TWI730230B - Lactulose glycoside compound, its preparation method and its use - Google Patents

Abstract

」表示乳果糖基通過α糖苷鍵或β糖苷鍵與苷元(G)中的雜原子連接。藥代試驗證明根據本發明的乳果糖糖苷化合物能夠通過哺乳動物胃腸道而不被胃腸道顯著吸收及不被哺乳動物宿主內源性酶顯著水解，因此該乳果糖糖苷化合物能到達哺乳動物結腸部位，在結腸菌群的作用下在結腸釋放出活性藥物。所述乳果糖糖苷化合物具有結腸定位釋藥的作用，可用於腸道疾病的預防或治療。 The present invention relates to the field of medicinal chemistry, in particular to a compound represented by formula (I), its preparation method and its medical use. In the compound represented by formula (I), the lactulose group is connected to the heteroatom in the aglycon (G) through a glycosidic bond, and the aglycon (G) is formed by removing a hydrogen atom from the heteroatom of the active drug molecule Group, and "

"It means that the lactulose group is connected to the heteroatom in the aglycon (G) through an α-glycosidic bond or β-glycosidic bond. Pharmacokinetic tests have proved that the lactulose glycoside compound according to the present invention can pass through the gastrointestinal tract of mammals without being significantly absorbed by the gastrointestinal tract and not significantly hydrolyzed by the endogenous enzymes of the mammalian host, so the lactulose glycoside compound can reach the colon of mammal , Under the action of colonic flora, active drugs are released in the colon. The lactulose glycoside compound has a colon-localized drug release effect, and can be used for the prevention or treatment of intestinal diseases.

Description

Lactulose glycoside compound, its preparation method and its use

The present invention relates to the field of medicinal chemistry, in particular to a lactulose glycoside compound with colonic positioning, transport and release, its preparation method and its medical use.

Most oral drugs are mainly absorbed through the upper digestive tract (stomach, small intestine) and enter the systemic circulation to play a therapeutic role. For diseases that exist in the lower end of the digestive tract (cecum, colon, rectum), it is difficult for the drug to reach the lesion directly, but only rely on the concentration of the drug circulating in the system to exert its efficacy. Such colon diseases include inflammatory bowel disease (ulcerative colitis, Krahn’s disease, etc.), intestinal infections (amoebic bowel disease), bowel cancer (colon cancer, rectal cancer), irritable bowel syndrome, chronic Constipation and so on. For the treatment of these diseases, most of the commonly used drugs such as glucocorticoids, non-steroidal anti-inflammatory drugs, immunomodulators, monoclonal antibodies, broad-spectrum cytotoxic drugs, etc., are administered orally or by injection, and exposed through the systemic system of drugs. The amount to treat local diseases. The obvious disadvantage is the unnecessary systemic side effects when the drug treatment concentration is locally reached. For example, for the treatment of ulcerative colitis, glucocorticoid treatment affects the hypothalamus-pituitary-adrenal glands, and immunomodulators are used. Lead to abnormal and disordered systemic immune function. The most ideal mode of administration is local administration in the colon, which can increase the local drug concentration, avoid or reduce the systemic exposure of the drug, thereby reducing systemic side effects.

In order to solve the above problems, researchers have provided different colon-targeted drug solutions. For example, balsalazide releases the active drug 5-aminosalicylic acid through colonic flora azoreductase to exert anti-inflammatory effects. Mesalazine enema and budesonide enema are directly used for enema treatment at the end of the digestive tract (rectum, sigmoid colon). Colon-targeted formulations prepared by various complex formulation technologies are also frequently reported. In addition, there are also reports of using prodrug technology to link active drugs with macromolecular carriers (glucan, xylan, cyclodextrin, etc.), hoping to release the active drug after the drug is delivered to the colon. For details, please refer to the following documents: Chinese Pharmacological Bulletin, (2002), 18(3), 328-330; Pharmaceutical Journal of Chinese People's Liberation Army, (2001), 17(2), 62-64; Carbohydrate Polymers(2017), 157, 1442-1450; and Journal of Pharmaceutical Sciences (2001), 90(12), 2103-2112.

Therefore, there is a great need to develop a simple small molecule prodrug, which has a single molecular structure and precise composition, and this prodrug is an active drug connected to a small molecule carrier, which is not absorbed by the upper gastrointestinal tract or is absorbed little, and is not absorbed by endogenous sources. Due to the degradation of sex enzymes, most of the drugs can be localized and delivered to the colon, and then the active drugs are quickly released in the colon, thereby exerting a therapeutic effect.

The present invention is implemented for the above purpose, and specifically, the present invention discloses a class of lactulose glycoside compounds that pass through the gastrointestinal tract of mammals without being significantly absorbed by the gastrointestinal tract and are not significantly hydrolyzed by the endogenous enzymes of the mammalian host. Therefore, this type of lactulose glycoside compound is not significantly absorbed by the gastrointestinal tract and is not significantly hydrolyzed by the endogenous enzymes of the mammalian host. Lactulose glycoside compounds can reach the colon of mammals and release active drugs in the colon under the action of colonic flora. It has a colon-oriented drug release effect and is used for the prevention or treatment of intestinal diseases.

Lactulose is also called lactulose, lactose, and galactosyl fructoside. It is a synthetic disaccharide formed by the combination of galactose and fructose with β-1,4 glycosidic bonds. Lactulose has a good function of bifidobacteria proliferation, has the effect of regulating the intestinal microecological balance, and has a wide range of applications in medicine, food and animal feeding. Lactulose can reduce blood ammonia, catharsis, and treat constipation. It can also be used for the prevention and treatment of various liver diseases. It can also be used as a low-calorie sweetener and functional food additive in food and has a wide range of applications. However, up to now, there has been no report on the use of lactulose as a small molecule carrier to localize and deliver active drugs.

其中，乳果糖基通過糖苷鍵與苷元(G)中的雜原子連接，所述苷元(G)為從活性藥物分子的雜原子除去一個氫原子所形成的基團，並且「

」表示乳果糖基通 過α糖苷鍵或β糖苷鍵與苷元(G)中的雜原子連接。 According to one aspect of the present invention, the present invention discloses a lactulose glycoside prodrug compound, which is represented by the following formula (I):

Among them, the lactulose group is connected to the heteroatom in the aglycon (G) through a glycosidic bond, and the aglycon (G) is a group formed by removing a hydrogen atom from the heteroatom of the active drug molecule, and

"It means that the lactulose group is connected to the heteroatom in the aglycon (G) through an α-glycosidic bond or β-glycosidic bond.

」表示乳果糖基通過α糖苷鍵或β糖苷鍵與苷元(G)中的雜原子連接。即，所形成的乳果糖糖苷前藥化合物具有由下式(III)或(IV)表示的結構：

in the text,"

"It means that the lactulose group is connected to the heteroatom in the aglycon (G) through an α-glycosidic bond or β-glycosidic bond. That is, the formed lactulose glycoside prodrug compound has a structure represented by the following formula (III) or (IV):

The lactulose glycoside prodrug compound according to the present invention may be an α-glycosidic bond-bonded compound, a β-glycosidic bond-bonded compound, or a mixture of the two.

According to the present invention, the term "aglycon" has the ordinary meaning in the art, that is, the non-sugar moiety condensed with sugar in glycoside compounds.

The active drug according to the present invention is a chemical component that has a certain pharmacological activity and is used for the prevention or treatment of human diseases. The active drug may be a drug approved for marketing in various countries, or a candidate drug in the stage of human clinical trials.

According to the technical solution of the present invention, the heteroatom system is selected from any one of oxygen atoms, nitrogen atoms and sulfur atoms. And the aglycon (G) is a group formed by removing a hydrogen atom from the alcoholic hydroxyl, phenolic hydroxyl, amino, amido or NH on the heterocyclic ring in the active drug molecule.

According to the technical content of the present invention, the case where two or more lactulose groups are connected with aglycone is also within the scope of the present invention.

According to some embodiments of the present invention, the active drug molecule is selected from the group consisting of glucocorticoids, apremilast, prucalopride, mesalamine, Any one of Metronidazole, Azathioprine, 6-Mercaptopurine, 5-Fluorouracil, and Tofacitinib.

The glucocorticoid drugs refer to a class of synthetic drugs that have similar effects to the glucocorticoids secreted by the adrenal cortex in the human body. They have the effect of regulating the biosynthesis and metabolism of sugar, fat and protein, and also have anti-inflammatory properties. effect. Glucocorticoid drugs include not only the endogenous substances with the above-mentioned characteristics and activities, but also many artificially synthesized drugs with similar structures and activities that have been structurally optimized. Examples of glucocorticoid drugs include but are not limited to: cortisone acetate, prednisone, prednisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone succinate Sodium, hydrocortisone sodium phosphate, fludrocortisone, fludrocortisone acetate, prednisolone, prednisolone acetate, prednisolone sodium succinate, prednisolone sodium phosphate, beta Methosone, betamethasone acetate, betamethasone dipropionate, clobetasone butyrate, betamethasone valerate, betamethasone sodium phosphate, beclomethasone dipropionate, mometasone furoate, clobetasone propionate Dexamethasone, dexamethasone, dexamethasone acetate, dexamethasone sodium phosphate, p-flumethasone, fluticasone propionate, methylprednisolone, methylprednisolone acetate, flumetholone, triamcinolone, triamcinolone acetonide, triamcinolone triamcinolone Anonide, fluorosinide, ancinonide, hascinonide, ciclesonide, budesonide, and defcote.

The glucocorticoid drug is preferably selected from hydrocortisone (Hydrocortisone), prednisolone (Prednisolone), methylprednisolone (Methylprednisolone), dexamethasone (Dexamethasone), betamethasone (Betamethasone), triturate Triamcinolone and Budesonide.

The active drug is more preferably selected from prednisolone, dexamethasone, budesonide, aprost, prucalopride and mesalazine.

其中，R₁為H或F；R₂為H、F或-CH₃；R₃為-OH；並且R₄為H、-OH或-CH₃； 或者R₃和R₄連接成基團

、

或

。 In the formula (I) shown above, the aglycon (G) is a group represented by the following formula (II):

Wherein, R ₁ is H or F; R ₂ is H, F or -CH ₃ ; R ₃ is -OH; and R ₄ is H, -OH or -CH ₃ ; or R ₃ and R _{4 are} connected to form a group

,

or

.

其中Me表示-CH₃。 Preferably, the aglycon (G) is selected from any one of the following:

Where Me represents -CH ₃ .

其中Me表示-CH₃。 Preferably, the aglycon (G) is selected from any one of the following:

Where Me represents -CH ₃ .

其中Me表示-CH₃。 More preferably, the aglycon (G) is selected from any of the following:

Where Me represents -CH ₃ .

More specifically, the compound is selected from any one of the following: (11β)-11,17-dihydroxy-21-{[4-O-(β-D-galactopyranosyl)-D -Frutofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β)-11,17-dihydroxy-21-{[4-O-(β-D-pyran Galactosyl)-D-Frutofuranosyl]oxy}pregna-4-ene-3,20-dione; (6α,11β)-11,17-dihydroxy-6-methyl-21-{[4 -O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β,16α)-9-fluoro- 11,17-Dihydroxy-16-methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene- 3,20-Diketone; (11β,16β)-9-fluoro-11,17-dihydroxy-16-methyl-21-{[4-O-(β-D-galactopyranosyl)-D -Frutofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β,16α)-9-fluoro-11,16,17-trihydroxy-21-{[4-O -(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β,16α)-11-hydroxy-16, 17-(Butylenedioxy)-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,20 -Diketone; 2-methyl-5-nitro-1-{2-{[4-O-(β-D-galactopyranosyl)-D-frutofuranosyl]oxy}ethyl}imidazole; 6-[(1-methyl-4-nitro-1 H -imidazol-5-yl)sulfur]-9-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl ]-9 H -purine; 6-mercapto-9-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]-9 H -purine; 5-fluoro-1-[4 -O-(β-D-galactopyranosyl)-D-fructofuranosyl]pyrimidine-2,4(1H,3H)-dione; N -{2-[(S)-1-(3- Ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl)-1,3-dioxo-2,3-dihydro-1 H -isoindole-4- Group} -N' -[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]acetamide; 4-amino-5-chloro-2,3-dihydro- N- [1-(3-Methoxypropyl)-4-pyridinyl] -N' -[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]-7-benzene Parafuranoformamide; 5-chloro-2,3-dihydro-4-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]amino} -N -[1 -(3-Methoxypropyl) -4-Pridinyl]-7-benzofuranocarbamide; 5-amino-2-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}benzene Formic acid; 2-hydroxy-5-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]amino}benzoic acid; 17-hydroxy-21-{[4-O-( β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,11,20-trione; and 4-{ N -methyl- N'- [(3R,4R)-4-methyl-1-(2-cyanoacetoxy)pyridin-3-yl]amino)7-[4-O-(β-D-galactopyranosyl) -D-Frutofuranosyl]-7 H -pyrrolo[2,3- d ]pyrimidine.

According to a second aspect of the present invention, a pharmaceutical composition is provided, which contains the lactulose prodrug compound as described above and a pharmaceutically acceptable carrier.

According to the third party of the present invention, the use of the compound as described above for the preparation of a medicament for the prevention or treatment of intestinal diseases, wherein the intestinal diseases are selected from ulcerative colitis, Crohn’s disease, and infectious enteritis , Irritable bowel syndrome, chronic constipation, amoebic bowel disease, colon cancer and rectal cancer, and more preferably, the intestinal disease is selected from ulcerative colitis, Crohn's disease and chronic constipation.

As demonstrated in the attached examples, the lactulose glycoside compound according to the present invention can pass through the gastrointestinal tract of mammals without being significantly absorbed by the gastrointestinal tract and is not significantly hydrolyzed by the endogenous enzymes of the mammalian host, so the lactulose glycoside compound can It reaches the colon of mammals and releases active drugs in the colon under the action of colonic flora. The lactulose glycoside compound has the effect of colon-localized drug release, and can be used for the prevention or treatment of intestinal diseases.

The following will further illustrate the implementation of the present invention in conjunction with the drawings. The following examples are used to illustrate the features and applications of the present invention, rather than to limit the scope of the present invention. Anyone familiar with the art will not depart from Within the spirit and scope of the present invention, some changes and modifications can be made. Therefore, the protection scope of the present invention shall be subject to those defined by the attached patent scope.

The following figures are provided to describe in detail the embodiments according to the present invention, in which: Figure 1 shows the changes in the concentration of prednisolone in the contents of the anterior 1/3 of the rat colon (including the cecum) after intragastric administration; Figure 2 shows Changes in the concentration of prednisolone in the second third segment of the rat colon after intragastric administration; Figure 3 shows the plasma concentration-time curve of prednisolone after intragastric administration in different groups of rats; Figure 4 shows the weight change curve of rats in different groups after intragastric administration; Figure 5 shows the comparison of the weight changes of rats in different groups on the 8th day of modeling; Figure 6 shows the weight changes of rats in different groups on the 8th day of modeling Comparison of the colon coefficient; and Figure 7 shows the comparison of the histopathological score results of different groups of rats on the 8th day of modeling.

Specifically, the example compounds provided by the present invention are selected from the structures shown in Table 1 below.

Next, the method for preparing the compound of the present invention will be described.

The compound of the present invention can be prepared, for example, by the following preparation method.

(Preparation method A)

In pyridine (Py) solvent, in the presence of dichloromethane (CH ₂ Cl ₂ ), lactulose is reacted with benzyl chloride (BzCl) to obtain octabyl lactulose (A), Then, under the action of trimethylsilyl trifluoromethanesulfonate (TMSOTf), it undergoes glycosylation condensation reaction with the 21-position primary hydroxyl group of glucocorticoid drugs (B), and the intermediate ( C). Finally, the benzyl group on the sugar group is removed by the ammonia methanol solution to obtain the target product (D). The typical examples are compounds 1-7 shown in the above table. Similarly, the octabyl lactulose (A) and metronidazole are subjected to a glycosylation reaction, and then the benzyl group is removed to obtain the target compound 8. Drug molecules with primary hydroxyl groups can be used to prepare corresponding target compounds using the above methods.

(Preparation method B)

Lactulose is acetylated with acetic anhydride/pyridine to obtain octaacetyl lactulose (compound F).

Azathioprine, 6-mercaptopurine, 5-fluorouracil (5-Fu) or apremilast are silylated with N,O-bis(trimethylsilyl)acetamide (abbreviated as BSA) in acetonitrile solvent , Under the action of trimethylsilyl trifluoromethanesulfonate (TMSOTf), it undergoes glycosylation condensation reaction with octaacetyllaculose (F), and the intermediate (G) is obtained after purification by column chromatography. The target product (H) (compounds 9, 10, 11, 12) was obtained by alkali treatment to remove the acetyl group on the sugar group and preparation and purification.

The corresponding lactulose glycoside (compound 18) can be obtained by using Tofacitinib using a synthetic method similar to the above.

(Preparation method C)

Octabenzyllactulose (A) is converted into chlorinated compound (K) by hydrogen chloride or hydrogen bromide, and prucalopride reacts with chlorinated compound (K) to form intermediate (M). The crude product is purified by column chromatography , Then alkali treatment to remove the benzyl group on the sugar group and preparation and purification to obtain compound 13.

After silylation of prucalopride by N,O-bis(trimethylsilyl)acetamide (abbreviated as BSA), under the action of tert-butyldimethylsilyl trifluoromethanesulfonate (TBSOTf) , Glycosylation reaction with octabyl lactulose (A), the intermediate (N) is obtained after purification by column chromatography, and then the benzyl group on the sugar group is removed by alkali treatment and the compound is prepared and purified 14.

(Preparation method D)

5-Aminosalicylic acid (mesalazine) is methylated to obtain mesalazine methyl ester (P), and the amino group is protected by benzyl chloroformate to obtain compound (Q). The phenolic hydroxyl group of compound (Q) undergoes glycosylation reaction with chlorosugar (K) to obtain mesalazine O-glycoside compound (R), and finally the benzyloxycarbonyl group is removed by hydrogenation, and methyl ester and benzoic acid are removed by alkaline hydrolysis. Esters to give compound 15.

After mesalazine methyl (P) is protected by di-tert-butyl dicarbonate (Boc ₂ O) to protect the amino group, it reacts with benzyl chloride to protect the phenolic hydroxyl group, and then the Boc protecting group is removed to obtain compound (S). The amino group of compound (S) then undergoes glycosylation reaction with chlorosugar (K) to obtain mesalazine N-glycoside compound (T). Finally, the benzyl group is removed by hydrogenation, and the methyl ester and benzoate are removed by alkaline hydrolysis. , Compound 16 was obtained.

The compound of structural formula (I) of the present invention can be mixed with a pharmaceutically acceptable carrier to prepare various oral pharmaceutical compositions. After mixing the compound of structural formula (I) with acceptable auxiliary additives such as disintegrants, excipients, lubricants, binders, fillers, etc., they can be made into tablets, pills, and capsules according to conventional methods. Oral drugs in solid dosage forms such as a variety of corresponding sustained-release agents, controlled-release agents, etc.; and commonly used surfactants, diluents, preservatives, and stabilizers such as solubilizers, emulsifiers, wetting agents, foaming or defoaming agents, etc. Mixing agents, flavoring agents, thickeners, etc., according to the corresponding conventional methods, can be made into liquid preparations such as liquids, syrups and other oral drugs.

The invention also provides methods for in vitro stability tests, drug metabolism and pharmacodynamic tests of the compounds of the examples. The results show that the compound of the present invention has a good colon-specific drug release effect and advantages in the treatment of intestinal diseases, and is especially suitable for medication treatment of the cecum and colon.

Therefore, the present invention also provides the use of a compound of formula (I) in the preparation of a pharmaceutical composition for the prevention or treatment of intestinal diseases or disorders, the diseases or disorders being selected from ulcerative colitis, Crohn Disease, infectious enteritis, irritable bowel syndrome, chronic constipation, amoebic bowel disease, colon cancer, rectal cancer.

The intestinal disease or disorder is preferably selected from ulcerative colitis, Crohn's disease, and chronic constipation.

Example

The preparation method of the compound according to the present invention is specifically illustrated below through examples. It should be pointed out that the technical solution according to the present invention is not limited to the described embodiment.

In the following examples, lactulose was purchased from Bailingwei Technology (content 99.0%). Prednisone (content 99.0%), prednisolone (content 99%), hydrocortisone (content 98.8%), dexamethasone (content 98.8%), betamethasone (content 99.8%), triamcinolone (Content 99.0%) and budesonide (content 98.5%) were purchased from Shandong Taihua Biological Technology Co., Ltd. Methylprednisolone (content 98%) was purchased from Wuhan Dongkangyuan Technology Co., Ltd., and metronidazole (content 98.0%) was purchased from Saen Chemical Technology Co., Ltd. Azathioprine (content 98%) was purchased from Aladdin reagent. 6-Mercaptopurine (content 98.0%) and 5-fluorouracil (98.0%) were purchased from Shanghai Bi De Pharmaceutical Technology Co., Ltd. Apsite (content 98.0%) was purchased from Adamas Reagent Co., Ltd. Procapride (99.2% content) was purchased from Zhiwei Chemical Technology Co., Ltd. Mesalazine (content 99.7%) was purchased from Hubei Xing Yinhe Chemical Co., Ltd.

In this application, unless specifically indicated otherwise, other reagents used are commercially available.

In addition, in this specification, unless specifically indicated otherwise, "%" means% by weight.

Preparation Example 1 (11β)-11,17-Dihydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3 ,20-Diketone (Compound 1)

step one:

Dissolve lactulose (10g, 0.029mol) in 100ml of pyridine, add 60ml of dichloromethane, stir and cool to 0°C, add benzyl chloride (61.1g, 0.435mol) dropwise, after the addition, warm to room temperature Reaction for 16 hours.

Concentrate the reaction solution under reduced pressure to dryness, add 200ml of dichloromethane to dissolve, wash with citric acid solution (pH>4) (120ml*3 times), saturated sodium bicarbonate (120ml*1 time), saturated sodium chloride Wash the solution (20ml*1 time), dry the organic phase with anhydrous sodium sulfate for 30 minutes, filter, concentrate under reduced pressure and purify by column chromatography to obtain 30.0g of white solid as the product of step 1 (compound A), the yield is 87.2 %. ESI(+) m/z: 1192.3 [M+NH ₄ ] ⁺ 1197.2 [M+Na] ⁺ .

Step two:

Under the protection of argon, dissolve the product (10.1g, 8.59mmol) of the above step 1 and prednisolone (2.6g, 7.16mmol) in 400ml of dichloromethane, add 5g of 4A molecular sieve, turn on the stirring and reduce the temperature to- At 35°C, TMSOTf (2.7g, 12.2mmol) was added dropwise, maintained at this temperature for 2 hours and then heated to -15°C, and reacted for 2-3 hours.

Trimethylpyridine (2.2 g, 17.9 mmol) was added dropwise to the reaction mixture, and the temperature was raised to 25° C. and stirring was continued for 1 hour. Then the reaction solution was washed with citric acid solution (pH>4) (200ml*3 times), saturated sodium carbonate (200ml*1 time), saturated sodium chloride solution (200ml*1 time), and the organic phase After drying with anhydrous sodium sulfate for 30 minutes, filtering, the filtrate was concentrated and purified by column chromatography to obtain 7.5 g of white solid as the product of step two, with a yield of 61.5%. ESI(+)m/z: 1435.2[M+Na] ⁺ .

Step 3: Preparation of compound 1

The product (5.4 g, 3.8 mmol) of the above step 2 was dissolved in 10 ml of tetrahydrofuran, 40 ml of ammonia methanol (10M) was added, and the mixture was stirred at room temperature for 72 hours.

Concentrate the reaction solution to dryness under reduced pressure, add water to dissolve, and then concentrate to dryness, repeat three times. 30 ml of water and 10 ml of methyl tert-butyl ether were added to the residue, dissolved and separated, the aqueous phase was concentrated under reduced pressure, and the remaining water was removed with anhydrous ethanol, the obtained solid was suspended in 30 ml of acetone and stirred for 50 minutes, and filtered. The filter cake was rinsed with a small amount of acetone and drained. The filter cake was recrystallized and purified with methanol/water (v/v)=1/1 to obtain 1.5 g of a white solid, which is the title compound (compound 1), with a yield of 57.7% . ESI(+)m/z: 707.4[M+Na] ⁺ .

¹ H-NMR(DMSO-d6,500M,BRUKER AV-500): δ(ppm)7.32(1H,d,=CH), 6.19(1H,dd,J ₁ =1.5Hz,J ₂ =10Hz,=CH ), 5.91(1H,s,=CH), 5.23(1H,s,CH(O) ₂ -), 5.09(1H,d,O-CH ₂ -CO-), 4.82-4.79(1H,t), 4.74-4.71(2H,m), 4.69(1H,d,O-CH ₂ -CO-), 4.62(1H,s), 4.53(1H,d), 4.38(1H,d), 4.29-4.23(3H) ,m), 4.14-4.10(2H,m), 3.84-3.81(1H,m), 3.78-3.75(1H,m), 3.61-3.59(2H,m), 3.56-3.48(3H,m), 3.46 -3.42(1H,m), 3.40-3.36(2H,m), 3.32-3.30(2H,m), 2.56-2.53(2H,m), 2.30-2.28(1H,m), 2.04-2.02(2H, m), 1.89-1.85(1H,dd,J ₁ =3Hz,J ₂ =13.5Hz), 1.66-1.60(3H,m), 1.41-1.39(1H,m), 1.38(3H,s,-CH ₃ ), 1.30-1.23 (1H, m), 1.02-0.99 (1H, m), 0.91-0.88 (1H, dd, J ₁ = 3Hz, J ₂ =10.5 Hz), 0.779 (3H, s, -CH ₃ ) .

Preparation Example 2 (11β)-11,17-Dihydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy)pregna-4-ene-3,20- Dione (Compound 2)

Preparation Example 2 was implemented in a similar manner to Preparation Example 1, except that hydrocortisone was used instead of prednisolone to obtain 0.45 g of the title compound. ESI(+)m/z: 709.3[M+Na] ⁺ .

¹ H-NMR(DMSO-d6,400M, BRUKER AV-400): δ(ppm)5.56(1H,s,=CH), 5.29(1H,s), 5.16(1H,d), 4.87-4.78(5H) ,m), 4.68-4.63(1H,m), 4.46(1H,m), 4.35-4.34(1H,m), 4.29-4.22(3H,m), 4.18-4.11(2H,m), 3.82-3.80 (1H,m), 3.77-3.74(1H,m), 3.59(2H,m), 3.56-3.42(4H,m), 3.40-3.34(2H,m), 3.30(2H,m), 2.57-2.54 (1H, m), 2.45-2.34 (2H, m), 2.21-2.17 (2H, m), 2.10-2.07 (1H, m), 1.93-1.88 (3H, m), 1.81-1.75 (1H, m) , 1.66-1.55(3H,m), 1.42-1.40(1H,m), 1.36(3H,s,-CH ₃ ), 1.28-1.24(1H,m), 1.06-0.98(1H,m), 0.88- 0.85 (1H, d), 0.75 (3H, s, -CH ₃ ).

Preparation Example 3 (6α,11β)-11,17-Dihydroxy-6-methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy)pregna-1 ,4-Diene-3,20-dione (Compound 3)

Preparation Example 3 was implemented in a similar manner to Preparation Example 1, except that methylprednisolone was used instead of prednisolone to obtain 0.73 g of the title compound. ESI(+)m/z: 721.3[M+Na] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 7.33 (1H, d, = CH), 6.19 (1H, d), 5.82 (1H, s, = CH), 5.28 ( 1H,s), 5.15(1H,d), 4.89-4.86(1H,m), 4.83-4.78(3H,m), 4.67-4.62(1H,m,), 4.59(1H,s), 4.46(1H) ,d), 4.36-4.33(1H,m), 4.27-4.22(2H,m), 4.14-4.10(2H,m), 3.81(1H,m), 3.77(1H,m), 3.61-3.59(2H) ,m), 3.56-3.46(3H,m), 3.43(1H,m), 3.39-3.36(2H,m), 3.29-3.25(2H,m), 2.66-2.65(1H,m), 2.57-2.53 (1H,m), 2.11-2.02(2H,m), 1.62-1.59(3H,m), 1.42(1H,m), 1.38(3H,s,-CH ₃ ), 1.31-1.29(1H,m) , 1.05-1.04 (3H, d, -CH ₃ ), 0.87-0.84 (1H, m), 0.77 (3H, s, -CH ₃ ), 0.72-0.66 (1H, m).

Preparation Example 4 (11β,16α)-9-fluoro-11,17-dihydroxy-16-methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy) Pregnancy-1,4-diene-3,20-dione (Compound 4)

Preparation Example 4 was implemented in a similar manner to Preparation Example 1, except that dexamethasone was used instead of prednisolone to obtain 0.95 g of the title compound. ESI(+)m/z: 739.3[M+Na] ⁺ .

¹ H-NMR(DMSO-d6,400M, BRUKER AV-400): δ(ppm) 7.30(1H,d,=CH), 6.24(1H,d,=CH), 6.01(1H,s,=CH) , 5.32-5.27(1H,m), 5.16(1H,d), 5.08(1H,s), 4.88-4.80(4H,m), 4.72-4.64(2H,m), 4.47(1H,s), 4.36 (1H,m), 4.27-4.22(1H,m), 4.15-4.13(3H,m), 3.83-3.79(2H,m), 3.60-3.53(5H,m), 3.44(2H,m), 3.29 (2H,m), 2.92(1H,m), 2.67-2.59(1H,m), 2.39-2.31(2H,m), 2.16-2.06(2H,m), 1.76(1H,m), 1.66-1.58 (1H,m), 1.52(1H,m), 1.49(3H,s,-CH ₃ ), 1.36-1.34(1H,m), 1.06(1H,m), 0.87(3H,s,-CH ₃ ) , 0.79 (3H, d, -CH ₃ ).

Preparation Example 5 (11β,16β)-9-fluoro-11,17-dihydroxy-16-methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy) Pregnancy-1,4-diene-3,20-dione (Compound 5)

Preparation Example 5 was implemented in a similar manner to Preparation Example 1, except that betamethasone was used instead of prednisolone to obtain 0.58 g of the title compound. ESI(+)m/z: 739.3[M+Na] ⁺ .

¹ H-NMR(DMSO-d6,400M, BRUKER AV-400): δ(ppm) 7.30(1H,d,=CH), 6.23(1H,d,=CH), 6.01(1H,s,=CH) , 5.24(1H,s), 5.20(1H,d), 5.14-5.12(1H,d), 4.88-4.77(4H,m), 4.60-4.55(1H,m), 4.47(1H,d), 4.40 -4.35(1H,m), 4.29-4.24(1H,m), 4.14-4.10(3H,m), 3.85-3.83(1H,m), 3.79-3.77(1H,m), 3.60-3.46(5H, m), 3.45-3.42(1H,m), 3.40-3.36(2H,m), 3.30(2H,m), 2.66-2.60(1H,m), 2.46-2.32(2H,m), 2.09-2.07( 2H,m), 1.98-1.82(3H,m), 1.49(3H,s,-CH ₃ ), 1.40-1.34(2H,m), 1.04-1.02(2H,m), 0.95(3H,s,- CH ₃ ).

Preparation Example 6 (11β,16α)-9-fluoro-11,16,17-trihydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy)pregna- 1,4-Diene-3,20-dione (Compound 6)

Preparation Example 6 was implemented in a similar manner to Preparation Example 1, except that triamcinolone was used instead of prednisolone to obtain 0.38 g of the title compound. ESI(+)m/z: 741.3[M+Na] ⁺ .

Preparation Example 7 (11β,16α)-11-hydroxy-16,17-(butylene dioxy)-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy)pregnancy Steroid-1,4-diene-3,20-dione (Compound 7)

Preparation Example 7 was implemented in a similar manner to Preparation Example 1, except that budesonide was used instead of prednisolone to obtain 1.2 g of the title compound. ESI(+)m/z: 777.3[M+Na] ⁺ .

¹ H-NMR(DMSO-d6,400M, BRUKER AV-400): δ(ppm) 7.32(1H,d,=CH), 6.18(1H,d,=CH), 5.92(1H,s,=CH) , 5.19-5.17(1H,m), 5.14(1H,m), 5.05(1H,d), 4.81-4.75(4H,m), 4.71(1H,m), 4.64-4.62(1H,d), 4.59 -4.57(1H,m), 4.45(1H,m), 4.35(2H,m), 4.31-4.24(2H,m), 4.15-4.13(2H,m), 3.82-3.75(2H,m), 3.60 (3H,m), 3.57-3.53(3H,m), 3.45-3.43(3H,m), 3.40-3.37(2H,m), 3.29(2H,m), 2.77-2.67(1H,m), 2.31 (1H,m), 2.09(1H,s), 1.98(2H,m), 1.79-1.72(3H,m), 1.61-1.52(4H,m), 1.42(2H,m), 1.38(3H,s) ,-CH ₃ ), 1.35-1.23(2H,m), 1.15-1.11(1H,m), 1.07-1.04(3H,t,-CH3), 1.01-0.93(2H,m), 0.88((1H, m), 0.86 (3H, s, -CH ₃ ), 0.81 (1H, s).

Preparation Example 8 2-Methyl-5-nitro-1-{2-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}ethyl}imidazole (Compound 8)

Preparation Example 8 was implemented in a similar manner to Preparation Example 1, except that metronidazole was used instead of prednisolone to obtain 1.9 g of the title compound. ESI(+)m/z: 518.2[M+Na] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 8.03 (1H, s, Ar-H), 5.06-5.04 (1H, d), 4.87-4.85 (1H, d), 4.82-4.79(1H,d), 4.76(2H,m), 4.47-4.43(3H,m), 4.35-4.32(1H,t), 4.09-4.07(1H,d), 3.94-3.91(1H,t) ), 3.81-3.79(2H,t), 3.72-3.69(1H,m), 3.60(1H,m), 3.54-3.49(2H,m), 3.48-3.39(5H,m), 3.34-3.27(3H) , m), 2.48 (3H, s, -CH ₃ ).

Preparation Example 9 6-[(1-methyl-4-nitro-1H-imidazol-5-yl)sulfur]-9-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl] -9H-purine (Compound 9)

step one:

Add 10.0g lactulose (0.029mol) to the reaction flask at room temperature, add 45.0g acetic anhydride (0.435mol), 34.4g pyridine (0.43mol) in turn, stir for about 1h, then dissolve it, stir at room temperature for 14h, reduce The acetic acid, pyridine and acetic anhydride were removed by pressure concentration, and the residue was purified by petroleum ether/ethyl acetate column chromatography to obtain octaacetyl lactulose (compound F), weighing 16.5 g, and the yield was 83.2%.

Step two:

Under argon atmosphere, mix and stir azathioprine (1.5g, 5.4mmol), N,O-bis(trimethylsilyl)acetamide (BSA) (1.3g, 6.48mmol), and acetonitrile (50ml) , Heated to reflux and reacted for 3 hours, then cooled to 0°C, added the above compound F (5.5 g, 8.1 mmol), and then added TMSOTf (2.2 g, 10 mmol) dropwise. After the addition, the temperature was raised to room temperature and reacted for 4 hours.

Add 130ml of saturated sodium bicarbonate solution and 100ml of dichloromethane to the reaction solution, separate the liquids, extract the aqueous phase with dichloromethane (50ml*2 times), combine the organic phases, dry and purify by column chromatography to obtain compound 2.24g , The yield is 46.3%. ESI(+)m/z: 918.3[M+Na] ⁺ .

Step 3: Preparation of compound 9

The product (2.0 g, 2.2 mmol) of the above step 2 was dissolved in 20 ml of methanol, 10 ml of lithium hydroxide aqueous solution (1M) was added, and the mixture was stirred at room temperature overnight.

The reaction solution was neutralized to neutrality and concentrated to dryness under reduced pressure. 30ml of water and 10ml of methyl tert-butyl ether were added to the residue, dissolved and separated. The aqueous phase product was prepared by C18 reverse phase to obtain 0.51g of solid. It is the title compound (Compound 9) with a yield of 38.0%. ESI(+)m/z: 602.2[M+H] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 8.60-8.55 (2H, Ar-H), 8.25 (1H, s, Ar-H), 5.05 (1H, d), 4.86(1H,d), 4.83-4.76(3H,m), 4.50-4.32(2H,m), 4.08(1H,d), 3.92(1H,t), 3.71-3.69(4H,m), 3.60- 3.50 (3H, m), 3.50-3.25 (8H, m).

Preparation Example 10 6-Mercapto-9-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]-9H-purine (compound 10)

Preparation Example 10 was carried out in a similar manner to Preparation Example 9, except that 6-mercaptopurine was used instead of azathioprine to obtain 1.1 g of the title compound. ESI(+)m/z: 477.2[M+H] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 8.38 (1H, s, Ar-H), 8.18 (1H, s, Ar-H), 5.04 (1H, d), 4.85(1H,d), 4.85-4.75(3H,m), 4.50-4.30(2H,m), 4.08(1H,d), 3.90(1H,t), 3.72(1H,m), 3.60-3.50( 3H, m), 3.50-3.25 (8H, m).

Preparation Example 11 5-Fluoro-1-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]pyrimidine-2,4(1H,3H)-dione (Compound 11)

Preparation Example 11 was implemented in a similar manner to Preparation Example 9, except that 5-fluorouracil was used instead of azathioprine to obtain 0.65 g of the title compound. ESI(-)m/z: 453.1[MH] ^- .

Preparation Example 12 N-{2-[(S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl]-1,3-dioxo-2 ,3-Dihydro-1H-isoindol-4-yl}-N'-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]acetamide (Compound 12)

Preparation Example 12 was carried out in a similar manner to Preparation Example 9.

step one:

Same as Step 1 of Preparation Example 12.

Step two:

Add 0.67g aprester (1.45mmol), BSA (2.18mmol) and 3ml acetonitrile to the reaction flask at room temperature, heat to reflux and react for 3h, then cool to room temperature, add 1.0g octaacetyl lactulose (compound F, the product of Step 1 of Preparation Example 9) (1.45 mmol), dissolved after stirring for 5 minutes, 0.55 g of TMSOTf (2.47 mmol) was slowly added dropwise, and stirred at room temperature for 14 hours after the addition.

At 0°C, add 10ml of saturated sodium bicarbonate solution and 20ml of dichloromethane to the reaction solution, separate the liquids, extract the aqueous phase with dichloromethane (10ml*2 times), combine the organic phases, dry and concentrate to dryness under reduced pressure. The residue was purified with methylene chloride/methanol system column chromatography to obtain the solid product, namely heptaacetyl lactulose alpster glycoside, weighing 263 mg, and the yield was 16.7%. ESI(+)m/z: 1101.4[M+Na] ⁺ .

Step 3: Preparation of compound 12

Dissolve 100mg of the second product in the above step with 5ml methanol, then add 5ml magnesium methoxide methanol solution (7~8%), stir at room temperature for 5h, the reaction solution is neutralized with saturated ammonium chloride solution, concentrated to dryness, dissolved with 5ml water, filtered Insoluble matter was removed, and the filtrate was purified by C18 reverse phase preparation (methanol-water elution). The eluate was concentrated to dryness under reduced pressure to obtain 45 mg of the title compound (compound 9) as a solid with a yield of 61.8%. ESI(+)m/z: 807.3[M+Na] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 8.0-7.8 (3H, m Ar-H), 7.2-6.9 (3H, m, Ar-H), 5.79 (1H, m), 5.0-4.53(5H,m), 4.50-4.20(4H,m), 4.20-3.83(6H,m), 3.74(3H,s), 3.67-3.10(10H,m), 3.0(3H, m), 2.51 (3H, s), 1.32 (3H, t).

Preparation Example 13 17-hydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,11,20-trione (Compound 17)

Preparation Example 13 was implemented in a similar manner to Preparation Example 1, except that prednisolone was used instead of prednisolone to obtain 1.5 g of the title compound. ESI(+)m/z: 705.3[M+Na] ⁺ .

Preparation Example 14 4-Amino-5-chloro-2,3-dihydro- N -[1-(3-methoxypropyl)-4-pyridinyl]- N' -[4-O-(β-D- Galactopyranosyl)-D-Frutofuranosyl)-7-benzofuranoformamide (Compound 13)

Step 1: Preparation of compound K

Dissolve 2.0 g of octabyl lactulose (compound A, preparation example 1) in 2.5M hydrogen chloride glacial acetic acid solution (10ml), stir at room temperature for 4 hours, TLC monitors the disappearance of the raw materials, concentrate under reduced pressure at 50°C and evaporate the solvent The residue was slurried with ether and filtered to obtain the crude compound (K) weighing 1.8 g, which was directly used in the next reaction.

Step 2: Preparation of Compound M

At room temperature, 1.8 g of crude compound K, 0.54 g (1.47 mmol) of prucalopride and 10 ml of toluene were mixed and stirred, 3 drops of triethylamine were added dropwise, and the reaction was stirred at 70° C. for 8 hours. The reaction solution was concentrated to dryness under reduced pressure, and purified by silica gel column chromatography (dichloromethane/methanol) to obtain 0.7 g of product (Compound M) with a yield of 33.6%. ESI(+)m/z: 1442.5[M+Na] ⁺ .

Step 3: Preparation of compound 13

0.68 g of compound M (0.48 mmol) was dissolved in 10 ml of tetrahydrofuran at room temperature, 20 mg (0.37 mmol) of sodium methoxide was added, and the reaction was stirred at room temperature for 4 hours. After the reaction is completed, the reaction solution was concentrated and evaporated to dryness at 45°C under reduced pressure, and then dissolved in 5ml of water, washed with dichloromethane for 5ml*3, the aqueous phase was adjusted to pH=8 with dilute acetic acid, and washed with dichloromethane for 5ml*2. The resulting aqueous product solution was purified by C18 reversed phase preparation (methanol-water elution), and the collected fractions were concentrated and evaporated to dryness at 50°C under reduced pressure to obtain 0.18 g of white solid as the title compound (compound 13) with a yield of 54.2%. ESI(+)m/z: 692.3[M+H] ⁺ . Chemical purity: 95.0% (HPLC method).

HPLC method:

Chromatographic column: octadecylsilane bonded silica gel column; column temperature: 35℃; detection wavelength: 240nm

Mobile phase: A MeOH: CH ₃ CN (2:1)

B 10mmol/L dipotassium hydrogen phosphate (phosphoric acid adjusts the pH to 7.1)

Gradient program: 0→20min (B: 70%→45%); 20→25min (B: 45%→70%); 25→29min (B: 70%→70%)

Retention time: 24.1min

Preparation Example 15 5-chloro-2,3-dihydro-4-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]amino} -N -[1-(3-methoxy Propyl)-4-pyridinyl)-7-benzofuranocarbamide (compound 14)

Step 1: Preparation of compound N

At room temperature, 20.0 g octabyl lactulose (16.3 mmol, compound A, preparation example 1), 5.0 g prucalopride (13.6 mmol), 40 g anhydrous sodium sulfate and 100 ml acetonitrile were sequentially added to the reaction flask. Stir under the protection of argon for 1 h, add tert-butyl dimethylsilyl triflate (TBSOTf, 7.1 g, 27.2 mmol) dropwise, and react at room temperature for 3 h after the addition is complete. Take 4.1g of trimethylpyridine diluted with 10ml of dichloromethane and slowly add dropwise to the reaction system to quench the reaction, stir for 30min, filter, and wash with dichloromethane. The filtrate is concentrated under reduced pressure at 45°C and evaporated to dryness. Silica gel column chromatography Purified (dichloromethane/methanol) to obtain 7.5 g of product (Compound N) with a yield of 38.8%. ESI(+)m/z: 1442.5[M+Na] ⁺ .

Step 2: Preparation of compound 14

Dissolve the product (Compound N, 1.8g, 1.27mmol) of the above step 1 at room temperature in 15ml of tetrahydrofuran, add 30mg (0.56mmol) of sodium methoxide and stir for 4h at room temperature. After the reaction is completed, the reaction solution was concentrated and evaporated to dryness at 45°C under reduced pressure, and then dissolved in 5ml of water, washed with dichloromethane for 5ml*3, the aqueous phase was adjusted to pH=8 with dilute acetic acid, and washed with dichloromethane for 5ml*2. The resulting aqueous product solution was purified by C18 reverse phase preparation (methanol-water elution), and the collected fractions were concentrated and evaporated to dryness at 50°C under reduced pressure to obtain 0.36 g of white solid as the title compound (Compound 14) with a yield of 40.9%. The chemical purity is 96.0% (HPLC method). ESI(+)m/z: 692.3[M+H] ⁺ .

¹ H-NMR (DMSO-d6,400M, BRUKER AV-400): δ (ppm) 7.57-7.45 (2H, m), 5.66 (1H, s), 5.41 (1H, s), 4.75-4.60 (4H, m), 4.34 (1H, m), 4.08 (1H, m), 4.0-3.82 (3H, m), 3.80-3.70 (2H, m), 3.70-3.62 (2H, m), 3.62-3.30 (12H, m), 3.24(2H,m), 3.21(3H,s), 2.70(2H,m), 2.30(2H,m), 2.04(2H,m), 1.79(2H,m), 1.64(2H,m) ), 1.45 (2H, m).

The HPLC method is the same as that of Preparation Example 14, and the retention time is 13.9 min.

Preparation Example 16 4-{ N -methyl- N '-[(3R,4R)-4-methyl-1-(2-cyanoacetoxy)piridin-3-yl]amino}7-[4-O- (β-D-galactopyranosyl)-D-fructofuranosyl)-7 H -pyrrolo[2,3- d ]pyrimidine (Compound 18)

Preparation Example 16 was carried out in a similar manner to Preparation Example 9, except that tofacitinib was substituted for azathioprine to obtain 0.25 g of the title compound. ESI(+)m/z: 637.5[M+H] ⁺ .

Hereinafter, various preparations (for example, tablets, enteric-coated granules, capsules, etc.) are prepared using the compound represented by formula (I) according to the present invention, and the formula and specific preparation methods are shown in the following examples.

Formulation Example 1

With Compound 1 prepared according to the present invention as the active ingredient of the medicament, tablets were prepared according to the formula shown in Table 2 below.

Among them, in the preparation of tablet formulations 1, 3, and 4, all the components of the tablet core are mixed and then compressed.

In the preparation of tablet formulation 2, in addition to magnesium stearate and microcrystalline cellulose, an aqueous solution (5%) of hydroxypropyl cellulose was mixed and granulated by wet method, dried, and finally magnesium stearate was added to the mixture and then compressed into tablets. .

In the preparation of tablet formulation 3, Opadry (gastric dissolving film coating powder, Shanghai Colorcon Co., Ltd.) water dispersion was used for coating. A conventional high-efficiency coating pan is sufficient.

In the preparation of tablet formulation 4, with regard to the preparation of the coating, add 20 g of Eudragit L30D-55 (Evonik_Rhom, Germany), 1.8 g of talc, and 0.5 g of triethyl citrate. Dilute with water, stir evenly, and prepare it in a high-efficiency coating pan according to the conventional coating process parameters.

Formulation Example 2

With Compound 1 prepared according to the present invention as the active ingredient of the medicament, enteric-coated granules were prepared according to the formula shown in Table 3 below.

Take the compound 1, hypromellose, and talc in the drug formulation to dissolve and disperse in water as the drug solution; place the pellet core in a fluidized bed (WBF-1G Chongqing Yingge Granulation Coating Technology Co., Ltd.) Medium fluidization, set parameters (air inlet 50m ³ /h, temperature 40~50℃, atomization pressure 1.0bar), spray the medicine solution on the bottom, flow rate 2~3g/min, control the material temperature 30±3℃. Encapsulate the enteric-coated layer after application of the medicine.

Take the formula of the enteric layer and mix it uniformly, and coat it according to the above process parameters to obtain enteric granules.

Formulation Example 3

Using Compound 7 prepared according to the present invention as the active ingredient of the drug, capsules were prepared according to the formula shown in Table 4 below.

In the preparation of the medicine, after the above contents are uniformly mixed, the capsule shell is filled to obtain a capsule.

Formulation Example 4

With compound 12 prepared according to the present invention as the active ingredient of the medicament, tablets were prepared according to the formula shown in Table 5 below.

In the preparation process of the medicine, the tablets are all granulated by the wet method. Among them, the tablet core components are mixed first, and then an aqueous solution of the binder is added for wet granulation, and after drying, the disintegrant and lubricant are added, mixed together, and finally compressed.

The coating adopts the conventional coating process.

Compound performance test

In this application, the compound represented by formula (I) according to the present invention has been tested in terms of stability in vitro, system pharmacokinetics in animals, and local gastrointestinal pharmacokinetic characteristics.

In vitro media stability test

At 37°C, compound 17 was incubated in different media (buffer solutions with different pH, simulated intestinal juice containing pancreatin, and simulated gastric juice containing pepsin). Samples were taken at intervals as shown in Table 6 below, and the percentages of compound 17 and its degradation products (ie, the original form, prednisone) were detected by the HPLC method, in which the content of each component was calculated by the normalization method. In the analysis results, the higher the proportion of compound 17 and the lower the proportion of degradation products, it indicates that compound 17 is more stable. The results in Table 6 show that when the pH is above 2.0, within 1.5 hours, the residual percentage of compound 17 is greater than 80%, and the degradation product is less than 20%, and it has a greater ability to tolerate gastric acid.

Stability in the presence of colonic flora

A phosphate buffer with a pH of 7.0 was prepared, and CO ₂ was introduced into it to adjust its pH to 6.8. Then, take the cecal contents of male SD rats (body weight 180-220g), in a 37℃ water bath, under the _{protection of N 2} added to the above-prepared pH 6.8 phosphate buffer to make the concentration 20% (w /v). Keep the buffer in an oxygen-free environment, then add test compound 17 and stir it evenly.

As shown in Table 7 below, take 0.5ml of the sample solution regularly, add 1.0ml of acetone to it to precipitate the protein, mix well, leave it for 2min, and then centrifuge (12000rpm, 5min). The supernatant was taken for HPLC analysis, and the content of each component was calculated by the normalization method. As shown in Table 7, compound 17 is easily degraded in a phosphate buffer of pH 6.8, and only 11.3% remains at 3 h, which is about 90% degraded. The above results indicate that compound 17 can rapidly degrade and remove glycosidic bonds in the colonic flora environment, produce active drugs, and exert its efficacy in time.

The stability of other typical example compounds was tested according to the above method, and the test results are shown in Table 8 below.

Test of systemic pharmacokinetics and local gastrointestinal pharmacokinetic characteristics after oral administration of compound 1 in SD rats

The systemic pharmacokinetics and local gastrointestinal tract of SD rats after intragastric administration of compound 1 (preparation example 1), compound 1 enteric-coated granules (preparation example 2) and its active ingredient (prednisolone) Comparative study of pharmacokinetics to further evaluate the absorption and transformation process of compound 1 in rats and the main parts of the transformation and release of active ingredients, and to explore whether the application of enteric-coated preparation technology can overcome the pH of compound 1 in in vitro tests. Defects that can be partially degraded in a lower acidic environment.

According to the experiment, 112 healthy SD male rats (body weight 180-220 grams) were selected and divided into 4 groups (prednisolone group, compound 1 group, compound 1 enteric granule group and blank control group), among which prednisolone There were 36 animals in each group of Songlong group, Compound 1 group and Compound 1 enteric-coated granule group, and 4 animals in the blank control group. See Table 9 below for the detailed dosage regimen.

For the experimental animals of group 1, group 2 and group 3, 4 animals were randomly selected at each time point of 30min, 1h, 2h, 3h, 4h, 5h, 6h, 8h and 10h after administration They were sacrificed and blood samples were collected, and the contents of the stomach and various segments of the intestine (the front half of the small intestine, the second half of the small intestine, the front 1/3 of the colon (including the cecum) and the back 2/3 of the colon) were taken and weighed. The LC-MS/MS method was used to determine the concentration of prednisolone and compound 1 in the plasma and contents, and the contents of prednisolone and compound 1 in each segment of the digestive tract were calculated based on the weight of the contents. The 4 animals in the 4th group were sacrificed at 10 hours, and the blood and the contents of each segment of the gastrointestinal tract were used as a blank control.

The distribution of prednisolone in the contents of the gastrointestinal tract of rats at different time points in the prednisolone group is shown in Table 10 below. From the results in Table 10, it can be seen that after rats were given prednisolone by gavage, the prednisolone in the gastrointestinal tract was mainly distributed in the stomach and small intestine, with the highest concentration in the stomach; while the contents of the cecum and colon had the highest concentration. The concentration of Nisonerone is very low.

The distribution of prednisolone in the contents of the gastrointestinal tract of the rats in the compound 1 group and the compound 1 enteric-coated granule group after intragastric administration is shown in Table 11 and Table 12, respectively. Compared with the prednisolone group, the gastrointestinal distribution of the compound 1 group and the compound 1 enteric-coated granule group was significantly reduced, while the maximum local prednisolone concentration (Cmax) in the cecum and colon increased 65 to 360 times (results) (See Table 13), the total exposure (AUC) is also much higher than the prednisolone prototype drug group (drug-time curve shown in Figure 1 and Figure 2). Because the enteric-coated granules were used for intragastric gavage to avoid the partial release of compound 1 in the acidic environment of the stomach, the compound 1 enteric-coated granules group basically did not release prednisolone in the stomach and the anterior part of the small intestine (the concentrations are all below the detection line. Detected), and the local prednisolone concentration in the colon was slightly higher than that of the compound 1 group. The above results indicate that the main release sites of the oral compound 1 enteric-coated particles in the body are in the cecum and colon, followed by the posterior small intestine close to the cecum, which is consistent with the purpose of the compound of the present invention.

The plasma prednisolone concentration change curve of rats in the prednisolone group, compound 1 group and compound 1 enteric granule group after intragastric administration is shown in Fig. 3. From the drug-time curve, it can be seen that, compared with the free prednisolone group, the Cmax values of the compound 1 group and the compound 1 enteric-coated granule group are very low, and the time to peak is also significantly delayed.

Compound 1 rats in the compound 1 group could detect a lower concentration of compound 1 (10.4 ng/ml) at only one time point of 0.5 hours, and compound 1 was not detected at all time points after administration of compound 1 enteric-coated particles. The results of this study show that compound 1 is only slightly absorbed in the stomach or the upper end of the small intestine in rats, and the absorption of compound 1 can be effectively avoided after the enteric-coated preparation technology is used to avoid the release from the stomach.

Pharmacodynamic effects of compound 1 on Wistar rat ulcerative colitis model induced by TNBS enema by intragastric administration

By carrying out a comparative study on the therapeutic effects of compound 1 and prednisolone (prednisolone tablets, produced by Wuhan Yuancheng Gongchuang Technology Co., Ltd.) on Wistar rats with ulcerative colitis model by intragastric administration of compound 1 and prednisolone, further To evaluate the improvement of the main efficacy indicators of compound 1 in the treatment of ulcerative colitis by intragastric administration and systematically compare it with the treatment group of the marketed preparation of equal-molar prednisolone, so as to provide pharmacodynamics for the further development of compound 1. support.

According to this experiment, 40 male Wistar rats of 180-220 grams were purchased and randomly divided into four groups (saline control group, model control group, prednisolone group and compound 1 group), with 10 animals in each group. Except for the normal saline control group, the other three groups of experimental animals all used a 3.2ml/kg 2.5% TNBS-50% ethanol mixed solution (containing 80mg/kg TNBS) to create animal models of ulcerative colitis. The normal saline control group received a one-time enema with 3.2ml/kg normal saline. 24 hours after the modeling and administration, the corresponding vehicle control or therapeutic drugs (qd×7d (administered once a day, continuous administration for 7 days)) were given by intragastric administration according to the animal groups. For detailed grouping and administration information, please refer to Table 14 below .

The main observation indicators during the research process include: animal death, animal clinical observation, body weight, colon coefficient, histopathological examination, etc. The animals in each experimental group were sacrificed on the 8th day after modeling, the colon tissue was taken to calculate the colon coefficient (colon coefficient = colon weight (g) / animal weight (g) * 100%), and then histopathological examination and score (tissue The pathology scoring criteria are shown in Table 15 below).

During the study, only one animal in the model control group died on the 6th day after modeling, and no abnormal death of animals was found in the other groups.

The clinical symptoms of the modeled animals are mainly: orange/yellow liquid around the anus, blood around the nasal cavity, blood stains at the corners of the eyes, arched hairs, loose stools, lack of energy, reduced activity, slow movement, yellow/orange loose stools , Brown loose stools, bloody loose stools and no stools.

As shown in Figure 4, the body weight of the animals in the negative control group maintained a continuous growth trend throughout the test period; on the contrary, the model control, prednisolone and compound 1 groups had an average of the animals in each group in the first 3 days after modeling. The body weight showed an obvious downward trend. The body weight of the compound 1 group began to rise from the 4th day of the modeling and the other two groups began to rise from the 5th day of the modeling. Among them, the weight recovery rate of compound 1 group was faster than that of the other two groups. A one-way analysis of variance was further used to statistically compare the weight changes on the 8th day of the different experimental groups (weight change = weight on day 8 (g)-weight on day 1 (g)) (see Figure 5, where * indicates p<0.05, compared with the negative control group; # means p<0.05, compared with the model control group), the results show that the weight gain of the three groups of animals modeled with TNBS was significantly lower than that of the negative control group modeled with saline; and model Compared with the group, the weight gain of the prednisolone group and the compound 1 group was higher than that of the model group, but only the difference between the compound 1 group and the model group was statistically significant. This result shows that the same molar number of compound 1 has a positive effect on TNBS. The therapeutic effect of the rat model of ulcerative colitis caused by enema is better than that of the prednisolone prototype.

The colon coefficient index was obtained after the animal was planned to be dissected on the 8th day after the model was created. The colon coefficient of each experimental group was compared by one-way analysis of variance, and the result was more consistent with the analysis result of weight change (see Figure 6, where * means p<0.05, compared with the negative control group; # indicates p<0.05, compared with the model control group). Compared with the negative control group, the colon coefficient of each model group was significantly increased (p<0.05), the model control group was the most obvious, followed by the prednisolone group, and the compound 1 group had the lowest increase. Compared with the compound 1 group and the model control group, the colon coefficient of the compound 1 group was significantly lower than that of the model control group (p<0.05), while the difference between the prednisolone group and the model control group did not reach statistical significance.

On the 8th day after TNBS modeling, the colonic histopathological changes mainly manifested as focal mucosal necrosis in the descending colon, neutrophil infiltration of the mucosal layer and submucosa, edema, vasodilation, and hyperemia.

The results of one-way analysis of variance of colonic histopathological scores in each experimental animal group are shown in Figure 7 (where * indicates p<0.05, compared with the negative control group; # indicates p<0.05, compared with the negative control group). Compared with the group, the histopathological scores of each model group were significantly higher (p<0.05), with the model control group being the most obvious, followed by the prednisolone group, and the compound 1 group had the lowest increase; further combining prednisolone and Compared with the model control group, the histopathology of the compound 1 group was significantly lower than that of the model control group (p<0.05), while the difference between the prednisolone group and the model control group did not reach statistical significance. This result is consistent with the aforementioned analysis results of body weight and colon coefficient, further suggesting that the oral administration of compound 1 in an equal molar amount is more effective than prednisolone in the rat model of ulcerative colitis.

The above studies have been conducted on compound 1 in terms of animal body system pharmacokinetics, gastrointestinal local pharmacokinetics and pharmacodynamic effects. The results prove that the lactulose glycoside compound 1 according to the present invention can pass through the gastrointestinal tract of mammals. It is not significantly absorbed by the gastrointestinal tract and is not significantly hydrolyzed by the endogenous enzymes of the mammalian host. Therefore, the lactulose compound 1 can reach the colon of the mammal and release the active drug in the colon under the action of the colonic flora. The lactulose glycoside compound 1 has a colon-specific drug release effect, and can be used for the prevention or treatment of intestinal diseases.

It should be pointed out that the present invention also studies other compounds (ie, compounds 2-17 shown in Table 1) in terms of system pharmacokinetics, gastrointestinal local pharmacokinetics, and pharmacodynamic effects in animals. Experimental research. The results prove that the lactulose compound 2-17 according to the present invention can also pass through the gastrointestinal tract of mammals without being significantly absorbed by the gastrointestinal tract and not significantly hydrolyzed by the endogenous enzymes of the mammalian host. Therefore, the lactulose compound 2-17 It can reach the colon of mammals and release active drugs in the colon under the action of colonic flora. The lactulose glycoside compound 2-17 has a colon-localized drug release effect, and can be used for the prevention or treatment of intestinal diseases.

Although the present invention has been described in detail above with reference to the embodiments and comparative examples, it should be pointed out that the present invention is not limited to the embodiments and comparative examples, and changes or modifications can be made to the present invention within the scope not departing from the spirit of the present invention.

Claims (14)

A compound represented by formula (I),

Among them, the lactulose group is connected to the heteroatom in the aglycon (G) through a glycosidic bond, and the aglycon (G) is a group formed by removing a hydrogen atom from the heteroatom of the active drug molecule, and

”Means that the lactulose group is connected to the heteroatom in the aglycon (G) through an α-glycosidic bond or β-glycosidic bond; wherein the active drug molecule is selected from the group consisting of glucocorticoid drugs, aprost, procabbit Any one of Li, mesalazine, metronidazole, azathioprine, 6-mercaptopurine, 5-fluorouracil, and tofacitinib. The compound according to the first item of the scope of patent application, wherein the heteroatom is selected from any one of oxygen atom, nitrogen atom and sulfur atom. The compound described in item 1 of the scope of the patent application, wherein the aglycon (G) is obtained by removing a hydrogen atom from the alcoholic hydroxyl, phenolic hydroxyl, amino, amido or heterocyclic NH in the active drug molecule The group formed. The compound according to item 1 of the scope of the patent application, wherein the glucocorticoid drug is selected from the group consisting of cortisone acetate, prednisone, prednisone acetate, hydrocortisone, hydrocortisone acetate, butyric acid Hydrocortisone, Hydrocortisone Sodium Succinate, Hydrocortisone Sodium Phosphate, Fludrocortisone, Fludrocortisone Acetate, Prednisolone, Prednisolone Acetate, Prednisolone Succinate Sodium, prednisolone sodium phosphate, betamethasone, betamethasone acetate, betamethasone dipropionate, clobetasone butyrate, betamethasone valerate, betamethasone sodium phosphate, beclomethasone dipropionate , Mometasone furoate, clobetasol propionate, dexamethasone propionate, dexamethasone acetate, dexamethasone sodium phosphate, p-flumethasone, fluticasone propionate, methylprednisolone, methylprednisolone acetate, fluorometholone, Qu Any one of Ancilone, Triamcinolone Acetonide, Triamcinolone Acetonide Acetate, Flucinonide, Ancinonide, Hascinonide, Ciclesonide, Budesonide, and Difcote. The compound according to item 1 of the scope of patent application, wherein the glucocorticoid drug is selected from hydrocortisone, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, and buddysone Any of Ned. The compound described in item 1 of the scope of patent application, wherein the aglycon (G) is a group represented by the following formula (II):

Wherein, R ₁ is H or F; R ₂ is H, F or -CH ₃ ; R ₃ is -OH; and R ₄ is H, -OH or -CH ₃ ; or R ₃ and R _{4 are} connected to form a group

,

or

. The compound described in item 6 of the scope of patent application, wherein the aglycon (G) is selected from any one of the following:

Where Me represents -CH ₃ . The compound described in item 1 of the scope of the patent application, wherein the aglycon (G) is selected from any one of the following:

Where Me represents -CH ₃ . The compound described in item 1 of the scope of the patent application, wherein the aglycon (G) is selected from any one of the following:

Where Me represents -CH ₃ . The compound described in item 1 of the scope of the patent application, wherein the compound is selected from any one of the following: (11β)-11,17-dihydroxy-21-{[4-O-(β-D -Galactosyl)-D-Frutofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β)-11,17-dihydroxy-21-{[4- O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-4-ene-3,20-dione; (6α,11β)-11,17-dihydroxy-6 -Methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,20-dione; ( 11β,16α)-9-fluoro-11,17-dihydroxy-16-methyl-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregnancy Steroid-1,4-diene-3,20-dione; (11β,16β)-9-fluoro-11,17-dihydroxy-16-methyl-21-{[4-O-(β-D -Galactopyranosyl)-D-fructofuranosyl]oxy}pregnant-1,4-diene-3,20-dione; (11β,16α)-9-fluoro-11,16,17-three Hydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,20-dione; (11β, 16α)-11-hydroxy-16,17-(butylene dioxy)-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy)pregna-1 ,4-Diene-3,20-dione; 2-methyl-5-nitro-1-{2-{[4-O-(β-D-galactopyranosyl)-D-frutofuranose Yl]oxy}ethyl}imidazole; 6-[(1-methyl-4-nitro-1 H -imidazol-5-yl)sulfur]-9-[4-O-(β-D-pyran half Lactosyl)-D-Frutofuranosyl]-9 H -purine; 6-Mercapto-9-[4-O-(β-D-galactopyranosyl)-D-Frutofuranosyl]-9 H -purine ; 5-Fluoro-1-[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]pyrimidine-2,4(1H,3H)-dione; N -{2-[ (S)-1-(3-ethoxy-4-methoxyphenyl)-2-(methylsulfonyl)ethyl)-1,3-dioxo-2,3-dihydro- 1 H -isoindol-4-yl} -N' -[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]acetamide; 4-amino-5-chloro- 2,3-Dihydro- N -[1-(3-Methoxypropyl)-4-pyridinyl] -N' -[4-O-(β-D-galactopyranosyl)-D -Frutofuranosyl]-7-benzofuranoformamide; 5-Chloro-2,3-dihydro-4-{[4-O-(β-D-galactopyranosyl)-D-Frutofuranose基]amino} -N -[1-(3-Methoxypropyl)-4-pyridinyl]-7-benzofuran carboxamide; 5-amino-2-{[4-O-(β-D-pyran half Lactosyl)-D-fructofuranosyl]oxy}benzoic acid; 2-hydroxy-5-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]amino}benzoic acid; 17-hydroxy-21-{[4-O-(β-D-galactopyranosyl)-D-fructofuranosyl]oxy}pregna-1,4-diene-3,11,20-trione ; And 4-{ N -methyl- N '-[(3R,4R)-4-methyl-1-(2-cyanoacetoxy)pyridin-3-yl]amino}7-[4- O-(β-D-galactopyranosyl)-D-fructofuranosyl]-7 H -pyrrolo[2,3- d ]pyrimidine. A pharmaceutical composition containing the compound described in any one of items 1 to 10 in the scope of the patent application and a pharmaceutically acceptable carrier. A use of the compound described in any one of items 1 to 10 in the scope of the patent application for preparing a medicine for preventing or treating intestinal diseases. The use according to item 12 of the scope of patent application, wherein the intestinal disease is selected from the group consisting of ulcerative colitis, Crohn's disease, infectious enteritis, irritable bowel syndrome, chronic constipation, amoebic intestinal disease, Colon cancer and rectal cancer. The use according to item 12 of the scope of patent application, wherein the intestinal disease is selected from ulcerative colitis, Crohn's disease and chronic constipation.

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