Application of MK2 inhibitor in preparation of medicine for preventing and / or treating diseases

Abstract

Relates to medical application of compounds 1 and 2 and pharmaceutically acceptable salts thereof, including application in preparation of medicines for preventing or treating atopic dermatitis and asthma.

Description

Application of an MK2 inhibitor in the preparation of drugs for preventing and / or treating diseases Technical Field

[0001] The present application belongs to the field of medical application technology, and relates to the medical application of compounds 1, 2 and their pharmaceutically acceptable salts, including their application in the preparation of drugs for preventing or treating atopic dermatitis and asthma, the pharmaceutical application of compounds 1 and 2 in combination with methotrexate, raw materials of compounds 1 and 2 containing limited amounts of impurities and their pharmaceutical compositions, as well as their application in the preparation of drugs for preventing or treating the above-mentioned diseases. Background Art

[0002] As the body's first line of defense and largest organ, the skin constantly participates in the body's functions, maintaining the balance between the body and the natural environment. Abnormalities can also be reflected on the skin's surface. The skin possesses nearly perfect physiological protective functions, such as barrier function, sensory function, temperature regulation, absorption, secretion, and excretion, playing a vital role in maintaining the body's health.

[0003] Once the physiological functions of the skin are damaged, skin diseases will occur. There are many factors that cause skin diseases, such as internal factors and external factors. Internal factors include chronic infection lesions (chronic cholecystitis, tonsillitis, intestinal parasitic diseases, etc.), endocrine and metabolic changes (menstrual disorders, pregnancy, etc.), blood circulation disorders (varicose veins in the lower legs, etc.), neuropsychiatric factors, genetic factors, etc.; external factors include food intake (such as fish, shrimp, beef and mutton, etc.), inhaled substances (such as pollen, dust mites, etc.), living environment (such as cold, hot, dry, etc.), animal fur, and various physical and chemical substances (such as cosmetics, soap, synthetic fibers, etc.). All of the above can induce or aggravate skin diseases.

[0004] Studies have shown that inflammatory cytokines and chemokines, such as IL-4 and IL-13, may induce atopic dermatitis, asthma, etc.

[0005] Among them, the relationship between cytokines and asthma:

[0006] Asthma is a chronic airway inflammatory disease, and its pathogenesis is related to the imbalance of multiple cytokines. IL-4 and IL-13 are two important Th2 cytokines that play a key role in the occurrence and development of asthma. A large number of studies have shown that increased levels of IL-4 and IL-13 are positively correlated with the severity of asthma. For example, a study by Lee et al. [1] found that IL-4 and IL-13 were significantly elevated in the airways and peripheral blood of asthma patients. IL-4 and IL-13 can promote the secretion of IgE by B lymphocytes, stimulate mucus secretion, and lead to airway hyperresponsiveness and remodeling. In addition, IL-4 and IL-13 can also directly act on smooth muscle cells, causing their contraction and proliferation [2]. Therefore, these two cytokines are considered to be key cytokines in the onset of asthma, and inhibiting IL-4 and IL-13 signaling or their biological activity may become a new target for asthma treatment [3].

[0007] [1]Lee CG, Homer RJ, Zhu Z et al. Interleukin-13 induces tissue fibrosis by selectively stimulating and activating transforming growth factor beta(1). J Exp Med 2001;194:809-821.

[0008] [2]Hirst SJ.Regulation of airway smooth muscle cell immunomodulatory function:role in asthma.Respir Physiol Neurobiol 2003;137:309-326.

[0009] [3]Wills-Karp M, Luyimbazi J, Xu X et al. Interleukin-13: central mediator ofallergic asthma. Science 1998; 282:2258-2261.

[0010] Among them, the relationship between cytokines and atopic dermatitis:

[0011] Atopic dermatitis is a chronic, recurrent inflammatory skin disease whose pathogenesis is related to the activation of Th2 immune responses. IL-4 and IL-13 are two important Th2 cytokines that play an important role in the development and progression of atopic dermatitis. Some studies have shown that the expression levels of IL-4 and IL-13 in skin lesions and peripheral blood of patients with atopic dermatitis are significantly increased [4]. IL-4 and IL-13 can promote excessive proliferation and differentiation of epidermal keratinocytes, stimulate the production of large amounts of IgE antibodies in skin tissue, and lead to skin inflammation [5]. In addition, IL-4 and IL-13 can also promote angiogenesis and participate in the remodeling process of skin tissue [6]. Therefore, IL-4 and IL-13 are considered to be key pathogenic factors of atopic dermatitis. Inhibiting IL-4 / IL-13 signaling or its biological activity may become a new strategy for the treatment of atopic dermatitis [7].

[0012] [4]Leung DY,et al.New insights into atopic dermatitis.J Clin Invest 2004;113:651-657.

[0013] [5]Bieber T.Atopic dermatitis.N Engl J Med 2008;358:1483-1494.

[0014] [6]Uchida T, et al. The innate immune system in atopic dermatitis. Curr Opin Immunol 2008; 20:666-672.

[0015] [7]Ruzicka T, et al. Anti-interleukin-4and 13antibodies for atopic eczema. N Engl J Med 2017;376:826-835.

[0016] Methotrexate is a dihydrofolate reductase inhibitor that inhibits the metabolism of purine and pyrimidine. It has the effects of reducing antigen-dependent T lymphocyte proliferation, increasing extracellular adenosine release, and promoting adenosine-mediated inhibition of inflammatory responses. It is easy to take and inexpensive. It is currently the first-line drug for the clinical treatment of rheumatoid arthritis. However, long-term use can cause toxic side effects such as liver and kidney damage.

[0017] Clinical studies have shown that long-term use of methotrexate can cause liver damage in patients. Therefore, lowering the therapeutic dose of rheumatoid arthritis drugs, while reducing and alleviating side effects, while maintaining or improving efficacy, is of great significance to patients.

[0018] International application PCT / US2022 / 022525 discloses a series of MK2 compounds. The present invention prepares targeted target impurities, establishes a detection method for target impurities, and effectively controls the quality of an MK2 compound raw material and pharmaceutical composition.

[0019] Summary of the Invention

[0020] In view of the problems existing in the prior art, the present application first relates to the medical uses of compounds 1, 2 and their pharmaceutically acceptable salts, including their use in the preparation of drugs for preventing or treating atopic dermatitis and asthma.

[0021] In one aspect, the present invention provides a method for preventing or treating atopic dermatitis using a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0022] In another aspect, the present invention provides a method for preventing or treating atopic dermatitis using a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0023] Inflammatory factors IL-13 and IL-4 can induce diseases such as atopic dermatitis and asthma. As a preferred technical solution of the present invention, the compounds of the present invention are used to treat atopic dermatitis, asthma and other diseases by inhibiting the inflammatory factors IL-13 and IL-4.

[0024] The present invention also provides a method for preventing or treating asthma using a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0025] The present invention also provides a method for preventing or treating asthma using a compound represented by the following formula or a pharmaceutically acceptable salt thereof:

[0026] The present invention also provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising a compound shown in the following formula and a pharmaceutically acceptable salt thereof:

[0027] The present invention also provides a pharmaceutical composition for preventing or treating atopic dermatitis, comprising a compound shown in the following formula and a pharmaceutically acceptable salt thereof:

[0028] The present invention also provides a pharmaceutical composition for preventing or treating asthma, comprising a compound shown in the following formula and a pharmaceutically acceptable salt thereof:

[0029] The present invention also provides a pharmaceutical composition for preventing or treating asthma, comprising a compound shown in the following formula and a pharmaceutically acceptable salt thereof:

[0030] The medical use described herein (ie, a method for preventing or treating atopic dermatitis and / or asthma) and the pharmaceutical composition for said medical use comprise Compound 1 or 2 in an amount of about 1 mg to about 300 mg, and the pharmaceutical composition is preferably administered orally.

[0031] In any embodiment, compound 1 or 2 is administered at about 1 mg to about 300 mg, about 5 mg to about 300 mg, about 7.5 mg to about 300 mg, about 10 mg to about 300 mg, about 12.5 mg to about 300 mg, about 15 mg to about 300 mg, about 17.5 mg to about 300 mg, about 20 mg to about 300 mg, about 22.5 mg to about 300 mg, about 25 mg to about 300 mg, about 27.5 mg to about 300 mg, about 30 mg to about 300 mg, about 30 mg to about 300 mg, about 31.5 mg to about 300 mg, about 32.5 ...3.5 mg to about 300 mg, about 34.5 mg to about 300 mg, about 35.5 mg to about 300 mg, about 36.5 mg to about 300 mg, about 37.5 mg to about 300 mg, about 38.5 mg to about 300 mg, about 39.5 mg to about 300 mg, about 40.5 mg to about 300 mg, about 41.5 mg to about 300 mg, about 42.5 mg to about 300 mg, about 43.5 mg to about 300 mg, about 44.5 mg to about 300 mg, about 45.5 mg to about 300 mg, about 46.5 mg to about 300 mg, about 47.5 mg to about 300 mg, about 48.5 mg to about 300 mg, about 49.5 mg to about about 32.5 mg to about 300 mg, about 35 mg to about 300 mg, about 37.5 mg to about 300 mg, about 40 mg to about 300 mg, about 42.5 mg to about 300 mg, about 45 mg to about 300 mg, about 47.5 mg to about 300 mg, about 50 mg to about 300 mg, about 50 mg to about 290 mg, about 50 mg to about 280 mg, about 50 mg to about 270 mg, about 50 mg to about 260 mg, about 50 mg to about 250 mg, about 50 mg to about 240 mg, about 50 mg to about 230 mg, about 50 mg to about 220 mg, about 50 mg to about 210 mg, about 50 mg to about 200 mg, about 50 mg to about 190 mg, about 50 mg In an amount ranging from about 100 mg to about 150 mg, about 50 mg to about 140 mg, about 50 mg to about 130 mg, about 50 mg to about 120 mg, about 50 mg to about 110 mg, about 50 mg to about 100 mg, about 50 mg to about 90 mg, about 50 mg to about 80 mg, about 50 mg to about 70 mg, about 50 mg to about 60 mg, about 40 mg to about 50 mg, about 30 mg to about 60 mg, about 20 mg to about 70 mg, about 15 mg to about 80 mg, about 10 mg to about 90 mg, about 5 mg to about 100 mg, or any amount therebetween, may be present in a pharmaceutical composition as described herein.

[0032] In any embodiment, compound 1 or 2 is administered at about 1 mg, 2.5 mg, 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 17.5 mg, 20 mg, 22.5 mg, 25 mg, 27.5 mg, 30 mg, 32.5 mg, 35 mg, 37.5 mg, 40 mg, 42.5 mg, 45 mg, 47.5 mg, 50 mg, 52.5 mg, 55 mg, 57.5 mg, 60 mg, 62.5 mg, 65 mg, 67.5 mg, 70 mg, 72.5 mg, 75 mg, 77.5 mg, 80 mg, 82.5mg, 85mg, 87.5mg, 90mg, 92.5mg, 95mg, 97.5mg, 100mg, 105mg, 107.5mg, 110mg, 112.5mg, 115mg, 117.5mg, 120mg, 122 .5mg, 125mg, 127.5mg, 130mg, 132.5mg, 135mg, 137.5mg, 140mg, 142.5mg, 145mg, 147.5mg, 150mg, 152.5mg, 155mg, 157.5mg, 1 60mg, 162.5mg, 165mg, 167.5mg, 170mg, 172.5mg, 175mg, 177.5mg, 180mg, 182.5mg, 185mg, 187.5mg, 190mg, 192.5mg, 195mg, 197.5mg, 200mg, 200mg, 205mg, 207.5mg, 210mg, 212.5mg, 215mg, 217.5mg, 220mg, 222.5mg, 225mg, 227.5mg, 230mg, 232.5mg, or 300 mg.

[0033] In preferred embodiments, the oral pharmaceutical combinations described herein include Compound 1 or 2 in an amount of about 10 mg to about 240 mg.

[0034] In preferred embodiments, Compound 1 or 2 is present in the pharmaceutical compositions described herein in an amount of 10 mg, 25 mg, 50 mg, 80 mg, 100 mg, 120 mg, 160 mg, or 240 mg.

[0035] The present invention provides methods for treating the disease using the compound or compound 2 or a pharmaceutical composition containing the compound. In some embodiments, treatment of a subject with the condition according to the methods and compositions comprising compound 1 or compound 2 as described herein is effective for achieving a circulating plasma concentration of compound 1 or compound 2 (i.e., AUC last ) is considered effective when the expected circulating concentration is reached, for example, >100 h*ng / mL is effective.

[0036] The dosage administered is a therapeutically effective amount of the composition sufficient to cause relief of a symptom or symptoms and may vary according to known factors such as the pharmacodynamic characteristics of the active ingredient and its mode of administration; the age, sex, health and weight of the recipient; the nature and extent of the symptoms; the type of concurrent treatment, the frequency of treatment and the desired effect.

[0037] In some embodiments, the subject can be administered once (e.g., with a single dose or single application) an oral composition including compound 1 or compound 2 as described herein. In some embodiments, the oral composition of the embodiments herein is administered at least once a day (e.g., at least twice, three times, or four times a day). In some embodiments, the oral composition of the embodiments herein can be administered as needed or additionally according to the guidance of a physician every day, twice a day, three times a day, weekly, twice a week, every two weeks, every three weeks, or monthly. The oral composition of the embodiments herein can be administered at any interval to achieve the desired effect in treatment, such as inducing or maintaining the alleviation, prevention, or relief of symptoms or multiple symptoms. In some embodiments, the oral composition of the embodiments herein can be administered to the subject for a period of 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, about one week, about two weeks, about three weeks, about four weeks, about five weeks, about six weeks, about two months, about three months, about four months, about five months, about six months, or within the range of any two of these values. In some embodiments, treatment can continue for at least one week, one month, one year, or additionally according to the guidance of a physician. In some embodiments, treatment can continue for many years, the duration of the disease, or the lifetime of the subject. In some embodiments, the oral compositions of the embodiments herein can be administered to a subject in need once or twice daily for a period of about two to about twenty-eight days, or from about seven to about ten days. The oral compositions of the embodiments herein can also be administered to a subject once, twice, or three times daily for a period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0038] In some embodiments, an oral composition comprising Compound 1 or Compound 2 as described herein is administered before, after, or with a meal. In some embodiments, an oral composition as described herein is administered before, after, or with a high-fat meal.

[0039] In some embodiments, the oral compositions described herein are administered after an overnight fast. In some embodiments, the overnight fast is at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, or at least about 10 hours. For example, the oral compositions described herein can be administered after a high-fat meal, a high-calorie meal, and after an overnight fast of at least 10 hours.

[0040] Combination therapy

[0041] One or more compounds of the present invention and one or more other pharmaceutically active compounds can be administered simultaneously (in the same dosage form or in separate dosage forms) or sequentially. Thus, in one embodiment, the present invention includes a method of treating a condition by administering to a subject a therapeutically effective amount of an oral pharmaceutical composition as described herein and one or more additional pharmaceutically active compounds.

[0042] Combination methods, compositions and preparations are not limited to the use of only two medicaments, and the use of multiple therapeutic combinations is also contemplated. It should be understood that the dosage regimen for treating, preventing or alleviating one or more conditions for which relief is sought is optionally altered according to a variety of factors. These factors include the disorder from which the subject suffers, as well as the subject's age, weight, sex, diet and physical condition. Therefore, in some embodiments, the dosage regimen actually employed varies widely and is therefore different from the dosage regimen set forth herein.

[0043] In any case, multiple therapeutic compositions (at least one of which is a composition disclosed herein) may be administered in any order or even simultaneously. If administered simultaneously, the multiple therapeutic compositions may be provided in a single, unified form or in multiple forms (as a single pill or as two separate pills, by way of example only). One of the therapeutic compositions may be administered in multiple doses, or both pharmaceutical compositions may be administered in multiple doses. If not administered simultaneously, the time between multiple doses may be any duration from a few minutes to eight weeks, or at any interval suitable for maintaining the desired therapeutic efficacy. In some embodiments, the time between multiple doses may be one minute, one hour, six hours, one day, two days, three days, four days, five days, six days, one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, or eight weeks.

[0044] Thus, in another aspect, certain embodiments provide a method for treating a condition in a human or animal subject in need of such treatment, the method comprising administering to the subject an oral composition comprising Compound 1 or Compound 2 as disclosed herein in an amount of 5 mg / day to 300 mg / day in combination with at least one additional agent known in the art for treating the disorder to reduce or prevent the condition in the subject. In a related aspect, certain embodiments provide a combination of a therapeutic composition disclosed herein with one or more additional pharmaceutically active compounds for treating the condition, and a pharmaceutically acceptable carrier.

[0045] The oral compositions described herein comprising Compound 1 or Compound 2 are also optionally used in combination with other therapeutic agents, which are selected based on the therapeutic value of the condition to be treated. Typically, in embodiments employing combination therapy, the compositions described herein and other agents do not have to be administered in the same pharmaceutical composition and may be administered by different routes due to different physical and chemical properties. Initial administration is typically performed according to established protocols, and then the dosage, mode of administration, and time of administration are subsequently modified based on the observed effects.

[0046] In some embodiments, the one or more additional pharmaceutically active compounds are selected from the group consisting of anti-inflammatory drugs, anti-atherosclerotic drugs, immunosuppressive drugs, immunomodulatory drugs, cytostatic drugs, antiproliferative agents, angiogenesis inhibitors, kinase inhibitors, cytokine blockers, and inhibitors of cell adhesion molecules.

[0047] In some embodiments, an oral composition comprising Compound 1 or Compound 2 is administered to a subject having or at risk of having the condition, along with one or more agents or compositions, in any combination, known to be useful for treating the condition.

[0048] Specific non-limiting examples of possible combination therapies for any of the conditions listed above include combinations of oral compositions comprising Compound 1 or Compound 2 as described herein with: (1) corticosteroids, including but not limited to cortisone, dexamethasone, and methylprednisolone; (2) nonsteroidal anti-inflammatory drugs (NSAIDs), including but not limited to ibuprofen, naproxen, acetaminophen, aspirin, fenoprofen (NALFON™), flurbiprofen (ANSAID™), ketoprofen, oxaprozin (DAYPRO™), diclofenac sodium (VOLTARENT™), diclofenac potassium (CAFENAC™), and fenac serotonin (FENAC). TAFLAM™), etodolac (LODINET™), indomethacin (INDOCINT™), ketorolac (TORADOL™), sulindac (CLINORIL™), tolmetin (TOLECTINT™), meclofenamic acid (MECLOMENT™), mefenamic acid (PONSTEL™), nabumetone (RELAFENT™) and piroxicam (FELDENET™); (3) immunosuppressants, including but not limited to methotrexate (RHEUMATREXT™), leflunomide (ARAVA™), thiothreitol Purine (IMURANTM), cyclosporine (NEORALTM, SANDIMMUNETM), tacrolimus and cyclophosphamide (CYTOXANTM); (4) CD20 blockers, including but not limited to rituximab (RITUXANTM); (5) tumor necrosis factor (TNF) blockers, including but not limited to etanercept (ENBRELTM), infliximab (REMICADETM) and adalimumab (HUMIRATM); (6) interleukin-1 receptor antagonists, including but not limited to anakinra (KINERETTM) TM), rilonacept (ARCALYSTTM) and canakinumab (ILARISTM); (7) interleukin-6 inhibitors, including but not limited to tocilizumab (ACTEMRATM); (8) interleukin-17 inhibitors, including but not limited to secukinumab, ixekizumab and brodalimumab; (9) Janus kinase inhibitors, including but not limited to tofacitinib; and (10) SYK inhibitors, including but not limited to fostamatinib.

[0049] Where the subject has the condition, examples of therapeutic agents / treatments include, but are not limited to, nonsteroidal anti-inflammatory drugs (NSAIDs), steroids (e.g., prednisone), corticosteroids, and disease modifying drugs (DMARDs) (e.g., methotrexate, leflunomide, hydroxychloroquine, sulfasalazine), Janus kinase (JAK) inhibitors (e.g., tofacitinib, upadacitinib, baricitinib, filgotinib, ruxolitinib, oclacitinib, peficitinib, fedratinib, cerdulatinib, gandotinib, lesarturtinib, momelotinib, parvotinib, These include, but are not limited to, pacritinib, abrocitinib, and BMS-986165), tumor necrosis factor inhibitors (e.g., adalimumab, etanercept, golimumab, infliximab, certolizumab), anti-B cell antibodies (e.g., rituximab), anti-IL-6 antibodies (e.g., sarilumab, tocilizumab), interleukin-1 receptor (IL-1) antagonists (e.g., anakinra), and T cell activation inhibitors (e.g., abatacept).

[0050] In any embodiment, suitable JAK inhibitors for use in combination with Compound 1 or Compound 2 include, but are not limited to, tofacitinib, upadacitinib, baricitinib, filgotinib, ruxolitinib, oclacitinib, peftinib, fizotinib, seletinib, gadotinib, lestaurinib, molotinib, pacitinib, abrocitinib, and BMS-986165.

[0051] Secondly, the present invention provides a combination use for preventing and / or treating inflammatory diseases, comprising the combined use of compound 1A or a salt thereof and methotrexate, or compound 2A or a salt thereof and methotrexate:

[0052] Furthermore, the compound 1A is selected from compound 1, or 1B, or a mixture of the two in any ratio, Or the compound 2A is selected from compound 2, or 2B, or a mixture of the two in any ratio,

[0053] Furthermore, the combined use of Compound 1 or its salt and methotrexate, Compound 2 or its salt and methotrexate has a good synergistic effect in inhibiting inflammatory diseases such as foot swelling, and can reduce the occurrence of side effects while treating inflammatory diseases.

[0054] As a preferred embodiment of the present invention, the dosage of the compound or its salt in the combined use is 0.01-500 mg / day, preferably 0.1-400 mg / day, more preferably 1-200 mg / day.

[0055] As a preferred embodiment of the present invention, the dosage of the compound or its salt is 0.01-500 mg / day, preferably 0.1-400 mg / day, more preferably 1-200 mg / day.

[0056] As a preferred embodiment of the present invention, the dosage of the compound or its salt is 10 mg / day, 15 mg / day, 20 mg / day, 25 mg / day, 30 mg / day, 35 mg / day, 40 mg / day, 45 mg / day, 50 mg / day, 55 mg / day, 60 mg / day, 65 mg / day, 70 mg / day, 75 mg / day, 80 mg / day, 85 mg / day, 90 mg / day, 95 mg / day, 100 mg / day day, 105 mg / day, 110 mg / day, 115 mg / day, 120 mg / day, 125 mg / day, 130 mg / day, 135 mg / day, 140 mg / day, 145 mg / day, 150 mg / day, 155 mg / day, 160 mg / day, 165 mg / day, 170 mg / day, 175 mg / day, 180 mg / day, 185 mg / day, 190 mg / day, 195 mg / day, 200 mg / day.

[0057] As a preferred embodiment of the present invention, the compound or its salt is used once a day, twice a day, or three times a day.

[0058] As a preferred embodiment of the present invention, the dosage of methotrexate is a clinical dosage.

[0059] As a preferred embodiment of the present invention, the frequency of use of methotrexate is 1 time / week, 2 times / week, or 3 times / week.

[0060] As a preferred embodiment of the present invention, the dosage of methotrexate is 2.5 mg-25 mg / week, for example, 2.5 mg / week, 5 mg / week, 7.5 mg / week, 10 mg / week, 12.5 mg / week, 15 mg / week, 17.5 mg / week, 20 mg / week, 22.5 mg / week, and 25 mg / week.

[0061] As a preferred embodiment of the present invention, 7.5 mg / time, once a week, or 2.5 mg / time, once every 12 hours, 3 times a week is preferred.

[0062] As a preferred embodiment of the present invention, the inflammatory diseases include: arthritis, cryoporphyrin-associated periodic syndromes (CAPS) (including Muckle-Wells syndrome (MWS), neonatal-onset multisystem inflammatory disease (NOMID), familial cold autoinflammatory syndrome (FCAS)), psoriasis (such as plaque psoriasis), colitis caused by inflammatory bowel disease (IBD) (such as Crohn's disease or ulcerative colitis) and pericarditis, etc.

[0063] As a preferred embodiment of the present invention, the arthritis includes: rheumatoid arthritis (RA), spinal arthritis (such as ankylosing spondylitis and psoriatic arthritis), juvenile rheumatoid arthritis (JIA) or idiopathic arthritis (JIA) (including systemic JIA (SJIA)) and gout).

[0064] Furthermore, the present invention provides a drug package for preventing and / or treating inflammatory diseases, comprising the combined use of Compound 1A or a salt thereof and methotrexate, and Compound 2A or a salt thereof and methotrexate:

[0065] Furthermore, the compound 1A is selected from compound 1, or 1B, or a mixture of the two in any ratio, Or the compound 2A is selected from compound 2, or 2B, or a mixture of the two in any ratio,

[0066] As a preferred embodiment of the present invention, the combined use of the present invention can be a treatment method, by combining the compound or its salt with methotrexate, wherein the combination can be administered simultaneously or in different time periods according to specific needs.

[0067] Furthermore, the present invention provides an impurity of MK2 compound 1, selected from impurities 1, 2, 3 of compound 1 or a mixture thereof in any proportion: Wherein, the impurity 1 and the impurity 2 are a pair of atropisomers, and the impurity 3 is a racemate of the impurities 1 and 2.

[0068] The present invention provides an impurity of MK2 compound 2, selected from impurities 1, 2, 3 of compound 2 or a mixture thereof in any proportion: Wherein, the impurity 1 and the impurity 2 are a pair of atropisomers, and the impurity 3 is a racemate of the impurities 1 and 2.

[0069] The present invention further provides a method for preparing impurity 1, impurity 2 or impurity 3 of the MK2 compound 1 or 2, comprising the following steps: synthesizing the impurity 1, impurity 2 or impurity 3 through the synthesis process of compound 1 or 2, or obtaining the impurity 1, impurity 2 or impurity 3 through liquid phase separation of the raw material of compound 1 or 2.

[0070] The present invention further provides a use of the impurity 1, impurity 2 or impurity 3 of the MK2 compound 1 or 2 as a reference substance for quality research of the compound 1 or 2 raw material and its pharmaceutical composition.

[0071] The present invention further provides a pharmaceutical composition, the active ingredient of which is compound 1 or 2, wherein the content of impurity 1 and / or impurity 2, and / or impurity 3 of compound 1 or 2 is less than 1% (preferably less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%), and the pharmaceutical composition further contains one or more pharmaceutically acceptable carriers.

[0072] For the sake of clarity, general terms used in the description of the compounds are defined herein.

[0073] Unless otherwise indicated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered as undefined or unclear in the absence of a specific definition, but should be understood according to its ordinary meaning. When a trade name appears in this article, it is intended to refer to its corresponding commercial product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to those compounds, materials, compositions and / or dosage forms that are suitable for use in contact with human and animal tissues within the scope of sound medical judgment without excessive toxicity, irritation, allergic reaction or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0074] The term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention, which is prepared by reacting the compound discovered in the present invention with a pharmaceutically acceptable acid or base and the specified substituents.

[0075] In addition to the form of salts, the compounds provided by the present invention also exist in prodrug form. The prodrugs of the compounds described herein easily undergo chemical changes under physiological conditions to be converted into the compounds of the present invention. In addition, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an in vivo environment.

[0076] The term "pharmaceutically acceptable carrier" refers to any formulation carrier or medium that can deliver an effective amount of the active substance of the present invention, does not interfere with the biological activity of the active substance, and has no toxic side effects on the host or patient. Representative carriers include water, oils, vegetables and minerals, cream bases, lotion bases, ointment bases, etc. These bases include suspending agents, viscosity increasing agents, transdermal enhancers, etc. Their preparations are well known to those skilled in the art of cosmetics or topical medicine. For additional information about carriers, reference can be made to Remington: The Science and Practice of Pharmacy, 21st Ed., Lippincott, Williams & Wilkins (2005), the contents of which are incorporated herein by reference.

[0077] The term "excipient" generally refers to the carrier, diluent, and / or medium required to formulate an effective pharmaceutical composition. Excipients include, but are not limited to, absorption enhancers, anti-adherents, defoaming agents, antioxidants, binders, buffers, carriers, coatings, colorants, delivery enhancers, delivery polymers, detergents, dextran, dextrose, diluents, disintegrants, emulsifiers, bulking agents, fillers, flavorings, glidants, wetting agents, oils, polymers, preservatives, saline, salts, solvents, sugars, surfactants, suspending agents, sustained-release matrices, sweeteners, thickeners, tonicity agents, vehicles, waterproofing agents, wetting agents, lubricants such as sodium lauryl sulfate and magnesium stearate, flavoring agents, and fragrances.

[0078] With respect to a drug or pharmacologically active agent, the term "effective amount" or "therapeutically effective amount" refers to a non-toxic amount of the drug or agent sufficient to achieve the intended effect. For the oral dosage forms of the present invention, an "effective amount" of an active substance in the composition means the amount required to achieve the intended effect when used in combination with another active substance in the composition. The determination of an effective amount varies from person to person, depending on the age and general condition of the recipient, as well as the specific active substance. The appropriate effective amount in each individual case can be determined by those skilled in the art through routine experimentation.

[0079] The terms "active ingredient," "therapeutic agent," "active substance," or "active agent" refer to a chemical entity that is effective in treating a target disorder, disease, or condition.

[0080] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0081] The compounds of the present invention can be prepared by a variety of synthetic methods well known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthesis methods, and equivalent substitutions well known to those skilled in the art. Preferred embodiments include but are not limited to the examples of the present invention.

[0082] Beneficial results of the present invention include:

[0083] The present invention provides the medical uses of compounds 1 and 2 and pharmaceutically acceptable salts thereof, including their use in preparing drugs for preventing or treating atopic dermatitis and asthma, the pharmaceutical use of compounds 1 and 2 in combination with methotrexate, raw materials of compounds 1 and 2 containing limited amounts of impurities and pharmaceutical compositions thereof, as well as their use in preparing drugs for preventing or treating the above-mentioned diseases.

[0084] The present invention prepared for the first time a new substance, MK2 compound A and its isomers' impurities 1, impurity 2, and impurity 3, which met the requirements of quality research. The related substances of compound A and its isomers were accurately quantitatively controlled and can be used as reference substances for quality research of raw materials and pharmaceutical compositions containing the drugs.

[0085] The present invention provides a pharmaceutical composition containing trace impurities (limited impurities), the quality of which is controllable and is beneficial for specific use. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] Figure 1 is a HPLC spectrum of the separation of substances in Example 14 of the present invention.

[0087] Figure 2 is an HPLC spectrum of impurities in the degradation test of Example 14 of the present invention. DETAILED DESCRIPTION

[0088] The present application is further described in detail below with reference to examples, but the implementation methods of the present application are not limited thereto.

[0089] Example 1

[0090] Compound 1A and its isomers were prepared by prior art PCT / US2022 / 022525:

[0091] The isomers are compounds 1 and 1B:

[0092] Specifically, the synthetic route is as follows:

[0093] Step 1: Synthesis of 2'-acetyl-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one.

[0094] At room temperature, dissolve 2'-acetyl-3-chloro-3'-fluoro-4-hydroxy-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one (14.9 g, 47.9 mmol) and potassium carbonate (13.3 g, 95.8 mmol) in N,N-dimethylformamide (96 ml). Cool the reaction system to 0°C and slowly add 2-bromomethyl-3,5-difluoropyridine (13.0 g, 62.3 mmol) dropwise. Return the reaction system to room temperature and react for 2 hours.

[0095] After the reaction was complete, ethyl acetate (200 mL) was added for dilution. The organic phase was washed with water (100 mL x 3 times), then washed with saturated sodium chloride solution (100 mL x 1 time), dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 1 / 2). 20.1 g of 2'-acetyl-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one was obtained as a yellow powdery solid (yield: 96%). LC-MS: RT = 3.81 min, [M+H] + =438.1.

[0096] Step 2: Synthesis of (E)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2'-(3-dimethylamino)acryloyl-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one.

[0097] Dissolve 2'-acetyl-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one (2.0 g, 4.7 mmol) in N,N-dimethylformamide dimethyl acetal (18.0 ml). Heat the mixture to 100°C and allow to react for 2 hours.

[0098] After the reaction was complete, the reaction mixture was concentrated to yield a crude yellow solid product, (E)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2'-(3-dimethylamino)acryloyl-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one. This product was directly used in the next reaction (Step 3) without purification.

[0099] Step 3: Synthesis of 3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-2'-(2-(2-hydroxypropyl-2-yl)pyrimidin-4-yl)-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one

[0100] The crude yellow solid (E)-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2'-(3-dimethylamino)acryloyl-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one, 2-hydroxy-2-methylpropionamidine hydrochloride (3.2 g, 23.3 mmol), and potassium carbonate (1.9 g, 14.0 mmol) were dissolved in N,N-dimethylformamide (45 ml) at room temperature. The mixture was heated to 80°C and reacted for 3 hours.

[0101] After the reaction was complete, water (30 ml) was added to quench the reaction. The product was extracted with ethyl acetate (30 ml × 3 times), washed with saturated sodium chloride solution (50 ml × 1 time), dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: ethyl acetate / methanol = 10 / 1). 1.8 g of 3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-2'-(2-(2-hydroxypropyl-2-yl)pyrimidin-4-yl)-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one was obtained as a yellow powdery solid (yield: 75%). LC-MS: RT = 3.24 min, [M+H] + =532.1.

[0102] Step 4: Split

[0103] CHIRALPAK IC CSP (20 μm) was used as chiral packing (Dacel) and dichloromethane-methanol (80:20) was used as the mobile phase for separation to first obtain compound 1 and then the isomer of compound 1 (compound 1B).

[0104] Compound 1: 1 H NMR(400MHz,DMSO-d6)δ9.05(d,J=5.2Hz,1H),8.80(s,1H),8.63(d,J=2.4Hz,1H),8.25–7.97(m,2H ),6.94(s,1H),5.57(d,J=2.1Hz,2H),5.12(s,1H),2.22(s,3H),2.10(s,3H),1.57(s,6H).HRMS(ESI + ):m / z for C 25 H 21 ClF3N5O3Na[M+Na] + calcd 554.1177, found 554.1108.

[0105] Example 2

[0106] Compound 2A and its isomers were prepared by the prior art PCT / US2022 / 022525:

[0107] The isomers are compounds 2 and 2B:

[0108] The specific synthetic route is as follows:

[0109] Step 1: Synthesis of 2'-bromo-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one

[0110] At room temperature, dissolve 2'-bromo-3-chloro-3'-fluoro-4-hydroxy-5',6-dimethyl-2H-[1,4'-bipyridinyl]-2-one (75.0 g, 196.3 mmol) and potassium carbonate (54.3 g, 392.7 mmol) in N,N-dimethylformamide (393 ml). Cool the reaction system to 0°C and slowly add 2-bromomethyl-3,5-difluoropyridine (53.1 g, 255.3 mmol) dropwise. Return the reaction system to room temperature and react for 2 hours.

[0111] After the reaction was completed, water (2 L) was added to the reaction system. A large amount of brown solid precipitated. Filter. The solid was dissolved in ethyl acetate (80 ml), and n-hexane (120 ml) was added and stirred at room temperature for 1 hour. Filtration gave 93.2 g of brown powdery solid 2'-bromo-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one (yield: >99%). LC-MS: RT = 2.97 min, [M+H] + =475.9.

[0112] Step 2: Synthesis of 3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-2'-(3-(2-hydroxypropyl-2-yl)-1H-pyrazol-1-yl)-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one

[0113] 2'-Bromo-3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy-3'-fluoro-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one (69.5 g, 146.4 mmol), 2-(1H-pyrazol-3-yl)propan-2-ol (36.9 g, 292.7 mmol), potassium carbonate (40.5 g, 292.7 mmol), cuprous iodide (5.6 g, 29.3 mmol), and N,N'-dimethylethylenediamine (5.2 g, 58.6 mmol) were dissolved in 1,4-dioxane (586 ml) at room temperature under a nitrogen atmosphere. The mixture was heated to 100°C and reacted for 2 hours.

[0114] After the reaction was complete, water (300 ml) was added to quench the reaction. The product was extracted with dichloromethane (500 ml x 3 times), washed with saturated sodium chloride solution (500 ml x 1 time), dried over anhydrous sodium sulfate, and concentrated. The resulting residue was purified by silica gel column chromatography (eluent: n-hexane / ethyl acetate = 3 / 7). 39.6 g of 3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-3'-fluoro-2'-(3-(2-hydroxypropyl-2-yl)-1H-pyrazol-1-yl)-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one was obtained as a yellow solid (yield: 52%). LC-MS: RT = 2.73 min, [M+H] + =520.1.

[0115] Step 3: Split

[0116] CHIRALPAK IC CSP (20 μm) was used as chiral packing (Dacel) and dichloromethane-methanol (80:20) was used as the mobile phase to perform separation, first obtaining compound 2 and then obtaining the isomer of compound 2 (compound 2B).

[0117] Compound 2: 1 H NMR (400MHz, DMSO-d6) δ8.58(d,J=2.4Hz,1H),8.47(s,1H),8.34(d,J=2.6Hz,1H),8.11–8.00(m,1H),6.88 (s,1H),6.58(d,J=2.6Hz,1H),5.51(s,2H),5.11(s,1H),2.09(s,3H),2.05(s,3H),1.46(s,6H).HRMS(ESI + ):m / z for C 24 H 21 ClF3N5O3Na[M+Na] + calcd 542.1177, found 542.0941.

[0118] Comparative Example 1

[0119] The structure of the compound 3-chloro-4-((3,5-difluoropyridin-2-yl)methoxy)-2'-(2-(2-hydroxypropane-2-yl)pyrimidin-4-yl)-5',6-dimethyl-2H-[1,4'-bipyridyl]-2-one is:

[0120] The synthetic route of the compound of Comparative Example 1 refers to the synthetic route of the compound with specification number 49 in Chinese patent number CN201480032278.5.

[0121] The racemic compound of Comparative Example 1 was separated using supercritical fluid chromatography (OD-H column) with a mobile phase of carbon dioxide and ethanol, and the atropisomers of Comparative Example 1A and 1B were eluted successively:

[0122] Comparative Example 1A compound: RT = 4.47 min (SFC, OD-H, 0.46 cm ID × 15 cm L column, 40% ethanol isocratic method, flow rate 2.5 mL / min and cycle time 10 min); [a] D 25 -0.66° (MeOH, Rudolph Autopol I rotator).

[0123] Compound of Comparative Example 1B: RT = 4.68 min (SFC, OD-H, 0.46 cm ID x 15 cm L column, 40% ethanol isocratic method, flow rate 2.5 mL / min and cycle time 10 min). [a] D25 + 0.68° (MeOH, Rudolph Autopol I polarimeter).

[0124] Example 3: LPS-induced TNFα release experiment in U937

[0125] Reagents and instruments:

[0126] 1640 Medium, Catalog No. A10491-01, Gibco. Penicillin-Streptomycin, Catalog No. 15140-122, Gibco. Fetal Bovine Serum, Catalog No. 10099-141C, Gibco. PBS, Catalog No. 10010-031, Gibco. LPS, Catalog No. L2880, Sigma. PMA, Catalog No. P1585, Sigma. Dimethyl Sulfoxide, Catalog No. D8418-1L, Sigma. TNFα Kit, Catalog No. K151QWD-4, MSD.

[0127] 96-well plate, Catalog No. 3599, Corning. Plate shaker, Catalog No. QB-9002, Qilinbeier. Centrifuge, Catalog No. 5810R, Eppendorf. CO2 incubator, Catalog No. 371, Thermo. Counter, Catalog No. C10281, Gibco. Microscope, Catalog No. CKX41, Olympus. MSD plate reader, 1201MESO SECTOR 600, MSD.

[0128] Experimental cells:

[0129] U937, ATCC, catalog number CRL-1593.2.

[0130] Drug preparation:

[0131] Weigh approximately 2 mg of a drug and prepare a 10 mM stock solution in DMSO. Dilute the stock solution 10-fold to 1 mM, then dilute it 4-fold to 250 μM, 62.5 μM, 15.6 μM, 3.9 μM, 0.97 μM, 0.24 μM, and 0.061 μM. Then, dilute the DMSO solution 20-fold in culture medium to prepare the working solution.

[0132] Experimental methods:

[0133] On Day 0, well-grown U937 cells were harvested and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded. The cells were resuspended in 1640 medium supplemented with 75 ng / ml PMA and the cell density was adjusted to 30 × 10⁴ cells / ml. These cells were cultured in a culture flask for 48 hours to allow differentiation.

[0134] On Day 2, follow these steps:

[0135] 1. Remove the supernatant of differentiated U937 cells, wash once with PBS, and then digest with digestion solution for 2 minutes.

[0136] 2. Add serum-containing culture medium to terminate the reaction, then remove the supernatant by centrifugation and count the cells.

[0137] 3. 80,000 cells / 100 μl were seeded into a 96-well plate.

[0138] 4. Add 2 μl of compound containing a final concentration of 1% DMSO, and then incubate at 37°C and 5% CO2 for 30 minutes.

[0139] 5. Add 2 μl LPS (final concentration 100 ng / ml) to stimulate the cells and culture them at 37°C and 5% CO2 for 4 hours.

[0140] 6. Centrifuge and collect the supernatant. Measure the TNFα content in the supernatant using an ELISA kit.

[0141] When performing statistical analysis, the standard curve in the kit was used to calculate the TNFα content in each well. The IC value of the compound was calculated using the GraphPad nonlinear fitting formula. 50 The experimental results are recorded in Table 1.

[0142] Table 1 IC of the compounds of the present invention for inhibiting TNFα production 50 value

[0143] From the experimental results in Table 1 above, it can be seen that Compound 1 of the present invention has significant inhibitory activity on TNFα production and has the potential to treat inflammation-related diseases.

[0144] Example 4: Pharmacokinetic study of compound in rats

[0145] (1) Experimental materials

[0146] SD rats: male, 180-250 g, purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd.

[0147] Reagents: DMSO (dimethyl sulfoxide), PEG-400 (polyethylene glycol 400), normal saline, heparin, acetonitrile, formic acid, and propranolol (internal standard) are all commercially available.

[0148] Instrument: Thermo Fisher LC-MS (U300 UPLC, TSQ QUANTUMN ULTRA triple quadrupole mass spectrometer).

[0149] (2) Experimental methods

[0150] Weigh the compound and dissolve it in DMSO-PEG-400-saline (5:60:35, v / v / v). After intravenous or oral administration, collect 200 μL of venous blood into a heparinized EP tube at 15 minutes, 30 minutes, 1 hour, 2 hours, 5 hours, 7 hours, and 24 hours (5 minutes additionally for the IV group). Centrifuge at 10,000 rpm for 2 minutes, and freeze the plasma at -80°C until testing. Accurately weigh a certain amount of the test compound and dissolve it in DMSO to 2 mg / mL to prepare the stock solution. Accurately pipette an appropriate amount of the stock solution and dilute it with acetonitrile and water (1:1, v / v) to prepare the standard curve working solution and quality control working solution. Accurately pipette 20 μL of each of the above working solutions, add 180 μL of blank plasma, and vortex to mix thoroughly. Standard curve plasma samples equivalent to plasma concentrations of 0.3, 1, 3, 10, 30, 100, 300, 1000, and 3000 ng / mL, as well as quality control plasma samples at concentrations of 9, 240, and 2400 ng / mL, are prepared. Duplicate samples are analyzed at each concentration to establish the standard curve and quality control. Take 30 μL of plasma (plasma diluted 5-fold 5 minutes, 15 minutes, and 30 minutes after intravenous administration) and add 200 μL of a solution of methanol and acetonitrile (1:1, v / v) containing the internal standard propranolol (50 ng / mL). After vortex mixing, centrifuge at 4000 rpm for 5 minutes. Transfer 100 μL of the supernatant to a new 96-well plate and add 100 μL of ultrapure water to mix thoroughly. LC-MS analysis. LC-MS detection conditions are as follows:

[0151] Chromatographic column: Thermo Fisher HYPERSIL GOLD C-18 UPLC column, 100*2.1 mm, 1.7 μm.

[0152] Mobile phase: Water (0.1% formic acid)-acetonitrile Gradient elution according to the table below

[0153] Table 2

[0154] 3. Data Processing

[0155] After LC-MS detection of blood drug concentration, WinNonlin 6.1 software was used to calculate the pharmacokinetic parameters using the non-compartmental model method. The results are shown in Table 3.

[0156] Table 3

[0157] From the experimental results in Table 3, it can be seen that compounds 1 and 2 exhibited good PK effects and had good oral exposure and bioavailability.

[0158] Example 5 Study on cytokine IL-4

[0159] Human PBMCs were obtained from TPCS. PBMCs were resuspended in complete RPMI1640 medium (containing RPMI1640, 10% inactivated FBS, 1% penicillin-streptomycin, and 55 μM 2-mercaptoethanol) and adjusted to a density of 2.5E6 cells / mL. Subsequently, the cells were plated into 96-well plates, with each well containing 2.5E5 cells and 100 μL of medium. According to a pre-defined layout, the cells were pretreated with 50 μL of compound or vehicle (in 0.1% DMSO) at room temperature for 10 minutes at nine serial concentrations.

[0160] After pretreatment, 50 μL of 80 μg / mL PHA-L was added to each well to stimulate the cells. The cells were then cultured for 48 hours at 37°C and 5% CO2.

[0161] After the incubation period, the culture plate was removed from the incubator and centrifuged at 350 g for 5 minutes, and the supernatant was collected. Finally, IL-4 in the supernatant was detected using ELISA. The results are shown in Table 4.

[0162] Table 4

[0163] As shown in Table 4, Compound 1 and Compound 2 have a good inhibitory effect on IL-4 releasing factor and can be used for diseases related to this cytokine, such as atopic dermatitis or asthma.

[0164] Example 6 Study on cytokine IL-13

[0165] Human PBMCs were obtained from TPCS. PBMCs were resuspended in complete RPMI1640 medium (containing RPMI1640, 10% inactivated FBS, 1% penicillin-streptomycin, and 55 μM 2-mercaptoethanol) and adjusted to a density of 2.5E6 cells / mL. Subsequently, the cells were plated into 96-well plates, with each well containing 2.5E5 cells and 100 μL of medium. According to a pre-defined layout, the cells were pretreated with 50 μL of compound or vehicle (in 0.1% DMSO) at room temperature for 10 minutes at nine serial concentrations.

[0166] After pretreatment, 50 μL of 80 μg / mL PHA-L was added to each well to stimulate the cells. The cells were then cultured for 48 hours at 37°C and 5% CO2.

[0167] After the incubation period, the culture plate was removed from the incubator and centrifuged at 350 g for 5 minutes, and the supernatant was collected. Finally, IL-13 in the supernatant was detected using ELISA. The results are shown in Table 5.

[0168] Table 5

[0169] As shown in Table 5, Compound 1 and Compound 2 have a good inhibitory effect on IL-13 releasing factor and can be used for diseases related to this cytokine, such as atopic dermatitis or asthma.

[0170] Example 7 Experimental study on atopic dermatitis

[0171] Experimental reagents and instruments are shown in Table 6:

[0172] Table 6 Experimental reagents and instruments

[0173] Reagent preparation:

[0174] (1) Preparation of modeling agent:

[0175] Acetone and olive oil were evenly mixed in a volume ratio of 3:1 and set aside. Then, in a fume hood, 2% DNCB and 1% DNCB solutions were prepared using the above mixed solution.

[0176] (2) Compound preparation:

[0177] Compound solvent preparation: Heat 1000 mL of PBS to 70°C, add 5 g of methyl cellulose (MC) while stirring, cool to room temperature, add 0.25 mL of Tween-20, and stir to obtain a PBS solvent containing 0.5% MC and 0.025% Tween-20. Store at 4°C until ready to use.

[0178] Positive control drug (ATI450), compound 1 and compound 2 (0.01 mg / mL) were prepared by weighing compound 1 and compound 2 (91.1%), adding the solvent, mixing thoroughly, and sonicating to obtain a clear solution with a final concentration of 0.01 mg / mL.

[0179] The experimental animals were 70 female Balb / c mice, aged 6-7 weeks. They were obtained from Zhejiang Weitonglihua Laboratory Animal Technology Co., Ltd., with a qualification certificate number of 44829700020235. After one week of adaptive feeding, the mice were randomly divided into five groups according to body weight for the experiment.

[0180] Model establishment and drug administration

[0181] The day before the experiment, Balb / c mice were hairless on their backs using an electric razor. Mice in the normal control group were sensitized and challenged with acetone / olive oil (volume ratio 3:1). Mice in the model group were sensitized and challenged with DNCB solution. The challenge procedure involved applying 100 μL of 1% DNCB to the back and auricles of the mice on days 1-2, and 120 μL of 2% DNCB to the back and auricles of the mice daily on days 3-4. On days 12-13, 100 μL of 1% DNCB was applied to the back and auricles of the mice, and on days 14-15, 120 μL of 2% DNCB was applied to the back and auricles of the mice daily. Thereafter, 120 μL of 2% DNCB was applied to the back and auricles of the mice twice weekly. During the experiment, mice in each group were observed for clinical signs of atopic dermatitis, such as redness, swelling, roughness, thickening, and lichenification. A successful model was established when the score was ≥4. Inflammation scoring was based on the Scoring Atopic Dermatitis (SCORAD) index, with lesion severity assessed weekly based on symptoms. The total atopic dermatitis score (indicating dermatitis severity in mice) was calculated as the sum of all individual scores, ranging from 0 to 12, as shown in Table 7.

[0182] Table 7

[0183] The animals with successful modeling were randomly divided into groups according to the atopic dermatitis score. The drug was administered orally the next day, twice a day (0.1 mg / kg). The atopic dermatitis score was calculated based on the back symptoms of the animals. The ear thickness of each group of animals was measured with a vernier caliper, and the data were processed as follows:

[0184] Calculate the inhibition rate of atopic dermatitis, inhibition rate = [(inflammatory score 模型 —Inflammation score 给药 ) / (inflammatory score 模型 —Inflammation score 空白 )]×100%

[0185] Calculate the ear thickness suppression rate, suppression rate = [(ear thickness 模型 - Ear thickness 给药 ) / (ear thickness 模型 - Ear thickness 空白 )]×100%

[0186] Calculate the AUC inhibition rate of atopic dermatitis and ear thickness, inhibition rate = [AUC 模型(D1-D14) —AUC 给药(D1-D14) ) / (AUC 模型(D1-D14) —AUC 空白(D1-D14) )]×100%

[0187] The statistical experimental results are shown in Table 8.

[0188] Table 8

[0189] Compared with the model group, both compound 1 and compound 2 could significantly reduce atopic dermatitis and ear thickness in mice.

[0190] Example 8: Pharmacological Efficacy Study

[0191] This study primarily tested the effects of different test articles on the CIA model in female Lewis rats. After an acclimation period, 7-week-old female Lewis rats were immunized with an intradermal injection of 500 μL of an emulsion (a 1:1 mixture of type II collagen and adjuvant) at two points on the back (200 μL each) and one point at the base of the tail (100 μL). Immunizations were performed twice, one week apart. After the second immunization, continuous observation was continued. After the onset of disease, rats with a clinical score greater than or equal to 1.1 cm were selected. 3 Animals were enrolled and divided into 7 groups, with 10 rats in each group. In addition, 5 unimmunized rats served as the normal control group (NC). This experiment was conducted approximately 1 week after the second emulsion immunization modeling. The day of enrollment was set as Day 1. According to the dosing method in Table 1, the normal group and the model group were orally administered with the solvent (PBS with 0.5% methylcellulose and 0.025% Tween 20). The other treatment groups were orally administered with the corresponding drugs in Table 1. Oral treatment was started and continued for 14 days. The dosage volume was 5 mL / kg / time.

[0192] During the experiment (days 1, 4, 8, 11, and 14), the animals' health was monitored, and body weight, arthritis clinical scores, and hindlimb swelling volume were recorded. At the endpoint, both hindlimbs were collected and fixed in formalin. Hematoxylin-eosin (HE) staining was performed, and toe joint histopathology was scored according to the scoring criteria.

[0193] Table 9 Experimental groups and dosages

[0194] The experimental results showed that compared with the NC group, the rats after two immunizations showed obvious clinical symptoms of arthritis such as erythema and swelling in the joints and metatarsals, and the swelling volume of the feet was greater than or equal to 1.1 cm 3 , indicating that the rat CIA model was successful.

[0195] After 14 days of treatment, the foot swelling scores, foot swelling volumes and pathological scores of each treatment group were significantly improved compared with the MC group.

[0196] Among them, methotrexate + compound 1 (low-dose group) and methotrexate + compound 1 (high-dose group) have a good inhibitory effect on foot swelling while treating arthritis.

[0197] qw means once a week, bid means twice a day.

[0198] The specific results are as follows:

[0199] Table 10 Experimental results of hind paw swelling score in rat CIA model

[0200] ***p<0.001vs model control group, AUC 1-14Day Inhibition rate = (AUC 模型,1-14Day -AUC 给药,1-14Day ) / AUC 模 型,1-14Day ×100%

[0201] Compared with the model control group, the compound 1 0.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group can significantly reduce the hind limb disease score of CIA rats and improve the hind limb inflammation after arthritis, indicating that the combination of MTX and compound 1 has a good synergistic effect in inhibiting hind limb inflammation.

[0202] Table 11 Results of hind paw volume experiment in rat CIA model

[0203] **p<0.001vs model control group, AUC 1-14Day Inhibition rate = (AUC 模型,1-14Day -AUC 给药,1-14Day ) / AUC 模型,1-14Day ×100% Compared with the model control group, the compound 1 0.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group can significantly inhibit the hind limb swelling of CIA rats, indicating that the combination of MTX and compound 1 has a good synergistic effect in inhibiting hind limb swelling.

[0204] The compound 10.01 mg / kg (bid) + MTX 0.8 mg / kg (qw) group and the compound 10.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group showed consistent synergistic effects.

[0205] Example 9: Pharmacological Efficacy Study

[0206] This study primarily tested the effects of different test articles on the CIA model in female Lewis rats. After an acclimation period, 7-week-old female Lewis rats were immunized with an intradermal injection of 500 μL of an emulsion (a 1:1 mixture of type II collagen and adjuvant) at two points on the back (200 μL each) and one point at the base of the tail (100 μL). Immunizations were performed twice, one week apart. After the second immunization, continuous observation was continued. After the onset of disease, rats with a clinical score greater than or equal to 1.1 cm were selected. 3Animals were enrolled and divided into 7 groups, with 10 rats in each group. In addition, 5 unimmunized rats served as the normal control group (NC). This experiment was conducted approximately 1 week after the second emulsion immunization modeling. The day of enrollment was set as Day 1. According to the dosing method in Table 1, the normal group and the model group were orally administered with the solvent (PBS with 0.5% methylcellulose and 0.025% Tween 20). The other treatment groups were orally administered with the corresponding drugs in Table 1. Oral treatment was started and continued for 14 days. The dosage volume was 5 mL / kg / time.

[0207] During the experiment (days 1, 4, 8, 11, and 14), the animals' health was monitored, and body weight, arthritis clinical scores, and hindlimb swelling volume were recorded. At the endpoint, both hindlimbs were collected and fixed in formalin. Hematoxylin-eosin (HE) staining was performed, and toe joint histopathology was scored according to the scoring criteria.

[0208] Table 12 Experimental groups and dosages

[0209] The experimental results showed that compared with the NC group, the rats after two immunizations showed obvious clinical symptoms of arthritis such as erythema and swelling in the joints and metatarsals, and the swelling volume of the feet was greater than or equal to 1.1 cm 3 , indicating that the rat CIA model was successful.

[0210] After 14 days of treatment, the foot swelling scores, foot swelling volumes and pathological scores of each treatment group were significantly improved compared with the MC group.

[0211] Among them, methotrexate + compound 2 (low-dose group) and methotrexate + compound 2 (high-dose group) have a good inhibitory effect on foot swelling while treating arthritis.

[0212] qw means once a week, bid means twice a day.

[0213] The specific results are as follows:

[0214] Table 13 Experimental results of hind paw swelling score in rat CIA model

[0215] ***p<0.001 vs model control group, AUC 1-14Day Inhibition rate = (AUC 模型,1-14Day -AUC 给药,1-14Day ) / AUC 模 型,1-14Day ×100%

[0216] Compared with the model control group, the compound 2 0.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group can significantly reduce the hind limb disease score of CIA rats and improve the hind limb inflammation after arthritis, indicating that the combination of MTX and compound 2 has a good synergistic effect in inhibiting hind limb inflammation.

[0217] Table 14 Results of hind paw volume experiment in rat CIA model

[0218] ***p<0.001 vs model control group, AUC 1-14Day Inhibition rate = (AUC 模型,1-14Day -AUC 给药,1-14Day ) / AUC 模 型,1-14Day ×100%

[0219] Compared with the model control group, the compound 2 0.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group significantly inhibited the hind limb swelling of CIA rats, indicating that the combination of MTX and compound 2 has a good synergistic effect in inhibiting hind limb swelling.

[0220] The compound 2 0.01 mg / kg (bid) + MTX 0.8 mg / kg (qw) group and the compound 2 0.005 mg / kg (bid) + MTX 0.8 mg / kg (qw) group showed consistent synergistic effects.

[0221] Example 10 Preparation of Impurities 1, 2, and 3 of p38α / MK2 Inhibitor Compound 1

[0222] Compound 1 and its isomer impurity 3 were prepared by referring to the following synthetic route:

[0223] Impurity 3 was separated by chiral column to obtain impurities 1 and 2 of compound 1 respectively:

[0224] Example 11 Preparation of Impurities 1, 2, and 3 of p38α / MK2 Inhibitor Compound 1

[0225] Compound 1 was prepared according to the prior art PCT / US2022 / 022525, and then subjected to light destruction and / or acid destruction, followed by separation and purification by high performance liquid chromatography to obtain impurities 1, 2, and 3 of compound 1:

[0226] Example 12: Application of impurities obtained by the present invention in quality research of raw materials

[0227] Operation method: Determine according to the 2020 edition of the Chinese Pharmacopoeia, General Chapter 0512 High Performance Liquid Chromatography.

[0228] The obtained determination method has good separation between compound 1 and impurities, the theoretical plate number and tailing factor of each impurity meet the requirements, the analysis time is short, the efficiency is high, and the cost is saved. It meets the requirements of the "Chinese Pharmacopoeia" and can effectively determine the quality of an MK2 inhibitor compound raw material and pharmaceutical composition.

[0229] Example 13 Investigation of the stability of the raw material drug

[0230] Compound 1 and its isomer 1B were prepared by referring to the method of the prior art PCT / US2022 / 022525. After 0 day (or 0h), illumination for several days (naked) and / or acid destruction for several hours, compound 1A and its isomers 1 and 1B were subjected to the method of Example 14 for determination of impurities 1, impurity 2, and impurity 3 to determine the content of the relevant impurities.

[0231] Therefore, analysis and detection of impurities 1, 2, and 3 are crucial for controlling the quality of compound 1 and its isomer raw materials.

[0232] Example 14 Preparation of Impurities 1, 2, and 3 of p38α / MK2 Compound 2

[0233] Compound 2 and its isomers were prepared according to the prior art PCT / US2022 / 022525, and then destroyed by light and / or acid and / or base. Then, impurities 1, 2, and 3 were separated and purified by high performance liquid chromatography:

[0234] Specifically, the impurity preparation steps are:

[0235] The compound with a retention time of 3.082 min in Example 14 was dissolved in 5 volumes of acetonitrile and 5 volumes of water in a reaction flask, and 1.0 M HCl (aq) was added. The mixture was heated to 50°C and allowed to react for 1 to 3 hours. Upon completion of the reaction, the reaction solution was neutralized with saturated sodium bicarbonate solution, extracted three times with ethyl acetate, washed with saturated sodium chloride solution, dried over anhydrous sodium sulfate, filtered, and concentrated. Degraded impurities were isolated and purified by HPLC. The peak times of the pre-degraded substance (compound with a retention time of 3.082 min, shown in Figure 1) and the post-degradation substance with the above structure were 14.749 min and 21.793 min, respectively, as shown in Figure 2.

[0236] HPLC analysis conditions are as follows:

[0237] Octadecylsilane bonded silica gel was used as the filler; water-methanol-formic acid (90:10:0.01) was used as mobile phase A, and methanol (containing 0.01% formic acid) was used as mobile phase B. Gradient elution was performed according to the table below; the flow rate was 1.0 ml per minute; the column temperature was 30°C; the detection wavelength was 220 nm; and the injection volume was 10 μl.

[0238] Table 15

[0239] The identification data of the compound with a retention time of 21.793 min are as follows:

[0240] 1 H NMR (400MHz, DMSO-d6) δ8.90–8.23(m,3H),8.26–7.89(m,1H),6.90(s,2H),5.68(s,1H),5.53(s,2H),5.22(s,1H),2.34–1.69(m,9H).

[0241] HRMS(ESI + ):m / z for C 24 H 19 ClF3N5ONa[M+Na] + calcd 524.1072, found 524.1095.

[0242] Example 15: Application of the impurities obtained by the present invention in the quality research of raw materials

[0243] Operation method: According to the 2020 edition of the Chinese Pharmacopoeia, General Chapter 0512 High Performance Liquid Chromatography, the specific analysis method is as shown in Example 14.

[0244] The obtained determination method has good separation between compound 2 and its isomers and impurities, the theoretical plate number and tailing factor of each impurity meet the requirements, the analysis time is short, the efficiency is high, and the cost is saved. It meets the requirements of the "Chinese Pharmacopoeia" and can effectively determine the quality of an MK2 compound raw material and pharmaceutical composition.

[0245] Example 16 Investigation of the stability of the raw material drug

[0246] Compound 2 and its isomer 2B were prepared by referring to the method of the prior art PCT / US2022 / 022525. After 0 day (or 0 h), several days of light exposure (naked) and / or several hours of acid destruction, the compound 2A and its isomers 2 and 2B were subjected to the method of Example 14 for determination of impurities 1, impurity 2, and impurity 3 to determine the content of the relevant impurities.

[0247] Therefore, analysis and detection of impurities 1, 2, and 3 are crucial for controlling the quality of compound 2 and its isomer raw materials.

[0248] Example 17 Activity Investigation

[0249] Test materials

[0250] Table 16

[0251] Test equipment

[0252] Table 17

[0253] Experimental methods:

[0254] 1. Human histiocytic lymphoma U937 cells were provided by Beina Biotechnology.

[0255] 2. Resuspend U937 cells in RPMI1640 complete medium (RPMI1640, 10% FBS, 1% penicillin-streptomycin) to a density of 1.05×10^5 cells / mL.

[0256] 3. U937 cells were dispensed into a 96-well plate at 1.0 × 10^4 cells / 95 μL / well. Ten concentrations of compound or vehicle (1% DMSO) were added in 5 μL volume according to the plate layout. The plates were incubated at 37°C, 5% CO2 for 48 hours.

[0257] 4. Remove the plate, add CCK8 11 μL / well, incubate at 37°C for 2.5 h, and measure the absorbance at 450 nm.

[0258] 5. Calculate cell viability or inhibition rate after reading at 450 nm on a microplate reader. Inhibition rate (%) = (OD control - OD experimental) / (OD control - OD blank) * 100%.

[0259] Experimental results:

[0260] Table 18

[0261] It can be seen from the above table that impurity 1 of compound 2 exhibits stronger anti-tumor activity than compound 2.

[0262] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be considered as equivalent replacement methods and are included in the scope of protection of the present invention.

Claims

1. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating atopic dermatitis, characterized in that: The compound is selected from:

2. Use of a compound or a pharmaceutically acceptable salt thereof in the preparation of a drug for preventing or treating asthma, characterized in that: The compound is selected from:

3. A pharmaceutical composition for preventing or treating atopic dermatitis, characterized in that: Comprising Compound 1 or a pharmaceutically acceptable salt thereof, or Compound 2 or a pharmaceutically acceptable salt thereof:

4. A pharmaceutical composition for preventing or treating asthma, characterized in that: Comprising Compound 1 or a pharmaceutically acceptable salt thereof, or Compound 2 or a pharmaceutically acceptable salt thereof:

5. Use of a compound or a pharmaceutically acceptable salt thereof in combination with methotrexate in the preparation of a medicament for preventing and / or treating inflammatory diseases, characterized in that: The compound is selected from:

6. The use according to claim 5, characterized in that The compound 1A is selected from compound 1, or 1B, or a mixture of the two in any ratio, Or the compound 2A is selected from compound 2, or 2B, or a mixture of the two in any ratio, 7. An impurity of compound 1, characterized in that Impurities 1, 2, 3 or mixtures thereof in any proportion selected from compound 1: Wherein: the impurity 1 and the impurity 2 are a pair of atropisomers, and the impurity 3 is a racemate of the impurities 1 and 2.

8. Use of an impurity of Compound 1 according to claim 7 as a reference substance for quality research of Compound 1 API and its pharmaceutical composition, characterized in that: The impurities are selected from impurities 1, 2, 3 of compound 1 or a mixture thereof in any proportion.

9. A pharmaceutical composition, characterized in that The active ingredient is compound 1, wherein the content of impurities 1, 2, 3 or a mixture thereof in any proportion of compound 1 is less than 1%.

10. An impurity of compound 2, characterized in that Impurities 1, 2, 3 or mixtures thereof in any proportion selected from compound 2: Wherein: the impurity 1 and the impurity 2 are a pair of atropisomers, and the impurity 3 is a racemate of the impurities 1 and 2.

11. Use of an impurity of Compound 2 according to claim 10 as a reference substance for quality research of Compound 2 API and its pharmaceutical composition, characterized in that: The impurities are selected from impurities 1, 2, 3 of compound 2 or a mixture thereof in any proportion.

12. A pharmaceutical composition, characterized in that The active ingredient is compound 2, wherein the content of impurities 1, 2, 3 or a mixture thereof in any proportion of compound 2 is less than 1%.

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