CN102276521A - Kukoamine B analogue, and pharmaceutically acceptable salt, preparation method and application thereof - Google Patents

Abstract

式(I)其中，R₁，R₂，R₃，R₄可以独立的为H，3碳以下的烷基，甲酰基，乙酰基，苄基等羟基的保护基；X_1-3可以独立的为C原子或者N原子，当其中一个为N原子时其余两个为C原子；X_4-6可以独立的为C原子或者N原子，当其中一个为N原子时其余两个为C原子；n_1-5可以独立为4碳以下烷基。The present invention relates to acrosamine B analogs with the structure of formula (1) or pharmaceutically acceptable salts thereof, their preparation methods and uses.

Formula (I) wherein, R ₁ , R ₂ , R ₃ , and R ₄ can independently be H, an alkyl group with 3 carbons or less, formyl, acetyl, benzyl and other hydroxyl protecting groups; X _1-3 can be independently X _4-6 can be C atoms or N atoms independently, and when one of them is N atom, the other two are C atoms; n _1-5 can be independently an alkyl group with 4 carbons or less.

Description

Kuchrysamine B analogue and pharmaceutically acceptable salt thereof, preparation method and application thereof

technical field

The invention belongs to the technical field of medicines, and in particular relates to a variety of chrysamine B analogs or pharmaceutically acceptable salts thereof and a preparation method thereof, and the use of the chrysamine B analogs and salts thereof for preparing medicines for preventing and treating sepsis. the

Background technique

Sepsis is a systemic inflammatory response syndrome (SIRS) mediated by infectious factors. It is a common complication in patients with burns, trauma, tumors and infectious diseases. The leading cause of death in (ICU) patients. The existing clinical treatment methods for sepsis are mainly the early use of antibiotics and the correction of ischemic hypoxic damage, and the conventional treatment of organ failure and shock. There is no specific treatment. Drug treatment is usually based on empirical use of non-specific drugs such as glucocorticoids, insulin, and immunomodulators, but the efficacy has not been confirmed. In the 1990s, the anti-lipid A monoclonal antibody HA-1A (Centoxin) was used in the treatment of sepsis after war wounds and burns in the US military during the Gulf War. The drug was also clinically used in some European countries and Japan, but Because it may have adverse effects on septic shock, it failed to obtain the approval of the US FDA in 1992, and then disappeared from the European market. Recombinant human activated protein C (rhAPC) is currently the only FDA-approved drug for the treatment of sepsis (trade name Xigris). The results of clinical trials showed that rhAPC could reduce the 28-day mortality rate of patients with sepsis, and it was approved for marketing by FDA in November 2001. However, the results of the second clinical trial in 2005 showed no difference in the 28-day mortality rate between the rhAPC group and the control group. In the clinical trial in 2007, rhAPC not only did not improve the survival rate of patients with sepsis, but also had side effects of causing severe bleeding tendencies in patients. Therefore, the institution that organized this clinical trial suggested not to recommend its use as a drug for the treatment of sepsis. clinical drug. the

The discovery of pathogen-associated molecular patterns (pathogen-associated molecular patterns, PAMPs) and its pattern recognition receptors (pattern recognition receptors, PRRs) has made a qualitative leap in human understanding of sepsis. It has been confirmed that the pathogenesis of sepsis is that after pathogens invade the body, the PAMPs (mainly including lipopolysaccharide/endotoxin (LPS), bacterial genomic DNA (CpG DNA) and peptideglycan (peptideglycan, PGN), etc.) are recognized by the corresponding PRRs on the inflammatory response cell membrane/intracellular in the non-specific immune system of the body, leading to the activation of inflammatory response cells, releasing inflammatory mediators and triggering systemic inflammatory response, which in turn leads to tissue and organ damage. Therefore, when previous treatment measures such as antagonizing important effector molecules in the inflammatory response, correcting coagulation and complement system disorders, and single antagonizing LPS failed, it is necessary to look for drugs that simultaneously antagonize multiple major PAMPs (LPS, CpG DNA, PGN, etc.) Drugs, blocking the occurrence of sepsis from the source may bring a breakthrough in its treatment. the

Kukoamine B (kukoamine B), also known as Digupi B, is a naturally occurring alkaloid compound isolated from the traditional Chinese medicine Digupi. Its chemical structure is as follows:

Kuchrysamine B was first discovered by Shinji Funayama from Japan in 1995 from Digus bark, and exists in the form of free base (S.Funayama, G. Zhang and S.Nozoe.Phytochemistry.1995; 38:1529-1531). Zheng Jiang et al. of the Third Military Medical University disclosed the use of Kuchrysamine B in the preparation of drugs for the prevention and treatment of sepsis and autoimmune diseases (Chinese Patent Application No. 201010156503.X). However, it did not synthesize the analogues of Kuchrysamine B to find the best compound and its pharmaceutical form that are better and more suitable for drug use. the

Contents of the invention

Aiming at the above shortcomings, the present invention designs and synthesizes a series of Kuchrysamine B analogs and pharmaceutically acceptable salts thereof for preventing and treating sepsis and autoimmune diseases. The present invention also provides the application of the pharmaceutical composition of these compounds in the preparation of medicines for preventing and treating sepsis. The use of these compounds and pharmaceutical compositions comprising these compounds provides the basis for some new alternative drugs for the treatment of sepsis. the

Technical scheme of the present invention is:

An analogue of Kuchrysamine B or a pharmaceutically acceptable salt thereof is provided, characterized in that: the analogue of Kuchrysamine B has the following chemical structure

Formula (I)

Among them, R ₁ , R ₂ , R ₃ , and R ₄ can independently be H, an alkyl group with less than 3 carbons, formyl, acetyl, benzyl and other hydroxyl protecting groups; X _1-3 can be independently a C atom Or N atoms, when one of them is an N atom, the other two are C atoms; X _4-6 can be independently C atoms or N atoms, and when one of them is an N atom, the other two are C atoms; n _1-5 Can be independently an alkyl group with 4 carbons or less.

The analogue of preferred Kuchrysamine B is:

Compound 1

Compound m

Compound n

Compound o

compound k

The most preferred analogue of Kucosamine B is compound k and its pharmaceutically acceptable salts. the

The pharmaceutically acceptable salts of the analogues of chrysamine B of the present invention are mineral acids of anaerobic or oxyacids or carboxylic acids or hydroxyacids or sulfonic acids or organic acids of acidic amino acids. the

The anaerobic acid is any one of hydrochloric acid and hydrobromic acid. the

The oxyacid is any one of sulfuric acid, phosphoric acid and nitric acid. the

The inorganic acid is any one of hydrochloric acid, hydrobromic acid, sulfuric acid and phosphoric acid. the

The carboxylic acid is acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid, adipic acid, benzoic acid, phenylpropionic acid, cinnamic acid, stearic acid, trifluoroacetic acid, maleic acid, fumaric acid any one of acid, niacin and palmitic acid. the

The hydroxy acid is any one of malic acid, citric acid, lactic acid, hydroxybutyric acid, lactobionic acid, tartaric acid, mandelic acid, gluconic acid, glucuronic acid and ascorbic acid. the

The sulfonic acid is any one of methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and camphorsulfonic acid. the

The acidic amino acid is any one of glutamic acid and aspartic acid. the

The organic acid is any one of acetic acid, maleic acid, succinic acid, malic acid, lactic acid, tartaric acid, methanesulfonic acid, p-toluenesulfonic acid, glutamic acid and aspartic acid. the

The acids in the salts of the analogues of chrysanthemum B are widely selected from a variety of inorganic acids and/or organic acids, mainly by selecting 2 to 3 representative acids from the various acids that are allowed to be used in medicine Illustrates the common problem of preparing picroamine B salt, including selecting hydrochloric acid and hydrobromic acid from anaerobic acids in inorganic acids, sulfuric acid and phosphoric acid from oxoacids, and acetic acid and horseradish from carboxylic acids in organic acids. Toric acid and succinic acid, malic acid, lactic acid and tartaric acid are selected among the hydroxy acids, methanesulfonic acid and p-toluenesulfonic acid are selected among the sulfonic acids, and glutamic acid and aspartic acid are selected among the acidic amino acids. Although the applicant's experiment has only proposed these four kinds of inorganic acids and ten kinds of organic acids, other inorganic acids and/or organic acids (comprising the present invention's exemplified and unenumerated acids) all can prepare picric acid in the same way. Chrysamine B salt. the

Preferred salts of the crocodamine B analogues of the present invention are hydrochloride, hydrobromide, sulfate, phosphate, acetate. the

Most preferred are hydrochloride and hydrobromide. the

The most preferred compounds of the present invention are therefore compound k and its hydrochloride or hydrobromide salts. the

The present invention also provides a pharmaceutical composition containing chrysanthemum B analogs and salts thereof, and their application in the preparation of drugs for the prevention and treatment of sepsis. The pharmaceutical composition of the present invention can be prepared into any pharmaceutical dosage form, For example, dosage forms that can be administered through the gastrointestinal tract, such as powders, tablets, granules, capsules, solutions, emulsions, suspensions, etc.; or parenteral dosage forms, such as injections Drug administration, cavity administration, mucosal administration and other methods of administration. For adults, the dosage is preferably 0.1-15 mg per kilogram of body weight per day, and the dosage can be once or several times a day. the

The pharmaceutical composition of Kuchrysamine B analogs and salts thereof of the present invention contains Kuchrysamine B analogs and salts thereof as active ingredients and pharmaceutically acceptable carriers and/or diluents. the

The Kuchrysamine B analogue and its salts described in the present invention can be used for the preparation of drugs for the prevention and treatment of sepsis. the

The applicant has shown through pharmacological activity experiments:

The binding ability of the active center lipid A of Kuchrysamine B analog of the present invention and LPS is stronger than that of Kuchrysamine B, and some analogues even far exceed the binding ability of Kuchrysamine B; these have stronger The analogs with binding ability neutralized LPS in a dose-dependent manner in vitro; these analogs with stronger binding ability inhibited the release of inflammatory mediators induced by LPS and CpG DNA in RAW264.7 cells in a dose-dependent manner, respectively. the

The pharmaceutical composition of the present invention uses the Kuchrysamine B analogue and its salt, which is different from existing drugs such as glucocorticoids, insulin, anti-inflammatory mediator drugs, anticoagulant drugs, anti-LPS polypeptides and lipid A monoclonal antibodies; good curative effect , can significantly inhibit the inflammatory response induced by antagonizing multiple pathogenic molecules at the same time, providing more optional small molecule compounds for the treatment of sepsis. the

In the present invention, a variety of inorganic acids and/or organic acids are widely selected to illustrate the common problems of preparing the pharmaceutically acceptable salts of chrysanthemum B analogues. Those skilled in the field of chemistry and pharmacy can understand that using similar inorganic acids and/or organic acids or organic acids can be prepared in the same manner as the pharmaceutically acceptable salts of Kuchrysamine B analogues by methods well known in the chemical field. Therefore, it should be noted that substituting other inorganic acids and/or organic acids for the inorganic acids and/or organic acids listed in the present invention should all belong to the technical solution content and patent protection scope of the present invention. Any changes made to the implementation of the present invention are within the scope of the claims of the present application. the

LPS and CpG DNA are the key pathogenic factors leading to the onset of sepsis, and the antagonism of drugs against LPS and CpG DNA reflects its preventive effect on sepsis. the

Detailed ways

The following example is a detailed description of one of the analog compounds k of the present invention, through which the main synthetic routes and experimental methods used for other compounds of the present invention are revealed. It should be understood that the following descriptions are only preferred embodiments of the present invention, which do not limit the present invention in any form. It should be noted that, unless departing from the spirit and scope of the present invention, modifications or equivalent replacements to the present invention shall be included in the protection scope of the claims of the present invention. the

Embodiment 1: the synthesis of the bitter chrysamine B analogue that pyridine ring replaces

compound k

1.1 Experimental method: (1) Dissolve 20g of compound a in 60ml of DMF, add 66g of potassium carbonate and 45ml of chlorinated chloride and raise the temperature to 80°C for reaction. TLC monitoring, after the reaction is complete, filter directly, extract with ethyl acetate, wash with water, wash with saturated brine, dry over anhydrous sodium sulfate, filter with suction, and concentrate to obtain compound b. Dissolve 8g of sodium hydroxide in 80ml of water, add 43g of compound b to it, then add 80ml of methanol, and heat up to 90°C for reflux reaction. TLC monitoring, after the reaction is complete, rotary evaporated to dryness, poured into a beaker, added concentrated HCl to strong acidity, filtered, collected filter cake, dried to obtain silver yellow compound c32g, yield 72%, chemical reaction equation is as follows:

(2) Weigh 13.1g of compound c and dissolve it in 40ml of anhydrous DCM, add 0.1ml of DMF, add 5ml of thionyl chloride, heat up to reflux for 2h, concentrate and rotary evaporate to dry DCM and excess thionyl chloride to obtain Compound d was directly used in the next reaction. Another 13.8g of compound e was weighed and dissolved in 50ml of DCM, 12ml of triethylamine was added, and then the compound d obtained above was dissolved with 20ml of DCM and added dropwise to it. After the reaction was complete, 50ml of water was added, and the organic phase was sequentially washed with saturated potassium carbonate solution, HCl solution, water, saturated brine, dried over anhydrous sodium sulfate, left standing, suction filtered and spin-dried to obtain 25 g of red oily substance, yield 90%, the chemical reaction equation is as follows:

(3) Weigh 13g of compound f and dissolve it in 20ml of DCM, add 10ml of TFA, and stir at room temperature. TLC monitoring, after the reaction was complete, concentrated and rotary evaporated to dry DCM, added 30ml of ethyl acetate and 20ml of water, washed the organic phase with water, washed with saturated brine, dried over anhydrous sodium sulfate, stood still, suction filtered, and concentrated to obtain 12g of oily matter, The yield is 90%, and the chemical reaction equation is as follows:

(4) Weigh 10g of compound g and dissolve it in 20ml of MeOH, add 4ml of triethylamine, raise the temperature to 60°C, and slowly drop in a mixed solution of 1.2ml of acrylonitrile and 15ml of methanol during stirring. After the dropwise addition was completed, the reaction was gradually lowered to room temperature and stirred. TLC monitoring, after the reaction is complete, direct concentration and rotary evaporation to remove the organic solvent to obtain compound h, the chemical reaction equation is as follows:

(5) Another 19.6 g of compound g was weighed and dissolved in 50 ml of DCM, 12 ml of triethylamine was added, and cooled in an ice-water bath. Compound d freshly prepared from 10.5 g of compound c was dissolved in 20 ml of DCM and added dropwise to the above solution. TLC monitoring, after the reaction is complete, filter with suction, concentrate, extract with ethyl acetate, wash with saturated potassium carbonate solution, 1mol/L HCl solution, water, saturated brine, dry over anhydrous sodium sulfate, stand still, and filter with suction , and evaporated to dryness to obtain an oil. Column chromatography can obtain compound i18g, the yield is 75%, and the chemical reaction equation is as follows:

(6) Weigh 20 g of compound i into an autoclave, add 400 ml of a saturated ammonia solution in methanol:THF at a ratio of 3:1, ventilate to keep the reaction system at 1-2 MPa, and stir at 50°C for reaction. TLC monitoring, after the reaction is complete, suction filtration, concentration to obtain the blue oily compound j19g, yield 94%, the chemical reaction equation is as follows:

(7) Weigh 1g of compound j and add it to 30% methanol, add 0.2g Pd/C catalyst, ventilate, make the reaction system under the hydrogen pressure of 10MPa, heat up to 45°C for reaction, TLC thin layer monitoring, after the reaction is complete, Suction filtration, concentrated to obtain oil 0.5g, yield 95%, chemical reaction equation is as follows:

The preparation method of other bitter chrysamine B analogues is as follows:

Formula (I)

Other picrochrysamine B analogs are shown in formula (I), wherein R ₁ , R ₂ , R ₃ , and R ₄ can be independently H, alkyl groups with less than 3 carbons, formyl groups, acetyl groups, benzyl groups, etc. A protecting group for hydroxyl; X _1-3 can be independently a C atom or an N atom, and when one of them is an N atom, the other two are C atoms; X _4-6 can be independently a C atom or an N atom, when one of them is an N atom When it is an N atom, the remaining two are C atoms; n _1-5 can be independently an alkyl group with 4 carbons or less. After the above changes, the synthesis steps of these picrylamine B analogs are the same as in Example 1.

Embodiment 2: Synthesis of Kuchrysamine B analog organic acid salt

Experimental method: Dissolve the Kuchrysamine B analog obtained in Example 1 in ethanol, then drop in the corresponding organic acid dissolved in ethanol, adjust the pH value to 7, evaporate the ethanol to dryness, and recrystallize the obtained solid with ethanol Get the corresponding organic acid salts, such as malate, lactate, succinate, methanesulfonate, etc. the

Embodiment 3: the synthesis of the inorganic acid salt of the Kuchrysamine B analogue

Experimental method: dissolve the bitter chrysamine B analogue obtained in Example 1 in ethanol, then drop the corresponding inorganic acid diluted with water, adjust the pH value to 7, evaporate the ethanol to dryness, and recrystallize the obtained solid with ethanol to obtain Corresponding organic acid salts, such as hydrochloride, sulfate, phosphate hydrobromide, etc. the

Example 4: Affinity determination of salts of Kuchrysamine B analogues and lipid A

Experimental method: get the bitter chrysamine B analog that embodiment 1 obtains, prepare following hydrochloride, hydrobromide, sulfate, phosphate, acetate, maleic acid with the method for embodiment 2,3 Salt, succinate, malate, lactate, tartrate, methanesulfonate, p-toluenesulfonate, glutamate and aspartate 1 mg each, add PBS (0.01M, pH 7.4 ) 1ml fully dissolved. Take 5 μl of each of the above solutions and add them to the sample pool of the affinity sensor that has been pre-coated with lipid A (the sample pool contains 45 μl of PBS) to react and record the physiological curve. the

The experimental results show that most of the salts of Kuchrysamine B analogs can combine with lipid A in vitro, and the binding ability of the salt of compound k is better than that of other analogs. Among the various salts of k, hydrochloride, hydrogen Bromate, sulfate, phosphate, acetate have the strongest binding capacity. the

Table 1 The binding data when reacting with lipid A for 3 minutes

LPS value Hydrochloride hydrobromide Sulfate Phosphate Acetate Malate Lactate Compound k 2230 2456 2130 1896 1635 1564 1325 Kucosamine B 998 926 540 2570 1480 1368 1765 Compound l 1874 2086 1907 2015 1532 1435 1247 Compound m 1986 2165 1654 1635 1765 1232 1202 Compound n 1765 1985 1746 2145 1543 1098 980 Compound o 1954 2342 1436 1789 1345 1123 1109

Example 5: In vitro neutralization of LPS by salts of Kuchrysamine B analogues

Experimental method: The experiment was carried out according to the operating instructions of the 32Well Kinetic Tube Reader (ATi321-06) endotoxin detector, and the dynamic turbidimetric method was used to detect the LPS value of each group, and the detection was repeated 3 times for each concentration;

The experimental results show that the salts of most of the analogues of Kuchrysamine B can neutralize LPS in vitro, and the neutralization ability of the salt of compound k is better than that of the other analogues. Among the various salts of k, hydrochloride, hydrogen bromide Salt, sulfate, phosphate, acetate have the strongest binding capacity. the

Table 2

the Hydrochloride hydrobromide Sulfate Phosphate Acetate Malate Lactate Compound k 0.18 0.13 0.14 0.15 0.15 0.24 0.26 Kucosamine B 0.24 0.18 0.26 0.17 0.15 0.18 0.19 Compound l 0.21 0.16 0.18 0.19 0.17 0.21 0.26 Compound m 0.22 0.20 0.22 0.14 0.16 0.19 0.19 Compound n 0.19 0.14 0.16 0.15 0.18 0.23 0.27 Compound o 0.25 0.17 0.17 0.15 0.14 0.21 0.22

Embodiment 6: the salt of Kuchrysamine B analog suppresses the experiment that LPS induces RAW264.7 cell to release inflammatory mediator

17.1 Experimental method: Dilute RAW264.7 cells to 1×10 ⁶ /ml with DMEM culture medium, add to a 96-well plate (200 μl/well), and incubate at 37°C and 5% CO ₂ for 4 hours. After adhering to the wall, replace the cell supernatant with 200 μl of serum-free DMEM culture medium, then add LPS (final concentration 100 ng/ml), and add various bitter chrysamine B analogs with final concentrations of 0, 50, 100, and 200 μM respectively. Salts (including chrysamine B malate, succinate, lactate, tartrate, mesylate, p-toluenesulfonate, glutamate, acetate, hydrochloride and sulfate), The blank control group (Medium) was incubated without adding LPS for 4 hours, the supernatant was taken, and the concentration of TNF-α was detected according to the operation instructions of the ELISA kit.

The experimental results show that most of the salts of Kuchrysamine B analogues can inhibit the release of inflammatory mediator TNF-α from RAW264.7 cells induced by LPS, and the inhibitory ability of the salt of compound k is better than that of other analogues. Among them, hydrochloride, hydrobromide, sulfate, phosphate and acetate have the strongest binding ability. the

table 3

100um Hydrochloride hydrobromide Sulfate Phosphate Acetate Malate Lactate Compound k 2892 3123 3051 2985 3203 3578 3980 Kucosamine B 4012 3876 4250 4104 4435 2786 2810 Compound l 3024 3249 3420 3568 3762 3980 3782 Compound m 2980 3428 3523 3123 3324 3643 3653 Compound n 3214 3543 3245 3213 3423 3214 3657 Compound o 2789 3143 3124 3215 3521 3425 3823

Claims (10)

1. An analog of Kuchrysamine B or a pharmaceutically acceptable salt thereof, characterized in that: the Kuchrysamine B analog has the following chemical structure

Formula (I) Among them, R ₁ , R ₂ , R ₃ , and R ₄ can independently be H, an alkyl group with less than 3 carbons, formyl, acetyl, benzyl and other hydroxyl protecting groups; X _1-3 can be independently a C atom Or N atoms, when one of them is an N atom, the other two are C atoms; X _4-6 can be independently C atoms or N atoms, and when one of them is an N atom, the other two are C atoms; n _1-5 It can be independently an alkyl group with 4 carbons or less, wherein the pharmaceutically acceptable salt is an oxygen-free acid or an oxygen-containing inorganic acid or a carboxylic acid or a hydroxy acid or a sulfonic acid or an organic acid of an acidic amino acid. 2. The analog of Kuchrysamine B according to claim 1 or its pharmaceutically acceptable salt, is characterized in that, the analog of Kuchrysamine B is the following compound Compound 1 Compound m

compound n

Compound o

compound k

3. The analogue of Kuchrysamine B or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the anaerobic acid is any one of hydrochloric acid and hydrobromic acid. 4. The analogue of Kuchrysamine B or a pharmaceutically acceptable salt thereof according to claim 1 or 2, wherein the oxyacid is any one of sulfuric acid, phosphoric acid and nitric acid. 5. The analogue or pharmaceutically acceptable salt thereof of Kuchrysamine B according to claim 1 or 2, wherein the carboxylic acid is acetic acid, propionic acid, butyric acid, oxalic acid, malonic acid, succinic acid , adipic acid, benzoic acid, phenylpropionic acid, cinnamic acid, stearic acid, trifluoroacetic acid, maleic acid, fumaric acid, niacin and palmitic acid. 6. The analogue of Kuchrysamine B or its pharmaceutically acceptable salt according to claim 1 or 2, wherein the hydroxy acid is malic acid, citric acid, lactic acid, hydroxybutyric acid, lactobionic acid, tartaric acid , mandelic acid, gluconic acid, glucuronic acid and ascorbic acid in any one. 7. The analogue of kuchrysamine B or its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that: the sulfonic acid is methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid and camphorsulfonic acid any of the 8. The analogue of Kuchrysamine B or its pharmaceutically acceptable salt according to claim 1 or 2, characterized in that: the acidic amino acid is any one of glutamic acid and aspartic acid. 9. A pharmaceutical composition comprising the analogue of Kuchrysamine B or a pharmaceutically acceptable salt thereof according to claim 1 or 2. 10. The use of the analogue of Kuchrysamine B or its pharmaceutically acceptable salt according to claim 1 or 2 and the composition containing them in the preparation of drugs for the prevention and treatment of sepsis.