CN116675653B - Aminoalkyl substituted 1,2,4-thiadiazolidine-3,5-dione compound and preparation method and application thereof - Google Patents

Abstract

The invention discloses an aminoalkyl substituted 1,2, 4-thiadiazolidine-3, 5-dione compound, and a preparation method and application thereof, and belongs to the technical field of pharmaceutical chemistry. The invention also discloses application of the aminoalkyl-substituted 1,2, 4-thiadiazolidine-3, 5-dione derivative in medicines for treating PTPN2 mediated diseases, wherein the aminoalkyl-substituted 1,2, 4-thiadiazolidine-3, 5-dione derivative is a compound with a structure shown as a general formula (I) or pharmaceutically acceptable salt thereof. The compound disclosed by the invention has remarkable activity on PTPN2 phosphatase, most of synthesized compounds keep the IC ₅₀ value of PTPN2 phosphatase at mu M level, have important influence on the occurrence and development of tumors and immune response, can be used in combination with immunosuppressants to treat related immune diseases, can be developed into antitumor drugs with high activity, good selectivity and small toxic and side effects, and has the characteristics of novel skeleton, strong plasticity and great future transformation potential.

Description

Aminoalkyl substituted 1,2,4-thiadiazolidine-3,5-dione compounds and their preparation methods and applications

This application is a divisional application of the Chinese invention patent with an application date of July 22, 2022, application number 2022108610700, and invention name "2,4-thiadiazolidine-3,5-dione compounds, preparation methods and applications thereof".

Technical Field

The present invention belongs to the field of medicinal chemistry and relates to 1,2,4-thiadiazolidine-3,5-dione derivatives, and specifically to compounds as shown in formula (I) or pharmaceutically acceptable salts thereof, drug combinations thereof, and uses thereof in treating PTPN2-mediated diseases.

Background Art

PTPNs are important regulators of numerous signaling pathways involved in major cellular processes such as cell growth, proliferation, and differentiation. PTPN2 is a widely expressed cytoplasmic tyrosine phosphatase. PTPN2 negatively regulates the JAK/STAT signaling pathway by dephosphorylating JAK/STAT proteins (such as STAT1 or JAK1) that are phosphorylated at different tyrosines. In addition to STAT1, PTPN2 can also dephosphorylate STAT3 and STAT5 and negatively regulate their activation. The JAK/STAT pathway plays a crucial role in the cancer process, and abnormal activation of STAT signaling is involved in the occurrence of many cancers.

PTP1B is one of the most important members of the PTPN family and plays an important role in a variety of cellular functions. So far, PTP1B has been reported to be involved in the development of a variety of diseases such as diabetes, cancer and cardiovascular disease. PTP1B is highly homologous to PTPN2, so many selective inhibitors of PTP1B have inhibitory activity against PTPN2. However, these compounds lack selectivity for PTPN2, resulting in possible toxic side effects; at the same time, the structure contains multiple highly polar carboxyl and phosphate groups, ortho-dicarbonyl groups and other groups, and its drugability needs to be optimized. So far, no PTPN2 selective small molecule inhibitors have been reported. Therefore, the discovery of small molecule inhibitors of PTPN2 can provide an important theoretical basis for the development of anti-tumor drugs with anti-tumor activity, good selectivity and low toxicity.

Summary of the invention

Purpose of the invention: The technical problem to be solved by the present invention is to develop a small molecule inhibitor with PTPN2 inhibitory activity based on 1,2,4-thiadiazolidine-3,5-dione as the parent core.

The technical problem that the present invention needs to solve is to provide the use of the above-mentioned 1,2,4-thiadiazolidine-3,5-dione derivatives in drugs for treating PTPN2-mediated diseases.

Technical solution: To solve the above technical problems, the present invention provides the following technical solutions:

A 1,2,4-thiadiazolidine-3,5-dione derivative or a pharmaceutically acceptable salt thereof, characterized in that the 1,2,4-thiadiazolidine-3,5-dione derivative is a compound having a structure as shown in general formula (I) or a pharmaceutically acceptable salt thereof:

Wherein, m=1-4; Y is O;

R ₁ is selected from in:

L is a covalent bond (i.e., not present) or O;

R is a C ₆ -C ₁₀ aryl group, a 4-7 membered heteroaromatic ring having 1-3 heteroatoms selected from N and O, or a 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O;

_Ra and _Rb are each independently selected from hydrogen, hydroxyl, aldehyde, carbonyl, carboxyl, nitro, cyano, _C1 - _C6 alkyl, _C1 - _C6 carboxyalkyl, C1- _C6 alkylcarbonyl, C1- _C6 alkylsulfonyl, _C3 - _C8 cycloalkylcarbonyl, _C6 - _C10 aryl, _4-7 membered heteroaromatic ring having 1-3 heteroatoms selected from N and O, _{C1 - C6} alkyl substituted with 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O;

R _c and R _d are each independently selected from hydrogen, hydroxy, aldehyde, carbonyl, carboxyl, nitro, cyano, and optionally substituted C ₁ -C ₆ alkyl; the substituent is selected from hydrogen, hydroxy, aldehyde, carboxyl, carbonyl, nitro, cyano, and C ₆ -C ₁₀ aryl.

In some embodiments, R is a 5-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O;

_Ra and _Rb are each independently selected from hydrogen, hydroxyl, aldehyde, carbonyl, carboxyl, nitro, cyano, _C1 - _C4 alkyl, _C1 - _C4 carboxyalkyl _{, C1} - _C4 alkylcarbonyl, _C1 - _C4 alkylsulfonyl, C3- _C8 cycloalkylcarbonyl, _C6 - _C10 aryl, a 4-7 membered heteroaromatic ring having 1-3 heteroatoms selected from N and O, and a _C1 - _C3 alkyl substituted by a 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O.

In some more specific examples,

R is

_Ra and _Rb are each independently selected from hydrogen, hydroxyl, aldehyde, carbonyl, carboxyl, nitro, cyano, _C1 - _C4 alkyl, _C1 - _C4 carboxyalkyl _{, C1} - _C4 alkylcarbonyl, _C1 - _C4 alkylsulfonyl, C3- _C8 cycloalkylcarbonyl, _C6 - _C10 aryl, a 4-7 membered heteroaromatic ring having 1-3 heteroatoms selected from N and O, and a _C1 - _C3 alkyl substituted by a 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O.

In some more specific examples, when R is not When , _Ra is hydrogen.

In some examples, R _c and R _d are each independently selected from hydrogen, hydroxyl, aldehyde, carbonyl, carboxyl, nitro, cyano or an optionally substituted C ₁ to C ₄ alkyl group; the substituent is selected from hydrogen, hydroxyl, aldehyde, carboxyl, carbonyl, nitro, cyano or C ₆ to C ₁₀ aryl. In some more specific examples, R _c and R _d are each independently selected from hydrogen or an optionally substituted C ₁ to C ₃ alkyl group; the substituent is selected from hydrogen, hydroxyl, aldehyde, carboxyl, carbonyl, nitro, cyano or a benzene ring.

When m=2, R ₁ is selected from -NHR ₂ , -NR ₃ R ₄ , 6-7 membered azacycloalkyl, wherein the 6-7 membered azacycloalkyl is

When _{m=4, R1 is selected from -Br, -NHR8, -NR9R10 , 6-7 membered} heterocycloalkyl, alkylphenoxy, wherein the 6-7 membered heterocycloalkyl is Alkylphenoxy is -O-Ph-R ₁₂ .

Preferably,

When m=2, R ₁ is selected from -Br, -NHR ₂ , -NR ₃ R ₄ , 6-7 membered azacycloalkyl, alkylphenoxy, wherein the 6-7 membered azacycloalkyl is Alkylphenoxy is -O-Ph-R ₇ ;

wherein R ₂ is selected from -CH(CH ₃ )-C(O)OH, -CH(CH ₂ Ph)-C(O)OH,

-NR ₃ R ₄ is selected from -N(CH ₃ )CH ₂ C(O)OH,

R ₅ is selected from -CH ₂ -C(O)OH, -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(O)CH ₃ , _R6 is selected from _-CH2- C(O)OH,

When m=4, R ₁ is selected from -NHR ₈ , -NR ₉ R ₁₀ , 6-7 membered azacycloalkyl, wherein the 6-7 membered azacycloalkyl is

wherein R ₈ is selected from -CH(CH ₃ )-C(O)OH, -CH(CH ₂ Ph)-C(O)OH,

-NR ₉ R ₁₀ is selected from -N(CH 3 ) CH 2 C(O) OH,

-R ₁₁ is selected from -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(O)CH ₃ ,

In some examples, the present application also provides compounds with the following specific structures:

The pharmaceutically acceptable salts described in the present invention are acid addition salts of the compounds of general formula (I), wherein the acids used for salt formation include inorganic acids and organic acids, wherein the inorganic acids include hydrochloric acid, sulfuric acid, phosphoric acid and methanesulfonic acid, and the organic acids include acetic acid, trifluoroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid and tartaric acid.

Preferably, the pharmaceutically acceptable salt described in the present invention is hydrochloride or trifluoroacetate.

The present invention also discloses a method for preparing the compound of general formula (I):

wherein Y, m, and _R1 are as defined above.

The present invention also discloses a pharmaceutical composition, comprising the compound of the general formula (I) or a pharmaceutically acceptable salt or an isomer thereof, and a pharmaceutically acceptable carrier.

Pharmaceutically acceptable carriers refer to excipients or diluents that do not cause significant irritation to organisms and do not interfere with the biological activity and properties of the administered compound. The excipients include adhesives, fillers, disintegrants, lubricants, preservatives, antioxidants, flavoring agents, aromatics, cosolvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants, etc., and the diluents include physiological saline, starch, dextrin, sucrose, lactose, etc.

A method for treating a PTPN2-mediated disease, comprising administering an effective amount of a compound of formula (I) or a pharmaceutically acceptable salt thereof.

The use of the compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a PTPN2-mediated disease is within the scope of protection of the present invention.

In some embodiments of the present invention, the PTPN2-mediated disease is selected from diseases mediated by regulating the JAK/STAT signaling pathway. In some embodiments of the present invention, the PTPN2-mediated disease includes cancer, inflammation, infection, immune disease, organ transplantation, viral disease, diabetes, cardiovascular disease or metabolic disease.

In some embodiments of the present invention, the cancer includes, but is not limited to, lung cancer, head and neck cancer, breast cancer, prostate cancer, esophageal cancer, rectal cancer, colon cancer, nasopharyngeal cancer, uterine cancer, pancreatic cancer, lymphoma, blood cancer, osteosarcoma, melanoma, kidney cancer, gastric cancer, liver cancer, bladder cancer, thyroid cancer or colorectal cancer. More specifically, acute myeloid leukemia (AML).

In some embodiments of the present invention, the cancer is selected from first-line cancer.

In some preferred embodiments of the present invention, the disease is selected from PTPN2-mediated diseases selected from pancreatic cancer.

Unless otherwise specified, the terms used in the present invention generally have the following meanings.

As used herein, the term "[CH ₂ ] _1-4 " means that the moiety has 1 to 4 carbon atoms.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "C _1-6 alkyl" refers to saturated straight and branched hydrocarbon groups having 1 to 6 carbon atoms, including but not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl.

The term "heterocycloalkyl" refers to a cycloalkane having one or more non-C heteroatoms such as N, O, S, etc., including but not limited to tetrahydropyrrole, piperidine, morpholine, piperazine, pyrazine, N-methylpiperazine, N-ethylpiperazine, etc.

The term "C(O)" refers to a carbonyl group, specifically a carbon-oxygen double bond.

The term "C(O)O" refers to an ester group, specifically a carbon-oxygen double bond plus a carbon-oxygen single bond.

The term "C ₁ -C ₆ carboxyalkyl" refers to a C ₁ -C ₆ alkyl group substituted by a carboxy group.

The term "C ₁ ～C ₆ alkylcarbonyl" refers to -C(O)-R', wherein R' is a C ₁ ～C ₆ alkyl group.

The term "C ₁ ～C ₆ alkylsulfonyl" refers to -S(O) ₂ -R', wherein R' is a C ₁ ～C ₆ alkyl group.

The term "C ₃ -C ₈ cycloalkylcarbonyl" refers to -C(O)-R", wherein R" is a C ₃ -C ₈ cycloalkyl group.

Beneficial effects:

The compounds disclosed in the present invention have significant activity against PTPN2 phosphatase, and the IC ₅₀ value of the synthesized compounds is maintained at the μM level. They can be used to have an important impact on the occurrence and development of tumors and immune responses, and can also be used in combination with immunosuppressants to treat related immune diseases. They can be developed into anti-tumor drugs with high activity, good selectivity, and low toxic and side effects. They have the characteristics of novel skeletons, strong plasticity, and great potential for future transformation.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is a study of compound X-18. (A) Body weight changes of mice in each group; (B) Weight changes of heart, liver, spleen, lung, and kidney of mice in each group;

Figure 2 shows the in vivo activity of compound X-18. (A) Changes in body weight of mice; (B) and (C) Changes in fluorescence intensity in mice on the first and ninth days of administration; ns means no significant difference, **p<0.01.

DETAILED DESCRIPTION

The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The present application is described in detail below in conjunction with specific embodiments.

Example 1: Synthesis of Intermediate K-1 (4-benzyl-2-(2-bromoethyl)-1,2,4-thiadiazolidine-3,5-dione)

Step 1. Synthesis of 1-bromo-2-isocyanatoethane (1b) and 4-benzyl-2-(2-bromoethyl)-1,2,4-thiadiazolidine-3,5-dione (K-1)

2-Bromoethylamine hydrobromide (20g, 1.0eq) and pyridine (32mL) were added to a three-necked flask and protected with nitrogen. The mixture was cooled to below -15 degrees, and then dichloromethane (150mL) was added. Triphosgene (13g, 0.5eq) was dissolved in dichloromethane (100mL) and slowly added dropwise to the reaction system in the three-necked flask, keeping the temperature not exceeding 0°C. After the addition, the mixture was stirred for 4-6 hours, during which the reaction was monitored by TLC plates. When the raw materials were basically reacted, the cooling bath was stopped. The reaction solution was washed twice with 0.5M dilute hydrochloric acid, the aqueous solution was washed twice with dichloromethane, the organic layers were combined, and then washed twice with saturated brine, dried, and concentrated under reduced pressure to obtain a yellow transparent oily substance, which was directly used in the next step. The yellow oily substance was dissolved in tetrahydrofuran (400 mL), and isothiocyanate (16 g, 1.0 eq) was added, cooled to 0 degrees, and then sulfonyl chloride (15 g, 1.0 eq) was slowly added dropwise, warmed to room temperature, and stirred overnight. The next day, the reaction was placed in the air and stirred for 30 minutes. After the reaction was completed, two products were detected by TLC plate. The product with greater polarity was K-1. After vacuum distillation and concentration, column chromatography was performed to obtain compound K-1 (12.1 g, yield 57%). ¹ H NMR (400 MHz, CDCl ₃ ) δ7.45–7.43 (m, 2H), 7.38–7.29 (m, 3H), 4.84 (s, 2H), 4.03–3.99 (m, 2H), 3.55–3.52 (m, 2H).

1. Synthesis of Compounds X-1-X-41

Example 2: 2-(2-(1,4-diaza-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-1)

Step 1. Synthesis of tert-butyl 4-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-1,4-diazane-1-carboxylate (2a)

Compound K-1 (300 mg, 1.0 eq) and tert-butyl 1,4-diazacycloheptane-1-carboxylate (195 mg, 1.03 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and potassium carbonate (329 mg, 2.5 eq) was added, and the mixture was reacted at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain compound 2a, a yellow oily liquid with a yield of 42%. MS (ESI) m/z 435.5 [M + H] ⁺ .

Step 2. Synthesis of 2-(2-(1,4-diaza-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-1)

The reactant 2a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-1) as a white solid with a yield of 90%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.36–7.30 (m, 5H), 4.73 (s, 2H), 4.46 (s, 1H), 4.06–4.02 (m, 2H), 3.80–3.61 (m, 4H), 3.38–3.17 (m, 6H), 2.17 (s, 2H), 1.23 (s, 1H).

Example 3: 2-(4-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-1,4-diazepin-1-yl)acetic acid (X-2)

Step 1. Synthesis of tert-butyl 2-(4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-1,4-diazepin-1-yl)acetate (2b)

X-1 (1.0 eq) and tert-butyl bromoacetate (1.0 eq) were dissolved in tetrahydrofuran, and then triethylamine (2.5 eq) was added to the reaction solution. The reaction was carried out at 65°C for 2 h and the reaction was monitored by TLC. After the reaction was completed, the reaction solution was concentrated under reduced pressure, dried over anhydrous sodium sulfate, and purified by rapid silica gel column to obtain compound 2b as a colorless oily liquid with a yield of 83%. MS (ESI) m/z 449.5 [M+H] ⁺ .

Step 2. Synthesis of 2-(4-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-1,4-diazepin-1-yl)acetic acid (X-2)

The intermediate 2b was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-2) as a white solid with a yield of 90%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.22 (m, 3H), 4.73 (s, 1H), 4.05 (t, J = 6.9 Hz, 1H), 3.78 (s, 2H), 3.43 (t, J = 7.0 Hz, 1H), 3.36 (d, J = 6.4 Hz, 1H), 2.25 (s, 1H), 1.24 (d, J = 3.7 Hz, 1H).

Example 4: 4-Benzyl-2-(2-(piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-3)

Step 1. Synthesis of tert-butyl 4-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)piperazine-1-carboxylate (2c)

Compound K-1 (300 mg, 1.0 eq) and tert-butyl piperazine-1-carboxylate (195 mg, 1.03 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and potassium carbonate (329 mg, 2.5 eq) was then added. The mixture was reacted at 80°C for 3-4 hours and monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain compound 2a. The yield of the yellow oily liquid was 51%. MS (ESI) m/z 421.5 [M + H] ⁺ .

Step 2. Synthesis of 4-benzyl-2-(2-(piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-4)

The intermediate 2c was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-3) as a white solid with a yield of 90%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.29 (m, 5H), 4.74 (s, 2H), 3.74 (t, J = 5.4 Hz, 2H), 3.08 (t, J = 4.9 Hz, 4H), 2.67–2.64 (m, 4H), 2.60 (d, J = 5.4 Hz, 2H).

Example 5: Synthesis of 2-(4-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)piperazin-1-yl)acetic acid (X-4)

Referring to the synthesis method of compound (X-2), intermediate 2d and compound X-4 were obtained. The yield of compound X-4 was 30%, white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.28 (m, 5H), 4.75 (s, 2H), 3.90–3.74 (m, 4H), 3.53 (s, 2H), 2.83 (t, J=41.7 Hz, 8H).

Example 6: Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-alanine (X-5)

Step 1. Synthesis of tert-butyl (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-alaninate (3a)

Compound K-1 (300 mg, 1.0 eq) and L-alanine tert-butyl ester hydrochloride (182 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and then anhydrous potassium carbonate (329 mg, 2.5 eq) was added, and the reaction was carried out at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain compound 2a, a yellow oily liquid with a yield of 60%, a colorless and transparent oily liquid. MS (ESI) m/z 380.4 [M + H] ⁺ .

Step 2. Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-alanine (X-5)

The reactant 3a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-5) as a white solid with a yield of 90%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.36–7.28 (m, 5H), 3.82–3.59 (m, 3H), 3.21 (d, J=7.0 Hz, 1H), 2.95–2.68 (m, 2H), 1.19 (d, J=7.0 Hz, 3H).

Example 7: Synthesis of N-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-N-methylglycine (X-6)

Step 1. Synthesis of tert-butyl N-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-N-methylglycinate (4a)

Referring to the synthesis method of intermediate 3a in step 1 of Example 6, the yield was 73%, colorless oily liquid. MS (ESI) m/z 380.4 [M+H] ⁺ .

Step 2. Synthesis of N-(2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-N-methylglycine (X-6)

The synthesis method of compound X-5 was referred to step 2 of Example 6, and the yield was 90%, white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 12.30 (s, 1H), 7.33 (dt, J = 22.1, 7.5 Hz, 5H), 4.74 (s, 2H), 3.70 (t, J = 5.4 Hz, 2H), 2.79 (t, J = 5.4 Hz, 2H), 2.35 (s, 3H).

Example 8: Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalanine (X-7)

Step 1. Synthesis of tert-butyl (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalaninate (5a)

Referring to the synthesis method of intermediate 3a in step 1 of Example 6, the yield was 51%, colorless oily liquid. MS (ESI) m/z 456.5 [M+H] ⁺ .

Step 2. Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalanine (X-7)

The synthesis method of compound X-5 was referred to step 2 of Example 6, with a yield of 90%, and the product was a white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.36–7.16 (m, 10H), 4.70 (s, 2H), 3.76–3.45 (m, 4H), 2.92–2.86 (m, 2H), 2.78 (dd, J=13.5, 7.4 Hz, 1H).

Example 9: Synthesis of 4-benzyl-2-(2-(4-ethylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-8)

Step 1. Synthesis of 4-benzyl-2-(2-(4-ethylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-9)

Compound K-1 (300 mg, 1.0 eq) and N-ethylpiperazine (116 mg, 1.05 eq) were dissolved in a sealed glass tube containing 1,4-dioxane (5 mL), and then DIPEA (307 mg, 2.5 eq) was added. The mixture was reacted at 100°C for 3-4 hours and monitored by TLC. After the reaction, the reaction solution was concentrated under reduced pressure and purified by a rapid silica gel column (DCM: MeOH = 94:6) to obtain compound X-8 as a white solid with a yield of 67%. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.39–7.29 (m, 3H), 4.74 (s, 1H), 3.52 (s, 1H), 3.11 (s, 1H), 1.25 (d, J = 7.3 Hz, 2H), 1.22 (d, J = 4.8 Hz, 1H).

Example 10: Synthesis of 4-benzyl-2-(2-(4-isopropylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-9)

Step 1. Synthesis of 4-benzyl-2-(2-(4-isopropylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-9)

Referring to the synthesis method of compound (X-8), N-ethylpiperazine was replaced by N-isopropylpiperazine. The yield was 68%, white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.28 (m, 5H), 4.73 (s, 2H), 4.11 (s, 2H), 4.03 (d, J＝7.1 Hz, 1H), 3.98 (s, 2H), 3.64 (s, 2H), 3.50 (d, J＝8.7 Hz, 2H), 1.29 (d, J＝6.6 Hz, 6H).

Example 11: Synthesis of 2-(2-(4-acetylpiperazin-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-10)

Step 1. Synthesis of 2-(2-(4-acetylpiperazin-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-10) hydrochloride

Referring to the synthesis method of compound (X-8), N-ethylpiperazine was replaced by 1-acetylpiperazine. The yield was 45%, white solid. ¹ H NMR (400 MHz, DMSO-d ₆ )δ11.36 (s, 1H), 7.38–7.29 (m, 5H), 4.74 (s, 2H), 4.08 (s, 2H), 4.01 (s, 2H), 3.56 (d, J＝12.3 Hz, 3H), 3.35 (s, 2H), 3.08 (d, J＝12.7 Hz, 2H), 2.96 (s, 1H), 2.04 (s, 3H).

Example 12: Synthesis of 4-benzyl-2-(2-(4-(methylsulfonyl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-11)

Step 1. Synthesis of 4-benzyl-2-(2-(4-(methylsulfonyl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-11)

Refer to the synthesis method of compound (X-8), replace N-ethylpiperazine with 1-methanesulfonylpiperazine. The yield is 81%, white solid. ¹ H NMR (300MHz, DMSO-d ₆ )δ11.25(s,1H),7.34(dt,J＝8.8,4.2Hz,5H),4.74(s,2H),4.06–4.05(m,2H),3.67(s,2H),3.00(s,3H).

Example 13: Synthesis of 4-benzyl-2-(2-(4-phenylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-12)

Step 1. Synthesis of 4-benzyl-2-(2-(4-phenylpiperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-12)

Referring to the synthesis method of compound (X-8), N-ethylpiperazine was replaced by N-phenylpiperazine. The yield was 56%, white solid. ¹ H NMR (300 MHz, DMSO-d ₆ )δ10.90(s,1H),7.37–7.24(m,7H),7.01(d,J＝8.1Hz,2H),6.87(t,J＝7.2Hz,1H),4.75(s,2H),4.06–4.00(m,8H),3.67(d,J＝10.7Hz,2H).

Example 14: Synthesis of 2-(2-(4-(Benzo[d][1,3]dioxin-5-ylmethyl)piperazin-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-13)

The synthetic route is as follows:

Step 1. Synthesis of 2-(2-(4-(Benzo[d][1,3]dioxin-5-ylmethyl)piperazin-1-yl)ethyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-13)

Referring to the synthesis method of compound (X-8), N-ethylpiperazine was replaced by 1-piperazine. The yield was 69%, white solid. ¹ H NMR (300MHz, DMSO-d ₆ )δ7.34 (qd, J＝7.8,7.3,4.9Hz,5H),7.23 (s,1H),7.06–6.98 (m,2H),6.07 (s,2H),4.74 (s,2H),3.84 (s,2H),3.22 (d, J＝69.5Hz,6H),1.24 (s,2H).

Example 15: Synthesis of 4-benzyl-2-(2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-19)

Step 1. Synthesis of 4-benzyl-2-(2-(4-(pyridin-2-yl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione (X-19)

Referring to the synthesis method of compound X-8, N-ethylpiperazine was replaced by 1-(2-pyridyl)piperazine to obtain product X-19, a white solid, with a yield of 67%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 8.13 (dd, J = 5.9, 1.8 Hz, 1H), 8.00 (t, J = 8.0 Hz, 1H), 7.38–7.28 (m, 6H), 7.00 (t, J = 6.5 Hz, 1H), 4.74 (s, 2H), 4.53 (s, 2H), 4.12 (t, J = 6.1 Hz, 2H), 3.39 (s, 2H), 3.24 (s, 2H), 2.51 (d, J = 3.8 Hz, 5H).

Example 16: Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-proline (X-20)

Step 1. Synthesis of tert-butyl (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-proline (7a)

Compound X-1 (300 mg, 1.0 eq) and L-proline tert-butyl ester (172 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and then anhydrous potassium carbonate (329 mg, 2.5 eq) was added, and the mixture was reacted at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain compound 7a, a yellow oily liquid, with a yield of 56%. MS (ESI) m/z 406.4 [M + H] ⁺ .

Step 2. Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-proline (X-20)

The intermediate 7a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by a rapid silica gel column to obtain compound (X-20) as a white solid with a yield of 89%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ7.39–7.29(m,5H),4.81–4.72(m,2H),4.19(dd,J＝14.7,7.2Hz,1H),3.88(d,J＝15.0Hz,1H),3.65(dd,J＝13.2,6.8Hz,2H),3.45– 3.25(m,3H),2.40(dt,J＝8.7,5.9Hz,1H),2.04(dq,J＝9.3,5.2Hz,2H),1.94–1.91(m,1H).

Example 17: Synthesis of 4-benzyl-2-(2-(4-(cyclopropylcarbonyl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-21)

Step 1. Synthesis of 4-benzyl-2-(2-(4-(cyclopropylcarbonyl)piperazin-1-yl)ethyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-21)

Referring to the synthesis method of compound X-8, N-ethylpiperazine was replaced by 1-cyclopropylcarbonylpiperazine to obtain product X-21, a white solid, with a yield of 68%. ¹ H NMR (300MHz, DMSO-d ₆ )δ11.15(s,1H),7.39–7.28(m,5H),4.75(s,2H),4.42(s,2H),4.07(s,2H),3.37(s,2H),2.02(dt,J＝10.7,4.1Hz,1H),0.75(d,J＝7.2Hz,4H).

Example 18: Synthesis of 4-benzyl-2-(4-(4-ethylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidin-3-one hydrochloride (X-23)

(1) Synthesis of 4-benzyl-2-(4-bromobutyl)-1,2,4-thiadiazolidine-3,5-dione (X-22)

The synthetic route is as follows:

Step 1. Synthesis of 4-benzyl-2-(4-bromobutyl)-1,2,4-thiadiazolidine-3,5-dione (X-22)

4-Bromo-1-butylamine hydrobromic acid (20g, 1.0eq) and pyridine (32mL) were added to a three-necked flask and protected with nitrogen. The mixture was cooled to below -15 degrees, and then dichloromethane (150mL) was added. Triphosgene (13g, 0.5eq) was dissolved in dichloromethane (100mL) and slowly added dropwise to the reaction system in the three-necked flask, keeping the temperature not exceeding 0°C. After the addition, the mixture was stirred for 4-6 hours, during which the reaction was monitored by TLC plates. When the raw materials were basically reacted, the cooling bath was stopped. The reaction solution was washed twice with 0.5M dilute hydrochloric acid, the aqueous solution was washed twice with dichloromethane, the organic layers were combined, and then washed twice with saturated brine, dried, and concentrated under reduced pressure to obtain a yellow transparent oily substance, which was directly used in the next step. The yellow oily substance was dissolved in tetrahydrofuran (400 mL), and isothiocyanate (16 g, 1.0 eq) was added, cooled to 0 degrees, and then sulfonyl chloride (15 g, 1.0 eq) was slowly added dropwise, warmed to room temperature, and stirred overnight. The next day, the reaction was placed in the air and stirred for 30 minutes. After the reaction was completed, two products were monitored by TLC plate, and the product with greater polarity was X-22. After vacuum distillation and concentration, column chromatography was performed to obtain compound X-23, a yellow oily liquid (11.1 g, yield 38%). ¹ H NMR (400MHz, CDCl ₃ ) δ7.44–7.42(m,2H),7.35–7.30(m,3H),4.82(s,2H),3.66(t,J＝6.8Hz,2H),3.42(t,J＝6.3Hz,2H),1.92–1.85(m,2H),1.84–1.75(m ,2H).

(2) The synthetic route is as follows:

Step 1. Synthesis of 4-benzyl-2-(4-(4-ethylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidin-3-one hydrochloride (X-23)

Compound X-22 (343 mg, 1.0 eq) and N-ethylpiperazine (120 mg, 1.05 eq) were dissolved in acetonitrile (5-10 mL), and potassium carbonate (345 mg, 2.5 eq) was then added, and the mixture was reacted at 80°C for 3 hours, and the reaction was monitored by TLC. After the reaction, the mixture was extracted with ethyl acetate and water for 3 times, and the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash silica gel column (DCM: MeOH = 94: 6) to obtain compound X-23 with a yield of 60%. ¹ H NMR (400MHz, DMSO-d ₆ ) δ11.69 (s, 1H), 7.39–7.29 (m, 5H), 4.76 (s, 2H), 3.71 (s, 2H), 3.66 (t, J = 6.7Hz, 2H), 3.36 (t, J = 7.2Hz, 2H), 1.81–1.58 (m, 4H), 1. 26(d,J＝7.2Hz,3H).

Example 19. Synthesis of 4-benzyl-2-(4-(4-isopropylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-24)

Step 1. Synthesis of 4-benzyl-2-(4-(4-isopropylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-24)

Referring to the synthesis method of compound (X-23), N-ethylpiperazine was replaced by N-isopropylpiperazine, yield 67%, white solid. ¹ H NMR (300 MHz, DMSO-d ₆ )δ11.76(s,1H),7.40–7.28(m,5H),4.76(s,2H),3.66(t,J＝6.6Hz,5H),1.72(s,2H),1.68–1.61(m,2H),1.29(d,J＝6.5Hz,6H).

Example 20. Synthesis of 2-(4-(4-acetylpiperazin-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-25)

Step 1. Synthesis of 2-(4-(4-acetylpiperazin-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-25)

Referring to the synthesis method of compound (X-23), N-ethylpiperazine was replaced by 1-acetylpiperazine, yield 38%, white solid. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.36–7.28 (m, 5H), 4.74 (s, 2H), 4.16 (dd, J＝169.3,

14.1Hz,2H),3.64(t,J＝6.8Hz,2H),3.61–3.56(m,1H),3.39(d,J＝11.8Hz,2H),3.12(d,J＝14.5Hz,1H) ,3.08–3.04(m,2H),2.99–2.80(m,2H),2.03(s,3H),1.75–1.71(m,2H),1.63–1.59(m,2H).

Example 21. Synthesis of 4-benzyl-2-(4-(methylsulfonyl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-26)

Step 1. Synthesis of 4-benzyl-2-(4-(methylsulfonyl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-26)

Referring to the synthesis method of compound (X-23), N-ethylpiperazine was replaced by 1-methanesulfonylpiperazine, yield 69%, white solid. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.28 (m, 3H), 4.75 (s, 1H), 3.63 (t, J＝6.9 Hz, 1H), 3.07 (d, J＝6.5 Hz, 2H), 2.86 (s, 2H), 2.41 (s, 2H), 2.36–2.29 (m, 1H), 1.57 (q, J＝7.2 Hz, 1H), 1.44–1.39 (m, 1H).

Example 22. Synthesis of 4-benzyl-2-(4-(4-phenylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-27)

Step 1. Synthesis of 4-benzyl-2-(4-(4-phenylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-28)

Referring to the synthesis method of compound (X-23), N-ethylpiperazine was replaced by N-phenylpiperazine, with a yield of 48%, and a white solid. ¹ H NMR (400 MHz, Chloroform-d) δ7.88 (d, J = 7.4 Hz, 2H), 7.58–7.49 (m, 3H), 7.45–7.42 (m, 2H), 7.38–7.31 (m, 3H), 4.84 (s, 2H), 4.74 (t, J = 12.3 Hz, 2H), 4.27 (s, 2H), 3.71 (t, J = 6.2 Hz, 2H), 3.62 (t, J = 15.2 Hz, 4H), 3.24 (s, 2H), 1.98 (s, 2H), 1.84 (d, J = 8.3 Hz, 2H).

Example 23. Synthesis of 2-(4-(4-(Benzo[d][1,3]dioxin-5-ylmethyl)piperazin-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-28)

Step 1. Synthesis of 2-(4-(4-(Benzo[d][1,3]dioxin-5-ylmethyl)piperazin-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-28)

Refer to the synthesis method of compound (X-23), replace N-ethylpiperazine with 1-piperazine. The yield is 69%, white solid. ¹ H NMR (400MHz, DMSO-d ₆ )δ7.38–7.27 (m, 6H), 7.08 (d, J＝8.0Hz, 1H), 6.99 (d, J＝7.9Hz, 1H), 6.07 (s, 2H), 4.75 (s, 2H), 4.27 (s, 2H), 3.64 (t, J＝6.1Hz, 4H), 3.53 (s, 4H), 3.37 (s, 2H), 3.11 (s, 2H), 1.69 (s, 2H), 1.62 (d, J＝7.3Hz, 2H).

Example 24. Synthesis of 4-benzyl-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-29)

Step 1. Synthesis of 4-benzyl-2-(4-(4-(pyridin-2-yl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-29)

Refer to the synthesis method of compound (X-23), replace N-ethylpiperazine with 1-(2-pyridyl)piperazine. The yield is 55%, white solid. ¹ H NMR (400MHz, Chloroform-d) δ8.19 (ddd, J = 4.9, 2.1, 0.9 Hz, 1H), 7.46 (ddd, J = 14.8, 8.2, 1.8 Hz, 3H), 7.36–7.30 (m, 3H), 6.65–6.60 (m, 2H), 4.82 (s, 2H), 3.67 (t, J = 7.0 Hz, 2H), 3.54–3.52 (m, 4H), 2.53–2.50 (m, 4H), 2.42–2.38 (m, 2H), 1.73–1.66 (m, 2H), 1.60–

1.52(m,2H).

Example 25: Synthesis of 4-benzyl-2-(4-morpholinobutyl)-1,2,4-thiadiazolidine-3,5-dione (X-30)

Step 1. Synthesis of 4-benzyl-2-(4-morpholinobutyl)-1,2,4-thiadiazolidine-3,5-dione (X-30)

Refer to the synthesis method of compound (X-23), replace N-ethylpiperazine with morpholine. The yield is 58%, white solid. ¹ H NMR (400MHz, Chloroform-d) δ7.45–7.42 (m, 2H), 7.36–7.30 (m, 3H), 4.82 (s, 2H), 3.70–3.68 (m, 4H), 3.65 (t, J = 7.0 Hz, 2H), 2.40 (t, J = 4.7 Hz, 4H), 2.36–2.32 (m, 2H), 1.66 (q, J = 7.3 Hz, 2H), 1.51 (ddd, J = 11.4, 5.7, 3.4 Hz, 2H).

Example 26: Synthesis of 4-benzyl-2-(4-(4-methylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-31)

Step 1. Synthesis of 4-benzyl-2-(4-(4-methylpiperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-31) hydrochloride

Refer to the synthesis method of compound (X-23), replace N-ethylpiperazine with N-methylpiperazine. The yield is 73%, white solid. ¹ H NMR (300MHz, DMSO-d ₆ )δ11.77(s,1H),7.40–7.28(m,5H),4.75(s,2H),3.66(t,J＝6.6Hz,4H),1.69(d,J＝9.9Hz,2H),1.64(d,J＝6.7Hz,2H).

Example 27: Synthesis of 4-benzyl-2-(4-(piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-35)

Step 1. Synthesis of tert-butyl 4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)piperazine-1-carboxylate (8a)

Compound X-22 (343 mg, 1.0 eq) and tert-butyl piperazine-1-carboxylate (195 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and potassium carbonate (329 mg, 2.5 eq) was added. The mixture was reacted at 80°C for 3-4 hours and monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain intermediate 8a as a yellow oily liquid with a yield of 52%. MS (ESI) m/z 449.8 [M+H] ⁺ .

Step 2. Synthesis of 4-benzyl-2-(4-(piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione (X-35)

The intermediate 8a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-35) as a white solid with a yield of 87%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.37–7.29 (m, 5H), 4.76 (s, 2H), 3.65 (d, J＝6.4 Hz, 2H), 3.47 (s, 2H), 3.07–3.04 (m, 8H), 1.74–1.62 (m, 4H).

Example 28: Synthesis of (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-alanine (X-36)

Step 1. Synthesis of tert-butyl (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-alaninate (9a)

Compound X-22 (343 mg, 1 eq) and L-alanine tert-butyl ester hydrochloride (191 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and then anhydrous potassium carbonate (345 mg, 2.5 eq) was added, and the reaction was carried out at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column (PE: EA = 1: 4) to obtain intermediate 9a, a yellow oily liquid, with a yield of 73%. MS (ESI) m/z 408.4 [M + H] ⁺ .

Step 2. Synthesis of (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-alanine (X-36)

Intermediate 9a was removed from tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane ( ^{10 ml/mmol) for 4 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-36) as a white solid with a yield of 87%. 1} H NMR (400 MHz, DMSO-d ₆ ) δ 7.38–7.28 (m, 5H), 4.75 (s, 2H), 3.62 (d, J = 6.4 Hz, 2H), 3.24 (d, J = 7.7 Hz, 1H), 2.82 (d, J = 7.2 Hz, 2H), 1.60 (d, J = 6.0 Hz, 4H), 1.27 (d, J = 7.0 Hz, 3H).

Example 29: Synthesis of (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-proline (X-37)

Step 1. Synthesis of tert-butyl (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-proline (10a)

Compound X-22 (343 mg, 1 eq) and L-proline tert-butyl ester (180 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and then anhydrous potassium carbonate (345 mg, 2.5 eq) was added, and the mixture was reacted at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain compound 10a, a yellow oily liquid, with a yield of 56%. MS (ESI) m/z 434.5 [M + H] ⁺ .

Step 2. Synthesis of (4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-L-proline (X-37)

Intermediate 10a was removed from tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane ( ^{10 ml/mmol) for 4 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-37) as a white solid with a yield of 89%. 1} H NMR (300 MHz, DMSO-d ₆ ) δ 7.39–7.27 (m, 5H), 4.75 (s, 2H), 3.62 (d, J = 6.7 Hz, 2H), 3.51–3.43 (m, 2H), 3.07–2.86 (m, 2H), 2.77 (td, J = 10.0, 7.1 Hz, 1H), 2.19–1.89 (m, 2H), 1.89–1.67 (m, 2H), 1.65–1.56 (m, 4H).

Example 30: Synthesis of 4-benzyl-2-(4-(4-(cyclopropylcarbonyl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-38)

Step 1. Synthesis of 4-benzyl-2-(4-(4-(cyclopropylcarbonyl)piperazin-1-yl)butyl)-1,2,4-thiadiazolidine-3,5-dione hydrochloride (X-38)

Referring to the synthesis method of compound X-23, N-ethylpiperazine was replaced by 1-cyclopropanecarbonylpiperazine to obtain product X-38, a white solid, with a yield of 68%. ¹ H NMR (400MHz, DMSO-d ₆ )δ10.81(s,1H),7.39–7.29(m,5H),4.76(s,2H),4.40(d,J＝14.2Hz,2H),3.66(t,J＝6.7Hz,2H),3.09(dd,J＝9.9,5.4Hz,4H),2.01(dd,J＝11.2,4.8Hz,1H),1.72(dd,J＝10.9,5.5Hz,2H),1.63(q,J＝7.1Hz,2H),0.75(d,J＝7.6Hz,4H).

Example 31: Synthesis of N-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-N-methylglycine (X-39)

Step 1. Synthesis of tert-butyl N-(4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-N-methylglycinate (11a)

Compound X-22 (343 mg, 1.0 eq) and glycine tert-butyl ester (138 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and then anhydrous potassium carbonate (345 mg, 2.5 eq) was added, and the mixture was reacted at 80°C for 3-4 hours, and the reaction was monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried with anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain intermediate 11a, with a yellow oily liquid yield of 78%, and a light yellow oily liquid. MS (ESI) m/z 408.4 [M + H] ⁺ .

Step 2. Synthesis of N-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-N-methylglycine (X-39)

The intermediate 11a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 4 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-39) as a white solid with a yield of 90%. ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.36–7.19 (m, 5H), 4.19 (d, J = 4.5 Hz, 2H), 3.63 (s, 2H), 3.32 (s, 2H), 2.86 (d, J = 8.4 Hz, 2H), 2.59 (s, 3H), 1.46 (d, J = 7.6 Hz, 4H).

Example 32: Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalanine (X-40)

Step 1. Synthesis of tert-butyl (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalaninate (12a)

Refer to the synthesis method of intermediate 11a in Example 40, and replace glycine tert-butyl ester with L-phenylalanine tert-butyl ester hydrochloride. Intermediate 12a is an oily liquid with a yield of 70%. MS (ESI) m/z 484.5 [M + H] ⁺ .

Step 2. Synthesis of (2-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)ethyl)-L-phenylalanine (X-40)

Refer to the synthesis method of compound X-39 in Example 40. The product (X-40) is a white solid with a yield of 85%. ¹ HNMR (300 MHz, DMSO-d ₆ ) δ 7.39–7.18 (m, 10H), 4.75 (s, 2H), 3.60–3.55 (m, 2H), 3.46 (t, J=6.4 Hz, 2H), 3.07–2.89 (m, 3H), 2.75–2.60 (m, 2H).

Example 33: Synthesis of 2-(4-(1,4-diaza-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-41)

Step 1. Synthesis of tert-butyl 4-(4-(4-benzyl-3,5-dioxy-1,2,4-thiadiazolidin-2-yl)butyl)-1,4-diazane-1-carboxylate (13a)

Compound X-22 (343 mg, 1.0 eq) and tert-butyl 1,4-diazacycloheptane-1-carboxylate (210 mg, 1.05 eq) were dissolved in a sealed glass tube containing acetonitrile (5 mL), and potassium carbonate (345 mg, 2.5 eq) was added. The mixture was reacted at 80°C for 3-4 hours and monitored by TLC. After the reaction, the reaction solution was extracted with ethyl acetate three times, the organic layers were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by rapid silica gel column to obtain intermediate 13a as a yellow oily liquid with a yield of 51%. MS (ESI) m/z 463.5 [M + H] ⁺ .

Step 2. Synthesis of 2-(4-(1,4-diazepin-1-yl)butyl)-4-benzyl-1,2,4-thiadiazolidine-3,5-dione (X-41)

The intermediate 13a was removed from the tert-butyl ester in a mixed solution of trifluoroacetic acid (10 ml/mmol) and dichloromethane (10 ml/mmol) for 3-4 hours. After the reaction was completed by monitoring by TLC, it was concentrated under reduced pressure and purified by rapid silica gel column to obtain compound (X-41) as a white solid with a yield of 83%. ¹ H NMR (300 MHz, DMSO-d ₆ ) δ 7.39–7.28 (m, 5H), 4.75 (s, 2H), 3.67–3.63 (m, 2H), 3.45 (s, 4H), 3.25 (t, J＝5.4 Hz, 4H), 3.05 (s, 2H), 2.07 (d, J＝5.8 Hz, 2H), 1.62–1.60 (m, 4H).

2. Biological Evaluation

(1) PTPN2 kinase activity analysis test method

The established in vitro phosphatase activity assay was used to evaluate the inhibitory activity of the compounds on PTPN2 and calculate the IC ₅₀ value. The enzyme activity buffer of the reaction system was 50 mM Tris-cl, pH 7.2, 10 mM NaCl, 10% glycerol, 0.1% bovine serum albumin (BSA), and the reaction was carried out in a 96-well plate. The total reaction system was 100 μL and was divided into three groups: experimental group, negative control group, and positive control group. In the experimental group, 48 μL of PTPN2 (obtained by artificial purification) and 2 μL of the compound (dissolved in DMSO) were added to each well in turn, and the mixture was mixed by shaking for 30 s, and incubated at 37°C for 15 min. Then, 50 μL of 50 nM PNPP was added, and the mixture was mixed by shaking for 30 s, and incubated at 37°C for 5 min. Then, 100 μL of 1 M sodium hydroxide was added, and the absorbance at a wavelength of 405 nm was measured by a microplate reader. The log (inhibitor) vs. response-Variable slope of the analysis software GraphPad Prism was used to fit the dose-effect curve, thereby obtaining the IC ₅₀ value of each compound on the enzyme activity.

The obtained IC ₅₀ values are shown in Table 1, and it can be seen that the synthesized example compounds all have good inhibitory activity against PTPN2.

Table 1 IC ₅₀ values of the inhibitory activity of the example compounds on PTPN2 phosphatase

Example IC ₅₀ (μM) Example IC ₅₀ (μM) Example IC ₅₀ (μM) X-1 8.77 X-4 8.51 X-5 4.84 X-6 4.60 X-7 8.45 X-8 4.34 X-9 7.10 X-10 4.92 X-11 6.12 X-12 4.04 X-13 5.29 X-19 4.91 X-20 2.63 X-21 2.61 X-23 8.74 X-24 8.62 X-25 8.15 X-26 7.31 X-27 5.07 X-28 7.71 X-29 1.53 X-30 1.63 X-35 7.49 X-36 4.65 X-37 1.80 X-38 4.75 X-40 1.62 X-41 1.63

As can be seen from the table, the compounds of the present invention have very good inhibitory activity against PTPN2, among which compound 18 has the strongest inhibitory activity against PTPN2, and further in vivo activity evaluation was performed on it.

(2) Acute toxicity test of compounds

Test animals: ICR mice (provided by Shanghai Slake Laboratory Animal Co., Ltd.); 18-22 g; female; 20 mice in total.

Test sample: Example X-18

Group dose setting: a total of four groups were set up, control group; 1000mg/kg group; 2500mg/kg group: 5000mg/kg group: gavage drugs, each group was given once, and the control group was given an equal amount of solvent, 5 mice in each group, and observed for 14 days;

Experimental results: As shown in Figure 1, no abnormality was observed in the animals within 12 hours after administration of the drug in each group of mice. No animal death was observed within 24 hours of administration, and no animal death was observed after the 14th day of administration. No other obvious abnormalities were observed. There was no significant difference in the body weight of the mice in the three administration groups and that in the control group. There was no significant difference in the weight of the heart, liver, spleen, lung and kidney of the mice in the three administration groups and that in the control group. Therefore, no toxic reaction was observed when the test drug was administered orally at 1000 mg/kg, 2500 mg/kg and 5000 mg/kg, and Example X-19 has good safety.

(3) Determination of the anti-acute myeloid leukemia (AML) activity of the compounds

Test sample: Implementation X-18

Experimental methods: MOLM13-Luciferase (5×10 ⁵ ) tumor cells were injected into the tail vein of M-NSG mice to establish a MOLM13-Luciferase transplant tumor model. On the second day after inoculation, the tumor-bearing mice were randomly divided into two groups according to the Flux value: a control group and a compound group (10 mg/kg), with 5 mice in each group. On the third day after inoculation, intraperitoneal injection was performed once a day for 10 consecutive days. The weight of the mice was recorded every day, and the changes in tumor fluorescence values and the death of mice were detected.

The experimental results showed that, whether in the control group or the drug-treated group, the mice lost weight significantly on the 9th day, and the control group died on the 10th day. We used small animal imaging to detect the fluorescence intensity of mice on the 2nd and 9th days. As shown in Figure 2, on the 9th day, the fluorescence intensity in the mice of the drug-treated group was significantly lower than that of the blank group. Therefore, Example X-18 has a significant inhibitory effect on the growth of human acute myeloid leukemia cell MOLM13-Luciferase transplanted tumors.

Claims (7)

1. A compound represented by general formula (I) or a pharmaceutically acceptable salt thereof: Wherein, m=2 or 4; Y is O; R ₁ is selected from in: R is _Ra and _Rb are each independently selected from hydrogen, hydroxyl, carboxyl, _C1 - _C4 alkyl, C1- _C4 carboxylalkyl, _C1 - _C4 alkylcarbonyl, _{C1 - C4} alkylsulfonyl, _C3 - _C8 cycloalkylcarbonyl, _C6 - _C10 aryl, a 4-7 membered heteroaromatic ring having 1-3 heteroatoms selected from N and O, and a _C1 - _C3 alkyl substituted by a 4-7 membered heterocyclic ring having 1-3 heteroatoms selected from N and O; L is a covalent bond; R _c and R _d are each independently selected from hydrogen or an optionally substituted C ₁ -C ₃ alkyl group; the substituent is selected from hydrogen, hydroxyl, carboxyl or benzene ring. 2. The compound represented by the general formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein: when m=2, R ₁ is selected from -NHR ₂ , -NR ₃ R ₄ , wherein R ₂ is selected from -CH(CH ₃ )-C(O)OH, -CH(CH ₂ Ph)-C(O)OH, -NR ₃ R ₄ is selected from -N(CH ₃ )CH ₂ C(O)OH, R ₅ is selected from -CH ₂ -C(O)OH, -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(O)CH ₃ , RR ₆ is selected from -CH ₂ -C(O)OH, When m=4, R ₁ is selected from -NHR ₈ , -NR ₉ R ₁₀ , wherein R ₈ is selected from -CH(CH ₃ )-C(O)OH, -CH(CH ₂ Ph)-C(O)OH, -NR ₉ R ₁₀ is selected from -N(CH ₃ )CH ₂ C(O)OH, -R ₁₁ is selected from -CH ₃ , -CH ₂ CH ₃ , -CH(CH ₃ ) ₂ , -C(O)CH ₃ , 3. A compound or a pharmaceutically acceptable salt thereof, characterized in that the compound is selected from the following structures: 4. Preparation method of the compound represented by general formula (I): wherein Y, m, and _R1 are as defined in any one of claims 1 to 3. 5. A pharmaceutical composition comprising the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers. 6. Use of the compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating a disease mediated by PTPN2. 7. The use according to claim 6, characterized in that the PTPN2-mediated disease is acute myeloid leukemia.