Patent Application
20250025438
The present invention relates to the field of biomedicine, specifically to a derivative of aspartic acid and use thereof in the treatment of liver fibrosis, non-alcoholic hepatitis and other metabolic diseases.
Fibrosis can occur in a variety of tissues and organs, its main pathological changes are the increase of fibrous connective tissue, the decrease of parenchymal cells in organ tissues, and its continued progression can cause organ structural damage and functional decline, or even failure, thereby seriously threatening human health and life. Worldwide, tissue fibrosis is the main cause of disability and death in many diseases. According to relevant statistics of the United States, nearly 45% of patients who die from various diseases in the United States can be attributed to diseases associated with tissue fibrogenesis.
Liver fibrosis is especially common in patients with tissue fibrosis. It is a reversible pathological phenomenon in which fibrous connective tissue is excessively deposited in the liver tissue during the body's repair process after liver damage. There are many causes of liver fibrosis. Patients with various chronic viral liver diseases are at high risk of developing liver fibrosis and cirrhosis. Alcoholics or long-term drinkers develop fatty liver in the early stage and can develop liver fibrosis and liver cirrhosis in the later stage, while other fatty livers caused by non-alcoholic factors such as obesity can also develop into liver fibrosis and liver cirrhosis; in addition, repeated infection with schistosomiasis can easily cause portal liver fibrosis; chronic cholestasis can produce biliary liver fibrosis; hepatolenticular degeneration and hemoglobin deposition can produce metabolic liver fibrosis; various toxic substances can cause toxic liver fibrosis; people who like low-protein diets and fatty fried foods can develop malnutrition-induced liver fibrosis; patients with chronic congestive heart failure can develop cardiogenic liver fibrosis. In addition, liver fibrosis is also an important pathological diagnostic indicator of non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH).
The mechanism of occurrence and development of liver fibrosis is very complex. Current research mainly focuses on the activation and transformation of hepatic stellate cells. The possible pathways are the activation of signal transduction pathways mediated by transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), tumor necrosis factor α (TNF-α), etc., due to various chronic stimuli, and the activation of hepatic stellate cells by prostaglandin cyclooxygenase-2 (COX-2), diffuse extracellular matrix (ECM) and oxidative stress, so that they are converted into myofibroblasts and fibroblasts, resulting in increased secretion or decreased degradation of extracellular matrix, thereby forming liver fibrosis. Since the mechanism of occurrence and development of liver fibrosis is not yet clear, the development of drugs to treat liver fibrosis has been relatively slow.
Currently, in the clinical treatment of liver fibrosis or cirrhosis caused by viral hepatitis (mainly hepatitis B or hepatitis C), nucleoside (acid) analogues or interferons are mainly used for antiviral treatment. By inhibiting viral replication, the response of inflammatory factors is controlled, and the progression of liver fibrosis or cirrhosis is slowed down. However, there is no effective treatment for liver fibrosis or cirrhosis caused by other factors such as alcohol, metabolism, drugs, etc., to which auxiliary treatment with traditional Chinese medicine or ready-prepared Chinese medicines is mainly applied.
Through suppression of subtractive hybridization and bioinformatics methods, NS3TP1 (HCV nonstructural protein 3-transactivated protein 1, NS3TP1) was screened and cloned for the first time, which is also named ASNSD1 (asparagine synthetase domain containing 1, ASNSD1), has registration number AY11696 in GenBank, and locates on human chromosome 2q32.2. The total length of the coding sequence of this gene is 1,932 nucleotides, and the coding product consists of 643 amino acid residues. NS3TP1 is widely distributed in the body, mainly in hepatocytes and gallbladder gland epithelial cells in the liver. Janine Meienberg et al. found through MLPA analysis that complete COL3A1 (encoding collagen III, a major component of liver fibrosis extracellular matrix deposition) hemizygous deletion affects the expression of ASNSD1, indicating that the expression of ASNSD1 may interact with COL3A1. Therefore, the development of NS3TP1 modulators can be a direction for the development of new drugs for NAFLD.
The purpose of the present invention is to obtain a compound having regulatory effect on NS3TP1, and to obtain a compound that having therapeutic effect on liver fibrosis and non-alcoholic fatty liver disease. The inventors have discovered through creative research that the aspartic acid derivatives of the present invention have therapeutic effects on liver fibrosis and non-alcoholic fatty liver disease, and these molecules have good pharmaceutical properties (e.g., solubility, AUC and/or oral bioavailability and other properties) and safety.
To this end, in the first aspect of the present invention, the present invention provides a use of a compound represented by the following Formula I, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, in the manufacture of a medicament or agent, and the medicament is used for at least one of the following:
A-(L1)x-A′ (Formula I)
A and A′ are the same or different;
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 1 or 2, A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 1 or 2 (or x is 1), A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
CH3—NH—, HOCOCH2NH—, —OCH(CH3)2, —CH3,
wherein, Trt represents trityl.
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
—OCH(CH3)2,
R3 and R4 are each independently selected from the group consisting of: hydrogen, hydroxyl,
—COCH2CH3, —COCH(CH3)2, —COCH(CH2)2, —COOCH3, —CON(CH3)2, —COCH(CH3)(NH2); wherein, Boc represents tert-butoxycarbonyl, Cbz represents benzyloxycarbonyl, and Fmoc represents fluorenylmethoxycarbonyl.
In some embodiments, in the above Formula I, when x is 1, A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 1, A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 2, A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 2, A is the same as or different from A′, wherein
In some embodiments, in the above Formula I, when x is 2, A is the same as or different from A′, wherein
In some embodiments, the present invention provides a use of a compound represented by the following Formula I, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, in the manufacture of a medicament or agent, and the medicament is used for at least one of the following:
A-(L1)x-A′ (Formula I)
A and A′ are the same or different;
In some embodiments, in the above Formula I, when x is 0, A′ is absent; for A, wherein:
In some embodiments, in the above Formula I, when x is 1, A is the same as or different from A′, wherein:
any one of R1 and R2 of a unit A is connected to any one of R1′ and R2′ of an adjacent unit A′ through L1;
In some embodiments, in the above Formula I, when x is 1, A is the same as or different from A′, wherein:
for A, wherein:
In some embodiments, in the above Formula I, when x is 1, A is the same as or different from A′, wherein:
In some embodiments, in the above Formula I, when x is 2, A is the same as or different from A′, wherein:
In some embodiments, in the above Formula I, when x is 2, A is the same as or different from A′, wherein:
In the second aspect of the present invention, the present invention provides a use of a compound represented by the following Formula I-4, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, in the manufacture of a medicament or agent, and the medicament is used for at least one of the following:
HO-p-Ph-CH2—CH(COORm)—, HO—CH2—CH(NH2)—C(═O)—, CH3—S—(CH2)2—CH(NH2)—C(═O)—, HN═C(NH2)—NH—(CH2)3—CH(NH2)—C(═O)—, (CH3)2CH—CH2—CH(NH2)—C(═O)—; wherein Ph represents phenyl, HO-p-Ph-CH2—CH(NH2)—C(═O)- represents p-hydroxybenzylaminomethylcarbonyl;
HO-p-Ph-CH2—CH(COORm)—, HO—CH2—CH(COORm)—, CH3—S—(CH2)2—CH(COORm)—, HN═C(NH2)—NH—(CH2)3—CH(COORm)—, (CH3)2CH—CH2—CH(COORm)—;
In some embodiments, in the above Formula I-4, Rn1 and Rn2 are each independently selected from the group consisting of: C1-C6 alkyl, —O—Ra4, —O-L2-O—Ra4, —NRa5Ra6;
HO-p-Ph-CH2—CH(NH2)—C(═O)—, HO—CH2—CH(NH2)—C(═O)—, CH3—S—(CH2)2—CH(NH2)—C(═O)—, HN═C(NH2)—NH—(CH2)3—CH(NH2)—C(═O)—, (CH3)2CH—CH2—CH(NH2)—C(═O)—;
HO-p-Ph-CH2—CH(COORm)—, HO—CH2—CH(COORm)—, CH3—S—(CH2)2—CH(COORm)—, HN═C(NH2)—NH—(CH2)3—CH(COORm)—, (CH3)2CH—CH2—CH(COORm)—;
In some embodiments, in the above Formula I-4, Rn1 and Rn2 are each independently selected from the group consisting of: C1-C6 alkyl, —O—Ra4, —O-L2—O—Ra4, —NRa5Ra6;
HO-p-Ph-CH2—CH(NH2)—C(═O)—, HO—CH2—CH(NH2)—C(═O)—, HN═C(NH2)—NH—(CH2)3—CH(NH2)—C(═O)—, CH3—S—(CH2)2—CH(NH2)—C(═O)—, (CH3)2CH—CH2—CH(NH2)—C(═O)—;
HO-p-Ph-CH2—CH(COORm)—, HO—CH2—CH(COORm)—, CH3—S—(CH2)2—CH(COORm)—, HN═C(NH2)—NH—(CH2)3—CH(COORm)—, (CH3)2CH—CH2—CH(COORm)—;
In some embodiments of the first and second aspects of the present invention, the compound represented by Formula I or I-1, I-2, I-3 or I-4 is selected from the group consisting of the following:
In some embodiments of the first and second aspects of the present invention, the compound represented by Formula I or I-1, 1-2, 1-3 or 1-4 is selected from the group consisting of the following:
In the third aspect of the present invention, the present invention provides a use of a peptide containing 2 to 10 or 2 to 3 amino acids, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, in the manufacture of a medicament or agent, the drug or agent is used for at least one of the following:
In the fourth aspect of the present invention, the present invention provides the compound represented by the above Formula I, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotope compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, or the compound represented by the above Formula I-4, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvent, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, or the peptide containing 2 to 10 or 2 to 3 amino acids, or pharmaceutical above acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, as described in the third aspect above,
In a fifth aspect of the present invention, the present invention provides a method for obtaining the following effects:
In the above-described first to fifth aspects of the present invention,
In the sixth aspect of the present invention, the present invention provides a use of a pharmaceutical composition in the manufacture of a medicament, in which the pharmaceutical composition comprising the compound represented by the above Formula I, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotope compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, or the compound represented by the above Formula I-4, or pharmaceutically acceptable salt, ester, prodrug, stereoisomer, hydrate, solvent, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, or the peptide containing 2 to 10 or 2 to 3 amino acids, or pharmaceutical above acceptable salt, ester, prodrug, stereoisomer, hydrate, solvate, isotopic compound, crystalline form, metabolite form thereof, or any combination or mixture thereof, as described in the third aspect above, and the medicament is used for at least one of the following:
In a seventh aspect of the present invention, the present invention provides a pharmaceutical composition, which is used for at least one of the following:
In an eighth aspect of the present invention, the present invention provides a method for obtaining the following effects:
In the above-mentioned sixth to eighth aspects of the present invention, in some embodiments, the liver fibrosis includes, but is not limited to: liver fibrosis caused by chronic viral liver diseases, liver fibrosis caused by alcoholism or long-term drinking, liver fibrosis caused by non-alcoholic factor such as obesity, portal liver fibrosis caused by repeated schistosomiasis infection, biliary liver fibrosis caused by chronic cholestasis, metabolic liver fibrosis caused by hepatolenticular degeneration and hemoglobin deposition, toxic liver fibrosis caused by various toxic substances, malnutrition-induced liver fibrosis caused by a preference for low-protein diets and fatty fried foods, and cardiogenic liver fibrosis caused by chronic congestive heart failure.
In some embodiments, the pharmaceutical composition further comprises an additional active ingredient: other amino acids for improving liver function (including but not limited to alanine, glutamic acid, cystine, glutamine, glycine, histidine, tyrosine, serine, methionine, arginine, leucine), cholesterol absorption inhibitors (e.g., Ezetimibe), HSC activation and proliferation inhibitors (e.g., Pirfenidone, Fluorofenidone, Pegbelfermin), PCSK9 inhibitors, PPAR agonists (e.g., Gemfibrozil, Fenofibrate, Clofibrate, Bezafibrate, Pemafibrate, Elafibranor), ACE inhibitors, CCR2/5 inhibitors, TLR4 inhibition agents, LOXL2 inhibitors, TIMP-1 inhibitors, FXR agonists, AT1R blockers, NOX inhibitors, calcium channel blockers, ARBs, diuretics, renin, GLP-1 or synthetic variants thereof, insulin or synthetic variants thereof, metformin, sulfonylurea compounds, thiazolidinediones (TZD), SGLT2 inhibitors, DPP-IV inhibitors, inhibitors of HMGCoA reductase, inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), Gemcabene (CI-1027), ACC inhibitors, ApoC-III inhibitors, ACL-inhibitors (e.g., bepedic acid), prescription fish oil, CETP inhibitors, ursodeoxycholic acid, obeticholic acid, polyene phosphatidylcholine, glucocorticoids, silymarin, glycyrrhizic acid preparations (e.g., magnesium isoglycyrrhizinate injection and diammonium glycyrrhizinate enteric-coated capsules), and combinations thereof.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments, the pharmaceutical composition is a solid preparation, an injection, a topical preparation, a spray, a liquid preparation or a compound preparation.
In the ninth aspect of the present invention, the present invention provides a compound represented by Formula II, pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate, crystalline form, and metabolism form thereof,
A1-(L1)x-A1′ Formula II
A1 and A1′ are the same or different;
hydroxyl, methoxy, and at the same time, R3 and R4 are independently selected from the group consisting of: hydrogen, —COCH2CH3 and —COCH(CH3)(NH2),
or one of R3 and R4 is-COCH(CH3)(NH2);
hydroxyl, methoxy,
hydroxyl,
methoxy,
or one of R3 and R4 is-COCH(CH3)(NH2), or
or one of R3 and R4 is —COCH(CH3)(NH2);
In some embodiments, the compound represented by Formula II or II-1, II-2, II-3 is selected from the group consisting of the following:
In the tenth aspect of the present invention, the present invention provides a pharmaceutical composition, which comprises the compound represented by the above Formula II, pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, and solvate, crystalline form, metabolite form thereof, or any combination or mixture thereof.
In the eleventh aspect of the present invention, the present invention provides a use of the compound represented by the above Formula II, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate, solvate, crystalline form, metabolism thereof, or any combination or mixture thereof, in the manufacture of a medicament or agent, the medicament is used for at least one of the following:
In a twelfth aspect of the present invention, the present invention provides a use of a pharmaceutical composition in the manufacture of a medicament or agent, the medicament is used for at least one of the following:
In the thirteenth aspect of the present invention, the present invention provides the compound represented by the above Formula II, pharmaceutically acceptable salt, prodrug, stereoisomer, hydrate, solvate, crystalline form, metabolism form thereof, or any combination or mixture thereof, or the pharmaceutical composition comprising the compound represented by Formula II, pharmaceutically acceptable salt or ester, prodrug, stereoisomer, hydrate, solvate, crystalline form, metabolite form thereof, or any combination or mixture thereof, which is used for at least one of the following:
In a fourteenth aspect of the present invention, the present invention provides a method for obtaining the following effects:
In the above-described ninth to fourteenth aspects of the present invention, in some embodiments, the liver fibrosis includes, but is not limited to: liver fibrosis caused by chronic viral liver diseases, liver fibrosis caused by alcoholism or long-term drinking, liver fibrosis caused by non-alcoholic factor such as obesity, portal liver fibrosis caused by repeated schistosomiasis infection, biliary liver fibrosis caused by chronic cholestasis, metabolic liver fibrosis caused by hepatolenticular degeneration and hemoglobin deposition, toxic liver fibrosis caused by various toxic substances, malnutrition-induced liver fibrosis caused by a preference for low-protein diets and fatty fried foods, and cardiogenic liver fibrosis caused by chronic congestive heart failure.
In some embodiments, the pharmaceutical composition further comprises an additional active ingredient: other amino acids for improving liver function (including but not limited to alanine, glutamic acid, cystine, glutamine, glycine, histidine, tyrosine, serine, methionine, arginine, leucine), cholesterol absorption inhibitors (e.g., Ezetimibe), HSC activation and proliferation inhibitors (e.g., Pirfenidone, Fluorofenidone, Pegbelfermin), PCSK9 inhibitors, PPAR agonists (e.g., Gemfibrozil, Fenofibrate, Clofibrate, Bezafibrate, Pemafibrate, Elafibranor), ACE inhibitors, CCR2/5 inhibitors, TLR4 inhibition agents, LOXL2 inhibitors, TIMP-1 inhibitors, FXR agonists, AT1R blockers, NOX inhibitors, calcium channel blockers, ARBs, diuretics, renin, GLP-1 or synthetic variants thereof, insulin or synthetic variants thereof, metformin, sulfonylurea compounds, thiazolidinediones (TZD), SGLT2 inhibitors, DPP-IV inhibitors, inhibitors of HMGCoA reductase, inhibitors of proprotein convertase subtilisin/kexin type 9 (PCSK9), Gemcabene (CI-1027), ACC inhibitors, ApoC-III inhibitors, ACL-inhibitors (e.g., bepedic acid), prescription fish oil, CETP inhibitors, ursodeoxycholic acid, obeticholic acid, polyene phosphatidylcholine, glucocorticoids, silymarin, glycyrrhizic acid preparations (e.g., magnesium isoglycyrrhizinate injection and diammonium glycyrrhizinate enteric-coated capsules), and combinations thereof.
In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient.
In some embodiments, the pharmaceutical composition is a solid preparation, an injection, a topical preparation, a spray, a liquid preparation or a compound preparation.
In the above aspects of the present invention and embodiments thereof,
The “C1-C6 alkyl” in “C1-C6 alkyl” and various composite groups involving “C1-C6 alkyl” (e.g., “halogenated C1-C6 alkyl”) can be replaced by “C1-C20 alkyl”, “C1-C12 alkyl”, “C1-C10 alkyl”, “C1-C8 alkyl”, “C1-C4 alkyl”, “C1-C3 alkyl” or “C1-C2 alkyl”;
Therein, “con” represents the control well, “P1” represents the positive control well (hydronidone), and “ASP-50” represents that the aspartic acid concentration was 50 μM; the remaining numbers are all in the form of compound number-concentration (μM): “6-50” represents that the concentration of Compound 6 was 50 μM, “6-100” represents that the concentration of Compound 6 was 100 μM, “6-200” represents that the concentration of Compound 6 was 200 μM; “47-50” represents that the concentration of Compound 47 was 50 μM, and “47-100” represents that the concentration of Compound 47 was 100 μM, “47-200” represents that the concentration of Compound 47 was 200 μM; “54-50” represents that the concentration of Compound 54 was 50 μM, “54-100” represents that the concentration of Compound 54 was 100 μM, “54-200” represents that the concentration of Compound 54 was 200μ.
Therein, “con” represents the control well, “P1” represents the positive control well (hydronidone), and “ASP-50” represents that the aspartic acid concentration was 50 μM; the remaining numbers are all in the form of compound number-concentration (μM).
Therein, “con” represents the control well, “P1” represents the positive control well (hydronidone), and “ASP-50” represents the aspartic acid concentration of 50 μM; the remaining numbers are all in the form of compound number-concentration (μM).
Therein, “40×” means that the microscope magnification was 40 times, and “100×” means that the microscope magnification was 100 times.
Therein, “40×” means that the microscope magnification was 40 times, and “100×” means that the microscope magnification was 100 times.
Therein, “100×” means that the magnification of the microscope was 100 times, and “200×” means that the magnification of the microscope was 200 times.
Therein, “100×” means that the magnification of the microscope was 100 times, and “200×” means that the magnification of the microscope was 200 times.
The examples of the present invention are described in detail below. The examples described below are exemplary and are intended to explain the present invention, but should not be understood as limiting the present invention.
As used herein, the term “effective amount” or “therapeutically effective amount” refers to an amount that is sufficient, within the scope of reasonable medical judgment, to treat or prevent a patient's disease but low enough to avoid serious side effects (at a reasonable benefit/risk ratio). The therapeutically effective amount of a compound will vary upon the specific compound selected (e.g., taking into account the potency, effectiveness, and half-life of the compound), the route of administration selected, the disease being treated, the severity of the disease being treated, the factors of patient being treated such as age, size, weight and medical illness, the medical history of the patient being treated, duration of treatment, the nature of concurrent therapies, the desired therapeutic effect, etc., but can still be routinely determined by those skilled in the art.
As used herein, the term “mammal” refers to a warm-blooded animal that suffers from or is at risk of developing the diseases described herein, including but not limited to guinea pig, dog, cat, rat, mouse, hamster, and primate, including human.
In addition, it should be pointed out that the specific dosage and usage of the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof are determined by many factors, including the patient's age, body weight, gender, natural health status, nutritional status, activity intensity of drug, administration time, metabolic rate, severity of disease, and the subjective judgment of physician. The preferred dosage used herein is 0.001 to 1000 mg/kg body weight/day.
The pharmaceutically acceptable salt of the compound represented by Formula I, Formula I-4 or Formula II of the present invention includes inorganic or organic acid salt thereof, as well as inorganic or organic alkali salt. The present invention relates to all forms of the above-mentioned salt, which includes, but is not limited to: sodium salt, potassium salt, calcium salt, lithium salt, meglumine salt, hydrochloride, hydrobromide, hydriodate, nitrate, sulfate, bisulfate, phosphate, hydrophosphate, acetate, propionate, butyrate, oxalate, trimethylacetate, adipate, alginate, lactate, citrate, tartrate, succinate, maleate, fumarate, picrate, aspartate, gluconate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate and pamoate, etc.
The pharmaceutical composition involved in the present invention may comprise a pharmaceutically acceptable carrier. The carrier includes but is not limited to: ion exchanger, aluminum oxide, aluminum stearate, lecithin, serum protein such as human albumin, and buffer substance such as phosphate, glycerin, sorbic acid, potassium sorbate, partial glyceride mixture of saturated vegetable fatty acid, water, salt or electrolyte such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salt, colloidal silicon oxide, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substance, polyethylene glycol, sodium carboxymethylcellulose, polyacrylate, beeswax, lanolin.
The pharmaceutical composition of the present invention can be prepared in various forms according to different administration routes.
According to the present invention, the pharmaceutical composition can be administered in any of the following ways: oral administration, spray inhalation, rectal administration, nasal administration, buccal administration, vaginal administration, topical administration, parenteral administration such as subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal and intracranial injection or infusion, or administration by means of an explanted reservoir. Therein, oral, intraperitoneal or intravenous administration is preferred.
When used for oral administration, the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can be made into any orally acceptable preparation forms, including, but not limited to, tablets, capsules, aqueous solutions or aqueous suspensions. Therein, commonly used carriers for tablets include lactose and corn starch, and lubricants such as magnesium stearate can also be added. Commonly used diluents for capsule formulations include lactose and dried corn starch. Aqueous suspension formulations usually consist of the active ingredient mixed with suitable emulsifying and suspending agents. If desired, some sweetening, flavoring or coloring agents may be added to the above oral preparation forms.
When used for rectal administration, the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can generally be made into a suppository form, which is prepared by mixing the drug with a suitable non-irritating excipient. The excipient is solid at room temperature and melts at rectal temperature to release the drug. Such excipient includes cocoa butter, beeswax and polyethylene glycol.
When used for topical administration, especially when treating affected surfaces or organs easily accessible by topical application, such as eye, skin or lower intestinal neurological diseases, the compound represented by Formula I, Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can be made into different topical preparation forms according to different affected surfaces or organs. The specific instructions are as follows:
When used for topical administration to eyes, the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can be formulated in the form of micronized suspension or solution, the carrier used is isotonic sterile saline with a certain pH, in which a preservative such as chlorobenzyl alkoxide may or may not be added. Additionally, for ophthalmic use, the compound can also be formulated in the form of ointment such as petroleum jelly.
When used for topical administration to skin, the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can be formulated in the form of suitable ointment, lotion or cream in which the active ingredient is suspended or dissolved in one or more carriers. The carriers that can be used for ointment include, but are not limited to: mineral oil, liquid vaseline, white vaseline, propylene glycol, polyoxyethylene, polyoxypropylene, emulsified wax and water; the carriers that can be used for lotion or cream include, but are not limited to: mineral oil, sorbitan monostearate, Tween 60, cetyl ester wax, hexadecene aromatic alcohol, 2-octyldodecanol, benzyl alcohol and water.
When used for topical administration to lower intestine, the compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can be formulated in the form of rectal suppository or suitable enema preparation as described above, and topical transdermal patch may also be used.
The compound represented by Formula I or Formula I-4 or Formula II, stereoisomer thereof or pharmaceutically acceptable salt and/or solvate thereof and/or hydrate thereof can also be administered in the form of sterile injection preparation, including sterile injectable aqueous or oily suspension, or sterile injectable solution. Therein, the carriers and solvents that can be used include water, Ringer's solution and isotonic sodium chloride solution. Alternatively, sterile nonvolatile oil such as monoglyceride or diglyceride may also be used as solvent or suspending medium.
The drugs in various dosage forms mentioned above can be prepared according to conventional methods in the pharmaceutical field.
In various parts of the present specification, substituents of the compounds disclosed herein are disclosed according to group types or ranges. In particular, the present invention comprises each and every individual subcombination of the individual members of these group types and ranges. For example, the term “C1-C6 alkyl” specifically refers to the independently disclosed methyl, ethyl, C3 alkyl, C4 alkyl, C5 alkyl and C6 alkyl, or “C1-C4 alkyl”, or “C1-C3 alkyl”.
In addition, it should be noted that, unless otherwise clearly stated, the description “ . . . independently selected from the group consisting of” used throughout herein should be understood in a broad sense, which refers to that the specific options expressed by different symbols do not affect each other and can be the same or different.
“Optionally” means that the situation mentioned may or may not exist. For example, when it is described that a certain structure is “optionally” substituted by certain groups, it means that the “substitution” may or may not exist.
Unless otherwise indicated, the structural formulas described in the present invention include all isomeric forms such as enantiomers, diastereomers, and geometric isomers (or conformational isomers): for example, R, S configurations that contain an asymmetric center, (Z), (E) isomers of double bond, and (Z), (E) conformational isomers. Therefore, the individual stereochemical isomers, diastereomers, or mixtures of geometric isomers (or conformational isomers) of the compound of the present invention all fall within the scope of the present invention.
Any asymmetric atom (e.g., carbon, etc.) of the compound disclosed in the present invention may exist in a racemic or enantioenriched form, such as (R), (S) or (R,S) configuration. In certain embodiments, each asymmetric atom has at least 50% enantiomeric excess, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess in the (R) or(S) configuration.
The term “prodrug” used in the present invention represents a compound that is converted into the compound represented by Formula (I) in vivo. Such conversion is affected by the hydrolysis of the prodrug in the blood or by the enzymatic conversion of the prodrug to the parent structure in the blood or tissue. The prodrug compound of the present invention can be an ester. In the existing invention, the ester that can be used as prodrug includes phenyl ester, aliphatic (C1-C24) ester, acyloxymethyl ester, carbonate ester, carbamate ester and amino acid ester.
“Metabolite” refers to a product obtained by metabolism of a specific compound or salt thereof in the body. The metabolites of a compound can be identified by techniques well known in the art, and the activity thereof can be characterized by assays as described herein. Such product can be obtained by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, deesterification, enzymatic cleavage, etc., of the compound administered. Accordingly, the present invention comprises a metabolite of the compound, which comprises a metabolite produced by sufficiently contacting the compound of the present invention with a mammal for a period of time.
“Solvate” as used herein refers to an association complex of one or more solvent molecules with the compound of the present invention. Solvents that form the solvate include, but are not limited to, water, isopropanol, ethanol, methanol, dimethyl sulfoxide, ethyl acetate, acetic acid, and aminoethanol. The term “hydrate” refers to an association complex formed with water as solvent molecules.
The terms “halogen” and “halo” are used interchangeably in the present invention and refer to fluorine (F), chlorine (C1), bromine (Br) or iodine (I).
The term “alkyl” used in the present invention refers to a saturated straight or branched monovalent hydrocarbonyl containing 1 to 20 carbon atoms (C1-C20 alkyl), wherein the alkyl can be independently optionally substituted with one or more substituents as described in the present invention. In some embodiments, the alkyl contains 1 to 12 carbon atoms (C1-C12 alkyl); in other embodiments, the alkyl contains 1 to 10 carbon atoms (C1-C10 alkyl); in other embodiments, the alkyl contains 1 to 8 carbon atoms (C1-C8 alkyl); in other embodiments, the alkyl contains 1 to 6 carbon atoms (C1-C6 alkyl); in other embodiments, the alkyl contains 1 to 4 carbon atoms (C1-C4 alkyl); in other embodiments, the alkyl contains 1 to 3 carbon atoms (C1-C3 alkyl); in other embodiments, the alkyl contains 1 to 2 carbon atoms (C1-C2 alkyl). Further examples of alkyl include, but are not limited to, methyl (Me, —CH3), ethyl (Et, —CH2CH3), n-propyl (n-Pr, —CH2CH2CH3), isopropyl (i-Pr, —CH(CH3)2), n-butyl (n-Bu, —CH2CH2CH2CH3), 2-methylpropyl or isobutyl (i-Bu, —CH2CH(CH3)2), 1-methylpropyl or sec-butyl (s-Bu, —CH(CH3) CH2CH3), tert-butyl (t-Bu, —C(CH3)3), n-pentyl (—CH2CH2CH2CH2CH3), 2-pentyl (—CH(CH3) CH2CH2CH3), 3-pentyl (—CH(CH2CH3)2), 2-methyl-2-butyl (—C(CH3)2CH2CH3), 3-methyl-2-butyl (—CH(CH3) CH(CH3)2), 3-methyl-1-butyl (—CH2CH2CH(CH3)2), 2-methyl-1-butyl (—CH2CH(CH3) CH2CH3), n-hexyl (—CH2CH2CH2CH2CH2CH3), 2-hexyl (—CH(CH3) CH2CH2CH2CH3), 3-hexyl (—CH(CH2CH3) (CH2CH2CH3)), 2-methyl-2-pentyl (—C(CH3)2CH2CH2CH3), 3-methyl-2-pentyl (—CH(CH3) CH(CH3) CH2CH3), 4-methyl-2-pentyl (—CH(CH3) CH2CH(CH3)2), 3-methyl-3-pentyl (—C(CH3) (CH2CH3)2), 2-methyl-3-pentyl (—CH(CH2CH3) CH(CH3)2), 2,3-dimethyl-2-butyl (—C(CH3)2CH(CH3)2), 3,3-dimethyl-2-butyl (—CH(CH3) C(CH3)3), n-heptyl, n-octyl, etc.
The term “C1-C6 alkyl” refers to any alkyl containing 1 to 6 carbon atoms, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, tert-pentyl, n-hexyl, etc.
The term “alkenyl” refers to a straight or branched monovalent hydrocarbonyl containing 2 to 12 carbon atoms (C2-C12 alkenyl), or 2 to 8 carbon atoms (C2-C8 alkenyl), or 2 to 6 carbon atoms (C2-C6 alkenyl), or 2 to 4 carbon atoms (C2-C4 alkenyl), wherein at least one position of C—C is an sp2 double bond, which comprises “cis”, “trans” or “Z”, “E” isomers. Specific examples thereof include, but are not limited to, vinyl (—CH═CH2), propenyl (—CH═CHCH3), allyl (—CH2CH═CH2), etc.
The term “C2-C6 alkenyl” refers to any alkenyl containing 2 to 6 carbon atoms and containing at least one double bond, and examples thereof include vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl, 3-butenyl, 2-pentenyl, 1-hexenyl, etc.
The term “alkynyl” refers to a straight or branched monovalent hydrocarbonyl containing 2 to 12 carbon atoms (C2-C12 alkynyl), or 2 to 8 carbon atoms (C2-C8 alkynyl), or 2 to 6 carbon atoms (C2-C6 alkynyl), or 2 to 4 carbon atoms (C2-C4 alkynyl), in which at least one position of C—C is an sp triple bond. Specific examples thereof include, but are not limited to, ethynyl (—C═CH), propargyl (—CH2C═CH), propynyl (—C═C—CH3), 1-butynyl (—CH2CH2C═CH), 2-butynyl (—CH2C═CCH3), 3-butynyl (—C═CCH2CH3), etc.
The term “C2-C6 alkynyl” refers to any alkynyl containing 2 to 6 carbon atoms and containing at least one triple bond, such as ethynyl, 2-propynyl, 4-pentynyl, etc.
The term “alkoxy” refers to that an alkyl attached to the remainder of molecule through an oxygen atom, wherein the alkyl has the meaning described herein. Unless otherwise specified, the alkoxy contains 1 to 12 carbon atoms and may be represented as C1-C12 alkoxy. In some embodiments, the alkoxy contains 1 to 8 carbon atoms, which may be represented by C1-C8 alkoxy; in other embodiments, the alkoxy contains 1 to 6 carbon atoms, which may be represented by C1-C6 alkoxy; in other embodiments, the alkoxy contains 1 to 4 carbon atoms, which may be represented by C1-C4 alkoxy; in yet other embodiments, the alkoxy contains 1 to 3 carbon atoms, which may be represented by C1-C3 alkoxy. Examples of alkoxy include, but are not limited to, methoxy (MeO, —OCH3), ethoxy (EtO, —OCH2CH3), 1-propoxy (n-PrO, n-propoxy, —OCH2CH2CH3), 2-propoxy (i-PrO, i-propoxy, —OCH(CH3)2), 1-butoxy (n-BuO, n-butoxy, —OCH2CH2CH2CH3), 2-methyl-1-propoxy (i-BuO, i-butoxy, —OCH2CH(CH3)2), 2-butoxy (s-BuO, s-butoxy, —OCH(CH3) CH2CH3), 2-methyl-2-propoxy (t-BuO, t-butoxy, —OC(CH3)3), 1-pentyloxy (n-pentyloxy, —OCH2CH2CH2CH2CH3), 2-pentyloxy (—OCH(CH3) CH2CH2CH3), 3-pentyloxy (—OCH(CH2CH3)2), 2-methyl-2-butoxy (—OC(CH3)2CH2CH3), 3-methyl-2-butoxy (—OCH(CH3) CH(CH3)2), 3-methyl-1-butoxy (—OCH2CH2CH(CH3)2), 2-methyl-1-butoxy (—OCH2CH(CH3)) CH2CH3), etc.
The term “alkylamino” or “alkylamino group” comprises “N-alkylamino” and “N,N-dialkylamino”, wherein the amino group is each independently substituted with one or two alkyl groups, wherein the alkyl has the meaning as described in the present invention. Suitable alkylamino may be monoalkylamino or dialkylamino, examples of which include, but are not limited to, N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylamino etc. The alkylamino is optionally substituted with one or more substituents described herein.
The term “haloalkoxy” means that an alkoxy is substituted with one or more halogen atoms, wherein the alkoxy has the meaning as described in the present invention, and such examples include, but are not limited to, —OCHF2, —OCF3, —OCHFCH2F, —OCF2CHF2, —OCH2CF3, —OCHFCH3, —OCH2CH2F, —OCF2CH3, —OCH2CF2CHF2, etc. In one embodiment, C1-C6 haloalkoxy comprises fluorine-substituted C1-C6 alkoxy; in another embodiment, C1-C4 haloalkoxy comprises fluorine-substituted C1-C4 alkoxy; in yet another embodiments, C1-C2 haloalkoxy comprises fluorine-substituted C1-C2 alkoxy.
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 (i.e., “C3-C20 cycloalkyl”), preferably 3 to 12 carbon atoms (i.e., “C3-C12 cycloalkyl”), more preferably 3 to 10 carbon atoms (i.e., “C3-C10 cycloalkyl”), most preferably 3 to 7 carbon atoms (i.e., “C3-C7 cycloalkyl”). Non-limiting examples of monocyclic cycloalkyl (e.g., “C3-C7 cycloalkyl”) include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, etc.; and polycyclic cycloalkyl comprises spiro-, fused- and bridged-cycloalkyl.
The term “halogenated C1-C6 alkyl” refers to a group in which C1-C6 alkyl skeleton is substituted with one or more halogens, such as monofluoromethyl, difluoroethyl, trifluoromethyl, etc.
The term “halogen” refers to fluorine, chlorine, bromine, and iodine.
The term “C6-C12 aryl” refers to a group of a carbocyclic aromatic system having 6 to 12 carbon atoms.
The term “C6-C10 aryl” refers to a group of a carbocyclic aromatic system having 6 to 10 carbon atoms, such as phenyl, naphthyl, etc.
The term “heteroalkyl” refers to a straight or branched alkyl (preferably an alkyl having 2 to 14 or 3 to 7 carbon atoms), in which one or more carbon atoms are independently replaced by heteroatoms selected from the group consisting of S, O, P and N (i.e., “2- to 14-membered heteroalkyl” or “3- to 7-membered heteroalkyl”). Exemplary heteroalkyl groups include alkyl ethers, alkylamines, secondary alkylamines, thioethers, and the like.
The term “heterocyclyl” refers to a saturated or partially unsaturated monocyclic or polycyclic cyclic hydrocarbon substituent containing 3 to 20 ring atoms (i.e., a “3- to 20-membered heterocyclyl”), in which one or more ring atoms are heteroatoms selected from the group consisting of nitrogen, oxygen and S(O) m (wherein m is an integer of 0 to 2), but it does not contain a ring part of —O—O—, —O—S—or —S—S—, and the remaining ring atoms are carbon atoms. Preferably, it contains 3 to 15 ring atoms (i.e., “3- to 15-membered heterocyclyl”) or 3 to 12 ring atoms (i.e., “3- to 12-membered heterocyclyl”), of which 1 to 4 are heteroatoms; more preferably, the heterocyclyl contains 3 to 10 ring atoms (i.e., “3- to 10-membered heterocyclyl”). Non-limiting examples of monocyclic heterocyclyl (e.g., 3- to 7-membered heterocyclyl) include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl includes spiro-, fused- or bridged-heterocyclyl.
The term “3- to 15-membered heterocyclyl” refers to 3—, 4-, 5-, 6- and 7-membered up to 15-membered saturated or partially unsaturated carbocyclic rings, in which one or more carbon atoms are replaced by heteroatoms such as nitrogen, oxygen and sulfur. “3- to 15-membered heterocyclyl” includes, for example, “3- to 7-membered heterocyclyl”, and non-limiting examples thereof include, such as, thiacyclobutyl, pyran, pyrrolidine, pyrroline, imidazoline, imidazolidine, pyrazolidine, pyrazoline, thiazoline, thiazolidine, dihydrofuran, tetrahydrofuran, 1,3-dioxolane, piperidine, piperazine, morpholine, morpholinyl, tetrahydropyrrolyl, thiomorpholinyl, et al.
The term “polycyclic aromatic hydrocarbonyl” refers to a group formed by fusing 1 to 2 C5-C6 aromatic rings and 1 to 2 aromatic rings or non-aromatic rings together, and non-limiting examples thereof include, such as indenyl, naphthalenyl, phenanthrenyl, fluorenyl, etc.
Composite structures such as “-(C1-C6 alkyl)n-O—(CO)—Rb3” refer to that any n C1-C6 alkyl groups (e.g., methyl, ethyl) and the like as described above are connected to Rb3 through oxygen (—O—) and carbonyl (—(C═O)—). The meanings of other similar composite structures can be understood with reference to the foregoing contents.
Peptide: The amino group of one amino acid condenses with the carboxyl group of another amino acid to form a peptide; for example: C#OOH—CH(N#H2)—CH2—C#OOH, which can form peptide bonds with other amino acids through one, two or three #-signed atoms.
Any structural formulas given herein are also intended to represent non-isotopically enriched as well as isotopically enriched forms of these compounds. Isotopically enriched compounds have the structure depicted by the general formula of the present invention, except that one or more atoms are replaced by atoms that have selected atomic weights or mass numbers. Exemplary isotopes that may be introduced into the compounds of the present invention include isotopes of hydrogen, carbon, nitrogen, oxygen, phosphorus, sulfur, fluorine and chlorine, such as 2H, 3H, 11C, 13C, 14C, 15N, 17O, 18O, 18F, 31P, 32P, 35S, 36Cl and 125I.
In *N═CH—NH—CH═*C—CH2—CH(NH2)—C(═O)—, N and C are marked with *, indicating that the two atoms marked with * are single bonded.
Commercial sources of some known compounds:
(S)-4-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (1 g, 3.096 mmol) was dissolved in dichloromethane (10 mL), added dropwise with acetyl chloride (490 mg, 6.19 mmol) to the reaction system, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated, then diluted by adding dichloromethane, the above solution was added dropwise into absolute ethanol (10 ml), and the reaction solution was stirred at room temperature for 1 hour. After it was detected that the reaction was completed, the reaction solution was washed with water, and the organic phase was separated, dried, and concentrated to obtain 900 mg of 1-benzyl 4-ethyl (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 352 [M+H]+.
1-Benzyl 4-ethyl (tert-butoxycarbonyl)-L-aspartic acid ester (900 mg, 2.56 mmol) was dissolved in ethyl acetate (5 mL) at room temperature, and a solution of hydrogen chloride in ethyl acetate (2M) (7.7 ml, 15.38 mmol) was added to the reaction system, the reaction solution was allowed to react at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 600 mg of 1-benzyl 4-ethyl L-aspartic acid ester. MS m/z (ESI): 252 [M+H]+.
1-Benzyl 4-ethyl L-aspartic acid ester (600 mg, 2.39 mmol) was dissolved in ethanol (5 mL), added with palladium-on-carbon (120 mg), stirred and reacted at room temperature under a hydrogen atmosphere for 16 h. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the filtrate was concentrated to obtain 200 mg of 4-ethyl L-aspartic acid ester (Compound 35). MS m/z (ESI): 162 [M+H]+. 1H NMR (400 MHZ, DMSO-d6) δ 13.92 (s, 1H), 8.49 (s, 2H), 4.17 (t, J=5.6 Hz, 1H), 4.13-4.06 (m, 2H), 2.98-2.86 (m, 2H), 1.18 (t, J=7.1 Hz, 3H).
(S)-4-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (1 g, 3.096 mmol) was dissolved in dichloromethane (10 mL), acetyl chloride (490 mg, 6.19 mmol) was added dropwise to the reaction system, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated and diluted with dichloromethane. The above solution was added dropwise to isopropanol (10 ml), and the reaction solution was stirred at room temperature for 1 hour. After it was detected by TLC that the reaction was completed, the reaction solution was washed with water, and the organic phase was separated, dried and concentrated to obtain 850 mg of 1-benzyl 4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 366 [M+H]+.
1-Benzyl 4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester (860 mg, 2.35 mmol) was dissolved in ethyl acetate (5 mL) at room temperature, then a solution of hydrogen chloride in ethyl acetate (2M) (7.07 ml, 14.14 mmol) was added, and the solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 500 mg of 1-benzyl 4-isopropyl L-aspartic acid ester. MS m/z (ESI): 266 [M+H]+.
1-Benzyl 4-isopropyl L-aspartic acid ester (500 mg, 1.88 mmol) was dissolved in ethanol (5 mL), added with palladium-on-carbon (100 mg), stirred and reacted at room temperature under a hydrogen atmosphere for 16 h. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the filtrate was concentrated to obtain 200 mg of 4-isopropyl L-aspartic acid ester (Compound 36). MS m/z (ESI): 176 [M+H]+. 1H NMR (400 MHZ, DMSO-d6) δ 8.90 (s, 2H), 4.99-4.93 (m, 1H), 4.17 (t, J=5.2 Hz, 1H), 2.92-2.89 (m, 2H), 1.18 (dd, J=6.2, 1.5 Hz, 6H).
(S)-4-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (2 g, 6.19 mmol), chloromethyl isopropyl carbonate (1.47 g, 9.29 mmol) and potassium carbonate (1.7 g, 12.38 mmol) were dissolved in DMF (10 mL), and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted by adding water, extracted with ethyl acetate, then the reaction solution was washed with brine, and the organic phase was separated, dried and concentrated to obtain 900 mg of 1-benzyl 4-(((isopropoxycarbonyl)oxy) methyl) (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 440 [M+H]+.
1-Benzyl 4-(((isopropoxycarbonyl)oxy) methyl) (tert-butoxycarbonyl)-L-aspartic acid ester (900 mg, 2.05 mmol) was dissolved in methanol (5 mL), added with palladium-on-carbon (180 mg), stirred and reacted for 16 h at room temperature under a hydrogen atmosphere. After it was monitored by TLC that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the filtrate was concentrated to obtain 500 mg of (S)-2-((tert-butoxycarbonyl)amino)-4-(((isopropoxycarbonyl)oxy) methoxy)-4-oxobutyric acid. MS m/z (ESI): 350 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-4-(((isopropoxycarbonyl)oxy) methoxy)-4-oxobutyric acid (500 mg, 1.43 mmol) was dissolved in ethyl acetate (5 mL), a solution of hydrogen chloride in ethyl acetate (2M) (4.3 ml, 8.6 mmol) was added, and the solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated, water was added to dilute the reaction solution, and sodium bicarbonate was added to adjust the pH of the solution to neutral. The mixture was extracted with ethyl acetate, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate and concentrated under reduced pressure to obtain 100 mg of 4-(((isopropoxycarbonyl)oxy) methyl)-L-aspartic acid (Compound 37). 1H NMR (300 MHZ, DMSO-d6) δ 13.93 (s, 1H), 8.66 (s, 2H), 5.69 (d, J=12.8 Hz, 2H), 4.79 (h, J=6.2 Hz, 1H), 4.18 (s, 1H), 3.16-2.94 (m, 2H), 1.23 (d, J=6.2 Hz, 6H). MS m/z (ESI): 250 [M+H]+.
(S)-3-Amino-4-methoxy-4-oxobutyric acid (100 mg, 0.68 mmol) was dissolved in tetrahydrofuran (3 ml), added with a solution of hydrogen chloride in ethyl acetate (0.34 ml, 0.68 mmol), and stirred at room temperature for 30 minutes, and then solid precipitation could be observed. The mixture was filtered to obtain the filter residue, and the filter residue was dried to obtain 80 mg of(S)-3-amino-4-methoxy-4-oxobutyric acid hydrochloride (Compound 49). 1H NMR (300 MHz, DMSO-d6) δ 9.60 (s, 1H), 4.23 (t, J=5.2 Hz, 1H), 3.70 (s, 3H), 2.94 (d, J=5.2 Hz, 2H). MS m/z (ESI): 184 [M+H]+.
(S)-4-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (1 g, 3.096 mmol) was dissolved in dichloromethane (10 mL), acetyl chloride (490 mg, 6.19 mmol) was added dropwise to the reaction system, and the reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated, diluted by adding dichloromethane, the above solution was added dropwise to isopropanol (10 ml), and the reaction solution was stirred at room temperature for 1 hour. After it was detected by TLC that the reaction was completed, the reaction solution was washed with water, and the organic phase was separated, dried, and concentrated to obtain 840 mg of 4-benzyl 1-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 366 [M+H]+.
4-Benzyl 1-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester (840 mg, 2.3 mmol) was dissolved in ethanol (5 mL), added with palladium-on-carbon (160 mg), stirred and reacted at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the filtrate was concentrated to obtain 300 mg of (S)-3-((tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyric acid. MS m/z (ESI): 276 [M+H]+.
(S)-3-((Tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyric acid (300 mg, 1.1 mmol) was dissolved in ethyl acetate (5 mL) at room temperature, a solution of hydrogen chloride in ethyl acetate (2M) (3.3 ml, 6.55 mmol) was added to the reaction system, and the solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 130 mg of 1-isopropyl L-aspartic acid ester hydrochloride (Compound 50). 1H NMR (300 MHz, DMSO-d6) δ 13.00 (s, 1H), 8.58 (s, 2H), 4.97 (p, J=6.3 Hz, 1H), 4.19 (t, J=5.2 Hz, 1H), 2.90 (dd, J=5.2, 1.8 Hz, 2H), 1.20 (dd, J=10.2, 6.2 Hz, 6H). MS m/z (ESI): 212 [M+H]+.
(S)-4-(Benzyloxy)-2-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (10 g, 30.96 mmol), chloromethyl isopropyl carbonate (5.65 g, 37.15 mmol) and potassium carbonate (8.54 g, 61.92 mmol) were dissolved in DMF (100 mL), and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted by adding water, extracted with ethyl acetate, washed with brine, and the organic phase was separated, dried and concentrated to obtain 4.2 g of 4-benzyl 1-(((isopropoxycarbonyl)oxy) methyl) (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 440 [M+H]+.
4-Benzyl 1-(((isopropoxycarbonyl)oxy) methyl) (tert-butoxycarbonyl)-L-aspartic acid ester (4.2 g, 9.57 mmol) was dissolved in methanol (50 mL), added with palladium-on-carbon (810 mg), stirred and reacted for 16 h at room temperature under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the filtrate was concentrated to obtain 3 g of (S)-3-((tert-butoxycarbonyl)amino)-4-(((isopropoxycarbonyl)oxy) methoxy methyl)-4-oxobutyric acid. MS m/z (ESI): 350 [M+H]+.
At room temperature, (S)-3-((tert-butoxycarbonyl)amino)-4-(((isopropoxycarbonyl)oxy) methoxy)-4-oxobutyric acid (3 g, 8.60 mmol) was dissolved in ethyl acetate (30 mL), and added with a solution of hydrogen chloride in ethyl acetate (2M) (25.8 ml, 51.58 mmol), and the solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 1.2 g of 1-(((isopropoxycarbonyl)oxy) methyl) L-aspartic acid ester hydrochloride (Compound 51). 1H NMR (400 MHZ, DMSO-d6) δ 8.80 (s, 2H), 5.79-5.68 (m, 2H), 4.80 (p, J=6.2 Hz, 1H), 4.34 (s, 1H), 2.97 (d, J=5.1 Hz, 2H), 1.23 (d, J=6.2 Hz, 6H). MS m/z (ESI): 286 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-4-(((isopropoxycarbonyl)oxy) methoxy)-4-oxobutyric acid (500 mg, 1.43 mmol) was dissolved in ethyl acetate (5 mL), added with a solution of hydrogen chloride in ethyl acetate (2M) (4.3 ml, 8.6 mmol), and the solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 130 mg of 4-(((isopropoxycarbonyl)oxy) methyl) L-aspartic acid ester hydrochloride (Compound 52). 1H NMR (300 MHZ, DMSO-d6) δ 13.93 (s, 1H), 8.66 (s, 2H), 5.69 (d, J=12.8 Hz, 2H), 4.79 (h, J=6.2 Hz, 1H), 4.18 (s, 1H), 3.16-2.94 (m, 2H), 1.23 (d, J=6.2 Hz, 6H). MS m/z (ESI): 286 [M+H]+.
(Tert-butoxycarbonyl)-L-phenylalanine (2 g, 7.55 mmol), chloromethyl isopropyl carbonate (1.38 g, 9.06 mmol) and potassium carbonate (2.08 g, 15.09 mmol) were dissolved in DMF (10 mL), the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted by adding water, extracted with ethyl acetate, then the reaction solution was washed with brine, and the organic phase was separated, dried, and concentrated to obtain 550 mg of ((isopropoxycarbonyl)oxy) methyl ester (tert-butoxycarbonyl)-L-phenylalanine. MS m/z (ESI): 382 [M+H]+.
((Isopropoxycarbonyl)oxy)methyl ester (tert-butoxycarbonyl)-L-phenylalanine (550 mg, 1.44 mmol) was dissolved in ethyl acetate (5 mL) at room temperature, then added with a solution of hydrogen chloride in ethyl acetate (2M) (4.33 ml, 8.66 mmol), and the above reaction solution was allowed to react at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 416 mg of ((isopropoxycarbonyl)oxy) methyl ester L-phenylalanine hydrochloride. MS m/z (ESI): 318 [M+H]+.
((Isopropoxycarbonyl)oxy)methyl ester L-phenylalanine hydrochloride (416 mg, 1.3 mmol), (S)-4-(tert-butoxy)-2-((tert-butyloxycarbonyl)amino)-4-oxobutyric acid (416 mg, 1.44 mmol), HATU (994 mg, 2.62 mmol) and DMAP(16 mg, 0.13 mmol) were dissolved in DMF (5 mL), added dropwise with DIPEA (506 mg, 3.9 mmol), stirred and reacted at room temperature for 2 h. After it was monitored by TLC that the reaction was completed, the reaction solution was diluted by adding water, extracted with ethyl acetate (5 mL*2), and separated to obtain an organic phase. The organic phase was dried, concentrated, and purified by column chromatography to obtain 500 mg of tert-butyl(S)-3-((tert-butoxycarbonyl)amino)-4-(((S)-1-(((isopropoxycarbonyl)oxy) methoxy)-1-oxo-3-phenylpropan-2-yl)amino)-4-oxobutyrate. MS m/z (ESI): 553 [M+H]+.
Tert-butyl(S)-3-((tert-butoxycarbonyl)amino)-4-(((S)-1-(((isopropoxycarbonyl)oxy) methoxy)-1-oxo-3-phenylpropan-2-yl)amino)-4-oxobutyrate (500 mg, 0.91 mmol) was dissolved in ethyl acetate (5 mL), and added with a solution of hydrogen chloride in ethyl acetate (2M) (2.72 ml, 5.43 mmol), and the above reaction solution was reacted at room temperature for 1 hour. After it was monitored by TLC that the reaction was completed, the reaction solution was concentrated to obtain 320 mg of (S)-3-amino-4-(((S)-1-(((isopropoxycarbonyl)oxy) methoxy)-1-oxo-3-phenylpropan-2-yl)amino)-4-oxobutyric acid hydrochloride (Compound 53). 1H NMR (400 MHZ, DMSO-d6) δ 12.86 (s, 1H), 9.17 (d, J=7.3 Hz, 1H), 8.31 (s, 2H), 7.31-7.22 (m, 5H), 5.74-5.63 (m, 2H), 4.81 (hept, J=6.2 Hz, 1H), 4.56 (ddd, J=8.6, 7.3, 5.6 Hz, 1H), 4.10-4.03 (m, 1H), 3.09-2.95 (m, 2H), 2.89-2.69 (m, 2H), 1.24 (d, J=6.2 Hz, 6H). MS m/z (ESI): 433 [M+H]+.
Boc-L-aspartic acid 4-methyl ester (1.2 g, 5.0 mmol) and tetrabutylammonium bisulfate (84.0 mg, 0.3 mmol) were dissolved in dichloromethane (5 mL), placed in an ice bath, stirred and cooled to 0° C. A solution of potassium carbonate (2.7 g, 20.0 mmol) in water (5 mL) and a solution of chloromethyl chlorosulfonate (1.0 g, 6.0 mmol) in dichloromethane (5 mL) were added dropwise to the above reaction system in sequence, stirred and slowly warmed to room temperature, and stirred overnight. After it was monitored by TLC that the reaction was completed, 5 mL of dichloromethane was added, washing was performed with water three times, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EA/PE gradient elution, 20:1 to 5:1) to obtain 1.1 g of 1-(chloromethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (colorless oil, yield 83%).
Sodium iodide (174 mg, 1.1 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in DMF (1.5 mL), placed in an ice bath, stirred and cooled to 0° C. Boc-L-aspartic acid 4-methyl ester (296.7 mg, 1.2 mmol) was dissolved in DMF (1.0 mL) and added dropwise into the above reaction system, placed in an ice bath and stirred for 30 minutes. 1-(Chloromethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (295.7 mg, 1.0 mmol) was dissolved in DMF (1.0 mL) and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight, and the reaction was monitored by TLC. After the reaction of the raw materials was completed, 10 mL of water was added, extracted with ethyl acetate three times, and washing was performed with water three times. The organic phases were separated and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EA/PE gradient elution, 10:1 to 3:1) to obtain 60 mg of 4-dimethyl O′1,01-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (white solid, yield 12%).
4-Dimethyl O′1,01-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (1.0 g, 2.0 mmol) was dissolved in dichloromethane (6 mL), placed in an ice bath, stirred and cooled to 0° C., added dropwise with 2N solution of hydrochloric acid in ethyl acetate (12 mL), stirred and slowly warmed to room temperature, then stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 530 mg of 4-dimethyl O′1,01-methylene (2S,2'S)-bis (2-aminosuccinate) hydrochloride (Compound 54) (white solid, yield 60%). 1H NMR (400 MHZ, CD3OD), δ 5.99 (s, 2H), 4.52 (t, J=6 Hz, 2H), 3.77 (s, 6H), 3.10 (m, 4H). MS m/z (ESI): 343 [M+H]+.
For the synthesis method of the titled compound, the synthesis of Compound 54 in Example 9 of the present application was referred to. Boc-L-aspartic acid 4-methyl ester was replaced by N-tert-butoxycarbonyl-L-aspartic acid 1-methyl ester in step 1 and step 2 to obtain 1-dimethyl O′4,04-methylene (2S,2'S)-bis (2-aminosuccinate) hydrochloride (Compound 56), yield 46%, as a white solid. 1H NMR (400 MHZ, CD3OD), δ 5.88 (s, 2H), 4.45 (t, J=8 Hz, 2H), 3.85 (s, 6H), 3.02 (m, 4H). MS m/z (ESI): 343 [M+H]+.
Tert-butoxycarbonyl-L-aspartic acid 4-tert-butyl ester (2.3 g, 8.0 mmol) and tetrabutylammonium bisulfate (136 mg, 0.4 mmol) were dissolved in dichloromethane (8 mL), placed in an ice bath, stirred and cooled to 0° C. A solution of potassium carbonate (4.4 g, 32 mmol) in water (8 mL) and a solution of chloromethyl chlorosulfonate (1.7 g, 10.0 mmol) in dichloromethane (8 mL) were added dropwise to the reaction system in sequence, stirred and slowly warmed to room temperature overnight. After it was monitored by TLC that the reaction was completed, 8 mL of dichloromethane was added, washing was performed with water three times, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EA/PE gradient elution, 20:1 to 5:1) to obtain 1.4 g of 1-(chloromethyl)4-n-butyl (tert-butoxycarbonyl)-L-aspartic acid ester (colorless oil, yield 53%).
Sodium iodide (349 mg, 2.1 mmol) and potassium carbonate (552 mg, 4.0 mmol) were dissolved in DMF (2 mL), placed in an ice bath, stirred and cooled to 0° C. 1-(Chloromethyl)4-n-butyl (tert-butoxycarbonyl)-L-aspartic acid ester (694.0 mg, 2.4 mmol) was dissolved in DMF (2 mL) and added dropwise into the above reaction system, placed in an ice bath and stirred for 30 minutes. 1-(Chloromethyl)4-n-butyl (tert-butoxycarbonyl)-L-aspartic acid ester (676.0 mg, 2.0 mmo) was dissolved in DMF (2 mL) and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred for about overnight. After it was monitored by TLC that the reaction was completed, 10 mL of water was added, extracted with ethyl acetate three times, then the organic phase was separated and washed with water, the organic phases were combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by a chromatographic column (EA/PE gradient elution, 10:1 to 3:1) to obtain 619 mg of 4-di (n-butyl) O′1,01-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (white solid, yield 52%).
4-Di (n-butyl)O′1,01-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (295.0 mg, 0.5 mmol) was dissolved in dichloromethane (2 mL), placed in an ice bath, stirred and cooled to 0° C., added dropwise with 2N solution of hydrochloric acid in ethyl acetate (6 mL), stirred and slowly warmed to room temperature and stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 95 mg of (3S,3'S)-4,4′-(methylenebis (oxy))bis (3-amino-4-oxobutyric acid) hydrochloride (Compound 57) (white solid, yield 61%). 1H NMR (400 MHZ, CD3OD), δ 5.99 (s, 2H), 4.16 (t, J=4 Hz, 2H), 3.05 (m, 4H). MS m/z (ESI): 315 [M+H]+.
For the synthesis method of the titled compound, the synthesis of Compound 57 in Example 11 of the present application was referred to. Tert-butoxycarbonyl-L-aspartic acid 4-tert-butyl ester was replaced by N-tert-butoxycarbonyl-L-aspartic acid 1-tert-butyl ester in step 1 and step 2 to obtain (2S,2'S)-4,4′-(methylenebis (oxy))bis (2-amino-4-oxobutyric acid) hydrochloride (Compound 55) (yield 54%, white solid). 1H NMR (400 MHZ, CD3OD), δ 5.89 (s, 2H), 4.36 (dd, J1=8 Hz, J2=4 Hz, 2H), 3.12 (m, 4H). MS m/z (ESI): 315 [M+H]+.
L-Aspartic acid dibenzyl ester (1 g, 3.19 mmol) and propionyl chloride (353 mg, 3.83 mmol) were dissolved in dichloromethane (10 mL), triethylamine (0.88 mL, 6.39 ml) was added dropwise into the reaction system, and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 980 mg of dibenzyl propionyl-L-aspartic acid ester. MS m/z (ESI): 370 [M+H]+.
Dibenzyl propionyl-L-aspartic acid ester (200 mg, 0.54 mmol) and palladium-on-carbon (40 mg) were dissolved in tetrahydrofuran (3 mL), added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated to obtain 80 mg of propionyl-L-aspartic acid (Compound 58). 1H NMR (500 MHZ, DMSO-d6) δ 12.48 (s, 2H), 8.07 (d, J=8.0 Hz, 1H), 4.51 (td, J=7.5, 5.7 Hz, 1H), 2.69-2.51 (m, 2H), 2.09 (q, J=7.6 Hz, 2H), 0.96 (t, J=7.6 Hz, 3H). MS m/z (ESI): 190 [M+H]+.
L-aspartic acid dibenzyl ester (1 g, 3.19 mmol) and isobutyryl chloride (406 mg, 3.83 mmol) were dissolved in dichloromethane (10 mL), then added dropwise with triethylamine (0.88 mL, 6.39 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 960 mg of dibenzyl isobutyryl-L-aspartic acid ester. MS m/z (ESI): 384 [M+H]+.
Dibenzyl isobutyryl-L-aspartic acid ester (200 mg, 0.52 mmol) and palladium-on-carbon (40 mg) were dissolved in tetrahydrofuran (3 mL), and added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated to obtain 85 mg of isobutyryl-L-aspartic acid (Compound 59). 1H NMR (500 MHZ, DMSO-d6) δ 12.47 (s, 2H), 8.02 (d, J=8.0 Hz, 1H), 4.50 (td, J=7.6, 5.7 Hz, 1H), 2.70-2.51 (m, 2H), 2.40 (p, J=6.8 Hz, 1H), 0.97 (t, J=6.5 Hz, 6H). MS m/z (ESI): 204 [M+H]+.
L-Aspartic acid dibenzyl ester (1 g, 3.19 mmol) and cyclopropylformyl chloride (398 mg, 3.83 mmol) were dissolved in dichloromethane (10 mL), then added dropwise with triethylamine (0.88 mL, 6.39 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 480 mg of dibenzyl (cyclopropylcarbonyl)-L-aspartic acid ester. MS m/z (ESI): 382 [M+H]+.
Dibenzyl (cyclopropylcarbonyl)-L-aspartic acid ester (200 mg, 0.52 mmol) and palladium-on-carbon (40 mg) were dissolved in tetrahydrofuran (3 mL), then added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated to obtain 90 mg of (cyclopropylcarbonyl)-L-aspartic acid (Compound 60). 1H NMR (500 MHz, DMSO-d6) δ 12.51 (s, 2H), 8.39 (d, J=8.0 Hz, 1H), 4.53 (td, J=7.4, 5.6 Hz, 1H), 2.70-2.52 (m, 2H), 1.66-1.59 (m, 1H), 0.68-0.61 (m, 4H). MS m/z (ESI): 202 [M+H]+.
L-Aspartic acid dibenzyl ester (1 g, 3.19 mmol) and methyl chloroformate (360 mg, 3.83 mmol) were dissolved in dichloromethane (10 mL), then added dropwise with triethylamine (0.88 mL, 6.39 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 600 mg of dibenzyl (methoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 372 [M+H]+.
Dibenzyl (methoxycarbonyl)-L-aspartic acid ester (500 mg, 1.35 mmol) and palladium-on-carbon (100 mg) were dissolved in tetrahydrofuran (5 mL), then added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated to obtain 130 mg of (methoxycarbonyl)-L-aspartic acid (Compound 61). 1H NMR (500 MHZ, DMSO-d6) δ 12.55 (s, 2H), 7.43 (d, J=8.4 Hz, 1H), 4.30 (td, J=8.2, 5.4 Hz, 1H), 3.53 (s, 3H), 2.73-2.51 (m, 2H). MS m/z (ESI): 192 [M+H]+.
L-Aspartic acid dibenzyl ester (1 g, 3.19 mmol) and dimethylcarbamoyl chloride (411 mg, 3.83 mmol) were dissolved in dichloromethane (10 mL), then added dropwise with triethylamine (0.88 mL, 6.39 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 940 mg of dibenzyl (methylcarbamoyl)-L-aspartic acid ester. MS m/z (ESI): 385 [M+H]+.
Dibenzyl (dimethylcarbamoyl)-L-aspartic acid ester (500 mg, 1.30 mmol) and palladium-on-carbon (100 mg) were dissolved in tetrahydrofuran (5 mL), then added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated to obtain 120 mg of (dimethylcarbamoyl)-L-aspartic acid (Compound 62). 1H NMR (500 MHZ, DMSO-d6) δ 12.38 (s, 2H), 6.47 (d, J=8.1 Hz, 1H), 4.39 (td, J=7.6, 5.7 Hz, 1H), 2.77 (s, 6H), 2.73-2.53 (m, 2H). MS m/z (ESI): 205 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyric acid (9 g, 36.44 mmol), benzyl bromide (7.47 g, 43.72 mmol) and potassium carbonate (10.06 g, 72.87 mmol) were dissolved in DMF (10 mL), and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted by adding water, and extracted with ethyl acetate, the reaction solution was washed with brine, and the organic phase was dried, and concentrated to obtain 9 g of 1-benzyl 4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 338 [M+H]+.
1-Benzyl 4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (9 g, 26.71 mmol) was dissolved in dichloromethane (20 mL) at room temperature, then added with a solution of hydrogen chloride in ethyl acetate (2M) (80 ml, 160.24 mmol), and the solution was reacted at room temperature for 1 hour. After it was monitored that the reaction was completed, the precipitated solid was collected by filtration to obtain 6 g of 1-benzyl 4-methyl L-aspartic acid ester. MS m/z (ESI): 238 [M+H]+.
1-Benzyl 4-methyl L-aspartic acid ester (1 g, 4.22 mmol) and acetyl chloride (397 mg, 5.06 mmol) were dissolved in dichloromethane (10 mL), and then added dropwise with triethylamine (1.17 mL, 8.44 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 900 mg of 1-benzyl 4-methyl acetyl-L-aspartic acid ester. MS m/z (ESI): 280 [M+H]+.
1-Benzyl 4-methyl acetyl-L-aspartic acid ester (900 mg, 3.23 mmol) and palladium-on-carbon (180 mg) were dissolved in tetrahydrofuran (5 mL), and added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated, and purified by column chromatography to obtain 50 mg of 4-methyl acetyl-L-aspartic acid ester (Compound 63). 1H NMR (400 MHZ, DMSO-d6) δ 12.68 (s, 1H), 8.22 (d, J=8.0 Hz, 1H), 4.54 (ddd, J=8.0, 7.3, 5.9 Hz, 1H), 3.59 (s, 3H), 2.78-2.59 (m, 2H), 1.82 (s, 3H). MS m/z (ESI): 190 [M+H]+.
1-Benzyl 4-methyl L-aspartic acid ester (1 g, 4.22 mmol) and propionyl chloride (468 mg, 5.06 mmol) were dissolved in dichloromethane (10 mL), and then added dropwise with triethylamine (1.17 mL, 8.44 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 860 mg of 1-benzyl 4-methyl propionyl-L-aspartic acid ester. MS m/z (ESI): 294 [M+H]+.
1-Benzyl 4-methyl propionyl-L-aspartic acid ester (860 mg, 2.93 mmol) and palladium-on-carbon (180 mg) were dissolved in tetrahydrofuran (5 mL), and added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated, and purified by column chromatography to obtain 65 mg of 4-methyl propionyl-L-aspartic acid ester (Compound 64). 1H NMR (400 MHZ, DMSO-d6) δ 12.70 (s, 1H), 8.12 (d, J=8.0 Hz, 1H), 4.55 (td, J=7.6, 6.0 Hz, 1H), 3.59 (s, 3H), 2.79-2.58 (m, 2H), 2.09 (q, J=7.6 Hz, 2H), 0.96 (t, J=7.6 Hz, 3H). MS m/z (ESI): 204 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-1-methoxy-1-oxobutyric acid (9 g, 36.44 mmol), benzyl bromide (7.47 g, 43.72 mmol) and potassium carbonate (10.06 g, 72.87 mmol) were dissolved in DMF (10 mL), and the reaction solution was stirred at room temperature for 16 hours. After the reaction was completed, the reaction solution was diluted by adding water, and extracted with ethyl acetate, the reaction solution was washed with brine, and the organic phase was separated, dried and concentrated to obtain 12 g of 4-benzyl 1-methyl (tert-butoxycarbonyl)-L-aspartic acid ester. MS m/z (ESI): 338 [M+H]+.
4-Benzyl 1-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (12 g, 35.6 mmol) was dissolved in dichloromethane (50 mL) at room temperature, then added with a solution of hydrogen chloride in ethyl acetate (2M) (106 ml, 213.65 mmol), and the solution was reacted at room temperature for 1 hour. After it was monitored that the reaction was completed, the precipitated solid was collected by filtration to obtain 9 g of 4-benzyl 1-methyl L-aspartic acid ester. MS m/z (ESI): 238 [M+H]+.
4-Benzyl 1-methyl L-aspartic acid ester (1 g, 4.22 mmol) and acetyl chloride (397 mg, 5.06 mmol) were dissolved in dichloromethane (10 mL), and then added dropwise with triethylamine (1.17 mL, 8.44 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 600 mg of 4-benzyl 1-methyl acetyl-L-aspartic acid ester. MS m/z (ESI): 280 [M+H]+.
4-Benzyl 1-methyl acetyl-L-aspartic acid ester (600 mg, 2.15 mmol) and palladium-on-carbon (120 mg) were dissolved in tetrahydrofuran (5 mL), and added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, the organic phase was concentrated, and purified through column chromatography to obtain 40 mg of 1-methyl acetyl-L-aspartic acid ester (Compound 65). 1H NMR (400 MHZ, DMSO-d6) δ 12.43 (s, 1H), 8.31 (d, J=7.8 Hz, 1H), 4.56 (td, J=7.4, 5.6 Hz, 1H), 3.60 (s, 3H), 2.72-2.53 (m, 2H), 1.82 (s, 3H). MS m/z (ESI): 190 [M+H]+.
4-Benzyl 1-methyl L-aspartic acid ester (1 g, 4.22 mmol) and propionyl chloride (468 mg, 5.06 mmol) were dissolved in dichloromethane (10 mL), and then added dropwise with triethylamine (1.17 mL, 8.44 ml), and the reaction solution was stirred at room temperature for 2 h. The reaction solution was added with an appropriate amount of water, and extracted with dichloromethane. The organic phase was separated. The organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 650 mg of 4-benzyl 1-methyl propionyl-L-aspartic acid ester. MS m/z (ESI): 294 [M+H]+.
4-Benzyl 1-methyl propionyl-L-aspartic acid ester (650 mg, 2.22 mmol) and palladium-on-carbon (130 mg) were dissolved in tetrahydrofuran (5 mL), and added with two drops of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, the organic phase was concentrated, and purified by column chromatography to obtain 37 mg of 1-methyl propionyl-L-aspartic acid ester (Compound 66). 1H NMR (400 MHZ, DMSO-d6) δ 12.40 (s, 1H), 8.21 (d, J=7.8 Hz, 1H), 4.57 (td, J=7.5, 5.8 Hz, 1H), 3.60 (s, 3H), 2.72-2.54 (m, 2H), 2.10 (q, J=7.6 Hz, 2H), 0.97 (t, J=7.6 Hz, 3H). MS m/z (ESI): 204 [M+H]+.
1-Benzyl 4-methyl L-aspartic acid ester (500 mg, 2.11 mmol), (tert-butoxycarbonyl)-L-alanine (479 mg, 2.5 mmol), HATU (962 mg, 2.53 mmol)) and DMAP(26 mg, 0.21 mmol) were dissolved in DMF (5 mL), added dropwise with DIPEA (506 mg, 3.9 mmol), and then stirred and reacted at room temperature for 2 h. After it was monitored that the reaction was completed, the reaction solution was diluted by adding water, and extracted with ethyl acetate (5 mL*2), the organic phase was washed with water, dried, concentrated, and purified by column chromatography to obtain 400 mg of 1-benzyl 4-methyl (tert-butoxycarbonyl)-L-alanyl-L-aspartic acid ester. MS m/z (ESI): 409 [M+H]+.
1-Benzyl 4-methyl (tert-butoxycarbonyl)-L-alanyl-L-aspartic acid ester (400 mg, 0.98 mmol) was dissolved in dichloromethane (5 mL), a solution of hydrogen chloride in ethyl acetate (2M) (2.9 ml, 5.88 mmol) was added to the reaction system, and the solution was reacted at room temperature for 1 hour. After it was monitored that the reaction was completed, the reaction solution was concentrated to obtain 100 mg of 1-benzyl 4-methyl L-alanyl-L-aspartic acid ester. MS m/z (ESI): 309 [M+H]+
1-Benzyl 4-methyl L-alanyl-L-aspartic acid ester (100 mg, 0.32 mmol) and palladium-on-carbon (50 mg) were dissolved in tetrahydrofuran (3 mL), and added with one drop of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, and the organic phase was concentrated, and purified by column chromatography to obtain 20 mg of (S)-2-((S)-2-aminopropionamido)-4-methoxy-4-oxobutyric acid hydrochloride (Compound 67). 1H NMR (400 MHZ, DMSO-d6) δ 12.60 (s, 1H), 8.94 (d, J=8.0 Hz, 1H), 8.35 (s, 2H), 4.62-4.55 (m, 1H), 3.84 (dd, MS m/z (ESI): 255 [M+H]+.
To a solution of 1-chloroethyl chloroformate (3.02 mL, 28.0 mmol) in anhydrous dichloromethane (40 mL), chlorosulfonic acid (3.72 mL, 56.0 mmol) was slowly added at 0° C. within 10 minutes. The mixture was stirred at 0° C. for 4 h under nitrogen atmosphere. Ice water was added to the reaction system to quench the reaction, and dichloromethane (200 mL) was used for extraction. The organic layer was separated, washed with brine solution, dried over anhydrous sodium sulfate, and concentrated to obtain 4.0 g of 1-chloroethylsulfonyl chloride as a light yellow liquid.
(S)-2-((Tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyric acid and tetrabutylammonium bisulfate were dissolved in dichloromethane, stirred and cooled in an ice bath to 0° C. An aqueous solution of potassium carbonate and a dichloromethane solution of 1-chloroethylsulfonyl chloride were added dropwise to the reaction system in sequence, stirred and slowly heated to room temperature, and stirred overnight. After it was monitored by TLC that the reaction was completed, dichloromethane was added, and washed three times with water, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EA/PE gradient elution, 20:1 to 5:1) to obtain 1.0 g of 1-(1-chloroethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester as a colorless oil.
Sodium iodide and potassium carbonate were dissolved in DMF, placed in an ice bath, stirred and cooled to 0° C. Boc-L-aspartic acid 4-methyl ester was dissolved in DMF and added dropwise into the above reaction system, placed in an ice bath, and stirred for 30 minutes. 1-(1-Chloroethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester was dissolved in DMF and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight, and the reaction was monitored TLC. After the reaction of the raw materials was completed, 10 mL of water was added, and ethyl acetate was used to perform extraction three times, and washing was performed with water three times. The organic phases were separated and combined, then dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography (EA/PE gradient elution, 10:1 to 3:1) to obtain 200 mg of O′1,O1-(ethane-1,1-diyl)4-dimethyl (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) as a white solid.
O′1,O1-(Ethan-1,1-diyl)4-dimethyl (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) was dissolved in dichloromethane, placed in an ice bath, stirred and cooled to 0° C., then added dropwise with 2N solution of hydrochloric acid in ethyl acetate, stirred and slowly warmed to room temperature, and stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 105 mg of O′1,O1-(ethan-1,1-diyl)4-dimethyl (2S,2'S)-bis (2-aminosuccinate) hydrochloride (Compound 68), as a white solid. MS m/z (ESI): 357 [M+H]+.
(S)-2-(((Benzyloxy) carbonyl)amino)-4-methoxy-4-oxobutyric acid and 2,2-dibromopropane were dissolved in acetonitrile, added dropwise with DIPEA, stirred and reacted at room temperature for 2 h. After it was monitored that the reaction was completed, the reaction solution was diluted by adding water, the reaction solution was extracted with ethyl acetate, the organic phase was separated, and washed with water, then dried, concentrated, and purified by column chromatography to obtain 4-dimethyl O′1,O1-(propan-2,2-diyl) (2S,2'S)-bis (2-((((benzyloxy) carbonyl)amino) succinate).
(S)-4-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (5 g), EDCI, triethylamine, DMAP and ethanol were added into dichloromethane, and stirred at room temperature, and the reaction was monitored by TLC. After it was monitored that the reaction was completed, saturated sodium bicarbonate solution was added to the reaction solution, and the organic layer was separated. The organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 4.8 g of 1-benzyl 4-ethyl (tert-butoxycarbonyl)-L-aspartic acid ester.
1-Benzyl 4-ethyl (tert-butoxycarbonyl)-L-aspartic acid ester (4.8 g) and palladium-on-carbon were dissolved in tetrahydrofuran, and added with one drop of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, the organic phase was concentrated, and purified by column chromatography to obtain 3.2 g of(S)-2-((tert-butoxycarbonyl)amino)-4-ethoxy-4-oxobutyric acid.
Sodium iodide and potassium carbonate were dissolved in DMF, placed in an ice bath, stirred and cooled to 0° C. (S)-2-((Tert-butoxycarbonyl)amino)-4-ethoxy-4-oxobutyric acid (1.2 g) was dissolved in DMF and added dropwise into the above reaction system, placed in an ice bath and stirred for 30 minutes. 1-(Chloromethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester was dissolved in DMF and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight. The reaction was monitored by TLC. After the reaction of the raw materials was completed, water was added, extracted with ethyl acetate three times, and washing was performed with water three times. The organic phases were separated and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 720 mg of 1-((((S)-2-((tert-butylcarbonyl)amino)-4-ethoxy-4-oxobutyryl)oxy)methyl) 4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester.
1-((((S)-2-((Tert-butylcarbonyl)amino)-4-ethoxy-4-oxobutyryl)oxy)methyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (720 mg) was dissolved in dichloromethane, stirred in an ice bath and cooled to 0° C., then added dropwise with 2N solution of hydrochloric acid in ethyl acetate, stirred and slowly warmed to room temperature, and stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, and the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 225 mg of 1-((((S)-2-amino-4-ethoxy-4-oxobutyryl)oxy)methyl) 4-methyl L-aspartic acid ester hydrochloride (Compound 70). MS m/z (ESI): 357 [M+H]+.
(S)-4-(Benzyloxy)-3-((tert-butoxycarbonyl)amino)-4-oxobutyric acid (10 g), EDCI, triethylamine, DMAP and isopropanol alcohol were added to dichloromethane, stirred at room temperature, and the reaction was monitored by TLC. After it was monitored that the reaction was completed, a saturated sodium bicarbonate solution was added to the reaction solution, the organic layer was separated, and the organic layer was dried over anhydrous sodium sulfate, filtered, concentrated, and purified to obtain 1-benzyl 4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester (8.8 g).
1-Benzyl 4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester (8.8 g) and palladium-on-carbon were dissolved in tetrahydrofuran, added with one drop of concentrated hydrochloric acid, and the reaction solution was stirred at room temperature for 16 h under a hydrogen atmosphere. After it was monitored that the reaction was completed, the palladium-on-carbon was filtered out using diatomaceous earth, the organic phase was concentrated, and purified by column chromatography to obtain 6.0 g of (S)-2-((tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxybutyric acid.
Sodium iodide and potassium carbonate were dissolved in DMF, placed in an ice bath, stirred and cooled to 0° C. (S)-2-((Tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyric acid (1.6 g) was dissolved in DMF and added dropwise into the above reaction system, placed in an ice bath, and stirred for 30 minutes. 1-(Chloromethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester was dissolved in DMF and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight. The reaction was monitored by TLC. After the reaction of the raw materials was completed, water is added, extracted three times with ethyl acetate, and washing was performed three times with water, the organic phases were separated and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by chromatography to obtain 870 mg of 1-((((S)-2-((tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyryl)oxy)methyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester.
1-((((S)-2-((Tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyryl)oxy)methyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (570 mg) was dissolved in dichloromethane, placed in an ice bath, stirred and cooled to 0° C., added dropwise with 2N solution of hydrochloric acid in ethyl acetate, stirred and slowly heated to room temperature, stirred for about 2 h. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, and the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 140 mg of 1-((((S)-2-amino-4-isopropoxy-4-oxobutyryl)oxy)methyl) 4-methyl L-aspartic acid ester hydrochloride (Compound 71). MS m/z (ESI): 371 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-4-ethoxy-4-oxobutyric acid (3.2 g) and tetrabutylammonium bisulfate were dissolved in dichloromethane, placed in an ice bath, stirred and cooled to 0° C. An aqueous solution of potassium carbonate and a dichloromethane solution of 1-chloromethylsulfonyl chloride were added dropwise to the reaction system in sequence, stirred and slowly warmed to room temperature, and stirred overnight. After it was monitored by TLC that the reaction was completed, dichloromethane was added, washed three times with water, and the organic phase was separated. The organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 1.6 g of 1-(chloromethyl)4-ethyl (tert-butyloxycarbonyl)-L-aspartic acid ester.
Sodium iodide and potassium carbonate were dissolved in DMF, placed in an ice bath, stirred and cooled to 0° C. (S)-2-((Tert-butoxycarbonyl)amino)-4-ethoxy-4-oxobutyric acid was dissolved in DMF and added dropwise into the above reaction system, placed in an ice bath and stirred for 30 minutes. 1-(Chloromethyl)4-ethyl (tert-butoxycarbonyl)-L-aspartic acid ester (1.6 g) was dissolved in DMF and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight. The reaction was monitored by TLC. After the reaction of the raw materials was completed, water was added, extracted with ethyl acetate three times, and washing was performed with water three times. The organic phases were separated and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 850 mg of 4-diethyl O′1,O1-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate).
4-Diethyl O′1,O1-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (850 mg) was dissolved in dichloromethane, placed in an ice bath, stirred and cooled to 0° C., then added dropwise with 2N solution of hydrochloric acid in ethyl acetate, stirred and slowly warmed to room temperature, and stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtered by suction, the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 174 mg of (S)-2-((tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyric acid hydrochloride (Compound 72). MS m/z (ESI): 371 [M+H]+.
(S)-2-((Tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyric acid (4.0 g) and tetrabutylammonium bisulfate were dissolved in dichloromethane, placed in an ice bath, stirred and cooled to 0° C. An aqueous solution of potassium carbonate and a dichloromethane solution of 1-chloromethylsulfonyl chloride were added dropwise to the reaction system in sequence, stirred and slowly warmed to room temperature, and stirred overnight. After it was monitored by TLC that the reaction was completed, dichloromethane was added, washed with water three times, the organic phase was separated, and the organic phase was dried over anhydrous sodium sulfate, filtered, concentrated, and purified by column chromatography to obtain 4.0 g of 1-(chloromethyl)4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester.
Sodium iodide and potassium carbonate were dissolved in DMF, placed in an ice bath, stirred and cooled to 0° C. (S)-2-((Tert-butoxycarbonyl)amino)-4-isopropoxy-4-oxobutyric acid was dissolved in DMF and added dropwise into the above reaction system, placed in an ice bath and stirred for 30 minutes. 1-(Chloromethyl)4-isopropyl (tert-butoxycarbonyl)-L-aspartic acid ester (2.5 g) was added in DMF and added dropwise into the reaction system, stirred and slowly warmed to room temperature, and stirred about overnight. The reaction was monitored by TLC. After the reaction of the raw materials was completed, water was added, extracted with ethyl acetate three times, and washing was performed with water three times. The organic phases were separated and combined, dried over anhydrous sodium sulfate, filtered, concentrated, and purified by chromatography to obtain 1.3 g of 4-diisopropyl O′1,O1-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate).
4-Diisopropyl O′1,O1-methylene (2S,2'S)-bis (2-((tert-butoxycarbonyl)amino) succinate) (0.8 g) was dissolved in dichloromethane, placed an ice bath, stirred and cooled to 0° C., added dropwise with 2N solution of hydrochloric acid in ethyl acetate, stirred and slowly warmed to room temperature, and stirred for about 2 hours. After it was monitored by HPLC that the reaction was completed, the reaction solution was filtrated by suction, the filter cake was washed with a small amount of ethyl acetate, and the filter cake was dried to obtain 270 mg of 4-diisopropyl O′1,O1-methylene (2S,2'S)-bis (2-aminosuccinate) hydrochloride (Compound 73). MS m/z (ESI): 399 [M+H]+.
For the synthesis method of the titled compound, the synthesis of Compound 54 in Example 9 of the present application was referred to. Boc-L-aspartic acid 4-methyl ester was replaced by N-tert-butoxycarbonyl-L-aspartic acid 1-methyl ester in step 2 to obtain 4-((((S)-2-amino-4-methoxy-4-oxobutyryl)oxy)methyl)1-methyl L-aspartic acid ester hydrochloride (Compound 74) as a white solid. MS m/z (ESI): 343 [M+H]+.
Ethylene glycol (0.3 g, 4.84 mmol), S-2-(tert-butoxycarbonyl)amino-4-methoxy-4-oxobutyric acid (2.5 g, 10.16 mmol), EDCI (2.32 g, 12.1 mmol) and DIPEA (3.12 g, 24.2 mmol) were dissolved in DMF (20 mL), the reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was completed, an appropriate amount of ice water and ethyl acetate were added, the organic phase was separated. The organic phase was washed with water, dried, and purified by column chromatography (PE:EA=3:1) to obtain 1.5 g of O′1,O1-(ethan-1,2-diyl)4-dimethyl (2S,2'S)-bis (2-(tert-butoxycarbonyl)amino) succinic acid). MS (ESI): mass calcd. For C22H36N2O12 520.2 m/z found 521.2 [M+H]+.
At room temperature, O′1,O1-(ethan-1,2-diyl)4-dimethyl (2S,2'S)-bis (2-(tert-butoxycarbonyl)amino) succinic acid) (1.5 g, 2.88 mmol) was dissolved in dichloromethane (20 mL), and then added with a solution of hydrochloric acid in ethyl acetate (17.29 mL, 2M, 34.58 mmol) under ice bath, and the solution was reacted at room temperature for 1 hour. After it was monitored that the reaction was completed, the supernatant was discarded, and the solid was dried to obtain 800 mg of O′1,O1-(ethan-1,2-diyl)4-dimethyl (2S,2'S)-bis (2-aminosuccinate) hydrochloride. MS (ESI): mass calcd. For C12H2ON2O8+2HCl, 320.1 m/z found 321.1 [M+H]+. 1H NMR (DMSO-d6) δ: 8.88 (br s, 6H), 4.32-4.42 (m, 6H), 3.66 (s, 6H), 3.00-3.12 (m, 4H).
S-2-(Tert-butoxycarbonyl)amino-4-methoxy-4-oxobutyric acid (1.9 g, 7.68 mmol), 2-bromoethanol (1.06 g, 8.45 mmol), EDCI (2.21 g, 11.53 mmol) and DIPEA (3.82 ml, 23.05 mmol) were dissolved in DMF (20 mL), the reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was completed, an appropriate amount of ice water and ethyl acetate were added, and the organic phase was separated. The organic phase was washed with water, dried, and purified by column chromatography (PE:EA=4:1) to obtain 1.1 g of 1-(2-bromoethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester, MS (ESI): mass calcd. For C12H2OBrNO6 353.0 m/z found 354.0 [M+H]+.
1-(2-Bromoethyl)4-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (1 g, 2.82 mmol), (S)-3-(tert-butoxycarbonyl)amino-4-methoxy-4-oxobutyric acid (837 mg, 3.39 mmol), potassium iodide (938 mg, 5.65 mmol), and potassium carbonate (780 mg, 5.65 mmol) were dissolved in DMF (20 mL), and the reaction solution was stirred at room temperature for 2 hours under a nitrogen atmosphere. After the reaction was completed, an appropriate amount of ice water and ethyl acetate were added, and the organic phase was separated. The organic phase was washed with water, dried, and purified by column chromatography (PE:EA=3:1) to obtain 460 mg of 4-(2-((S)-2-(tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyryl)oxy)ethyl)1-methyl (tert-butoxycarbonyl)-L-aspartic acid ester, MS (ESI): mass calcd. For C22H36N2O12 520.2 m/z found 521.2 [M+H]+.
At room temperature, 4-(2-((S)-2-(tert-butoxycarbonyl)amino)-4-methoxy-4-oxobutyryl)oxy)ethyl)1-methyl (tert-butoxycarbonyl)-L-aspartic acid ester (460 mg, 0.88 mmol) was dissolved in dichloromethane (10 mL), a solution of hydrochloric acid in ethyl acetate (5.3 mL, 2M, 10.6 mmol) was added to the reaction system under an ice bath, the solution was reacted at room temperature for 1 hour. After it was monitored that the reaction was completed, the supernatant was poured off, and the solid was dried to obtain 180 mg of 4-(2-(S)-2-amino-4-methoxy-4-oxobutyryl)ethyl)1-methyl L-aspartic acid ester hydrochloride, MS (ESI): mass calcd. For C12H2ON2O8+2HCl, 320.1 m/z found 321.1 [M+H]+. 1H NMR (DMSO-d6) δ:8.87 (br s, 6H), 4.33-4.42 (m, 4H), 4.26-4.32 (m, 2H), 3.74 (s, 3H), 3.66 (s, 3H), 2.98-3.14 (m, 4H).
1. Recovery of LX2 cells:
2. Replacement of cell medium:
After the cells were recovered, the complete medium was replaced according to the cell growth status, degree of adhesion, and color of the medium. LX2 cells were larger in size. After adhesion, the cells were pleomorphic and mainly spindle-shaped. The cells had good refractive index under the microscope and the cells proliferated significantly on the 2nd to 3rd days. Replacement with fresh cell culture medium was carried out according to the density and status of LX2 cells in the culture dish. Note: Before replacement of medium, fresh complete culture medium should be placed in a 37° C. water bath and preheated for 10 to 15 minutes. A negative pressure suction device was used to suck out the old culture medium, the cells were washed twice with 4 mL of 1×PBS buffer, and then the preheated complete culture medium was used for replacement. The cells were placed in a 37° C. incubator and continuously cultured.
3. Cell passage: When the cell confluence reached about 90%, cell passage culture could be carried out.
4. Western blotting to detect protein expression level
4.1 Extraction of total cell protein: The cells were taken out, the original culture medium was discarded, 1 to 2 mL of 1×PBS was added, washing was performed twice, digestion was performed with trypsin, the supernatant was discarded, the cells at the bottom of the tube were gently moved by flicking, and then 1 mL of PBS was added again to wash the cells twice; after being centrifuged at 1200 rpm for 5 minutes, the supernatant was completely discarded, the cells at the bottom of the tube was mixed well by flicking. Depending on the amount of cells, 50 to 100 μL of protein lysis buffer containing protease inhibitor and phosphatase inhibitor was added, the cells were mixed thoroughly by pipetting, placed on ice and lysed for 30 minutes, then centrifuged at 12,000 rcf for 15 minutes at 4° C., and the supernatant was pipetted and transferred into a new EP tube to obtain the total cell protein, which could be directly used for subsequent determination of protein concentration or stored at −80° C.
4.2 Determination of protein concentration by BCA method: BCA reagent solution A and solution B were mixed well at a ratio of 50:1 to prepare a working solution; 5 μL of the sample to be tested was taken, added into 95 μL of deionized water, mixed, and diluted 20 times, and 20 μL of the diluted sample to be tested was taken and added to a 96-well plate; after 200 μL of the working solution was added, the 96-well plate was placed in a 37° C. incubator and incubated for 30 min, and then tested on the machine.
4.3 Protein denaturation: 100 μg of each of samples was taken respectively, supplemented with protein lysis buffer to reach the volume of each sample, then added with 4× loading buffer at 1/3 of the total volume and mixed thoroughly, underwent denaturation at 99° C. for 10 min, centrifuged and stored at −80° C.
4.4 Protein electrophoresis: The precast gel was installed into the electrophoresis tank, added with 1×MOPS electrophoresis solution, the comb on the top of the precast gel was removed, the loading well was gently pipetted to remove the residual gel in the well; pre-stained protein Maker and equal quality of the protein sample were carefully added into the gel well; the voltage was adjusted to 60 V to 80 V. After 30 minutes, the voltage was adjusted to 110 V to 120 V. When bromophenol blue reached the groove at the bottom of the gel, the power supply was disconnected to terminate electrophoresis, and the entire electrophoresis process took about 2 h to 2.5 h.
4.5 Electro-transfer: PVDF membrane was immersed in anhydrous methanol to activate for 1 minute; the transfer clamp was placed black-side down, sponge-filter paper-PAGE gel-PVDF membrane-filter paper-sponge were placed in order from bottom to top, and air bubbles between glue/membrane and filter paper were carefully driven out, the transfer clamp was then fasten and put into the transfer tank, with the PAGE gel facing the negative electrode and the PVDF membrane facing the positive electrode; it was placed in an ice box, and pre-cooled 1× electro-transfer solution was poured therein; the power supply was adjusted to 100 V, and membrane transferring was carried out for 1 h to 1.5 h.
4.6 Blocking: After the membrane transferring was completed, the PVDF membrane was taken out, and washed several times with 1×TBST. After the residual electro-transfer solution on the membrane was removed, the membrane was immersed in 5% skim milk for blocking for 2 hours at room temperature, or overnight at 4° C.
4.7 Incubation of primary antibody: The primary antibodies GAPDH and HCBP6 were diluted according to 1/1000; according to the molecular weight of the protein, the PVDF membrane at the corresponding position and the above antibodies were sealed in a hybridization bag, and incubated at 4° C. overnight.
4.8 Washing membrane: The hybridization bag was cut to recover the primary antibodies, the PVDF membrane was taken out, placed in 1×TBST, and washed three times on a shaker, 10 minutes each time.
4.9 Incubation of secondary antibody: The secondary antibodies were diluted at a ratio of 1/5,000 to 1/10,000, the above PVDF membrane was immersed in the corresponding secondary antibody, placed on a shaker, and incubated at room temperature for 45-60 minutes.
4.10 Washing membrane: The secondary antibodies were recovered, the PVDF membrane was taken out, placed into 1×TBST, and washed three times on a shaker, 10 minutes each time; when phosphorylated protein was detected, it was necessary to appropriately reduce the washing times and washing time.
4.11 Exposure and color development: The membrane was placed in the dark box of Fusion Solo imager, added dropwise with ECL exposure solution to perform color development.
5. Q-PCR detection of target gene expression
5.1 Extraction of total cellular RNA
Test compounds were added to the LX-2 cells respectively, and total RNA in the cells was extracted 48 hours later. This experiment was performed strictly in accordance with the instructions of the Total RNA Kit. The steps were as follows (note: this experiment was performed on ice throughout the entire process, and special RNase-free tips were used throughout the entire process):
5.2 Reverse transcription of RNA into cDNA: According to the instructions of PrimeScript™ RT reagent Kit (Perfect Real Time), the specific experimental steps were as follows:
5.2.1 Preparation of reverse transcription reaction system (operated on ice):
5.2.2 Reverse transcription reaction conditions on machine were as follows:
5.2.3 The obtained product was cDNA, which could be directly used in subsequent Q-PCR experiment or stored at −20° C.
5.3 Q-PCR detection of target gene expression: According to the instructions of Power SYBR® Green PCR Master Mix, the specific operations were as follows:
5.3.1 The Q-PCR reaction system was prepared according to the instructions as follows:
5.3.2 The ABI PRISM® 7500 system was used and the following two-step standard amplification procedure was adopted:
After the reaction was completed, the amplification curve and dissolution curve of Q-PCR were confirmed, and the relative quantification method (2-44° C., method) was used to perform data analysis on the experimental results.
5.3.3 The primer sequences used were as follows:
Long-term intraperitoneal injection of carbon tetrachloride (CCl4) induced reversible liver fibrosis, which was often used to screen and evaluate anti-hepatic fibrosis drugs. C57BL/6 wild-type mice were randomly divided into control group, carbon tetrachloride group and treatment group. The treatments were as follows:
Mice in the carbon tetrachloride group: The animals were intraperitoneally injected with carbon tetrachloride in a total amount of 0.5 μL/g, diluted with corn oil to 10 μL/g, 3 times/week. After 4 weeks of modeling, the carbon tetrachloride group was randomly divided into groups, plus the negative control group:
The animals were treated for 4 weeks, and blood was collected from the eyeballs 48 hours after the last injection of carbon tetrachloride. About 1 mL of whole blood per animal was collected in an anticoagulant tube, and immediately placed on ice and allowed to stand for 1 hour. Centrifugation was performed at 4° C., 3000 rpm for 15 min. About 0.4 mL of the supernatant was taken and added into an EP tube, in which it should be carefully avoided to pipet the lower layer liquid, and then it was stored in a −80° C. refrigerator. The mice were euthanized, and fresh liver tissue was taken, part of which was placed in a cryopreservation tube and stored in liquid nitrogen for later use; the other part was fixed in 4% paraformaldehyde solution for 16 h to 24 h for subsequent experiments.
(2) Mice in the negative control group (n=10): The animals were fed with normal diet, and 200 μL of drinking water was administered by gavage, bid.
Mice in HFD or CHOL group (n=10): The animals were fed with HFD or CHOL diet, and 200 μL of normal saline was administered by gavage at the same time, bid. (Note: HFD (60% high fat) feed (Whitby Technology Development (Beijing) Co., Ltd.) (D12492), CHOL (40% high fat +1.25% cholesterol) feed (Whitby Technology Development (Beijing) Co., Ltd.) (D12108C)) (3) Mice in Compound 6-low dose group (n=10): The animals were fed with HFD or CHOL diet, and administrated simultaneously with Compound 6 at a dose of 10 mg/kg by gavage, bid.
(4) Mice in Compound 6-middle dose group (n=10): The animals were fed with HFD or CHOL diet, and administrated simultaneously with Compound 6 at a dose of 50 mg/kg by gavage, bid.
(5) Mice in Compound 6-high dose group (n=10): The animals were fed with HFD or CHOL diet, and administrated simultaneously with Compound 6 at a dose of 150 mg/kg by gavage, bid.
After 8 weeks of administration, blood was collected by enucleating the eyeballs. About 1 mL of whole blood per animal was collected in an anticoagulant tube, and immediately allowed to stand on ice. Centrifugation was performed at 4° C., 3000 rpm for 15 min. 0.4 ml of the supernatant was taken and added into an EP tube, and it should be carefully avoided to pipette the lower layer liquid, and then it was stored in a −80° C. refrigerator to detect biochemical indicators such as blood lipid and transaminase. The mice were euthanized, and fresh liver tissue was taken and weighed, part of which was placed in a cryopreservation tube and stored in liquid nitrogen for later use, and the other part was fixed in 4% paraformaldehyde solution for 16 h to 24 h, and underwent histological HE staining and oil red O staining to determine the severity of fatty liver.
(6 The slide was placed horizontally on a 70° C. slide dryer for preliminary drying, and then placed in a 70° C. oven to be baked for about 1 hour.
Masson staining is one of the classic and authoritative staining methods for collagen fiber staining. Collagen fibers appear red. The staining steps were as follows:
Conventional baking and dewaxing (steps were the same as before); oxidization was performed with potassium permanganate solution for 5 minutes, washing was performed with water; staining was performed with Masson staining solution for 5 minutes; 0.2% acetic acid solution for 2 to 3 seconds; 5% phosphotungstic acid solution for 5 minutes; aniline blue solution for 7 minutes; washing was performed 3 times with 0.2% acetic acid solution; dehydration, transparency of section, sealing of section, natural drying, and storage at room temperature were performed by routine methods.
An imaging system was used to collect images of stained tissue on the section, and an analysis software was used to automatically read the tissue measurement area, calculate the positive area and tissue area in the measurement area, and calculate the proportion of positive area.
Mouse serum was taken to detect the related levels of ALT and AST in the serum.
(1) Extraction of total tissue RNA
According to the instructions of PrimeScript™ RT reagent Kit (Perfect Real Time), the specific experimental steps were as follows:
The obtained product was cDNA, which could be directly used in subsequent Q-PCR experiment or stored at −20° C.
According to the instructions of Power SYBR® Green PCR Master Mix, the specific operations were as follows:
After the reaction was completed, the amplification curve and dissolution curve of Q-PCR were confirmed, and the relative quantification method (2-44CT method) was used to perform data analysis on the experimental results.
Several portions of the drug was taken, and used to prepare a series of solutions from an unsaturated solution to a saturated solution, which were oscillated under constant temperature conditions until equilibrium was reached, and filtered through a membrane, the filtrate was taken for analysis to determine the actual concentration S of the drug in the solution, and a graph for the concentration c of the prepared solution was drawn, and the turning point A of the curve in the graph was the equilibrium solubility of the drug.
Thermodynamic solubility was measured using the shake flask method (pH=7.4) and analyzed using high-performance liquid chromatography-diode array detector (HPLC-DAD) or high-performance liquid chromatography-tandem mass spectrometry (LC/MS/MS).
Rats were used as test animals to study the pharmacokinetic behavior of the following compounds in rat plasma.
The drugs were self-made according to the examples of the present invention.
SD rats, 6-8 weeks old, 3 for each administration method/example, male.
2.3 Eating status:
The animals were fasted overnight, ate food 8 hours after taking the drug, and drank water freely.
2.4 Sample collection:
Blood was collected before administration and at 5 min, 15 min, 30 min, 1 hr, 2 hr, 4 hr, 8 hr and 24 hr after administration.
The blood was placed on wet ice and centrifuged to obtain plasma sample (2000 g, 5 min at 4° C.).
The LC-MS/MS method was used to determine the blood drug concentration. The HPLC conditions were: mobile phase A: H2O-0.5% FA; mobile phase B: ACN-0.5% FA;
WinNonlin 8.2 non-compartmental model was used to estimate pharmacokinetic parameters (PK parameters, including but not limited to peak concentration (Cmax), time to peak (Tmax), terminal elimination rate (Ke), terminal elimination half-life (T1/2), area under drug-time curve (AUC), clearance rate (CL), apparent volume of distribution (Vd), mean residence time (MRT), bioavailability (F), etc.).
LX2 cells were passaged and plated normally. After 12 hours of adherent growth, 200 UM of the compound to be tested was added. After 48 hours, the cells were collected, total RNA was extracted, and the expression of NS3TP1 gene was detected using Q-PCR method.
The results (Table 1) show that the compounds of the present invention could up-regulate the mRNA expression level.
LX2 cells were passaged and plated normally. After adherent growth for 12 hours, 50 UM of the test compound was added. After 48 hours, the cells were collected, total RNA was extracted, and Q-PCR was used to detect the changes in the mRNA expression levels of liver fibrosis-related genes (ACTA2 encoding α-SMA, COL1A1 and COLIA2 encoding collagen I, COL3A1 encoding collagen III, SMAD3 encoding Smad3) and inflammation-related genes (IL1β encoding IL-1β, TNFα encoding TNFα) (for specific methods, see 5. Q-PCR detection of target gene expression as described above). α-SMA was a marker of hepatic stellate cell activation, and collagen I and collagen III were the main components of extracellular matrix deposition.
The results (Table 2) showed that the compounds of the present invention could inhibit the mRNA expression levels of liver fibrosis-related genes. Among them, Compound 2, Compound 4, Compound 6, Compound 8, Compound 10, Compound 12, Compound 14, Compound 15, Compound 18, Compound 19, Compound 21, Compound 22, Compound 26, Compound 28, Compound 30, Compound 33, Compound 35, Compound 38, Compound 40, Compound 42, Compound 44, Compound 47, Compound 48, Compound 50, Compound 52, Compound 53, Compound 54, Compound 59, Compound 61, Compound 63, Compound 66, Compound 70, Compound 71, Compound 72, Compound 74, etc., could inhibit the expression of collagen I and collagen III at the mRNA level; Compound 4, Compound 6, Compound 26, Compound 28, Compound 33, Compound 53, Compound 54, Compound 68, Compound 70, Compound 73, etc., could inhibit the expression of α-SMA at the mRNA level; Compound 1, Compound 4, Compound 6, Compound 9, Compound 12, Compound 15, Compound 22, Compound 26, and Compound 28 could inhibit the expression of Smad3 at the mRNA level. Overall, Compound 4, Compound 6, Compound 12, Compound 26, Compound 47, Compound 48, Compound 52, and Compound 54 had stronger effects to inhibit the mRNA expression levels of liver fibrosis-related genes.
(c) Evaluation of Drug Efficacy in In-Vitro Liver Fibrosis Model in Activated LX2 Cells-qPCR LX2 cells were passaged and plated normally. After 12 hours of adherent growth, TGFβ1 (5 ng/mL) was administered to activate the cells, and a compound to be tested with concentration of 25 μM was added. After 24 hours, the cells were collected, the total RNA was extracted, and the real-time PCR was used to detect the expression levels of liver fibrosis-related genes and inflammation-related genes. α-SMA was a marker of hepatic stellate cell activation, and collagen 1 and collagen 3 were the main components of extracellular matrix deposition.
The results showed that in this test, the compounds of the present invention could inhibit the mRNA expression levels of liver fibrosis-related genes.
LX2 cells were passaged and plated normally. After 12 hours of adherent growth, a compound to be tested was added at different concentrations (50 μM, 100 μM, 200 μM, 400 μM). After 48 hours, the cells were collected, proteins were extracted, and Western blot was used to detect the changes in expression levels of proteins (α-SMA, collagen I, collagen III, FN) (for specific methods, see 4. Detection of protein expression level by protein immunoblotting) of liver fibrosis-related genes. α-SMA was a marker of hepatic stellate cell activation, collagen I and collagen III were the main components of extracellular matrix deposition, and fibronectin (FN) was an adhesion glycoprotein mainly involved in cell adhesion interaction.
The results showed that the compounds of the present invention could inhibit the mRNA expression levels of liver fibrosis-related genes. The results were not fully displayed (con was the control well, P1 was the positive control well, “ASP-50” was 50 μM aspartic acid, and the other numbers were all in the form of compound number-concentration (μM). Among them, Compound 6, Compound 47, and Compound 54 had stronger inhibitory effects on collagen I and collagen III protein expression (
LX2 cells were passaged and plated normally. After 12 hours of adherent growth, TGFβ1 (5 ng/mL) was administered to activate the cells, and a compound to be tested was added at different concentrations. After 24 hours, the cells were collected, proteins were extracted, and Western blot was used to detect the changes in expression levels of proteins (α-SMA, collagen I) of liver fibrosis-related genes (for specific methods, see 4. Detection of protein expression level by protein immunoblotting). α-SMA was a marker of hepatic stellate cell activation, and collagen I was the main component of extracellular matrix deposition.
The results showed that after stimulation of LX2 cells by TGFβ, the expression of collagen I in the cells increased. After the administration of Compounds 54, 77a, and 78a, the expression of Collagen I and α-SMA in the cells significantly decreased (
After CCl4 induction, the expression of NS3TP1 in mouse liver tissue decreased significantly, indicating that there could be a correlation between mouse liver fibrosis and abnormal expression of NS3TP1. After treatment with different doses of Compound 6, compared with the untreated group, the expression of NS3TP1 in the liver tissue of mice gradually increased in a dose-dependent manner (
Observation and computer analysis of Masson-stained sections showed that collagen (blue) was obviously deposited in mouse liver tissue after CCl4 induction. After treatment with Compound 4 (20 mg/kg/d), Compound 6 (25 mg/kg/d), Compound 47 (20 mg/kg/d), and Compound 54 (40 mg/kg/d), compared with the untreated group and the aspartic acid group (30 mg/kg/d), collagen deposition in the mouse liver tissue was reduced, indicating that the compounds of the present invention (e.g., Compound 4, Compound 6, Compound 47, Compound 54) could have the ability to reduce collagen deposition and inhibit liver fibrosis (
Observation and computer analysis of Masson-stained sections showed that collagen (blue) was obviously deposited in mouse liver tissue after CCl4 induction. After treatment with Compound 6 (10 mg/kg/bid, 50 mg/kg/bid, 150 mg/kg/bid), compared with the untreated group, collagen deposition in the mouse liver tissue decreased, indicating that the compounds of the present invention (e.g., Compound 6) could have the ability to reduce collagen deposition and inhibit liver fibrosis (
After CCl4 induction, the levels of aminotransferases ALT and AST in mice significantly increased, and obvious liver damage occurred. After administration of the compounds of the present invention, compared with the untreated group, the ALT in the plasma of mice decreased, indicating that the compounds of the present application had protective effects on liver cells, and could reduce liver damage and improve liver function in mice.
It could be seen from the activity results that,
1. in the mouse model of liver fibrosis induced by CCl4, the expression level of NS3TP1 in liver tissue decreased, indicating that there could be a correlation between liver fibrosis and abnormal expression of NS3TP1 in mice.
2. from the activity characterization results, it could be seen that the compounds of the present invention had the effect of up-regulating the expression of NS3TP1 in the cell experiment and the in vivo experiment.
3. from the activity characterization results, it could be seen that the compounds of the present invention also showed the effect of alleviating the symptoms of liver fibrosis in liver fibrosis models at the cellular level and animal level.
Observation and computer analysis of H&E stained sections showed that after being fed with HFD diet, a large number of lipid droplets accumulated in the liver tissue of mice, and the liver tissue showed soapy diffuse fatty change. After treatment with Compound 6, compared with the untreated group, lipid droplet aggregation in the liver tissue of mice was reduced in a dose-dependent manner, indicating that the compounds of the present invention (e.g., Compound 6) could have the effect of inhibiting the formation of fatty liver (
Observation and computer analysis of H&E stained sections showed that after being fed CHOL diet, a large number of lipid droplets accumulated in the liver tissue of mice, and the liver tissue showed soapy diffuse fatty change. After treatment with Compound 6, compared with the untreated group, lipid droplet aggregation in the liver tissue of mice was reduced in a dose-dependent manner, indicating that the compounds of the present invention (e.g., Compound 6) could have the effect of inhibiting the formation of fatty liver (
Some compounds of the present invention showed good solubility, which was obviously better than that of aspartic acid.
After aspartic acid and Compound 6 were administered to rats respectively (PO 20 mg/kg), it could be seen that the Cmax and AUC of the rats administrated with Compound 6 were significantly higher than those of rats administrated with aspartic acid, suggesting that Compound 6 had a significant increase in in vivo exposure amount as compared with aspartic acid. At the same time, it could be seen that the Tmax of rats administered with Compound 6 was significantly smaller than that of rats administered with aspartic acid, suggesting that Compound 6 could reach the highest concentration faster.
The results showed that the compounds of the present invention had good pharmacokinetic parameters, performed better than aspartic acid in multiple pharmacokinetic parameters such as AUC, Cmax and/or bioavailability, and had better druggability.
| Number | Date | Country | Kind |
|---|---|---|---|
| 202111341140.1 | Nov 2021 | CN | national |
| 202210661248.7 | Jun 2022 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2022/131311 | 11/11/2022 | WO |
