CN118108676B

CN118108676B - Benzenesulfonamide compounds, pharmaceutical compositions and applications thereof

Info

Publication number: CN118108676B
Application number: CN202410236108.4A
Authority: CN
Inventors: 查晓明; 延秋铭; 王丹丹
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2024-03-01
Filing date: 2024-03-01
Publication date: 2025-02-07
Anticipated expiration: 2044-03-01
Also published as: CN118108676A

Abstract

The invention discloses a benzenesulfonamide compound, a pharmaceutical composition and application thereof. The compound has the function of promoting melanin generation, has obvious effect and low toxicity, and can be used for preparing drugs for treating skin pigment loss diseases or diseases related to pigment loss, wherein the skin pigment loss diseases comprise leucoderma, sarcoidosis, aegilia, mottle disease, eyelid albinism syndrome, phenylketonuria, idiopathic punctate hypopigmentation, comfetti speckle, vagabond's leukosis and the like.

Description

Benzenesulfonamide compound, pharmaceutical composition and application thereof

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to a benzenesulfonamide compound, a pharmaceutical composition and application thereof.

Background

Depigmentation (Depigmentation) is a disease caused by hypopigmentation or depigmentation of the skin, and the skin or mucous membrane of a patient is marked by patches lighter than normal skin tone, which can occur in any part of the whole body, with the majority of the parts such as the face, neck, back of the hand, etc. Vitiligo (Vitiligo) is the most effective skin disease among all depigmentation, the most well known skin disease has incidence rate far higher than that of cancer, and though the disease is not fatal, the disease can damage appearance, so that psychological burden of patients is heavy, and life of the patients is seriously affected. It is good for young people, and has similar incidence rate of male and female, and higher incidence rate in spring and summer, and is often occurred at exposed and rubbed parts, and sunburn, mental and traumatic factors are common inducing factors of vitiligo. Clinical dermatologists diagnose vitiligo more easily, but treat more difficult, and the disease has adverse effects on physical and mental health of patients to a certain extent.

Melanin production is closely related to the pathogenesis of depigmentation including vitiligo. Melanocytes are cells responsible for the production of skin pigments, and melanotropin stimulates the activity of these cells, affecting skin pigmentation. However, in depigmentation, the function of these melanocytes is impaired or destroyed, leading to the appearance of white spots on the skin. Although the specific mechanisms have not been fully elucidated, studies have shown that abnormal levels of melanogenesis or abnormal melanocytes may be associated with the occurrence of depigmentation.

Therefore, the promotion of the connection between melanin generation and pigment loss diseases becomes an important direction of the current research, and the development of a high-efficiency low-toxicity medicament for promoting the melanin generation has important scientific research significance and application value in treating the pigment loss diseases such as vitiligo and the like.

Disclosure of Invention

The invention aims at providing a benzenesulfonamide compound shown as a general formula I or pharmaceutically acceptable salt thereof:

Wherein,

X is selected from-NH, -O, or (CH ₂)_n;

n=0, 1, 2 or 3;

R ₁ is selected from nitro, alkoxy, haloalkyl or halogen;

R ₂、R₃、R₄ is each independently selected from hydrogen, halogen, hydroxy, cyano, nitro, carboxy, C ₁-C₈ alkyl, alkoxy, haloalkyl, amino, aminomethyl, aminoethyl, aminoisopropyl, dimethylamino, -NHC (O) OC ₁-C₆ alkyl, carboxamido, aminomethylcarboxamide or methoxycarbonyl.

In certain preferred embodiments of the present invention,

X is selected from-NH, -O, or (CH ₂)_n;

n=0 or 1;

r ₁ is selected from nitro;

R ₂ is selected from hydrogen or C ₁-C₃ alkoxy;

r ₃ is selected from hydrogen, halogen, hydroxy, alkoxy, carboxy, carboxamido, aminomethylcarboxamide or methoxycarbonyl;

R ₄ is selected from hydrogen, halogen, hydroxy, cyano, nitro, carboxy, C ₁-C₃ alkyl, C ₁-C₃ alkoxy, haloalkyl, amino, aminomethyl, aminoethyl, aminoisopropyl, dimethylamino, -NHC (O) OC (CH ₃)₃ group, carboxamide group, aminomethylcarboxamide or methoxycarbonyl group.

In some of the more preferred embodiments of the present invention,

X is selected from-NH, -O, or (CH ₂)_n;

n=0 or 1;

r ₁ is selected from nitro;

r ₂ is selected from hydrogen or methoxy;

R ₃ is selected from hydrogen, fluoro, hydroxy, methoxy, carboxy, carboxamido, aminomethylcarboxamide or methoxycarbonyl;

R ₄ is selected from hydrogen, fluoro, hydroxy, cyano, nitro, carboxy, methyl, methoxy, trifluoromethyl, amino, aminomethyl, aminoethyl, aminoisopropyl, dimethylamino, -NHC (O) OC (CH ₃)₃ group, carboxamide group, aminomethylcarboxamide group or methoxycarbonyl group.

In some preferred embodiments, the pharmaceutically acceptable salts include acid addition salts of the compounds of formula I with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and also include acid salts of the compounds of formula I with inorganic bases.

In some more preferred embodiments, the pharmaceutically acceptable salts include basic metal cation salts, alkaline earth metal cation salts, and ammonium cation salts. Further, the ions used for salification include sodium, potassium, calcium, zinc, magnesium or aluminum.

The compounds of the general formula I according to the invention are preferably the following compounds:

The compounds of the general formula I according to the invention mentioned above may also be present in the form of their salts, which are converted in vivo into compounds of the general formula I. For example, within the scope of the present invention, the compounds of the present invention are converted to pharmaceutically acceptable salt forms and used in salt form according to procedures known in the art.

The present invention also provides a process for the preparation of a compound having the general formula I comprising the reaction steps as shown in scheme 1:

Scheme 1

Compound I (a) is prepared by reacting intermediate NA with intermediate NB under the action of a base. Wherein R is selected from hydrogen, hydroxyl, cyano, nitro, methyl, methoxy, trifluoromethyl, amino, aminomethyl, aminoethyl, aminoisopropyl, dimethylamino, -NHC (O) OC (CH ₃)₃ group; R ₁ is as defined in formula I).

The preparation method of the compound with the general formula I further comprises the reaction steps as shown in scheme 2:

Scheme 2

The compound I (B) is prepared by coupling reaction of an intermediate NC and an intermediate ND under the action of a palladium catalyst. Wherein R ₁、R₃、R₄ is as defined in formula I.

The preparation method of the compound with the general formula I further comprises the reaction steps as shown in scheme 3:

scheme 3

Compound I (C) is prepared by reacting intermediate NG with intermediate NA in the presence of a base. Wherein R ₁、R₃、R₄ and X are defined as in the general formula I.

The compounds of the general formula I can be prepared by the preparation method or the preparation method similar to the preparation method, and corresponding starting materials are selected according to different substituents and different substituent positions. Those skilled in the art will recognize that the above routes are helpful in understanding the present invention, but do not limit the invention unless otherwise specified, the variables are as defined in formula I.

It is another object of the present invention to provide a pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

The pharmaceutical compositions of the present invention may be administered in a variety of known ways, such as orally, parenterally, by inhalation spray, or via an implanted reservoir. The pharmaceutical composition of the invention can be administered alone or in combination with other drugs. The oral composition may be any orally acceptable dosage form including, but not limited to, tablets, capsules, emulsions, suspensions, dispersions, and solutions.

Pharmaceutically acceptable carrier refers to excipients or diluents that do not cause significant irritation to the organism and do not interfere with the biological activity and properties of the compound being administered. Common pharmaceutically acceptable carriers include stabilizers, diluents, surfactants, lubricants, antioxidants, binders, colorants, fillers, emulsifiers, and the like. Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like. Topical compositions may be formulated as oils, lotions, creams and the like. Carriers for the compositions include vegetable or mineral oils, animal fats, and high molecular weight alcohols, among others. A pharmaceutically acceptable carrier is a carrier in which the active ingredient is soluble.

The actual dosage level of the active ingredient in the pharmaceutical compositions of the present invention may be varied to obtain an amount of active ingredient that is effective to achieve the desired therapeutic response for the particular patient, composition and mode of administration, and that is non-toxic to the patient. The dosage level selected will depend on a variety of factors including the activity of the particular compound of the invention or salt thereof employed, the route of administration, the time of administration, the rate of excretion of the particular composition employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the age, sex, weight, general health and past medical history of the patient being treated, and like factors well known in the medical arts.

Another object of the present invention is to provide the use of a compound of formula I or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment of skin depigmentation or a disorder associated with depigmentation. The medicine is a melanin generation promoting medicine containing benzenesulfonamide compounds. The skin pigment loss disease comprises vitiligo, tuberous sclerosis, aegilsonia, mottle disease, eyelid albinism syndrome, phenylketonuria, idiopathic punctate pigment reduction disease, comfetti speckle or Vagabond's leukosis.

The terms used in the present invention generally have the following meanings unless specifically indicated:

Me is methyl, et is ethyl, and Boc is t-butoxycarbonyl.

The term "alkyl" denotes a straight or branched saturated hydrocarbon group having the stated number of carbon atoms.

The term "C ₁-C₈ alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 8 carbon atoms. C ₁-C₈ alkyl includes, but is not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, n-pentyl, isopentyl, neopentyl, n-hexyl, isohexyl, 2-dimethylbutyl, 2, 3-dimethylbutyl, and the like.

The term "C ₁-C₃ alkyl" refers to a straight or branched chain saturated hydrocarbon group having 1 to 3 carbon atoms.

The term "alkoxy" denotes an O-alkyl group. The term "C ₁-C₃ alkoxy" refers to an alkyl group having O-C ₁-C₃.

The term "halogen" is fluorine, chlorine, bromine or iodine. Fluorine, chlorine and bromine are preferred.

The beneficial effects are that:

The benzenesulfonamide compound with the general formula I and the pharmaceutically acceptable salt thereof have the function of promoting melanin generation, and have obvious effect and low toxicity. Therefore, the compound can be used for preparing medicines for treating depigmentation or depigmentation-related diseases.

Drawings

FIG. 1 is a graph showing the effect of compound I-1~I-15 of the present invention on B16F10 cell viability;

FIG. 2 shows the effect of the compounds I-16 to I-30 of the present invention on B16F10 cell viability;

FIG. 3 shows the activation of tyrosinase by the compound I-1~I-15 of the present invention;

FIG. 4 shows the activation effect of the compounds I-16-I-25 and I-28-I-30 on tyrosinase;

FIG. 5 is a graph showing the effect of compound I-1~I-15 of the present invention on B16F10 cell melanin content;

FIG. 6 shows the effect of the compounds I-16 to I-30 of the present invention on B16F10 cell melanin content;

FIG. 7 shows the effect of compound I-21 of the present invention on tyrosinase, MITF protein expression;

FIG. 8 shows the therapeutic effect of the compound I-21 of the present invention on mice with vitiligo model.

Detailed Description

The process for the preparation of the compounds of the general formula I according to the invention is described below in connection with specific examples, which, however, do not constitute any limitation of the invention. The compounds of the present invention may also be conveniently prepared by optionally combining the various synthetic methods described in this specification or known in the art, such combinations being readily apparent to those skilled in the art to which the present invention pertains. The starting materials, reagents, etc. used in the examples of the present invention are all commercially available. The invention can be prepared into salt form by adopting a salt forming method commonly used in the field, for example, the compound is dissolved in ethanol hydrochloride at room temperature to react to form hydrochloride, or benzenesulfonic acid is added into the compound to react to form benzenesulfonate.

Synthesis of intermediates:

n- (2-Chloropyrimidin-4-yl) -4-nitrobenzenesulfonamide (1 c)

2-Chloro-4-aminopyrimidine (500 mg,3.86 mmol) was dissolved in 8mL THF and sodium tert-butoxide (294 g,3.09 mmol) was added in portions with stirring on an ice bath (0 ℃ C.) and stirred on an ice bath (0 ℃ C.) for 0.5 hours. 4-Nitrophenyl sulfonyl chloride (570 mg,2.57 mmol) was then added in portions and the solution stirred at room temperature for 0.5 h. TLC monitored to the end of the reaction. The reaction solution was poured into 50mL of ice water, the reaction solution was extracted three times with ethyl acetate, 50mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, the organic phase was dried over anhydrous sodium sulfate overnight, anhydrous sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a white crude product. Flash column chromatography (PE: ea=2:1, v:v) afforded 602mg of white solid in yield 74％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)12.15(s,1H),8.53-8.38(m,3H),8.31-8.19(m,2H),7.01(d,J＝5.7Hz,1H).

(S) -1- ([ 1,1' -biphenyl ] -4-yl) ethan-1-amine (1 b)

(S) -1- (4-bromophenyl) ethan-1-amine (328 mg,1.64 mmol), phenylboronic acid (300 mg,2.46 mmol), pd [ P (Ph) ₃]₄ (95 mg,0.082 mmol) were dissolved in 6mL acetonitrile, and then K ₂CO₃ aqueous solution (1.5 mL, 5M) was added thereto, and the mixture was heated to reflux under nitrogen protection and stirred for 12 hours. TLC monitored to the end of the reaction. The reaction solution was cooled to room temperature, insoluble matter was removed by suction filtration, the reaction was dissolved with ethyl acetate, and the organic phase was slowly added dropwise to the mixed solution with concentrated hydrochloric acid to adjust the pH to 1, followed by stirring in an ice bath (0 ℃ C.) for about 1.5 hours. The mixed solution was washed 3 times with 50mL of water each time and the aqueous phases were combined. The aqueous pH was adjusted to 10 using 5M NaOH. The mixed solution was extracted three times with ethyl acetate, about 50mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure and separated by flash column chromatography (PE: ea=1:1, v: v) to give 372mg of white oil, yield 93％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.63-7.58(m,4H),7.50-7.43(m,4H),7.40-7.34(m,1H),4.20(q,J＝6.6Hz,1H),1.46(d,J＝6.6Hz,3H).

(S) -1- (4 '- (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) ethan-1-amine (2 b)

Synthesis and treatment procedure of Compound 2b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-trifluoromethylphenyl boronic acid (427 mg,2.25 mmol) was charged and flash column chromatography (PE: EA=1:1, v: v) was performed to give yellow solid 2b 372mg in yield 94％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.38-8.25(m,2H),7.99-7.92(m,2H),7.77-7.67(m,2H),7.58-7.48(m,2H),4.05(q,J＝6.6Hz,1H),1.87(s,2H),1.28(d,J＝6.6Hz,3H).

(S) -1- (4 '-Nitro- [1,1' -biphenyl ] -4-yl) ethan-1-amine (3 b)

Synthesis and treatment procedure of Compound 3b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-nitrobenzoic acid (378 mg,2.25 mmol) were charged and flash column chromatography (PE: EA=1:1, v: v) was performed to isolate 3b341mg as a yellow solid in yield 92％.¹H NMR(400MHz,Chloroform-d)δ(ppm)8.29(d,J＝8.8Hz,2H),7.73(d,J＝8.8Hz,2H),7.60(d,J＝8.3Hz,2H),7.49(d,J＝8.2Hz,2H),4.21(q,J＝6.6Hz,1H),1.43(d,J＝6.6Hz,3H).

(S) -4'- (1-aminoethyl) - [1,1' -biphenyl ] -4-carbonitrile (4 b)

Synthesis and treatment procedure of Compound 4b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-cyanophenylboronic acid (330 mg,2.25 mmol) was added and flash column chromatography (PE: EA=1:1, v:v) was performed to afford 4b289mg as a yellow solid in yield 84％.¹H NMR(400MHz,Chloroform-d)δ(ppm)8.38-8.26(m,2H),7.81-7.70(m,2H),7.67-7.59(m,2H),7.56-7.45(m,2H),4.23(q,J＝6.6Hz,1H),1.46(d,J＝6.6Hz,3H).

(S) -1- (4 '-methyl- [1,1' -biphenyl ] -4-yl) ethan-1-amine (5 b)

Synthesis and treatment procedure of Compound 5b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-methylphenylboronic acid (306 mg,2.25 mmol) was charged and flash column chromatography (PE: EA=1:1, v: v) was performed to isolate yellow solid 5b291mg in yield 92％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.60-7.55(m,2H),7.53-7.50(m,2H),7.46-7.41(m,2H),7.27(d,J＝8.0Hz,2H),4.19(q,J＝6.6Hz,1H),2.42(s,3H),1.46(d,J＝6.6Hz,3H).

(S) -4'- (1-aminoethyl) - [1,1' -biphenyl ] -4-amine (6 b)

Synthesis and working up of Compound 6b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), pinacol 4-aminophenylborate (493 mg,2.25 mmol) were charged and flash column chromatography (PE: EA=1:1, v: v) was carried out to give yellow solid 6b 289mg, yield 91％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.45(d,J＝8.2Hz,2H),7.39-7.28(m,4H),6.67-6.58(m,2H),5.17(s,2H),3.99(q,J＝6.6Hz,1H),1.26(d,J＝6.6Hz,3H).

N-methyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (7 a)

4- (4, 5-Tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (500 mg,2.28 mmol) was dissolved in 10mL DMF and K ₂CO₃ (473 mg,3.42 mmol) and methyl iodide (324 mg,2.28 mmol) were added. The mixture was stirred at 50 ℃ for 14 hours and TLC monitored to the end of the reaction. The reaction solution was returned to room temperature, poured into 50mL of water, extracted three times with ethyl acetate, 50mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, the organic phases were dried over anhydrous sodium sulfate overnight, the anhydrous sodium sulfate was removed by filtration, and the filtrate was concentrated under reduced pressure to give a crude product as a white oil. Purification on a flash silica gel column (PE: ea=15:1, v:v) gave 387mg of white solid in yield 72％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.43-7.36(m,2H),6.51-6.45(m,2H),6.04(q,J＝4.9Hz,1H),2.68(d,J＝5.0Hz,3H),1.25(s,12H).

(S) -4'- (1-aminoethyl) -N-methyl- [1,1' -biphenyl ] -4-amine (7 b)

Synthesis and working up of Compound 7b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 7a (349 mg,1.50 mmol) was taken up and flash column chromatography (PE: EA=1:1, v: v) to afford 7b 317mg as a grey solid in yield 93％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.52-7.44(m,2H),7.44-7.38(m,2H),7.38-7.31(m,2H),6.64-6.55(m,2H),5.76(d,J＝5.0Hz,1H),4.00(q,J＝6.4Hz,1H),2.71(d,J＝5.1Hz,3H),1.27(d,J＝6.6Hz,3H).

N-ethyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (8 a)

Synthesis and treatment procedure of Compound 8a As in Compound 7a, 4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (500 mg,2.28 mmol), iodoethane (356 mg,2.28 mmol) was added and flash column chromatography (PE: EA=15:1, v: v) was carried out to give white solid 8a 3991 g, yield 69％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.41-7.36(m,2H),6.54-6.47(m,2H),6.00-5.89(m,1H),3.05(qd,J＝7.2,5.3Hz,2H),1.25(s,12H),1.15(t,J＝7.1Hz,3H).

(S) -4'- (1-aminoethyl) -N-ethyl- [1,1' -biphenyl ] -4-amine (8 b)

Synthesis and working up of Compound 8b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300.00 mg,1.5 mmol), 8a (375 mg,1.50 mmol) were added and flash column chromatography (PE: EA=1:1, v: v) gave 8b 302mg as a grey solid in yield 87％.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.61-7.56(m,2H),7.49-7.43(m,2H),7.34-7.28(m,2H),6.73-6.67(m,2H),4.32(tq,J＝5.9,5.0Hz,1H),3.11(pd,J＝5.6,1.6Hz,2H),1.41(d,J＝4.9Hz,3H),1.19(t,J＝5.6Hz,3H).

N-isopropyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (9 a)

Synthesis and working up of Compound 9a As in Compound 7a, 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline (500 mg,2.28 mmol), 2-iodopropane (3838 mg,2.28 mmol) were added and flash column chromatography (PE: EA=15:1, v: v) was carried out to give 9a 416mg as a white solid in yield 70％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.40-7.34(m,2H),6.54-6.47(m,2H),5.80(d,J＝7.9Hz,1H),1.25(s,12H),1.13(d,J＝6.3Hz,6H).

(S) -4'- (1-aminoethyl) -N-isopropyl- [1,1' -biphenyl ] -4-amine (9 b)

Synthesis and working up of Compound 9b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300.00 mg,1.5 mmol), 9a (399mg, 1.50 mmol) were added and flash column chromatography (PE: EA=1:1, v: v) afforded grey solid 9b 347mg in yield 91％.¹H NMR(300 MHz,DMSO-d₆)δ(ppm)7.52-7.41(m,4H),7.37-7.31(m,4H),6.62(d,J＝8.6Hz,1H),3.96(q,J＝6.6 Hz,1H),1.22(t,J＝6.4 Hz,6H),1.15(d,J＝6.3 Hz,3H).

N, N-dimethyl-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) aniline (10 a)

Synthesis and treatment procedure of Compound 10a As in Compound 7a, 4- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) aniline (500 mg,2.28 mmol), methyl iodide (809 mg,5.71 mmol) was added, and flash column chromatography (PE: EA=20:1, v: v) was carried out to obtain a white solid 10a 391 mg, yield 68％.¹H NMR(400 MHz,Chloroform-d)δ(ppm)7.75-7.69(m,2H),6.74-6.69(m,2H),3.01(s,6H),1.35(s,12H).

(S) -4'- (1-aminoethyl) -N, N-dimethyl- [1,1' -biphenyl ] -4-amine (10 b)

Synthesis and treatment procedure of Compound 10b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300.00 mg,1.5 mmol), 10a (370 mg,1.50 mmol) were charged and flash column chromatography (PE: EA=1:1, v: v) separated to afford 10b 331 mg as a gray solid in yield 91％.¹H NMR(400 MHz,Chloroform-d)δ(ppm)7.63-7.46(m,4H),7.46-7.34(m,2H),6.89-6.77(m,2H),4.17(q,J＝6.6 Hz,1H),3.02(s,6H),1.45(d,J＝6.6 Hz,3H).

(S) - (4 '- (1-aminoethyl) - [1,1' -biphenyl ] -4-yl) carbamic acid tert-butyl ester (11 b)

Synthesis and treatment procedure for Compound 11b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300.00 mg,1.5 mmol), (4- ((tert-butoxycarbonyl) amino) phenyl) boronic acid (353 mg,1.50 mmol) was charged and flash column chromatography (PE: EA=1:1, v: v) was performed to isolate 390 mg of 11b as a gray solid in yield 83％.¹H NMR(400 MHz,DMSO-d₆)δ(ppm)9.42(s,1H),7.59-7.50(m,6H),7.45-7.39(m,2H),4.02(q,J＝6.7 Hz,1H),1.49(s,9H),1.27(d,J＝6.6 Hz,3H).

(S) -4'- (1-aminoethyl) - [1,1' -biphenyl ] -4-ol (12 b)

Synthesis and treatment procedure of Compound 12b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-hydroxyphenylboronic acid (308 mg,2.25 mmol) was charged and flash column chromatography (PE: EA=1:1, v:v) was performed to isolate yellow solid 12b301mg, yield 94％.¹H NMR(400 MHz,Chloroform-d)δ(ppm)9.52(s,1H),7.54-7.43(m,4H),7.42-7.35(m,2H),6.88-6.79(m,2H),4.01(q,J＝6.6 Hz,1H),1.27(d,J＝6.6 Hz,3H).

(S) -1- (4 '-methoxy- [1,1' -biphenyl ] -4-yl) ethan-1-amine (13 b)

Synthesis and treatment procedure of Compound 13b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 4-methoxyphenylboronic acid (342 mg,2.25 mmol) was charged, and flash column chromatography (PE: EA=1:1, v:v) was performed to isolate 318mg of white solid 13b, yield 93％.¹H NMR(400MHz,Chloroform-d)δ(ppm)7.61-7.50(m,4H),7.45-7.39(m,2H),7.04-6.96(m,2H),4.18(q,J＝6.6Hz,1H),3.87(s,3H),1.45(d,J＝6.6Hz,3H).

(S) -1- (3 '-methoxy- [1,1' -biphenyl ] -4-yl) ethan-1-amine (14 b)

Synthesis and working up of Compound 14b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 3-methoxyphenylboronic acid (349mg, 2.25 mmol) was taken up and flash column chromatography (PE: EA=1:1, v:v) gave 14b 309mg as a white solid in yield 90％.¹H NMR(400MHz,Chloroform-d)δ(ppm)7.56-7.51(m,2H),7.44-7.39(m,2H),7.34(ddt,J＝6.3,4.6,2.1Hz,2H),7.08-6.99(m,2H),4.18(q,J＝6.8Hz,1H),3.84(s,3H),1.46(d,J＝6.6Hz,3H).

(S) -1- (2 '-methoxy- [1,1' -biphenyl ] -4-yl) ethan-1-amine (15 b)

Synthesis and working up of Compound 15b As in Compound 1b, (S) -1- (4-bromophenyl) ethan-1-amine (300 mg,1.5 mmol), 2-methoxyphenylboronic acid (349mg, 2.25 mmol) was taken up and flash column chromatography (PE: EA=1:1, v: v) gave 15b 292 mg as a white solid in yield 86％.¹H NMR(400MHz,Chloroform-d)δ(ppm)7.66-7.53(m,2H),7.51-7.41(m,2H),7.38(t,J＝7.9Hz,1H),7.27-7.07(m,2H),6.91(ddd,J＝8.3,2.6,1.0Hz,1H),4.20(q,J＝6.6Hz,1H),3.89(s,3H),1.45(d,J＝6.7Hz,3H).

2-Hydroxy-5- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (16 a)

5-Bromo-2-hydroxybenzoamide (500 mg,2.31 mmol), potassium acetate (454 mg,4.63 mmol), pd (dppf) Cl ₂ (85 mg,0.115 mmol) and pinacol diboronate (882 mg,3.47 mmol) were dissolved in 20mL of 1, 4-dioxane. The reaction was monitored by TLC to endpoint by stirring with heating at 90 ℃ for 12 hours under nitrogen protection. The reaction solution was cooled to room temperature, the solvent was dried under reduced pressure, 100mL of water was poured into the reaction solution, extracted three times with ethyl acetate, about 50mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, dried over night over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, the filtrate was concentrated under reduced pressure, and separated by flash column chromatography (PE: ea=10:1, v: v) to give 16a 1.18g of white solid in yield 98％.¹H NMR(400MHz,Chloroform-d)δ(ppm)12.54(s,1H),7.90-7.85(m,2H),7.00(d,J＝8.4Hz,1H),1.36(s,12H).

2-Hydroxy-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzamide (17 a)

Synthesis and workup of Compound 17a same as 16a, 4-bromo-2-hydroxybenzoamide (600 mg,2.78 mmol), pinacol diboronate (1.06 g,4.17 mmol) were added and flash column chromatography (PE: EA=10:1, v: v) gave 17a as a white solid 550mg, yield 74％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)12.77(s,1H),8.41(s,1H),7.93(s,1H),7.85(d,J＝8.1Hz,1H),7.15-7.10(m,2H),1.30(s,12H).

2-Hydroxy-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzoic acid methyl ester (18 a)

Synthesis and treatment of Compound 18a As in 16a, methyl 5-bromo-2-hydroxybenzoate (1.00 g,4.33 mmol), pinacol biborate (1.65 g,6.49 mmol) were added and flash column chromatography (PE: EA=10:1, v: v) was performed to afford 1.18g as a white solid, in yield 98％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.77(s,1H),8.12(d,J＝1.6Hz,1H),7.76(dd,J＝8.3,1.7Hz,1H),6.99(d,J＝8.3Hz,1H),3.91(s,3H),1.29(s,12H).

2-Hydroxy-4- (4, 5-tetramethyl-1, 3, 2-dioxaborane-2-yl) benzoic acid methyl ester (19 a)

Synthesis and workup of Compound 19a As in 16a, methyl 4-bromo-2-hydroxybenzoate (2.00 g,8.66 mmol), pinacol biborate (3.30 g,12.98 mmol) were added and flash column chromatography (PE: EA=10:1, v: v) was carried out to afford 19a as a white solid, 2.17g, in yield 90％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.38(s,1H),7.76(d,J＝7.8Hz,1H),7.25-7.17(m,2H),3.89(s,3H),1.30(s,12H).

(S) -N- (2- ((1- (4-bromophenyl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (16 c)

1C (1.0 g,3.18 mmol), (S) -1- (4-bromophenyl) ethan-1-amine (1.27 g,6.36 mmol), DIPEA (954 mg,4.77 mmol) was dissolved in 15mL of n-butanol, strictly evacuated under nitrogen protection, heated to reflux to 110℃and reacted for 36h with TLC monitoring to the end of the reaction. The reaction solution was cooled to room temperature, the solvent was dried in vacuo, and flash column chromatography on silica gel (DCM: meoh=100:1, v:v) gave 513mg of yellow solid in yield 33％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.89(s,1H),8.23(d,J＝7.7Hz,2H),8.14-7.86(m,2H),7.65(d,J＝19.8Hz,2H),7.46(d,J＝8.0Hz,2H),7.22(d,J＝8.1Hz,2H),6.01(s,1H),4.83(s,1H),1.33(s,3H).

2, 6-Trimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (20 a)

5-Methyl salicylic acid (1.0 g,6.57 mmol) was dissolved in 5mL of anhydrous acetone, cooled at-8℃for 30 minutes, 1.5mL of acetic anhydride and two drops of concentrated sulfuric acid were slowly added dropwise with stirring at low temperature, and stirring was continued at-8℃for 12 hours. TLC monitored to the end of the reaction. The reaction solution was slowly added dropwise to 100mL of saturated sodium bicarbonate solution, extracted three times with ethyl acetate, 50mL each time, the organic phases were combined, saturated brine was washed three times, 50mL each time, dried over night over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 1.14g of a white solid which was directly put into the next step .¹H NMR(300MHz,Chloroform-d)δ(ppm)7.77(d,J＝2.6Hz,1H),7.37(dd,J＝8.3,2.3Hz,1H),6.87(d,J＝8.4Hz,1H),2.35(s,3H),1.74(s,6H).

2, 7-Trimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (21 a)

Synthesis and working up of Compound 21a same as 20a, 4-methyl salicylic acid (2.0 g,10.00 mmol) was charged to give 21a 2.32g as a white solid with a yield of 92%, which was directly charged to the next step .¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.75(d,J＝8.0Hz,1H),7.03(ddd,J＝7.9,1.6,0.7Hz,1H),6.96-6.93(m,1H),2.37(s,3H),1.68(s,6H).

6- (Bromomethyl) -2, 2-dimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (20 b)

20A (1.0 g,5.20 mmol), N-bromosuccinimide (1.11 g,6.24 mmol), benzoyl peroxide (75% hydrate, 353g,1.09 mmol) was dissolved in 10mL carbon tetrachloride and heated to 75℃under nitrogen protection and stirred for 2 hours. TLC monitored to the end of the reaction. The reaction was cooled to room temperature, the reaction solution was poured into 80mL of water, extracted three times with dichloromethane, 100mL each time, the organic phases were combined, washed with saturated brine three times, 100mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 839mg of white solid by flash silica gel column chromatography (PE: ea=20:1, v: v) in yield 65％.¹H NMR(400MHz,Chloroform-d)δ(ppm)8.00(d,J＝2.2Hz,1H),7.62(dd,J＝8.5,2.3Hz,1H),6.98(d,J＝8.4Hz,1H),4.50(s,2H),1.76(s,6H).

7- (Bromomethyl) -2, 2-dimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (21 b)

Synthesis and workup of Compound 21b same as 20b, 21a (1.0 g,5.20 mmol) was added thereto, and flash column chromatography over silica gel (PE: EA=20:1, v: v) afforded 789mg of white solid 21a, yield 57％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.86(d,J＝8.0Hz,1H),7.28(dd,J＝8.0,1.6Hz,1H),7.21(d,J＝1.6Hz,1H),4.72(s,2H),1.69(s,6H).

(S) - (1- (4-bromophenyl) ethyl) carbamic acid tert-butyl ester (20 c)

(S) -1- (4-bromophenyl) ethan-1-amine (2.00 g,10.00 mmol), triethylamine (1.21 g,12.00 mmol) were dissolved in 50mL dry CH ₂Cl₂, and slowly added dropwise to a solution of Boc ₂ O (2.62 mg,12.00 mmol) in dry CH ₂Cl₂ (10 mL) in ice bath (0 ℃ C.) and stirred at room temperature for 24 hours. TLC monitored to the end of the reaction. The reaction mixture was poured into 200mL of water, extracted three times with dichloromethane, the organic phases were combined, washed three times with saturated brine, each time with 50mL of anhydrous sodium sulfate, dried overnight, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 2.96g of white solid by flash column chromatography (PE: ea=20:1, v:v), yield 98％.¹H NMR(400MHz,Chloroform-d)δ(ppm)7.50-7.43(m,2H),7.24-7.16(m,2H),4.77(s,1H),1.43(s,12H).

(S) - (1- (4, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) ethyl) carbamic acid tert-butyl ester (20 d)

Synthesis and workup of Compound 20d same as 16a, 20c (500 mg,1.67 mmol) was charged, and pinacol diboronate (634 g,2.50 mmol) was isolated by flash column chromatography (PE: EA=20:1, v: v) to afford 20d 348 g as a colorless oil, yield 59％.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.86-7.73(m,2H),7.32(d,J＝7.7Hz,2H),4.82(s,1H),1.44(s,9H),1.36(s,12H),1.28(d,J＝2.4Hz,3H).

(S) - (1- (4- ((2, 2-dimethyl-4-oxo-4H-benzo [ d ] [1,3] dioxin-6-yl) methyl) phenyl) ethyl) carbamic acid tert-butyl ester (20 e)

Synthesis and workup of Compound 20e same as 1b, 20b (600 mg,2.22 mmol), 20d (803 mg,2.32 mmol) were added and flash column chromatography (PE: EA=20:1, v: v) was carried out to afford 20e 620mg as a white solid in yield 68％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.70(d,J＝2.3Hz,1H),7.56(dd,J＝8.4,2.3Hz,1H),7.24-7.18(m,4H),7.05(d,J＝8.4Hz,1H),4.63-4.52(m,1H),3.94(s,2H),1.67(s,6H),1.36(s,9H),1.27(d,J＝7.0Hz,3H).

(S) - (1- (4- ((2, 2-dimethyl-4-oxo-4H-benzo [ d ] [1,3] dioxin-7-yl) methyl) phenyl) ethyl) carbamic acid tert-butyl ester (21 e)

Synthesis and workup of Compound 21e same as 1b, 21b (750 mg,2.76 mmol), 20d (1.00 g,2.90 mmol) were charged and flash column chromatography (PE: EA=20:1, v:v) was carried out to afford 21e as a white solid 778mg, yield 69％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.77(d,J＝8.0Hz,1H),7.26-7.18(m,4H),7.08(dd,J＝8.0,1.6Hz,1H),6.96(s,1H),4.67-4.51(m,1H),3.97(s,2H),1.67(s,6H),1.36(s,9H),1.27(d,J＝7.0Hz,3H).

(S) - (1- (4- (4-fluoro-3-methylbenzyl) phenyl) ethyl) carbamic acid tert-butyl ester (22 e)

Synthesis and workup of Compound 22e in the same manner as 1b, 4-fluoro-3-methylbenzyl bromide (200 mg,0.98 mmol), 20d (319 mg,1.03 mmol) and flash column chromatography (PE: EA=15:1, v: v) gave 22e 719 mg as a white solid in yield 97％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.34(d,J＝7.9Hz,1H),7.19(d,J＝8.2Hz,2H),7.14(d,J＝8.3Hz,3H),7.03(d,J＝9.4Hz,1H),4.60-4.51(m,1H),3.84(s,2H),2.18(d,J＝2.1Hz,3H),1.36(s,9H),1.25(s,3H).

(S) -6- (4- (1-aminoethyl) benzyl) -2, 2-dimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (20 f)

20E (560 mg,1.84 mmol) was dissolved in 6mL of anhydrous CH ₂Cl₂, stirred for 15 min in an ice bath (0 ℃) and 2mL of trifluoroacetic acid were slowly added dropwise and stirred for 4 h in an ice bath (0 ℃). TLC monitored to the end of the reaction. The reaction was allowed to return to room temperature, the reaction solution was poured into 30mL of water, the pH was adjusted to 10 with 5M NaOH solution, extracted three times with dichloromethane, 50mL each time, the organic phases combined, saturated brine washed three times, 50mL each time, dried over night over anhydrous sodium sulfate, filtered to remove anhydrous sodium sulfate, the filtrate concentrated under reduced pressure, and flash column chromatography (DCM: meoh=120:1, v:v) afforded 400mg of yellow solid in yield 98％.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.81(d,J＝2.3Hz,1H),7.39(dd,J＝8.4,2.3Hz,1H),7.31(d,J＝1.9Hz,1H),7.17(d,J＝8.0Hz,2H),6.90(d,J＝8.4Hz,1H),4.13(q,J＝6.6Hz,1H),3.96(s,2H),1.74(s,6H),1.41(d,J＝6.6Hz,3H).

(S) -7- (4- (1-aminoethyl) benzyl) -2, 2-dimethyl-4H-benzo [ d ] [1,3] dioxin-4-one (21 f)

The synthesis and workup of compound 21f was followed by 20f, 21e (760 mg,1.84 mmol) and flash column chromatography (DCM: meOH=120:1, v:v) to afford 21f as a yellow solid 520mg in yield 90％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)7.77(d,J＝8.0Hz,1H),7.30(d,J＝8.0Hz,2H),7.19(d,J＝8.0Hz,2H),7.07(dd,J＝8.0,1.5Hz,1H),6.96(d,J＝1.5Hz,1H),3.97(s,2H),3.93(d,J＝6.5Hz,1H),1.66(s,6H),1.22(d,J＝6.6Hz,3H).

(S) -1- (4- (4-fluoro-3-methylbenzyl) phenyl) ethan-1-amine (22 f)

The synthesis and workup of compound 22f was followed in the same manner as 20f by addition of 22e (760 mg,1.84 mmol) and flash column chromatography (DCM: meOH=120:1, v:v) to afford 21f as a yellow solid 520mg in yield 66％.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.30(d,J＝1.9Hz,1H),7.27(s,1H),7.20-7.12(m,2H),7.04-6.88(m,3H),4.11(q,J＝6.6Hz,1H),3.91(s,2H),2.25(d,J＝2.0Hz,3H),1.40(d,J＝6.6Hz,3H).

4- (4-Fluoro-3-methylphenoxy) benzaldehyde (24 a)

4-Fluoro-3-methylphenol (3.00 g,15.86 mmol), 4-fluorobenzaldehyde (1.97 g,15.86 mmol), K ₂CO₃ (4.38 g,31.71 mmol) were dissolved in 15mL DMF and heated to 120℃under nitrogen and stirred for 12 hours. TLC monitored to the end of the reaction. The reaction was cooled to room temperature, the reaction solution was poured into 50mL of water, extracted three times with ethyl acetate, 50mL each time, the organic phases were combined, washed with saturated brine three times, 50mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 3.08g of a yellow liquid by flash silica gel column chromatography (PE: ea=20:1, v: v) in yield 84％.¹H NMR(400MHz,Chloroform-d)δ(ppm)9.94(d,J＝2.5Hz,1H),7.98-7.81(m,2H),7.17-7.00(m,3H),7.00-6.85(m,2H),2.31(d,J＝2.3Hz,3H).

(S, E) -N- (4- (4-fluoro-3-methylphenoxy) benzylidene) -2-methylpropane-2-sulfinamide (24 b)

24A (2.00 mg,8.96 mmol), (S) -tert-butylsulfinamide (1.58 g,13.06 mmol), cs ₂CO₃ (8.49 g,26.06 mmol) were dissolved in 30mL 1, 2-dichloroethane, nitrogen-protected, stirred at 80℃under reflux for 5h, cooled to 0℃after the reaction was completed, saturated ammonium chloride solution was added, and dichloromethane extracted. The organic phases were combined and dried over anhydrous Na ₂SO₄ and concentrated under reduced pressure. Flash column chromatography (PE: ea=10:1, v:v) gave 24b as a white solid in yield 91％.¹H NMR(400MHz,Chloroform-d)δ(ppm)8.54(s,1H),7.87-7.78(m,2H),7.08-6.97(m,3H),6.90(ddt,J＝16.0,8.6,3.1Hz,2H),2.29(d,J＝2.1Hz,3H),1.27(s,9H).

(S) -N- ((S) -1- (4- (4-fluoro-3-methylphenoxy) phenyl) ethyl) -2-methylpropane-2-sulfinamide (24 c)

24B (2.00 g,6.00 mmol) was dissolved in 30mL of anhydrous DCM, the reaction solution was placed at-78℃and 7.5mL of a solution of methylmagnesium bromide (894.09 mg,7.50 mmol) in anhydrous THF (1 mol/L) was slowly added dropwise, after 2h of reaction, the temperature was raised to-20℃and the reaction was continued for 2h. The reaction was quenched by slowly dropping saturated aqueous NH ₄ Cl, extracted with DCM, the combined organic phases were dried over anhydrous Na ₂SO₄ and separated by flash column chromatography (PE: ea=10:1, v: v) to give 2.05g of a colorless oily substance in yield 98％.¹H NMR(400MHz,Chloroform-d)δ(ppm)7.30(s,1H),7.27(s,1H),6.98(t,J＝8.9Hz,1H),6.95-6.90(m,2H),6.88(dd,J＝6.3,2.9Hz,1H),6.85-6.79(m,1H),4.14(q,J＝7.1Hz,1H),2.27(d,J＝2.0Hz,3H),1.55(d,J＝6.7Hz,3H),1.22(s,9H).

(S) -1- (4- (4-fluoro-3-methylphenoxy) phenyl) ethan-1-amine (24 d)

24C (1.90 g,5.44 mmol) was added to a concentrated hydrochloric acid/methanol (v: v=1:2) system and stirred at room temperature for 2h, after TLC monitored to completion, the methanol was removed by concentration under reduced pressure, the residue was diluted with water, pH was adjusted to 10, extracted with dichloromethane, the organics were combined and concentrated under reduced pressure, flash column chromatography (PE: EA=3:1, v: v) afforded 1.02g as a colorless oily liquid in yield 76％.¹H NMR(300MHz,Chloroform-d)δ(ppm)7.30(d,J＝1.9Hz,1H),7.27(s,1H),7.20-7.12(m,2H),7.04-6.88(m,3H),4.11(q,J＝6.6Hz,1H),2.25(d,J＝2.0Hz,3H),1.40(d,J＝6.6Hz,3H).

(S) -N- (2- ((1- (4- ((2, 2-dimethyl-4-oxo-4H-benzo [ d ] [1,3] dioxin-6-yl) methyl) phenyl) ethyl)) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (20 g)

20F (360 mg,1.15 mmol), 1C (400 mg,1.25 mmol), DIPEA (436 mg,0.11 mmol) were dissolved in 8mL of n-butanol, strictly evacuated under nitrogen protection, heated to reflux to 110℃and reacted for 48 hours. TLC monitored to the end of the reaction. The reaction solution was cooled to room temperature, the solvent was dried in vacuo, dissolved in ethyl acetate, washed three times with water, washed three times with saturated brine, dried over anhydrous sodium sulfate, and the solvent was dried by flash column chromatography on silica gel (DCM: meoh=80:1, v:v) to give 291mg of pale yellow solid in yield 41％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.78(s,1H),8.23(s,2H),8.02(s,3H),7.69(d,J＝2.3Hz,2H),7.54(dd,J＝8.4,2.3Hz,1H),7.19(s,4H),7.05(d,J＝8.4Hz,1H),6.00(s,1H),4.76(s,1H),3.95(s,2H),1.67(s,6H),1.32(d,J＝5.7Hz,3H).

(S) -N- (2- ((1- (4- ((2, 2-dimethyl-4-oxo-4H-benzo [ d ] [1,3] dioxin-7-yl) methyl) phenyl) ethyl)) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (21 g)

Synthesis and workup of Compound 21g As with Compound 20g, 21f (460 mg,1.48 mmol), 1c (310 mg,1.61 mmol) was added and flash column chromatography (DCM: meOH=70:1, v:v) was carried out to afford 20g 310mg as a pale yellow solid in yield 35％.¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.86(s,1H),8.22(s,2H),7.99(s,2H),7.77(d,J＝8.0Hz,2H),7.58(s,1H),7.19(s,4H),7.06(dd,J＝8.0,1.6Hz,1H),6.96(d,J＝1.6Hz,1H),5.98(s,1H),4.73(s,1H),3.97(s,2H),1.67(s,6H),1.24(s,3H).

Example 1 (S) -N- (2- ((1- ([ 1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-1)

1B (200 mg,1.01 mmol), 1C (213 mg, 0.6755 mmol), DIPEA (436 mg,0.11 mmol) were dissolved in 8mL of n-butanol and heated to reflux under a strict vacuum nitrogen atmosphere at 110℃for 48 hours. TLC monitored to the end of the reaction. The reaction solution was cooled to room temperature, the solvent was dried in vacuo, dissolved in ethyl acetate, washed three times with water, washed three times with saturated brine, dried over anhydrous sodium sulfate, and dried over flash silica gel column chromatography (DCM: meoh=80:1, v: v) to give 80mg of pale yellow solid, yield 25%, calculated value C ₂₄H₂₁N₅O₄S,[M+H]⁺ m/z of HPLC purity :99.08％.Mp:184 -185℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.24(s,3H),8.03(s,2H),7.88(s,1H),7.64(dd,J＝7.1,1.5Hz,2H),7.61-7.55(m,2H),7.47(t,J＝7.7Hz,2H),7.36(ddt,J＝10.0,6.7,2.5Hz,3H),6.03(s,1H),4.88(s,1H),1.38(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)166.90,164.61,162.16,153.79,149.13,142.89,140.52,140.37,139.41,139.03,133.44,129.35,128.36,127.82,127.73,127.14,127.10,127.07,127.05,126.80,124.36,110.59,49.66,22.26.HRMS(ESI):, 476.1387, found 476.1384.

EXAMPLE 2 (S) -4-nitro-N- (2- ((1- (4 '- (trifluoromethyl) - [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) benzenesulfonamide (I-2)

Reference example 1 synthesis with 43% yield and HPLC purity :97.27％.Mp:214-216℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.95(br,1H),8.20(s,2H),7.99(s,2H),7.92-7.78(m,5H),7.78-7.57(m,3H),

7.45-7.31(m,2H),6.02(s,1H),4.89(s,1H),1.36(d,J=16.4Hz,3H)。¹³C NMR(101MHz,

DMSO-d₆)δ(ppm)173.10,162.84,161.32,160.14,153.39,151.36,148.31,144.86,143.76,138.30,137.81,134.49,134.01,128.92,128.60,128.40,128.04,127.77,126.80,126.77,124.95,124.07,110.88,49.57,22.10.

HRMS (ESI) calculated C ₂₅H₂₀F₃N₅O₄S,[M+H]⁺ m/z,544.1261, found 544.1257.

Example 3 (S) -4-nitro-N- (2- ((1- (4 '-nitro- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) benzenesulfonamide (I-3)

The synthesis method of reference example 1 gave a yield of 27% and an HPLC purity :98.41％.Mp:194-195℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.23(s,1H),8.35-8.29(m,2H),8.21(s,2H),8.10-7.90(m,4H),7.82(s,1H),7.75-7.62(m,3H),7.42(d,J＝7.4Hz,2H),6.04(s,1H),4.93(s,1H),1.40(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)167.47,160.17,156.87,152.20,149.15,147.61,147.32,140.99,138.95,137.46,129.35,128.93,128.72,128.65,128.26,127.98,127.86,125.19,125.11,124.93,124.30,111.01,60.80,21.80.HRMS(ESI): calculated as C ₂₄H₂₀N₆O₆S,[M+H]⁺ m/z,521.1238, found 521.1227.

EXAMPLE 4 (S) -N- (2- ((1- (4 '-cyano- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-4)

The synthesis method of reference example 1 gave a yield of 30% and an HPLC purity :96.85％.Mp:134-135℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.22(s,1H),8.21(s,2H),8.01(s,2H),7.95-7.90(m,2H),7.90-7.84(m,2H),7.71-7.66(m,2H),7.53-7.45(m,1H),7.39(d,J＝7.7Hz,2H),7.22(d,J＝8.3Hz,1H),6.03(s,1H),4.91(s,1H),1.39(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)160.84,159.29,157.50,151.32,149.41,145.41,140.91,139.59,137.79,133.85,132.71,132.06,130.10,129.29,128.46,128.01,127.92,127.80,124.82,124.31,122.76,119.97,110.90,50.38,21.52.HRMS(ESI): calculated as C ₂₅H₂₀N₆O₄S,[M+H]⁺ m/z,501.1340, found 501.1341.

Example 5 (S) -N- (2- ((1- (4 '-methyl- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-5)

The synthesis method of reference example 1 gave a yield of 36% and an HPLC purity :98.73％.Mp:157-158℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.22(s,2H),8.00(s,2H),7.82(s,1H),7.63(s,1H),7.54(dd,J＝10.5,7.9Hz,4H),7.28(t,J＝10.9Hz,4H),6.00(s,1H),4.83(s,1H),2.34(s,3H),1.36(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)166.03,160.39,157.88,157.12,152.89,149.47,147.57,144.96,142.77,139.83,137.94,137.61,135.15,132.44,130.43,128.99,127.49,127.37,127.31,127.16,124.87,109.29,58.20,30.79,21.61.HRMS(ESI): calculated as C ₂₅H₂₃N₅O₄S,[M+H]⁺ m/z,490.1544, found 490.1539.

Example 6 (S) -N- (2- ((1- (4 '-amino- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-6)

The synthesis method of reference example 1 gave a yield of 30% and an HPLC purity of 99.60%. Mp 169-171 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.25(s,2H),8.04(s,2H),7.68(s,2H),7.57-7.39(m,4H),7.34-7.17(m,2H),6.87-6.72(m,2H),6.02(s,1H),4.83(s,1H),1.35(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)161.03,159.54,158.53,150.84,149.62,141.56,140.11,137.40,135.72,129.51,128.83,128.40,128.35,128.03,127.48,127.21,126.44,126.15,124.91,124.69,113.67,110.96,60.40,23.13.HRMS(ESI): Calculated C ₂₄H₂₂N₆O₄S,[M+H]⁺ m/z,491.1496, found 491.1485.

EXAMPLE 7 (S) -N- (2- ((1- (4 '- (methylamino) - [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-7)

The synthesis method of reference example 1 gave a yield of 32% and an HPLC purity of 98.49%. MP, 146-148 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.24(s,3H),8.01(s,2H),7.82(s,1H),7.50-7.43(m,2H),7.43-7.37(m,2H),7.23(s,2H),6.66-6.56(m,2H),5.98(s,1H),5.80(s,1H),4.76(s,1H),2.71(s,3H),1.27(d,J＝25.7Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)163.20,160.07,158.40,150.37,149.16,144.70,139.65,131.31,130.58,130.12,128.40,127.93,127.62,127.54,126.99,126.86,125.97,125.78,124.43,124.30,113.19,110.93,55.09,40.61,19.89.HRMS(ESI): Calculated C ₂₅H₂₄N₆O₄S,[M+H]⁺ m/z,505.1580, found 505.1587.

Example 8 (S) -N- (2- ((1- (4 '- (ethylamino) - [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-8)

The synthesis method of reference example 1 gave a yield of 25% and an HPLC purity of 97.86%. MP 129-130 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.36-8.15(m,3H),7.68(d,J＝29.6Hz,1H),7.50-7.42(m,2H),7.42-7.34(m,2H),7.24(s,4H),6.69-6.56(m,2H),6.01(s,1H),5.70(s,1H),4.80(s,1H),3.07(q,J＝7.1Hz,2H),1.32(d,J＝15.2Hz,3H),1.18(t,J＝7.1Hz,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)165.86,162.62,159.34,150.30,149.50,145.24,140.45,133.66,132.43,130.53,128.04,128.00,127.84,127.36,126.20,126.07,124.93,124.84,124.77,124.60,113.21,110.11,54.46,38.25,23.04,15.38.HRMS(ESI): Calculated C ₂₆H₂₆N₆O₄S,[M+H]⁺ m/z,519.1736, found 519.1738.

Example 9 (S) -N- (2- ((1- (4 '- (isopropylamino) - [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-9)

The synthesis method of reference example 1 gave a yield of 21% and an HPLC purity of 98.10%. MP at 136-138 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.26(s,2H),7.46(d,J＝8.2Hz,2H),7.41-7.34(m,2H),7.24(s,6H),6.66-6.59(m,2H),6.02(s,1H),5.56(s,1H),4.79(s,1H),3.58(s,1H),1.30(d,J＝45.0Hz,3H),1.15(d,J＝6.3Hz,6H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)161.06,160.20,153.50,151.27,148.24,142.81,136.47,135.83,130.16,128.93,127.65,127.17,126.95,126.91,125.74,124.40,123.62,123.47,123.12,117.62,113.21,111.94,68.14,43.19,25.21,22.47.HRMS(ESI): Calculated C ₂₇H₂₈N₆O₄S,[M+H]⁺ m/z,533.1893, found 533.1896.

Example 10 (S) -N- (2- ((1- (4 '- (dimethylamino) - [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-10)

The synthesis method of reference example 1 gave a yield of 26% and an HPLC purity of 99.47%. Mp 169-170 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.25(s,2H),8.02(s,2H),7.80(s,1H),7.62(s,1H),7.55-7.42(m,4H),7.26(s,2H),6.84-6.76(m,2H),6.01(s,1H),4.79(s,1H),2.94(s,6H),1.34(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)164.55,159.65,156.39,150.79,145.38,140.07,135.56,131.76,130.53,128.37,128.08,127.95,127.48,127.41,126.39,126.22,124.88,124.84,124.71,124.71,113.61,109.19,61.13,41.03,21.57.HRMS(ESI): Calculated C ₂₆H₂₆N₆O₄S,[M+H]⁺ m/z,519.1736, found 519.1735.

EXAMPLE 11 tert-butyl- (S) - (4 '- (1- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -4-yl) carbamate (I-11)

The synthesis method of reference example 1 gave a yield of 22% and an HPLC purity of 97.84%. MP, 164-165 ℃.

¹H NMR(300MHz,DMSO-d₆)δ(ppm)9.45(s,1H),8.24(d,J＝9.0Hz,2H),8.02(s,2H),7.76(s,1H),7.67(s,1H),7.53(d,J＝5.0Hz,6H),7.30(d,J＝7.9Hz,2H),6.02(s,1H),4.85(s,1H),1.50(s,9H),1.25(d,J＝4.3Hz,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)166.19,161.97,159.53,154.61,153.72,149.50,140.01,139.95,139.62,139.57,134.39,132.23,128.75,127.70,127.53,127.46,127.09,127.03,124.90,124.86,119.51,119.37,110.40,80.07,49.65,29.09,23.04.HRMS(ESI): Calculated C ₂₉H₃₀N₆O₆S,[M+H]⁺ m/z,591.1948, found 591.2021.

Example 12 (S) -N- (2- ((1- (4 '-hydroxy- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-12)

The synthesis method of reference example 1 gave a yield of 21% and an HPLC purity of 98.57%. MP:138-139 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)9.53(s,1H),8.22(d,J＝8.6Hz,3H),8.00(s,2H),7.47(dd,J＝15.2,8.0Hz,5H),7.26(s,2H),6.84(d,J＝8.1Hz,2H),5.97(s,1H),4.78(s,1H),1.34(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)162.42,160.32,158.11,153.45,147.92,139.38,137.49,137.20,136.05,134.74,133.95,133.11,132.23,130.63,130.03,128.59,128.20,127.07,126.96,126.89,126.76,124.45,58.54,21.21.HRMS(ESI): Calculated C ₂₄H₂₁N₅O₅S,[M+H]⁺ m/z,492.1263, found 492.1268.

EXAMPLE 13 (S) -N- (2- ((1- (4 '-methoxy- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-13)

The synthesis method of reference example 1 gave a yield of 24% and an HPLC purity of 98.01%. Mp is 150-152 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.22(s,1H),8.24(s,2H),8.02(s,2H),7.73(d,J＝28.5Hz,2H),7.61-7.55(m,1H),7.55-7.50(m,1H),7.50-7.44(m,2H),7.30(d,J＝8.2Hz,1H),7.22(d,J＝8.1Hz,2H),7.08-6.96(m,1H),6.04(s,1H),4.84(s,1H),3.80(s,3H),1.34(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)163.24,161.26,159.40,156.69,143.24,141.45,139.12,132.82,132.26,131.62,131.47,129.65,128.98,128.84,128.45,128.24,127.09,126.65,124.39,120.37,114.88,102.31,55.72,49.92,21.06.HRMS(ESI): Calculated C ₂₅H₂₃N₅O₅S,[M+H]⁺ m/z,506.1420, found 506.1489.

EXAMPLE 14 (S) -N- (2- ((1- (3 '-methoxy- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-14)

The synthesis method of reference example 1 gave a yield of 35% and an HPLC purity of 98.74%. MP, 123-125 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.94(s,1H),8.23(s,2H),8.01(s,2H),7.85(s,1H),7.72-7.49(m,3H),7.36(q,J＝12.0,10.0Hz,3H),7.22-7.14(m,2H),6.94(dd,J＝8.4,2.6Hz,1H),6.01(s,1H),4.86(s,1H),3.83(s,3H),1.35(d,J＝7.3Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)164.70,162.40,160.72,155.31,151.98,149.66,147.73,142.45,139.84,135.35,130.97,128.63,127.76,127.54,127.34,124.92,124.72,123.05,121.84,119.97,113.97,113.14,56.10,49.58,31.44.HRMS(ESI): Calculated C ₂₅H₂₃N₅O₅S,[M+H]⁺ m/z,506.1420, found 506.1499.

Example 15 (S) -N- (2- ((1- (2 '-methoxy- [1,1' -biphenyl ] -4-yl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-15)

The synthesis method of reference example 1 gave a yield of 32% and an HPLC purity of 98.99%. MP, 135-136 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.41-8.13(m,4H),8.02(s,1H),7.48-7.16(m,7H),7.10(d,J＝8.2Hz,1H),7.03(t,J＝7.4Hz,1H),6.02(s,1H),4.83(s,1H),3.75(s,3H),1.37(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)163.07,157.11,153.65,149.54,148.13,146.31,143.37,142.18,141.34,140.64,137.97,131.31,130.41,130.25,129.88,129.70,128.80,126.72,125.32,124.98,121.75,112.63,56.40,39.87,22.1.HRMS(ESI): Calculated C ₂₅H₂₃N₅O₅S,[M+H]⁺ m/z,506.1420, found 506.1497.

EXAMPLE 16 (S) -4-hydroxy-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -3-carboxamide (I-16)

16A (100 mg,0.38 mmol), 16c (165 mg,0.35 mmol) and Pd (dppf) Cl ₂ (13 mg, 17. Mu. Mol) were dissolved in 4mL of 1, 4-dioxane, and a solution of cesium carbonate (450 mg,1.38 mmol) in water (1 mL) was added. The reaction was monitored by TLC to endpoint by stirring for 6 hours under heat at 100 ℃ under stringent nitrogen protection. The reaction solution was cooled to room temperature, the pH was adjusted to 1 with 0.1M dilute hydrochloric acid, the insoluble matter was removed by suction filtration with celite, the liquid was collected, 1, 4-dioxane was swirled off under reduced pressure, 30mL of water was added, extraction was performed three times with ethyl acetate, 50mL each time, the organic phases were combined, washed three times with saturated saline, 50mL each time, dried over night over anhydrous sodium sulfate, and the anhydrous sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and flash column chromatography (DCM: meOH=60:1, v: v) afforded 85mg of white solid, yield 46%, calculated C ₂₅H₂₂N₆O₆S,[M+H]⁺ M/z of HPLC purity :97.88％.Mp:183-184℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)13.11(s,1H),8.57(s,1H),8.23(s,2H),8.15(d,J＝2.3Hz,1H),7.98(s,1H),7.72(dd,J＝8.5,2.2Hz,1H),7.61(d,J＝8.0Hz,2H),7.40-7.29(m,4H),6.96(d,J＝8.6Hz,1H),6.00(s,1H),4.82(s,1H),1.28(d,J＝16.0Hz,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)173.13,163.05,161.62,158.25,149.59,148.08,141.25,139.00,133.07,131.23,128.86,127.45,127.20,127.13,127.01,124.92,124.86,122.47,118.88,118.84,115.60,115.51,109.50,55.37,23.04.HRMS(ESI):, 535.1322; measured value 535.1328.

Example 17 (S) -3-hydroxy-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -4-carboxamide (I-17)

The synthesis of reference example 16 gave a yield of 66% and an HPLC purity of 98.58%. MP:174-175 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)13.18(s,1H),10.75(s,1H),8.44(s,1H),8.12-7.76(m,3H),7.63(d,J＝8.0Hz,3H),7.45(d,J＝7.9Hz,2H),7.21-7.09(m,2H),6.58(d,J＝8.4Hz,2H),6.07(d,J＝5.4Hz,3H),5.11(q,J＝7.6Hz,1H),1.43(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)172.71,161.21,158.05,155.63,153.76,151.77,149.06,145.09,144.29,138.54,132.67,130.77,129.78,128.33,127.03,126.79,126.70,126.57,124.44,118.46,117.98,115.02,110.40,59.84,22.63.HRMS(ESI): Calculated C ₂₅H₂₂N₆O₆S,[M+H]⁺ m/z,535.1322, found 535.1328.

Example 18 (S) -4-hydroxy-N-methyl-4 '- (1- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -3-carboxamide (I-18)

18A (300 mg,1.08 mmol), 16c (469 mg,0.98 mmol) and Pd (dppf) Cl2 (39 mg, 51. Mu. Mol) were dissolved in 8mL of 1, 4-dioxane, and a solution of cesium carbonate (1.35 g,4.14 mmol) in water (2 mL) was added. The reaction was monitored by TLC to endpoint by stirring for 6 hours under heat at 100 ℃ under stringent nitrogen protection. The reaction solution was cooled to room temperature, pH was adjusted to 1 with 0.1M dilute hydrochloric acid, insoluble matter was removed by suction filtration with celite, the liquid was collected, 1, 4-dioxane was swirled off under reduced pressure, 30mL of water was added, extraction was performed three times with ethyl acetate, about 50mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, dried over night over anhydrous sodium sulfate, anhydrous sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and 131mg of pale yellow solid was isolated by flash column chromatography (DCM: meoh=40:1, v: v) in 25% yield with HPLC purity of 98.04%.

The pale yellow solid (120 mg,0.24 mmol), methylamine hydrochloride (40 mg,0.60 mmol) and DIPEA (154 mg,1.20 mmol) were dissolved in 3mL of anhydrous DMF, and HATU (170 mg,0.44 mmol) was added under stirring in an ice bath (0 ℃), followed by stirring at room temperature for 3 hours. TLC monitored the reaction to endpoint. The reaction solution was poured into 30mL of water, extracted three times with ethyl acetate, about 30mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 91mg of white solid which was isolated by flash silica gel column chromatography (DCM: meoh=70:1, v: v) in 80% yield, calculated as C26H24N6O6S, [ m+h ] +m/z,549.1478, found 549.1475 by HPLC purity :99.21％.Mp:143-144℃.1H NMR(400MHz,DMSO-d6)δ(ppm)12.83(s,1H),8.97(s,1H),8.21(s,3H),8.12(s,1H),7.99(s,1H),7.70(d,J＝8.1Hz,1H),7.66-7.52(m,3H),7.43-7.26(m,3H),6.98(d,J＝8.6Hz,1H),5.97(s,1H),4.79(s,1H),2.85(d,J＝4.4Hz,3H),1.27(d,J＝16.3Hz,3H).13C NMR(126MHz,DMSO-d6)δ(ppm)170.08,162.39,160.25,159.70,157.44,148.06,142.05,138.55,132.43,132.13,130.83,128.20,126.98,126.75,126.63,126.58,125.86,125.76,124.39,124.35,118.40,115.63,110.56,59.13,29.47,26.44.HRMS(ESI):.

Example 19 (S) -3-hydroxy-N-methyl-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -4-carboxamide (I-19)

The synthesis of reference example 18 gave a yield of 78% and an HPLC purity of 98.78%. MP at 156-158 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)12.88(s,1H),8.85(q,J＝4.6Hz,1H),8.21(s,2H),8.00(d,J＝8.2Hz,2H),7.88(d,J＝8.4Hz,1H),7.63(d,J＝8.1Hz,2H),7.35(d,J＝9.4Hz,4H),7.22-7.09(m,2H),6.01(s,1H),4.86(s,1H),2.84(d,J＝4.5Hz,3H),1.37(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)170.44,161.62,159.12,156.34,154.30,149.54,145.87,144.63,140.85,138.51,130.56,128.91,128.77,127.74,127.64,127.57,127.51,124.86,124.83,117.91,115.94,114.82,112.45,60.09,29.99,26.96.HRMS(ESI): Calculated C ₂₆H₂₄N₆O₆S,[M+H]⁺ m/z,549.1478, found 549.1479.

Example 20 (S) -4-hydroxy-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -3-carboxylic acid (I-20)

18A (300 mg,1.08 mmol), 16c (469 mg,0.98 mmol) and Pd (dppf) Cl ₂ (39 mg, 51. Mu. Mol) were dissolved in 8mL of 1, 4-dioxane, and a solution of cesium carbonate (1.35 g,4.14 mmol) in water (2 mL) was added. The reaction was monitored by TLC to endpoint by stirring for 6 hours under heat at 100 ℃ under stringent nitrogen protection. The reaction solution was cooled to room temperature, the pH was adjusted to 1 with 0.1M dilute hydrochloric acid, the insoluble matter was removed by suction filtration with celite, the liquid was collected, 1, 4-dioxane was swirled off under reduced pressure, 30mL of water was added, extraction was performed three times with ethyl acetate, about 50mL each time, the organic phases were combined, washed three times with saturated saline, 50mL each time, dried over night over anhydrous sodium sulfate, and the anhydrous sodium sulfate was removed by filtration, the filtrate was concentrated under reduced pressure, and 131mg of pale yellow solid was isolated by flash column chromatography (DCM: meOH=40:1, v: v) in 25% yield, calculated as HPLC purity 98.04％.Mp:199-200℃.¹H NMR(300MHz,DMSO-d₆)δ(ppm)8.20(s,2H),8.01(s,4H),7.85-7.70(m,1H),7.54(d,J＝7.9Hz,2H),7.30(s,3H),7.04(d,J＝8.6Hz,1H),6.01(s,1H),4.84(s,1H),1.36(s,3H).¹³C NMR(126MHz,DMSO-d₆)δ(ppm)172.93,172.81,162.44,161.56,160.51,151.27,149.55,143.07,138.82,135.71,134.58,131.82,128.83,128.69,127.57,127.40,127.06,126.97,125.75,124.86,118.66,114.63,110.29,60.70,21.99.HRMS(ESI): C ₂₅H₂₁N₅O₇S,[M+H]⁺ M/z,536.1162; observed value 536.1233.

Example 21 (S) -3-hydroxy-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -4-carboxylic acid (I-21)

The synthesis method of reference example 20 gave a yield of 34% and an HPLC purity of 97.01%. MP 203-204 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.33(s,1H),8.19(s,2H),7.98(s,2H),7.85(d,J＝8.4Hz,1H),7.64(d,J＝8.0Hz,2H),7.34(s,2H),7.30-7.26(m,1H),7.22(d,J＝5.5Hz,3H),6.00(s,1H),4.86(s,1H),1.36(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)175.54,173.02,164.33,162.58,161.10,149.58,147.41,138.57,133.55,131.82,128.72,127.91,127.83,127.62,127.45,125.27,124.93,124.18,121.06,118.36,116.58,115.54,113.70,58.15,22.08.HRMS(ESI): Calculated C ₂₅H₂₁N₅O₇S,[M+H]⁺ m/z,536.1162, found 536.1163.

EXAMPLE 22 methyl (S) -4-hydroxy-4 '- (1- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -3-carboxylate (I-22)

Compound I-20 (120 mg,0.24 mmol) was dissolved in 3mL of anhydrous methanol, thionyl chloride (52 mg,0.44 mmol) was slowly added dropwise, and the mixture was stirred under heating at 65℃for 4 hours, followed by TLC monitoring the reaction to the end point. The reaction solution was cooled to room temperature, the solvent was dried under reduced pressure, and flash column chromatography (DCM: meoh=100:1, v: v) was performed to obtain 91mg of a white solid in 88% yield, and HPLC purity :100.00％.Mp:151-153℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.53(s,1H),8.18(d,J＝8.4Hz,2H),8.03-7.91(m,3H),7.81(dd,J＝8.6,2.5Hz,1H),7.54(d,J＝8.0Hz,2H),7.42-7.35(m,1H),7.35-7.22(m,3H),7.09(d,J＝8.7Hz,1H),5.97(s,1H),4.81(s,1H),3.92(s,3H),1.35(d,J＝13.1Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)175.04,169.53,159.87,158.81,153.89,147.64,140.78,140.21,138.16,134.66,134.24,131.68,128.37,128.18,127.17,126.66,124.78,124.45,123.70,123.25,118.63,116.12,114.14,53.07,49.14,22.62.HRMS(ESI): calculated as C ₂₆H₂₃N₅O₇S,[M+H]⁺ m/z,550.1318, found 550.1317.

EXAMPLE 23 methyl (S) -3-hydroxy-4 '- (1- ((4- ((4-nitrophenyl) sulfonylamino) pyrimidin-2-yl) amino) ethyl) - [1,1' -biphenyl ] -4-carboxylate (I-23)

The synthesis of reference example 22 gave a yield of 64% and an HPLC purity of 98.79%. Mp 167-168 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)10.59(s,1H),8.21(s,2H),8.00(s,2H),7.89-7.82(m,1H),7.69-7.60(m,2H),7.38(d,J＝11.1Hz,3H),7.25(dd,J＝6.2,1.6Hz,3H),6.02(s,1H),4.89(s,1H),3.92(s,3H),1.37(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)170.17,166.10,161.47,153.30,151.88,149.46,147.85,145.60,138.19,136.95,131.58,128.73,127.94,127.94,127.68,127.49,125.30,124.94,124.79,118.87,118.72,115.94,112.71,53.52,49.59,30.82.HRMS(ESI): Calculated C ₂₆H₂₃N₅O₇S,[M+H]⁺ m/z,550.1318, found 550.1316.

Example 24 (S) -2-hydroxy-5- (4- (1- ((4- ((4-nitrophenyl) sulfamido) pyrimidin-2-yl) amino) ethyl) benzyl) benzoic acid (I-24)

20G (280 mg,0.46 mmol) of the compound was dissolved in 6mL of trifluoroacetic acid/water (9/1, v/v) and stirred at room temperature for 12 hours. TLC monitored the reaction to endpoint. The reaction solution was poured into 20mL of water, extracted three times with dichloromethane, 20mL each time, the organic phases combined, washed three times with saturated brine, 20mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate concentrated under reduced pressure to give 216mg of a brown solid by flash column chromatography (DCM: meoh=50:1, v: v) in 82% yield, calculated HPLC purity :97.63％.Mp:205-206℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.13(s,1H),8.33-8.12(m,4H),7.99(s,2H),7.85(s,1H),7.63(d,J＝2.3Hz,1H),7.36(dd,J＝8.5,2.3Hz,1H),7.14(s,3H),6.88(d,J＝8.4Hz,1H),6.01(s,1H),4.73(s,1H),3.86(s,2H),1.28(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)172.40,161.79,158.97,158.65,150.16,148.92,139.50,136.56,130.90,129.27,129.07,127.97,127.87,127.20,126.69,125.84,124.42,124.22,120.35,119.07,117.44,116.10,111.43,49.13,41.16,18.54.HRMS(ESI): C ₂₆H₂₃N₅O₇S,[M+H]⁺ m/z,550.1318; found 550.1314.

Example 25 (S) -2-hydroxy-5- (4- (1- ((4- ((4-nitrophenyl) sulfamido) pyrimidin-2-yl) amino) ethyl) benzyl) benzamide (I-25)

Compound I-24 (100 mg,0.18 mmol), pyBOP (142 mg,0.26 mmol), HOBt (38 mg,0.26 mmol) were dissolved in 3mL of anhydrous DMF and stirred in an ice bath (0 ℃ C.), DIPEA (48 mg,0.36 mmol), and amine chloride (20 mg,0.36 mmol) were added sequentially and stirring was resumed for 3 hours at room temperature. TLC monitored the reaction to endpoint. The reaction solution was poured into 30mL of water, extracted three times with ethyl acetate, about 30mL each time, the organic phases were combined, washed three times with saturated brine, 50mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove the anhydrous sodium sulfate, and the filtrate was concentrated under reduced pressure to give 78mg of a white solid by flash silica gel column chromatography (DCM: meoh=50:1, v:v), 78% yield, calculated HPLC purity :99.13％.Mp:172-173℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)12.77(s,1H),11.22(s,1H),8.35(s,1H),8.22(s,2H),8.07-7.92(m,2H),7.84(s,1H),7.78(d,J＝2.2Hz,1H),7.76-7.69(m,1H),7.62(s,1H),7.22(dd,J＝8.5,2.1Hz,1H),7.15(s,4H),6.80(d,J＝8.4Hz,1H),6.00(s,1H),4.72(s,1H),3.82(s,2H),1.29(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)172.88,160.28,157.38,154.54,145.43,144.03,141.39,139.74,135.46,132.10,129.43,129.15,128.67,127.15,126.91,125.11,124.93,124.12,118.44,115.25,109.73,55.95,41.13,40.92,30.04,23.12.HRMS(ESI): C ₂₆H₂₄N₆O₆S,[M+H]⁺ m/z,549.1478; found 549.1475.

EXAMPLE 26 (S) -2-hydroxy-4- (4- (1- ((4- ((4-nitrophenyl) sulfamido) pyrimidin-2-yl) amino) ethyl) benzyl) benzoic acid (I-26

The synthesis method of reference example 24 gave a yield of 77% and an HPLC purity of 99.49%. MP, 192-193 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)11.22(s,1H),8.22(s,2H),8.00(s,2H),7.82(s,1H),7.73-7.60(m,2H),7.17(s,4H),6.83-6.74(m,2H),6.02(s,1H),4.75(s,1H),3.89(s,2H),1.31(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)172.85,162.25,159.42,159.11,151.36,150.61,146.26,139.96,134.78,131.36,129.73,129.54,128.43,128.33,127.67,127.16,124.88,124.68,120.81,119.55,117.90,116.58,111.92,54.51,41.63,21.27.HRMS(ESI): Calculated C ₂₆H₂₃N₅O₇S,[M+H]⁺ m/z,550.1318, found 550.1379.

EXAMPLE 27 (S) -2-hydroxy-4- (4- (1- ((4- ((4-nitrophenyl) sulfamido) pyrimidin-2-yl) amino) ethyl) benzyl) benzamide (I-27)

With reference to the synthesis of example 25, the yield was 81%, the HPLC purity :97.40％.Mp:178-179℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)13.01(s,1H),11.81(s,1H),8.30(s,1H),8.22(d,J＝7.4Hz,2H),8.02(s,2H),7.80(s,1H),7.74(d,J＝8.1Hz,1H),7.63(s,2H),7.16(s,4H),6.76-6.67(m,2H),4.77(s,1H),3.86(s,2H),1.28(d,J＝10.7Hz,3H.¹³C NMR(75MHz,DMSO-d₆)δ(ppm)172.55,162.79,161.74,160.68,159.88,148.43,143.67,140.45,138.94,135.07,130.46,129.17,128.61,127.80,126.64,124.43,124.39,119.65,119.48,117.88,117.71,112.63,110.16,59.26,40.98,21.54.HRMS(ESI): was calculated as C ₂₆H₂₄N₆O₆S,[M+H]⁺ m/z,549.1478, found 549.1475.

EXAMPLE 28 (S) -N- (2- ((1- (4- (4-fluoro-3-methylbenzyl) phenyl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-28)

The synthesis method of reference example 1 gave a yield of 30% and an HPLC purity of 99.59%. MP 119-121 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.20(s,2H),7.97(s,2H),7.74(s,1H),7.60(dd,J＝19.9,7.8Hz,1H),7.41(dt,J＝42.6,8.6Hz,1H),7.13(d,J＝2.8Hz,4H),7.07-6.97(m,2H),5.96(s,1H),4.69(s,1H),3.84(s,2H),2.18(s,3H),1.23(s,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)161.44,159.05,157.46,150.80,149.38,141.16,138.06,138.03,132.74,132.69,129.91,129.51,128.73,128.65,127.63,127.60,127.14,125.07,124.90,115.85,115.63,110.68,59.22,49.73,23.12,15.15.HRMS(ESI): Calculated C ₂₆H₂₄FN₅O₄S,[M+H]⁺ m/z,522.1533, found 522.1535.

Example 29 (S) -N- (2- ((1- (4- ((4-fluoro-3-methylphenyl) amino) phenyl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-29)

4-Fluoro-3-methylaniline (700 mg,0.56 mmol), 16C (400 mg,1.33 mmol), pd ₂dba₃ (5 mg, 5. Mu. Mol), XPhos (9 mg, 19. Mu. Mol), sodium t-butoxide (67 mg,0.70 mmol) were dissolved in 8mL toluene and heated to 95℃under nitrogen and stirred for 15 hours. TLC monitored to the end of the reaction. The reaction was cooled to room temperature, the reaction solution was poured into 50mL of saturated ammonium chloride solution, extracted three times with ethyl acetate, 50mL each time, the organic phases combined, washed three times with saturated brine, 50mL each time, dried over anhydrous sodium sulfate overnight, filtered to remove anhydrous sodium sulfate, and the filtrate concentrated under reduced pressure to give 86mg of brown solid by flash column chromatography on silica gel (PE: ea=1:3, v: v) in 31% yield, calculated C ₂₅H₂₃FN₆O₄S,[M+H]⁺ m/z HPLC purity :99.49％.Mp:151-152℃.¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.28(d,J＝8.3Hz,2H),8.03(s,3H),7.86(s,1H),7.69(s,1H),7.08(s,2H),7.00(t,J＝9.1Hz,1H),6.96-6.90(m,3H),6.86(dt,J＝8.1,3.6Hz,1H),6.03(s,1H),4.64(s,1H),2.18(s,3H),1.25(d,J＝9.6Hz,3H).¹³C NMR(101MHz,DMSO-d₆)δ(ppm)160.10,156.74,154.40,149.08,143.64,139.88,138.83,131.65,130.35,128.79,128.37,128.11,127.50,125.18,125.00,124.45,120.71,116.91,116.32,115.92,115.69,109.83,61.14,31.68,14.94.HRMS(ESI):, 523,1486; measured 523.1485.

Example 30 (S) -N- (2- ((1- (4- (4-fluoro-3-methylphenoxy) phenyl) ethyl) amino) pyrimidin-4-yl) -4-nitrobenzenesulfonamide (I-30)

The synthesis method of reference example 1 gave a yield of 22% and an HPLC purity of 99.49%. MP:174-175 ℃.

¹H NMR(400MHz,DMSO-d₆)δ(ppm)8.24(s,2H),7.93(d,J＝5.9Hz,1H),7.37(s,3H),7.23(s,2H),7.15(t,J＝9.1Hz,1H),6.96(dd,J＝6.5,3.1Hz,1H),6.91-6.84(m,2H),6.37(d,J＝5.9Hz,1H),5.97(s,1H),4.73(s,1H),2.21(s,3H),1.26(d,J＝25.7Hz,3H).

¹³C NMR(101MHz,DMSO-d₆)δ(ppm)165.72,160.32,158.61,157.23,156.81,156.23,152.54,150.89,148.93,138.57,128.18,126.62,126.43,124.46,122.53,122.48,118.57,118.49,118.15,116.67,116.43,104.53,60.29,21.58,14.75.HRMS(ESI): Calculated C ₂₅H₂₂FN₅O₅S,[M+H]⁺ m/z,524,1326, found 524.1326.

EXAMPLE 31 biological evaluation experiment

(1) Analysis and detection method for influence of compound on cell viability

The effect of the compounds of the examples on B16F10 cell viability was examined using the MTT method. The MTT assay was performed on cells in the logarithmic growth phase, and murine B16F10 melanoma cells were inoculated at 1X 10 ⁴ per well in 96-well plates, incubated at 5% CO ₂ and 37℃for 24 hours, and the supernatant was removed. The experimental groups were each charged with 200. Mu.L of DMEM medium at a drug concentration of 0.25 or 1. Mu.M, and 6 duplicate wells were set at a concentration of 50. Mu.M for 8-MOP (positive control). 200 μl of DMEM medium is directly added as a control group, incubated for 48h, 20 μl of MTT solution (5 g/L) is added to each well, incubation is continued for 4h at 37 ℃ under 5% CO ₂, the supernatant is removed, 150 μl of DMSO is added to each well, shaking is performed for 10min, and absorbance values of each well are measured under a 490nm microplate reader.

Cell proliferation rate= (average absorbance value of each concentration of the screened substance/average absorbance value of the control group) ×100%.

The results of the effect of the compounds of the examples on cell viability are shown in FIGS. 1 and 2. From the graph, it can be seen that the compounds of the examples of the present invention have weak cytotoxicity to B16F10 in addition to the compound I-10, and further in vitro activity evaluation can be performed.

(2) Analysis and detection method for influence of compound on tyrosinase activity

The influence of the compound of the example on tyrosinase activity was examined, and in vitro enzyme activity detection was performed. Tyrosinase was formulated as 250U/mL potassium phosphate buffer (ph=6.5, 50 mM), 0.06mL potassium phosphate buffer (ph=6.5, 50 mM), 0.04mL tyrosinase solution (250U/mL) and 2 μl of drug-containing DMSO solution were added to 96-well plates, after incubation for 5 min at 30 ℃, 0.1mL L-tyrosine (2 mM) was added, and incubation for another 30min at 30 ℃. Absorbance values for each well were measured under a 490nm microplate reader and activation Rates (RA) were calculated according to the formula:

RA=[(C-D)-(A-B)]/(A-B)×100%。

A is the absorbance at 490nm of a tyrosinase-only system;

b is the absorbance at 490nm of the system without compound and tyrosinase;

c is the absorbance at 490nm of a system containing the compound and tyrosinase;

d is the absorbance at 490nm of the compound-only system.

The measured activation rate of tyrosinase by the compound of the example is shown in fig. 3 and 4. From the graph, it can be seen that the compounds I-2, I-4, I-11, I-13, I-14, I-15, I-17, I-21, I-24 and I-30 in the examples have certain activation effect on tyrosinase, and further in vitro activity evaluation is performed.

(3) Analysis and detection method for influence of compound on melanin content of cells

The melanogenesis promoting effect of the compound of example on B16F10 cells was examined, and intracellular melanin content was measured. Mouse B16F10 melanoma cells were incubated on a 60mm diameter petri dish at 37℃and 5% CO ₂ for 24h, the supernatant was removed, the control group was supplemented with DMEM medium alone, and the experimental group was supplemented with medium containing a drug concentration, respectively. The positive control group was 8-MOP at 50. Mu.M, each group was repeated 2 times, two concentrations (0.25, 1. Mu.M) were selected, and the cells were cultured for 48 hours, then treated with 0.25% pancreatin, washed twice with PBS, 1ml of NaOH at 1M was added to the precipitate, incubated at 80℃for 1 hour, 4ml of distilled water was added, naOH was diluted to 0.2M, and absorbance at 400nm was measured under a spectrophotometer.

Melanin content= [ (average value of absorbance/cell number of screened group)/(average value of absorbance/cell number of control group) ]100%.

The effect of the compounds of the examples on the melanin content of B16F10 cells is shown in FIGS. 5 and 6. As can be seen, the compound I-1、I-2、I-3、I-4、I-5、I-6、I-7、I-8、I-9、I-11、I-12、I-13、I-14、I-15、I-16、I-17、I-20、I-21、I-23、I-24、I-25、I-26、I-27、I-29、I-30 of the present invention has a very good melanogenesis promoting effect on B16F10 cells. The melanogenesis promoting effect of example compound I-21 was strongest and was evaluated for further in vitro activity.

(4) Method for detecting expression level of tyrosinase and MITF protein by using compound

The influence of the compound I-21 of the example on the contents of tyrosinase and MITF protein was examined and analyzed by Western blot.

(A) Cell culture the suspension of B16F10 cells (DMEM medium) was inoculated into six-well plates at a density of 2X 10 ⁵/well, the solution was changed after 24 hours, and the cells were harvested for protein extraction after 2 days of action with the addition of the drug (compound I-21 at a concentration of 0.25uM or 1uM, positive control 8-MOP at a concentration of 50 uM).

(B) Protein extraction, namely collecting cells, washing adherent cells in a culture dish once by PBS, scraping all the cells by the cells, collecting the cells collected in each hole into an EP tube of 1.5ml respectively, washing 3 times by 1ml of precooled PBS, centrifuging at 2000rpm for 5min, carefully discarding the supernatant, leaving cell sediment for later use, adding 200ul of RIPA lysate (RIPA: PMSF=100:1) into each tube, carrying out ice lysis for 30min, carrying out vortex shaking for 3 times, centrifuging for 30min under the condition of 4 ℃ and 15000rpm, and carefully sucking the supernatant, namely the total protein containing the whole cells, and measuring the protein by a BCA method.

(C) Protein denaturation by taking a 10ug protein-containing solution in a 0.5mL EP tube, adding 5 XSOD loading buffer to a final concentration of 1 XSOD, double steaming to a constant volume of 1 μg/μl, and heating in a 100deg.C water bath for 10min to denature the protein.

(D) And (3) loading the protein marker, the sample and the protein marker from left to right, loading 20ul of sample per hole, and simultaneously adding the electrophoresis buffer solution.

(E) Electrophoresis, namely using EPS-300 electrophoresis apparatus (Shanghai Tian energy technology Co., ltd.) to concentrate gel voltage 80V, transferring to 120V after 30min, and running to the bottom of the separation gel.

(F) Transferring film, namely taking out the electrophoresis gel, transferring film according to the sequence of sponge, three layers of filter paper, gel, PVC film, three layers of filter paper and sponge, wherein no bubbles exist between the layers during the process, and transferring film conditions are 200mA,90min and ice water bath.

(G) Incubation the membranes were washed 3 times with TBST on a shaker for 10min each time at room temperature, transferred to a blocking solution (purchased from Biotechnology Co., ltd.) and shaking bed for 2h at room temperature, incubation with primary antibody (abcam, 170905,303530), overnight at 4℃and 3 times with TBST on a shaker for 10min each time, incubation with secondary antibody (abcam, 6721), 2h at room temperature and 3 times with TBST for 10min each time, and chemiluminescent reactions were performed.

(H) Color development and detection, developing with reference to ECL kit, fixing, and analyzing the strips with a gel image processing system.

The effect of compound I-21 on tyrosinase, MITF protein expression is shown in FIG. 7. Wherein, the left graph shows the expression level of tyrosinase and MITF protein detected by Western blot, and the right graph shows the result of standardized treatment of data.

As can be seen from the figure, the positive control 8-methotrexate (8-MOP) has promoting effect on tyrosinase and MITF protein expression compared with the blank control group (Ctrl). The compound I-21 with different concentrations has the function of promoting the expression of tyrosinase proteins in a concentration-dependent manner, meanwhile, the expression quantity of MITF proteins is increased along with the increase of the concentration of the compound I-21, and the promoting function of the compound I-21 on the expression of two proteins is obviously superior to that of a positive control. From the results, it was found that the compound I-21 of the present invention had an effect of promoting protein expression, and was subjected to further in vivo activity evaluation.

(5) Determination of anti-leukoplakia effect of Compounds

Sample preparation example Compound I-21 and Tacrolimus (Tacrolimus), a positive drug

The experimental method is that after C57BL/6 mice are purchased, the mice are fed into SPF-class animal laboratories, and the animals are adapted to the environment for 7 days. On day 4, the electric shaver removed the back hair in a 2cm x 2cm area and the small hair was removed with depilatory cream, modeling was started after 24 hours with a modeling period of 20 days. The C57BL/6 mice were randomly divided into (I) blank group, (II) hydroquinone model group, (III) tacrolimus (0.1%) ointment group, (IV) compound I-21 (0.1%) ointment low dose group and (V) compound I-21 (0.3%) ointment high dose group after numbering in sequence. The (I) group was applied with 0.5 ml/day of sterile distilled water, the other groups were applied with 2.5% hydroquinone ointment at a dose of 100 mg/kg.d on the test area, the (II) hydroquinone model group, (III) tacrolimus (0.1%) ointment group, (IV) compound I-21 (0.1%) ointment low dose group, and the (V) compound I-21 (0.3%) ointment high dose group were applied for 21 days, and the mice were applied with a thin layer of cream on the skin, gently rubbed and completely covered. The skin condition was photographed once a day and the changes in skin color of the mice were closely observed during the experiment. The experimental result shows that the blank control group does not have any pigment loss, while the mice of the hydroquinone model group have serious pigment loss at the back. In terms of the degree of leucoderma mice pigment loss, the high-dose (0.3%) treatment effect of the compound I-21 is better than that of a positive drug tacrolimus (0.1%) treatment group, and the mice pigment loss is improved to a certain extent.

FIG. 8 shows the therapeutic effect of the compound I-21 of the present invention on mice with vitiligo model. Experimental results show that the compound I-21 has remarkable effect of promoting melanin generation and has the potential of treating vitiligo.

As described above, although the present invention has been shown and described with reference to certain preferred embodiments, it is not to be construed as limiting the invention itself. Various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims.

Claims

1. A benzenesulfonamide compound, characterized in that it is selected from the group consisting of:

2. a pharmaceutical composition comprising a compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable salt comprises an acid addition salt of the compound of claim 1 with hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, and an acid salt of the compound of claim 1 with an inorganic base.

4. The pharmaceutical composition of claim 2, wherein the pharmaceutically acceptable salt comprises a basic metal cation salt, an alkaline earth metal cation salt, and an ammonium cation salt.

5. Use of a compound according to claim 1 for the manufacture of a medicament for the treatment of depigmentation or a disorder associated with depigmentation of the skin.

6. The use according to claim 5, wherein the medicament is a melanogenesis-promoting medicament comprising a benzenesulfonamide-type compound.

7. The use according to claim 5, wherein the skin depigmentation disorder comprises vitiligo, sarcoidosis, aegillosis, mottle disease, eyelid albinism syndrome, phenylketonuria, idiopathic punctate hypopigmentation, comfetti spots or Vagabond's leukosis.