CN111763218B

CN111763218B - Thienopyrimidinone thioglycolic acid derivative and preparation method and application thereof

Info

Publication number: CN111763218B
Application number: CN202010672542.9A
Authority: CN
Inventors: 刘新泳; 艾炜; 展鹏; 庞建新; 吴婷; 孙卓森; 赵彤; 张健; 董悦; 梁瑞鹏
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2020-07-14
Filing date: 2020-07-14
Publication date: 2021-05-28
Anticipated expiration: 2040-07-14
Also published as: CN111763218A

Abstract

The invention relates to a thienopyrimidone thioglycolic acid derivative and a preparation method and application thereof. The compound has a structure shown in formula I or II. The invention also relates to a preparation method and a pharmaceutical composition of the compound containing the structure of the formula I or II. The invention also provides application of the compound in preparing anti-gout drugs.

Description

Thienopyrimidinone thioglycolic acid derivative and preparation method and application thereof

Technical Field

The invention belongs to the technical field of organic compound synthesis and medicine application, and particularly relates to a thienopyrimidone thioglycolic acid derivative and a preparation method and application thereof.

Background

Gout is a metabolic disease caused by increased uric acid production or decreased excretion due to purine metabolic disorder, and formed mono-natrium urate crystals are deposited in joint tissues, and hyperuricemia is the main clinical manifestation of the gout. The final product of purine metabolism in vivo is uric acid, and hyperuricemia is a metabolic disease caused by purine metabolic abnormality in vivo. In recent decades, the number of patients with gout and hyperuricemia is increasing, which seriously affects the life quality of human beings and brings heavy burden to society. China is a high-incidence country of gout, the number of gout patients is about 1% -3%, and the number of hyperuricemia patients reaches 5.0% -23.4%. At present, the drugs clinically used for treating gout and hyperuricemia mainly comprise anti-inflammatory drugs, uric acid generation inhibitors mainly comprising xanthine oxidase inhibitors and uric acid excretion promoting drugs, but the clinical use is greatly limited due to poor effect or serious toxic and side effects of the drugs after healing. Urate transporter 1(URAT1) is a novel target of the current drugs for promoting uric acid excretion, and the increase of URATI activity or gene expression caused by gene mutation is one of the important pathogenesis of hyperuricemia. Lesinurad is a recently marketed oral drug for increasing uric acid excretion for treating gout, and can inhibit the kidney proximal tubule uric acid transporter URAT 1. However, the compound has serious hepatotoxicity, so that modification of the chemical structure has great significance for finding novel anti-gout drugs which are efficient, low-toxicity and have independent intellectual property rights.

Disclosure of Invention

Aiming at the defects of the prior art, the invention provides a thienopyrimidone thioglycollic acid derivative and a preparation method thereof, and also provides an activity screening result of the compound as an anti-gout drug and application thereof.

The technical scheme of the invention is as follows:

mono-and thienopyrimidone thioglycolic acid derivatives

The thienopyrimidone thioglycolic acid derivative has a structure shown in the following general formula I or II:

wherein the content of the first and second substances,

r is-H, -CH₃Or

R₁is-CH₂-,-^*CH(CH₃) -or-C (CH)₃)₂-; represents a chiral carbon atom;

R₂is-OH or-OCH₃。

Preferred derivatives of thienopyrimidinone thioglycolic acid according to the invention are one of the following:

TABLE 1 structural formulas of Compounds M1-T8

Preparation method of di-and thienopyrimidone thioglycolic acid derivatives

The preparation method of the thienopyrimidone thioglycollic acid derivative comprises the following steps:

(1) preparation method of target compound (I) of 4-oxo-3, 4-dihydrothieno [3,2-d ] pyrimidine

The preparation method of the target compound of 4-oxo-3, 4-dihydrothieno [3,2-d ] pyrimidine comprises the following steps:

4-bromo-1-naphthylamine is used as an initial raw material to perform Suzuki coupling reaction with cyclopropylboronic acid to generate an intermediate 4-cyclopropyl-1-naphthylamine a, then the intermediate b is reacted with 1,1' -thiocarbonyldiimidazole to obtain an intermediate b, then the intermediate b is reacted with 3-aminothiophene-2-methyl formate containing different substituents in absolute ethyl alcohol or pyridine solution to obtain an intermediate I-1(a-c), and then K is added into DMF solution₂CO₃With substituted bromoacetic acid A under the catalysis ofAnd (3) carrying out nucleophilic substitution reaction on the ester to obtain a target product I-2(a-I), and finally hydrolyzing the target product I-2(a-I) in a mixed solution of tetrahydrofuran and methanol by using lithium hydroxide or sodium hydroxide.

The synthesis route one is as follows:

reagent and conditions (i) K₃PO₄，Pd(PPh₃)₄Cyclopropyl boronic acid, toluene: water 25:2v/v, N₂Protection, 100 ℃, 12 h; (ii) CH (CH)₂

Cl

₂1,1' -thiocarbonyl diimidazole at room temperature for 12 h; (iii) 3-aminothiophene-2-methyl formate or 3-amino-5-methylthiophene-2-methyl formate, absolute ethanol, 90 ℃, 4h, 3-amino-4-methylthiophene-2-methyl formate, pyridine, 50 ℃, 12h, NaOH, 90 ℃, 12 h; (iv) k₂CO₃DMF, methyl 2-bromoisobutyrate or methyl 2-bromopropionate or methyl bromoacetate, 100 ℃ for 3 h; (v) methanol, tetrahydrofuran, lithium hydroxide/sodium hydroxide, normal temperature, 12h.

(2) Preparation method of target compound (II) of 4-oxo-3, 4-dihydrothieno [2,3-d ] pyrimidine

The preparation method of the target compound of 4-oxo-3, 4-dihydrothieno [2,3-d ] pyrimidine comprises the following steps:

4-bromo-1-naphthylamine is used as an initial raw material to perform Suzuki coupling reaction with cyclopropylboronic acid to generate an intermediate 4-cyclopropyl-1-naphthylamine a, then the intermediate b is reacted with 1,1' -thiocarbonyldiimidazole to obtain an intermediate b, the intermediate b is reacted with methyl 2-aminothiophene-3-carboxylate containing different substituents in pyridine or absolute ethyl alcohol to generate an intermediate II-1(a-e), and then the intermediate II-1(a-e) is reacted in a DMF solution in K₂CO₃Carrying out nucleophilic substitution reaction with various esters under catalysis to obtain a corresponding target product II-2(a-o), and finally hydrolyzing with lithium hydroxide or sodium hydroxide in a mixed solution of tetrahydrofuran and methanol to obtain a target product II-3 (a-o).

The second synthetic route is as follows:

Cl

₂1,1' -thiocarbonyl diimidazole at room temperature for 12 h; (iii) various substituted 2-aminothiophene-3-methyl formate, absolute ethyl alcohol, 90 ℃,4 hours, pyridine, 50 ℃, 12 hours, NaOH, 90 ℃, 12 hours; (iv) k₂CO₃DMF, methyl 2-bromoisobutyrate or methyl 2-bromopropionate or methyl bromoacetate, 100 ℃ for 3 h; (v) methanol, tetrahydrofuran, lithium hydroxide or sodium hydroxide, normal temperature, 12h.

The room temperature of the invention is 20-30 ℃.

Application of tri-thienopyrimidone thioglycolic acid derivatives

The invention discloses screening results of blood uric acid reducing activity of thienopyrimidone thioglycolic acid derivatives and application of the thienopyrimidone thioglycolic acid derivatives as anti-gout drugs for the first time. Experiments prove that the thienopyrimidone thioglycollic acid derivative can be applied as a medicine for reducing blood uric acid. In particular to a compound for reducing blood uric acid and preparing an anti-gout drug. The invention also provides application of the compound in preparing anti-gout drugs.

Anti-gout Activity of Compounds of interest

48 compounds (the structural formula of the compounds is shown in table 1) synthesized according to the method are screened for in vitro target inhibition activity of carboxylic acid compounds, the target inhibition activity data of the compounds are listed in table 2, and Lesinurad is used as a positive control. As shown in Table 2, the compounds I-3a, I-3b, I-3e, II-3a, II-3b and II-3h all showed better URAT1 inhibitory activity and were all stronger than the positive control drugs.

Therefore, the thienopyrimidone thioglycolic acid derivative is a series of URAT1 inhibitors with novel structures, and can be used as a lead compound for resisting gout.

The thienopyrimidone thioglycolic acid derivative can be used as a URAT1 inhibitor. In particular to a URAT1 inhibitor used for preparing anti-gout drugs.

An anti-gout pharmaceutical composition comprises the thienopyrimidone thioglycolic acid derivative and one or more pharmaceutically acceptable carriers or excipients.

Drawings

Figure 1 is a plot of mean plasma concentration versus time for oral and injection of compound II-3 a.

Detailed Description

The present invention will be understood by reference to the following examples, in which all the numbers of the objective compounds are the same as those in Table 1, but the contents of the present invention are not limited thereto.

The synthetic route is as follows:

EXAMPLE 1 preparation of intermediate 1-cyclopropyl-4-isothiocyanatonaphthalene (b)

Preparation of intermediate 4-cyclopropyl-1-naphthylamine (a):

the starting material 4-bromo-1-naphthylamine (3.0g, 13.50mmol) was mixed with cyclopropylboronic acid (1.5g, 17.55mmol) and placed in a 250mL two-necked flask, and K was added to the flask₃PO₄(10.2g, 47.10mmol) and tetrakistriphenylphosphine palladium (1.5g, 1.35mmol), mixing, adding about 60mL of toluene and 5mL of water as solvent, sealing, and performing N₂Protection, heating and refluxing at 100 ℃ for reaction for 12 hours; monitoring by TLC, after the reaction is finished, cooling the reaction liquid to room temperature, filtering, washing with ethyl acetate (20mL × 3), collecting filtrate, drying the filtrate to obtain a dark brown residue, adding about 30mL of water into the residue, extracting with ethyl acetate (40mL × 3), combining organic phases, adding a proper amount of anhydrous magnesium sulfate for drying, filtering and drying after 2h to obtain a crude product a, and purifying by column chromatography (petroleum ether: ethyl acetate ═ 6:1) to obtain a corresponding intermediate, wherein the product is a brownish red oily substance, and the yield is as follows: 60 percent.

¹H NMR(400MHz,DMSO-d₆)δ8.34–8.21(m,1H),8.02(d,J＝8.3Hz,1H),7.48(ddd,J＝8.2,6.6,1.3Hz,1H),7.42(ddd,J＝8.3,6.7,1.4Hz,1H),7.00(d,J＝7.7Hz,1H),6.57(d,J＝7.7Hz,1H),5.58(s,2H),2.16(ddd,J＝13.5,8.4,5.3Hz,1H),1.00–0.85(m,2H),0.63–0.49(m,2H).

Preparation of intermediate 1-cyclopropyl-4-isothiocyanatonaphthalene (b)

Intermediate a (0.62g, 3.36mmol) was added to a 250mL round bottom flask, and then about 40mL of dichloromethane was added to dissolve, followed by the addition of 1,1' -thiocarbonyldiimidazole (1.0g, 5.6mmol) and stirring at room temperature for 12h, monitored by TLC, after the reaction was completed the solvent was evaporated to dryness, and eluted (pure petroleum ether) and purified to give intermediate b as a colorless oil in 80.0% yield.¹H NMR(400MHz,DMSO-d₆)δ8.54–8.42(m,1H),8.02(dd,J＝7.9,2.0Hz,1H),7.74(td,J＝7.4,6.7,3.5Hz,2H),7.55(d,J＝7.7Hz,1H),7.24(d,J＝7.7Hz,1H),2.42(td,J＝8.4,4.3Hz,1H),1.13–1.02(m,2H),0.73(dd,J＝5.5,1.8Hz,2H).

EXAMPLE 2 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercaptothieno [3,2-d ] pyrimidin-4 (3H) -one (I-1a)

Dissolving the intermediate b (0.507g, 2.25mmol) and methyl 3-aminothiophene-2-carboxylate (0.307g, 1.95mmol) in 20mL of absolute ethanol, and heating and refluxing at 90 ℃ for 4 hours; monitoring by TLC, cooling to room temperature after complete reaction, precipitating crystals, filtering, taking filter cakes, adding a small amount of ethanol for recrystallization, filtering, taking out solids, adding 30mL of 2mol/L KOH solution, heating and refluxing at 90 ℃ until compounds are dissolved, stopping heating, cooling to room temperature, precipitating a small amount of floccules, adjusting the pH to about 3 by using 3mol/L HCl, precipitating a large amount of white solids, slowly filtering, washing by using clear water (10mL multiplied by 2), and drying to obtain an intermediate I-1a which is a white solid with the yield of 18.3%; melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.63(s,1H),8.48(d,J＝8.4Hz,1H),8.26(d,J＝5.3Hz,1H),7.72–7.57(m,2H),7.54–7.46(m,1H),7.44–7.30(m,2H),7.15(d,J＝5.3Hz,1H),2.47(dd,J＝8.3,5.5Hz,1H),1.12(d,J＝8.4Hz,2H),0.90–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ176.91,157.34,146.57,140.18,138.29,134.64,134.07,129.88,127.22,127.03,126.53,125.17,123.51,123.29,117.98,115.74,13.34,7.51,7.23.ESI-MS:m/z 349.3[M-H]^-,C₁₉H₁₄N₂OS₂[350.05]

EXAMPLE 3 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-7-methylthio-thieno [3,2-d ] pyrimidin-4 (3H) -one (I-1b)

Dissolving the intermediate b (0.224g, 0.99mmol) and a reactant methyl 3-amino-5-methylthiophene-2-carboxylate (0.135g, 0.79mmol) in about 5mL of pyridine solution, heating at 45 ℃ for reaction for 12h, monitoring by TLC, cooling to room temperature after the reaction is completed, adding about 40mL of ice water, extracting with dichloromethane (40mL multiplied by 3), combining organic phases, drying with anhydrous magnesium sulfate, filtering, taking a filtrate, evaporating to dryness, heating 30mL of 1% NaOH solution at 90 ℃ for reflux for 12h, cooling to room temperature, filtering, taking a filtrate, adjusting the pH to about 3 with 1mol/L hydrochloric acid, precipitating a large amount of white solid, filtering, washing a filter cake with clear water to obtain an intermediate I-1b, wherein the yield of the white solid is 53.0%; melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.40(s,1H),8.48(d,J＝8.5Hz,1H),7.90(s,1H),7.64–7.54(m,2H),7.52–7.46(m,1H),7.36(q,J＝7.6Hz,2H),2.47(dd,J＝8.3,5.5Hz,1H),2.39(s,3H),1.18–1.09(m,2H),0.83(q,J＝8.0,5.4Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ177.48,157.36,145.70,140.15,134.74,134.12,133.23,129.87,127.66,127.23,127.01,126.51,125.19,123.53,123.25,116.12,13.78,13.36,7.52,7.26.ESI-MS:m/z 363.3[M-H]^-,C₂₀H₁₆N₂OS₂[364.07].

EXAMPLE 4 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-6-methylthio [3,2-d ] pyrimidin-4 (3H) -one (I-1c)

The intermediate was prepared as in example 3 as a white solid in 50.0% yield; melting point: 209-211 ℃;¹H NMR(400MHz,DMSO-d₆)δ13.53(s,1H),8.47(d,J＝8.4Hz,1H),7.65–7.53(m,2H),7.51–7.45(m,1H),7.34(q,J＝7.6Hz,2H),6.91(s,1H),2.61(s,3H),2.48–2.42(m,1H),1.11(d,J＝8.6Hz,2H),0.81(tt,J＝12.1,7.6Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ176.88,156.81,152.88,146.72,140.14,134.69,134.09,129.91,127.24,127.00,126.49,125.17,123.47,123.25,116.53,113.54,16.66,13.33,7.50,7.24.ESI-MS:m/z 363.3[M-H]^-,C₂₀H₁₆N₂OS₂[364.07].

EXAMPLE 5 preparation of Compound I-2a

Intermediate I-1a (0.17mmol) was reacted with K₂CO₃(0.61mmol) is mixed in a 25mL round-bottom flask, then dissolved in about 8mL DMF, heated at 90 ℃ for reaction for 5min, then methyl bromoacetate (0.30mmol) is added dropwise, the mixture is continuously heated to 100 ℃, reflux reaction is carried out for 3h, TLC monitoring is carried out, after the reaction is completed, the system is cooled to room temperature, then ice water is slowly added into the reaction solution, the solution is clear and then turbid, dropwise added until no new solid is generated, filtration is carried out, a filter cake is washed by clear water, and the filter cake is dried to obtain a target product I-2 a; white solid, yield: 85.2%, melting point: 157 ℃ and 159 ℃.

Compound I-2a spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.4Hz,1H),8.28(d,J＝5.2Hz,1H),7.69(t,J＝7.6Hz,1H),7.66–7.52(m,2H),7.43(d,J＝7.9Hz,2H),7.38(d,J＝5.3Hz,1H),4.03–3.85(m,2H),3.64(s,3H),2.56(dq,J＝8.2,4.2,2.7Hz,1H),1.15(dq,J＝8.5,3.2,2.8Hz,2H),0.86(dq,J＝14.0,9.2,7.4Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.09,159.78,157.66,156.64,142.80,136.96,134.13,130.47,129.67,128.48,128.05,127.26,125.48,125.14,123.22,122.60,119.45,52.85,34.61,13.42,7.83,7.54.ESI-MS:m/z 423.3[M+H]⁺,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 6 preparation of Compound I-2b

The procedure is as in example 5, except that the ester used is methyl 2-bromopropionate, white solid, yield: 75.5%, melting point: 165-167 ℃.

Compound I-2b spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.4Hz,1H),8.29(d,J＝5.2Hz,1H),7.69(t,J＝7.5Hz,1H),7.60(dq,J＝14.0,7.0,6.3Hz,2H),7.50–7.31(m,3H),4.44(dq,J＝14.7,7.3Hz,1H),3.64(d,J＝18.9Hz,3H),2.63–2.54(m,1H),1.37(dd,J＝13.4,7.3Hz,3H),1.22–1.08(m,2H),0.87(ddd,J＝29.7,9.9,5.3Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.06,159.13,157.65,156.60,142.82,137.01,134.10,130.40,129.64,128.46,128.05,127.27,125.49,125.07,123.20,122.49,119.54,53.01,44.38,17.25,13.40,7.94,7.49.ESI-MS:m/z437.4[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 7 preparation of Compounds I-2c

The procedure is as in example 5, except that the ester used is methyl 2-bromoisopropanoate, white solid, yield 68.7%, melting point: 198 ℃ and 201 ℃.

Compound I-2c spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),8.27(d,J＝5.3Hz,1H),7.69(t,J＝7.6Hz,1H),7.59(dd,J＝7.6,4.1Hz,2H),7.39(dd,J＝7.9,4.5Hz,2H),7.27(d,J＝5.2Hz,1H),3.66(s,3H),2.56(dd,J＝9.6,4.3Hz,1H),1.52(s,3H),1.46(s,3H),1.22–1.08(m,2H),0.97–0.75(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.86,158.99,157.58,156.48,142.70,137.08,134.10,130.37,129.56,128.36,128.08,127.24,125.49,124.74,123.15,122.42,119.34,53.28,53.07,26.03,25.80,13.39,8.04,7.42.ESI-MS:m/z 451.3[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 8 preparation of Compounds I-2d

The procedure is as in example 5, except that intermediate I-1b, white solid, yield: 81.7%, melting point: 96-98 ℃.

Compound I-2d spectroscopic data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.5Hz,1H),7.93(s,1H),7.69(t,J＝7.6Hz,1H),7.64–7.54(m,2H),7.42(t,J＝7.8Hz,2H),3.90(d,J＝3.7Hz,2H),3.65(s,3H),2.56(dq,J＝8.2,4.2,2.8Hz,1H),2.33(s,3H),1.23–1.10(m,2H),0.87(h,J＝9.7,8.4Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.29,159.67,157.81,155.45,142.79,134.15,133.49,131.65,130.51,129.66,128.42,128.04,127.25,125.47,123.21,122.59,119.17,52.78,34.86,13.42,12.56,7.82,7.55.ESI-MS:m/z 437.4[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 9 preparation of Compounds I-2e

The procedure is as in example 8, except that the ester used is methyl 2-bromopropionate, white solid, yield: 80.0%, melting point: 152 ℃ and 154 ℃.

Compound I-2e spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.5Hz,1H),7.93(s,1H),7.69(t,J＝7.5Hz,1H),7.64–7.52(m,2H),7.47–7.32(m,2H),4.35(dq,J＝14.8,7.3Hz,1H),3.64(d,J＝11.5Hz,3H),2.62–2.53(m,1H),2.34(s,3H),1.39(t,J＝6.8Hz,3H),1.21–1.09(m,2H),0.96–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.34,158.99,157.77,155.48,142.77,134.12,133.56,131.63,130.47,129.63,128.41,128.07,127.25,125.49,123.18,122.47,119.25,52.95,44.41,16.47,13.40,12.62,7.94,7.50.ESI-MS:m/z 451.4[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 10 preparation of Compounds I-2f

The procedure is as in example 8, except that the ester used is methyl 2-bromoisopropanoate, white solid, yield: 83.0%, melting point: 130 ℃ and 132 ℃.

Compound I-2f spectral data:¹H NMR(400MHz,Chloroform-d)δ8.52(d,J＝8.5Hz,1H),7.60(t,J＝7.5Hz,1H),7.54–7.47(m,1H),7.42(d,J＝7.8Hz,3H),7.37(d,J＝7.6Hz,1H),3.71(s,3H),2.45(td,J＝8.3,4.0Hz,1H),2.39(s,3H),1.59(s,3H),1.56(s,3H),1.14(dd,J＝8.4,4.3Hz,2H),0.88(dq,J＝22.5,5.6Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.91,158.74,157.74,155.55,142.68,134.11,133.75,131.53,130.43,129.57,128.35,128.06,127.23,125.49,123.14,122.41,119.12,53.13,53.06,26.00,25.73,13.38,12.74,8.04,7.41.ESI-MS:m/z 465.3[M+H]⁺,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 11 preparation of Compound I-2g

The procedure is as in example 5, except that the intermediate used is I-1c, white solid, yield: 86.4%, melting point: 179 ℃ and 181 ℃.

Compound I-2g spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),7.69(q,J＝7.5Hz,1H),7.63–7.54(m,2H),7.40(t,J＝7.4Hz,2H),7.12(s,1H),3.98–3.84(m,2H),3.63(s,3H),2.63(s,3H),2.59–2.52(m,1H),1.15(d,J＝8.3Hz,2H),0.90–0.81(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.07,159.74,157.08,156.89,151.24,142.75,134.14,130.54,129.70,128.44,128.02,127.23,125.46,123.56,123.18,122.57,117.74,52.81,34.59,16.76,13.41,7.80,7.55.ESI-MS:m/z 437.3[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 12 preparation of Compound I-2h

The procedure is as in example 11, except that the ester used is methyl 2-bromopropionate, white solid, yield: 62.7%, melting point: 178 ℃ and 180 ℃.

Compound I-2h spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),7.68(t,J＝7.4Hz,1H),7.63–7.53(m,2H),7.38(dd,J＝18.1,7.5Hz,2H),7.12(s,1H),4.38(dt,J＝14.6,7.5Hz,1H),3.63(d,J＝18.4Hz,3H),2.63(s,3H),2.59–2.53(m,1H),1.43–1.27(m,3H),1.15(d,J＝8.3Hz,2H),0.86(d,J＝20.4Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.14,159.03,157.07,156.85,151.30,142.77,138.45,134.11,129.67,128.43,128.07,127.24,125.48,123.49,123.17,122.53,117.83,52.98,44.37,17.18,16.75,13.39,7.92,7.50.ESI-MS:m/z 451.4[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 13 preparation of Compound I-2I

The procedure is as in example 11, except that the ester used is methyl 2-bromoisopropanoate, white solid, yield: 66.5%, melting point: 176 ℃ and 178 ℃.

Compound I-2I spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.53(d,J＝8.4Hz,1H),7.68(t,J＝7.5Hz,1H),7.57(dd,J＝14.0,7.4Hz,2H),7.43–7.32(m,2H),7.04(s,1H),3.66(s,3H),2.62(s,3H),2.58–2.52(m,1H),1.50(s,3H),1.44(s,3H),1.15(d,J＝7.4Hz,2H),0.97–0.75(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.84,158.96,157.02,156.74,151.42,142.65,134.10,130.44,129.59,128.33,128.05,127.22,125.48,123.21,123.12,122.39,117.63 53.24,53.05,26.03,25.79,16.71,13.37,8.02,7.43.ESI-MS:m/z 465.3[M+H]⁺,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 14 preparation of Compound I-3a

Dissolving a compound I-2a (40mg) in a mixed solution of 5mL of methanol and 2.5mL of THF, stirring to dissolve, adding a newly prepared LiOH solution (210mg of LiOH is dissolved in 5mL of water to prepare a 2M lithium hydroxide solution) by about 1-2mL, stirring at normal temperature to react for 12h, monitoring by TLC, adding 5mL of clear water after the reaction is completed, evaporating methanol and THF in a system, adjusting the pH of the solution to about 3 by using 1mol/L of HCl, precipitating a solid, filtering, washing by using the clear water, and drying to obtain a target compound, namely a white solid, wherein the yield is as follows: 88.3%, melting point: 134 ℃ and 136 ℃.

Compound I-3a spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.86(s,1H),8.55(d,J＝8.4Hz,1H),8.27(d,J＝5.3Hz,1H),7.68(t,J＝7.5Hz,1H),7.62(d,J＝7.6Hz,1H),7.59–7.54(m,1H),7.42(dt,J＝10.0,4.5Hz,3H),3.88(s,2H),2.56(dq,J＝8.2,4.2,2.8Hz,1H),1.15(q,J＝10.0Hz,2H),0.86(tt,J＝9.8,5.5Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.69,160.07,157.71,156.74,142.72,136.88,134.13,130.57,129.72,128.47,128.01,127.22,125.47,125.16,123.24,122.64,119.38,35.22,13.43,7.81,7.53.ESI-MS:m/z 407.4[M-H]^-,C₂₁H₁₆N₂O₃S₂[408.06].

EXAMPLE 15 preparation of Compound I-3b

The same procedure as in example 14 was followed, except that the hydrolyzed compound gave I-2b as a white solid in yield: 91.8%, melting point: 152 ℃ and 155 ℃.

Compound I-3b spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.92(s,1H),8.55(d,J＝8.5Hz,1H),8.28(d,J＝5.2Hz,1H),7.68(t,J＝7.5Hz,1H),7.58(dt,J＝13.4,7.3Hz,2H),7.49–7.31(m,3H),4.41(p,J＝7.0Hz,1H),2.64–2.53(m,1H),1.40(dd,J＝24.6,7.2Hz,3H),1.15(d,J＝8.4Hz,2H),0.98–0.74(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.83,159.33,157.72,156.72,142.71,136.87,134.10,130.50,129.69,128.46,128.05,127.22,125.48,125.13,123.23,122.57,119.50,45.01,17.87,13.41,7.89,7.48.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 16 preparation of Compounds I-3c

The procedure is as in example 14, except that the compound to be hydrolyzed is I-2 c. White solid, yield: 86.6%, melting point: 158-.

Compound I-3c spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.65(s,1H),8.54(d,J＝8.5Hz,1H),8.26(d,J＝5.3Hz,1H),7.68(t,J＝7.6Hz,1H),7.57(dd,J＝7.5,4.7Hz,2H),7.39(t,J＝7.7Hz,2H),7.28(d,J＝5.2Hz,1H),2.55(dq,J＝8.3,4.2,2.8Hz,1H),1.52(s,3H),1.48(s,3H),1.13(dd,J＝15.0,8.6Hz,2H),0.98–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.73,159.38,157.65,156.58,142.57,136.88,134.10,130.51,129.63,128.35,127.98,127.18,125.48,124.81,123.17,122.44,119.28,53.53,26.14,25.89,13.38,8.02,7.41.ESI-MS:m/z 435.3[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 17 preparation of Compounds I-3d

The procedure is as in example 14, except that the compound to be hydrolyzed is I-2d, white solid, yield: 91.4%, melting point: 139 ℃ and 141 ℃.

Compound I-3d spectroscopic data:¹H NMR(400MHz,DMSO-d₆)δ12.75(s,1H),8.48(d,J＝8.5Hz,1H),7.86(s,1H),7.61(t,J＝7.6Hz,1H),7.56–7.45(m,2H),7.35(dd,J＝7.7,5.6Hz,2H),3.77(s,2H),2.53–2.46(m,1H),2.29(s,3H),1.13–1.03(m,2H),0.79(tt,J＝9.8,6.0Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.87,159.87,157.89,155.55,142.69,134.13,133.58,131.57,130.60,129.69,128.39,128.00,127.22,125.47,123.23,122.63,119.04,35.39,13.42,12.73,7.81,7.54.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 18 preparation of Compounds I-3e

The procedure is as in example 14, except that the compound to be hydrolyzed is I-3e, white solid, yield: 95.7%, melting point: 210 ℃ and 213 ℃.

Compound I-3e spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.79(s,1H),8.48(d,J＝8.5Hz,1H),7.86(s,1H),7.61(t,J＝7.6Hz,1H),7.51(q,J＝9.0,8.2Hz,2H),7.33(dd,J＝13.1,8.7Hz,2H),4.24(dq,J＝14.7,7.3Hz,1H),2.49(dq,J＝8.2,4.2,2.7Hz,1H),2.30(s,3H),1.33(dd,J＝18.0,7.3Hz,3H),1.07(dd,J＝13.4,8.7Hz,2H),0.88–0.70(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.12,159.25,157.86,155.59,142.68,134.11,133.64,131.53,130.56,129.67,128.39,127.98,127.22,125.48,123.23,122.55,119.13,45.36,17.19,13.41,12.78,7.90,7.49.ESI-MS:m/z435.4[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 19 preparation of Compounds I-3f

The procedure is as in example 14, except that the compound to be hydrolyzed is I-2 f. White solid, yield: 93.4%, melting point: 242 ℃ and 245 ℃.

Compound I-3f spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.54(s,1H),8.54(d,J＝8.4Hz,1H),7.90(s,1H),7.68(t,J＝7.6Hz,1H),7.56(t,J＝8.0Hz,2H),7.38(dd,J＝14.4,8.0Hz,2H),2.56(d,J＝5.1Hz,1H),2.35(s,3H),1.51(d,J＝19.9Hz,6H),1.19–1.11(m,2H),0.95–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.76,159.05,157.83 155.70,142.57,134.10,133.94,131.39,130.53,129.60,128.31,127.99,127.20,125.49,123.18,122.44,118.91,53.50,26.10,25.81,13.38,12.79,8.01,7.41.ESI-MS:m/z 449.4[M-H]^-,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 20 preparation of Compound I-3g

The procedure is as in example 14, except that the compound to be hydrolyzed is I-2g, white solid, yield: 51.7%, melting point: 218 ℃ and 221 ℃.

Compound I-3g spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.84(s,1H),8.55(d,J＝8.4Hz,1H),7.73–7.65(m,1H),7.58(dd,J＝14.2,7.3Hz,2H),7.41(dd,J＝7.9,3.0Hz,2H),7.15(s,1H),3.87(s,2H),2.63(s,3H),2.56(dt,J＝10.8,5.8Hz,1H),1.16(d,J＝8.6Hz,2H),0.95–0.79(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.67,159.99,157.13,156.98,151.15,142.67,134.13,130.62,129.74,128.44,127.98,127.20,125.45,123.59,123.20,122.60,117.69,35.13,16.76,13.41,7.79,7.54.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 21 preparation of Compound I-3h

The procedure is as in example 14, except that the compound to be hydrolyzed is I-2h, white solid, yield: 89.9%, melting point: 230 ℃ and 233 ℃.

Compound I-3h spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.97(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.57(q,J＝6.7Hz,2H),7.39(t,J＝8.4Hz,2H),7.15(s,1H),4.37(p,J＝6.8Hz,1H),2.63(s,3H),2.59–2.53(m,1H),1.38(dd,J＝24.3,7.2Hz,3H),1.14(d,J＝8.4Hz,2H),0.95–0.78(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.92,159.28,157.14,156.97,151.22,142.66,134.10,130.55,129.70,128.44,127.98,127.21,125.49,123.57,123.18,122.53,117.75,44.96,17.79,16.77,13.41,7.87,7.49.ESI-MS:m/z 435.3[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 22 preparation of Compound I-3I

The procedure is as in example 14, except that the hydrolyzed compound is I-2I, a white solid, yield: 95.2%, melting point: 241 ℃ and 244 ℃.

Compound I-3I spectral data:¹H NMR(400MHz,DMSO-d₆)δ¹H NMR(400MHz,DMSO-d₆)δ12.65(s,1H),8.53(d,J＝8.4Hz,1H),7.67(t,J＝7.3Hz,1H),7.55(dd,J＝14.2,7.7Hz,2H),7.36(dd,J＝14.0,8.0Hz,2H),7.04(s,1H),2.62(s,3H),2.58–2.53(m,1H),1.49(d,J＝12.7Hz,6H),1.14(d,J＝8.0Hz,2H),0.94–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.75,159.73,157.11,156.87,151.16,142.47,134.10,130.60,129.65,128.32,127.96,127.16,125.48,123.34,123.17,122.42,117.53,67.77,26.11,23.29,16.71,13.38,7.99,7.41.ESI-MS:m/z 449.4[M-H]^-,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 23 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercaptothieno [2,3-d ] pyrimidin-4 (3H) -one (II-1a)

The procedure is as in example 2, except that methyl 2-aminothiophene-3-carboxylate, white solid, yield: 15.1%, melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.92(s,1H),8.47(d,J＝8.3Hz,1H),7.67–7.54(m,2H),7.52–7.44(m,1H),7.35(q,J＝7.7Hz,3H),7.26(d,J＝5.6Hz,1H),2.46(dd,J＝8.3,5.5Hz,1H),1.11(d,J＝8.4Hz,2H),0.91–0.70(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ175.81,157.46,151.92,140.10,134.75,134.09,129.86,127.12,126.97,126.51,125.15,123.51,123.33,122.89,120.50,118.79,13.34,7.50,7.23.ESI-MS:m/z 349.4[M-H]^-,C₁₉H₁₄N₂OS₂[350.05].

EXAMPLE 24 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-6-methylthio-thieno [2,3-d ] pyrimidin-4 (3H) -one (II-1b)

The procedure is as in example 2, except that methyl 2-amino-5-methylthiophene-3-carboxylate, white solid, yield: 16.5%, melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.89(s,1H),8.47(d,J＝8.5Hz,1H),7.64–7.53(m,2H),7.51–7.45(m,1H),7.34(s,2H),6.98(s,1H),2.48(s,1H),2.46(s,3H),1.12(d,J＝8.6Hz,2H),0.87–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ175.34,157.07,150.47,140.05,134.78,134.09,133.87,129.83,127.12,126.97,126.49,125.15,123.50,123.28,120.13,118.51,15.31,13.34,7.49,7.22.ESI-MS:m/z 363.3[M-H]^-,C₂₀H₁₆N₂OS₂[364.07].

EXAMPLE 25 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-5-methylthio-thieno [2,3-d ] pyrimidin-4 (3H) -one (II-1c)

The procedure is as in example 2, except that 2-amino-4-methylthio-benzene is usedThiophene-3-carboxylic acid methyl ester, white solid, yield: 16.3%, melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.85(s,1H),8.47(d,J＝8.6Hz,1H),7.65–7.57(m,2H),7.52–7.46(m,1H),7.39–7.31(m,2H),6.95(s,1H),2.47(dd,J＝8.4,5.4Hz,1H),2.32(s,3H),1.12(d,J＝8.5Hz,2H),0.80(dq,J＝24.7,5.8Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ175.68,158.06,152.86,139.99,134.78,134.69,134.08,129.96,127.13,126.94,126.46,125.11,123.47,123.39,116.85,114.74,19.04,15.96,13.33,7.19.ESI-MS:m/z 363.3[M-H]^-,C₂₀H₁₆N₂OS₂[364.07].

EXAMPLE 26 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-5, 6-dimethylthieno [2,3-d ] pyrimidin-4 (3H) -one (II-1d)

The procedure is as in example 2, except that methyl 2-amino, 4, 5-dimethylthiophene-3-carboxylate is used as a white solid in yield: 14.5%, melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.77(s,1H),8.46(d,J＝8.5Hz,1H),7.65–7.57(m,1H),7.55(d,J＝8.3Hz,1H),7.52–7.42(m,1H),7.32(s,2H),2.45(dd,J＝8.3,5.5Hz,1H),2.32(s,3H),2.23(s,3H),1.09(dd,J＝18.8,7.7Hz,2H),0.89–0.71(m,2H)，ESI-MS:m/z 377.4[M-H]^-,C₂₁H₁₈N₂OS₂[378.09].

EXAMPLE 27 preparation of intermediate 3- (4-Cyclopropylnaphthalen-1-yl) -2-mercapto-5, 6,7, 8-tetrahydrobenzo [4,5] thieno [2,3-d ] pyrimidin-4 (3H) -one (II-1e)

The procedure is as in example 3, except that 2-amino-4, 5,6, 7-tetrahydrobenzo [ b ] is used]Thiophene-3-carboxylic acid methyl ester, white solid, yield: 35.9%, melting point:>250℃；¹H NMR(400MHz,DMSO-d₆)δ13.84(s,1H),8.47(d,J＝8.5Hz,1H),7.59(dd,J＝18.0,8.3Hz,2H),7.52–7.45(m,1H),7.33(s,2H),2.71(s,4H),2.47(d,J＝7.0Hz,1H),1.88–1.67(m,4H),1.13(t,J＝9.7Hz,2H),0.91–0.72(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ175.09,157.59,139.96,134.79,134.08,131.56,129.93,128.98,127.12,126.94,126.46,125.12,123.47,123.35,116.46,25.27,24.47,22.94,22.03,13.32,7.50,7.18.ESI-MS:m/z 403.5[M-H]^-,C₂₃H₂₀N₂OS₂[404.10].

EXAMPLE 28 preparation of the object Compound II-2a

The procedure is as in example 5, except that this example uses intermediate II-1a, a white solid, in yield: 88.2%, melting point: 142 ℃ and 144 ℃.

Compound II-2a spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.5Hz,1H),7.69(t,J＝7.5Hz,1H),7.65–7.52(m,3H),7.49–7.32(m,3H),4.01–3.84(m,2H),3.64(s,3H),2.62–2.52(m,1H),1.23–1.08(m,2H),0.87(dt,J＝9.6,5.0Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.03,163.61,158.93,157.79,142.76,134.14,130.58,129.56,128.34,128.03,127.26,125.47,123.49,123.23,122.59,122.57,120.98,52.89,34.60,13.41,7.82,7.55.ESI-MS:m/z 423.3[M+H]⁺,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 29 preparation of Compound II-2b

The procedure is as in example 28 except that methyl 2-bromopropionate is used in this example. White solid, yield: 67.0%, melting point: 200 ℃ and 203 ℃.

Compound II-2b spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.55(d,J＝8.4Hz,1H),7.69(t,J＝7.3Hz,1H),7.61(d,J＝8.1Hz,1H),7.57(d,J＝5.7Hz,2H),7.46(d,J＝8.4Hz,1H),7.40(d,J＝5.7Hz,2H),4.40(dq,J＝14.6,7.2Hz,1H),3.64(d,J＝17.0Hz,3H),2.59–2.52(m,1H),1.37(dd,J＝10.8,7.3Hz,3H),1.15(d,J＝8.2Hz,2H),0.96–0.78(m,2H).¹³C NMR(100MHz,Chloroform-d)δ172.58,172.18,158.24,142.74,134.59,127.50,127.44,127.28,126.70,125.41,123.61,123.42,122.73,122.16,121.98,121.48,121.07,52.72,44.26,16.82,13.57,7.03,6.65.ESI-MS:m/z 437.4[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 30 preparation of Compound II-2c

The procedure is as in example 28 except that methyl 2-bromoisopropanoate is selected for this example. White solid, yield: 76.8%, melting point: 204 ℃ and 206 ℃.

Compound II-2c spectral data:¹H NMR(400MHz,Chloroform-d)δ8.52(d,J＝8.4Hz,1H),7.63–7.57(m,1H),7.54–7.49(m,1H),7.46–7.41(m,2H),7.41–7.35(m,2H),7.13(d,J＝5.7Hz,1H),3.77(s,3H),2.44(p,J＝8.4Hz,1H),1.55(d,J＝10.9Hz,6H),1.19–1.10(m,2H),0.95–0.82(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.78,163.32,158.11,157.73,142.66,134.11,130.45,129.46,128.25,128.06,127.24,125.48,123.47,123.16,122.57,122.43,120.99,53.41,53.14,26.06,25.84,13.38,8.03,7.42.ESI-MS:m/z 451.4[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 31 preparation of Compound II-2d

The procedure is as in example 5, except that this example uses intermediate II-1b, a white solid, yield: 68.5%, melting point: 194 ℃ and 196 ℃.

Compound II-2d spectroscopic data:¹H NMR(400MHz,DMSO-d₆)δ8.52(d,J＝8.5Hz,1H),7.66(t,J＝7.6Hz,1H),7.56(t,J＝8.8Hz,2H),7.39(t,J＝7.2Hz,2H),7.08(s,1H),3.97–3.81(m,2H),3.61(s,3H),2.54(dd,J＝8.4,5.4Hz,1H),2.49–2.45(m,3H),1.24–1.03(m,2H),0.83(tt,J＝9.4,5.7Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.04,162.52,158.08,157.32,142.70,136.81,134.14,130.64,129.54,128.29,128.01,127.24,125.47,123.21,122.56,121.18,119.95,52.86,34.56,15.96,13.41,7.80,7.54.ESI-MS:m/z 437.4[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 32 preparation of Compound II-2e

The operation is as in example 31, except that methyl 2-bromopropionate, white solid, yield: 70.1%, melting point: 100-102 ℃.

Compound II-2e spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.52(d,J＝8.5Hz,1H),7.66(t,J＝7.6Hz,1H),7.55(dd,J＝11.7,5.7Hz,2H),7.45–7.30(m,2H),7.08(s,1H),4.42–4.29(m,1H),3.61(d,J＝17.2Hz,3H),2.54(dd,J＝8.5,5.2Hz,1H),2.48(s,3H),1.33(dd,J＝10.0,7.4Hz,3H),1.13(q,J＝5.1Hz,2H),0.93–0.75(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.10,172.00,162.41,157.31,142.69,136.91,134.11,130.51,129.52,128.28,128.01,127.25,125.48,123.20,122.45,121.29,119.96,53.02,44.34,17.13,15.97,13.39,7.93,7.48.ESI-MS:m/z 451.2[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 33 preparation of Compound II-2f

The operation is as in example 31, except that methyl 2-bromoisopropanoate, white solid, yield: 73.22%, melting point: 182 ℃ and 184 ℃.

Compound II-2f spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.53(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.57(dd,J＝15.5,7.7Hz,2H),7.44–7.33(m,2H),7.09(s,1H),3.65(s,3H),2.56(dd,J＝8.3,5.5Hz,1H),2.51(s,3H),1.47(d,J＝26.2Hz,6H),1.15(q,J＝6.6Hz,2H),0.97–0.74(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.77,162.22,157.27,142.61,136.84,134.12,130.53,129.45,128.19,128.03,127.22,125.48,123.13,122.39,121.19,119.93,53.36,53.09,26.07,25.85,15.95,13.37,8.02,7.41.ESI-MS:m/z 465.3[M+H]⁺,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 34 preparation of Compound II-2g

The procedure is as in example 5, except that this example uses intermediate II-1g, white solid, yield: 59.1%, melting point: 108-110 ℃.

Compound II-2g spectral data:¹H NMR(400MHz,Chloroform-d)δ8.51(d,J＝8.4Hz,1H),7.60(t,J＝7.4Hz,1H),7.52(q,J＝8.3Hz,2H),7.46–7.35(m,2H),6.73(s,1H),3.96–3.78(m,2H),3.73(s,3H),2.51(s,3H),2.42(td,J＝8.3,4.3Hz,1H),1.14(dt,J＝8.1,3.8Hz,2H),0.97–0.74(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.01,164.16,158.67,158.41,142.66,134.42,134.14,130.56,129.69,128.37,128.00,127.21,125.43,123.19,122.65,119.26,117.78,52.87,34.51,16.49,13.40,7.80,7.56.ESI-MS:m/z 437.3[M+H]⁺,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 35 preparation of Compound II-2h

The procedure is as in example 34, except that methyl 2-bromopropionate is used in this example. White solid, yield: 75.2%, melting point: 101-103 ℃.

Compound II-2h spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.58(q,J＝8.7,7.5Hz,2H),7.51–7.37(m,2H),7.14(s,1H),4.39(dq,J＝14.6,7.2Hz,1H),3.63(d,J＝16.6Hz,3H),2.55(dq,J＝8.3,4.2,2.9Hz,1H),2.40(s,3H),1.36(dd,J＝10.1,7.4Hz,3H),1.15(q,J＝5.1Hz,2H),0.97–0.74(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.07,164.07,158.41,158.02,142.65,134.44,134.11,130.50,128.35,129.66,128.01,127.22,125.45,123.15,122.54,119.38,117.86,52.99,44.12,17.16,16.49,13.38,7.92,7.51.ESI-MS:m/z 451.4[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 36 preparation of Compound II-2i

The procedure is as in example 34 except that methyl 2-bromoisopropanoate is selected for this example. White solid, yield: 73.0%, melting point: 183 ℃ and 185 ℃.

Compound II-2i spectral data:¹H NMR(400MHz,Chloroform-d)δ8.51(d,J＝8.4Hz,1H),7.60(t,J＝7.5Hz,1H),7.57–7.44(m,2H),7.43–7.34(m,2H),6.71(s,1H),3.77(s,3H),2.49(s,3H),2.43(td,J＝8.4,4.3Hz,1H),1.56(s,6H),1.22–1.05(m,2H),0.97–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.78,163.87,158.37,157.90,142.58,134.46,134.11,130.44,129.58,128.27,128.04,127.20,125.45,123.12,122.48,119.29,117.74,53.34,53.12,26.09,25.87,16.47,13.36,8.02,7.43ESI-MS:m/z 465.2[M+H]⁺,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 37 preparation of Compound II-2j

The procedure is as in example 5, except that ethyl 2-chloropropionate is used as intermediate. White solid, yield: 84.7%, melting point: 99-102 ℃.

Compound II-2j spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.3Hz,1H),7.79–7.63(m,1H),7.63–7.50(m,2H),7.41(d,J＝7.6Hz,2H),4.00–3.77(m,2H),3.62(s,3H),2.55(s,1H),2.38(s,3H),2.32(s,3H),1.15(d,J＝7.4Hz,2H),0.94–0.68(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.05,161.23,158.16,157.61,142.61,134.14,130.68,129.67,129.33,128.99,128.32,127.99,127.20,125.44,123.18,122.61,119.76,52.85,34.46,13.40,13.22,13.07,7.79,7.55.ESI-MS:m/z 451.4[M+H]⁺,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 38 preparation of Compound II-2k

The procedure is as in example 37, except that the ester used in this example is methyl 2-bromopropionate. White solid, yield: 95.8%, melting point: 108-110 ℃.

Compound II-2k spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),7.68(t,J＝7.4Hz,1H),7.57(dt,J＝16.9,7.7Hz,2H),7.46–7.36(m,2H),4.42–4.31(m,1H),3.62(d,J＝17.0Hz,3H),2.56(dd,J＝8.7,5.0Hz,1H),2.39(s,3H),2.31(s,3H),1.44–1.29(m,3H),1.21–1.10(m,2H),0.92–0.78(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.02,161.13,158.15,155.89,142.62,134.11,130.63,129.65,129.36,129.09,128.31,128.00,127.21,125.46,123.17,122.58,119.88,53.00,44.04,17.13,13.38,13.22,13.08,7.90,7.49.ESI-MS:m/z 465.3[M+H]⁺,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 39 preparation of Compound II-2l

The procedure is as in example 37, except that the ester used in this example is methyl 2-bromoisopropanoate. White solid, yield: 61.9%, melting point: 175 ℃ and 178 ℃.

Compound II-2l spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.53(d,J＝8.4Hz,1H),7.68(t,J＝7.6Hz,1H),7.59(t,J＝7.6Hz,1H),7.53(d,J＝7.6Hz,1H),7.38(d,J＝7.8Hz,2H),3.65(s,3H),2.60–2.52(m,1H),2.37(s,3H),2.30(s,3H),1.49(s,3H),1.43(s,3H),1.15(q,J＝6.6Hz,2H),0.95–0.73(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.80,160.93,158.11,156.84,142.52,134.10,130.55,129.55,129.35,129.00,128.22,128.03,127.19,125.46,123.11,122.43,119.79,53.27,53.09,26.10,25.87,13.36,13.21,13.07,8.00,7.42.ESI-MS:m/z 479.3[M+H]⁺,C₂₆H₂₆N₂O₃S₂[478.14].

EXAMPLE 40 preparation of Compound II-2m

The procedure is as in example 5, except that this example uses intermediate II-1 e. White solid, yield: 74.1%, melting point: 114 ℃ and 116 ℃.

Compound II-2m spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),7.68(t,J＝7.6Hz,1H),7.58(dd,J＝11.9,7.8Hz,2H),7.41(d,J＝7.2Hz,2H),3.97–3.82(m,2H),3.62(s,3H),2.82–2.74(m,4H),2.55(d,J＝5.0Hz,1H),1.81(s,2H),1.74(s,2H),1.15(d,J＝8.3Hz,2H),0.83(dd,J＝16.1,6.8Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.03,161.94,157.87,157.77,142.62,134.14,131.93,131.44,130.63,129.66,128.32,127.99,127.21,125.44,123.19,122.63,118.95,52.84,34.49,25.60,24.93,22.93,22.19,13.40,7.80,7.53.ESI-MS:m/z 477.2[M+H]⁺,C₂₆H₂₄N₂O₃S₂[476.12].

EXAMPLE 41 preparation of Compound II-2n

The procedure is as in example 40, except that methyl 2-bromopropionate is used in this example. White solid, yield: 81.4%, melting point: 108-110 ℃.

Compound II-2n spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.53(d,J＝8.4Hz,1H),7.68(t,J＝7.3Hz,1H),7.56(dt,J＝17.0,7.8Hz,2H),7.47–7.35(m,2H),4.37(dq,J＝14.7,7.1Hz,1H),3.62(d,J＝16.6Hz,3H),2.83–2.71(m,4H),2.59–2.53(m,1H),1.80(s,2H),1.74(s,2H),1.35(t,J＝8.0Hz,3H),1.14(d,J＝8.2Hz,2H),0.94–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.08,161.84,157.86,157.11,142.61,134.11,132.03,131.46,130.58,129.64,128.30,128.04,127.22,125.45,123.18,122.53,119.07,53.00,44.26,25.61,24.94,22.93,22.19,17.13,13.39,7.89,7.48.ESI-MS:m/z 491.4[M+H]⁺,C₂₇H₂₆N₂O₃S₂[490.14].

EXAMPLE 42 preparation of Compound II-2o

The procedure is as in example 40, except that methyl 2-bromoisopropanoate is used in this example. White solid, yield: 86.2%, melting point: 188 ℃ and 192 ℃.

Compound II-2o spectral data:¹H NMR(400MHz,DMSO-d₆)δ8.54(d,J＝8.4Hz,1H),7.68(t,J＝7.5Hz,1H),7.59(t,J＝7.3Hz,1H),7.51(d,J＝7.6Hz,1H),7.39(d,J＝7.6Hz,2H),3.65(s,3H),2.83–2.72(m,4H),2.55(d,J＝4.8Hz,1H),1.78(d,J＝25.9Hz,4H),1.47(d,J＝23.9Hz,6H),1.16(d,J＝7.8Hz,2H),0.95–0.75(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ173.76,161.65,157.82,156.99,142.52,134.12,131.92,131.45,130.52,129.57,128.15,127.92,127.10,125.38,123.05,122.42,118.96,53.26,52.94,25.98,25.76,25.57,24.89,22.90,22.16,13.29,7.84,7.27.ESI-MS:m/z 505.4[M+H]⁺,C₂₈H₂₈N₂O₃S₂[504.15].

EXAMPLE 43 preparation of Compound II-3a

The procedure is as in example 14, except that in this example the compound hydrolysed is II-2a, white solid, yield: 25.5%, melting point: 142 ℃ and 144 ℃.

Compound II-3a spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.80(s,1H),8.55(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.57(dd,J＝18.6,6.8Hz,3H),7.49–7.32(m,3H),3.86(s,2H),2.55(dq,J＝8.2,4.3,2.8Hz,1H),1.14(t,J＝8.9Hz,2H),0.96–0.73(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.58,163.72,159.72,157.83,142.67,134.15,130.68,129.63,128.33,127.96,127.21,125.45,123.34,123.24,122.63,122.54,120.94,35.16,13.41,7.80,7.54.ESI-MS:m/z407.4[M-H]^-,C₂₁H₁₆N₂O₃S₂[408.06].

example 44 preparation of Compound II-3b

The procedure is as in example 14, except that the hydrolyzed compound in this example is II-2 b. White solid, yield: 33.6%, melting point: 152 ℃ and 154 ℃.

Compound II-3b spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.91(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.58(q,J＝6.8,5.3Hz,3H),7.42(dt,J＝14.2,7.2Hz,3H),4.35(p,J＝7.2Hz,1H),2.55(dd,J＝10.9,5.7Hz,1H),1.39(dd,J＝22.1,7.3Hz,3H),1.21–1.09(m,2H),0.96–0.78(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.83,163.66,158.57,158.46,157.85,142.67,134.11,130.61,129.59,128.34,128.03,127.22,125.47,123.44,123.22,122.55,121.04,45.00,17.68,13.40,7.88,7.48.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 45 preparation of Compound II-3c

The procedure is as in example 14, except that the compound hydrolyzed is II-2 c. White solid, yield: 88.5%, melting point: 155 ℃ and 157 ℃.

Compound II-3c spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.72(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.5Hz,1H),7.56(q,J＝6.6,5.6Hz,3H),7.48–7.30(m,3H),2.55(dq,J＝8.4,4.2,3.0Hz,1H),1.50(s,3H),1.46(s,3H),1.22–1.09(m,2H),0.97–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.68,163.39,158.39,157.80,142.55,134.11,130.56,129.50,128.25,128.00,127.20,125.49,123.36,123.19,122.50,122.42,120.93,53.44,26.15,25.88,13.38,8.03,7.41.ESI-MS:m/z 435.4[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 46 preparation of Compounds II-3d

The procedure is as in example 14, except that the compound hydrolyzed is II-2 d. White solid, yield: 94.7%, melting point: 139 ℃ and 142 ℃.

Compound II-3d spectroscopic data:¹H NMR(400MHz,DMSO-d₆)δ12.89(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.56(t,J＝9.0Hz,2H),7.41(d,J＝7.7Hz,2H),7.10(s,1H),3.84(s,2H),2.54(s,1H),2.52(s,3H),1.15(d,J＝8.5Hz,2H),0.90–0.80(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.62,162.67,158.43,157.38,142.60,136.64,134.14,130.75,129.60,128.28,127.96,127.20,125.46,123.24,122.61,121.13,119.93,35.28,15.97,13.41,7.79,7.52.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 47 preparation of Compound II-3e

The procedure is as in example 14, except that the compound hydrolyzed is II-2 e. White solid, yield: 91.3%, melting point: 162 ℃ and 165 ℃.

Compound II-3e spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.94(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.56(t,J＝7.8Hz,2H),7.46–7.34(m,2H),7.10(s,1H),4.32(p,J＝7.2Hz,1H),2.56(dd,J＝8.6,5.5Hz,1H),2.52–2.46(m,3H),1.38(dd,J＝23.6,7.3Hz,3H),1.15(p,J＝4.7Hz,2H),0.96–0.77(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.83,162.60,157.67,157.38,142.60,136.72,134.12,130.65,129.58,128.27,127.94,127.19,125.48,123.20,122.54,121.24,119.94,45.08,17.79,15.97,13.40,7.86,7.47.ESI-MS:ESI-MS:m/z 435.4[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 48 preparation of Compound II-3f

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2 f. White solid, yield: 94.1%, melting point: 158-.

Compound II-3f spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.69(s,1H),8.53(d,J＝8.5Hz,1H),7.67(t,J＝7.6Hz,1H),7.56(dd,J＝14.9,7.6Hz,2H),7.37(dd,J＝14.1,8.0Hz,2H),7.08(s,1H),2.56(dd,J＝8.4,5.4Hz,1H),2.50(s,3H),1.46(d,J＝14.7Hz,6H),1.15(q,J＝6.4Hz,2H),0.96–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.65,162.32,157.52,157.33,142.49,136.70,134.11,130.65,129.50,128.23,127.96,127.18,125.48,123.16,122.40,121.17,119.86,53.35,26.17,25.89,15.98,13.37,8.01,7.40.ESI-MS:m/z 449.4[M-H]^-,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 49 preparation of Compound II-3g

The procedure is as in example 14, except that in this example II-2g of the compound to be hydrolyzed are used. White solid, yield: 75.14%, melting point: 132 ℃ and 134 ℃.

Compound II-3g spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.86(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.57(t,J＝6.5Hz,2H),7.43(dd,J＝15.6,8.0Hz,2H),7.13(s,1H),3.84(s,2H),2.55(dq,J＝8.1,4.2,2.6Hz,1H),2.40(s,3H),1.13(dd,J＝17.6,5.9Hz,2H),0.84(dq,J＝11.8,6.7,6.1Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.60,164.30,159.03,158.47,142.57,134.39,134.14,130.68,129.74,128.36,127.96,127.17,125.43,123.21,122.69,119.20,117.66,35.23,16.51,13.41,7.79,7.55.ESI-MS:m/z 421.3[M-H]^-,C₂₂H₁₈N₂O₃S₂[422.08].

EXAMPLE 50 preparation of Compound II-3h

The procedure is as in example 14, except that the compound to be hydrolyzed in this example is II-2 h. White solid, yield: 85.7%, melting point: 221-224 ℃.

Compound II-3h spectral data:¹H NMR(400MHz,DMSO-d₆)δ13.00(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.6Hz,1H),7.62–7.52(m,2H),7.50–7.34(m,2H),7.14(s,1H),4.32(p,J＝7.0Hz,1H),2.55(dq,J＝8.2,4.2,2.9Hz,1H),2.40(s,3H),1.38(dd,J＝23.7,7.2Hz,3H),1.15(d,J＝8.4Hz,2H),0.95–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.85,164.25,158.49,158.35,142.54,134.39,134.11,130.62,129.71,128.34,127.94,127.17,125.44,123.21,122.62,119.29,117.70,45.02,17.75,16.51,13.40,7.86,7.50.ESI-MS:m/z 435.4[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 51 preparation of Compound II-3i

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2 i. White solid, yield: 93.7%, melting point: 238 deg.C and 240 deg.C.

Compound II-3i spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.62(s,1H),8.47(d,J＝8.4Hz,1H),7.61(t,J＝7.6Hz,1H),7.56–7.42(m,2H),7.33(t,J＝7.2Hz,2H),7.05(s,1H),2.55–2.45(m,1H),2.32(s,3H),1.40(d,J＝12.8Hz,6H),1.14–1.01(m,2H),0.88–0.67(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.66,163.93,158.43,158.16,142.46,134.36,134.10,130.55,129.64,128.27,127.96,127.15,125.45,123.14,122.48,119.24,117.65,53.35,26.17,25.91,16.50,13.36,8.00,7.42.ESI-MS:m/z 449.4[M-H]^-,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 52 preparation of Compound II-3j

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2j, white solid, yield: 87.8%, melting point: 162 ℃ and 165 ℃.

Compound II-3j spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.95(s,1H),8.54(d,J＝8.5Hz,1H),7.68(t,J＝7.5Hz,1H),7.56(t,J＝7.7Hz,2H),7.41(dd,J＝7.6,5.3Hz,2H),3.82(s,2H),2.60–2.52(m,1H),2.37(d,J＝7.0Hz,3H),2.31(d,J＝6.6Hz,3H),1.21–1.07(m,2H),0.85(tt,J＝11.7,6.3Hz,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.61,161.44,158.23,158.19,142.45,134.14,130.85,129.74,129.28,128.74,128.29,127.92,127.14,125.42,123.22,122.67,119.67,35.77,13.40,13.23,13.07,7.76,7.52.ESI-MS:m/z 435.5[M-H]^-,C₂₃H₂₀N₂O₃S₂[436.09].

EXAMPLE 53 preparation of Compound II-3k

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2k, white solid, yield: 95.18%, melting point: 150 ℃ and 153 ℃.

Compound II-3k spectral data:¹H NMR(400MHz,DMSO-d₆)δ13.00(s,1H),8.53(d,J＝8.5Hz,1H),7.67(t,J＝7.6Hz,1H),7.61–7.49(m,2H),7.46–7.34(m,2H),4.31(dt,J＝12.8,7.1Hz,1H),2.55(dt,J＝10.8,5.6Hz,1H),2.38(s,3H),2.31(s,3H),1.37(dd,J＝25.5,7.2Hz,3H),1.20–1.09(m,2H),0.95–0.76(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.99,161.34,158.23,157.30,142.46,134.11,130.74,129.70,129.32,128.92,128.30,127.98,127.15,125.45,123.18,122.58,119.83,45.12,17.89,13.39,13.23,13.08,7.84,7.48.ESI-MS:m/z 449.4[M-H]^-,C₂₄H₂₂N₂O₃S₂[450.11].

EXAMPLE 54 preparation of Compound II-3l

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2l, white solid, yield: 86.2%, melting point: 163 ℃ and 165 ℃.

Compound II-3l spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.68(s,1H),8.53(d,J＝8.4Hz,1H),7.67(t,J＝7.4Hz,1H),7.63–7.48(m,2H),7.46–7.29(m,2H),2.55(dd,J＝9.2,4.1Hz,1H),2.38(s,3H),2.31(s,3H),1.48(d,J＝8.7Hz,3H),1.44(d,J＝5.3Hz,3H),1.22–1.07(m,2H),0.97–0.72(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.70,161.03,158.18,157.06,142.38,134.10,130.69,129.62,129.25,128.86,128.23,127.95,127.14,125.46,123.14,122.44,119.76,53.24,26.10,25.87,13.37,13.23,13.10,7.99,7.40.ESI-MS:m/z 463.4[M-H]^-,C₂₅H₂₄N₂O₃S₂[464.12].

EXAMPLE 55 preparation of Compound II-3m

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2m, white solid, yield: 85.8%, melting point: 158 ℃ and 162 ℃.

Compound II-3m spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.82(s,1H),8.47(d,J＝8.5Hz,1H),7.61(t,J＝7.6Hz,1H),7.49(t,J＝8.6Hz,2H),7.40–7.31(m,2H),3.77(s,2H),2.80–2.65(m,4H),2.56–2.47(m,1H),1.75(s,2H),1.68(s,2H),1.08(d,J＝8.5Hz,2H),0.87–0.70(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ169.63,162.09,158.12,157.92,142.52,134.13,131.78,131.40,130.73,129.71,128.31,127.95,127.17,125.46,123.22,122.67,118.90,35.21,25.61,24.93,22.94,22.20,13.41,7.78,7.52.ESI-MS:m/z 461.4[M-H]^-,C₂₅H₂₂N₂O₃S₂[462.58].

EXAMPLE 56 preparation of Compound II-3n

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2n, white solid, yield: 92.9%, melting point: 155 ℃ and 157 ℃.

Compound II-3n spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.90(s,1H),8.47(d,J＝8.4Hz,1H),7.60(t,J＝7.5Hz,1H),7.54–7.43(m,2H),7.34(dd,J＝13.1,8.3Hz,2H),4.26(p,J＝6.9Hz,1H),2.75–2.65(m,4H),2.52–2.46(m,1H),1.74(s,2H),1.67(s,2H),1.30(dd,J＝23.3,7.2Hz,3H),1.14–1.02(m,2H),0.87–0.69(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ172.85,162.01,157.93,157.42,142.53,134.10,131.91,131.43,130.62,129.68,128.32,128.01,127.17,125.45,123.21,122.60,119.06,44.91,25.62,24.94,22.94,22.20,17.62,13.39,7.86,7.47.ESI-MS:m/z 475.4[M-H]^-,C₂₆H₂₄N₂O₃S₂[476.12].

EXAMPLE 57 preparation of Compound II-3o

The procedure is as in example 14, except that in this example the compound to be hydrolyzed is II-2o, a white solid, yield: 80.7%, melting point: 172 ℃ and 174 ℃.

Compound II-3o spectral data:¹H NMR(400MHz,DMSO-d₆)δ12.71(s,1H),8.52(d,J＝8.5Hz,1H),7.67(t,J＝7.5Hz,1H),7.60–7.54(m,1H),7.50(d,J＝7.5Hz,1H),7.37(d,J＝7.2Hz,2H),2.84–2.70(m,4H),2.53(s,1H),1.77(d,J＝28.1Hz,4H),1.47(s,3H),1.44(s,3H),1.19–1.09(m,2H),0.95–0.74(m,2H).¹³C NMR(100MHz,DMSO-d₆)δ174.68,161.72,157.88,157.23,142.40,134.10,131.81,131.37,130.65,129.61,128.21,127.94,127.14,125.45,123.14,118.95,53.30,26.18,25.90,25.61,24.96,22.95,22.21,13.36,8.00,7.38.ESI-MS:m/z 489.4[M-H]^-,C₂₇H₂₆N₂O₃S₂[490.14].

example 58 in vitro target inhibition Activity assay of Compounds of interest (in two batches)

Principle of testing

In HEK293T cells stably expressing hURAT1 protein¹⁴C-labeled substrate uric acid, detecting the effect of the compound and a positive control drug Racinonide on hURAT 1-mediated substrate uric acid uptake at different concentrations, and measuring the radioactive intensity of uric acid taken up by cells to calculate the inhibition effect (IC) of each compound on the protein₅₀)。

Experimental Material

pcDNA3.1(+) -hURAT1-T2A-eGFP plasmid (Shenzhen Qianjiang Dongzhe Biotech, Inc.); plasmid extraction kit (OMEGA biotech); fat powder Agar (OXOID, USA); yeast Extract (OxOID, USA); peptone Tryptone (OXOID, usa); ampicillin (Sigma, usa); purifying the water; glycerol (bi yun sky biotechnology limited); fetal bovine serum (Corning, usa); DMEM medium (Corning, usa); DMSO (Sigma, usa); 96-well microplate (Corning, usa); PBS (Corning corporation, usa); HEPES (Sigma, USA);¹⁴C-Uric acid (American radio laboratory Chemicals, USA).

Test method

The 96-well plate was preincubated with poly-D-lysine solution (0.1mg/mL) for 12 hours to obtain better cell adhesion. Then inoculating the cells into a flat plate, when the cells are fused to 90%, uniformly mixing the opti and the lip 3000 in a hole of 5 mu L/hole and a hole of 0.15 mu L/hole respectively, and standing for 5 min; simultaneously mixing opti, P3000 and plasmid DNA respectively at 5 μ L/well, 0.2 μ L/well and 500 ng/well, standing for 5 min; mixing the above two liquids, and standing at room temperature for 15 min; add to 96-well plates with complete medium replaced. Culturing at 37 deg.C in an incubator containing 5% CO2 for 16-20h, observing the expression of EGFP (green fluorescent protein) with a fluorescence inverted microscope to verify whether transfection is successful, removing the culture medium after successful, and washing cells twice with PBS. To evaluate drug inhibition of URAT1The effect is that the drug is primarily screened (20uM) by taking Raxinder as a positive drug, and the absorption rate is (adding drug CPM-blank CPM)/(model CPM-blank CPM). And carry out IC₅₀The measurement of (1). Prior to the absorption experiment, the wells were aspirated, 50. mu.l each containing compounds at specific concentrations (20. mu.M, 10. mu.M, 5. mu.M, 2.5. mu.M, 1.25. mu.M) were added to each well, and the model group and blank group were not dosed. After 15 minutes incubation, the cells were aspirated off and 50. mu.M solution was added¹⁴Uric acid absorption buffer of C-uric acid to start uric acid absorption, and incubation at 37 ℃ for 15 min. The wells were aspirated and washed three times with 100. mu.L of ice-cold DPBS, and 40. mu.L of 0.1M NaOH was added to each well to lyse the cells. After lysis at room temperature for 30 minutes, 0.2mL of scintillation fluid was added to each well and the plate was placed on a plate shaker and shaken at 260rpm/min for 15 minutes. Measurement with liquid scintillation counter¹⁴The C-uric acid emission value (CPM) was measured in triplicate and averaged.

TABLE 2 in vitro target inhibition Activity of I series Compounds and II series Compounds

And (4) conclusion: as can be seen from Table 2, in the I series, the compounds I-3a, I-3b and I-3e and the compounds II-3a, II-3b and II-3h of the II series all present significant in vitro target inhibition activities, which are superior to or equal to the positive control drug Raschild; in the II series, the activity of II-3a and II-3b is far superior to that of the drug lesinurad, and an unexpected effect is achieved. Can be further developed as a drug lead with a brand-new structure.

Example 59 in vivo anti-gout Activity of Compounds of interest.

Test materials and methods

(1) Experimental animals: male Kunming mice, provided by the Experimental animals center of Shandong university.

(2) Sample treatment: selecting a compound to be tested with target inhibition activity obviously superior to lesinurad and corresponding ester thereof, and preparing the compound and the corresponding ester into proper concentration by using CMC-Na before use.

(3) Molding medicine: xanthine and potassium oxonate.

(4) Positive control drug: lesinurad.

(5) The test method comprises the following steps: feeding male Kunming mice of about 20g adaptively for 1 week, randomly dividing the mice into a blank group and a model group, feeding the blank group with an intragastric 5% CMC-Na solution of 0.2mL, feeding the model group with an intragastric 600mg/Kg hypoxanthine suspension of 0.2mL, subcutaneously injecting 400mg/Kg oteracil potassium suspension of 0.2mL, carrying out eyeball taking and blood taking after 4 hours, separating supernatant, and carrying out blood uric acid concentration detection.

TABLE 3 results of in vivo activity in animals

And (4) conclusion: as can be seen from Table 3, the in vivo uric acid reducing activity of most compounds is obviously superior to that of lesinurad, particularly, the target inhibition activity of the compound II-3a in cell experiment screening reaches 3.27 mu M, the blood uric acid reduction rate in animal in vivo activity screening experiment reaches 90.2 percent, and the in vivo uric acid reducing activity of the compound II-3a is obviously superior to that of lesinurad (target inhibition activity IC)₅₀The rate of serum uric acid decline was 31.4% at 15.34 μ M, worth further study.

EXAMPLE 60 pharmacokinetic experiments with the Compound of interest II-3a

The test principle is as follows:

compound II-3a was administered to SD rats by single intravenous (iv) and oral (po) administration, respectively, blood was collected at different time points, plasma drug concentrations of II-3a in rats were measured, and relevant pharmacokinetic parameters were calculated.

The test method comprises the following steps:

rats were evaluated for health at a temperature of 20-26 deg.CThe culture is adaptively cultured for at least three days under the conditions of 12h light and shade alternation at the temperature of 30-70 percent of relative humidity. The rats in the gavage group were fasted for 12h before dosing and were returned to diet 4h after dosing, and the rats in the i.v. group were free to diet in the experiment. Test drug II-3a was dissolved in DMSO: PEG 400: the mixed solution of normal saline (3: 60: 37) is mixed by ultrasound to obtain clear solution or even suspension. The rats were given 20mg/kg intragastric administration (n-3) and 1mg/kg intravenous administration (n-3) based on body weight, 250 μ L of venous blood was taken at 0.083, 0.25, 0.5, 1, 2, 4, 6, 10 and 24h after administration, respectively. Blood samples were collected into heparin sodium treated centrifuge tubes and placed on ice until plasma treatment. Blood samples will be immediately processed within the second half hour of collection by centrifugation at about 4 ℃ at 3200g for 10 minutes. Plasma samples will be stored in polypropylene tubes, snap frozen on dry ice, and maintained at around-70 ± 10 ℃ until LC/MS analysis is performed. At the end of the trial, all surviving animals were sacrificed according to the institutional ethical guidelines of the committee on pharmaceutical sciences for animal experimentation. AUC was calculated using pharmacokinetic software Phoenix 8.0_0-t、AUC_0-∞、MRT_0-tAnd T_1/2And the like.

And (3) testing results:

TABLE 4 pharmacokinetic study of Compound II-3a

And (4) conclusion: as can be seen from the results of Table 4 and FIG. 1, the peak time was 2.33h after oral administration (20mg/kg), and II-3a had a moderate plasma half-life (t-time)_1/23.67h) and has higher bioavailability (II-3a: 95.6%). At the same time, the maximum concentration of II-3a in plasma (C) after oral administration_max) 18938ng/mL, and the average residence time was 5.98 h.

After the injection administration of II-3a (1mg/kg), the maximum blood concentration is 11213ng/mL, the half-life period is 3.88h, the average residence time is 2.72h, and the apparent distribution volume and the clearance rate are 0.425L/kg and 2.64mL/min/kg respectively. II-3a has better pharmacokinetic parameters, and experimental results preliminarily prove the reasonability of the design and are worthy of further research.

Claims

1. A thienopyrimidone thioglycolic acid derivative is characterized by having a structure shown as the following general formula I or II:

wherein the content of the first and second substances,

r is-H, -CH₃Or

R₁is-CH₂-,-^*CH(CH₃) -or-C (CH)₃)₂-; represents a chiral carbon atom;

R₂is-OH or-OCH₃。

2. A thienopyrimidinone thioglycolic acid derivative characterized by being one of the compounds of the following structure:

3. the process for preparing thienopyrimidinone thioglycolic acid derivatives as claimed in claim 2, characterized in that the process for preparing I-3(a-I) is as follows:

4-bromo-1-naphthylamine is used as an initial raw material to perform Suzuki coupling reaction with cyclopropylboronic acid to generate an intermediate 4-cyclopropyl-1-naphthylamine a, then the intermediate is reacted with 1,1' -thiocarbonyldiimidazole to obtain an intermediate b, and the intermediate b is then reacted with 3-aminothiophene-2-methyl formate containing different substituents in the absence of methyl formateReacting in water ethanol or pyridine solution to obtain intermediate I-1(a-c), and then in DMF solution K₂CO₃Carrying out nucleophilic substitution reaction with substituted methyl bromoacetate under catalysis to obtain a target product I-2(a-I), and finally hydrolyzing with lithium hydroxide or sodium hydroxide in a mixed solution of tetrahydrofuran and methanol to obtain a target product I-3 (a-I);

the synthesis route one is as follows:

reagent and conditions (i) K₃PO₄，Pd(PPh₃)₄Cyclopropyl boronic acid, toluene: water 25:2v/v, N₂Protection, 100 ℃, 12 h; (ii) CH (CH)₂Cl₂1,1' -thiocarbonyl diimidazole at room temperature for 12 h; (iii) 3-aminothiophene-2-methyl formate or 3-amino-5-methylthiophene-2-methyl formate, absolute ethanol, 90 ℃,4 hours, NaOH, 90 ℃, 12 hours or 3-amino-4-methylthiophene-2-methyl formate, pyridine, 50 ℃, 12 hours, NaOH, 90 ℃, 12 hours; (iv) k₂CO₃DMF, methyl 2-bromoisobutyrate or methyl 2-bromopropionate/methyl bromoacetate, 100 ℃ for 3 h; (v) methanol, tetrahydrofuran, lithium hydroxide/sodium hydroxide, normal temperature, 12h.

4. The process for preparing thienopyrimidinone thioglycolic acid derivatives as claimed in claim 2, characterized in that the process for preparing II-3(a-o) is as follows:

4-bromo-1-naphthylamine is used as an initial raw material to perform Suzuki coupling reaction with cyclopropylboronic acid to generate an intermediate 4-cyclopropyl-1-naphthylamine a, then the intermediate b is reacted with 1,1' -thiocarbonyldiimidazole to obtain an intermediate b, the intermediate b is reacted with methyl 2-aminothiophene-3-carboxylate containing different substituents in pyridine or absolute ethyl alcohol to generate an intermediate II-1(a-e), and then the intermediate II-1(a-e) is reacted in a DMF solution in K₂CO₃Carrying out nucleophilic substitution reaction with various esters under catalysis to obtain a corresponding target product II-2(a-o), and finally hydrolyzing with lithium hydroxide or sodium hydroxide in a mixed solution of tetrahydrofuran and methanol to obtain a target product II-3 (a-o);

the second synthetic route is as follows:

reagent and conditions (i) K₃PO₄，Pd(PPh₃)₄Cyclopropyl boronic acid, toluene: water 25:2v/v, N₂Protection, 100 ℃, 12 h; (ii) CH (CH)₂Cl₂1,1' -thiocarbonyl diimidazole at room temperature for 12 h; (iii) the substituted 2-aminothiophene-3-methyl formate, absolute ethyl alcohol, 90 ℃,4 hours, NaOH, 90 ℃, 12 hours or the substituted 2-aminothiophene-3-methyl formate, pyridine, 50 ℃, 12 hours, NaOH, 90 ℃, 12 hours; (iv) k₂CO₃DMF, methyl 2-bromoisobutyrate or methyl 2-bromopropionate or methyl bromoacetate, 100 ℃ for 3 h; (v) methanol, tetrahydrofuran, lithium hydroxide or sodium hydroxide, normal temperature, 12h.

5. Use of the thienopyrimidinone thioglycolic acid derivative according to claim 1 or 2 for the preparation of a medicament against gout.

6. An anti-gout pharmaceutical composition comprising the thienopyrimidone thioglycolic acid derivative of claim 1 or 2 and one or more pharmaceutically acceptable carriers or excipients.