into a compound of structural formula (I), or a compound of chemical structural formula (I). A salt or solution of a compound.

In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Accordingly, the terms "consisting essentially of" and "consisting of" are included in the term "comprising".

Preparation

Those skilled in the art will understand that, after knowing the structure of the compound of the present invention, the compound of the present invention can be obtained by various methods well known in the art, using known raw materials, such as chemical synthesis or from organisms (such as microorganisms, especially is the method for extracting in Streptomyces), and these methods are all included in the present invention.

As a preferred mode of the present invention, a method for preparing a compound represented by formula (I) of the present invention is provided, the method comprising: cultivating Streptomyces HO 1518 Streptomyces sp.HO1518, obtaining a culture product (preferably culturing supernatant), from which the compound of formula (I) is isolated. Afterwards, the culture product is purified, and different compounds of formula (I) are separated from different eluents. The collection and purification of products are well known in the art, for example, resin adsorption and elution methods can be used.

Other methods of preparing compounds of formula (I) are also included in the present invention, such as linking sugar units by chemical synthesis. The synthesized compound can be further purified by column chromatography, high performance liquid chromatography and the like.

combination

The present invention also provides a composition comprising the compound of formula (I) described herein or its pharmaceutically or food-acceptable salt, isomer, racemate, solvate, hydrate or precursor, and pharmaceutically or food-acceptable excipients. The composition can be a food, a health care product, or a pharmaceutical composition.

In the present invention, "food or pharmaceutically acceptable excipients" are used to combine the compound of formula (I) of the present invention or its pharmaceutically or food acceptable salts, isomers, racemates, solvates A pharmaceutically or food-acceptable carrier, solvent, suspending agent or excipient for delivering substances, hydrates or precursors to animals or humans. Excipients can be liquid or solid and include but are not limited to: pH adjusters, surfactants, carbohydrates, adjuvants, antioxidants, chelating agents, ionic strength enhancers, preservatives, carriers, glidants, sweeteners , dye/colorant, flavor enhancer, wetting agent, dispersant, suspending agent, stabilizer, isotonic agent, solvent or emulsifier. In some embodiments, pharmaceutically acceptable excipients may include one or more inactive ingredients, including but not limited to: stabilizers, preservatives, additives, adjuvants, sprays, compressed air or other suitable gases, Or other suitable inactive ingredients used in combination with medicinal compounds. More specifically, suitable excipients may be excipients commonly used in the art for administration of plant extract components or nucleic acids.

Typically, the composition will contain a therapeutically effective amount of an agent described herein. A therapeutically effective amount refers to a dose that can achieve treatment, prevention, alleviation and/or alleviation of a disease or condition in a subject. The therapeutically effective dose can be determined according to factors such as the patient's age, sex, disease and its severity, and other physical conditions of the patient. A therapeutically effective amount may be administered as a single dose, or may be administered in multiple doses in accordance with an effective treatment regimen. Herein, a subject or a patient generally refers to a mammal, especially a human. Exemplarily, the composition contains, for example, 0.001-50% by weight, preferably 0.01-30%, more preferably 0.05-10% of the compound represented by formula (I) or its pharmaceutically or food-acceptable Salts, isomers, racemates, solvates, hydrates or precursors.

Compositions described herein may be used in combination with other agents for weight management or therapeutic benefit from reduced fat absorption. Dosages of other agents to be administered can be determined by those skilled in the art.

In one or more embodiments, the composition may also contain protein, lipid, carbohydrate, dietary fiber, vitamins and the like. Proteins such as milk protein (whole milk protein, sodium caseinate, calcium caseinate, etc.) and their hydrolysates, other animal proteins (egg protein, gelatin, etc.) things. The composition of the present invention may contain protein as a main ingredient. The total protein content can be appropriately determined, but if the purpose is to ingest 1/3 of the daily required intake, it is preferably about 13 to 30 g/400 ml. Vitamins can also be added vitamins A, B1, B2, B6, B12, C, D, E, K2, niacin, pantothenic acid, folic acid, etc., which can be added alone or mixed. The composition of the present invention may also contain dry yeast (eg brewer's yeast, baker's yeast, etc.).

The dosage form of the composition of the present invention can be varied, as long as the active ingredient can effectively reach the mammalian body, it can be made into a unit dosage form. Dosage forms can be selected from, for example, gels, aerosols, tablets, capsules, powders, granules, syrups, solutions, suspensions, injections, powders, pills, controlled immediate releases, infusions, suspensions, and the like. According to the type of disease to be treated by the compound of the present invention, those skilled in the art can choose a convenient dosage form. From the standpoint of ease of preparation and storage, preferred compositions are solid compositions, especially tablets and solid-filled or liquid-filled capsules. The compounds of the present invention or compositions thereof may also be stored in sterile devices suitable for injection or infusion. The compounds of the present invention or compositions thereof may also be stored in suitable containers and placed in kits or kits.

The composition can also be a daily chemical product, such as shampoo, shower gel, cosmetics, washing powder and the like.

method and use

The present inventors found in the research that the compound represented by formula (I) has a high-efficiency inhibitory effect on lipase, so it is a good lipase inhibitor. As lipase inhibitors, the compounds can control body weight, improve fat utilization, or prevent, alleviate or treat diseases or conditions that would benefit from reduced fat absorption. As used herein, "disease or condition benefiting from reduced fat absorption" generally refers to a disease, disorder or condition caused by accumulation of fat in an organ, tissue or cell due to excessive fat absorption by an organism, such as hyperlipidemia, obesity, obesity Type 2 diabetes and obesity-induced cancers (such as liver cancer, pancreatic cancer, colorectal cancer, ovarian cancer, etc.), cardiovascular diseases (coronary heart disease, hypertension, heart failure, etc.), etc. Obesity is a common complication of type 2 diabetes. It will significantly increase insulin resistance and pancreatic β-cell load, reduce the body's ability to metabolize glucose, lead to further increase in blood sugar, and seriously affect the quality of life of diabetic patients. At the same time, overweight is also an important risk factor for inducing cancer, cardiovascular disease and other diseases, which makes the body of diabetic patients weaker and seriously affects their health. The compound of the present invention inhibits the lipase activity in the gastrointestinal tract, prevents the degradation of fat in food, excretes the undecomposed lipase, reduces the intake of fat, thereby controlling or reducing the patient's weight and improving the patient's physical condition .

Therefore, in a non-diagnostic or therapeutic aspect, the present invention provides a method for inhibiting lipase activity, comprising: making the compound of formula (I) described herein or its pharmaceutically or food acceptable salt, isomer, external The racemate, solvate, hydrate or precursor is contacted with the lipase-containing sample, thereby inhibiting the activity of the lipase. In addition, the present invention also provides a method for controlling body weight, improving fat utilization, or preventing, alleviating or treating diseases or conditions benefiting from reduced fat absorption, comprising: administering an effective amount of the formula (I) described herein to a subject in need thereof The indicated compound or its pharmaceutically or food acceptable salt, isomer, racemate, solvate, hydrate or precursor.

Specifically, "effective amount" refers to the injection amount that can produce therapeutic functions for humans or animals and can be accepted by animals and humans. For example, in a liquid combination drug, the concentration of the peptide can be 20 ng/mL Above, 50ng/mL, above 100ng/mL, etc. The effective amount will vary with the mode of administration and the severity of the disease being treated. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as the exigencies of the therapeutic situation dictate.

The administration methods of the compounds of the present invention may include, but are not limited to, subcutaneous injection, transdermal injection, implantation, topical administration, intramuscular injection, sustained release administration, oral administration, and the like. Those skilled in the art are aware of other agents needed to administer the drug to a subject under different modes of administration, doses, sites of administration, and the like. Such as dressings, solvents (such as water), etc.

The kit for implementing the above method comprises the compound represented by formula (I) described herein or its pharmaceutically or food-acceptable salt, isomer, racemate, solvate, hydrate or precursor, or The compositions, and optionally other items required for their administration, and optionally instructions. Such other items are, for example, measuring instruments, containers such as syringes, etc. required for using or administering the composition in various dosage forms. The instructions are for directing the use or administration process. Therefore, the present invention also provides the compound of formula (I) described herein or its pharmaceutically or food acceptable salt, isomer, racemate, solvate, hydrate or precursor in the preparation of inhibiting lipase activity , use in a product for controlling body weight, improving fat utilization, or preventing, alleviating or treating a disease or condition that would benefit from reduced fat absorption. The product can be daily chemical products, food, health products, pharmaceutical compositions or kits.

The present invention is described in further detail by referring to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Accordingly, the invention should in no way be construed as limited to the following examples, but rather should be construed to encompass any and all variations that become apparent as a result of the teaching presented herein. The methods and reagents used in the examples, unless otherwise stated, are conventional methods and reagents in the art.

Example

Example 1, Global Prediction of Alcaftadine Family Compounds in Streptomyces strain HO 1518 Streptomyces sp.HO1518 Using High Resolution Liquid Mass Spectrometry (LC-MS/MS)

Inoculate an appropriate amount of HO 1518 seed liquid into 10ml Pharmamedia liquid medium, 3 parallels, 28°C, 200rpm fermentation culture for 7 days, centrifuge and discard the bacteria, collect the supernatant; the supernatant is passed through the macroporous resin XAD-16 adsorption column layer The total extract of HO1518 was obtained by eluting with 95% ethanol. Using LC-MS/MS liquid-mass spectrometry technology, the metabolomics analysis of the HO 1518 strain was carried out. In the end, 98 compounds of the alcaftidine family were predicted, and the structures are shown in Figure 4 and Table 5 for details.

Table 5 from Streptomyces HO1518 Streptomyces sp. Prediction of alcatathidine family compounds

Aca, acarviostatin; Ac, acetyl; Hac, hydroxyacetyl; Pr, propionyl; Hpr, hydroxypropionyl; Bu, butyryl; isoBu, isobutyryl; Hbu, hydroxybutyryl; Mbu, 2-methyl-butyryl; hexanoyl; Hhe, hydroxyhexanoyl; diHva, dihydroxyvaleryl. Acarviostatins, marked with “-” in Ref.column, are potential new compounds.

Embodiment 2, the fermentation culture of bacterial strain and the preparation of compound

Streptomyces strain HO 1518 Streptomyces sp.HO1518 was collected from marine sediments in the 50-100m deep sea area near Rizhao, Shandong, China in 2010, and its preservation number in the China Center for Type Culture Collection is CCTCC NO:M 2018176.

Add 100mL 3% TSB solution (purchased from BD Company, USA) to a 500mL Erlenmeyer flask, sterilize with high pressure steam at 121°C for 20min, cool to room temperature, inoculate Streptomyces HO 1518 slant spores, and place on a constant temperature shaker at 28°C and 250rpm , cultured for 48h, as the seed solution.

Accurately weigh the following inorganic salts (g/L): NaCl 24.4770, Na ₂ SO ₄ 3.9170, KCl 0.664, SrCl ₂ ^. 6H ₂ O 0.0404, MgCl ₂ ^. 6H ₂ O 4.981, CaCl ₂ 0.9482, NaHCO ₃ 0.192, H ₃ BO ₃ 0.026, NaF 0.004, dissolved in tap water and fixed to a certain volume to prepare artificial seawater; accurately weigh trace element metal salts (g/L): MnCl ₂ 0.389, NiSO ₄ ^. 6H ₂ O 0.056, LiCl 0.028, K ₂ Cr ₂ O ₇ 0.15, Na ₂ EDTA 1.00, FeCl ₃ ^. 6H ₂ O 2.00, AlCl ₃ 0.05, CuCl ₂ 0.02, CoCl ₂ 6H ₂ O 0.005, ZnCl ₂ 0.06, Na ₂ MoO ₄ 2H ₂ O 0.074, KI 0.08, BaCl ₂ 2H ₂ O 0.05, use tap water to make up to the corresponding volume , Preparation of trace element solution. Use artificial seawater to prepare PH medium (Pharmamedia powder 1%, soluble starch 1%, glucose 1.2%, corn steep liquor dry powder 0.5%), adjust the pH to 7.2, add 400mL PH medium (containing 400μL trace element solution), sterilized by autoclaving at 121°C for 20 minutes, inoculated with HO 1518 seed solution after cooling, and cultured in a fermenter at 28°C and 200 rpm for 7 days, with a total of 35 L fermented.

The fermentation broth of strain HO1518 was centrifuged at high speed, and the supernatant was collected; the metabolites in the supernatant were adsorbed by macroporous resin XAD-16, and the crude extract of HO1518 was obtained. The crude extract was subjected to C ₁₈ reversed-phase silica gel column chromatography, and was eluted with different concentrations of methanol/water (5:95→100:0) gradient, and was divided into 6 fractions (Fr.1-Fr.6).

We performed targeted separation of the target compounds in fractions Fr.1 and Fr.2. Fraction Fr.1 was subjected to ODS-C ₁₈ reverse phase silica gel column chromatography, methanol/water gradient elution (5:95→100:0), and was divided into 6 subcomponents (Fr.1-1～Fr.1- 6). Subfraction Fr.1-1 was subjected to MCI column chromatography, and then purified by full preparative HPLC (MeCN/H ₂ O, 6:94, 8mL/min), and the known compound 1 (alcaftadine II03, Aca II03, 10.0 mg, t _R 10.5 min) and new compound 2 (alcaftadine II02, Aca II02, 1.6 mg, t _R 9.6 min). Subfraction Fr.1-3 was passed through the MCI column, and then purified by full preparative HPLC (MeCN/H ₂ O, 10:90, 8mL/min) to obtain the known compound 11 (β-hydroxybutyrylalcaftadine II03 , Hbu-Aca II03, 4.5 mg, t _R 15.1 min). Subcomponents Fr.1-4 were subdivided by MCI and ODS-C ₁₈ column chromatography, and then purified by HPLC (MeCN/H ₂ O, 10:90, 6mL/min) to obtain a new compound 6 (propionyl Alcaftadine II03, Pr-Aca II03, 13.2mg, t _R 40.5min). Subfractions Fr.1-5 were purified by reverse-phase silica gel and MCI column chromatography, followed by two HPLC preparative purifications (MeCN/H ₂ O, 10:90, 6 mL/min; MeCN/H ₂ O, 16:84 , 8mL/min); Obtain known compound 9 (alcastatine I03, Aca I03, 2.2mg, t _R 6.0min) and 10 (butyryl alcastatine I03, Bu-Aca I03, 10.2mg, t _R 14.3min). Subfraction Fr.1-6 was purified by MCI column chromatography, and then purified by HPLC twice (MeCN/H ₂ O, 10:90, 6mL/min; MeCN/H ₂ O, 18:82, 8mL/min ); Obtain new compound 8 (2-methylbutyryl alcatadine II03, Mbu-Aca II03, 4.2 mg, t _R 12.2min) and known compound 12 (isovaleryl alcatadine II03, isoVa-Aca II03, 6.2 mg, _tR 12.8 min).

Fraction Fr.2 was subjected to gradient elution (MeOH/H ₂ O, 5:95→100:0) using an MCI column, and was subdivided into 4 subcomponents. Subfraction Fr.2-2 was purified by reverse-phase silica gel column chromatography, and then purified by HPLC twice (MeCN/H ₂ O, 10:90, 6mL/min; MeCN/H ₂ O, 16:84, 8mL /min), obtain new compound 7 (butyryl alcastatine II03, Bu-Aca II03, 5.9mg, t _R 11.9min) and 4 (isobutyryl alcastatine II02, isoBu-Aca II02, 1.9mg, t _R 10.4min). Subfraction Fr.2-3 was purified twice by HPLC (MeCN/H ₂ O, 10:90, 6mL/min; MeCN/H ₂ O, 12:88, 8mL/min) to obtain the new compound 3 (Propionylalcatadine II02, Pr-Aca II02, 2.2mg, _tR 17.0min). Subfraction Fr.2-4 was purified by full preparative HPLC (MeCN/H ₂ O, 6:94, 8mL/min) to obtain a new compound 5 (isovalerylalcastatine II02, isoVa-Aca II02, 2.0mg , t _R 44.3min).

The full preparative column is SilGreen C ₁₈ , 250×20mm; semi-preparative column is TSK-gel 100V C ₁₈ , 250×10mm; the analytical detection column is Phenomenex/Luna C ₁₈ (2) 100A, 4.6×250mm; UV detection wavelength is 210nm.

Embodiment 3, the chemical structure of the new compound of acyl hepatidine family

The seven new compounds obtained above are all colorless transparent solids, easily soluble in water, and the ultraviolet absorption is terminal absorption.

Alcaftadine II 02 (corresponding to compound 2) has a molecular formula of C ₅₀ H ₈₄ N ₂ O ₃₅ and a molecular weight of 1273.4927.

Propionatadine II 02 (corresponding to compound 3) has a molecular formula of C ₅₃ H ₈₈ N ₂ O ₃₆ and a molecular weight of 1329.5190.

Isobutyrate II 02 (corresponding to compound 4) has a molecular formula of C ₅₄ H ₉₀ N ₂ O ₃₆ and a molecular weight of 1343.5346.

Isovaleratadine II02 (corresponding to compound 5) has a molecular formula of C ₅₅ H ₉₂ N ₂ O ₃₆ and a molecular weight of 1357.5503.

Propionatadine II 03 (corresponding to compound 6) has a molecular formula of C ₅₉ H ₉₈ N ₂ O ₄₁ and a molecular weight of 1491.5718.

Butyrastatidine II 03 (corresponding to compound 7) has a molecular formula of C ₆₀ H ₁₀₀ N ₂ O ₄₁ and a molecular weight of 1505.5872.

2-Methylbutyrazidine II 03 (corresponding to compound 8) has a molecular formula of C ₆₁ H ₁₀₂ N ₂ O ₄₁ and a molecular weight of 1519.6031.

Table 1 Hydrogen spectrum and carbon spectrum data of compounds 2 and 3.

Table 2 Hydrogen spectrum and carbon spectrum data of compounds 4 and 5.

Table 3 Hydrogen spectrum and carbon spectrum data of compounds 6-8.

Example 4, Mass Spectrometry Fragmentation Rules of New Compounds of Acyl Hepatidine Family

1. Alcaftadine family Ⅱ 02 skeleton type

The new compounds 1-4 of acyltadine belong to the skeleton type of alcaftadine family Ⅱ 02, and they form common fragment ion peaks m/z 304(b2), 466(b3), 624(b4) in the fragmentation mode of positive ion mass spectrometry and 769(b5). Compared with the precursor compound 1 (alcaftadine Ⅱ 02), the fragment ion peaks and molecular ion peaks of compound 2-4 at m/z y5 and b3-b5 are 56, 70 and 84 degrees different from alcaftadine Ⅱ 02, respectively unit of mass. The above shows that the acyl substitution positions of acyltadine compounds 1-4 are all on the D ring. The detailed fragmentation modes of the four compounds are shown in Figure 1A, and their HRESIMS/MS spectra are shown in Figures 1B-1E.

2. Alcaftadine family II03 skeleton type

The new compound 6-8 of acyltadine belongs to the skeleton type II03 of the alcaftadine family, and in the fragmentation mode of positive ion mass spectrometry, it forms common fragment ion peaks m/z 304(b2), 466(b3), 624(b4) and 769(b5), which are the same as the mass spectrometry fragments of the precursor compound - alcaftadine II03; and the fragment ion peaks and molecular ion peaks at m/z y5-y8 and b6-b8 are the same as the mass spectrometry fragments of alcaftadine II03 The difference is 56, 70 and 84 mass units. As explained above, the acyl substitution position of the acyltadine compound M-1b is also on the D ring. The detailed fragmentation mode of compound M-1b is shown in Figure 2A, and its HRESIMS/MS spectra are shown in Figures 2B-2D.

Example 5. Inhibition of pancreatic lipase activity by acyltadine compounds at the enzymatic level

The acyltadine compounds obtained above belong to amino-oligosaccharide compounds. It is reported that such compounds have significant α-amylase inhibitory activity, but there is no literature report on their lipase inhibitory activity. The inventor chose orlistat as a positive control, whose mechanism of action is to inhibit the activity of lipase in the gastrointestinal tract, prevent the hydrolysis of fat in food into free fatty acids and monoacylglycerols that can be absorbed by the human body, and directly discharge undecomposed fat In vitro, to prevent fat absorption from the source, so as to achieve the goal of easy weight loss.

With p-nitrophenyl laurate as substrate and 50% ethanol water as negative control, test the above 12 compounds Lipase inhibitory activity of compounds 1-12. In a 1.5 mL centrifuge tube, add 50 μL of different concentrations of the test compound (inhibitor, dissolved in 50% ethanol water), 150 μL of 10 mg/mL pancrelipase solution and 350 μL of 0.1M Tris-HCL buffer (pH=8.2), Mix well and incubate at 37°C for 10min. Next, add 450 μL of p-nitrophenyl laurate solution (substrate, dissolved in 5 mmol/L sodium acetate solution), react at 37°C for 0.5 h, inactivate in boiling water for 1 min, and cool to room temperature. Finally, centrifuge at 12,000 r/min for 3 min, and add 100 μL of supernatant to a 96-well plate. The absorbance value of each sample ( _sample A) was measured at 405 nm, and the parallel measurement was performed 3 times. Under the same conditions, the absorbance value of the sample blank group (A _{sample empty} ) with Tris-HCl buffer instead of pancreatic lipase solution, the absorbance value of the negative control group without sample solution (A _negative ), without sample, enzyme solution The absorbance value of the blank control group (A _empty ).

According to the absorbance value of sample solution and blank solution of different concentrations, calculate its lipase inhibition rate, the calculation formula of its enzyme inhibition is as follows:

Inhibition rate of pancreatic lipase activity/%=[1-(A _sample -A _{sample empty} )/(A _negative -A _empty )]×100

Finally, use GraphPad Prism 5.0 to calculate the IC ₅₀ value of each sample, see Figure 3 and Table 4.

The inhibitory activity of table 4 acyl hepatidine compound to pancreatic lipase

It can be seen from Table 4 that all the 12 compounds of the present invention can inhibit porcine pancreatic lipase, and their IC ₅₀ values range from 1.34 to 31.56 μM. Among the seven new oligosaccharides (2-8), compounds 5 and 8 had the strongest inhibitory activity, with IC ₅₀ values of 1.56 and 1.34 μM, which were basically equivalent to those of the positive control orlistat (IC ₅₀ =0.34 μM). The IC ₅₀ of compounds 2, 9 and 10 were greater than 10 μM, indicating that their inhibitory ability to lipase was not significant enough. The results of the structure, performance and biological activity of oligosaccharides show that the lipase inhibitory activity of compounds containing two pseudotrisaccharide structural active units (1-8, 11 and 12) is better than that of compounds with only one pseudotrisaccharide structural active unit ( 9 and 10), indicating that pseudotriose knots The extension of the structural unit can improve the biological activity of the compound; similarly, the length of the side chain acyl group of the compound will also affect its lipase inhibitory activity. By comparing the inhibitory activities of 12 oligosaccharides, it was found that the longer the acyl side chain, the stronger the lipase inhibitory ability.

Claims

A compound of formula (I) or its pharmaceutically or food-acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor,

Wherein, n is 2, 3 or 4;

m is 2, 3 or 4;

Each R 1 , R 2 , R 3 , R 4 , R 5 is each independently selected from the following group: hydrogen, C 1 -C 4 alkyl or -OZ; wherein each Z is independently selected from the following group: hydrogen, C 3 -C 6 acyl; and, at least one of R 1 , R 2 , R 3 , R 4 , R 5 is -OZ;

Each R 1 ′, R 2 ′ , R 3 ′, R 4 ′, R 1 ″, R 2 ″, R 3 ″, R 4 ″, Ra are each independently selected from the following group: hydrogen, hydroxyl, C 1 -C 4 alkyl, halogen;

Each Y 2 and Y 3 are independently selected from the following group: -O-, -NH-;

When m is 3 and n is 2, R is not hydroxyl or
The compound of formula (I) or its pharmaceutically or food acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor as claimed in claim 1, is characterized in that, each each R 3 is independently -OZ; and each Z is independently a C 3 -C 6 acyl group.

In another preference, said R is selected from the following group:
The formula (I) compound as claimed in claim 1 or its pharmaceutical or food acceptable salt or ester, isomer Body, racemate, solvate, hydrate or precursor, characterized in that each R 1 , R 2 , R 4 , R 1 ', R 2 ', R 4 ', R 1 ", R 2 " , R 4 ″, Ra are each independently selected from the group consisting of hydrogen, C 1 -C 2 alkyl or hydroxyl.

In another preferred example, each R 2 is independently C 1 -C 2 alkyl.
The compound of formula (I) or its pharmaceutically or food acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor as claimed in claim 1, characterized in that, Described formula (I) compound has the structure shown in formula (I-1) or formula (I-2),

Wherein, the definition of R is as described in claim 1.
The compound of formula (I) or its pharmaceutically or food acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor as claimed in claim 1, characterized in that, Described compound has the structure that is selected from following group:
A composition characterized in that it comprises:

(1) The compound of formula (I) as described in any one of claims 1-5 or its pharmaceutically acceptable salt or ester, isomer, racemate, solvate, hydrate or precursors; and

Optional (2) pharmaceutically or food-acceptable excipients.

In another preferred example, the composition is selected from the group consisting of daily chemical products, food, health products or pharmaceutical compositions.
A test kit, characterized in that it comprises:

(1) The compound of formula (I) described in any one of claims 1-5 or its pharmaceutically acceptable salt or ester, isomer, racemate, solvate, hydrate or a precursor, or a composition as claimed in claim 6; and

Optionally (2) using or administering other agents as needed; and

Optional (3) instructions.
Use of the compound as claimed in any one of claims 1-5 or its pharmaceutically or food-acceptable salt or ester, isomer, racemate, solvate, hydrate or precursor, which It is characterized in that it is used for the preparation of a product for inhibiting lipase activity, controlling body weight, improving fat utilization, or preventing, alleviating or treating a disease or condition benefiting from reduced fat absorption.

In another preferred example, the product is selected from the group consisting of daily chemical products, food, health care products, pharmaceutical compositions or kits.
A non-diagnostic, non-therapeutic method for inhibiting lipase activity, comprising: formula (I) compound or pharmaceutically acceptable salt or ester thereof as described in any one of claims 1-5, The isomer, racemate, solvate, hydrate or precursor, or the composition of claim 6 is contacted with a lipase-containing sample.
A method for improving the inhibitory activity of amino oligosaccharides against lipase, comprising: adding at least one acyl group to the amino oligosaccharides.