CN106660974A - Sulfamyl-containing 1,2,5-oxadiazole derivative, preparation method therefor and use thereof in pharmaceuticals - Google Patents

Abstract

Disclosed are a sulfamyl-containing 1,2,5-oxadiazole derivative, a preparation method therefor, and a use thereof in pharmaceuticals. Specifically disclosed are a sulfamyl-containing 1,2,5-oxadiazole derivative of generic formula (I), a preparation method thereof, a pharmaceutical composition containing the derivative, and use thereof as an IDO (Indoleamine-pyrrole-2,3-dioxygenase) inhibitor, and the sulfamyl-containing 1,2,5-oxadiazole derivative can be applied to treating diseases having IDO-mediated tryptophan metabolic pathway pathological features, including tumors, cancers, Alzheimer's Disease, autoimmune diseases, depression, anxiety disorder, cataract, psychological disorder and AIDS. Wherein various substituents in generic formula (I) have the same meanings as defined in the description.

Description

1,2, 5-oxadiazole derivative containing sulfamoyl, preparation method and application thereof in medicine Technical Field

The invention belongs to the field of medicines, and relates to a synthesis method of 1,2, 5-oxadiazole derivatives containing sulfamoyl and application thereof in medicines. The invention discloses a pharmaceutical composition which is used as an IDO inhibitor and is used for treating diseases with IDO mediated tryptophan metabolic pathway pathological characteristics, wherein the diseases comprise cancer, Alzheimer disease, autoimmune diseases, depression, anxiety, cataract, psychological disorder and AIDS.

Background

Tumors are one of the major diseases that seriously harm human life, and more than half occur in developing countries. The incidence of malignant tumors in China generally tends to rise, the incidence is increased at a speed of 3% -5% per year, and 400 million people in China are expected to have cancer and 300 million people die of cancer by 2020, and the main reasons are that: aging, urbanization, industrialization and change of living habits. In the drug market of Chinese hospitals, the sales scale of the antitumor drugs is steadily increased in recent years, the sales scale of the antitumor drugs reaches 664.2 hundred million yuan in 2012 and is increased by 13.07 percent on a par, and the market scale of the antitumor drugs is estimated to reach 1055.7 hundred million yuan and is increased by 7.57 percent on a par by 2017.

Due to unlimited growth, infiltration and metastasis of malignant tumors, three conventional treatment methods (surgery, radiotherapy and chemotherapy) clinically adopted at present cannot completely remove or completely kill tumor cells, so that tumor metastasis or recurrence often occurs. The biological treatment of tumor is a new treatment method for preventing and treating tumor by applying modern biotechnology and related products, and is a fourth mode of tumor treatment after surgery, radiotherapy and chemotherapy due to the characteristics of safety, effectiveness, low adverse reaction and the like, and the anti-tumor effect is obtained by mobilizing the natural defense mechanism of host (such as IDO-mediated tumor immune escape mechanism) or giving naturally-generated substances with strong targeting property.

Indoleamine-pyrrole-2,3-dioxygenase (IDO) is a monomeric iron-containing heme protein consisting of 403 amino acid residues and including two folded alpha-helical domains, a large domain containing a catalytic pocket, and a substrate capable of interacting with IDO in the catalytic pocket in a hydrophobic manner. IDO is an enzyme catalyzing the conversion of tryptophan into formylkynurenine, is widely distributed in tissues of human and other mammals (rabbits and mice) except liver, is the only rate-limiting enzyme capable of catalyzing the catabolism of tryptophan except liver, and tryptophan is an amino acid necessary for cell maintenance, activation and proliferation and is also an essential component for constituting proteins. IDO is closely related to various cytokines such as Interferon (IFN), Interleukin (IL), tumor necrosis factor and the like, and can activate IDO under certain conditions. On one hand, IDO causes local tryptophan depletion, so that T-cells are arrested in the middle stage of G1, and the proliferation of the T-cells is inhibited; on the other hand, the main product of canine urea produced by IDO catalysis of tryptophan metabolism induces T-cell apoptosis by oxygen free radical mediated changes in intracellular oxidants and antioxidants, which is an inherent immunosuppressive mechanism present in the body. A large number of studies at present show that IDO is highly expressed in leukemia cells, so that local T cell proliferation is inhibited, T-cell mediated immune reaction is inhibited, T-cell activation signal transduction is inhibited, and the attack of tumor cells escaping from an immune system is mediated. It has been found that most human tumors constitutively express IDO. Therefore, IDO is a potential target for cancer immunotherapy.

1-methyltryptophan (1-methyltryptophan) is an oral small molecule IDO inhibitor developed by NewLink Genetics for the treatment of metastatic breast cancer and solid tumors, currently entering clinical stage ii in the united states. Studies show that 1-methyltryptophan can enhance the sensitivity of tumor cells to T-cell immunostimulation in vitro, can delay the growth of tumor cells and strengthen the anti-tumor effect of chemotherapeutic drugs in vivo animal models, and has an effect on almost all spontaneous tumors. In addition, Incyte is developing a series of oral IDO small molecule inhibitors, INCB-24360 is also in clinical secondary trials, mainly used for treating various cancers including myelodysplastic syndrome.

Published inhibitor patent applications for selective inhibition of IDO include WO2004094409, WO2006122150, WO2007075598, WO2010005958, and WO2014066834, among others.

The IDO inhibitor has good application prospect in the pharmaceutical industry as a medicine, but no good IDO inhibitor can be used as a medicine on the market at present, and in order to achieve the purpose of better tumor treatment effect and better meet the market demand, a new generation of high-efficiency and low-toxicity selective IDO inhibitor is hoped to be developed. The present invention is to provide a selective IDO inhibitor of a novel structure, and to find that a compound having such a structure exhibits excellent effects and actions, particularly excellent drug absorption activity.

Disclosure of Invention

The invention aims to provide a compound shown in a general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture form thereof, or a pharmaceutically acceptable salt thereof, wherein the structure of the compound shown in the general formula (I) is as follows:

wherein:

selected from the cis isomer, trans isomer or a mixture of cis and trans isomers;

R¹selected from the group consisting of alkyl, cycloalkyl and alkoxy, wherein said alkyl, cycloalkyl, alkoxy are each independently optionally further substituted with one or more substituents selected from the group consisting of hydroxy, alkyl, alkoxy and haloalkyl;

R²selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group and an alkoxy group, wherein said alkyl group, cycloalkyl group and alkoxy group are each independently optionally further substituted with one or more substituents selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group and a haloalkyl group;

or R¹And R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group, wherein said cycloalkyl, heterocyclyl group is optionally further selected from the group consisting of alkyl, halogen, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁶R⁷、-C(O)NR⁶R⁷、-NHS(O)_mR⁸、-S(O)_pR⁸、-NHC(O)R⁸、-OR⁸、-C(O)R⁸、-OC(O)R⁸and-C (O) OR⁸Is substituted with one or more substituents of (1);

R³and R⁴Each independently selected from the group consisting of hydrogen, alkyl, cyano, amino, halogen, alkenyl, alkynyl, hydroxyl, nitro, cycloalkyl, heterocyclyl, aryl, and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl, or heteroaryl, each independently, is optionally further substituted with one or more substituents selected from the group consisting of hydroxyl, halogen, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl, and heteroaryl;

R⁵selected from alkyl and amino groups, said alkyl and amino groups optionally being further selected from hydroxy, halogen, amino, cyano, alkyl, alkoxy, cycloalkaneSubstituted with one or more substituents selected from the group consisting of aryl, heteroaryl, heterocyclyl, and heteroaryl; the alkyl group is preferably C_1～4An alkyl group;

R⁶and R⁷Each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl are each independently optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

R⁸selected from the group consisting of hydrogen atoms, alkyl groups, amino groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups, wherein said alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups are each independently optionally further substituted with one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, amino, cyano, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl groups;

m is 0, 1, 2;

n is 0, 1, 2; and is

p is 0, 1, 2.

In a preferred embodiment of the present invention, the compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein R is³And R⁴Each independently selected from a hydrogen atom, a halogen, preferably fluorine, chlorine or bromine, an alkyl, preferably C, or a haloalkyl_1～4Preferably, the haloalkyl group is trifluoromethyl.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is a compound represented by the general formula (II) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

R¹is alkyl or alkoxy, said alkyl or alkoxy being further substituted by one or more hydroxy or alkoxy groups; wherein R is preferred¹Is alkyl substituted by hydroxy or alkoxy;

R³～R⁵m and n are as defined in formula (I).

Preferred compounds of the invention are represented by the general formula (II) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

R¹is alkyl or alkoxy, said alkyl or alkoxy being further substituted with one or more hydroxy or alkoxy groups;

R³is halogen, alkyl or haloalkyl, more preferably halogen;

R⁴is halogen, alkyl or haloalkyl, more preferably halogen;

R⁵is amino;

m is an integer of 0 to 2, more preferably 0;

n is an integer of 0 to 2, and more preferably 1.

In a preferred embodiment of the present invention, the compound represented by the general formula (I) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, is a compound represented by the general formula (III) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

a is a 3-8 membered ring, preferably a 3-6 membered ring, wherein the ring is selected from a carbocyclic ring or a heterocyclic ring;

R³～R⁵m and n are as defined in formula (I).

Preferred compounds of the invention are represented by the general formula (III) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

a is cycloalkyl, more preferably cyclopropyl;

R³is halogen, alkyl or haloalkyl, more preferably halogen;

R⁴is halogen, alkyl or haloalkyl, more preferably halogen;

R⁵is amino;

m is an integer of 0-2;

n is an integer of 1 to 2.

The present invention also provides a process for preparing a compound of the general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, the process comprising:

the compound of the general formula (IV) is subjected to ring opening under alkaline conditions to obtain a compound of the general formula (I), wherein a reagent under the alkaline conditions is potassium carbonate or sodium hydroxide;

wherein:

R¹～R⁵m and n are as defined in formula (I).

The invention also relates to a compound shown in the general formula (IV) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

R¹～R⁵m and n are as defined in formula (I).

In a preferred embodiment of the present invention, a compound represented by the general formula (IV) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which is a compound represented by the general formula (V) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

R¹、R³～R⁵m and n are as defined in formula (I).

In a preferred embodiment of the present invention, a compound represented by the general formula (IV) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof, which is a compound represented by the general formula (VI) or a tautomer, a mesomer, a racemate, an enantiomer, a diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof:

wherein:

a is a 3-8 membered ring, wherein the ring is selected from a carbocyclic ring or a heterocyclic ring;

R³～R⁵m and n are as defined in formula (I).

The present invention synthesizes typical compounds of general formula (I), (II), (III) through a series of reactions, including, but not limited to:

another aspect of the present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I), (II) and (III) or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

The present invention also relates to a process for preparing the above composition, which comprises mixing the compounds represented by the general formulae (I), (II) and (III) or tautomers, mesomers, racemates, enantiomers, diastereomers, or mixtures thereof, or pharmaceutically acceptable salts thereof, with a pharmaceutically acceptable carrier, diluent or excipient.

The invention further relates to the use of a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for the preparation of a medicament for the prophylaxis and/or treatment of a disease characterized by a pathology of the IDO-mediated tryptophan metabolic pathway. IDO inhibitors may be useful for the inhibition of cardiac disorders and for the treatment of other diseases with pathological features of IDO-mediated tryptophan metabolic pathway, including viral infections such as AIDS, cellular infections such as lyme disease and streptococcal infections, neurodegenerative disorders (e.g., alzheimer's disease, huntington's disease and parkinson's disease), autoimmune diseases, depression, anxiety, cataracts, psychological disorders, AIDS, cancer (including T-cell leukemia and colon cancer), ocular disease states (e.g., cataracts and age-related yellowing), and autoimmune diseases, wherein the cancer may be selected from breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, Ovarian tumors, peritoneal tumors, stage IV melanoma, solid tumors, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, head and neck tumors, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

The invention also relates to a compound of general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same, for use in the prevention and/or treatment of a disease characterized by a pathology of the IDO-mediated tryptophan metabolic pathway. These diseases include infection by viruses such as AIDS, cell infections such as Lyme disease and streptococcal infection, neurodegenerative disorders (e.g., Alzheimer's disease, Huntington's disease and Parkinson's disease), autoimmune diseases, depression, anxiety, cataracts, psychological disorders, AIDS, cancer (including T-cell leukemia and colon cancer), ocular disease states (e.g., cataracts and age-related yellowing), and autoimmune diseases, wherein the cancer may be selected from breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, solid tumor, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, prostate tumor, kidney tumor, prostate tumor, and prostate tumor, Head and neck tumors, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

The present invention also relates to a method for the treatment and/or prophylaxis of diseases which are characterized by pathologies of the IDO-mediated tryptophan metabolic pathway, which comprises administering to a patient a therapeutically effective dose of a compound of the general formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition comprising same. These diseases include infection by viruses such as AIDS, cell infections such as Lyme disease and streptococcal infection, neurodegenerative disorders (e.g., Alzheimer's disease, Huntington's disease and Parkinson's disease), autoimmune diseases, depression, anxiety, cataracts, psychological disorders, AIDS, cancer (including T-cell leukemia and colon cancer), ocular disease states (e.g., cataracts and age-related yellowing), and autoimmune diseases, wherein the cancer may be selected from breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, solid tumor, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, prostate tumor, kidney tumor, prostate tumor, and prostate tumor, Head and neck tumors, leukemia, lymphoma, myeloma, and non-small cell lung cancer.

Another aspect of the present invention relates to a method for treating cancer, which comprises administering to a patient a therapeutically effective amount of a compound of formula (I) of the present invention, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof. The method shows outstanding therapeutic effects and fewer side effects, wherein the cancer may be selected from breast cancer, cervical cancer, colon cancer, lung cancer, stomach cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, solid tumor, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer, preferably fallopian tube tumor, peritoneal tumor, stage IV melanoma, myeloma, and breast cancer, more preferably breast cancer.

The pharmaceutical compositions containing the active ingredient may be in a form suitable for oral use, for example, as tablets, troches, lozenges, aqueous or oily suspensions, dispersible powders or granules, emulsions, hard or soft capsules, or syrups or elixirs. Oral compositions may be prepared according to any method known in the art for preparing pharmaceutical compositions, and such compositions may contain one or more ingredients selected from the group consisting of: sweetening agents, flavoring agents, coloring agents and preserving agents in order to provide a pleasant to the eye and palatable pharmaceutical preparation. Tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets. These excipients may be inert excipients, such as calcium carbonate, sodium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as microcrystalline cellulose, croscarmellose sodium, corn starch or alginic acid; binding agents, for example starch, gelatin, polyvinylpyrrolidone or acacia, and lubricating agents, for example magnesium stearate, stearic acid or talc. These tablets may be uncoated or they may be coated by known techniques which mask the taste of the drug or delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, water soluble taste masking substances such as hydroxypropylmethyl cellulose or hydroxypropyl cellulose, or time extending substances such as ethyl cellulose, cellulose acetate butyrate may be used.

Oral formulations may also be presented as hard gelatin capsules wherein the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin, or as soft gelatin capsules wherein the active ingredient is mixed with a water soluble carrier, for example polyethylene glycol, or an oil vehicle, for example peanut oil, liquid paraffin or olive oil.

Aqueous suspensions contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients are suspending agents, for example sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone and acacia; dispersing or wetting agents may be a naturally occurring phosphatide, for example lecithin, or condensation products of an alkylene oxide with fatty acids, for example polyoxyethylene stearate, or condensation products of ethylene oxide with long chain aliphatic alcohols, for example heptadecaethyleneoxycetanol (heptadecaethyleneoxy cetanol), or condensation products of ethylene oxide with partial esters derived from fatty acids and a hexitol such as polyethylene oxide sorbitol monooleate, or condensation products of ethylene oxide with partial esters derived from fatty acids and hexitol anhydrides, for example polyethylene oxide sorbitan monooleate. The aqueous suspensions may also contain one or more preservatives, for example ethyl or n-propyl paraben, one or more colouring agents, one or more flavouring agents and one or more sweetening agents, such as sucrose, saccharin or aspartame.

Oil suspensions may be formulated by suspending the active ingredient in a vegetable oil, for example arachis oil, olive oil, sesame oil or coconut oil, or in a mineral oil such as liquid paraffin. The oil suspension may contain a thickening agent, for example beeswax, hard paraffin or cetyl alcohol. Sweetening agents such as those set forth above, and flavoring agents may be added to provide a palatable preparation. These compositions can be preserved by the addition of antioxidants such as butylated hydroxyanisole or alpha-tocopherol.

Dispersible powders and granules suitable for preparation of an aqueous suspension by the addition of water provide the active ingredient in admixture with a dispersing or wetting agent, suspending agent or one or more preservatives. Suitable dispersing or wetting agents and suspending agents are illustrative of the examples given above. Other excipients, for example sweetening, flavoring and coloring agents, may also be present. These compositions are preserved by the addition of an antioxidant such as ascorbic acid.

The pharmaceutical compositions of the invention may also be in the form of oil-in-water emulsions. The oily phase may be a vegetable oil, for example olive oil or arachis oil, or a mineral oil, for example liquid paraffin or mixtures thereof. Suitable emulsifying agents may be naturally-occurring phosphatides, for example soy bean lecithin, and esters or partial esters derived from fatty acids and hexitol anhydrides, for example sorbitan monooleate, and condensation products of the said partial esters with ethylene oxide, for example polyethylene oxide sorbitol monooleate. The emulsions may also contain sweetening agents, flavouring agents, preservatives and antioxidants. Syrups and elixirs may be formulated with sweetening agents, for example glycerol, propylene glycol, sorbitol or sucrose. Such formulations may also contain a demulcent, a preservative, a colorant and an antioxidant.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous solution. Among the acceptable vehicles and solvents that may be employed are water, ringer's solution and isotonic sodium chloride solution. The sterile injectable preparation may be a sterile injectable oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then treated to form a microemulsion by adding to a mixture of water and glycerol. The injection solution or microemulsion may be injected into the bloodstream of a patient by local bulk injection. Alternatively, it may be desirable to administer the solutions and microemulsions in a manner that maintains a constant circulating concentration of the compounds of the present invention. To maintain such a constant concentration, a continuous intravenous delivery device may be used. An example of such a device is an intravenous pump model Deltec CADD-PLUS. TM.5400.

The pharmaceutical compositions may be in the form of sterile injectable aqueous or oleaginous suspensions for intramuscular and subcutaneous administration. The suspension may be formulated according to the known art using those suitable dispersing or wetting agents and suspending agents which have been mentioned above. The sterile injectable preparation may also be a sterile injectable solution or suspension prepared in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1, 3-butanediol. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any blend fixed oil may be used, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid find use in the preparation of injectables.

The compounds of the present invention may be administered in the form of suppositories for rectal administration. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum and therefore will melt in the rectum to release the drug. Such materials include cocoa butter, glycerogelatin, hydrogenated vegetable oils, polyethylene glycols of various molecular weights and mixtures of fatty acid esters of polyethylene glycols.

As is well known to those skilled in the art, the dosage of a drug to be administered depends on a variety of factors, including, but not limited to: the activity of the particular compound employed, the age of the patient, the weight of the patient, the health condition of the patient, the patient's integument, the patient's diet, the time of administration, the mode of administration, the rate of excretion, the combination of drugs, and the like; in addition, the optimal treatment regimen, such as mode of treatment, daily amount of the compound of formula (la) or type of pharmaceutically acceptable salt, can be verified according to conventional treatment protocols.

Detailed description of the invention

Unless stated to the contrary, terms used in the specification and claims have the following meanings.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably an alkyl group containing 1 to 12 carbon atoms, more preferably an alkyl group containing 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2, 3-dimethylpentyl, 2, 4-dimethylpentyl, 2-dimethylpentyl, 3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2, 3-dimethylhexyl, 2, 4-dimethylhexyl, 2, 5-dimethylhexyl, 2-dimethylhexyl, 3-dimethylhexyl, 4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2-methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2-dimethylpentyl, 2-dimethylhexyl, 3-dimethylpentyl, 2-ethylhexyl, 3-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-dimethylhexyl, 2-ethylhexyl, 2-ethyl, 2-2, 2-2, 2-2, or, 2, 2-diethylpentyl, n-decyl, 3-diethylhexyl, 2-diethylhexyl, and various branched isomers thereof. More preferred are lower alkyl groups having 1 to 6 carbon atoms, non-limiting examples of which include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2, 3-dimethylbutyl.And the like. Alkyl groups may be substituted OR unsubstituted, and when substituted, substituents may be substituted at any available point of attachment, preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halo, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent, and the cycloalkyl ring contains 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and further preferably 3 to 6 carbon atoms. Non-limiting examples of monocyclic cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like; polycyclic cycloalkyl groups include spiro, fused and bridged cycloalkyl groups.

The term "spirocycloalkyl" refers to a 5 to 20 membered polycyclic group sharing one carbon atom (referred to as a spiro atom) between monocyclic rings, which may contain one or more double bonds, but none of the rings have a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. Spirocycloalkyl groups are classified into a single spirocycloalkyl group, a double spirocycloalkyl group or a multi spirocycloalkyl group, preferably a single spirocycloalkyl group and a double spirocycloalkyl group, according to the number of spiro atoms shared between rings. More preferably 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered. Non-limiting examples of spirocycloalkyl groups include:

the term "fused cyclic alkyl" refers to a 5 to 20 membered all carbon polycyclic group in which each ring in the system shares an adjacent pair of carbon atoms with other rings in the system, wherein one or more of the rings may contain one or more double bonds, but none of the rings has a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused ring alkyls according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicycloalkyl. Non-limiting examples of fused ring alkyl groups include:

the term "bridged cycloalkyl" refers to a 5 to 20 membered all carbon polycyclic group in which any two rings share two carbon atoms not directly attached, which may contain one or more double bonds, but none of the rings have a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. They may be classified as bicyclic, tricyclic, tetracyclic or polycyclic bridged cycloalkyl groups, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic, depending on the number of constituent rings. Non-limiting examples of bridged cycloalkyl groups include:

the cycloalkyl ring may be fused to an aryl, heteroaryl or heterocycloalkyl ring, where the ring to which the parent structure is attached is cycloalkyl, non-limiting examples of which include indanyl, tetrahydronaphthyl, benzocycloheptanyl, and the like. Cycloalkyl groups may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "heterocyclyl" refers to a saturated or partially unsaturated mono-or polycyclic cyclic hydrocarbon substituent containing from 3 to 20 ring atoms wherein one or more of the ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer from 0 to 2) but excludes the ring moiety of-O-O-, -O-S-, or-S-S-, the remaining ring atoms being carbon. Preferably 3 to 12 ring atoms, of which 1 to 4 are heteroatoms; more preferably from 3 to 10 ring atoms; more preferably from 3 to 8 ring atoms; more preferably from 3 to 6 ring atoms. Non-limiting examples of monocyclic heterocyclyl groups include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclic groups include spiro, fused and bridged heterocyclic groups.

The term "spiroheterocyclyl" refers to a 5-to 20-membered polycyclic heterocyclic group in which one atom (referred to as the spiro atom) is shared between monocyclic rings, and in which one or more ring atoms is selected from nitrogen, oxygen, or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. It may contain one or more double bonds, but no ring has a completely conjugated pi-electron system. Preferably 6 to 14, more preferably 7 to 10. The spiro heterocyclic group is classified into a mono-spiro heterocyclic group, a di-spiro heterocyclic group or a multi-spiro heterocyclic group, preferably a mono-spiro heterocyclic group and a di-spiro heterocyclic group, according to the number of spiro atoms shared between rings. More preferred are 4-membered/4-membered, 4-membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered mono spiroheterocyclic groups. Non-limiting examples of spiro heterocyclic groups include:

the term "fused heterocyclyl" refers to a 5 to 20 membered polycyclic heterocyclic group in which each ring in the system shares an adjacent pair of atoms with other rings in the system, one or more rings may contain one or more double bonds, but none of the rings has a fully conjugated pi-electron system in which one or more ring atoms is selected from nitrogen, oxygen or S (O))_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclic groups according to the number of constituent rings, preferably bicyclic or tricyclic, more preferably 5-or 6-membered bicyclic fused heterocyclic groups. Non-limiting examples of fused heterocyclic groups include:

the term "bridged heterocyclyl" refers to a 5 to 14 membered polycyclic heterocyclic group in which any two rings share two atoms not directly attached which may contain one or more double bonds, but none of the rings have a fully conjugated pi-electron system in which one or more of the ring atoms is selected from nitrogen, oxygen or S (O)_m(wherein m is an integer of 0 to 2) and the remaining ring atoms are carbon. Preferably 6 to 14, more preferably 7 to 10. They may be classified into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclic groups according to the number of constituent rings, preferably bicyclic, tricyclic or tetracyclic, more preferably bicyclic or tricyclic. Non-limiting examples of bridged heterocyclic groups include:

the heterocyclyl ring may be fused to an aryl, heteroaryl or cycloalkyl ring, wherein the ring to which the parent structure is attached is heterocyclyl, non-limiting examples of which include:

the heterocyclyl group may be optionally substituted or unsubstituted, and when substituted, the substituents are preferably one or more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, cycloalkyl, nitro, cyano, nitro, alkoxy,Aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, oxo, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "aryl" refers to a 6 to 14 membered all carbon monocyclic or fused polycyclic (i.e., rings which share adjacent pairs of carbon atoms) group having a conjugated pi-electron system, preferably 6 to 10 membered, such as phenyl and naphthyl. The aryl ring may be fused to a heteroaryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is an aryl ring, non-limiting examples of which include:

aryl groups may be substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "heteroaryl" refers to a heteroaromatic system comprising 1 to 4 heteroatoms, 5 to 14 ring atoms, wherein the heteroatoms are selected from oxygen, sulfur and nitrogen. Heteroaryl is preferably 5 to 10 membered, more preferably 5 or 6 membered, for example furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring joined together with the parent structure is a heteroaryl ring, non-limiting examples of which include:

heteroaryl groups may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "alkoxy" refers to-O- (alkyl) and-O- (unsubstituted cycloalkyl), wherein alkyl is as defined above. Non-limiting examples of alkoxy groups include: methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted OR unsubstituted, and when substituted, the substituents are preferably one OR more groups independently selected from alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, mercapto, hydroxy, nitro, cyano, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocycloalkylthio, -C (O) OR⁷、-OC(O)R⁷、-NHS(O)_pR⁷、-C(O)R⁷、-NHC(O)R⁷、-NHC(O)OR⁷、-NR⁸R⁹、-OC(O)NR⁸R⁹or-C (O) NR⁸R⁹。

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens wherein the alkyl group is as defined above.

The term "hydroxyalkyl" refers to an alkyl group substituted with a hydroxy group, wherein the alkyl group is as defined above.

The term "hydroxy" refers to an-OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to the group-NH₂。

The term "cyano" refers to — CN.

The term "nitro" means-NO₂。

The term "oxo" refers to ═ O.

The term "carboxy" refers to-C (O) OH.

The term "carboxylate" refers to-C (O) O (alkyl) or-C (O) O (cycloalkyl), wherein alkyl is as defined above.

The term "acyl halide" refers to a compound containing a group that is-C (O) -halogen.

The term "vinyloxy" means CH₂＝CH-O-。

The term "methylene" refers to the radical-CH₂-。

The term "ethylene" refers to- (CH)₂)₂-。

The term "propylene" refers to- (CH)₂)₃-。

The term "butylene" refers to- (CH)₂)₄-。

The term "alkenyl" refers to-CH ═ CH-.

The term "alkynyl" refers to-C.ident.C-.

In the invention, different terms such as "X is selected from A, B or C", "X is selected from A, B and C", "X is A, B or C", "X is A, B and C" and the like all express the same meaning, that is, X can be any one or more of A, B, C.

"optional" or "optionally" means that the subsequently described event or circumstance may, but need not, occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl" means that an alkyl may, but need not, be present, and the description includes the case where the heterocyclic group is substituted with an alkyl and the heterocyclic group is not substituted with an alkyl.

"substituted" means that one or more, preferably up to 5, more preferably 1 to 3, hydrogen atoms in the group are independently substituted with a corresponding number of substituents. It goes without saying that the substituents are only in their possible chemical positions, and that the person skilled in the art is able to determine (experimentally or theoretically) possible or impossible substitutions without undue effort. For example, amino or hydroxyl groups having free hydrogen may be unstable in combination with carbon atoms having unsaturated (e.g., olefinic) bonds.

"pharmaceutical composition" means a mixture containing one or more compounds described herein or a physiologically/pharmaceutically acceptable salt or prodrug thereof in admixture with other chemical components, as well as other components such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate administration to an organism, facilitate absorption of the active ingredient and exert biological activity.

"pharmaceutically acceptable salts" refers to salts of the compounds of the present invention which are safe and effective for use in the body of a mammal and which possess the requisite biological activity.

Synthesis of the Compounds of the invention

In order to achieve the purpose of the invention, the invention adopts the following technical scheme:

the preparation method of the compound shown in the general formula (I) or the salt thereof comprises the following steps:

scheme one

Under acidic conditions, the compound of formula (Ia) is oxidized to a compound of formula (Ib); reacting the compound of the general formula (Ib) with the compound of the general formula (Ic) under alkaline conditions to obtain a compound of the general formula (Id); cyclizing the compound of the general formula (Id) under heating and alkaline conditions, wherein the base under the conditions is preferably N, N' -carbonyldiimidazole to obtain a compound of the general formula (Ie); removing the protecting group on the amino group of the cyclized compound of the general formula (Ie) under an acidic condition to obtain a compound of the general formula (If) or a salt thereof; reacting an alkaline solution of the compound of formula (If) or a salt thereof with an alcoholic solution of chlorosulfonyl isocyanate at low temperature to obtain a compound of formula (Ig), the alcoholic solution preferably being a tert-butanol solution; removing the protecting group on the amino group of the compound of the general formula (Ig) under an acidic condition to obtain a compound of a general formula (IV); the obtained compound of the general formula (IV) is subjected to ring opening under alkaline conditions to obtain the target compound of the general formula (I).

The reagents that provide basic conditions include organic bases including, but not limited to, sodium hexamethyldisilazide, triethylamine, N-diisopropylethylamine, N-butyllithium, potassium t-butoxide, tetrabutylammonium bromide, and inorganic bases including, but not limited to, sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, or cesium carbonate.

Oxidants used include, but are not limited to: hydrogen peroxide, potassium permanganate and manganese dioxide.

Solvents used include, but are not limited to: n, N-dimethylformamide, toluene, acetic acid, methanol, ethanol, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, 1, 4-dioxane or water.

Wherein:

R¹～R⁴m and n are defined as general formula (I).

The preparation method of the compound shown in the general formula (II) or the salt thereof comprises the following steps:

scheme two

Under acidic conditions, the compound of formula (Ia) is oxidized to a compound of formula (Ib); reacting the compound of the general formula (Ib) with a compound of the general formula (IIc) under alkaline conditions to obtain a compound of the general formula (IId); cyclizing the compound of the general formula (IId) under heating and alkaline conditions, wherein the base under the conditions is preferably N, N' -carbonyldiimidazole to obtain the compound of the general formula (IIe); removing the protecting group on the amino group of the cyclized compound of the general formula (IIe) under an acidic condition to obtain a compound of the general formula (IIf) or a salt thereof; reacting an alkaline solution of the compound of formula (IIf) or a salt thereof with an alcoholic solution of chlorosulfonyl isocyanate at low temperature to give the compound of formula (IIg), the alcoholic solution preferably being a tert-butanol solution; removing the protecting group on the amino group of the compound of the general formula (IIg) under an acidic condition to obtain a compound of a general formula (V); the obtained compound of the general formula (V) is subjected to ring opening under alkaline conditions to obtain the target compound of the general formula (II).

Reagents for basic conditions include organic bases including but not limited to sodium hexamethyldisilazide, triethylamine, N-diisopropylethylamine, N-butyllithium, potassium tert-butoxide, tetrabutylammonium bromide, and inorganic bases including but not limited to sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, or cesium carbonate.

Oxidants used include, but are not limited to: hydrogen peroxide, potassium permanganate and manganese dioxide.

Solvents used include, but are not limited to: n, N-dimethylformamide, toluene, acetic acid, methanol, ethanol, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, 1, 4-dioxane or water.

Wherein:

R¹、R³、R⁴m and n are defined as general formula (I).

The preparation method of the compound shown in the general formula (III) or the salt thereof comprises the following steps:

scheme three

Under acidic conditions, the compound of formula (Ia) is oxidized to a compound of formula (Ib); reacting the compound of the general formula (Ib) with the compound of the general formula (IIIc) under alkaline conditions to obtain a compound of the general formula (IIId); cyclizing the compound of the general formula (IIId) under heating and alkaline conditions, wherein the base under the conditions is preferably N, N' -carbonyldiimidazole to obtain a compound of the general formula (IIIe); removing the protecting group on the amino group of the cyclized compound of the general formula (IIIe) under an acidic condition to obtain a compound of the general formula (IIIf) or a salt thereof; reacting an alkaline solution of the compound of formula (IIIf) or a salt thereof with an alcoholic solution of chlorosulfonyl isocyanate at low temperature to give a compound of formula (IIIg), the alcoholic solution preferably being a tert-butanol solution; removing the protecting group on the amino group of the compound of the general formula (IIIg) under an acidic condition to obtain a compound of a general formula (VI); the obtained compound of the general formula (VI) is subjected to ring opening under alkaline conditions to obtain the target compound of the general formula (III).

Reagents for basic conditions include organic bases including but not limited to sodium hexamethyldisilazide, triethylamine, N-diisopropylethylamine, N-butyllithium, potassium tert-butoxide, tetrabutylammonium bromide, and inorganic bases including but not limited to sodium hydride, sodium carbonate, sodium bicarbonate, potassium carbonate, potassium bicarbonate, or cesium carbonate.

Oxidants used include, but are not limited to: hydrogen peroxide, potassium permanganate and manganese dioxide.

Solvents used include, but are not limited to: n, N-dimethylformamide, toluene, acetic acid, methanol, ethanol, tetrahydrofuran, dichloromethane, dimethyl sulfoxide, 1, 4-dioxane or water.

Wherein:

a is a 3-8 membered ring, wherein the ring is selected from a carbocyclic ring or a heterocyclic ring;

R³、R⁴m and n are defined as general formula (I).

Detailed Description

The present invention is further described below with reference to examples, which are not intended to limit the scope of the present invention.

Examples

The structure of the compounds is determined by Nuclear Magnetic Resonance (NMR) or Mass Spectrometry (MS). NMR was measured using a Bruker AVANCE-400 NMR spectrometer using deuterated dimethyl sulfoxide (DMSO-d)₆) Deuterated chloroform (CDCl)₃) Deuterated methanol (CD)₃OD), internal standard Tetramethylsilane (TMS), chemical shift is 10^-6(ppm) is given as a unit.

MS was determined using a FINNIGAN LCQAD (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

HPLC was carried out using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18150X 4.6mm column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18150X 4.6mm column).

Average inhibition rate of kinase and IC₅₀The values were determined with a NovoStar microplate reader (BMG, Germany).

The thin layer chromatography silica gel plate adopts HSGF254 of tobacco yellow sea or GF254 of Qingdao, the specification of the silica gel plate used by Thin Layer Chromatography (TLC) is 0.15 mm-0.2 mm, and the specification of the thin layer chromatography separation and purification product is 0.4 mm-0.5 mm.

The column chromatography generally uses 200-300 mesh silica gel of Taiwan yellow sea as a carrier.

Known starting materials of the present invention can be synthesized by or according to methods known in the art, or can be purchased from companies such as ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Shao Yuan Chemical technology (Accela ChemBio Inc), Darri Chemicals, and the like.

In the examples, the reaction was carried out under an argon atmosphere or a nitrogen atmosphere unless otherwise specified.

An argon atmosphere or nitrogen atmosphere means that the reaction flask is connected to a balloon of argon or nitrogen with a volume of about 1L.

The hydrogen atmosphere refers to a reaction flask connected with a hydrogen balloon with a volume of about 1L.

The pressure hydrogenation reaction used a hydrogenation apparatus of Parr 3916EKX type and a hydrogen generator of Qinglan QL-500 type or a hydrogenation apparatus of HC2-SS type.

The hydrogenation reaction was usually evacuated and charged with hydrogen and repeated 3 times.

The microwave reaction was carried out using a CEM Discover-S908860 type microwave reactor.

In the examples, the solution in the reaction is an aqueous solution unless otherwise specified.

In the examples, the reaction temperature was room temperature unless otherwise specified.

The room temperature is the optimum reaction temperature, and the temperature range is 20-30 ℃.

The progress of the reaction in the examples was monitored by Thin Layer Chromatography (TLC) using a developing solvent system of: a: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: petroleum ether and ethyl acetate system, D: the volume ratio of acetone and solvent is adjusted according to the polarity of the compound.

The system of eluents for column chromatography and developing agents for thin layer chromatography used for purifying compounds include: a: dichloromethane and methanol system, B: n-hexane and ethyl acetate system, C: n-hexane, ethyl acetate and dichloromethane system, D: petroleum ether and ethyl acetate system, E: the volume ratio of ethyl acetate and solvent is adjusted according to the polarity of the compound, and a small amount of triethylamine and acidic or basic reagent can be added for adjustment.

Example 1

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- (((1- (sulfamoylamino) cyclopropyl) methyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 1

First step of

4- (3-bromo-4-fluorophenyl) -3- (4-nitro-1, 2, 5-oxadiazol-3-yl) -1,2, 4-oxadiazol-5 (4H) -one 1b

3- (4-amino-1, 2, 5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 1a (264mg, 2.23mmol, prepared by the method disclosed in patent application "WO 2010005958") was added to 5mL of trifluoroacetic acid, 3mL of hydrogen peroxide (30%) was added, and the reaction was carried out at 45 ℃ for 16 hours. After the reaction was completed, the reaction was quenched by addition of saturated sodium sulfite solution, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 1B (210mg, yellow oil) in 78.6% yield.

MS m/z(LC-MS):342.9[M-1]

Second step of

(1- (((4- (N- (3-bromo-4-fluorophenyl) -N' -hydroxycarbamimidoyl) -1,2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) carbamic acid tert-butyl ester 1d

1b (210mg, 0.56mmol) was dissolved in 15mL of tetrahydrofuran, and tert-butyl (1- (aminomethyl) cyclopropyl) carbamate 1c (210mg, 1.13mmol, prepared by the method disclosed in patent application "WO 2013052394") was added, and 1.5mL of 2N sodium hydroxide solution was added and reacted at room temperature for 30 minutes. After the reaction was completed, 1N hydrochloric acid was added to adjust the pH of the reaction solution to 2, extraction was performed three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title product 1d (260mg, yellow solid) which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):485.2[M+1]

The third step

(1- (((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) carbamic acid tert-butyl ester 1e

Crude 1d (260mg, 0.535mmol) was dissolved in 20mL tetrahydrofuran, N' -carbonyldiimidazole (95mg, 0.586mmol) was added, and the reaction was carried out at 70 ℃ for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid, water and a saturated sodium chloride solution in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 1e (330mg, yellow solid), which was subjected to the next reaction without purification.

MS m/z(LC-MS):455.3 [M+1-56]

The fourth step

3- (4- (((1-aminocyclopropyl) methyl) amino) -1,2, 5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2, 4-oxadiazol-5 (4H) -one trifluoroacetate 1f

Crude 1e (330mg, 0.645mmol) was dissolved in 30mL of dichloromethane, 2mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude title product 1f (400mg, tan oil), which was directly subjected to the next reaction without purification.

The fifth step

N- (1- (((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) sulfamoylcarbamic acid tert-butyl ester 1g

Chlorosulfonyl isocyanate (0.793g, 5.6mmol, prepared by known method "Organic Synthesis, 1966,46, 23-7") was dissolved in 10mL of dichloromethane, cooled to 0 deg.C, t-butanol (0.43g, 5.8mmol) was added, and the reaction mixture was reacted at 0 deg.C for 1 hour to obtain reaction mixture A. Crude product 1f (400mg, 1.0mmol) was dissolved in 20mL of dichloromethane, and 0.75mL of triethylamine was added to obtain reaction solution B. The reaction mixture A was added to the reaction mixture B at 0 ℃ and reacted at 0 ℃ for 1 hour. After the reaction was completed, the reaction solution was quenched by addition of saturated sodium bicarbonate solution, separated, washed with water, the organic phase was washed successively with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to obtain 1g (120mg, white solid) of the title product in 20% yield.

MS m/z(LC-MS):607.1 [M+18]

The sixth step

N- {1- [ ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl ] -1,2, 5-oxadiazol-3-yl } amino) methyl ] cyclopropyl } sulfamide 1h

1g (120mg, 0.198mmol) was dissolved in 20mL of dichloromethane, 4mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 1h (160mg, brown dope), which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):490.0 [M+1]

Seventh step

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- (((1- (sulfamoylamino) cyclopropyl) methyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 1

The crude product was dissolved in 20mL of methanol for 1h (160mg, 0.27mmol), and reacted at 50 ℃ for 1h with the addition of potassium carbonate (230mg, 1.67 mmol). After the reaction was completed, saturated ammonium chloride solution was added to neutralize the reaction, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 1 (10mg, white solid) in 7.5% yield.

MS m/z(ESI):466.2[M+1]

¹H NMR(400MHz,DMSO-d₆)δ11.54(s,1H),8.89(s,1H),7.11-7.25(m,3H),6.78-6.79(m,1H),6.68(s,2H),6.23(t,1H),3.44(d,2H),0.94(s,2H),0.68(s,2H).

Example 2

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- (((1- ((sulfamoylamino) methyl) cyclopropyl) methyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 2

First step of

((1- (((4- (N- (3-bromo-4-fluorophenyl) -N' -hydroxycarbamimidoyl-1, 2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) methyl) carbamic acid tert-butyl ester 2b

1b (360mg, 0.968mmol) was dissolved in 30mL of tetrahydrofuran, and tert-butyl ((1- (aminomethyl) cyclopropyl) methyl) carbamate 2a (387mg, 1.935mmol, prepared by the method disclosed in patent application "WO 2009054468") was added thereto, and 2.9mL of 2N sodium hydroxide solution was added and reacted at room temperature for 30 minutes. After the reaction was completed, 1N hydrochloric acid was added to adjust the pH of the reaction solution to less than 7, extraction was performed three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title product 2b (650mg, tan solid), which was directly subjected to the next reaction without purification.

Second step of

((1- (((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) methyl) carbamic acid tert-butyl ester 2c

Crude 2b (650mg, 1.3mmol) was dissolved in 30mL tetrahydrofuran, N' -carbonyldiimidazole (222mg, 1.37mmol) was added, and the reaction was carried out at 70 ℃ for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid, water and a saturated sodium chloride solution in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 2c (715mg, tan solid), which was directly subjected to the next reaction without purification.

The third step

3- (4- (((1- (aminomethyl) cyclopropyl) methyl) amino) -1,2, 5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 2d

Crude 2c (715mg, 1.36mmol) was dissolved in 5mL of dichloromethane, 5mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 2d (1.02g, a reddish brown oil) which was used in the next reaction without purification.

MS m/z(LC-MS):427.0 [M+1]

The fourth step

N- ((1- (((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) methyl) cyclopropyl) methyl) sulfamoylcarbamic acid tert-butyl ester 2e

Chlorosulfonyl isocyanate (509mg, 3.59mmol) was dissolved in 10mL of dichloromethane, cooled to 0 ℃, t-butanol (267mg, 3.59mmol) was added, and the reaction mixture was reacted at 0 ℃ for 15 minutes to obtain reaction mixture A. Crude 2d (1.021g, 2.4mmol) was dissolved in 20mL of dichloromethane, and 1mL of triethylamine was added to prepare reaction solution B. The reaction mixture A was added to the reaction mixture B at 0 ℃ and reacted at 0 ℃ for 1 hour. After the reaction was completed, the reaction solution was quenched by addition of saturated sodium bicarbonate solution, separated, washed with water, the organic phase was washed successively with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 2e (28mg, brown solid) in 19% yield.

MS m/z(LC-MS):621.0[M+18]

The fifth step

N- ({1- [ ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl ] -1,2, 5-oxadiazol-3-yl } amino) methyl ] cyclopropyl } methyl) aminosulfonamide 2f

2e (282mg, 0.466mmol) was dissolved in 10mL of dichloromethane, 5mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude title product 2f (160mg, brown viscous substance), which was subjected to the next reaction without purification.

MS m/z(LC-MS):504.0[M+1]

The sixth step

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- (((1- ((sulfamoylamino) methyl) cyclopropyl) methyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 2

Crude 2f (386mg, 0.765mmol) was dissolved in 10mL of methanol, potassium carbonate (528mg, 3.86mmol) was added, and the reaction was carried out at 50 ℃ for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to neutralize the reaction, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 2(180mg, white solid) in 49% yield.

MS m/z(ESI):478.2[M+1]

¹H NMR(400MHz,DMSO-d₆)δ11.47(s,1H),8.89(s,1H),7.19(t,1H),7.10-7.12(m,1H),6.78-6.79(m,1H),6.77(t,1H),6.49(s,2H),6.14(t,1H),3.23(d,2H),2.86(d,2H),0.48(s,4H).

Example 3

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 3

First step of

(1- (2- ((4- (N- (3-bromo-4-fluorophenyl) -N' -hydroxycarbamimidoyl) -1,2, 5-oxadiazol-3-yl) amino) ethyl) cyclopropyl) carbamic acid tert-butyl ester 3b

1b (210mg, 0.564mmol) was dissolved in 20mL of tetrahydrofuran, tert-butyl ester (1- (2-aminoethyl) cyclopropyl) carbamic acid 3a (240mg, 1.19mmol, prepared by the method disclosed in patent application "WO 2013020993") was added, 1.5mL of 2N sodium hydroxide solution was added, and the reaction was carried out at room temperature for 30 minutes. After the reaction was completed, 1N hydrochloric acid was added to adjust the pH of the reaction solution to less than 7, extraction was performed three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title product 3b (370mg, pale yellow solid), which was subjected to the next reaction without purification.

MS m/z(LC-MS):497.1 [M-1]

Second step of

(1- (2- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) ethyl) cyclopropyl) carbamic acid tert-butyl ester 3c

Crude 3b (370mg, 0.74mmol) was dissolved in 15mL tetrahydrofuran, N' -carbonyldiimidazole (126mg, 0.78mmol) was added, and the reaction was carried out at 70 ℃ for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid, water and a saturated sodium chloride solution in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 3c (350mg, yellow solid), which was subjected to the next reaction without purification.

MS m/z(LC-MS):523.0[M-1]

The third step

3- (4- ((2- (1-aminocyclopropyl) ethyl) amino) -1,2, 5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 3d

Crude 3c (350mg, 0.666mmol) was dissolved in 5mL of dichloromethane, 3mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain the crude title product 3d (300mg, tan oil), which was directly subjected to the next reaction without purification.

The fourth step

N- (1- (2- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) ethyl) cyclopropyl) sulfamoylcarbamic acid tert-butyl ester 3e

Chlorosulfonyl isocyanate (145mg, 1.024mmol) was dissolved in 10mL of dichloromethane, cooled to 0 ℃, t-butanol (76mg, 1.025mmol) was added, and the reaction mixture was reacted at 0 ℃ for 15 minutes to obtain reaction mixture A. Crude 3d (500mg, 1.17mmol) was dissolved in 20mL of dichloromethane, and 1mL of triethylamine was added to prepare reaction solution B. The reaction mixture A was added to the reaction mixture B at 0 ℃ and reacted at 0 ℃ for 1 hour. After the reaction was completed, the reaction solution was quenched by addition of saturated sodium bicarbonate solution, separated, washed with water, the organic phase was washed successively with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 3e (180mg, brown solid) in a yield of 25%.

MS m/z(LC-MS):602.0[M-1]

The fifth step

N- {1- [2- ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl ] -1,2, 5-oxadiazol-3-yl } amino) ethyl ] cyclopropyl } sulfamide 3f

3e (180mg, 0.298mmol) was dissolved in 40mL of dichloromethane, 2mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure to obtain a crude title product 3f (223mg, brown yellow viscous substance), which was directly subjected to the next reaction without purification.

The sixth step

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 3

Crude 3f (223mg, 0.44mmol) was dissolved in 15mL of methanol, and potassium carbonate (380mg, 2.75mmol) was added to react at 50 ℃ for 1 hour. After the reaction was completed, saturated ammonium chloride solution was added to neutralize the reaction, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to obtain the title product 3(70mg, white solid) in 33% yield.

MS m/z(ESI):480.1[M+1]

¹H NMR(400MHz,DMSO-d₆)δ11.45(s,1H),8.89(s,1H),7.19(t,1H),7.07-7.10(m,2H),6.77-6.78(m,1H),6.60(s,2H),6.13(t,1H),3.38-3.43(m,2H),1.82(t,2H),0.88(t,2H),0.46(t,2H).

Example 4

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((3-hydroxy-2- (sulfamoylamino) propyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 4

First step of

4- (((4- (N- (3-bromo-4-fluorophenyl) -N' -hydroxycarbamimidoyl) -1,2, 5-oxadiazol-3-yl) amino) methyl) -2, 2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester 4b

1b (505mg, 1.54mmol) was dissolved in 30mL of tetrahydrofuran, and 4- (aminomethyl) -2, 2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester 4a (630mg, 2.73mmol, prepared by the method disclosed in patent application "WO 2013020993") was added, and 4mL of 2N sodium hydroxide solution was added and reacted at room temperature for 30 minutes. After the reaction was completed, a saturated ammonium chloride solution was added to adjust the pH of the solution to 9, extraction was performed three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title product 4b (667mg, brown solid) which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):529.1[M-1]

Second step of

4- (((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) methyl) -2, 2-dimethyloxazolidine-3-carboxylic acid tert-butyl ester 4c

Crude 4b (667mg, 1.26mmol) was dissolved in 20mL tetrahydrofuran, N' -carbonyldiimidazole (225mg, 1.38mmol) was added, and the reaction was carried out at 70 ℃ for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid, water and a saturated sodium chloride solution in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give a crude title product 4c (710mg, a tan oily substance) which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):555.1[M-1]

The third step

(1- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -3-hydroxypropan-2-yl) carbamic acid tert-butyl ester 4d

Crude 4c (710mg, 1.28mmol) was dissolved in 20mL of methanol, p-toluenesulfonic acid monohydrate (124mg, 0.65mmol) was added, and the reaction was carried out at room temperature for 12 hours. After the reaction was completed, saturated sodium bicarbonate solution was added to neutralize the reaction, the reaction solution was concentrated under reduced pressure, extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system a to give the title product 4d (310mg, tan oil) in 47% yield.

MS m/z(LC-MS):513.0[M-1]

The fourth step

3- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -2- ((tert-butoxycarbonyl) amino) propyl acetate 4e

4d (310mg, 0.60mmol) was dissolved in 30mL of dichloromethane, 0.18mL of triethylamine was added, cooled to 0 deg.C, acetyl chloride (0.066mL, 0.92mmol) was added dropwise, and the reaction was carried out at 0 deg.C for 1 hour. After the reaction was completed, a saturated sodium bicarbonate solution was added, liquid separation was performed, the organic phase was washed with water and a saturated sodium chloride solution in turn, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 4e (510mg, brown viscous substance) which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):556.0[M-1]

The fifth step

2-amino-3- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) propyl acetate 4f

Crude 4e (300mg, 0.538mmol) was dissolved in 5mL of dichloromethane, 3mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 4f (510mg, tan oil) which was carried on to the next step without purification.

The sixth step

3- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -2- ((N- (tert-butoxycarbonyl) sulfamoyl) amino) propyl acetate 4g

Chlorosulfonyl isocyanate (120mg, 0.848mmol) was dissolved in 10mL of dichloromethane, cooled to 0 ℃, t-butanol (63mg, 0.849mmol) was added, and the reaction mixture was reacted at 0 ℃ for 15 minutes to obtain reaction mixture A. Crude 4f (510mg, 1.11mmol) was dissolved in 10mL of dichloromethane, and 1mL of triethylamine was added to obtain reaction solution B. The reaction mixture A was added to the reaction mixture B at room temperature, and reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was quenched by addition of saturated sodium bicarbonate solution, the organic phase was washed successively with water and saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system a to obtain 4g (238mg, yellow solid) of the title product in 33% yield.

MS m/z(LC-MS):653.0[M+18]

Seventh step

3- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -2- (sulfamoylamino) propyl acetate 4h

4g (238mg, 0.37mmol) was dissolved in 5mL of dichloromethane, 3mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 4h (250mg, light brown viscous) which was directly subjected to the next reaction without purification.

Eighth step

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((3-hydroxy-2- (sulfamoylamino) propyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 4

The crude product was dissolved in 20mL of methanol for 4h (250mg, 0.666mmol), and reacted with potassium carbonate (600mg, 4.35mmol) at 50 ℃ for 1.5 hours. After completion of the reaction, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developer system a to give the title product 4(40mg, pale yellow solid) in 18% yield.

MS m/z(ESI):468.1[M+1]

¹H NMR(400MHz,DMSO-d₆)δ11.47(s,1H),8.87(s,1H),7.12-7.19(m,2H),6.77-6.78(m,2H),6.61(d,1H),6.56(s,1H),6.20-6.22(m,1H),4.82(t,1H),4.02-4.04(m,1H),3.44-3.57(m,3H),3.24-3.26(m,1H).

Example 5

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((3-methoxy-2- (sulfamoylamino) propyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 5

First step of

Tert-butyl (1- ((4- (N- (3-bromo-4-fluorophenyl) -N' -hydroxycarbamimidoyl) -1,2, 5-oxadiazol-3-yl) amino) -3-methoxypropan-2-yl) carbamate 5b

1b (756mg, 2.03mmol) was dissolved in 50mL tetrahydrofuran, tert-butyl (1-amino-3-methoxypropan-2-yl) carbamate 5a (800mg, 3.92mmol, prepared using the method disclosed in patent application "WO 2012002577") was added, 6mL of 2N sodium hydroxide solution was added, and the reaction was carried out at room temperature for 30 minutes. After the reaction was completed, 1N hydrochloric acid was added to adjust the reaction solution to pH <7, extraction was performed three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to give crude title product 5b (1g, yellow solid), which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):475.1 [M+1-28]

Second step of

(1- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -3-methoxypropane-2-ane) carbamic acid tert-butyl ester 5c

Crude 5b (1g, 1.99mmol) was dissolved in 30mL tetrahydrofuran, N' -carbonyldiimidazole (322mg, 1.99mmol) was added, and the reaction was carried out at 70 ℃ for 1 hour. After the reaction was completed, the reaction solution was concentrated under reduced pressure, the residue was dissolved in ethyl acetate, washed with 1N hydrochloric acid, water and a saturated sodium chloride solution in this order, the organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 5c (1g, yellow solid), which was subjected to the next reaction without purification.

The third step

3- (4- ((2-amino-3-methoxypropyl) amino) -1,2, 5-oxadiazol-3-yl) -4- (3-bromo-4-fluorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 5d

Crude 5c (1g, 1.89mmol) was dissolved in 10mL of dichloromethane, 10mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 5d (1.55g, tan oil) which was directly subjected to the next reaction without purification.

MS m/z(LC-MS):429.1[M+1]

The fourth step

N- (1- ((4- (4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) -3-methoxypropan-2-yl) sulfamoylcarbamic acid tert-butyl ester 5e

Chlorosulfonyl isocyanate (400mg, 2.83mmol) was dissolved in 30mL of dichloromethane, cooled to 0 ℃, and tert-butanol (210mg, 2.83mmol) was added to react the reaction mixture at 0 ℃ for 15 minutes to obtain reaction mixture A. Crude 5d (1.55g, 3.6mmol) was dissolved in 20mL of dichloromethane, cooled to 0 deg.C, and 2.5mL of triethylamine was added to obtain reaction solution B. The reaction mixture A was added dropwise to the reaction mixture B at room temperature, and the mixture was reacted at room temperature for 1 hour. After the reaction was completed, the reaction solution was quenched by addition of saturated sodium bicarbonate solution, separated, the organic phase was washed successively with water, saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 5e (1.3g, tan solid) in 59% yield.

MS m/z(LC-MS):625.0[M+18]

The fifth step

N- [1- ({4- [4- (3-bromo-4-fluorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl ] -1,2, 5-oxadiazol-3-yl } amino) -3-methoxypropan-2-yl ] sulfamide 5f

5e (410mg, 0.67mmol) was dissolved in 10mL of dichloromethane, 8mL of trifluoroacetic acid was added, and the reaction was carried out at room temperature for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 5f (574mg, brown oil) which was directly subjected to the next reaction without purification.

The sixth step

N- (3-bromo-4-fluorophenyl) -N' -hydroxy-4- ((3-methoxy-2- (sulfamoylamino) propyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 5

Crude 5f (574mg, 1.129mmol) was dissolved in 10mL tetrahydrofuran, and 3mL of 2N sodium hydroxide solution was added and reacted at room temperature for 15 minutes. After the reaction was completed, saturated ammonium chloride solution was added to neutralize the reaction, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developer system a to give the title product 5(150mg, white solid) in 27% yield.

MS m/z(ESI):484.1[M+1]

¹H NMR(400MHz,DMSO-d₆)δ11.45(s,1H),8.88(s,1H),7.12-7.19(m,2H),6.74-6.78(m,2H),6.59(s,2H),6.20-6.2(m,1H),3.61-3.63(m,1H),3.43-3.46(m,2H),3.25-3.36(m,2H),3.28(s,3H).

Example 6

N- (3-chlorophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 6

First step of

4-amino-N- (3-chlorophenyl) -N' -hydroxy-1, 2, 5-oxadiazole-3-carboxamidine 6b

3-chloroaniline (4.7g, 36.8mmol) was dissolved in 100mL of ethanol, 50mL of a prepared ethanol suspension of 4-amino-N-hydroxy 1,2, 5-oxadiazole-3-carbodiimido chloride 6a (5g, 30.8mmol, prepared by the well-known method "Journal of the American Chemical Society,2015,137(33), 10532-. After the reaction was completed, the reaction solution was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 6B (6.0g, pale yellow solid) in a yield of 76.9%.

Second step of

3- (4-amino-1, 2, 5-oxadiazol-3-yl) -4- (3-chlorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 6c

6b (5g, 19mmol) was dissolved in 100mL of tetrahydrofuran, and N, N' -carbonyldiimidazole (3.2g, 19mmol) was added to the solution to react at 70 ℃ for 1 hour. After the reaction, the reaction solution was cooled to room temperature, concentrated under reduced pressure, added with ethyl acetate and 1N hydrochloric acid, separated, the organic phase washed with saturated sodium chloride solution, dried with anhydrous sodium sulfate, filtered to remove the drying agent, the filtrate concentrated under reduced pressure, and slurried with ethyl acetate and N-hexane to give crude title product 6c (3.7g, pale yellowish brown solid), which was directly subjected to the next reaction without purification.

The third step

4- (3-chlorophenyl) -3- (4-nitro-1, 2, 5-oxadiazol-3-yl) -1,2, 4-oxadiazol-5 (4H) -one 6d

Crude 6c (800mg, 2.86mmol) was dissolved in 15mL of trifluoroacetic acid, and 7mL of hydrogen peroxide (30%) was added and reacted at 50 ℃ for 12 hours. After the reaction was completed, ethyl acetate was added, a saturated sodium sulfite solution was added, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered to remove the drying agent, concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 6d (320mg, pale yellow solid) in a yield of 36%.

The fourth step

(1- (2- ((4- (4- (3-chlorophenyl) -5-carbonyl-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) ethyl) cyclopropyl) carbamic acid tert-butyl ester 6e

6d (320mg, 1.03mmol) and 3a (227mg, 1.135mmol) were dissolved in 20mL of tetrahydrofuran, and 1.1mL of 1N sodium hydroxide solution was added to stir the reaction for 30 minutes. After the reaction was completed, 1N hydrochloric acid was added to adjust the pH of the reaction solution to less than 7, ethyl acetate was added, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude title product 6e (540mg, pale yellow solid), which was subjected to the next reaction without purification.

The fifth step

3- (4- ((2- (1-aminocyclopropyl) ethyl) amino) -1,2, 5-oxadiazol-3-yl) -4- (3-chlorophenyl) -1,2, 4-oxadiazol-5 (4H) -one 6f

Crude 6e (540mg, 1.17mmol) was dissolved in 15mL of dichloromethane and 10mL of trifluoroacetic acid was added and the reaction stirred for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 6f (900mg, brown oil) which was directly used in the next reaction without purification.

The sixth step

6g of tert-butyl N- (1- (2- ((4- (4- (3-chlorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl) -1,2, 5-oxadiazol-3-yl) amino) ethyl) cyclopropyl) sulfamoylcarbamate

Chlorosulfonyl isocyanate (215mg, 1.52mmol) was dissolved in 15mL of dichloromethane, cooled to 0 ℃, t-butanol (113mg, 1.52mmol) was added, and the reaction mixture was reacted at 0 ℃ for 15 minutes to obtain reaction mixture A. Crude 6f (900mg, 2.48mmol) was dissolved in 30mL of dichloromethane, and 1mL of triethylamine was added to prepare reaction solution B. The reaction mixture A was added to the reaction mixture B at 0 ℃ and reacted for 1 hour. After the reaction was completed, the reaction was quenched by addition of saturated sodium bicarbonate solution, liquid separation, washing of the organic phase with water successively, washing with saturated sodium chloride solution, drying over anhydrous sodium sulfate, filtration, concentration of the filtrate under reduced pressure, and purification of the resulting residue by silica gel column chromatography with eluent system B to obtain 6g (270mg, white solid) of the title product in 20% yield.

Seventh step

N- {1- [2- ({4- [4- (3-chlorophenyl) -5-oxo-4, 5-dihydro-1, 2, 4-oxadiazol-3-yl ] -1,2, 5-oxadiazol-3-yl } amino) ethyl ] cyclopropyl } aminosulfonamide 6h

6g (270mg, 0.498mmol) was dissolved in 10mL of dichloromethane, 5mL of trifluoroacetic acid was added, and the reaction was stirred for 1 hour. After the reaction was complete, the reaction was concentrated under reduced pressure to give the crude title product 6h (264mg, pale yellow solid) which was directly subjected to the next reaction without purification.

Eighth step

N- (3-chlorophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 6

The crude 6h (264mg, 0.597mmol) was dissolved in 30mL tetrahydrofuran, 1.2mL of 2.5M sodium hydroxide solution was added and the reaction stirred for 15 min. After the reaction was completed, a saturated ammonium chloride solution was added, liquid separation was performed, the aqueous phase was extracted three times with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with eluent system B to obtain the title product 6(50mg, white solid) in a yield of 20%.

MS m/z(ESI):414.3[M+1]

¹H NMR(400MHz,CD₃OD)δ7.17(t,1H),6.97-6.99(m,1H),6.86(t,1H),6.74-6.76(m,1H),3.56(t,2H),1.93(t,2H),1.00-1.03(m,2H),0.59-0.62(m,2H).

Example 7

N- (4-fluoro-3- (trifluoromethyl) phenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 7

Using the synthetic route from example 6, the starting material from the first step was replaced with 6a and 4-fluoro-3- (trifluoromethyl) aniline to give the title product 7(170mg, white solid).

MS m/z(ESI):468.3[M+1]

¹H NMR(400MHz,CD₃OD)δ7.14-7.20(m,2H),7.10-7.12(m,1H),3.56(t,2H),1.94(t,2H),1.00-1.03(m,2H),0.60-0.63(m,2H).

Example 8

N- (4-fluoro-3-methylphenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 8

Using the synthetic route from example 6, the starting material from the first step was replaced with 6a and 4-fluoro-3-methylaniline to give the title product 8(150mg, white solid).

MS m/z(ESI):414.3[M+1]

¹H NMR(400MHz,CD₃OD)δ6.87(t,1H),6.78-6.81(m,1H),6.68-6.72(m,1H),3.53(d,2H),2.20(d,3H),1.91(t,2H),0.99-1.02(m,2H),0.58-0.61(m,2H).

Example 9

N- (3-bromophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 9

Using the synthetic route to example 6, the starting material from the first step was replaced with 6a and 3-bromoaniline to afford the title product 9(50mg, white solid).

MS m/z(ESI):462.2[M+1]

¹H NMR(400MHz,CD₃OD)δ7.11-7.12(m,2H),7.01-7.02(m,1H),6.78-6.80(m,1H),3.55(t,2H),1.93(t,2H),1.00-1.03(m,2H),0.59-0.62(m,2H).

Example 10

N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -N- (m-tolyl) -1,2, 5-oxadiazole-3-carboxamidine 10

Using the synthetic route to example 6, the starting materials from the first step were replaced with 6a and m-toluidine to give the title product 10(250mg, white solid).

MS m/z(ESI):396.3[M+1]

¹H NMR(400MHz,CD₃OD)δ7.08(t,1H),6.84(d,1H),6.64(s,1H),6.63(d,1H),3.52(t,2H),2.27(s,3H),1.90(t,2H),0.98-1.01(m,2H),0.57-0.60(m,2H).

Example 11

N- (3-chloro-4-fluorophenyl) -N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -1,2, 5-oxadiazole-3-carboxamidine 11

Using the synthetic route from example 6, the starting material from the first step was replaced with 6a and 3-chloro-4-fluoroaniline, and the title product 11(30mg, brown solid) was obtained.

MS m/z(ESI):434.2[M+1]

¹H NMR(400MHz,CD₃OD)δ7.08(t,1H),6.98-6.99(m,1H),6.80-6.82(m,1H),3.55(t,2H),1.93(t,2H),1.01(s,2H),0.61(s,2H).

Example 12

N' -hydroxy-4- ((2- (1- (sulfamoylamino) cyclopropyl) ethyl) amino) -N- (3- (trifluoromethyl) phenyl) -1,2, 5-oxadiazole-3-carboxamidine 12

Using the synthetic route to example 6, the starting material from the first step was replaced with 6a and 3- (trifluoromethyl) aniline to give the title product 12(105mg, white solid).

MS m/z(ESI):450.3[M+1]

¹H NMR(400MHz,CD₃OD)δ7.38(t,1H),7.25(d,1H),7.10(s,1H),7.05(d,1H),3.57(t,2H),1.94(t,2H),1.00-1.03(m,2H),0.59-0.62(m,2H).

Biological evaluation

The present invention is further described and explained below in conjunction with test examples, which are not intended to limit the scope of the present invention.

Test example 1:

1. determination of inhibitory Activity of Compounds of the present invention against human IDO1 protease

In vitro human IDO1 protease activity was tested by the following method.

This method was used to determine the inhibitory effect of the compounds of the present invention on the activity of human IDO1 protease.

First, experimental material and instrument

1. Enzyme mark instrument (Synergy HT, BIOTEK)

2. Tryptophan (T0254-5G, Sigma-Aldrich)

3. Catalase was derived from bovine liver (C1345-1G, Sigma-Aldrich)

4. Methylene blue (M9140-25G, Sigma-Aldrich)

5. L-ascorbic acid sodium salt (A7631-25G, Sigma-Aldrich)

6. 4- (dimethylamino) benzaldehyde (D2004-25G, Sigma-Aldrich)

7. Trichloroacetic acid (T9159-100G, Aigma-Aldrich)

8. IDO1 gene (SC126221, Origene)

9. Rossetta (CW0811A, Beijing kang is century Biotechnology Co., Ltd.)

10. Vortex mixer (6776, Corning)

11. Mini plate centrifuge (mini-p25, ABSON life science equipment)

Second, the experimental procedure

Homemade IDO1 protease

Transferring the IDO1 gene into a PET30a plasmid by a gene cloning technology, and then transferring into competent escherichia coli rosseta; culturing in liquid LB (Luria-Bertani) culture medium (per liter of culture medium prepared according to the molecular cloning Experimental guidelines, J. SammBruker D.W. Lassel), collecting the thallus, ultrasonicating, passing through a hanging column, and eluting to obtain purified IDO1 protease.

Compound testing experiments:

100ul of the enzyme (IDO1) was diluted 30-fold with 50mM KPB to 3ml of a 26ng/ul enzyme solution, and 24ul of the enzyme solution was added to each well of a 96-well reaction plate (AXYGEN, PCR-96-FLT-C) (hereinafter referred to as a reaction plate). Blank wells were loaded with 24ul KPB buffer [ preparation of KPB buffer (50 mM): weighing KH with analytical balance₂PO₄6.805g is put into a 1000ml beaker, deionized water is added into a measuring cylinder to reach 900ml, the pH value is adjusted to 6.5 by using 1M KOH, and the mixture is led into a 1L measuring cylinder and is added with water to reach 1L. Storage at 4 deg.C]. 1ul of compound or DMSO was added to the corresponding well in the reaction plate. Preparing a solution A: 250ul of 500mM L-sodium ascorbate was added to 1437ul of KPB and mixed by vortexing for 3 seconds at maximum speed. And B, liquid B: 125ul of 10mM tryptophan acid plus 100ul of 100000 units/ml catalase, 5ul of 10mM methylene blue, and finally 1437ul of KPB, vortex mixer maximum speed mixing for 3 seconds. 1500ul of liquid A and 1500ul of liquid B were mixed together at maximum speed for 3 seconds at votex. This mixture was then added to the reaction plate at 24ul per well. Placing the reaction plate in a plate centrifuge at the highest speedCentrifuging for 15 seconds to ensure that the reaction liquid is gathered at the bottom, uniformly mixing for 30 seconds by using an oscillator, and incubating for 1h at 37 ℃ in a constant-temperature incubator. In the reaction plate, 10ul of 30% (W/V) trichloroacetic acid was added to each well, and incubated at 65 ℃ for 15 minutes in an incubator. The reaction plate was centrifuged at 4700RPM on a centrifuge at room temperature for 5 minutes. 40ul of supernatant was transferred from the reaction plate to a corresponding 96-well test plate (corning, #3599) using a line gun. 40ul of 2% (W/V) 4- (dimethylamino) benzaldehyde/glacial acetic acid solution was added to each well and mixed well for 1 min at maximum speed on a shaker. After incubation for 2 min at room temperature, the absorbance at 480nm was read on Synergy HT (BIOTEK).

The inhibitory activity of the compound of the present invention against human IDO1 protease was determined by the above assay, and the IC was determined₅₀The values are shown in Table 1.

TABLE 1 inhibition of human IDO1 protease Activity IC by the Compounds of the invention₅₀

Example numbering	IC50(nM)
2	78
3	18

4	57
5	68
6	28
7	98
8	15
9	9
11	77
12	70

And (4) conclusion: the compound of the invention has obvious inhibition effect on the activity of human IDO1 protease.

Test example 2 determination of IDO protease inhibitory Activity of Compounds of the present invention on Hela's intracellular IDO protease

The IDO protease activity in Hela cells was tested by the following method.

This method was used to determine the inhibitory effect of the compounds of the invention on the activity of IDO protease in Hela cells. (Note: HeLa cell line expresses indoleamine 2,3-dioxygenase (IDO) under induction of interferon gamma (INF-. gamma.))

First, experimental material and instrument

1. Enzyme mark instrument (Synergy HT, BIOTEK)

2. Tryptophan (T0254-5G, Sigma-Aldrich)

3. 4- (dimethylamino) benzaldehyde (D2004-25G, Sigma-Aldrich)

4. Trichloroacetic acid (T9159-100G, Aigma-Aldrich)

5. HeLa cell line (CCL-2, ATCC)

Second, the experimental procedure

HeLa cell suspension was prepared from fresh cell culture medium, 10000 cells/well were added to a 96-well cell culture plate of 100ul culture system, and cultured with 5% carbon dioxide at 37 ℃ for 24 hours. Removing supernatant, and adding 90ul of serum-free DMEM high-sugar culture medium into each well; then 10ul of the compound (final concentration: 100000, 10000, 1000, 100, 10, 1nM) formulated in INF-gamma and tryptophan-containing medium was added to each well, cultured with 48 hours 5% carbon dioxide at 37 ℃ and 80ul of the supernatant from the 96-well cell culture plate was taken to the 96-well round bottom plate, 16ul of 30% (W/V) trichloroacetic acid was added to each well, and incubated at 65 ℃ for 25 minutes in an incubator. The reaction plate was centrifuged at 4700RPM for 5 minutes in a centrifuge. Transfer 50ul of supernatant from the reaction plate to a 96-well flat-bottom transparent plate using a calandria, add 50ul of 2% (W/V) 4- (dimethylamino) benzaldehyde/glacial acetic acid solution per well, and mix well on a shaker for 1 minute. After incubation for 2 min at room temperature, the absorbance at 480nm was read on a Synergy HT Reader.

The inhibitory activity of the compound of the present invention against the IDO protease in Hela cells was determined by the above assay, and the IC was determined₅₀The values are shown in Table 2.

TABLE 2 inhibition of IDO protease Activity IC in Hela cells by the Compounds of the invention₅₀

Example numbering	IC50(nM)
1	73

3	3
4	16
5	73
6	7
7	24
8	6
9	5
10	59
11	14
12	15

And (4) conclusion: the compound of the invention has obvious inhibition effect on the activity of IDO protease in Hela cells.

Pharmacokinetic evaluation

Test example 3, pharmacokinetic testing of Compounds of examples 3, 6 and 8 of the invention

1. Abstract

With SD rats as test animals, the drug concentrations in plasma were determined at various times after intravenous administration of the compounds of examples 3, 6 and 8 to the rats by LC/MS/MS method. The pharmacokinetic behavior of the compounds of the invention in rats was studied and their pharmacokinetic profile was evaluated.

2. Test protocol

2.1 test drugs

Compounds of examples 3, 6 and 8

2.2 test animals

Healthy adult SD rats 12, male and female halves, purchased from shanghai sipel-bikeka laboratory animals ltd, animal production license number: SCXK (Shanghai) 2008-0016.

2.3 pharmaceutical formulation

Weighing appropriate amount of the medicine, adding 0.3ml of dimethyl acetamide to dissolve, adding 10% of 2-hydroxypropyl-beta-cyclodextrin to the final volume, and performing ultrasonic treatment to obtain 2.0mg/ml suspension.

2.4 administration

12 SD rats with half of male and female, which are divided into 3 groups on average; after fasting overnight, the administration was performed by gavage with a volume of 10 ml/kg.

3. Operation of

The intragastric administration group collected blood of 0.1ml from orbit before and after administration at 0.5, 1.0, 2.0, 4.0, 6.0, 8.0, 11.0, and 24.0h, placed in heparinized test tube, centrifuged at 3500rpm for 10min to separate plasma, stored at-20 deg.C, and taken 2h after administration.

The LC/MS/MS method is used for measuring the content of the compound to be measured in the plasma of rats after the intragastric administration of different compounds.

4. Pharmacokinetic parameter results

TABLE 3 pharmacokinetic parameters of the compounds of examples 3, 6 and 8 of the invention

And (4) conclusion: the compound of the invention has good drug absorption and obvious drug absorption effect.

Claims (16)

1. A compound of formula (I) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

   

   wherein:

   

   selected from the cis isomer, trans isomer or a mixture of cis and trans isomers;

   R¹selected from the group consisting of alkyl, cycloalkyl and alkoxy, wherein said alkyl, cycloalkyl, alkoxy are each independently optionally further substituted with one or more substituents selected from the group consisting of hydroxy, alkyl, alkoxy and haloalkyl;

   R²selected from the group consisting of a hydrogen atom, an alkyl group, a cycloalkyl group and an alkoxy group, wherein said alkyl group, cycloalkyl group and alkoxy group are each independently optionally further substituted with one or more substituents selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group and a haloalkyl group;

   or R¹And R²Together with the carbon atom to which they are attached form a cycloalkyl or heterocyclyl group, wherein said cycloalkyl, heterocyclyl group is optionally further selected from the group consisting of alkyl, halogen, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, -NR⁶R⁷、-C(O)NR⁶R⁷、-NHS(O)_mR⁸、-S(O)_pR⁸、-NHC(O)R⁸、-OR⁸、-C(O)R⁸、-OC(O)R⁸and-C (O) OR⁸Is substituted with one or more substituents of (1);

   R³and R⁴Each independently selected from the group consisting of hydrogen, alkyl, cyano, amino, halogen, alkenyl, alkynyl, hydroxyl, nitro, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl each independently optionally is further selected from the group consisting of hydroxyl, halogen, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroarylSubstituted with one or more substituents of (a);

   R⁵selected from the group consisting of alkyl and amino, said alkyl and amino optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halo, amino, cyano, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl; the alkyl group is preferably C_1～4An alkyl group;

   R⁶and R⁷Each independently selected from the group consisting of hydrogen, alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, wherein said alkyl, cycloalkyl, heterocyclyl, aryl or heteroaryl are each independently optionally further substituted with one or more substituents selected from the group consisting of hydroxy, halogen, alkyl, alkoxy, cycloalkyl, heterocyclyl, aryl and heteroaryl;

   R⁸selected from the group consisting of hydrogen atoms, alkyl groups, amino groups, cycloalkyl groups, heterocyclic groups, aryl groups, and heteroaryl groups, wherein said alkyl, cycloalkyl, heterocyclic, aryl, or heteroaryl groups are each independently optionally further substituted with one or more substituents selected from the group consisting of alkyl, halogen, hydroxy, amino, cyano, alkoxy, cycloalkyl, heterocyclic, aryl, and heteroaryl groups;

   m is 0, 1, 2;

   n is 0, 1, 2; and is

   p is 0, 1, 2.
2. The compound of formula (I) according to claim 1, wherein R is in the form of its tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof³And R⁴Each independently selected from a hydrogen atom, a halogen, preferably fluorine, chlorine or bromine, an alkyl, preferably C, or a haloalkyl_1～4Preferably, the haloalkyl group is trifluoromethyl.
3. The compound of the general formula (I) according to claim 1 or 2, which is a compound of the general formula (II) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

   

   wherein:

   R¹is alkyl or alkoxy, said alkyl or alkoxy being further substituted by one or more hydroxy or alkoxy groups; preferably R¹Is alkyl substituted by hydroxy or alkoxy;

   

   R³～R⁵m, n are as defined in claim 1.
4. A compound of formula (I) according to claim 3, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, wherein m is 0 and/or n is 1.
5. The compound of the general formula (I) according to claim 1 or 2, which is a compound of the general formula (III) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

   

   wherein:

   a is a 3-8 membered ring, wherein the ring is selected from a carbocyclic ring or a heterocyclic ring;

   

   R³～R⁵m, n are as defined in claim 1.
6. The compound of formula (I) according to claim 5, wherein a is cycloalkyl, preferably cyclopropyl, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.
7. The compound of formula (I) according to claim 6, wherein m is 0 or 1 and/or n is 1 or 2, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof.
8. The compound of formula (I) according to any one of claims 1 to 7, wherein the compound is selected from the group consisting of:

   
9. a process for the preparation of a compound of formula (I) according to claim 1 or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising:

   

   the compound of the general formula (IV) is subjected to ring opening under alkaline conditions to obtain a compound of a general formula (I);

   wherein:

   R¹～R⁵m, n are as defined in claim 1.
10. A compound of formula (IV) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

    

    wherein:

    R¹～R⁵m, n are as defined in claim 1.
11. The compound of formula (IV) according to claim 10, which is a compound of formula (V) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, according to formula (IV):

    

    wherein: r¹、R³～R⁵M, n are as defined in claim 10.
12. The compound of formula (IV) according to claim 10, which is a compound of formula (VI) or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, wherein:

    

    wherein:

    a is a 3-8 membered ring, wherein the ring is selected from a carbocyclic ring or a heterocyclic ring;

    R³～R⁵m, n are as defined in claim 10.
13. A pharmaceutical composition comprising a therapeutically effective amount of a compound of formula (I) according to any one of claims 1 to 8, or a tautomer, mesomer, racemate, enantiomer, or diastereomer thereof, or a mixture thereof, or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.
14. Use of a compound of general formula (I) according to any one of claims 1 to 8, or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, or a composition according to claim 13, for the preparation of a medicament for the prevention and/or treatment of a disease characterized by the pathology of an IDO-mediated tryptophan metabolic pathway.
15. The use according to claim 14, wherein the disease having pathological features of IDO mediated tryptophan metabolic pathway is selected from the group consisting of cancer, myelodysplastic syndrome, alzheimer's disease, autoimmune diseases, depression, anxiety, cataracts, psychological disorders and aids.
16. The use of claim 15, wherein the cancer is selected from the group consisting of breast cancer, cervical cancer, colon cancer, lung cancer, gastric cancer, rectal cancer, pancreatic cancer, brain cancer, skin cancer, oral cancer, prostate cancer, bone cancer, kidney cancer, ovarian cancer, bladder cancer, liver cancer, fallopian tube tumor, ovarian tumor, peritoneal tumor, stage IV melanoma, glioma, glioblastoma, hepatocellular carcinoma, papillary renal tumor, head and neck tumor, leukemia, lymphoma, myeloma, and non-small cell lung cancer.