Compound, Preparation Method Therefor, And Use Thereof Patent Application (2025)

U.S. patent application number 16/961690 was filed with the patent office on 2021-03-18 for compound, preparation method therefor, and use thereof. This patent application is currently assigned to MEDSPRING INTERNATIONAL LIMITED. The applicant listed for this patent is MEDSPRING INTERNATIONAL LIMITED. Invention is credited to Haifeng ZHAO.

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COMPOUND, PREPARATION METHOD THEREFOR, AND USE THEREOF

Abstract

The present disclosure provides a compound of formula I or apharmaceutically acceptable salt, a solvate, or a prodrug thereof,wherein M is a monovalent alkali metal. The present disclosure alsoprovides a method for preparing the compound or itspharmaceutically acceptable salt, solvate, or prodrug, and furtherprovides a pharmaceutical composition containing the compound andits use in the preparation of a medicine, which can be used for thetreatment of CCR4-mediated diseases. ##STR00001##

Foreign Application Data

Claims

1. A compound of formula I, or a pharmaceutical acceptable salt, asolvate, or a prodrug thereof: ##STR00021## wherein M is amonovalent alkali metal.

2. The compound, or the pharmaceutical acceptable salt, thesolvate, or the prodrug thereof according to claim 1, wherein M isLi, Na, K, Rb, or Cs.

3. The compound, or the pharmaceutical acceptable salt, thesolvate, or the prodrug thereof according to claim 2, wherein M isNa or K.

4-8. (canceled)

9. A method for preparing a compound of formula I: ##STR00022##wherein M is a monovalent alkali metal, the method comprising:mixing a compound 6 of formula: ##STR00023## with a strong alkaliof the monovalent alkali metal M in an organic solvent.

10. The method according to claim 9, wherein the strong alkali ofthe monovalent alkali metal M is a hydride of M or an alkoxide ofM.

11. The compound, or the pharmaceutical acceptable salt, thesolvate, or the prodrug thereof according to claim 1, wherein thecompound has a solubility of at least 10 mg/ml in water.

12. The compound, or the pharmaceutical acceptable salt, thesolvate, or the prodrug thereof according to claim 1, wherein thecompound has an IC50 less than 1.00.times.10.sup.-8 mol/L in an invitro CCR4 antagonistic activity test.

13. The compound, or the pharmaceutical acceptable salt, thesolvate, or the prodrug thereof according to claim 1, wherein thecompound has a P.sub.app of at least 10.times.10.sup.-6 in an invitro membrane permeability test.

14. A method for treating a CCR4-mediated disease in a subject,comprising: administering a pharmaceutical composition in thesubject, wherein the pharmaceutical composition comprises thecompound, or the pharmaceutical acceptable salt, the solvate, orthe prodrug thereof according to claim 1.

15. The method of claim 14, wherein the pharmaceutical compositionfurther comprises one or more pharmaceutically acceptableexcipients, wherein the one or more pharmaceutically acceptableexcipients are mixed with the compound, or the pharmaceuticalacceptable salt, the solvate, or the prodrug thereof in thepharmaceutical composition.

16. The method of claim 14, wherein the compound, or thepharmaceutical acceptable salt, the solvate, or the prodrug thereofhas a dosage of 0.1 mg-1 g in a single dosage form of thepharmaceutical composition.

17. The method of claim 14, wherein M is Na or K.

18. The method of claim 14, wherein the CCR4-mediated disease is: arespiratory disease; inflammation of bone or joints; psoriasis, orinflammatory dermatosis; blepharitis; conjunctivitis; or anotherdisease associated with CCR4, selected from gastrointestinalinflammation, genitourinary inflammation, and immune systeminflammation.

19. The method of claim 18, wherein the CCR4-mediated disease is arespiratory disease, wherein the respiratory disease is selectedfrom a group consisting of an obstructive disease of airway,chronic obstructive pulmonary disease (COPD), bronchitis, cysticfibrosis, hypersensitivity pneumonitis, lung fibrosis, vasculiticdisorder or thrombotic disorder of lung vasculature, pulmonaryhypertension, rhinitis, and acute viral infection.

20. The method of claim 19, wherein the CCR4-mediated disease isasthma, COPD, or rhinitis.

21. The method according to claim 9, wherein a molar ratio of thecompound 6 to the strong alkali of the monovalent alkali metal M isapproximately 1:0.5-2.

22. The method according to claim 21, wherein a molar ratio of thecompound 6 to the strong alkali of the monovalent alkali metal M isapproximately 1:1.

23. The method according to claim 9, wherein M is Na or K.

24. The method according to claim 10, wherein the alkoxide of M isa tert-butoxide of M.

25. The method according to claim 9, wherein the organic solvent isanhydrous tetrahydrofuran.

Description

[0001] The present application claims priority to the Chinesepatent application No. 201810032410.2, entitled "Compound,Preparation Method therefor, and Use thereof" and filed Jan. 12,2018, the whole content of which is hereby incorporated byreference in its entirety.

TECHNICAL FIELD OF THE INVENTION

[0002] The present invention relates to compounds, and theirpharmaceutically acceptable salts, solvates, or prodrugs, and alsorelates to a method for preparing the compounds or theirpharmaceutically acceptable salts, solvates, or prodrugs, andfurther relates to pharmaceutical compositions containing thecompounds and their application in the preparation of apharmaceutical drug, which can be used for the treatment ofCCR4-mediated diseases.

BACKGROUND

[0003] CCR4 (chemokine receptor 4) was first discovered byChristine A. power in 1995 (Christine A P et al. J. Biol. Chem.1995, 270 (8): 19495-19500). It belongs to the chemokine receptor(CCR) family and is a seven-time transmembrane G-protein coupledreceptor. It has two naturally existing specific ligands: MDC(macrophage derived chemokine) and TARC (thymus and activationregulated chemokine) (Sadatoshi Maeda et al., Veterinary Immunologyand Immunology 2002 (90): 145-154). A newly discovered chemokinelike factor 1 (CKLF1) is also one of its ligands (Han W L et al.,Biochem J. 2001, 357 (Pt1): 127-135).

[0004] CCR4 can be expressed in peripheral blood leukocytes,thymocytes, basophils, monocytes, macrophages, platelets,IL-activated NK cells, spleen, and brain, etc., and plays animportant role in many diseases. For example, upon onset of humanallergic dermatitis (AD), the expression of CCR4 on CD4+T cells isincreased in peripheral blood monocytes (PBMCs), and the serum TARClevel is also increased accordingly. This suggests that thechemotaxis of CCR4 expression in cells is induced by TARC andselectively causes Th2 cells to migrate to damaged skin in theallergic dermatitis. Drugs used to treat allergic dermatitis mainlyinclude antihistamines and bronchodilators, which nonetheless canonly improve symptoms, but have no effects on the development ofthe disease. In addition, corticosteroids also have certain effectson the allergic dermatitis, but there are potential safety risks.Studies have shown that antagonism against MDC or TARC can reducethe aggregation of T cells in inflammatory sites. CCR4 antagonistsmay be effective in the treatment of allergic inflammations, whichinclude, but are not limited to, allergic rhinitis and allergicdermatitis.

[0005] CCR4 has been found to be associated with lung diseases suchas chronic obstructive pneumonia, chronic bronchitis, and asthma.CCR4 can be limited to being expressed in cells involved in asthmaresponse, and thus is considered to be a good target for thetreatment of asthma. Therefore, the development of CCR4 antagonistshas a promising application prospect.

[0006] CN101370793A discloses compounds having the followingformula and their use as CCR4 antagonists, including theirapplication in the CCR4 mediated diseases

##STR00002##

wherein the various substituent groups are disclosed in the patentapplication.

[0007] CN102388039A discloses a class of indazole compounds andtheir use as CCR4 antagonists, including their application in thetreatment that is related to CCR4 antagonists

##STR00003##

wherein the various substituent groups are disclosed in the patentapplication.

[0008] In the field, better CCR4 inhibitors are still needed, whichare characterized by having, for example, a better CCR4 inhibitoryactivity, a higher solubility in water or other solvents, or ahigher bioavailability.

SUMMARY OF THE INVENTION

[0009] Provided in this disclosure includes a compound of formulaI, and its pharmaceutically acceptable salts, solvates, orprodrugs.

##STR00004##

[0010] Herein, M can be a monovalent alkali metal. For example, Mcan be Li, Na, K, Rb, or Cs, and preferably, M is Na or K.

[0011] In one aspect of the disclosure, the compound of formula Iis

##STR00005##

[0012] In another aspect of the disclosure, the compound havingformula I is

##STR00006##

[0013] In this present disclosure, the suitable pharmaceuticallyacceptable salt of the compound of formula I can be, for example,an acid addition salt of the compound of formula I, such as an acidaddition salt formed with an inorganic or organic acid (e.g.hydrochloric acid, hydrobromic acid, sulfuric acid, trifluoroaceticacid, citric acid, or maleic acid), or can be an ammonium salt, ora salt formed with organic bases (e.g. methylamine, dimethylamine,trimethylamine, piperidine, morpholine, or tri-(2-hydroxyethyl)amine).

[0014] In this present disclosure, the suitable pharmaceuticallyacceptable solvate for the compound of formula I can be, forexample, a hydrate, such as a hemihydrate, a monohydrate, adihydrate, a trihydrate, or a combination thereof.

[0015] In this present disclosure, the compound can be administeredin the form of prodrugs. Herein, the term "prodrug" is referred toas a compound which is decomposed or cracked in human body oranimal body to thereby release the compound provided in thispresent disclosure. Examples of prodrugs include in vivo cleavableester derivatives and in vivo cleavable amide derivatives. Theester derivatives can be formed on the carboxyl or hydroxyl groupsof the compound of formula I, and the amide derivatives can beformed on the carboxyl or amino groups of the compound of formulaI.

[0016] A pharmaceutically acceptable prodrug of the compound offormula I can be, for example, an ester or ether that is cleavableor decomposable in a body. The in vivo cleavable esters or ethersof the compound of formula I containing hydroxyl groups can bepharmaceutically acceptable esters or ethers, such as esters orethers that can be cleaved in humans and animals to produceparental hydroxyl compounds. With regard to hydroxyl groups,suitable pharmaceutically acceptable ester-forming groups caninclude inorganic esters such as phosphate esters (includingaminophosphocyclic esters). Further with regard to the hydroxylgroups, suitable pharmaceutically acceptable ester-forming groupscan also include (1-10C) alkanoyl groups (e.g. acetyl group,benzoyl group, phenylacetyl group and substituted benzoyl group,and phenylacetyl group), and (1-10C) alkoxycarbonyl groups (e.g.ethoxycarbonyl group, N, N-[di-(1-4C) alkyl] carbamoyl group,2-dialkylaminoacetyl group, and 2-carboxyacetyl group). Examples ofthe substituents on the phenylacetyl group and benzoyl group caninclude aminomethyl group, n-alkylaminomethyl group,N,N-dialkylaminomethyl group, morpholinomethyl group,piperazine-1-ylmethyl group, and 4-(1-4C) alkylpiperazine-1-ylmethyl group. Further with regard to the hydroxylgroups, suitable pharmaceutically acceptable ether-forming groupscan include .alpha.-acyloxyalkyl groups such as acetoxymethyl groupand pivaloyloxymethyl group.

[0017] In this present disclosure, the compound of formula I or itspharmaceutically acceptable salts, solvates, or prodrugs have anCCR4 inhibitor activity. As such, the compound of formula I or itspharmaceutically acceptable salts, solvates, or prodrugs can beused for the treatment of CCR4-mediated diseases, or can be usedfor the preparation of medicines for the treatment of CCR4-mediateddiseases. Specifically, the compound of formula I provided in thepresent disclosure, or its pharmaceutically acceptable salts,solvates or prodrugs can be used to treat diseases in which theregulation of CCR4 (e.g., inhibiting the activity of CCR4) isbeneficial to patients.

[0018] In one aspect of the present disclosure, a pharmaceuticalcomposition is provided, which comprises the compound of formula Idescribed above or a pharmaceutically acceptable salt, a solvate,or a prodrug thereof, and a pharmaceutically acceptable excipientmixed therewith, such as a diluent or a carrier. The pharmaceuticalcomposition can be used for the treatment of CCR4-mediateddiseases.

[0019] The pharmaceutical composition provided in the presentdisclosure can include a conventional pharmaceutical excipient thatis well known in the art, and can be obtained by a conventionalmethod. For example, an excipient included in a pharmaceuticalcomposition that is intended for oral administration can compriseone or more of a colorant, a sweetener, a flavoring agent, and/or apreservative, etc.

[0020] The pharmaceutical composition provided in the presentdisclosure can be used in a form that is suitable for oraladministration (e.g. tablets, pastilles, hard or soft capsules,water or oil suspensions, emulsions, dispersible powder orgranules, syrup, or elixir), in a form suitable for topicaladministration (e.g. cream, ointment, gel, water or oily solutionor suspension), in a form suitable for inhalation administration(e.g. finely dispersed powder or liquid aerosol), in a formsuitable for spray (e.g. fine dispersible powder), or in a formsuitable for parenteral administration (e.g., sterile water or oilysolution for intravenous, subcutaneous, intraperitoneal orintramuscular administration, or suppositories for rectaladministration).

[0021] The amount of the active ingredient that is combined withone or more excipients to form a single dosage form can changebased on needs, depending on the subject receiving the treatmentand the specific route of administration. For example, the dosageform intended for oral administration to human subjects generallycan contain, e.g. 0.1 mg-1 g (more preferably 1-250 mg, e.g., 1-100mg) of the active ingredient mixed with an appropriate andconvenient amount of excipients, which can account for about 5-98%of the total weight of the composition.

[0022] The dosage size of the compound of formula I or itspharmaceutically acceptable salts, solvates, or prodrugs fortherapeutic or preventive purposes typically can vary according towell-known medical principles, depending on the nature and severityof the disease, the age and sex of an animal or a patient, and theroute of administration.

[0023] In one aspect of the present disclosure, the compound offormula I as described above, or the pharmaceutically acceptablesalts, solvates or prodrugs thereof, are provided for the treatment(or prevention) of a CCR4-mediated disease.

[0024] In another aspect of the present disclosure, the compound offormula I as described above, or the pharmaceutically acceptablesalts, solvates or prodrugs thereof, are provided for use in thepreparation of drugs or medicines for the treatment (or prevention)of CCR4 mediated diseases.

[0025] The CCR4 mediated diseases can be diseases and conditionsassociated with inflammation or infection. The CCR4 mediateddiseases can include:

[0026] (1) respiratory diseases, such as obstructive diseases ofairways including: asthma, which includes bronchial, allergic,intrinsic, extrinsic, exercise-induced, drug-induced (includingaspirin and NSAID-induced), and dust-induced asthma, andintermittent and persistent asthma; chronic obstructive pulmonarydisease (COPD); bronchitis, which includes infectious andeosinophilic bronchitis; emphysema; bronchiectasis; cysticfibrosis; hypersensitivity pneumonitis; lung fibrosis, whichincludes cryptogenic fibrosing alveolitis, idiopathic interstitialpneumonias, and fibrosis complicating anti-neoplastic therapy andchronic infection; vasculitic and thrombotic disorders of the lungvasculature, and pulmonary hypertension; rhinitis, which includesacute and chronic rhinitis, and includes rhinitis medicamentosa,and vasomotor rhinitis, perennial and seasonal allergic rhinitisincluding rhinitis nervosa; acute viral infection including commoncold, and infection cased by respiratory syncytial virus,influenza, coronavirus and adenovirus;

[0027] (2) inflammation of bone and joints, such as arthritidesassociated with or including osteoarthritis or osteoarthrosis;cervical and lumbar spondylitis, low back pain, and neck pain;osteoporosis; rheumatoid arthritis; acute and chronic synovitis,including urate gout, calcium pyrophosphate deposition disease, andcalcium apatite-related tendon, bursal and synovial inflammation;inflammatory myopathies, including dermatomyositits andpolymyositis; polymalgia rheumatica; juvenile arthritis includingidiopathic inflammatory arthritides of whatever joint distributionand associated syndromes, and rheumatic fever and its systemiccomplications; and drug-induced arthalgias, tendonititides, andmyopathies;

[0028] (3) psoriasis and inflammatory dermatosis, such asdermatitis, eczema, atopic dermatitis, allergic contact dermatitis,rubella and pruritus;

[0029] (4) blepharitis and conjunctivitis, including perennialallergic conjunctivitis or spring allergic conjunctivitis; iritis;anterior and posterior uveitis; choroiditis; autoimmune eyediseases; degenerative or inflammatory disorders affecting theretina; ophthalmitis including sympathetic ophthalmitis;sarcoidosis; and eye infections, including viral, fungal, andbacterial infections; and

[0030] (5) other diseases associated with CCR4, such asgastrointestinal inflammation, genitourinary inflammation, andimmune system inflammation, etc.

[0031] In yet another aspect of the present disclosure, a methodfor preparing a compound of formula I is provided.

##STR00007##

[0032] Herein, M can be a monovalent alkali metal, such as Li, Na,K, Rb or Cs, with Na or K being preferred. The method comprises:stir-mixing the compound 6 of the formula:

##STR00008##

[0033] with a strong alkali of the monovalent alkali metal M in anorganic solvent, which is then filtered and dried. Preferably, themolar ratio of the compound 6 to the strong alkali of themonovalent alkali metal M is approximately 1:0.5-2. Morepreferably, the molar ratio of the compound 6 to the strong alkaliof the monovalent alkali metal M is approximately 1:1.

[0034] The reaction can be carried out in a suitable organicsolvent. For example, the organic solvent can be an ester solvent,an aromatic hydrocarbon solvent, an ether solvent, an alcoholsolvent, or another water-soluble polar solvent: the ester solventcan be methyl acetate, ethyl acetate, propyl acetate or butylacetate; the aromatic solvent can be benzene, toluene,ethylbenzene, isopropyl benzene or xylene; the ether solvent isether, isopropyl ether, methyl tert butyl ether, tetrahydrofuran,1,4-dioxane, ethylene glycol dimethyl ether, or diethylene glycoldimethyl ether; the alcohol solvent can be methanol, ethanol,propanol, n-butanol or ethylene glycol; the another water-solublepolar solvents can be acetone, butanone, N, N-dimethylformamide, N,N-dimethylacetamide or dimethyl sulfoxide. The strong alkali of themonovalent alkali metal M can be a hydride of M, such as NaH or KH.The strong alkali of the monovalent alkali metal M can also be analkoxide of M, such as sodium tert-butoxide or potassiumtert-butoxide. The strong alkali can also be an oxide, a hydroxideor a bicarbonate of the alkali metal M. In another aspect of thepresent invention, the method comprises: dissolving the compound 6in an organic solvent, adding MH or an alkoxide of M,stirring-mixing for a reaction (e.g., at a temperature of15.degree. C.-35.degree. C. for 1-3.5 hours), and filtering anddrying. Preferably, the molar ratio of the compound 6 to the MH orthe alkoxide of M is 1:0.5-2, and more preferably, the molar ratioof the compound 6 to the MH or the alkoxide of M is about 1:1.

[0035] In yet another aspect of the present disclosure, a methodfor preparing a compound of formula II shown below is provided.

##STR00009##

[0036] Herein, the method comprises: dissolving the compound 6 inan organic solvent, adding NaH, stirring-mixing for a reaction(e.g., at a temperature of 15.degree. C.-35.degree. C. for 1-3.5hours), and filtering and drying. Preferably, the molar ratio ofthe compound 6 to the NaH is 1:0.5-2, and more preferably is about1:1.

[0037] In yet another aspect of the present disclosure, a methodfor preparing a compound of formula III shown below isprovided.

##STR00010##

[0038] Herein, the method comprises: dissolving the compound 6 inan organic solvent, adding KH, stirring-mixing for a reaction(e.g., at a temperature of 15.degree. C.-35.degree. C. for 1-3.5hours), and filtering and drying. Preferably, the molar ratio ofthe compound 6 to the KH is 1:0.5-2, and more preferably, is about1:1.

[0039] In yet another aspect of the present disclosure, a methodfor preparing a compound of formula III shown below isprovided.

##STR00011##

[0040] Herein, the method comprises: dissolving the compound 6 inan organic solvent, adding potassium tert butyl alcohol,stirring-mixing for a reaction (e.g., at a temperature of15.degree. C.-35.degree. C. for 1-3.5 hours), and filtering anddrying. Preferably, the molar ratio of the compound 6 to thepotassium tert-butoxide is 1:0.5-2, and more preferably, is about1:1.

[0041] Currently existing CCR4 inhibitors have an issue of lowsolubility, which increases the difficulty in drug preparation,affects the release of active ingredients, causes lowbioavailability, and fails to reach an expected blood drugconcentration in vivo at a maximum safe dose, thereby affecting theefficacy of the drugs. The compound of formula I, especially thecompounds of formula II and formula III are better CCR4 inhibitors,which have advantages over the prior art, including: 1.Significantly enhanced solubility. Compared with compound 6,compound II and compound III have significantly enhanced solubilityunder the same conditions.

[0042] For example, in an aqueous solution at room temperature, thesolubility of compounds of formula II and formula III is 500 or 40mg/ml, whereas the solubility of compound 6 is 0.02 mg/ml under thesame conditions, thus the solubility of compounds II and III is2000-25000 times higher than that of compound 6.

[0043] 2. Better bioavailability. Based on a standard CaCO-2 invitro membrane permeability evaluation, results showed that theP.sub.app (apparent permeability coefficient) of compound 6 is0.13.times.10.sup.-6, indicating that compound 6 is adifficult-to-absorb compound, whereas the P.sub.app of compounds offormula II and formula III is 68.82-101.33.times.10.sup.-6 underthe same experimental conditions, which is 530-780 times higher interms of the permeability, indicating that compounds of formula IIand formula III are easy-to-absorb compounds.

[0044] 3. Better in vitro and in vivo activity. Radiolabelingexperiments show that the IC50 of the compounds of formula II andformula III are 2.26.times.10.sup.-9 mol/L-7.47.times.10.sup.-10mol/L, which are 6 and 210 times higher than that of compound 6under the same experimental conditions.

DETAILED DESCRIPTION OF THE INVENTION

[0045] In the following, with combination of embodiments,description of the substantive contents and the beneficial effectsof the present invention is further provided. It is noted that theembodiments serve only to illustrate, and are not intended to limitthe scope of, the present invention.

Embodiment 1 Preparation ofN-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methyl propionamide

[0046] Preparation of compound 6 having the following formula:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl) methyl]-2-hydroxy-2-methyl propionamide isprovided.

##STR00012##

Step 1 Preparation of an Intermediate Compound 1 (Show Below):4-(methoxy)-1H-indazole-3-amine

##STR00013##

[0048] 6-fluoro-2-methoxybenzonitrile (50 g, 0.33 mol) andhydrazine hydrate (50 g, 0.99 mol) were dissolved in 400 mln-butanol, and refluxed for 20 h under the protection of N.sub.2.After the solution was cooled down, 400 ml water was added, thereaction was stirred to mix evenly, the organic layer is separated,and the solid precipitated from the aqueous phase was collected.The organic layer solvent was removed by vacuum distillation, andthe residues were mixed with the aqueous phase and the solidprecipitated from the aqueous phase, and the mixture was extractedwith ethyl acetate (3.times.400 ml). The organic layer was washedwith water (2.times.400 ml) and dried with anhydrous magnesiumsulfate. The gray white solid (46.32 g, yield 85.8%) was obtainedby vacuum drying after solvent removal by vacuum distillation. MSESI+164 (M+H).sup.+.

Step 2 Preparation of an Intermediate Compound 2 (Show Below):3-{[3-amino-4-(methoxy)-1H-indazole-1-yl]methyl}benzonitrile

##STR00014##

[0050] Potassium hydroxide (9 g, 160.7 mmol) and DMSO (200 ml) weremixed and stirred evenly. Then 4-(methoxy)-1H-indazole-3-amine(10.48 g, 64.3 mmol) was added, and the solution turned darkpurple. After stirring for 10 min at room temperature,3-(chloromethyl)benzonitrile (11.8 g, 64.3 mmol) was added andstirred to mix for 2 h. 500 ml water was then added to the reactionsolution, and the reaction changes into an emulsion, which furtherunderwent dichloromethane extraction (3.times.400 ml), waterwashing with water (2.times.400 ml), anhydrous magnesium sulfatedrying, column chromatography (dichloromethane/ethyl acetate=5/1),solvent removal by vacuum distillation, and vacuum drying. A lightyellow solid (13.22 g, yield 73.9%) was obtained. MS ESI+279(M+H).sup.+.

Step 3 Preparation of an Intermediate Compound 3 (Shown Below):5-chloro-N-{1-[(3-cyanophenyl)methyl]-4-(methoxy)-1H-indazole-3-yl}-2-thi-ophene sulphonamide

##STR00015##

[0052] Pyridine (12 ml) solution of 5-chloro-2-thiophenesulfonylchloride (7.96 g, 36.7 mmol) was dropped into a two-mouthed bottlecontaining3-{[3-amino-4-(methoxy)-1H-indazole-1-yl]methyl}benzonitrile (10.2g, 36.7 mmol) at room temperature and under a nitrogen atmosphere.The reaction system turns dark red. One hour later, the reactionsolution was poured into 500 ml hydrochloric acid (2 mol/L), andthe reaction further underwent dichloromethane extraction(4.times.500 ml), washing with water (3.times.500 ml), anhydrousmagnesium sulfate drying, column chromatography(dichloromethane/ethyl acetate=100/1), solvent removal by vacuumdistillation, and vacuum drying. A white solid (13.43 g, yield79.9%) was obtained. MS ESI+ 459 (M+H).sup.+.

Step 4 Preparation of an Intermediate Compound 4 (Shown Below):N-[1-{[3-(aminomethyl)phenyl]methyl}-4-(methoxy)-1H-indazole-3-yl]-5-chlo-ro-2-thiophene sulfonamide hydrochloride

##STR00016##

[0054]5-chloro-N-{1-[(3-cyanophenyl)methyl]-4-(methoxy)-1H-indazole-3-yl}--2-thiophene sulfonamide (15.75 g, 34.4 mmol) was dissolved in 200ml tetrahydrofuran and placed at 0.degree. C. Under the protectionof nitrogen, 1.0 M lithium aluminum hydride solution in THE (86 ml,86 mmol) was added dropwise into the reaction system, and thetemperature of the system was kept below 10.degree. C. Afterdropping, the system was transferred to room temperature andstirred to mix for 2 h. A quenching reaction was carried out byadding 10 ml water and 60 ml 2 mol/L sodium hydroxide solution.After stirring to mix for 30 min, the solid was filtered out, thesolid residue was washed with THF, and the filtrate was combined.The filtrate was further treated with a silica gel column, filteredand washed with methanol, and then washed with 10% 2N hydrochloricacid in 2.5 L. A white solid (12.6 g, yield 73.6%) was obtainedafter vacuum drying. MS ESI+463 (M+H).sup.+.

Step 5 Preparation of an Intermediate Compound 5 (Shown Below):2-{[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole--1-yl]methyl}phenyl)methyl]amino}-1,1-dimethyl-2-oxoethylacetate

##STR00017##

[0056]N-[1-{[3-(aminomethyl)phenyl]methyl}-4-(methoxy)-1H-indazole-3-yl]--5-chloro-2-thiophene sulfonamide hydrochloride (15.6 g, 31.3 mmol),triethylamine (9.6 g, 95 mmol) and dichloromethane (400 m) weremixed and stirred evenly in ice water bath. Then2-chloro-1,1-dimethyl-2-oxoethyl acetate (5.2 g, 31.3 mmol) wasadded dropwise. After dropping, stirring was continued for 2 hoursat room temperature and under nitrogen protection. Washing wascarried out with 2 mol/L hydrochloric acid (150 ml), saturatedsodium bicarbonate solution (150 ml) and saturated salt water (150ml), followed by anhydrous magnesium sulfate drying, columnchromatography (petroleum ether/ethyl acetate=1/1), solvent removalby vacuum distillation, and vacuum drying to thereby obtain a whitefoam solid (14.1 g, yield 76.3%). MS ESI+591 (M+H).sup.+.

Step 6 Preparation of an Intermediate Compound 6 (Shown Below):N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide

##STR00018##

[0058] Potassium carbonate (4.6 g, 33.3 mmol) was added to methanol(350 ml) solution containing2-{[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole--1-yl]methyl}phenyl)methyl]amino}-1,1-dimethyl-2-oxoethyl acetate(6.55 g, 11.1 mmol). After stirring for 4 h at room temperature, alarge amount of solid was precipitated from the system. After thesolvent is evaporated for removal by vacuum (i.e. solvent removalby vacuum distillation), 100 ml water is added, and the pH isadjusted to acid with 2 mol/L hydrochloric acid. Ethyl acetate wasextracted (200 ml.times.2), and the extraction solution was washedwith water (200 ml.times.2) until the water layer was neutral. Theorganic layer was dried with anhydrous magnesium sulfate. Thencolumn chromatography (petroleum ether/ethyl acetate=1/1.5),solvent removal by vacuum distillation, and vacuum drying werecarried out to thereby obtain a white solid (5.08 g, yield 83.5%).MS ESI+549 (M+H).sup.+; 1H-NMR (400 MHz, DMSO-d6): 10.40 (s, 1H),8.13 (t, 1H), 7.33 (d, 1H), 7.24-7.08 (m, 6H), 6.93 (d, 1H), 6.46(d, 1H), 5.45 (s, 2H), 5.33 (s, 1H), 4.17 (d, 2H), 3.72 (s, 3H),1.20 (s, 6H).

Embodiment 2 Preparation of the Compound of Formula II:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide sodium

##STR00019##

[0060] The compound of formula 6:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide (2.5 g,4.56 mmol) was dissolved in 30 ml of anhydrous tetrahydrofuran andstirred to mix until the solution was clear. NaH (60%) (182 mg,4.56 mmol) was added into the system and stirred to mix for 1.0 hat 35.degree. C. A large amount of a white solid was produced inthe system. The white solid was obtained by filtration, which wasthen washed with tetrahydrofuran and transferred to a vacuum dryingoven for drying. A white solid (2.09 g, yield 80.4%) was obtained.1H-NMR (400 MHz, DMSO-d6) 8.08 (t, 1H), 7.20 (d, 1H), 7.14-6.78 (m,7H), 6.20 (d, 1H), 5.33 (s, 1H), 5.19 (s, 2H), 4.16 (d, 2H), 3.74(s, 3H), 1.21 (s, 6H).

Embodiment 3 Preparation of the Compound of Formula III:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamidepotassium

##STR00020##

[0062] Method 1: The compound of formula 6:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)meth yl]-2-hydroxy-2-methylpropionamide (2.5 g,4.56 mmol) was dissolved in 30 ml of anhydrous tetrahydrofuran andstirred to mix evenly until the solution was clear. KH (30%) (600mg, 4.56 mmol) was added into the system and stirred to mix at15.degree. C. for 3.5 h. A large amount of a white solid wasproduced in the system. The white solid was obtained by filtration,washed with tetrahydrofuran, and transferred to a vacuum dryingoven for drying. A white solid (2.21 g, yield 83.1%) was thenobtained. 1H-NMR (400 MHz, DMSO-d6): 8.10 (t, 1H), 7.22 (d, 1H),7.17-6.79 (m, 7H), 6.21 (d, 1H), 5.35 (s, 1H), 5.19 (s, 2H), 4.18(d, 2H), 3.75 (s, 3H), 1.22 (s, 6H).

[0063] Method 2: The compound of formula 6:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)meth yl]-2-hydroxy-2-methylpropionamide (1.37 g,2.5 mmol) was dissolved in 15 ml of anhydrous tetrahydrofuran andstirred to mix evenly until the solution was clear. Potassiumtert-butoxide (280 mg, 2.5 mmol) was added into the system andstirred to mix for 10 min at room temperature. A large amount of awhite solid was produced in the system. The white solid wasobtained by filtration, washed with tetrahydrofuran, and thentransferred to a vacuum drying oven for drying. A white solid (1.35g, yield 92.1%) was obtained. 1H-NMR (400 MHz, DMSO-d6): 8.09 (t,1H), 7.20 (d, 1H), 7.13-6.78 (m, 7H), 6.20 (d, 1H), 5.34 (s, 1H),5.18 (s, 2H), 4.16 (d, 2H), 3.74 (s, 3H), 1.21 (s, 6H).

Embodiment 4 Solubility Tests

[0064] Solubility of compounds was determined according to themethod established in the general instructions of Part II ofPharmacopoeia 2010 edition. Respectively, 10, 100, and 1000 mg ofcompound 6 that was prepared in the Embodiment 1:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide wereaccurately weighed, arranged in 500 ml volumetric flasks at25.degree. C..+-.2.degree. C., and diluted with water to the scale,which were shaken vigorously for 30 seconds every 5 minutes. Thedissolution was observed within 30 minutes. Only the sample addedwith 10 mg of compound 6 showed no visible solute. The solubilityin water was determined to be 0.02 mg/ml.

[0065] Respectively, 10, 100, 1000, and 2000 mg of the compoundwith formula II that was prepared in the Embodiment 2:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide sodiumwere accurately weighed, arranged in 2 ml volumetric flasks at25.degree. C..+-.2.degree. C., and diluted with water to the scale,which were shaken vigorously for 30 seconds every 5 minutes. Thedissolution was then observed within 30 minutes. No visible solutewas observed for all samples except the 2000 mg sample, and thesolubility in water was determined to be 500 mg/ml.

[0066] Respectively, 8, 80, 800, and 1600 mg of the compound offormula III that was prepared by the method described in theEmbodiment 3:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide potassiumwere accurately weighed, arranged in 2 ml volumetric flasks at25.degree. C..+-.2.degree. C., and diluted with water to the scale,which were shaken vigorously for 30 seconds every 5 minutes. Thedissolution was then observed within 30 minutes. No visible solutewas observed for all samples except the 800 mg and 1600 mg sample,and the solubility in water was determined to be 40 mg/ml.

[0067] The results are shown in Table 1.

TABLE-US-00001 TABLE 1 Solubility in water Compound under testunder 25.degree. C. .+-. 2.degree. C. Compound 6 0.02 mg/mlCompound of formula II 500 mg/ml Compound of formula III 40mg/ml

[0068] Compared with the above compound 6:N-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1-yl]methyl}phenyl)methyl]-2-hydr-oxy-2-methylpropionamide, compounds of formula II and formula IIIhave significantly enhanced solubility under the same conditions.In an aqueous solution and at room temperature, the solubility ofcompounds of formula II and formula III is 500 mg/ml and 40 mg/ml,respectively, whereas under the same conditions, the solubility ofN-[(3-{[3-{[(5-chloro-2-thienyl)sulfonyl]amino}-4-(methoxy)-1H-indazole-1--yl]methyl}phenyl)methyl]-2-hydroxy-2-methylpropionamide is 0.02mg/ml. Thus the solubility is 2000-25000 times higher in relativeterms.

Embodiment 5 In Vitro CCR4 Antagonistic Activity Tests

[0069] The potency of antagonists was tested by a competitive testof [35S]-GTP.gamma.S. Briefly, CHO membranes expressing CCR4 werehomogenized through a 23 G needle. These membranes were thenattached to WGA-coated Leadseeker SPA beads in a testing buffer (20mM HEPES, 10 mM MgCl.sub.2, 100 mM NaCl, 0.05% BSA, 40 ug/mlsaponin, adjusted to pH 7.4 with KOH 5M) to produce 3 .mu.g/wellfinal acceptance criteria (FAC) membrane and 250 .mu.g/well FACbead solution. After 60 minutes of pre-coupling on ice, GDP wasadded to obtain 4.4 uM FAC. Then [35S]-GTPS, which was prepared inthe testing buffer, was added into the bead/membrane solution tothereby obtain 0.33 nM FAC. Human MDC was added to thebead/membrane/[35S]-GTP.gamma.S suspension to obtain 80% (EC80) ofFAC which showed the largest agonist response. The suspension ofbeads/membranes/[35S]-GTP.gamma.S/agonist was distributed into awhite Greiner polypropylene 384-pore plate (45 .mu.L/well), whichcontained 0.5 .mu.l of compound. The final testing solution (45.5.mu.L) was sealed, centrifuged and incubated at room temperaturefor 3-6 hours. Then, the plate was read with Viewlux and theluminescence was plotted as the percentage of the maximuminhibitory response induced by IC.sub.100 standard antagonist. Thetest results are shown in Table 2.

TABLE-US-00002 TABLE 2 Compound under test IC50 Compound 6 3.54.times. 10.sup.-8 mol/L Compound of formula II 7.47 .times.10.sup.-10 mol/L.sup. Compound of formula III 2.26 .times.10.sup.-9 mol/L

Embodiment 6 In Vitro Membrane Permeability Test

[0070] Caco-2 cell model is a human colonic adenocarcinoma cellline having a structure and function similar to differentiatedintestinal epithelial cells. It has microvilli and otherstructures, and contains enzyme system related to intestinal brushborder epithelium. It can be used to simulate intestinal transportin vivo, and has become a standard in vitro screening tool forpredicting drug absorption in human small intestine and studyingthe drug transport mechanisms. The method to determine the drugabsorption capacity can be P.sub.app (apparent permeabilitycoefficient), with P.sub.app>2.times.10.sup.-6 determined to bea good-absorption drug.

[0071] 1. Experimental Materials and Instruments

[0072] Caco-2 cells (gifted by School of Pharmacy, ZhejiangUniversity), DMEM medium (GIBCO-BRL, USA), standard fetal bovineserum (FBS, Hydroxyne, USA), and nonessential amino acids (NEAA,Hyclone, USA), penicillin (Huabei Pharmaceutical Co., Ltd.),streptomycin (Huabei Pharmaceutical Co., Ltd.), HEPES (Amresco,USA), EDTA-Na (Beijing Chemical Plant), trypsin (1:250, Amresco,USA), glutamine (Sigma, USA), G418 (Merck, USA), and MilliPoreultra pure water.

[0073] Millicell insert-type culture dish (polycarbonate membrane,0.4 .mu.m, 12 mm diameter, MilliPore, USA), cell culture plate(COSTAR, USA), cell resistance meter (Millicell-ERS, MilliPore,USA), ultra clean bench (SW-CJ-IFD, Suzhou Antai Air TechnologyCo., Ltd.), carbon dioxide incubator (Sanyo, Japan), invertedmicroscope (CK, Olympus, Tokyo), culture dish (Corning, USA), ultrapure water machine (Simplicity, MilliPore, USA), fluorescencespectrophotometer (F-4500, Hitachi, Japan), electronic balance (BS110S, Beijing Saiduolis Balance Co., Ltd.), LC-MS (Agilent 1100,LC-MSD_VL, DE, USA).

[0074] 2. Experimental Methods

[0075] 2.1 Solution Preparation

[0076] Preparation of culture medium: 10% FBS, 1% glutamine, 100UmL.sup.-1 penicillin and streptomycin double antibiotics solution,1% nonessential amino acids, and 1.2 mgL.sup.-1 G418 were added toDMEM when used.

[0077] Preparation of digestive solution: 1 g trypsin and 80 mgEDTA were added into 400 ml phosphate buffer solution, which wasfurther filtered with 0.22 .mu.M filter membrane for sterilization,and was frozen storaged at -20.degree. C. for standby use.

[0078] Preparation of glutamine stock solution: glutamine 2.92 gwas added into 100 ml PBS buffer, which was further filtered with0.22 .mu.M filter membrane for sterilization, sub-packaged in 1 mLand frozen storaged at -20.degree. C.

[0079] The preparation of penicillin stock solution: 0.8 million Uof penicillin was added into 20 ml of physiological saline; 1million U of streptomycin was added into 25 ml physiologicalsaline, which were mixed in 1:1, and was then filtered with 0.22.mu.M filter membrane for sterilization, sub-packaged in 1 mL, andfrozen storaged at -20.degree. C.

[0080] HBSS solution preparation: NaCl 8.0 g, KC 0.4 g,Na.sub.2IPO.sub.4.H.sub.2O 0.0475 g, KH.sub.2PO.sub.4 0.06 g, andhapes 6 g were added to ultrapure water to dissolve, the pH valuewas adjusted to 7.2-7.4, and water was added to a final 1 L, whichwas then filtered with 0.22 .mu.M filter membrane forsterilization, and storaged at -20.degree. C.

[0081] 2.2 Cell Culture

[0082] The cryopreserved Caco-2 cells were thawed in 37.degree. C.water bath. After resuscitation, the cells were added into DMEMmedium containing 10% FBS, and cultured in an incubator with37.degree. C., 5% CO.sub.2 and 90% relative humidity. The culturemedium was changed every other day. After 1-2 days of growth, thecells were digested with 0.25% trypsin-EDTA (0.2%) mixed digestivesolution at 37.degree. C. and passaged in a certain proportion. Thecell generation used in the experiments was 40-60 passages.

[0083] When cells reached 80% confluency, they were digested andsuspended in complete medium and seeded on Millicell plate at1.times.10.sup.6 cellsml.sup.-1. After that, the culture medium waschanged once every 2 days and after 1 week, was changed once every1 day. After 5 days of culture, the resistance value reachesplateau (>200 .OMEGA.cm.sup.2), which can be then used formembrane permeability experiment.

[0084] 2.3 Quality Control of Caco-2 Monolayer Cells:

[0085] 2.3.1 Measurement of Transmembrane Epithelial Cell ElectricResistance (TEER).

[0086] When measuring the transmembrane resistance, an electrodewas immersed in DMEM culture medium for 24 hours, then it was takenout and immersed in 70% alcohol for disinfection for 15 minutes.After that, the electrode was placed at room temperature and driednaturally, and then put into sterile DMEM culture medium for 15minutes. During the experiment, the two ends of the electrode wereinserted into the upper and lower pools of each hole of the 24-wellMillicell culture plate to detect the resistance value. Theresistance value was measured three times at any point of eachhole, and the resistance value of the blank hole was measured atthe same time.

[0087] The transmembrane resistance value (TEER) was calculatedaccording to the following formula:

TEER=(R.sub.t-R.sub.0).times.S.

[0088] Herein, R.sub.t is the measured resistance; R.sub.0 is theresistance of the blank hole; and S is the effective film area.

[0089] 2.3.2 Quality Control with Positive Control Compound:

[0090] Rho-123 was used as a positive control compound. Thecompound was diluted to 5 .mu.molL.sup.-1 by HBSS. Beforeexperiments, the culture medium in each well was discarded, washedtwice with HBSS solution at 37.degree. C., and then incubated in a37.degree. C. incubator. Rho-123 was added in an upper pool, andHBSS solution was added in a lower pool, and incubated in aconstant-temperature shaker at 0, 30, 90, 120 min at each timepoint. Fluorescence spectrophotometer was used to detect the amountof Rho-123 in the lower pool. The emission wavelength was set at430 nm and the excitation wavelength was set at 530 nm. TheP.sub.app value of Rho-123 in this experiment is consistent withthat reported in literature.

[0091] 2.4 Drug Membrane Penetration Test.

[0092] Before experiments, Millicell inoculated with cells wassoaked in HBSS solution at 37.degree. C. for an appropriate timeperiod, and the Millicell was washed slightly to remove theattachment on the cell surface. The permeability from cavitysurface to basal surface: 0.35 ml HBSS solution containing drug wasadded to the top side (AP) and 1.2 ml blank HBSS solution was addedto the bottom side (BL). The Millicell was incubated while shakingat 50 rmin.sup.-1 at 37.degree. C., a 50 .mu.L sample was taken inthe lower layer at 0, 30, 90 and 120 min, and a same volume ofblank HBSS solution as added back. 3 holes were repeated for eachconcentration. 50 .mu.L internal standard solution and 350 .mu.Lethyl acetate were added to the sample, which was shaken and mixed,centrifuged at 12000 rpm for 5 min. 300 .mu.L of supernatant wastaken out, dried by evaporation, and re-dissolved in 50 .mu.Lacetonitrile. 10 .mu.L sample was taken out for testing. For thepermeability from the base surface to the cavity surface, the drugwas added to the bottom side (BL), and the blank HBSS solution wasadded to the top side (AP). The following steps are the same as thepermeability test operation from the cavity surface to the basesurface.

[0093] The apparent permeability coefficient (P.sub.app) of drugsreflects the ability of drugs to pass through monolayer cells andthe speed and degree of drug absorption. It can be calculated bythe following formula:

P a p p = .DELTA. Q .DELTA. t A C 0 . ##EQU00001##

[0094] Herein, .DELTA.Q is the amount of drug penetration duringthe time period of .DELTA.t, A is the cell surface area, which isthe area of supporting membrane (0.6 cm.sup.2) in this model, andCo is the initial concentration. The unit of P.sub.app is usuallyexpressed in cm/h (cmh.sup.-1) or cm/s (cms.sup.-1).

[0095] 2.5 Sample Testing

[0096] LC/MS was used for testing. The standard curve (50 nm-10000nM) was used to quantify the concentration of each sample.

[0097] 3. Experimental Results

TABLE-US-00003 Compound under test P.sub.app (.times.10.sup.-6)Rho-123 4.89 Compound 6 0.13 Compound of formula II 68.82 Compoundof formula III 101.33

[0098] The inventor of the present application unexpectedlydiscovered and provided a compound of formula I as a CCR4inhibitor, which has significantly enhanced solubility, betterbioavailability and better in vivo and in vitro inhibitory activityof CCR4 compared with the prior art, which solves the issues thatthe existing CCR4 inhibitors have low solubility, affect therelease of active pharmaceutical ingredients and causes lowbioavailability, and fail to reach an expected blood drugconcentration in vivo at a maximum safe dose.

[0099] The above is the description of the present disclosure,which shall not be regarded as a limitation of the inventionprovided therein. Unless otherwise pointed out, the implementationof the invention can use conventional technologies such as organicchemistry, polymer chemistry, biotechnology, etc. It is obviousthat the invention can be realized in other ways besides thespecific description in the above disclosure and embodiments. Otheraspects and improvements within the scope of the disclosure will beapparent to those skilled in the art. Based on the teachings of thepresent disclosure, many changes and variations are feasible, whichare therefore covered within the scope of the presentdisclosure.

[0100] Unless otherwise specified, the temperature unit "degree" inthis present disclosure is referred to as Celsius degree, i.e.,.degree. C.

[0101] The references cited in this present disclosure areincorporated in their entirety.

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