CN112209884A - 1-H benzimidazole derivative, preparation method and application thereof - Google Patents
1-H benzimidazole derivative, preparation method and application thereof Download PDFInfo
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- 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
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- A—HUMAN NECESSITIES
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P35/02—Antineoplastic agents specific for leukemia
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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
- C07D235/18—Benzimidazoles; Hydrogenated benzimidazoles with aryl radicals directly attached in position 2
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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 derivative A, B, C, E is determined to be a main relevant substance of the 1-H benzimidazole derivative NL-101 to be marketed, and can be used as a standard substance in the NL-101 approval, analysis, identification and quality control research process. Also disclosed is a method of analyzing NL-101.
Description
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 first chemotherapeutic drugs used to treat malignant tumors. 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 agent 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, causing the elimination of negatively charged acetyl groups on histones, leading to tight binding of DNA duplexes and histones (Kim HJ, et al. American journal of molecular biology.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.12003; 63(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.12005; 11(13): 4912-22; Chen CS, et al. Cancer research.Jun 12007; 67(11):5318-27), rendering 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 the precise analysis of the structure-activity relationship between bendamustine and vorinostat, a novel small molecule compound NL-101 with both DNA alkylation and HDAC inhibition 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.
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, in which CN102186842B of the national family is now licensed and the patent right is Hangzhou TNO medicine science and technology Co. 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 conditionally marketed in the United states, and is clinically developed smoothly 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, a substance A, B, C, E and a method for synthesizing the same, the structural formula of which is as follows:
the compound A, C is a new compound synthesized for the first time, and although the compound B, E has been disclosed, the compound has been subjected to experimental studies such as improvement of a synthetic route, purification, identification and the like, so that the compound completely meets the requirements of application as a standard product in the processes of approval, analysis and quality control research as the compound A, 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 relevant substances has a crucial influence on the safety and effectiveness of the final shipped drug. 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-10150IC with value of 25.85. mu.M, TN-11150The value was 1.17. mu.M.
2. Better metabolic stability, TN-431 compounds withComparable antitumor activity to NL-101, but with 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 slower in metabolism than NL-101, t1/2About 18 minutes and 10 minutes, respectively; (2) the TN-431 metabolite has no beta-oxidation product found therein; (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. Higher safety. 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 150 mg/kg.
The following classes of compounds were obtained:
a compound having the following structural formula, or a pharmaceutically acceptable salt thereof:
wherein R is2Is CH2CH2Cl；
R1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or is methyl;
R4is composed of
n is optionally 0, 1,2, 3, 4, 5, 6, 7, 8, 9, n is preferably 5, 6, 7;
when R is1Is CH2CH2Cl or CH2CH2OH，R4Is composed of
And when n is 6, R3Is not methyl;
when R3 is methyl, R1Is CH2CH2Cl or CH2CH2OH, and R4Is composed of
When n is not 2 or 6.
A compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl，
R3Is H or is methyl;
n is optionally 3, 4, 5, 6 or 7;
when R is1Is CH2CH2Cl or CH2CH2OH, and when n is 6, R3Is not methyl.
A compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or methyl;
n is optionally 3, 4, 5, 6 or 7;
R3is methyl; and R is1Is CH2CH2Cl or CH2CH2OH, n is not 2 or 6.
A compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or methyl;
R4is composed of
Drawings
FIG. 11-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 given only as a way of preparing such compounds and are not intended to be limiting.
Example 1
TN-101 was synthesized as follows.
Synthesis of Compound 1:
100.00g (0.654mol) of 4-nitro-1, 2-phenylenediamine and 182.00g (1.045mol) of suberic acid are added into 500mL of 6M hydrochloric acid, stirred and refluxed for 5 hours, 500mL of water is added, the pH value is adjusted to 11-12 by KOH under stirring, filtration is carried out, the pH value 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 drying is carried out at 65 ℃ to obtain 116.94g of compound 1, wherein the yield is 61.47%.
Synthesis of Compound 2:
116.94g (0.401mol) of compound 1, 660mL of absolute ethanol and 35mL of concentrated sulfuric acid are stirred and refluxed for 4 hours, the reaction solution is slowly poured into 2200g of 10% potassium carbonate solution precooled to about 10 ℃, the solution is filtered, the solid is washed by water for a plurality of times and dried at 50 ℃, and the dried solid is recrystallized by isopropyl ether and ethanol to obtain 71.43g of compound 2, wherein the yield is 61.68%.
Synthesis of Compound 3:
70.00g (0.219mol) of Compound 2 was added to 1200mL of tetrahydrofuran, dissolved by stirring, 14.0g of 5% palladium on charcoal (50% wet with water) was added, hydrogen was replaced by evacuation, and then hydrogenation was carried out under stirring for 8 hours or more. 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 (0.110mol) 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 (0.571mol) of ethylene oxide, and the mixture was allowed to slowly warm 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:
dissolving 37.75g (0.100mol) of compound 4 in 250mL of dichloromethane, adding 0.1mL of N, N-dimethylformamide, dropwise adding 30.93g (0.260mol) of thionyl chloride while cooling in an ice water bath, reacting at room temperature for 12-24 hours after the addition, concentrating, adding 50mL of absolute ethyl alcohol, heating to dissolve, adding 500mL of ethyl acetate for crystallization, filtering under the protection of nitrogen, washing the solid with 100mL of ethyl acetate, and drying in vacuum at 45 ℃ for 10-12 hours to obtain 33.31g of quasi-white solid compound 5 hydrochloride with the yield of 73.89%.
Synthesis of TN-101:
under an ice-water bath, 34.50g (0.615mol) of potassium hydroxide is dissolved in 150mL of methanol, added into 33.31g (0.479mol) of hydroxylamine hydrochloride and 100mL of methanol, stirred for 2-3 hours at room temperature, filtered, the filtrate is cooled to 0-10 ℃, 13.53g (0.030mol) of compound 5 hydrochloride is added, stirred and reacted for 1 hour, glacial acetic acid is used for adjusting the pH value to 6-8, 250mL of water is added, and the mixture is filtered, washed with water and dried in vacuum at 50 ℃ for 10-12 hours to obtain 10.64g of off-white solid TN-101, 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.
The hydrochloride salt of compound 1 was obtained from example 1.
Synthesis of TN-102:
9.02g (0.0200mol) of Compound 1 hydrochloride and 50mL of concentrated hydrochloric acid were stirred and added for 5 hours, the solvent was evaporated under reduced pressure, the residue was dissolved in 80mL of methanol, the pH was adjusted to 4-5 with 10% KOH solution cooled in an ice-water bath, the filtrate was filtered, the solid was washed with 30 mL. times.2 and dried under vacuum at 65 ℃ for 10-12 hours to give 6.46g of TN-102 as an off-white solid with a yield of 83.56% and a purity of 98.36% 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.
Synthesis of Compound 1:
Synthesis of Compound 2:
11.58g (0.040mol) 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 (0.104mol) 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 separated and purified by a silica gel column to obtain 5.08g of Compound 2 with a yield of 37.48%.
Synthesis of hydrochloride salt of compound 3:
dissolving 3.39g (0.010mol) of compound 2 in 30mL of dichloromethane, adding 2 drops of N, N-dimethylformamide, dropwise adding 1.79g (0.015mol) 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 (0.154mol) of potassium hydroxide is dissolved in 40mL of methanol, then added into a mixture of 8.63g (0.124mol) 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 (6.44mmol) of compound 4 hydrochloride is added, the mixture is stirred and reacted for 0.5 hour, the pH is adjusted to 6-8 by glacial acetic acid, 65mL of water is added, the mixture is filtered, washed and dried in vacuum at 50 ℃ for 10-12 hours, 1.79g of off-white TN-103 is obtained, the yield is 82.03%, and the purity is 98.74% calculated by an HPLC area normalization method.
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.
Synthesis of hydrochloride salt of compound 1:
synthesized as in example 3.
Synthesis of TN-104:
synthesized in a similar manner to example 2, TN-104 was an off-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.
Synthesis of hydrochloride salt of compound 1:
synthesized according to the method of example 3
Synthesis of Compound 2:
2.68g (6.90mmol) 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 (17.93mmol) 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 (0.154mol) of potassium hydroxide is dissolved in 40mL of methanol, then added into a mixture of 8.63g (0.124mol) of hydroxylamine hydrochloride and 25mL of methanol, stirred for 2-3 hours at room temperature, filtered, 15mL of filtrate is cooled to 0-10 ℃, 0.73g (1.84mmol) of compound 2 is taken, stirred and reacted for 0.5 hour, the pH is adjusted to 6-8 by glacial acetic acid, 15mL of water is added, filtered, washed with water and dried in vacuum for 10-12 hours at 50 ℃, 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.
Synthesis of Compound 1:
synthesized as in example 4.
Synthesis of TN-106:
1.62g (5.00mmol) 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 (12.94mmol) 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 ℃ under stirring, and after completion of the addition, the mixture was 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.
Synthesis of Compound 1:
33.44g (0.200mol) of N1-methyl-4-nitrobenzene-1, 2-diamine is added into 250mL of dichloromethane, 22.02g (0.220mol) of succinic anhydride is added in batches under stirring, reaction is carried out for 12 to 24 hours at room temperature, filtration is carried out, the solid is washed by 100mL of isopropyl ether, and 52.73g of compound 1 is obtained after drying at 65 ℃, and the yield is 98.66%.
Synthesis of Compound 2:
48.10g (0.180mol) 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 (0.165mol) 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 (0.165mol) 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 (0.859mol) of ethylene oxide was added thereto, 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:
dissolving 33.54g (0.100mol) of compound 4 in 250mL of dichloromethane, adding 0.1mL of N, N-dimethylformamide, dropwise adding 28.55g (0.240mol) of thionyl chloride while cooling in an ice water bath, reacting at room temperature for 12-24 hours after the addition, concentrating, adding 250mL of dichloromethane, stirring to dissolve, washing an organic phase with 250mL of water and 250mL of saturated sodium bicarbonate solution respectively, drying over anhydrous sodium sulfate, filtering, concentrating a filtrate, and recrystallizing isopropyl ether to obtain 22.60g of compound 5 with the yield of 60.71%.
Synthesis of TN-111:
under an ice-water bath, 34.50g (0.615mol) of potassium hydroxide is dissolved in 150mL of methanol, then added into a mixture of 34.50g (0.496mol) 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 (0.0269mol) 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 with water, 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% calculated by an HPLC area normalization method.
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.
Synthesis of Compound 1:
synthesized by the method of example 7
Synthesis of Compound 2:
synthesized in a similar manner 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 with a yield of 92.49%.
Synthesis of TN-112:
synthesized in a similar manner to example 3, TN-112 was an off-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.
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 an off-white solid compound with 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.
Synthesis of Compound 1:
synthesized as in example 8.
Synthesis of Compound 2:
2.33g (8.00mmol) of Compound 1, 20mL of diglyme, 1.49g (12.00mmol) of 2- (2-chloroethoxy) ethanol, and 2.21g (16.00mmol) of potassium carbonate were refluxed for 12 to 24 hours. Filtration was carried out, and the filtrate was concentrated and then 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 (6.00mmol) of Compound 2 was dissolved in 20mL of dichloromethane, 2 drops of mLN, N-dimethylformamide were added, 1.86g (0.156mol) 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 separated and purified by a silica gel column to obtain 1.81g of compound 3 with a yield of 72.46%.
Synthesis of TN-114:
under an ice-water bath, 8.63g (0.154mol) of potassium hydroxide is dissolved in 40mL of methanol, then added into a mixture of 8.63g (0.124mol) 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 (1.20mmol) of compound 3 is added, stirred and reacted for 0.5 hour, the pH 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 to obtain 0.37g of off-white solid TN-114, the yield is 76.45%, and the purity is 97.29% calculated by HPLC area normalization.
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.
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 an off-white 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.
Synthesis of Compound 1:
synthesized by 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 an off-white solid compound in a yield of 83.61% and a purity of 97.53% as calculated 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.
Synthesis of Compound 1:
synthesized by 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.
Synthesis of Compound 1:
34.76g (0.179mol) of monoethyl terephthalate, 0.1mL of N, N-dimethylformamide and 100.00g (0.841mol) 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 (0.179mol) of N1-methyl-4-nitrobenzene-1, 2-diamine, 25.85g (0.200mol) of N, N-diisopropylethylamine and 500mL of methylene chloride with stirring and cooling. After the completion of 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, 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 with a yield of 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 with a yield of 61.03%.
Synthesis of TN-411:
under an ice water bath, 26.62g (0.474mol) of potassium hydroxide is dissolved in 120mL of methanol, added into a mixture of 25.52g (0.367mol) of hydroxylamine hydrochloride and 80mL of methanol, stirred at room temperature for 2-3 hours, filtered, 7.34g (0.0175mol) of compound 5 and 50mL of dichloromethane are added into filtrate, stirred and reacted for 1 hour, the pH is adjusted to 6-8 by glacial acetic acid, 250mL of water is added, filtered, washed with 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.
Synthesis of Compound 1:
synthesized as in 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 with 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.
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, yield 73.82%.
Synthesis of TN-413:
synthesized in a similar manner to example 14, TN-413 was a pale yellow solid in 88.46% yield 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.
Synthesis of hydrochloride salt of compound 1:
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 88.46% yield and had a purity of 97.61% as calculated 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.
Synthesis of Compound 1:
16.72g (0.100mol) of N1-methyl-4-nitrobenzene-1, 2-diamine, 21.36g (0.110mol) of p-phenylenediacetic acid, 25.85g (0.200mol) of N, N-diisopropylethylamine and 200ml of N, N-dimethylformamide are stirred and mixed uniformly, 45.51g (0.120mol) of O-benzotriazole-tetramethyluronium Hexafluorophosphate (HBTU) is added, and the mixture is stirred and reacted for 24 hours at room temperature. Pouring the mixture into 1000mL of water under stirring, and adjusting the pH value to 11-12 by using KOH. And filtering, adjusting the pH 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, wherein the yield is 31.92%.
Synthesis of Compound 2:
synthesized in a similar manner to example 7 with a yield of 73.04%.
Synthesis of Compound 3:
synthesized in a similar manner to example 7 in approximately 100% yield.
Synthesis of Compound 4:
synthesized in a similar manner 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.
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 with a yield of 89.58%. Synthesis of TN-432:
synthesized in a similar manner to example 14, TN-432 was a pale yellow solid with 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.
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.
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.
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 hydrochloride salt of compound 3:
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 an off-white solid in 74.58% yield and 98.77% purity 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.
Synthesis of compound 1 hydrobromide salt:
synthesized by the method of example 22
Synthesis of Compound 2:
synthesized in a similar manner to example 10, yield 74.39%.
Synthesis of Compound 3:
synthesized in a similar manner 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.
Synthesis of hydrochloride salt of compound 1:
synthesized in a similar manner to example 1, starting from N1-methyl-4-nitrophenyl-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.
Synthesis of hydrochloride salt of compound 1:
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 DMF60ml, stirring, dissolving at normal temperature, dropwise adding ethyl acetate solution of corresponding acid (such as hydrochloric acid) with the molar ratio of 1:1.1, stirring to separate out salt, 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 above examples, a compound having the following general structural formula, or a pharmaceutically acceptable salt thereof, can be prepared:
wherein R is2Is CH2CH2Cl；
R1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or is methyl;
R4is composed of
n is optionally 0, 1,2, 3, 4, 5, 6, 7, 8, 9, n is preferably 5, 6, 7;
a compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl，
R3Is H or is methyl;
n is optionally 3, 4, 5, 6 or 7.
A compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or methyl;
n is optionally 3, 4, 5, 6 or 7.
A compound having the structural formula:
wherein R is1Is H, CH2CH2Cl、CH2CH2OH or CH2CH2OCH2CH2Cl；
R3Is H or methyl;
R4is composed of
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
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 (4.6mm × 150mm,3 μm) as filler; gradient elution was performed with 0.1% trifluoroacetic acid aqueous solution as mobile phase a and trifluoroacetic acid-water-acetonitrile (0.8:50:950) as mobile phase B according to the following table; the flow rate is 1.0 ml/min; the detection wavelength is 231 nm; 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 × 104One/ml, seeded in 96-well plates and 100. mu.l of cell suspension added per well. At 37 deg.C, 5% CO2Cultivation in incubator with saturated humidity 24h, after most cells grow well and are completely attached to the wall, adding the tested sample to make the final concentration form several gradients (0.5, 2.5, 12.5, 50 and 100 mu M respectively), setting 4 multiple wells for each concentration, and taking 48h without drug-containing culture medium as a blank control. Mu.l of MTT (5mg/mL) culture solution was added to each well and the incubation was continued in the incubator for 4 hours. And (3) absorbing the culture medium, adding 150 mu l of DMSO into each hole, and placing on a horizontal shaking table to shake at low speed for 10-15 min at room temperature. Measuring absorbance OD values at 570nm and 630nm with multifunctional microplate reader, respectively calculating relative cell activity and cell inhibition rate according to L1-L2, and calculating IC with GraphPad Prism 5.050The value is obtained.
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 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 into an EP tube containing 5 mul heparin sodium anticoagulation and precooling, manually reversing for at least 5 times, centrifuging at 4 ℃ for 5min at 5000g, and collecting plasma; taking the supernatant to a new EP tube, measuring the volume, adding 4 Xvolume of acetonitrile solution, and precipitating for 20 min; centrifuging at 13000g for 10min at 4 ℃ within 1h, taking supernatant, identifying and determining a test sample and main metabolites 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 metabolites.
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 being about 18 and 10 minutes, respectively; (2) the TN-431 metabolite has no beta-oxidation product found therein; (3) the single chloride hydrolysis impurity 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.
Claims (11)
1. A compound having the following structural formula, or a pharmaceutically acceptable salt thereof:
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having the following structural formula:
3. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having the following structural formula:
4. The compound of claim 1, or a pharmaceutically acceptable salt thereof, having the following structural formula:
5. The compound of claims 1,2, 3, or a pharmaceutically acceptable salt thereof, characterized as a compound having the following structural formula:
6. the compound according to claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein the salt is a hydrochloride, acetate, citrate, tartrate, succinate, malate, maleate, lactate, besylate, sulfate, phosphate, glutamate, glutarate, or salicylate.
7. Use of a compound according to any one of claims 1 to 6, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of a tumour or an immune system disorder.
8. The use according to claim 7, 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, multiple myeloma, malignant melanoma, basal cell carcinoma, squamous cell carcinoma, bladder cancer, renal cancer, sarcoma, mesothelioma, thymoma, myelodysplastic syndrome, or myeloproliferative disease.
9. Use of a compound according to any one of claims 1 to 5 or a pharmaceutically acceptable salt thereof and a compound having the following structural formula as standards during NL-101 approval, analysis and quality control studies.
10. The use of compound A, B, C, E as a standard substance in NL-101 approval, analysis, and quality control studies is defined in claim 9, or a pharmaceutically acceptable salt thereof.
An analytical method of NL-101, characterized in that 3 μm phenyl bonded silica gel is used as a filler, a column of 4.6mm X150 mm, a 0.1% aqueous trifluoroacetic acid solution as a mobile phase A, and trifluoroacetic acid-water-acetonitrile (0.8:50:950) as a mobile phase B, at a flow rate of 1.0 ml/min; the detection wavelength is 231 nm; the column temperature was 30 ℃ and the sample introduction temperature was 6 ℃ and gradient elution was carried out as follows.
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