CN107973764B - Andrographolide compound, preparation method thereof, pharmaceutical composition and application - Google Patents
Andrographolide compound, preparation method thereof, pharmaceutical composition and application Download PDFInfo
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- C07D307/02—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings
- C07D307/34—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
- C07D307/56—Heterocyclic compounds containing five-membered rings having one oxygen atom as the only ring hetero atom not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
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- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
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Abstract
The invention discloses an andrographolide compound, a preparation method, a pharmaceutical composition and application thereof. The andrographolide compound (I), isomer, prodrug, solvate or pharmaceutically acceptable salt thereof has the following structure. The andrographolide compound has good effect of treating inflammatory diseases, including but not limited to various inflammatory diseases possibly caused by pathogens such as bacteria, viruses and the like or caused by autoimmunity.
Description
Technical Field
The invention relates to an andrographolide compound, an isomer, a prodrug, a solvate or pharmaceutically acceptable salt thereof, a pharmaceutical composition, a preparation method and application thereof.
Background
Andrographolide (Andrographolide) is one of the highest content ingredients in Andrographis paniculata Nees extract. The andrographolide has a multi-target action mechanism, so that the andrographolide has wide pharmacological activities such as antipyresis, anti-inflammatory, analgesic, antibacterial and hypoglycemic effects, and the like, and along with the continuous and deep research on the pharmacological effects of andrographolide, the andrographolide is found to have wide application in aspects such as immunoregulation, antiviral and antitumor (Dai Guifu, and the like, chinese patent medicine 2006,28 (7): 1032). The andrographolide has the following specific structure:
Currently, there are literature and patent reports (Lin HQ, et al biol. Pharm. Bull.2006,29 (2): 220, NC 101012110B, NC 1666985A, U.S. 20050215628A 1), andrographolide and derivatives thereof can inhibit LPS-induced expression of TNF-alpha, IL1 beta and IL-6, thereby inhibiting inflammatory response in the body. TNF- α is a precursor inflammatory cytokine involved in many inflammatory processes. TNF-alpha inhibition is useful in the treatment of various autoimmune diseases (rheumatoid arthritis, crohn's disease, systemic lupus erythematosus, psoriasis, etc.), neurological diseases (Alzheimer's disease, parkinson's disease, AIDS dementia complex, depression), cancer, respiratory tract viral infections, etc. (Ogata H, hibi T.et al curr Pharm Des.2003,9 (14): 1107;Sack M.et al.Pharmacol Ther.2002,94 (1-2): 123-135;Barnes PJ.Et al.Annu Rev Pharmacol Toxicol.2002,42:81;Goldring MB.Et al.Expert Opin Biol Ther.2001Sep;1 (5): 817). IL1 beta is a cytokine produced by monocytes, macrophages, dendritic cells, fibroblasts, and the like, and can stimulate proliferation and differentiation of T cells and B cells, and participate in inflammatory reactions. Inhibition of IL 1. Beta. Is useful in the treatment of a variety of inflammatory responses including viral infections (Taylor PC et al Curr Pharm Des 2003;9 (14): 1095;Dellinger RP et al Clin Infect Dis.2003May 15;36 (10): 1259). IL-6, also known as B cell stimulatory factor, is capable of producing IL-6 spontaneously or under other stimuli by a variety of cells, which play an important role in peripheral and central nervous system development, differentiation, regeneration and degeneration.
In addition, andrographolide and its derivatives have been found to have a certain effect on antiviral infections in recent years, and some progress has been made in the treatment and prevention of aids, hepatitis and hand-foot-and-mouth disease virus infections. For example, CN103739575A and CN103739597a disclose the anti-hepatitis b virus effect of andrographolide derivatives. CN104042621a discloses that andrographolide-xiyanping has the serious effect of preventing and slowing down the virus infection of hand-foot-and-mouth disease, but xiyanping is a sulfonated product of andrographolide and is not a single structure, so that a small molecular compound with a single structure is adopted to further define the mechanism, and has important significance for the research on the efficacy and toxicology of the andrographolide.
In summary, although andrographolide compounds have achieved some research and development results, there is no particularly effective andrographolide analogue in clinical use in the fields of anti-inflammatory and antiviral treatment for various diseases. Upon searching, it was not found that the 17-double bond of andrographolide was rearranged into the diterpene ring and an ester bond or ether bond was introduced at the 14-hydroxyl group to form a new analogue, and was used in reports of anti-inflammatory and antiviral fields.
Disclosure of Invention
The invention aims to solve the technical problem of providing a novel andrographolide compound, a preparation method, a pharmaceutical composition and application thereof. The andrographolide compound has good anti-inflammatory effect and can effectively treat inflammatory diseases. Inflammatory diseases to which the present invention refers include, but are not limited to, various inflammatory diseases that may be caused by pathogens such as bacteria, viruses, etc., by autoimmunity, or radiation damage.
The invention provides an andrographolide compound (I), an isomer, a prodrug, a solvate or pharmaceutically acceptable salt thereof;
wherein, the liquid crystal display device comprises a liquid crystal display device,
a and b are each independently a single bond or a double bond, and a and b are not both double bonds or single bonds;
x is-OR 5 or-SR 6 ；
Y and Z are each independently selected from-O-or-NH-;
R 1 and R is 2 Each independently selected from hydrogen or alkyl;
R 3 and R is 4 Are each independently selected from hydrogen, alkyl, haloalkyl, -Si (R) c ) 3 、-C(O)R c 、-SO 3 M; m is hydrogen, sodium or potassium; or R is 3 And R is 4 Are linked to each other to form a 5-8 membered heterocycloalkyl group, which may further contain 1 to 2 groups selected from Si, S (O) 0-2 Or a heteroatom or group of N atoms; the heterocycloalkyl is unsubstituted or further substituted in any position with 1 to 4 substituents selected from alkyl, halogen, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, amino, aminoalkyl or oxo;
R 5 is a substituted or unsubstituted alkyl group, a substituted or unsubstituted cycloalkyl group, a substituted or unsubstituted heterocycloalkyl group, a substituted or unsubstituted aryl group, a substituted or unsubstituted heteroaryl group, a substituted or unsubstituted cycloalkylalkyl group, a substituted or unsubstituted heterocycloalkylalkyl group, a substituted or unsubstituted arylalkyl group, a substituted or unsubstituted heteroarylalkyl group, or R a 、-C(O)R b 、-C(O)NR b R c 、-C(O)OR c 、-S(O) 2 R b 、-S(O) 2 NR c R d 、-NR c R d 、-(CH 2 ) n OR a ；
R 6 Is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl, -C (O) R b 、-C(O)NR b R c 、-C(O)OR c Or- (CH) 2 ) n OR a ；
R 5 Or R is 6 Wherein 1 to 4 groups selected from halogen, C when the substituted alkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted cycloalkylalkyl, substituted heterocycloalkylalkyl, substituted arylalkyl or substituted heteroarylalkyl are substituted 1-4 Alkyl, halogenated C 1-3 Alkyl, C 1-3 Alkoxy, halo C 1-3 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, -NO 2 、-CN、-OR c 、-SR c 、-OC(O)R c 、-OC(O)NR c R d 、-NR c R d 、-N(R c )C(O)R d 、-N(R c )S(O) 2 R d 、-N(R c )S(O) 2 NR c R d 、-N(R c )C(O)NR c R d 、-S(O) 2 NR c R d 、-S(O) 0-2 R c 、-OP(O)(O-R c ) 2 、-C(O)R c 、-C(O)OR c or-C (O) NR c R d Is substituted at any position; n is an integer of 1 to 6;
R a is glycyl-, L-alanyl-, L-leucyl-, L-valyl-, L-isoleucyl-, etc. amino acid residues;
R b is hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted cycloalkylalkyl, substituted or unsubstituted heterocycloalkylalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroarylalkyl; when the substituted alkyl, substituted cycloalkyl, substituted heterocycloalkyl, substituted aryl, substituted heteroaryl, substituted cycloalkylalkyl, substituted heterocycloalkylalkyl, substituted arylalkyl or substituted heteroarylalkyl is substituted, it may be substituted with 1 to 4 groups selected from halogen, C 1-4 Alkyl, halogenated C 1-3 Alkyl, C 1-3 Alkoxy, halo C 1-3 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, -NO 2 、-CN、-OR c 、-SR c 、-OC(O)R c 、-OC(O)NR c R d 、-NR c R d 、-N(R c )C(O)R d 、-N(R c )S(O) 2 R d 、-N(R c )S(O) 2 NR c R d 、-N(R c )C(O)NR c R d 、-S(O) 2 NR c R d 、-S(O) 0-2 R c 、-OP(O)(O-R c ) 2 、-C(O)R c 、-C(O)OR c or-C (O) NR c R d Is substituted at any position;
R c and R is d Are independently selected from hydrogen, C 1-6 Alkyl, halogenated C 1-3 Alkyl, C 1-3 Alkoxy, halo C 1-3 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, or 5-6 membered heteroaryl.
Alternatively, R c And R is b Or R is d Together with the N atom to which they are attached, form a 3-8 membered heterocycloalkyl, which may further contain 1 to 3 groups selected from N, O, S, S (O) 2 The 3-8 membered heterocycloalkyl is unsubstituted orFurther substituted at any position with 1 to 3 substituents selected from alkyl, halogen, haloalkyl, hydroxyalkyl, alkoxyalkyl, alkoxy, amino, aminoalkyl or oxo.
The R is 1 Preferably hydrogen or C 1-6 An alkyl group; the R is 1 More preferably H.
The R is 2 Preferably hydrogen or C 1-6 An alkyl group; the R is 2 More preferably methyl.
The R is 3 Preferably hydrogen, C 1-6 Alkyl, halogenated C 1-3 Alkyl, -Si (R) c ) 3 、-C(O)R c 、-SO 3 M; m is hydrogen, sodium or potassium; the R is 3 More preferably hydrogen.
The R is 4 Preferably hydrogen, C 1-6 Alkyl, halogenated C 1-3 Alkyl, -Si (R) c ) 3 、-C(O)R c 、-SO 3 M; m is hydrogen, sodium or potassium; the R is 4 More preferably hydrogen.
Or said R 3 And R is 4 Are linked to each other to form a 5-8 membered heterocycloalkyl group, which may further contain 1-2 groups selected from Si, S (O) 0-2 Or a heteroatom of an N atom; said heterocycloalkyl being unsubstituted or further substituted by 1 to 3 members selected from C 1-4 Alkyl, halogen, halogenated C 1-3 Alkyl, hydroxy C 1-3 Alkyl, C 1-3 Alkoxy C 1-3 Alkyl, C 1-3 Alkoxy, amino C 1-3 One or more of the substituents of the alkyl or oxo group are substituted at any position.
The R is 5 Preferably substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted C 3-8 Cycloalkyl C 1-3 Alkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl C 1-3 Alkyl, substituted or unsubstituted phenyl C 1-3 Alkyl, substituted or unsubstituted 5-6 membered heteroaryl C 1-3 Alkyl, R a 、-C(O)R b 、-C(O)NR b R c 、-C(O)OR c 、-S(O) 2 R b 、-S(O) 2 NR c R d 、-NR c R d Or- (CH) 2 ) n OR a 。
The R is 6 Preferably substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted C 3-8 Cycloalkyl C 1-3 Alkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl C 1-3 Alkyl, substituted or unsubstituted phenyl C 1-3 Alkyl, substituted or unsubstituted 5-6 membered heteroaryl C 1-3 Alkyl, -C (O) R b 、-C(O)NR b R c 、-C(O)OR c Or- (CH) 2 ) n OR a 。
The R is b Preferably substituted or unsubstituted C 1-6 Alkyl, substituted or unsubstituted C 3-8 Cycloalkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl, substituted or unsubstituted phenyl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted C 3-8 Cycloalkyl C 1-3 Alkyl, substituted or unsubstituted 3-8 membered heterocycloalkyl C 1-3 Alkyl, substituted or unsubstituted phenyl C 1-3 Alkyl, or substituted or unsubstituted 5-6 membered heteroaryl C 1-3 An alkyl group.
An andrographolide compound shown as a formula (I), an isomer, a prodrug, a hydrate, a solvate or a pharmaceutically acceptable salt thereof;
in one preferred embodiment, when a is a double bond and b is a single bond;
in one preferred embodiment, when a is a single bond and b is a double bond.
In a preferred embodiment of the present invention, the andrographolide compound (I), an isomer, a prodrug, a solvate or a pharmaceutically acceptable salt thereof, has the following general formula:
wherein, is represented as R configuration or S configuration;
R 2 、R 3 、R 4 And R is 5 Is as defined above;
included in the definition of structural formula (IA) are the following:
in one preferred embodiment, R 2 Is CH 3 ；
In one preferred embodiment, R 3 Is H, R 4 H.
In a preferred embodiment of the present invention, the andrographolide compound (I), an isomer, a prodrug, a solvate or a pharmaceutically acceptable salt thereof, has the following general formula:
wherein, is represented as R configuration or S configuration;
the A ring is benzene ring or 5-6 membered heteroaromatic ring;
R 7 is halogen, C 1-4 Alkyl, halogenated C 1-3 Alkyl, C 1-3 Alkoxy, halo C 1-3 Alkoxy, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-6 Cycloalkyl, 3-6 membered heterocycloalkyl, phenyl, 5-6 membered heteroaryl, -NO 2 、-CN、-OR c 、-SR c 、-OC(O)R c 、-OC(O)NR c R d 、-NR c R d 、-N(R c )C(O)R d 、-N(R c )S(O) 2 R d 、-N(R c )S(O) 2 NR c R d 、-N(R c )C(O)NR c R d 、-S(O) 2 NR c R d 、-S(O) 0-2 R c 、-OP(O)(O-R c ) 2 、-C(O)R c 、-C(O)OR c or-C (O) NR c R d ；
t is 1, 2 or 3;
R 1 、R 2 、R 4 、R c and R is d Is defined as before.
Included in the definition of structural formula (IB) are the following:
in one preferred embodiment, R 2 Is CH 3 ；
In one preferred embodiment, R 3 Is H, R 4 H.
In a preferred embodiment of the present invention, the andrographolide compound (I), an isomer, a prodrug, a solvate or a pharmaceutically acceptable salt thereof, has the following general formula:
Wherein, is represented as R configuration or S configuration;
R 2 、R 3 、R 4 、R b and R is c Is as defined above;
the following cases are included in the definition of structural formula (IC) or (ID):
in one preferred embodiment, R 2 Is CH 3 ；
In one preferred embodiment, R 3 Is H, R 4 H.
The andrographolide compound (I), isomer, prodrug, solvate or pharmaceutically acceptable salt thereof is optimally any one of the following structures:
the invention also provides a preparation method of the andrographolide compound (I), an isomer, a prodrug, a solvate or pharmaceutically acceptable salt thereof, which is any one of the following methods:
method 1:
wherein R 'is phenyl and R' is H; or R 'and R' are both methyl; * R, R b Or R is c Is defined as before.
The method 1 comprises the following steps: step 1, in a solvent, reacting a compound 1.2 with substituted carbamoyl chloride or acyl chloride in an aprotic solvent under an alkaline condition to obtain a compound I-1 or I-2; and 2, in a solvent, under an acidic condition, removing the dihydroxyl protecting group from the I-1 or I-2 to obtain a compound Ia or Ib.
In method 1, the conditions and steps of the reaction may be conventional in the art, and the following reaction conditions are particularly preferred in the present invention: in step 1, the base is preferably triethylamine, the aprotic solvent is preferably dichloromethane, the amount of the aprotic solvent is preferably 1-50 mL/mmol of compound 1.2, the temperature is preferably 0-room temperature, the reaction time is preferably 0-24 hours, and the reaction time is preferably 0.2 and N (R) b )(R c ) COCl or R b The molar ratio of COCl is preferably 1:1-1:3, the molar ratio of 1.2 to triethylamine is preferably 1:1-1:10, and 0.1-1 equivalent of 4-Dimethylaminopyridine (DMAP) can be added into the reaction system to accelerate the reaction; step 2, the deprotection reaction is under deprotection conditions conventional in the art, e.g., removal can be performed under acidic conditions, and when R' and R "are both methyl, aqueous acetic acid is preferred; the acetic acid aqueous solution is preferably acetic acid aqueous solution with the volume percentage of 75-85%; the dosage of the acetic acid aqueous solution is preferably 5-20 mL/mmol of the compound I-2 or I-3, the reaction temperature is preferably 20-50 ℃ and the reaction is carried outThe time is preferably 0.2 to 2 hours. When R 'is phenyl and R' is H, the deprotection reaction is preferably carried out in ethanol solution in the presence of paratoluenesulfonic acid monohydrate, wherein the ethanol is preferably used in an amount of 1-50 mL/mmol of compound I-1 or I-2; the molar ratio of the compound I-1 or I-2 to the p-toluenesulfonic acid monohydrate is preferably 1:1 to 1:3, more preferably 1:2; the reaction temperature is preferably 20-70 ℃, and the reaction time is preferably 1-3 days.
Method 2:
wherein R 'is phenyl and R' is H; or R 'and R' are both methyl; * Ring A, R 7 And t is as defined above.
The method 2 comprises the following steps: step 1, in solvent, compounds 1.2 andobtaining a compound I-3 through Mitsunobu reaction; and 2, in a solvent, under an acidic condition, removing the dihydroxyl protecting group from the I-3 to obtain a compound Ic.
In method 2, the conditions and steps of the reaction may be conventional in the art, and the following reaction conditions are particularly preferred in the present invention: step 1, the Mitsunobu reaction is preferably a combination of triphenylphosphine and diisopropyl azodicarboxylate (DIAD), compound 1.2 andthe molar ratio is preferably 1:0.9-1:2; the triphenylphosphine, DIAD andthe molar ratio is preferably 1:1:1-1:2:2; the reaction temperature is preferably 0-room temperature; step 2, the deprotection reaction is under deprotection conditions conventional in the art, e.g., removal can be performed under acidic conditions, and when R' and R "are both methyl, aqueous acetic acid is preferred; the acetic acid aqueous solution is preferably acetic acid aqueous solution with the volume percentage of 75-85 percentThe method comprises the steps of carrying out a first treatment on the surface of the The amount of the aqueous acetic acid solution is preferably 5-20 mL/mmol of the compound I-2 or I-3, the reaction temperature is preferably 20-50 ℃, and the reaction time is preferably 0.2-2 hours. When R 'is phenyl and R' is H, the deprotection reaction is preferably carried out in ethanol solution in the presence of paratoluenesulfonic acid monohydrate, wherein the ethanol is preferably used in an amount of 1-50 mL/mmol of compound I-1 or I-2; the molar ratio of the compound I-1 or I-2 to the p-toluenesulfonic acid monohydrate is preferably 1:1 to 1:3, more preferably 1:2; the reaction temperature is preferably 20-70 ℃, and the reaction time is preferably 1-3 days.
In one or two methods, the compound 1.2 synthesis method is as follows:
wherein R 'is phenyl and R' is H; or R 'and R' are both methyl;
the synthesis method of the compound 1.2 comprises the following steps: step 1, in a solvent, carrying out rearrangement reaction on 17-position double bonds of andrographolide under the action of hydrogen bromide to obtain a compound 1.1; step 2, hydroxy groups in positions 3 and 19 of compound 1.1 are protected, generally preferably with an acetone and 2, 2-dimethoxypropane system, or a benzaldehyde dimethyl acetal and pyridine p-toluenesulfonate hydrochloride system.
In the compound 1.2 synthesis process, the reaction conditions and steps may be those conventional in the art, with the following reaction conditions being particularly preferred in the present invention: step 1, the solvent is ethanol, the dosage of the solvent is 1-50 mL/mmol andrographolide, the hydrogen bromide is 46-48% hydrogen bromide aqueous solution, the volume ratio of the hydrogen bromide aqueous solution to the solvent is 0.5:1-1:1, the reaction temperature is 0-room temperature, and the reaction time is 1-24 hours; step 2, the hydroxy protection reaction is under deprotection conditions conventional in the art. Preferably, an acetone and 2, 2-dimethoxypropane system is used, the dosage of the acetone is preferably 0.5-5 mL/mmol of compound 1.1, the dosage of the 2, 2-dimethoxypropane is preferably 1-10 mL/mmol of compound 1.1, the temperature is preferably 30-60 ℃, and the reaction time is preferably 2-10 hours; preferably using benzaldehyde dimethyl acetal and pyridine p-toluenesulfonate hydrochloride system, wherein the solvent is preferably dichloromethane; the molar ratio of the benzaldehyde dimethyl acetal to the compound 1.1 is preferably 1:1-1:3, more preferably 1:2; the dosage of the pyridine p-toluenesulfonate hydrochloride is catalytic amount, the molar ratio of the pyridine p-toluenesulfonate hydrochloride to the compound 1.1 is preferably 1:0.02-1:0.1, the temperature is preferably room temperature, and the reaction time is preferably 10-24 hours;
Pharmaceutically acceptable salts of andrographolide compounds (I) can be synthesized by a general chemical method.
In general, salts can be prepared by reacting the free base or acid with an equivalent stoichiometric or excess of an acid (inorganic or organic) or base (inorganic or organic) in a suitable solvent or solvent composition.
The invention also provides a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and pharmaceutically acceptable excipients; the active component comprises one or more of andrographolide compounds (I), isomers, prodrugs, solvates and pharmaceutically acceptable salts thereof.
In the pharmaceutical composition, the active ingredient may further include other therapeutic agents that inhibit inflammation, or viral infection, and the like.
In the pharmaceutical composition, the pharmaceutically acceptable excipients may include pharmaceutically acceptable carriers, diluents and/or excipients.
Depending on the purpose of treatment, the pharmaceutical composition may be formulated into various types of administration unit dosage forms such as tablets, pills, powders, liquids, suspensions, emulsions, granules, capsules, suppositories, and injections (solutions and suspensions), etc., preferably liquids, suspensions, emulsions, suppositories, and injections (solutions and suspensions), etc.
For shaping pharmaceutical compositions in tablet form, any excipient known and widely used in the art may be used. For example, carriers such as lactose, white sugar, sodium chloride, dextrose, urea, starch, calcium carbonate, kaolin, crystalline cellulose, silicic acid, and the like; binders such as water, ethanol, propanol, ordinary syrup, dextrose solution, starch solution, gelatin solution, carboxymethyl cellulose, shellac, methyl cellulose and potassium phosphate, polyvinyl pyrrolidone, and the like; disintegrants such as dry starch, sodium alginate, agar powder and kelp powder, sodium bicarbonate, calcium carbonate, fatty acid esters of polyethylene sorbitan, sodium lauryl sulfate, monoglyceride of stearic acid, starch, lactose, etc.; disintegration inhibitors such as white sugar, glycerol tristearate, coconut oil and hydrogenated oil; adsorption promoters such as quaternary ammonium bases and sodium lauryl sulfate; wetting agents such as glycerin, starch, and the like; adsorbents such as starch, lactose, kaolin, bentonite, colloidal silicic acid, etc.; and lubricants such as pure talc, stearate, boric acid powder, polyethylene glycol, and the like. The composition can be made into sugar coated tablet, tu Ming film coated tablet, enteric coated tablet, film coated tablet, double-layer film tablet and multilayer tablet by selecting common coating materials as required.
For shaping pharmaceutical compositions in the form of pellets, any known and widely used excipients in the art may be used, for example, carriers such as lactose, starch, coconut oil, hardened vegetable oils, kaolin and talc, etc.; binders such as gum arabic powder, tragacanth powder, gelatin, ethanol and the like; disintegrating agents such as agar and kelp powder.
For shaping pharmaceutical compositions in the form of suppositories, any of the excipients known and widely used in the art can be used, for example polyethylene glycol, coconut oil, higher alcohols, esters of higher alcohols, gelatin, semisynthetic glycerides and the like.
For preparing pharmaceutical compositions in the form of injections, the solutions or suspensions may be sterilized (preferably by adding appropriate amounts of sodium chloride, glucose or glycerol, etc.), and then prepared into injections isotonic with blood. In preparing the injection, any carrier commonly used in the art may be used. For example, water, ethanol, propylene glycol, ethoxylated isostearyl alcohol, polyoxylated isostearyl alcohol, fatty acid esters of polyethylene sorbitan, and the like. In addition, usual dissolving agents, buffers, analgesics, etc. may be added.
In the present invention, the content of the composition in the pharmaceutical composition is not particularly limited, wherein the andrographolide compound (I), its isomer, prodrug, solvate, or pharmaceutically acceptable salt thereof may be selected within a wide range, and the safe and effective dose thereof is determined according to the age, weight, condition, course of disease, administration route, etc. of the subject, and may be generally 5 to 95% by mass, preferably 30 to 80% by mass.
In the present invention, the administration method of the pharmaceutical composition is not particularly limited. The formulation of each dosage form may be selected for administration depending on the age, sex and other conditions and symptoms of the patient. For example, tablets, pills, solutions, suspensions, emulsions, granules or capsules for oral administration; the injection can be administered alone or mixed with injectable delivery solution (such as glucose solution and amino acid solution) for intravenous injection; suppositories are administered into the rectum.
The invention also provides application of the andrographolide compound (I), isomer, prodrug, solvate or pharmaceutically acceptable salt thereof or the pharmaceutical composition in preparing medicaments for treating inflammation, including inflammation caused by pathogen infection of bacteria, viruses and the like, inflammatory diseases caused by autoimmune reaction, inflammation caused by radiation injury and the like. Wherein the virus comprises: various viruses of hand-foot-and-mouth disease (e.g., enterovirus EV71, coxsackievirus CA16, etc. most commonly causing hand-foot-and-mouth disease), various hepatitis viruses (e.g., hepatitis a, hepatitis b, hepatitis c virus), aids virus, etc.
Inflammatory diseases caused by said autoimmune reaction, comprising: rheumatoid arthritis, systemic lupus erythematosus, mixed Connective Tissue Disease (MCTD), systemic scleroderma (including CREST syndrome), dermatomyositis, nodular vasculitis, kidney disease (including lung nephritis syndrome, acute glomerulonephritis, primary membranous proliferative glomerulonephritis, etc.), primary biliary cirrhosis, autoimmune cholangitis, autoimmune hepatitis, primary sclerosing cholangitis, inflammatory Bowel Disease (IBD) (including Crohn's Disease (CD) and Ulcerative Colitis (UC)), chronic obstructive pulmonary disease, etc.
The radiation inflammatory disease comprises: radiation esophagitis, radiation enteritis, radiation thyroiditis, radiation osteomyelitis, radiation stomatitis, radiation pleurisy, radiation dermatitis, radiation pneumonitis, radiation colpitis, radiation cystitis, and the like.
The invention also provides the andrographolide compound (I), isomer, prodrug, solvate or pharmaceutically acceptable salt thereof and one or more other kinds of therapeutic agents for treating inflammation, viral infection, autoimmune diseases and/or radioactive injury.
The invention also provides application of the andrographolide compound (I), isomer, prodrug, solvate or pharmaceutically acceptable salt thereof and one or more other kinds of therapeutic agents in combination for preparing medicaments for treating inflammation, viral infection, autoimmune diseases or radiation injury.
The other kinds of therapeutic agents can be prepared into single administration therapeutic dosage forms or respectively successive administration therapeutic dosage forms together with the andrographolide compound (I), an isomer, a prodrug, a solvate or pharmaceutically acceptable salt thereof or the pharmaceutical composition.
In the present invention, unless otherwise indicated, the definition of a substituent name preceded by "substituted or unsubstituted" refers to the case of unsubstituted, for example: "alkyl" refers to unsubstituted alkyl, and "cycloalkyl" refers to unsubstituted cycloalkyl.
Unless otherwise indicated, the following terms appearing in the present specification and claims have the following meanings:
the term "alkyl" refers to a saturated straight or branched hydrocarbon group containing 1 to 20 carbon atoms, preferably 1 to 10 carbon atoms, more preferably 1 to 8,1 to 6,1 to 4,1 to 3 carbon atoms, representative examples of alkyl groups include, but are not limited to: methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, 4-dimethylpentyl, 2, 4-trimethylpentyl, undecyl, dodecyl, and various isomers thereof and the like. When "alkyl" is used as the linking group for other groups, e.g., - (CH) 2 ) m -, which may be linear or branched, examples includeBut is not limited to-CH 2 -、-CH 2 CH 2 -、-CH 2 CH(CH 3 )-。
The term "cycloalkyl" refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) monocyclic or polycyclic group containing 3 to 20 carbon atoms. 3-12 membered cycloalkyl is preferred. "monocyclic cycloalkyl" is preferably 3-10 membered monocyclic alkyl, more preferably 3-8 membered monocyclic alkyl, for example: cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl, cyclododecyl, cyclohexenyl. Representative examples of "polycyclic cycloalkyl" include "bridged ring radicals", "fused cycloalkyl" and "spirocycloalkyl" and "bridged ring radicals" include, but are not limited to: a bornyl group, a bicyclo [2.2.1] heptenyl group, a bicyclo [3.1.1] heptyl group, a bicyclo [2.2.1] heptyl group, a bicyclo [2.2.2] octyl group, a bicyclo [3.2.2] nonyl group, a bicyclo [3.3.1] nonyl group, a bicyclo [4.2.1] nonyl group, an adamantyl group, and the like. "fused cycloalkyl" includes cycloalkyl rings fused to phenyl, cycloalkyl, or heteroaryl, fused cycloalkyl including, but not limited to: benzocyclobutene, 2, 3-dihydro-1-H-indene, 2, 3-cyclopentenopyridine, 5, 6-dihydro-4H-cyclopentyl [ B ] thiophene, decalin, and the like. Representative examples of "spirocycloalkyl" include, but are not limited to: spiro [2,4] heptyl, spiro [4,5] decyl, and the like. The monocyclic cycloalkyl or polycyclic cycloalkyl group may be linked to the parent molecule through any carbon atom on the ring.
The term "heterocycloalkyl" refers to a saturated or partially unsaturated (containing 1 or 2 double bonds) non-aromatic cyclic group consisting of carbon atoms and heteroatoms selected from nitrogen, oxygen or sulfur, which cyclic group may be a single ring or a multi-ring group, in the present invention, the number of heteroatoms in the heterocycloalkyl group is preferably 1,2,3 or 4, and the nitrogen, carbon or sulfur atoms in the heterocycloalkyl group may be optionally oxidized. The nitrogen atom may optionally be further substituted with other groups to form tertiary or quaternary ammonium salts. "monocyclic heterocycloalkyl" is preferably 3-10 membered monocyclic heterocycloalkyl, more preferably 3-8 membered monocyclic heterocycloalkyl. For example: aziridinyl, tetrahydrofuran-2-yl, morpholin-4-yl, thiomorpholin-S-oxide-4-yl, piperidin-1-yl, N-alkylpiperidin-4-yl, pyrrolidin-1-yl, N-alkylpyrrolidin-2-yl, piperazin-1-yl, 4-alkylpiperazin-1-yl, and the like. "polycyclic heterocycloalkyl" includes "fused heterocycloalkyl", "spiroheterocyclyl" and "bridged heterocycloalkyl". "fused heterocycloalkyl" includes monocyclic heterocycloalkyl rings fused to phenyl, cycloalkyl, heterocycloalkyl, or heteroaryl, fused heterocycloalkyl including, but not limited to: 2, 3-dihydrobenzofuranyl, 1, 3-dihydroisobenzofuranyl, indolinyl, 2, 3-dihydrobenzo [ b ] thienyl, dihydrobenzopyranyl, 1,2,3, 4-tetrahydroquinolinyl, and the like. The monocyclic heterocycloalkyl and polycyclic heterocycloalkyl groups can be linked to the parent molecule through any ring atom on the ring. The above-mentioned ring atoms are particularly carbon atoms and/or nitrogen atoms constituting the ring skeleton.
The term "cycloalkylalkyl" refers to a cycloalkyl group attached to the parent nucleus structure through an alkyl group. Thus, "cycloalkylalkyl" includes the definition of alkyl and cycloalkyl groups described above.
The term "heterocycloalkyl alkyl" refers to a heterocycloalkyl group attached to the parent structure through an alkyl group. Thus, "heterocycloalkyl alkyl" includes the definition of alkyl and heterocycloalkyl as described above.
The term "alkoxy" refers to a cyclic or acyclic alkyl group of the stated number of carbon atoms attached through an oxygen bridge, including alkyloxy, cycloalkyloxy, and heterocycloalkyloxy. Thus, "alkoxy" includes the definition of alkyl, heterocycloalkyl, and cycloalkyl described above.
The term "alkoxyalkyl" refers to an alkyl group in which any one of the hydrogen atoms is replaced with an alkoxy group. Thus, "alkoxyalkyl" includes the definition of alkyl and alkoxy groups described above.
The term "hydroxyalkyl" refers to any hydrogen atom on an alkyl group substituted with a hydroxyl group, including but not limited to: -CH 2 OH、-CH 2 CH 2 OH、-CH 2 CH 2 C(CH 3 ) 2 OH。
The term "alkenyl" refers to a straight, branched or cyclic non-aromatic hydrocarbon group containing at least 1 carbon-carbon double bond. Wherein 1-3 carbon-carbon double bonds may be present, preferably 1 carbon-carbon double bond is present. The term "C 2-4 Alkenyl "refers to alkenyl groups having 2 to 4 carbon atoms, the term" C 2-6 Alkenyl "means having 2 to 6Alkenyl groups of carbon atoms including ethenyl, propenyl, butenyl, 2-methylbutenyl and cyclohexenyl. The alkenyl group may be substituted.
The term "alkynyl" refers to a straight, branched or cyclic hydrocarbon group containing at least 1 carbon-carbon triple bond. Wherein 1-3 carbon-carbon triple bonds may be present, preferably 1 carbon-carbon triple bond. The term "C 2-6 Alkynyl "refers to alkynyl groups having 2 to 6 carbon atoms and includes ethynyl, propynyl, butynyl and 3-methylbutynyl.
The term "aryl" refers to any stable 6-10 membered monocyclic or bicyclic aromatic group, for example: phenyl, naphthyl, tetrahydronaphthyl, 2, 3-indanyl, biphenyl, or the like.
The term "heteroaryl" refers to an aromatic ring group formed by replacement of at least 1 carbon atom on the ring with a heteroatom selected from nitrogen, oxygen or sulfur, which may be a 5-7 membered monocyclic structure or a 7-12 membered bicyclic structure, preferably a 5-6 membered heteroaryl. In the present invention, the hetero atom number is preferably 1,2 or 3, and includes pyridyl, pyrimidinyl, pyridazin-3 (2H) -onyl, furyl, thienyl, thiazolyl, pyrrolyl, imidazolyl, pyrazolyl, oxazolyl, isoxazolyl, 1,2, 5-oxadiazolyl, 1,2, 4-triazolyl, 1,2, 3-triazolyl, tetrazolyl, indazolyl, isoindazolyl, indolyl, isoindolyl, benzofuryl, benzothienyl, benzo [ d ] [1,3] dioxolyl, benzothiazolyl, benzoxazolyl, quinolinyl, isoquinolinyl, quinazolinyl and the like.
The term "arylalkyl" refers to an aryl group attached to the parent nucleus through an alkyl group. Thus, "arylalkyl" includes the definition of alkyl and aryl groups described above.
The term "heteroarylalkyl" refers to a heterocycloalkyl group attached to the parent structure through an alkyl group. Thus, "heteroarylalkyl" includes the definition of alkyl and heteroaryl set forth above.
The term "halogen" means fluorine, chlorine, bromine or iodine.
The term "haloalkyl" refers to an alkyl group optionally substituted with halogen. Thus, "haloalkyl" includes the definition of halogen and alkyl above.
The term "haloalkoxy" refers to an alkoxy group optionally substituted with halogen. Thus, "haloalkoxy" includes the definition of halogen and alkoxy above.
The term "amino" refers to-NH 2 The term "alkylamino" refers to an amino group in which at least one hydrogen atom is replaced with an alkyl group, including but not limited to: -NHCH 2 、-NHCH 2 CH 3 . The term "aminoalkyl" refers to an alkyl group in which any one of the hydrogen atoms is replaced by an amino group, including, but not limited to: -CH 2 NH 2 、-CH 2 CH 2 NH 2 . Thus, "aminoalkyl" and "alkylamino" include the definition of alkyl and amino as described above.
The term "amino acid residue" refers to an amino-containing carboxylic acid fragment that is attached to the parent molecule via a carboxyl group to form an acyl group, and includes naturally or non-naturally occurring amino acid residues. Naturally occurring amino acid residues are preferred, including but not limited to: alanyl-, valyl-, L-alanyl-, L-leucyl-, L-isoleucyl-, prolyl-, phenylalanyl-, tryptophanyl-, egg-yl-, glycyl-, seryl-, threonyl-, cysteinyl-, tyrosyl-, aspartyl-, glutamyl-, lysyl-, arginyl-, histidyl-, aspartyl-, or glutamyl-.
The symbol "=" represents a double bond;
the term "room temperature" as used herein means 15-30deg.C.
By "prodrug" is meant a compound that is converted to the original active compound after metabolism in vivo. Typically, prodrugs are inactive substances or less active than the active parent compound, but may provide ease of handling, administration or improved metabolic characteristics.
"solvate" as used herein refers to a form of solvent addition that includes a stoichiometric or non-stoichiometric amount of solvent. Some compounds tend to trap a fixed molar proportion of the solvent molecules in the crystalline solid state, thus forming solvates. If the solvent is water, the solvate formed is a "hydrate", if the solvent is ethanol, the solvate formed isIs an ethanolate. The hydrate is formed by combining one or more water molecules with the substance, wherein the water molecules are in the state of H 2 O, such binding is capable of forming a hydrate comprising one or more water molecules.
The "pharmaceutically acceptable salts" of the present invention are discussed in Berge, et al, "Pharmaceutically acceptable salts," j.pharm.sci.,66,1-19 (1977), and are readily apparent to pharmaceutical chemists that are substantially non-toxic and provide desirable pharmacokinetic properties, palatability, absorption, distribution, metabolism, or excretion, and the like. The compounds of the present invention may have an acidic group, a basic group or an amphoteric group, and typical pharmaceutically acceptable salts include salts prepared by reacting the compounds of the present invention with an acid, for example: hydrochloride, hydrobromide, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, nitrate, acetate, propionate, decanoate, octanoate, formate, acrylate, isobutyrate, hexanoate, heptanoate, oxalate, malonate, succinate, suberate, benzoate, methylbenzoate, phthalate, maleate, methanesulfonate, p-toluenesulfonate, (D, L) -tartaric acid, citric acid, maleic acid, (D, L) -malic acid, fumaric acid, succinic acid, succinate, lactate, triflate, naphthalene-1-sulfonate, mandelate, pyruvate, stearate, ascorbate, salicylate. When the compounds of the present invention contain an acidic group, pharmaceutically acceptable salts thereof may further include: alkali metal salts, such as sodium or potassium salts; alkaline earth metal salts, such as calcium or magnesium salts; organic base salts such as salts with ammonia, alkylammons, hydroxyalkylamines, amino acids (lysine, arginine), N-methylglucamine and the like.
"isomers" in the sense of the present invention means that the compounds of formula (I) according to the invention may have asymmetric centers and racemates, racemic mixtures and individual diastereomers, all of which isomers, including stereoisomers and geometric isomers, are encompassed by the present invention. Among them, the "isomer" according to the present invention is preferably a "stereoisomer". In the present invention, the presence of a compound of formula (I) or a salt thereof in stereoisomeric forms (e.g., containing one or more asymmetric carbon atoms), individual stereoisomers (enantiomers and diastereomers) and mixtures thereof are included within the scope of the invention. The present invention also includes individual isomers of the compounds or salts represented by formula (I), as well as mixtures of isomers with one or more of the chiral centers reversed. The scope of the invention includes: mixtures of stereoisomers, and purified enantiomers or enantiomer/diastereomerically enriched mixtures. The present invention includes mixtures of stereoisomers of all enantiomers and all possible different combinations of non-corresponding isomers. The present invention includes all combinations and subsets of stereoisomers of all specific groups defined above. The invention also includes geometric isomers of the compounds of formula (I) or salts thereof, including cis and trans isomers.
The above preferred conditions can be arbitrarily combined on the basis of not deviating from the common knowledge in the art, and thus, each preferred embodiment of the present invention can be obtained.
The reagents and materials used in the present invention are commercially available.
Drawings
Fig. 1: andrographolide and the compound can inhibit IL6 and TNFα levels in BV2 cell culture liquid induced by LPS, and the compound can reduce IL6 and TNFα production to a certain extent at a concentration of 10 mu M, and has better effect than andrographolide.
Detailed Description
The invention is further illustrated by means of the following examples, which are not intended to limit the scope of the invention. The experimental methods, in which specific conditions are not noted in the following examples, were selected according to conventional methods and conditions, or according to the commercial specifications.
The structures of all the compounds of the invention can be changed by nuclear magnetic resonance 1 H NMR) and/or mass spectrometry detection (MS) identification.
1 H NMR chemical shifts (delta) were recorded in PPM (10 -6 )。NMRBy a Bruker AVANCE-400 spectrometer. A suitable solvent is deuterated chloroform (CDCl) 3 ) Deuterated methanol (CD) 3 OD), deuterated dimethyl sulfoxide (DMSO-d 6 ) Tetramethylsilane was used as an internal standard (TMS).
Low resolution Mass Spectrometry (MS) was determined by Agilent 1200HPLC/6120 mass spectrometer using XBridge C18, 4.6x50 mm,3.5 μm, gradient elution condition one: 80-5% solvent A 1 And 20-95% of solvent B 1 (1.8 minutes) then 95% solvent B 1 And 5% solvent A 1 (more than 3 minutes), the percentage is the volume percentage of a certain solvent in the total solvent volume. Solvent A 1 :0.01% trifluoroacetic acid (TFA) in water; solvent B 1 : acetonitrile solution of 0.01% trifluoroacetic acid; the percentage is the volume percentage of solute in the solution. Gradient elution condition II: 80-5% solvent A 2 And 20-95% of solvent B 2 (1.5 minutes) then 95% solvent B 2 And 5% solvent A 2 (more than 2 minutes), the percentage is the volume percentage of a certain solvent in the total solvent volume. Solvent A 2 :10mM ammonium bicarbonate in water; solvent B 2 : acetonitrile.
The compounds of the present invention may be purified by separation using conventional silica gel plates, silica gel column chromatography or by high performance liquid chromatography (prep-HPLC).
High performance liquid chromatography (prep-HPLC) was performed using Shimadzu LC-20 for liquid chromatography, column: waters xbridge Pre C18, 10um,19mm x 250mm. Mobile phase a:0.05% trifluoroacetic acid in water (percentage by volume), mobile phase B: acetonitrile; detection wavelength: 214nm &254nm; flow rate: 15.0 mL/min.
Thin layer silica gel plate (TLC) is a smoke table yellow sea HSGF254 or a peninsula GF254 silica gel plate. Silica gel column chromatography generally uses 200-300 mesh silica gel of yellow sea as carrier.
Example 1: synthesis of Compound 1-1
Step 1: andrographolide (40 g,0.11 mol) and ethanol (100 mL) were added to a three-necked flask (500 mL), stirred until the mixture became clear, cooled to 0-10℃and hydrogen bromide (70 mL, w/w: 48%) was added, the reaction was stopped under the temperature conditions, the reaction was stopped for 24 hours, aqueous sodium hydroxide solution (6.0M) was added to adjust the pH to between 6 and 7, ethyl acetate (350 mL) was added, stirred for 12 hours, a large amount of white solid was precipitated, filtered, the filter cake was recrystallized from ethyl acetate (100 mL), filtered, and the filter cake was dried to give Compound 1.1 (35 g, yield: 88%) as a white solid.
Step 2: compound 1.1 (35 g,0.10 mol), acetone (70 mL) and 2, 2-dimethoxypropane (175 mL) were added to a three-necked flask (500 mL), heated to 40-50 ℃, kept at that temperature, stirred for 5-6 hours, monitored by TLC (n-hexane/ethyl acetate=1/1) until the starting material disappeared, after the reaction was completed, the reaction solution was transferred to a single-necked flask (500 mL) and concentrated to dryness; ethyl acetate (175 mL) was added to the residue, heated to 50-60 ℃ for dissolution, n-hexane (175 mL) was added dropwise under heat-insulating conditions, after the addition was completed, the temperature was lowered to 20-30 ℃ to precipitate a large amount of white solid, and the mixture was filtered and the filter cake was dried to obtain compound 1.2 (20 g, yield: 51%) as a white solid powder.
Step 3: compound 1.2 (390 mg,1.00 mmol), triethylamine (303 mg,3.00 mmol) and DMAP (122 mg,1.00 mmol) were dissolved in methylene chloride (4 mL), and after the reaction system was stirred at room temperature for 0.5 hours, N-ethyl-N-methylformyl chloride (242 mg,2.00 mmol) was added, the reaction system was stirred at room temperature for 3 hours, and the reaction solution was concentrated to give crude compound 1.3, which was directly used in the next step.
Step 4: compound 1.3 was mixed with a mixed solvent of acetic acid (4 mL) and water (1 mL), the reaction system was stirred at room temperature for 20 minutes, the reaction solution was concentrated, and compound 1-1 (35 mg, two-step yield: 8%) was obtained as a white solid by purification with prep-HPLC.
1 H NMR(400MHz,CD 3 OD):δ6.81(td,J＝6.8,1.6Hz,1H),5.97(d,J＝5.6Hz,1H),4.80-4.86(m,2H,overlapped with water signal),4.58(dd,J＝11.0,6.0Hz,1H),4.31(d,J＝11.2Hz,1H),4.11(d,J＝11.2Hz,1H),3.33-3.42(m,2H),3.02-3.19(m,2H),2.91(d,J＝8.6Hz,3H),1.98-2.16(m,2H),1.68-1.88(m,4H),1.58(s,3H),1.43-1.53(m,1H),1.22-1.32(m,2H),1.20(s,3H),1.08-1.16(m,3H),0.98(s,3H)。
m/z:[M+NH 4 ] + 453.1
Example 2: synthesis of Compounds 1-2
Using the method for synthesizing Compound 1-1 of example 1, N-ethyl-N-methylformyl chloride in step 1 is replaced with 4-morpholinyl formyl chloride to give Compound 1-2:
1 H NMR(400MHz,CD 3 OD):δ6.82(td,J＝6.8,1.6Hz,1H),6.00(d,J＝5.8Hz,1H),4.58(dd,J＝11.2,5.8Hz,1H),4.34(dd,J＝11.1,1.8Hz,1H),4.11(d,J＝11.2Hz,1H),3.60-3.70(m,4H),3.35-3.50(m,6H),3.00-3.19(m,2H),2.05-2.11(m,2H),1.74-1.83(m,4H),1.57(s,3H),1.30-1.53(m,3H),1.21(s,3H),0.97(s,3H)。
m/z:[M-H] - 462.2
example 3: synthesis of Compound 2-1
Step 1: compound 1.2 (390 mg,1.00 mmol), triphenylphosphine (393 mg,1.50 mmol) and tert-butyl phenol p-formate (29 mg,1.50 mmol) were dissolved in dry tetrahydrofuran (4 mL), cooled to 0deg.C, DIAD (303 m g,1.50 mmol) was added dropwise, the reaction was stirred at room temperature overnight, the reaction concentrated, and the residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate=3:1) to give compound 2.1 (410 mg, yield: 72%) as a pale yellow solid.
Step 2: compound 2.1 (200 mg,0.353 mmol) was mixed with a mixed solvent of acetic acid (2 mL) and water (0.5 mL), the reaction system was stirred at room temperature for 20 minutes, the reaction solution was concentrated, and compound 2-1 (15 mg, yield: 6%) was obtained as a white solid by purification with prep-HPLC.
1 H NMR(400MHz,CD 3 OD):δ7.96(d,J＝8.8Hz,2H),7.02(d,J＝8.8Hz,2H),6.88(td,J＝6.4,1.2Hz,1H),5.85(d,J＝5.2Hz,1H),4.71(dd,J＝10.8,5.6Hz,1H),4.38(dd,J＝10.8,1.3Hz,1H),4.08(d,J＝11.2Hz,1H),3.32-3.40(m,2H),2.94-3.12(m,2H),1.98-2.10(m,2H),1.72-1.83(m,4H),1.59(s,9H),1.56(s,3H),1.38-1.50(m,1H),1.12-1.26(m,5H),0.89(s,3H)。
m/z:[M+NH 4 ] + 543.9
Example 4: synthesis of Compound 2-2
Using the synthetic method of compound 2-1 of example 3, tert-butyl phenol p-formate in step 1 was replaced with p-cyanophenol to give compound 2-2:
1 H NMR(400MHz,CD 3 OD):δ7.74(d,J＝6.8Hz,2H),7.12(d,J＝6.8Hz,2H),6.90(t,J＝6.8Hz,1H),5.87-5.91(m,1H),4.68-4.75(m,1H),4.37(d,J＝10.8Hz,1H),4.09(d,J＝10.4Hz,1H),3.48-3.32(m,2H),2.95-3.14(m,2H),2.04(s,2H),1.72-1.88(m,4H),1.56(s,3H),1.40-1.52(m,1H),1.15-1.25(m,5H),0.90(s,3H)。
m/z:[M+NH 4 ] + 468.9
example 5: synthesis of Compound 3.2
Step 1: to a solution of 4-hydroxybenzonitrile (2.0 g,0.02 mol) in N, N-dimethylformamide (50 mL) was added sequentially ammonium chloride (9.0 g,0.16 mol) and sodium azide (2.2 g,0.03 mol), and the reaction was stirred at 120℃overnight. The reaction system was cooled to room temperature, then poured into ice water, pH was adjusted to 2.5 with hydrochloric acid (1M), the resulting mixture was extracted with ethyl acetate (50 mL. Times.2), the organic phases were combined, washed with water and saturated brine, respectively, the organic phases were separated, dried over anhydrous sodium sulfate, filtered, and concentrated to give compound 3.1 (2.3 g, yield: 72%) as a beige solid.
Step 2: to compound 3.1 (2.0 g,12.3 mmol) in dichloromethane (20 mL)) Triethylamine (2.5 g,24.7 mmol) and Boc were added separately to the solution 2 O (3.0 g,13.7 mmol) and the reaction was stirred at room temperature for 12 hours. The reaction was then washed with water (10 mL. Times.2), the organic phase was separated, dried over anhydrous sodium sulfate, filtered, concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=1/1) to give compound 3.2 (1.6 g, yield: 50%) as a beige solid.
Example 6: synthesis of Compound 2-3
Step 1: compound 1.1 (8.0 g,22.8 mmol), benzaldehyde dimethyl acetal (6.95 g,45.6 mmol) and pyridine p-toluenesulfonate hydrochloride (PPTS) (0.29 g,1.4 mmol) were dissolved in dichloromethane (80 mL) and the reaction was stirred at room temperature for 18 hours. The reaction solution was then washed with water, and the organic phase was separated, dried over anhydrous sodium sulfate, filtered, and concentrated. The residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give compound 3.3 (5.1 g, yield: 51%) as a white solid.
Step 2: compound 3.3 (200 mg,0.46 mmol), triphenylphosphine (181 mg,0.69 mmol) and compound 3.2 (180 mg,0.69 mmol) were dissolved in dry tetrahydrofuran (5 mL), cooled to 0℃and then DIAD (139.5 mg,0.69 mmol) was added dropwise to the above mixture, the reaction system was stirred overnight at room temperature, then the reaction solution was concentrated, and the residue was purified by silica gel column chromatography (petroleum ether/ethyl acetate=3/1) to give compound 3.4 (124 mg, yield: 36%) as a pale yellow oil.
m/z:[M+H 2 O] + 699.8
Step 3: compound 3.4 (20 mg,0.029 mmol) and p-toluenesulfonic acid monohydrate (11 mg,0.06 mmol) were dissolved in ethanol, the reaction system was stirred at 70℃for 3 days, then the reaction solution was concentrated, and the residue was purified by prep-HPLC to give compound 2-3 (0.8 mg, yield: 6%) as a white solid.
m/z:[M+H 2 O] + 512.4
Biological test examples:
example 1: anti-inflammatory Activity in LPS-induced BV2 microglial inflammation model
1. Cell treatment
The method is used for gene expression detection and ELISA: laying cells for 16h in advance, and paving 24 pore plates and 20 ten thousand/pore plates; experimental group: 100ng/ml LPS+DMSO or the corresponding compound; control group: cells were treated with the same amount of DMSO as the experimental group; liquid volume of culture: 400 μl/well;
2. RNA extraction and RT-PCR
1. After 6h of compound treatment, the old culture broth was aspirated, cells were lysed by adding trizol, 500 μl/well, and transferred to EP tube;
2. adding 100 μl of CHCl3, mixing upside down for 30 times, and standing at room temperature for 10min;
3. centrifuging at 4deg.C, 12000rpm,15min;
4. 200 μl of supernatant was added to the new EP tube, 350 μl of isopropanol was added respectively, mixed upside down for 20 times, and left standing at-20deg.C for 20min;
5. centrifuging at 4deg.C, 12000rpm,15min;
6. the supernatant was discarded, 1ml of 75% ethanol was added, turned upside down several times, and the precipitate was washed;
7. Centrifuging at 4℃at 12000rpm for 5min;
8. discarding the supernatant, inverting and airing;
9. adding 30 μl DEPC water, stirring, standing on ice for dissolving precipitate, and measuring RNA concentration;
10.RT-PCR：
(1) RT-PCR formulation
| Reagent(s) | Volume of |
| oligo dN6(0.1μg/μl) | 1μl |
| 10mM dNTP | 1μl |
| Template RNA | 1μg |
| DEPC H2O | 13μl-V(RNA) |
| Totalizing | 15μl |
(2) 70 ℃ for 5min; placing on ice for 1min;
(3) Add 4. Mu.l of 5 XRT buffer, 1. Mu. l M-MLVR; mixing uniformly; standing at 25deg.C for 10min;
(4)42℃，50min；
(5)95℃，5min。
3. qPCR
Qpcr formulation:
| 2×PCR mix | 12.5μl |
| templite (RT product diluted 20 times) | 4μl |
| Primers F/R(2.5μM/2.5μM) | 4μl |
| Water and its preparation method | 4.5μl |
| Totalizing | 25μl |
2. The reaction procedure:
/>
4. ELISA (Dake for)
1. After 24h of compound treatment of the cells, transfer the culture to EP tubes;
2. centrifuging at 12000rpm for 5min, transferring the supernatant to a new EP tube;
3. subsequent procedures were performed according to the daceae instructions for IL6 and tnfα ELISA kit.
Experimental results: LPS treatment can cause an inflammatory response to BV2 (microglial cell line), one of which is typically manifested by increased production of inflammatory factors (il1β, IL6, etc.). Thus, the anti-inflammatory effect of the compounds of the invention was first evaluated by qPCR detection of the expression levels of IL1 beta and IL6 using the LPS-induced BV2 inflammation model. On this basis, the effect of the compounds of the invention on IL6 and TNFα production at 10. Mu.M was further examined by ELISA, and the results are shown in Table 1 and FIG. 1.
Table 1: the percentages are the expression levels of inflammatory factors in the compound treated group compared to the reference group; "++" is the best inhibitory effect on inflammatory factors, and "+" is next to "-" is that no significant effect is detected.
TABLE 1
Example 2: cytotoxicity test
For ATP-GLO detection: BV2 cells: spreading cells in advance for 16 hours, and paving cells in 96-well plates and 3 ten thousand/well plates; experimental group: treating the cells with the corresponding compound; control group: cells were treated with the same amount of DMSO as the experimental group; liquid volume of culture: 200 μl/well
ATP-GLO test:
1. after 24h of compound treatment, the old culture broth was aspirated, and ATP-GLO lysate was added, 100. Mu.l/well;
2. lysing cells at room temperature for 10min;
3. transfer lysates to white 96-well plates and assay Cell titer glo luminescence using a microplate reader.
Experimental results: the effect of the compounds of the present invention on BV2 cell activity was examined by ATP-GLO experiments, and the results showed that all the tested compounds had no significant effect on cell activity at 10. Mu.M and below, indicating that the compounds themselves were not significantly cytotoxic.
Claims (4)
1. An andrographolide compound or pharmaceutically acceptable salt shown in formula 1-2 or formula 2-1,
2. a pharmaceutical composition comprising a therapeutically effective amount of an active ingredient and pharmaceutically acceptable excipients; the active ingredient comprises the andrographolide compound or the pharmaceutically acceptable salt of claim 1.
3. The pharmaceutical composition of claim 2, wherein: in the pharmaceutical composition, the pharmaceutically acceptable auxiliary materials comprise pharmaceutically acceptable carriers.
4. Use of an andrographolide compound or a pharmaceutically acceptable salt as claimed in claim 1 or a pharmaceutical composition as claimed in claim 2 or 3 in the preparation of a medicament for treating inflammatory diseases.
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| CN101012211A (en) * | 2007-02-06 | 2007-08-08 | 中国药科大学 | Substituted andrographolide derivative, preparing method and pharmaceutical compound thereof |
| CN103145657A (en) * | 2012-04-12 | 2013-06-12 | 江西青峰药业有限公司 | 17-hydro-9-dehydroandrographolide compound and its preparation method and use in drug preparation |
| CN103224492A (en) * | 2013-04-15 | 2013-07-31 | 南京工业大学 | 14-aryl-ether andrographolide derivative and preparation method and application thereof |
| CN104856987A (en) * | 2015-01-22 | 2015-08-26 | 南京工业大学 | Application of 14-arylether andrographolide derivatives in antibiosis field |
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| CN101012211A (en) * | 2007-02-06 | 2007-08-08 | 中国药科大学 | Substituted andrographolide derivative, preparing method and pharmaceutical compound thereof |
| CN103145657A (en) * | 2012-04-12 | 2013-06-12 | 江西青峰药业有限公司 | 17-hydro-9-dehydroandrographolide compound and its preparation method and use in drug preparation |
| CN103224492A (en) * | 2013-04-15 | 2013-07-31 | 南京工业大学 | 14-aryl-ether andrographolide derivative and preparation method and application thereof |
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