CN112209884B - 1-H benzimidazole derivative, preparation method and application thereof - Google Patents

1-H benzimidazole derivative, preparation method and application thereof Download PDF

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CN112209884B
CN112209884B CN201910627148.0A CN201910627148A CN112209884B CN 112209884 B CN112209884 B CN 112209884B CN 201910627148 A CN201910627148 A CN 201910627148A CN 112209884 B CN112209884 B CN 112209884B
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蔡剑
何永富
方静芽
王树龙
葛求富
徐伟良
钟松鹤
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Hangzhou Tino Pharma Co ltd
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/06—Benzimidazoles; Hydrogenated benzimidazoles with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached in position 2
    • C07D235/16—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
    • C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
    • C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
    • C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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Abstract

The invention discloses 1-H benzimidazole derivatives shown in the figure, a preparation method and application thereof in the field of pharmacy, in particular to application in antitumor drugs. Meanwhile, the derivatives A, B, C and E are main related substances of the 1-H benzimidazole derivative NL-101 to be marketed, and can be used as standard substances in the NL-101 approval, analysis, identification and quality control research process. Also disclosed is a method of analyzing NL-101.

Description

1-H benzimidazole derivative, preparation method and application thereof
Technical Field
The invention belongs to the field of medicines, and relates to a 1-H benzimidazole derivative, and a preparation method and application thereof.
Background
Nitrogen mustard drugs were one of the earliest chemotherapeutic drugs used to treat malignancies. The main anti-tumor activity of the medicine is derived from the DNA damage effect of the medicine, and the strong DNA repair reaction can be activated by the crosslinking of a DNA double strand; if DNA repair is ineffective, it will cause strong p53 activation, eventually leading to apoptosis and necrosis. Bendamustine is a nitrogen mustard antineoplastic drug synthesized as early as 1963. Preclinical studies have shown that bendamustine is effective in inducing apoptosis in lymphoma cells, inhibiting the growth of malignant lymphoma, multiple myeloma, and various solid tumors. Bendamustine was approved by the FDA for the treatment of chronic lymphocytic leukemia at 3 months 2008, and approved for the treatment of non-hodgkin's lymphoma at 10 months in the same year. Bendamustine is also approved by the european drug administration for the treatment of multiple myeloma. The data show that bendamustine has also been clinically tested for a variety of solid tumors: soft tissue sarcoma, small cell lung cancer, breast cancer, cholangiocarcinoma, etc.
The nitrogen mustard medicines have definite clinical curative effects and are used for decades. However, in tumor cells HDACs are often overexpressed or abnormally activated, depleting the negatively charged acetyl groups on histones, leading to tight binding of DNA duplexes and histones (Kim HJ, et al. American journal of translational research.2011;3 (2): 166-79 Bartek J, et al. Nature reviews. Molecular cell biology.2004 5 (10): 792-804 Kim MS, et al. Cancer research.1 (21): 7291-300), rendering the DNA less susceptible to attack by alkylating agents; moreover, HDAC overexpression or abnormal activation is also involved in DNA damage repair (Munshi A, et al. Clinical Cancer research: an of the American Association for Cancer research.1 (13): 4912-22, chen CS, et al. Cancer research.Jun 1 2007 (11): 5318-27), making tumor cells susceptible to resistance or cross-resistance to such drugs, limiting the clinical efficacy of nitrogen mustard drugs. Compared with the traditional nitrogen mustard antineoplastic drugs, the patient has better tolerance to the modern molecular targeted HDAC inhibitor antineoplastic drugs, but the antineoplastic activity of the patient is not strong. Currently such drugs are mainly used for the treatment of T cell lymphomas.
Through precise analysis of the structure-activity relationship between bendamustine and vorinostat, a novel small molecule compound NL-101 having both DNA alkylation and HDAC inhibition effects is developed. NL-101 is one of the 1-H benzimidazole derivatives that has a bifunctional, potent inhibition of the HDAC pathway. NL-101 has the chemical formula, formula and molecular weight shown below, under the chemical name 7- [5- [ bis (chloroethyl) -amino ] -1-methylbenzimidazol-2-yl ] -N-hydroxy-heptanamide.
Figure BDA0002127460580000021
NL-101 is the first anti-malignant tumor compound with both DNA damage and Histone Deacetylase (HDAC) inhibition effect globally and was first disclosed in WO2010085377A2, the national homology CN102186842B of which is now authorized, and the patentee is Hangzhou Tinuo medicine science and technology Co., ltd. In 2016, phase I clinical trials were started in several countries such as the United states and Europe, and in 2017, clinical trials were approved in China.
Although NL-101 is a very excellent potential antitumor drug, NL-101 mainly has the defects of poor stability, fast in vivo metabolism, poor solubility, large toxicity, space improvement of antitumor activity and the like. Therefore, the intensive research on the compounds synthesizes a series of novel compounds with excellent potential.
Reference to substances refers to process impurities and degradation impurities that may occur during the manufacturing process. The process impurities refer to impurities generated by reaction in the process of synthesizing the raw material medicine and impurities carried by a solvent and a raw material used in the process of synthesizing, and the degradation impurities refer to impurities generated by chemical reactions of hydrolysis, oxidation, decomposition, isomerization, polymerization and the like of the medicine under the conditions of specific temperature, humidity, illumination and air. Any substance that affects the purity of a drug is collectively referred to as a related substance (i.e., an impurity).
It is known that: when registering medicines, the related substances with the content of more than 0.1 percent need to be researched, identified, controlled and formulated to the highest limit. NL-101 is on the market conditionally in the United states, and the clinical progress is smooth in China. Therefore, we studied NL-101-related substances and their preparation and detection.
Disclosure of Invention
Firstly, through a large number of experimental studies, relevant substance reference substances with the content of more than 0.1 percent, which are necessary for controlling the quality of NL-101 raw material medicines, are determined and obtained. NL-101 mainly relates to substances A, B, C and E and a synthesis method thereof, and the structural formula is as follows:
Figure BDA0002127460580000031
the compounds A and C are new compounds which are synthesized for the first time, and the compounds B and E are disclosed, but the experimental researches such as the improvement of a synthesis route, purification, identification and the like are carried out, so that the compounds A and C completely meet the application requirements of serving as standard products in the processes of approval, analysis and quality control research as the compounds A and C.
The identification and study of NL-101 related substances is an important part of the development of NL-101 drugs.
The control of the substances involved has a crucial influence on the safety and efficacy of the final drug product. Therefore, NL-101 related substances and preparation methods thereof are determined, and for establishing a detection method, the content of related substances is analyzed, and reasonable related substance limits are determined. Plays an important role in the quality control and the clinical medication safety of the NL-101.
Next, the structure of 1-hydrobenzimidazole (NL-101 is one of the 1-hydrobenzimidazole derivatives) was further modified. A large number of new compounds are obtained, and the pharmacological studies such as antitumor activity evaluation and the like show that part of the compounds have the following advantages compared with NL-101:
1. the better antitumor activity of the TN-111 compound is one order of magnitude higher than that of NL-101. In vitro Activity results on MDA-MB-231 cell line showed IC for NL-101 50 IC with value of 25.85. Mu.M, TN-111 50 The value was 1.17. Mu.M.
2. Better metabolic stability, TN-431 compound, has comparable antitumor activity with NL-101, but better metabolic stability. TN-431 is metabolized differently compared to NL-101. The hydroxamic acid hydrolysis products in the molecular structure are both one of the main metabolites of TN-431 and NL-101. (1) TN-431 is metabolized more slowly than NL-101, t 1/2 About 18 minutes and 10 minutes, respectively; (2) no beta-oxidation product is found in TN-431 metabolite; (3) The single chloro hydrolyzed impurity in the dichloroethylamino group is one of the major metabolites in whole blood, and no similar metabolite is found in NL-101 metabolism; (4) Glucuronic acid conjugates were not found in TN-431 metabolites.
3. And the safety is higher. The TN-901 compound has lower toxicity than NL-101 and has equivalent anti-tumor activity. NL-101 has an MTD of 50mg/kg in ICR mice, while TN-901 has an MTD of 150mg/kg.
The following classes of compounds were obtained:
a compound having the following structural formula:
Figure BDA0002127460580000041
wherein R is 2 Is CH 2 CH 2 Cl；
R 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H or is methyl;
R 4 is composed of
Figure BDA0002127460580000042
n is optionally 0, 1,2, 3, 4, 5, 6, 7, 8, 9, n is preferably 5, 6, 7;
when R is 1 Is CH 2 CH 2 Cl or CH 2 CH 2 OH，R 4 Is composed of
Figure BDA0002127460580000043
And n =6, R 3 Is not methyl;
when R3 is methyl, R 1 Is CH2CH2Cl or CH2CH2OH, and R 4 Is composed of
Figure BDA0002127460580000044
When n is not 2 or 6.
A compound having the structural formula:
Figure BDA0002127460580000045
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl，
R 3 Is H or is methyl;
n is optionally 3, 4, 5, 6 or 7;
when R is 1 Is CH 2 CH 2 Cl or CH 2 CH 2 OH, and n =6, R 3 Is not methyl.
A compound having the structural formula:
Figure BDA0002127460580000051
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H or methyl;
n is optionally 3, 4, 5, 6 or 7;
R 3 is methyl; and R is 1 Is CH 2 CH 2 Cl or CH 2 CH 2 OH, n is not 2 or 6.
A compound having the structural formula:
Figure BDA0002127460580000052
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H or methyl;
R 4 is composed of
Figure BDA0002127460580000053
Drawings
FIG. 1-H benzimidazole derivative structural formula
FIG. 2 NL-101 analysis of liquid phase spectrogram
DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION
The synthetic routes and examples are only given as a way of preparing such compounds and are not intended to be limiting.
Example 1
TN-101 was synthesized as follows.
Figure BDA0002127460580000061
Synthesis of Compound 1:
100.00g (0.654 mol) of 4-nitro-1, 2-phenylenediamine and 182.00g (1.045 mol) of suberic acid are added into 500mL of 6M hydrochloric acid, stirred and refluxed for 5 hours, 500mL of water is added, the pH is adjusted to 11-12 by KOH under stirring, filtration is carried out, the pH of filtrate is adjusted to 5-6 by glacial acetic acid, solid is precipitated, filtration is carried out, washing is carried out for a plurality of times by water, and then drying is carried out at 65 ℃, thus obtaining 116.94g of compound 1 with the yield of 61.47%.
Synthesis of Compound 2:
116.94g (0.401 mol) of Compound 1, 660mL of anhydrous ethanol and 35mL of concentrated sulfuric acid were stirred under reflux for 4 hours, the reaction mixture was slowly poured into 2200g of a 10% potassium carbonate solution precooled to about 10 ℃, filtered, the solid was washed with water several times, dried at 50 ℃ and recrystallized from isopropyl ether and ethanol to give 71.43g of Compound 2 with a yield of 61.68%.
Synthesis of Compound 3:
70.00g (0.219 mol) of Compound 2 was added to 1200mL of tetrahydrofuran, and dissolved by stirring, 14.0g of 5% palladium on charcoal (50% wet with water) was added, and after hydrogen gas was replaced by vacuum, hydrogenation was carried out under stirring for 8 hours or longer. After TLC detection, filtering, decompressing filtrate and evaporating solvent to obtain compound 3 as viscous liquid with yield near 100%.
Synthesis of Compound 4:
31.83g (0.110 mol) of Compound 3, 0.83g of sodium acetate, 57.00g of acetic acid and 170.00g of water were dissolved together with stirring, cooled to-10 to 0 ℃ and added with 25.17g (0.571 mol) of ethylene oxide, and the mixture was slowly warmed to room temperature to react for 24 to 48 hours. The reaction solution was poured into 1100g of a 10% potassium carbonate solution precooled to about 10 ℃ under stirring, and after completion of the addition, it was extracted with dichloromethane (400 mL. Times.2). The organic phase was washed with 500mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated to give 38.36g of Compound 4 in 92.38% yield.
Synthesis of compound 5 hydrochloride salt:
37.75g (0.100 mol) of the compound 4 is dissolved in 250mL of dichloromethane, 0.1mL of N, N-dimethylformamide is added, 30.93g (0.260 mol) of thionyl chloride is dropwise added under cooling of an ice water bath, reaction is carried out for 12-24 hours at room temperature after the addition, concentration is carried out, 50mL of absolute ethyl alcohol is added, heating and dissolution are carried out, 500mL of ethyl acetate is added for crystallization, filtration is carried out under the protection of nitrogen, the solid is washed by 100mL of ethyl acetate, and vacuum drying is carried out for 10-12 hours at 45 ℃, 33.31g of compound 5 hydrochloride of the off-white solid is obtained, and the yield is 73.89%.
Synthesis of TN-101:
under an ice-water bath, 34.50g (0.615 mol) of potassium hydroxide is dissolved in 150mL of methanol, added into a mixture of 33.31g (0.479 mol) of hydroxylamine hydrochloride and 100mL of methanol, stirred at room temperature for 2-3 hours, filtered, the filtrate is cooled to 0-10 ℃, 13.53g (0.030 mol) of compound 5 hydrochloride is added, stirred and reacted for 1 hour, the pH value is adjusted to 6-8 by glacial acetic acid, 250mL of water is added, filtered, washed by water and dried in vacuum at 50 ℃ for 10-12 hours, 10.64g of off-white solid TN-101 is obtained, the yield is 88.37%, and the purity is 99.02% calculated by an HPLC area normalization method.
1H-NMR(d6-DMSO)δ:12.09(s,1H)10.38(s,1H)；8.73(br,1H)；7.31～7.32(d,1H)；6.91-6.93(d,1H)；6.76～6.78(dd,1H)；3.67～3.72(m,8H)；2.75～2.78(t,2H)；1.93～1.96(t,2H)；1.68～1.74(m,2H)；1.47～1.52(m,2H)；1.26～1.39(m,4H)。
[M+H]+:401.1510
Example 2
TN-102 was synthesized as follows.
Figure BDA0002127460580000071
The hydrochloride salt of compound 1 was obtained from example 1.
Synthesis of TN-102:
9.02g (0.0200 mol) of the hydrochloride of the compound 1 and 50mL of concentrated hydrochloric acid are stirred, added and flowed for 5 hours, the solvent is evaporated under reduced pressure, the evaporation residue is dissolved by 80mL of methanol, the pH value of the KOH solution with 10 percent under the cooling of ice water bath is adjusted to 4-5, filtered, the solid is washed by 30mL of multiplied by 2, and dried in vacuum for 10-12 hours at 65 ℃ to obtain 6.46g of TN-102 as a white solid with the yield of 83.56 percent, and the purity of the TN-102 is 98.36 percent by HPLC area normalization.
1H-NMR(d6-DMSO)δ:11.93(s,1H)；10.36(s,1H)；7.30～7.32(d,1H)；6.91-6.93(d,1H)；6.76～6.79(dd,1H)；3.73(m,8H)；2.79～2.82(t,2H)；2.21～2.23(t,2H)；1.72～1.76(m,2H)；1.48～1.53(m,2H)；1.31～1.40(m,4H)。
[M+H]+:386.1398
Example 3
TN-103 was synthesized as follows.
Figure BDA0002127460580000081
Synthesis of Compound 1:
compound 1 was synthesized according to the procedure of example 1.
Synthesis of Compound 2:
11.58g (0.040 mol) of Compound 1, 0.30g of sodium acetate, 20.00g of acetic acid and 60.00g of water were dissolved by stirring, cooled to-10 to 0 ℃ and 4.58g (0.104 mol) of ethylene oxide was added, and the mixture was slowly warmed to room temperature to react for 12 to 24 hours. The reaction solution was poured into 400g of a 10% potassium carbonate solution precooled to about 10 ℃ under stirring, and after completion of the addition, the mixture was extracted with dichloromethane (100 mL. Times.2). The organic phase was washed with 100mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and then purified by silica gel column separation to obtain 5.08g of Compound 2 with a yield of 37.48%.
Synthesis of compound 3 hydrochloride salt:
dissolving 3.39g (0.010 mol) of compound 2 in 30mL of dichloromethane, adding 2 drops of N, N-dimethylformamide, dropwise adding 1.79g (0.015 mol) of thionyl chloride while cooling in an ice water bath, reacting at room temperature for 24 hours after the addition, concentrating, adding 10mL of absolute ethyl alcohol, heating for dissolution, adding 40mL of ethyl acetate for crystallization, filtering under the protection of nitrogen, washing the solid with 10mL of ethyl acetate, and drying in vacuum at 45 ℃ for 10-12 hours to obtain 3.55g of off-white solid compound 3 hydrochloride, wherein the yield is 91.42%.
Synthesis of TN-103:
under an ice-water bath, 8.63g (0.154 mol) of potassium hydroxide is dissolved in 40mL of methanol, added to a mixture of 8.63g (0.124 mol) of hydroxylamine hydrochloride and 25mL of methanol, stirred at room temperature for 2-3 hours, filtered, the filtrate is cooled to 0-10 ℃, 2.50g (6.44 mmol) of compound 4 hydrochloride is added, stirred and reacted for 0.5 hour, the pH is adjusted to 6-8 by glacial acetic acid, 65mL of water is added, filtered, washed with water, and dried under vacuum at 50 ℃ for 10-12 hours, so that 1.79g of off-white solid TN-103 is obtained, the yield is 82.03%, and the purity is 98.74% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:12.06(s,1H)；10.37(s,1H)；8.74(br,1H)；7.31～7.33(d,1H)；6.92-6.94(d,1H)；6.76～6.79(dd,1H)；5.43～5.45(t,1H)；3.67～3.72(m,4H)；2.74～2.77(t,2H)；1.94～1.96(t,2H)；1.68～1.74(m,2H)；1.44～1.49(m,2H)；1.25～1.38(m,4H)。
[M+H]+:339.1585
Example 4
TN-104 was synthesized as follows.
Figure BDA0002127460580000091
Synthesis of compound 1 hydrochloride salt:
synthesized as in example 3.
Synthesis of TN-104:
synthesized in a similar manner to example 2, TN-104 was a white solid compound in a yield of 78.46% and a purity of 98.16% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:11.86(s,1H)；10.33(s,1H)；7.31～7.33(d,1H)；6.92-6.94(d,1H)；6.74～6.78(dd,1H)；5.41～5.43(t,1H)；3.66～3.721(m,4H)；2.73～2.76(t,2H)；1.95～1.97(t,2H)；1.63～1.69(m,2H)；1.44～1.48(m,2H)；1.25～1.36(m,4H)。
[M+H]+:324.1480
Example 5
TN-105 was synthesized as follows.
Figure BDA0002127460580000101
Synthesis of hydrochloride salt of compound 1:
synthesized according to the method of example 3
Synthesis of Compound 2:
2.68g (6.90 mmol) of Compound 1 hydrochloride was dissolved in 20mL of dichloromethane, 30mL of a saturated sodium bicarbonate solution was added, stirring was carried out for 10 minutes, the mixture was allowed to stand for separation, and the organic phase was dried over anhydrous magnesium sulfate and filtered. The filtrate was evaporated under reduced pressure to remove the solvent, 3.60g of acetic acid and 12.00g of water were added, stirred and dissolved, cooled to-10-0 deg.C, 0.79g (17.93 mmol) of ethylene oxide was added, and the mixture was allowed to warm to room temperature and reacted for 24-48 hours. The reaction solution was poured into 70g of a 10% potassium carbonate solution precooled to about 10 ℃ under stirring, and after completion of the addition, the mixture was extracted with methylene chloride (20 mL. Times.2). The organic phase was washed with 20mL of water, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and then purified by silica gel column separation to obtain 1.27g of Compound 2 with a yield of 46.49%.
Synthesis of TN-105:
under an ice-water bath, 8.63g (0.154 mol) of potassium hydroxide is dissolved in 40mL of methanol, then added into a mixture of 8.63g (0.124 mol) of hydroxylamine hydrochloride and 25mL of methanol, stirred at room temperature for 2-3 hours, filtered, 15mL of filtrate is taken to be cooled to 0-10 ℃,0.73g (1.84 mmol) of compound 2 is stirred and reacted for 0.5 hour, the pH value is adjusted to 6-8 by glacial acetic acid, 15mL of water is added, filtered, washed and dried in vacuum at 50 ℃ for 10-12 hours, 0.61g of off-white solid TN-105 is obtained, the yield is 86.58%, and the purity is 97.86% calculated by an HPLC area normalization method.
1H-NMR(d6-DMSO)δ:12.09(s,1H)10.37(s,1H)；8.72(br,1H)；7.31～7.32(d,1H)；6.91-6.93(d,1H)；6.76～6.78(dd,1H)；4.65～4.68(t,1H)；3.68～3.73(m,4H)；3.54～3.56(q,2H)；3.43～3.45(q,2H)；2.74～2.76(t,2H)；1.94～1.97(t,2H)；1.68～1.74(m,2H)；1.47～1.52(m,2H)；1.26～1.39(m,4H)。
[M+H]+:383.1849
Example 6
TN-106 was synthesized as follows.
Figure BDA0002127460580000111
Synthesis of Compound 1:
synthesized according to the method of example 4.
Synthesis of TN-106:
1.62g (5.00 mmol) of Compound 1, 2.50g of acetic acid and 7.50g of water were dissolved with stirring, cooled to-10 to 0 ℃ and added with 0.57g (12.94 mmol) of ethylene oxide, and the mixture was allowed to warm to room temperature and reacted for 24 to 48 hours. The reaction solution was poured into 50g of a 10% potassium carbonate solution precooled to about 10 ℃ with stirring, and after completion of the addition, extracted with dichloromethane (25 mL. Times.2). The organic phase was washed with 20mL of water, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and then purified by silica gel column separation to obtain 0.78g of TN-106 with a yield of 46.04% and a purity of 98.39% by HPLC area normalization. 1H-NMR (d 6-DMSO). Delta.11.87 (s, 1H) 10.38 (s, 1H); 7.32 to 7.33 (d, 1H); 6.95-6.97 (d, 1H); 6.78-6.8 (dd, 1H); 4.66-4.69 (t, 1H); 3.70-3.75 (m, 4H); 3.55-3.57 (q, 2H); 3.44-3.46 (q, 2H); 2.76-2.78 (t, 2H); 1.95-1.98 (t, 2H); 1.68-1.73 (m, 2H); 1.48 to 1.53 (m, 2H); 1.25 to 1.38 (m, 4H).
[M+H]+:368.1740
Example 7
TN-111 was synthesized as follows.
Figure BDA0002127460580000121
Synthesis of Compound 1:
33.44g (0.200 mol) of N1-methyl-4-nitrobenzene-1, 2-diamine was added to 250mL of dichloromethane, 22.02g (0.220 mol) of succinic anhydride was added in portions with stirring, reacted at room temperature for 12 to 24 hours, filtered, the solid was washed with 100mL of isopropyl ether, and dried at 65 ℃ to obtain 52.73g of Compound 1, with a yield of 98.66%.
Synthesis of Compound 2:
48.10g (0.180 mol) of Compound 1, 300mL of anhydrous ethanol and 17mL of concentrated sulfuric acid were stirred under reflux for 4 hours, the reaction mixture was slowly poured into 1000g of a 10% potassium carbonate solution precooled to about 10 ℃, filtered, the solid was washed with water, dried at 50 ℃ and recrystallized from ethanol and isopropyl ether to give 46.31g of Compound 2 with a yield of 92.79%.
Synthesis of Compound 3:
adding 45.75g (0.165 mol) of compound 2 into 900mL of tetrahydrofuran, stirring for dissolution, adding 9.15g of 5% palladium-carbon (50% water wet), vacuumizing for replacing hydrogen, stirring for hydrogenation for more than or equal to pumping hours. After TLC detection, filtering, decompressing filtrate and evaporating solvent to obtain compound 3 as viscous liquid with yield near 100%.
Synthesis of Compound 4:
40.80g (0.165 mol) of Compound 2, 1.25g of sodium acetate, 85.00g of acetic acid and 250.00g of water were dissolved with stirring, cooled to-10 to 0 ℃ and 37.82g (0.859 mol) of ethylene oxide was added, and the mixture was slowly warmed to room temperature to react for 24 to 48 hours. The reaction solution was poured into 1600g of a 10% potassium carbonate solution precooled to about 10 ℃ under stirring, and after completion of the addition, it was extracted with methylene chloride (300 mL. Times.2). The organic phase was washed with 300mL of saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and recrystallized from ethyl acetate to give 43.87g of Compound 4 in 79.27% yield.
Synthesis of Compound 5:
33.54g (0.100 mol) of Compound 4 was dissolved in 250mL of dichloromethane, 0.1mL of N, N-dimethylformamide was added, 28.55g (0.240 mol) of thionyl chloride was added dropwise with cooling in an ice-water bath, and after completion of the addition, the reaction was carried out at room temperature for 12 to 24 hours, followed by concentration, 250mL of dichloromethane was added, stirring was carried out to dissolve the resulting mixture, the organic phase was washed with 250mL of each of water and a saturated sodium bicarbonate solution, dried over anhydrous sodium sulfate, filtered, and after concentration of the filtrate, isopropyl ether was recrystallized to give 22.60g of Compound 5, with a yield of 60.71%.
Synthesis of TN-111:
under ice-water bath, 34.50g (0.615 mol) of potassium hydroxide is dissolved in 150mL of methanol, added into a mixture of 34.50g (0.496 mol) of hydroxylamine hydrochloride and 100mL of methanol, stirred for 2-3 hours at room temperature, filtered, 200mL of filtrate is cooled to 0-10 ℃, 10.01g (0.0269 mol) of compound 5 is added, stirred and reacted for 0.5 hour, the pH is adjusted to 6-8 by glacial acetic acid, 200mL of water is added, filtered, washed, and dried in vacuum for 10-12 hours at 50 ℃ to obtain 8.88g of off-white solid TN-111, the yield is 91.89%, and the purity is 99.14% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.50(s,1H)；8.72(br,1H)；7.24～7.26(d,1H)；6.83-6.84(d,1H)；6.70～6.72(dd,1H)；3.64(s,3H)；3.53～3.55(t,4H)；3.42～3.44(t,4H)；2.76～2.79(t,2H)；2.53～2.58(t,2H)。
[M+H]+:359.1041
Example 8
TN-112 was synthesized as follows.
Figure BDA0002127460580000131
Synthesis of Compound 1:
synthesized according to the method of example 7
Synthesis of Compound 2:
synthesized by a similar method to example 3, yield 42.62%.
Synthesis of hydrochloride salt of compound 3:
synthesized in a similar manner to example 3, compound 3 hydrochloride was a white solid compound in a yield of 92.49%.
Synthesis of TN-112:
synthesized in a similar manner to example 3, TN-112 was a white solid compound in a yield of 81.53% and a purity of 98.79% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.36(s,1H)；8.63(br,1H)；7.34～7.37(d,1H)；6.92-6.95(d,1H)；6.74～6.77(dd,1H)；5.45～5.47(t,1H)；3.64(s,3H)；3.55～3.58(t,2H)；3.43～3.45(t,2H)；2.72～2.75(t,2H)；2.53～2.58(t,2H)。
[M+H]+:297.1118
Example 9
TN-113 was synthesized as follows.
Figure BDA0002127460580000141
Synthesis of hydrochloride salt of compound 1:
synthesized as in example 8.
Synthesis of TN-113:
synthesized in a similar manner to example 2, TN-113 was a white solid compound in a yield of 75.26% and a purity of 98.35% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.38(s,1H)；7.36～7.39(d,1H)；6.93-6.96(d,1H)；6.72～6.75(dd,1H)；5.47～5.49(t,1H)；3.64(s,3H)；3.58～3.61(t,2H)；3.44～3.46(t,2H)；2.73～2.76(t,2H)；2.55～2.60(t,2H)。
[M+H]+:282.1006
Example 10
TN-114 was synthesized as follows.
Figure BDA0002127460580000151
Synthesis of Compound 1:
synthesized as in example 8.
Synthesis of Compound 2:
2.33g (8.00 mmol) of Compound 1, 20mL of diglyme, 1.49g (12.00 mmol) of 2- (2-chloroethoxy) ethanol, and 2.21g (16.00 mmol) of potassium carbonate were refluxed for 12 to 24 hours. After filtration and concentration of the filtrate, the mixture was separated and purified by a silica gel column to obtain 2.41g of compound 2 with a yield of 79.39%.
Synthesis of Compound 3:
2.28g (6.00 mmol) of Compound 2 was dissolved in 20mL of dichloromethane, 2 drops of mLN, N-dimethylformamide were added, 1.86g (0.156 mol) of thionyl chloride was added dropwise with cooling in an ice-water bath, and after completion of the addition, the reaction was carried out at room temperature for 12 to 24 hours, the solvent was distilled off under reduced pressure, and 20mL of dichloromethane was added and dissolved, and the resulting solution was washed with 20mL of each of water and a saturated sodium bicarbonate solution. The organic phase was dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated and then purified by silica gel column separation to obtain 1.81g of compound 3 with a yield of 72.46%.
Synthesis of TN-114:
under an ice-water bath, 8.63g (0.154 mol) of potassium hydroxide is dissolved in 40mL of methanol, then added into a mixture of 8.63g (0.124 mol) of hydroxylamine hydrochloride and 25mL of methanol, stirred at room temperature for 2-3 hours, filtered, 12mL of filtrate is cooled to 0-10 ℃, 0.50g (1.20 mmol) of compound 3 is added, stirred and reacted for 0.5 hour, the pH value is adjusted to 6-8 by glacial acetic acid, 15mL of water is added, filtered, washed with water, and dried under vacuum at 50 ℃ for 10-12 hours, 0.37g of off-white solid TN-114 is obtained, the yield is 76.45%, and the purity is 97.29% calculated by an HPLC area normalization method.
1H-NMR(d6-DMSO)δ:10.46(s,1H)；8.76(br,1H)；7.26～7.28(d,1H)；6.83-6.85(d,1H)；6.72～6.74(dd,1H)；4.34～4.35(t,2H)；3.68(s,3H)；3.53～3.55(t,2H)；3.42～3.44(t,8H)；2.76～2.79(t,2H)；2.53～2.57(t,2H)。
[M+H]+:403.1298
Example 11
TN-115 was synthesized as follows.
Figure BDA0002127460580000161
Synthesis of Compound 1:
synthesized by the method of example 10
Synthesis of TN-115:
synthesized in a similar manner to example 2, TN-115 was a white-like solid compound in a yield of 78.66% and a purity of 97.49% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.43(s,1H)；7.24～7.26(d,1H)；6.82-6.84(d,1H)；6.73～6.75(dd,1H)；4.34～4.35(t,2H)；3.69(s,3H)；3.53～3.55(t,2H)；3.42～3.44(t,8H)；2.76～2.79(t,2H)；2.53～2.57(t,2H)。
[M+H]+:388.1196
Example 12
TN-116 was synthesized as follows.
Figure BDA0002127460580000162
Synthesis of Compound 1:
synthesized according to the method of example 8
Synthesis of Compound 2:
synthesized in a similar manner to example 5, yield 52.48%
Synthesis of TN-116:
synthesized in a similar manner to example 5, TN-116 was a off-white solid compound in a yield of 83.61% and a purity of 97.53% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.36(s,1H)；8.63(br,1H)；7.34～7.37(d,1H)；6.92-6.95(d,1H)；6.74～6.77(dd,1H)；4.66～4.69(t,1H)；3.67～3.72(m,2H)；3.65(s,3H)；3.53～3.55(t,2H)；3.42～3.44(t,4H)；2.78～2.81(t,2H)；2.55～2.60(t,2H)。
[M+H]+:341.1380
Example 13
TN-117 was synthesized as follows.
Figure BDA0002127460580000171
Synthesis of Compound 1:
synthesized according to the method of example 9
Synthesis of TN-117:
synthesized in a similar manner to example 6, TN-117 was a off-white solid compound in a yield of 42.48% and a purity of 98.26% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.38(s,1H)；7.32～7.35(d,1H)；6.91-6.94(d,1H)；6.73～6.75(dd,1H)；4.68～4.72(t,1H)；3.65～3.70(m,2H)；3.64(s,3H)；3.54～3.56(t,2H)；3.42～3.44(t,4H)；2.80～2.83(t,2H)；2.56～2.61(t,2H)。
[M+H] + :326.1272
Example 14
TN-411 was synthesized as follows.
Figure BDA0002127460580000181
Synthesis of Compound 1:
34.76g (0.179 mol) of monoethyl terephthalate, 0.1mL of N, N-dimethylformamide and 100.00g (0.841 mol) of thionyl chloride were heated under reflux for 3 hours, the excess thionyl chloride was evaporated under reduced pressure and dissolved in 75mL of methylene chloride, and the resulting solution was added dropwise to a mixture of 29.92g (0.179 mol) of N1-methyl-4-nitrobenzene-1, 2-diamine, 25.85g (0.200 mol) of N, N-diisopropylethylamine and 500mL of methylene chloride with stirring and cooling. After the addition, the reaction was stirred at room temperature for 5 hours, the solvent was distilled off under reduced pressure, 500mL of water was added, the mixture was filtered after stirring for 0.5 hour, and the solid was washed with water and dried to obtain 60.43g of Compound 1, with a yield of 98.33%.
Synthesis of Compound 2:
synthesized in a similar manner to example 7, yield 83.11%.
Synthesis of Compound 3:
synthesized in a similar manner to example 7, with a yield of approximately 100%.
Synthesis of Compound 4:
synthesized in a similar manner to example 7, yield 80.93%.
Synthesis of Compound 5:
synthesized in a similar manner to example 7, yield was 61.03%.
Synthesis of TN-411:
under an ice water bath, 26.62g (0.474 mol) of potassium hydroxide is dissolved in 120mL of methanol, added into a mixture of 25.52g (0.367 mol) of hydroxylamine hydrochloride and 80mL of methanol, stirred at room temperature for 2-3 hours, filtered, 7.34g (0.0175 mol) of compound 5 and 50mL of dichloromethane are added into filtrate, stirred and reacted for 1 hour, the pH value is adjusted to 6-8 by glacial acetic acid, 250mL of water is added, filtered, washed by water, and dried in vacuum at 65 ℃ for 10-12 hours, so that 6.64g of pale yellow solid TN-411 is obtained, the yield is 93.16%, and the purity is 98.88% calculated by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.56(s,1H)；8.78(br,1H)；7.93～7.95(d,2H)；7.80～7.82(d,2H)；7.37～7.39(d,1H)；6.98-7.01(d,1H)；6.85～6.87(dd,1H)；3.86(s,3H)；3.67～3.72(m,4H)；3.53～3.58(m,4H)。
[M+H]+:407.1039
Example 15
TN-412 was synthesized as follows.
Figure BDA0002127460580000191
Synthesis of Compound 1:
synthesized according to the method of example 14.
Synthesis of Compound 2:
synthesized in a similar manner to example 3, yield 34.27%.
Synthesis of hydrochloride salt of compound 3:
synthesized in a similar manner to example 3, compound 3 hydrochloride was a white solid compound in a yield of 90.58%. Synthesis of TN-412:
synthesized in a similar manner to example 14, TN-412 was a pale yellow solid TN-412 in a yield of 86.19% and a purity of 97.64% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.65(s,1H)；8.76(br,1H)；7.96～7.98(d,2H)；7.81～7.83(d,2H)；7.40～7.42(d,1H)；7.01-7.03(d,1H)；6.87～6.89(dd,1H)；5.74～5.77(t,1H)；3.93(s,3H)；3.64～3.69(m,2H)；3.50～3.55(m,2H)。
[M+H]+:345.1117
Example 16
TN-413 was synthesized as follows.
Figure BDA0002127460580000201
Synthesis of Compound 1:
synthesized according to the method of example 15.
Synthesis of Compound 2:
synthesized in a similar manner to example 10, yield 76.36%.
Synthesis of Compound 3:
synthesized in a similar manner to example 10, in 73.82% yield.
Synthesis of TN-413:
synthesized in a similar manner to example 14, TN-413 was a pale yellow solid in a yield of 88.46% and had a purity of 97.33% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.66(s,1H)；8.78(br,1H)；7.92～7.94(d,2H)；7.80～7.82(d,2H)；7.36～7.38(d,1H)；6.95-6.98(d,1H)；6.83～6.85(dd,1H)；4.31～4.32(t,2H)；3.84(s,3H)；3.52～3.54(t,2H)；3.40～3.42(t,8H)。
[M+H]+:451.1306
Example 17
TN-414 was synthesized as follows.
Figure BDA0002127460580000211
Synthesis of compound 1 hydrochloride salt:
synthesized according to the method of example 15.
Synthesis of Compound 2:
synthesized in a similar manner to example 5, yield 44.73%.
Synthesis of TN-115:
synthesized in a similar manner to example 14, TN-414 was a pale yellow solid in a yield of 88.46% and a purity of 97.61% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.62(s,1H)；8.72(br,1H)；7.96～7.98(d,2H)；7.81～7.83(d,2H)；7.40～7.42(d,1H)；7.01-7.03(d,1H)；6.87～6.89(dd,1H)；4.65～4.68(t,1H)；3.92(s,3H)；3.68～3.73(m,4H)；3.54～3.56(q,2H)；3.43～3.45(q,2H)；。
[M+H]+:389.1380
Example 18
TN-431 was synthesized as follows.
Figure BDA0002127460580000221
Synthesis of Compound 1:
16.72g (0.100 mol) of N1-methyl-4-nitrobenzene-1, 2-diamine, 21.36g (0.110 mol) of p-phenylenediacetic acid, 25.85g (0.200 mol) of N, N-diisopropylethylamine and 200mLN, N-dimethylformamide are stirred and mixed uniformly, 45.51g (0.120 mol) of O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) is added, and the mixture is stirred and reacted for 24 hours at room temperature. The mixture was poured into 1000mL of water with stirring and adjusted to pH 11-12 with KOH. Filtering, adjusting the pH value of the filtrate to 5-6 by using glacial acetic acid, filtering, washing the solid by using water, and airing to obtain 10.963g of the compound 1 with the yield of 31.92%.
Synthesis of Compound 2:
synthesized in a similar manner to example 7, yield 73.04%.
Synthesis of Compound 3:
synthesized in a similar manner to example 7 in approximately 100% yield.
Synthesis of Compound 4:
synthesized by a similar procedure to example 7, yield 69.53%.
Synthesis of Compound 5:
synthesized in a similar manner to example 7, yield 78.87%.
Synthesis of TN-431:
synthesized in a similar manner to example 14, TN-431 was a pale yellow solid in a yield of 90.68% and a purity of 98.83% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.34(s,1H)；8.72(br,1H)；7.41～7.43(d,2H)；7.28～7.30(d,1H)；7.16～7.18(d,2H)；6.86-6.87(d,1H)；6.73～6.76(d,1H)；4.01～4.03(s,2H)；3.82～3.85(s,2H)；3.73(s,3H)；3.65～3.67(m,4H)；3.53～3.58(m,4H)。
[M+H]+:435.1352
Example 19
TN-432 was synthesized as follows.
Figure BDA0002127460580000231
Synthesis of Compound 1:
synthesized as in example 18.
Synthesis of Compound 2:
synthesized in a similar manner to example 3, yield 40.63%.
Synthesis of hydrochloride salt of compound 3:
synthesized in a similar manner to example 3, compound 3 hydrochloride was a white solid compound in 89.58% yield. Synthesis of TN-432:
synthesized in a similar manner to example 14, TN-432 was a pale yellow solid in a yield of 90.68% and a purity of 97.94% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.35(s,1H)；8.74(br,1H)；7.42～7.45(d,2H)；7.30～7.32(d,1H)；7.13～7.15(d,2H)；6.88-6.90(d,1H)；6.72～6.75(d,1H)；5.70～5.73(t,1H)；4.03～4.05(s,2H)；3.89～3.91(s,2H)；3.83(s,3H)；3.63～3.69(m,2H)；3.50～3.55(m,2H)。
[M+H]+:373.1430
Example 20
TN-433 was synthesized as follows.
Figure BDA0002127460580000241
Synthesis of Compound 1:
synthesized as in example 19.
Synthesis of Compound 2:
synthesized in a similar manner to example 10, yield 73.33%.
Synthesis of Compound 3:
synthesized in a similar manner to example 10, yield 70.04%.
Synthesis of TN-433:
synthesized in a similar manner to example 14, TN-433 was a pale yellow solid with a yield of 86.63% and a purity of 96.85% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.40(s,1H)；8.78(br,1H)；7.42～7.44(d,2H)；7.36～7.38(d,1H)；7.18～7.22(d,2H)；6.93-6.96(d,1H)；6.80～6.83(d,1H)；4.33～4.35(t,2H)；3.84(s,3H)；3.52～3.54(t,2H)；3.38～3.41(t,8H)。
[M+H]+:479.1613
Example 21
TN-434 was synthesized as follows.
Figure BDA0002127460580000242
Synthesis of hydrochloride salt of compound 1:
synthesized as in example 19.
Synthesis of Compound 2:
synthesized in a similar manner to example 5, yield 40.58%.
Synthesis of TN-434:
synthesized in a similar manner to example 14, TN-434 was a pale yellow solid with a yield of 84.63% and a purity of 96.72% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.36(s,1H)；8.74(br,1H)；7.40～7.42(d,2H)；7.26～7.29(d,1H)；7.17～7.19(d,2H)；6.87～6.89(dd,1H)；6.75-6.78(d,1H)；4.65～4.67(t,1H)；4.01～4.03(s,2H)；3.95(s,3H)；3.82～3.85(s,2H)；3.68～3.73(m,4H)；3.54～3.56(q,2H)；3.43～3.45(q,2H)；。
[M+H]+:417.1691
Example 22
TN-901 was synthesized as follows.
Figure BDA0002127460580000251
Synthesis of Compound 1:
synthesized in a similar manner to example 1, starting from N1-methyl-4-nitrophenyl-1, 2-diamine.
Synthesis of Compound 2:
synthesized in a similar manner to example 3, yield 38.92%.
Synthesis of compound 3 hydrochloride salt:
synthesized in a similar manner to example 3, compound 3 hydrochloride was an off-white solid in 92.08% yield.
Synthesis of TN-901:
synthesized in a similar manner to example 3, TN-901 was a white solid in a yield of 74.58% and a purity of 98.77% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.35(s,1H)；8.69(br,1H)；7.17～7.19(dd,1H)；6.70(s,1H)；6.59～6.61(dd,1H)；5.43～5.45(t,1H)；3.72～3.74(t,2H)；3.63(s,3H)；3.38～3.41(q,2H)；2.74～2.77(t,2H)；1.93～1.96(t,2H)；1.67～1.73(m,2H)；1.46～1.52(m,2H)；1.26～1.39(m,4H)。
[M+H]+:337.185
Example 23
TN-902 was synthesized as follows.
Figure BDA0002127460580000261
Synthesis of compound 1 hydrobromide salt:
synthesized by the method of example 22
Synthesis of Compound 2:
synthesized by a similar procedure to example 10, yield 74.39%.
Synthesis of Compound 3:
synthesized by a similar method to example 10, yield 72.62%.
Synthesis of TN-902:
synthesized in a similar manner to example 14, TN-902 was an off-white solid in a yield of 78.29% and a purity of 96.73% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.37(s,1H)；8.73(br,1H)；7.28～7.30(d,1H)；6.86-6.87(d,1H)；6.73～6.76(dd,1H)；4.66～4.69(t,1H)；3.67～3.73(m,8H)；3.62(s,3H)；3.59～3.61(t,2H)；3.44～3.47(t,2H)；2.76～2.79(t,2H)；1.94～1.97(t,2H)；1.69～1.75(m,2H)；1.47～1.53(m,2H)；1.27～1.40(m,4H)。
[M+H]+:459.1928
Example 24
TN-903 was synthesized as follows.
Figure BDA0002127460580000271
Synthesis of compound 1 hydrochloride salt:
synthesized in a similar manner to example 1, starting from N1-methyl-4-nitrobenzene-1, 2-diamine. Synthesis of TN-903:
synthesized in a similar manner to example 2 in 84.73% yield and 98.42% purity by HPLC area normalization.
1H-NMR(d6-DMSO)δ:11.99(s,1H)；7.30～7.32(d,1H)；6.91-6.92(d,1H)；6.76～6.78(dd,1H)；3.70(s,8H)；3.66(s,3H)；2.77～2.80(t,2H)；2.19～2.22(t,2H)；1.70～1.76(m,2H)；1.48～1.54(m,2H)；1.31～1.40(m,4H)。
[M+H]+:400.1558
Example 25
TN-904 was synthesized as follows.
Figure BDA0002127460580000272
Synthesis of compound 1 hydrochloride salt:
synthesized by the method of example 22
Synthesis of Compound 2:
synthesized in a similar manner to example 5, yield 44.58%.
Synthesis of TN-904:
synthesized in a similar manner to example 5, TN-903 was an off-white solid in a yield of 85.44% and a purity of 97.44% by HPLC area normalization.
1H-NMR(d6-DMSO)δ:10.35(s,1H)；8.69(br,1H)；7.26～7.28(d,1H)；6.85-6.86(d,1H)；6.71～6.73(dd,1H)；4.66～4.69(t,1H)；3.67～3.72(m,4H)；3.65(s,3H)；3.53～3.55(q,2H)；3.42～3.44(q,2H)；2.75～2.78(t,2H)；1.93～1.96(t,2H)；1.68～1.74(m,2H)；1.47～1.52(m,2H)；1.26～1.39(m,4H)。
[M+H]+:397.2004
Example 26
Salt formation of compound
Taking 0.01mol of target compound (such as TN-111) with qualified purity, adding 60ml of DMF, stirring, dissolving at normal temperature, dropwise adding an ethyl acetate solution of corresponding acid (such as hydrochloric acid) with the molar ratio of the acid being 1.1, precipitating salt under stirring, performing suction filtration, washing a filter cake with a small amount of ethyl acetate, and performing vacuum drying to obtain the salt of the target product.
In combination with the process of the above examples, a compound having the following general structural formula, or a pharmaceutically acceptable salt thereof, may be prepared:
Figure BDA0002127460580000281
wherein R is 2 Is CH 2 CH 2 Cl；
R 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H orIs methyl;
R 4 is composed of
Figure BDA0002127460580000282
n is optionally 0, 1,2, 3, 4, 5, 6, 7, 8, 9, n is preferably 5, 6, 7;
a compound having the following structural formula:
Figure BDA0002127460580000283
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl，
R 3 Is H or is methyl;
n is optionally 3, 4, 5, 6 or 7.
A compound having the structural formula:
Figure BDA0002127460580000291
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H or methyl;
n is optionally 3, 4, 5, 6 or 7.
A compound having the structural formula:
Figure BDA0002127460580000292
wherein R is 1 Is H, CH 2 CH 2 Cl、CH 2 CH 2 OH or CH 2 CH 2 OCH 2 CH 2 Cl；
R 3 Is H or methyl;
R 4 is composed of
Figure BDA0002127460580000293
Example 27
NL-101 bulk drug related substance analysis
Based on NL-101 crude drug synthetic route and process, the main process impurities and degradation impurities are as shown in the table
Figure BDA0002127460580000294
Figure BDA0002127460580000301
Through a large number of experimental researches, impurity reference substances with the content of more than 0.1 percent, which are necessary for controlling the quality of NL-101 raw material medicines, are obtained. The synthesized novel 1-hydrobenzimidazole derivative can meet the standard sample requirement of NL-101 raw material medicine quality control.
Taking about 25mg of NL-101 sample, placing the NL-101 sample in a 50ml measuring flask, adding a proper amount of methanol to dissolve the NL-101 sample, diluting the NL-101 sample to a scale, and shaking up to obtain a sample solution; 1ml was measured precisely, placed in a 100ml measuring flask, diluted to the mark with methanol, shaken up as a control solution. And taking appropriate amount of NL-101 reference substance, impurity A reference substance, impurity B reference substance, impurity C reference substance and impurity E reference substance, respectively placing in different volumetric flasks, adding methanol to dissolve and dilute to prepare solutions containing NL-101 of 0.5mg, impurity A, impurity B and impurity E of 2.5 mu g and impurity C of 3.0 mu g in each 1ml, shaking up to obtain the system applicability test solution. Measuring by high performance liquid chromatography, using phenyl bonded silica gel (4.6 mm × 150mm,3 μm) as filler; gradient elution was performed with 0.1% trifluoroacetic acid in water as mobile phase a and trifluoroacetic acid-water-acetonitrile (0.8; the flow rate is 1.0ml/min; the detection wavelength is 231nm; the column temperature was 30 ℃ and the sample introduction temperature was 6 ℃.
Time (min) Mobile phase A (%) Mobile phase B (%)
0 85 15
1 85 15
8 75 25
24 40 60
35 0 100
35.1 85 15
40 85 15
NL-101 related substances are analyzed with a typical pattern shown in FIG. 2.
Example 28
Screening for antitumor Activity in vitro
Collecting tumor cells (such as breast cancer MDA-MB-231 cells) in logarithmic growth phase, subjecting to trypsinization and centrifugation, making into cell suspension, and adjusting concentration to 3 × 10 4 One/ml, seeded in 96-well plates and 100. Mu.l of cell suspension added per well. At 37 ℃ C, 5% CO 2 After culturing for 24h in an incubator with saturated humidity and after most cells grow well and are completely attached to the wall, the test sample is added to make the final concentration form several gradients (for example, 0.5, 2.5, 12.5, 50 and 100 μ M respectively), each concentration is provided with 4 multiple wells, and the culture medium without drugs is used as a blank control for 48h. Mu.l of MTT (5 mg/mL) culture solution was added to each well and the incubation in the incubator was continued for 4 hours. The culture medium is removed by suction, 150 mul DMSO is added into each hole, and the mixture is placed on a horizontal shaking table to shake for 10 to 15min at room temperature and low speed. Measuring absorbance OD values at 570nm and 630nm wavelength with multifunctional microplate reader, respectively calculating relative cell activity and cell inhibition rate according to L1-L2, and calculating IC with GraphPad Prism 5.0 50 The value is obtained.
Figure BDA0002127460580000311
Figure BDA0002127460580000321
Example 29
Pharmacokinetics study in rats
9 SD male rats were selected and tested in two groups, 2 groups each time, 2 and 1 control groups, and 3 test groups of 25 mg/kg. The tail vein injection is used for administration, the administration volume is 10ml/kg, and the injection is finished within 50-60 s. Blood is taken by tail tip bleeding method at 0.083h, 0.5h, 1h, 2h, 4h, 6h and 24h after administration, and the blood volume is 0.1-0.2ml each time. After blood sample collection, putting the blood sample into an EP tube which contains 5 mu l of heparin sodium, is anticoagulated and precooled, manually reversing the blood sample for at least 5 times, and centrifuging the blood sample at 4 ℃ for 5min at 5000g to collect blood plasma; taking the supernatant to a new EP tube, measuring the volume, adding 4 Xvolume of acetonitrile solution, and precipitating for 20min; centrifuging at 13000g for 10min at 4 ℃ within 1h, taking supernatant, identifying and determining the test sample and the main metabolite thereof in the whole blood sample by using an LC-MS method, and calculating the concentration trend of the substance to be detected according to the EIC peak area conditions of the test sample and the main metabolite.
TN-431 is metabolized differently compared to NL-101. The hydroxamic acid hydrolysis products in the molecular structure are both one of the main metabolites of TN-431 and NL-101. (1) TN-431 is slower in metabolism than NL-101, with t1/2 of about 18 and 10 minutes, respectively; (2) no beta-oxidation product is found in TN-431 metabolite; (3) A single chloride hydrolysis impurity is one of the major metabolites in whole blood, similar metabolites are not found in NL-101 metabolism; (4) Glucuronic acid conjugates were not found in TN-431 metabolites.

Claims (6)

1. A compound or a pharmaceutically acceptable salt thereof:
Figure FDA0003838153690000011
Figure FDA0003838153690000021
2. the compound according to claim 1, or a pharmaceutically acceptable salt thereof, which is:
Figure FDA0003838153690000022
Figure FDA0003838153690000031
3. the compound of claims 1-2, or a pharmaceutically acceptable salt thereof, wherein the salt is a hydrochloride, acetate, citrate, tartrate, succinate, malate, maleate, lactate, benzenesulfonate, sulfate, phosphate, glutamate, glutarate, or salicylate salt.
4. Use of a compound according to any one of claims 1-2, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the treatment of a tumour or an immune system disorder.
5. The use according to claim 4, wherein the neoplasm is lung cancer, head and neck cancer, central nervous system tumor, prostate cancer, testicular cancer, colorectal cancer, pancreatic cancer, liver cancer, gastric cancer, biliary tract cancer, esophageal cancer, gastrointestinal stromal tumor, breast cancer, cervical cancer, ovarian cancer, uterine cancer, leukemia, lymphoma, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, bladder cancer, renal cancer, sarcoma, mesothelioma, thymoma, myelodysplastic syndrome, or myeloproliferative disorder.
6. The use according to claim 5, wherein the neoplasm is lung cancer, breast cancer, multiple myeloma.
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