CN116574093B - Benzimidazole compound, and preparation method and application thereof - Google Patents
Benzimidazole compound, and preparation method and application thereof Download PDFInfo
- Publication number
- CN116574093B CN116574093B CN202310827474.2A CN202310827474A CN116574093B CN 116574093 B CN116574093 B CN 116574093B CN 202310827474 A CN202310827474 A CN 202310827474A CN 116574093 B CN116574093 B CN 116574093B
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- acid
- compound
- pharmaceutically acceptable
- acceptable salt
- hematopoietic stem
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- -1 Benzimidazole compound Chemical class 0.000 title abstract description 9
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- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 20
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 15
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- OFOBLEOULBTSOW-UHFFFAOYSA-N Propanedioic acid Natural products OC(=O)CC(O)=O OFOBLEOULBTSOW-UHFFFAOYSA-N 0.000 claims description 6
- 239000002253 acid Substances 0.000 claims description 6
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 6
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Classifications
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- C—CHEMISTRY; METALLURGY
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- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/14—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing three or more hetero rings
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P35/00—Antineoplastic agents
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- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
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- A—HUMAN NECESSITIES
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- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
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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
- C07D235/14—Radicals substituted by nitrogen atoms
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D405/00—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom
- C07D405/02—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings
- C07D405/12—Heterocyclic compounds containing both one or more hetero rings having oxygen atoms as the only ring hetero atoms, and one or more rings having nitrogen as the only ring hetero atom containing two hetero rings linked by a chain containing hetero atoms as chain links
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/55—Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Public Health (AREA)
- Life Sciences & Earth Sciences (AREA)
- Veterinary Medicine (AREA)
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Abstract
The application discloses a benzimidazole compound, a preparation method and application thereof, wherein the general formula of the benzimidazole compound is shown as formula (I) or pharmaceutically acceptable salt thereof, and the benzimidazole compound can selectively inhibit YTHDF2 so as to amplify hematopoietic stem cells, has the advantages of low toxicity, good safety, good treatment effect and the like, and can be used for preparing medicines for amplifying the hematopoietic stem cells.
Description
Technical Field
The application relates to a benzimidazole compound, a preparation method and application thereof, and belongs to the field of pharmaceutical chemistry.
Background
Hematopoietic Stem Cell Transplantation (HSCT) is widely used clinically for the treatment of various severe diseases such as malignant blood tumors, genetic metabolic diseases, autoimmune diseases, sickle cell anemia, and Severe Combined Immunodeficiency (SCID). To date, hematopoietic stem cell transplantation is the most effective and the only therapy that can eradicate the disease for some malignant hematological tumors, although there are many effective drugs that ameliorate some hematological diseases. There are four main sources of hematopoietic stem cells: bone marrow, peripheral mobilized blood, umbilical cord blood, and placental blood. Bone marrow blood and peripheral mobilized blood are the traditional and most common sources of hematopoietic stem cell transplantation. Unfortunately, about 30% of patients will have no Human Leukocyte Antigen (HLA) -matched donors due to the lack of HLA-matched donors. In addition, graft rejection (GVHD) remains a leading factor in graft-related death for patients receiving mismatched non-blood-related bone marrow donors.
In recent years, umbilical cord blood has been a source of increasingly attractive hematopoietic stem cell transplantation due to its significant advantages such as high HSC content, easy availability, low risk of viral transmission, tolerance to partial HLA mismatch, etc. More importantly, chronic GVHD is low in incidence of cord blood grafts, and chronic GVHD threatens the long-term survival of patients. However, the absolute number of hematopoietic stem cells in a single cord blood is small, and the dosage requirement of adult patients cannot be satisfied, so that the cord blood can only be applied to children. The low number of hematopoietic stem cells in a single cord blood directly results in slow transplantation, which is characterized by longer neutrophil and platelet recovery, increasing the risk of infection, graft failure, and recurrence. In recent years, the use of two cord blood sets has increased stem cell infusion, but the transplantation effect is similar to that of a single cord blood set, with a higher risk of developing GVHD. In order to obtain sufficient HSC numbers, in vitro expansion of hematopoietic stem cells is a promising therapeutic approach, and a variety of small molecule expanded cord blood is currently in clinical trials.
N6-methyladenosine (m 6A) is one of the most abundant epigenetic modifications on messenger RNA (mRNA) in eukaryotes. As a mode of post-transcriptional modification, m6A modification is closely related to biological processes such as formation, differentiation, leukemia generation, etc. of hematopoietic stem cells in embryonic stages. m6A modification regulates mRNA splicing, translation, transport and stability by RNA methylase (Writer) METTL3/METTL14, demethylase (Eraser) FTO and ALKBH5, and the recognition protein (Reader) YTH domain protein family (YTHDF 1/2/3 and YTHDC1/2 proteins). YTHDF2 is the earliest found m6A recognition protein that recognizes m 6A-tagged mRNA through a conserved aromatic hydrophobic pocket in the protein structure and subsequently regulates RNA degradation by recruiting CCR4-NOT deaminase complex. Recent studies revealed that following conditional knockout of YTHDF2, the number of hematopoietic stem cells in vivo increased and their function was normal, with no differential lineage differentiation and leukemia tendencies. More importantly, after knockout of YTDHF2 in human cord blood hematopoietic stem cells, the number of HSCs increased 10-fold, and the number of functional hematopoietic stem cells increased 8-fold. Transplanting YTDHF 2-knocked down HSCs can reconstitute the hematopoietic system of immunodeficient mice, and secondary transplantation can still stably exert normal hematopoietic function in immunodeficient mice without causing malignant hematological neoplasms. The mechanical study shows that the stability of m6A modified mRNA is increased after YTHDF2 is deleted, and the mRNA can code transcription factors with key regulation and control effects on self-renewal of hematopoietic stem cells, such asTal1And finally expanding the hematopoietic stem cells. Furthermore, it was confirmed from researchers at the university of Edinburgh that YTHDF2 is not necessary for normal hematopoietic stem cell function, but is necessary for survival of leukemic stem cells. These studies indicate that YTHDF2 is an effective and safe target for in vitro expansion of hematopoietic stem cells, and no small molecule inhibitors have been reported.
Disclosure of Invention
The application aims to: in order to solve the problem of insufficient content of hematopoietic stem cells in umbilical cord blood, the application can amplify the biological functions of the hematopoietic stem cells by utilizing the deletion of YTHDF2, and provides a benzimidazole compound or pharmaceutically acceptable salt thereof for inhibiting YTHDF2 in the hematopoietic stem cells, thereby amplifying the hematopoietic stem cells. The application also provides a specific preparation method of the compound and a medicine applied to the expansion of hematopoietic stem cells, which are expected to provide candidate compounds for clinical treatment.
The technical scheme is as follows: in order to solve the problems, the application provides a benzimidazole compound, such as a compound shown in a general formula (I) or pharmaceutically acceptable salt thereof:
；
wherein R is 1 Selected from the following groups:
。
wherein, the structural formulas of the compounds I-1 to I-5 are shown as follows:
。
further, the above pharmaceutically acceptable salts are acid addition salts of the compounds of the general formula (I), wherein the acid used for salt formation comprises an inorganic acid comprising hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid comprising acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid, tartaric acid or methanesulfonic acid.
Further, the pharmaceutically acceptable salt is hydrochloride.
The application also provides a preparation method of the compound shown in the general formula (I).
The application also provides a medicinal composition which comprises the compound of the general formula (I) or pharmaceutically acceptable salt or isomer thereof and a pharmaceutically acceptable carrier.
Pharmaceutically acceptable carrier refers to excipients or diluents that do not cause significant irritation to the organism and do not interfere with the biological activity and properties of the compound being administered.
The application relates to application of a compound or pharmaceutically acceptable salt thereof in preparation of YTDDF 2 target inhibitor drugs.
The application of the compound or the pharmaceutically acceptable salt thereof in preparing medicines for amplifying hematopoietic stem cells is provided.
The compound of the general formula (I) or pharmaceutically acceptable salt thereof has YTDDF 2 target inhibition activity and has therapeutic effect on amplifying hematopoietic stem cells.
The beneficial effects are that: compared with the prior art, the application has the following remarkable characteristics: the application provides a novel compound shown in the general formula (I) for the first time, can selectively inhibit YTHDF2 so as to amplify hematopoietic stem cells, has the advantages of low toxicity, good safety, good treatment effect and the like, and can be used for preparing medicines for amplifying hematopoietic stem cells.
Drawings
FIG. 1 shows the effect of the compounds of the application on the expansion of hematopoietic stem cells in vitro: FIG. 1A shows the frequency of hematopoietic stem cells of mouse LSK detected by flow cytometry 14 days after in vitro treatment of bone marrow mononuclear cells of mice with a compound of the application; FIG. 1B shows the absolute numbers of hematopoietic stem cells of the LSK of mice after 14 days of in vitro treatment of bone marrow mononuclear cells of mice with a compound of the application; FIG. 1C shows the detection of CD48 in mice by flow cytometry after 14 days of in vitro treatment of bone marrow mononuclear cells of mice with a compound of the application – CD150 + Hematopoietic stem cell frequency; FIG. 1D shows the detection of mouse CD48 by flow cytometry after 14 days of in vitro treatment of mouse bone marrow mononuclear cells with the compound of the present application – CD150 + Absolute number of hematopoietic stem cells;
FIG. 2 shows the effect of transplantation of murine, myeloid or B cells in vivo after treatment of hematopoietic stem cells with Compound I-5: FIG. 2A shows the proportion of CD45.2 mouse cells in CD45.1 receptor mice after in vitro treatment of CD45.2 mouse hematopoietic stem cells with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice, and examined by flow cytometry; FIG. 2B is the proportion of CD45.2 mouse myeloid cells in CD45.1 receptor mice detected by flow cytometry after in vitro treatment of CD45.2 mouse hematopoietic stem cells with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice; FIG. 2C shows the proportion of CD45.2 mouse B cells in CD45.1 receptor mice detected by flow cytometry after in vitro treatment of CD45.2 mouse hematopoietic stem cells with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice;
FIG. 3 is Lin 14 days after treatment of hematopoietic stem cells with Compound I-5 – Ratio of cells and LSK cells to total mononuclear cells and absolute number: FIG. 3A shows the flow cytometry detection of Lin in CD45.1 receptor mice after in vitro treatment of hematopoietic stem cells of CD45.2 mice with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice – The proportion and absolute number of cells to total mononuclear cells; FIG. 3B shows the proportion and absolute number of LSK cells in total mononuclear cells in vitro treated with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice;
FIG. 4 shows the proportion and absolute number of hematopoietic stem cells in total mononuclear cells of CD45.1 receptor mice tested by flow cytometry after 14 days of in vitro treatment of CD45.2 mice hematopoietic stem cells with DMSO and Compound I-5, transplanted into lethally irradiated CD45.1 receptor mice;
FIG. 5 shows the proportion of various hematopoietic progenitor cells to total mononuclear cells in a CD45.1 receptor mouse, as determined by flow cytometry, transplanted into a lethally irradiated CD45.1 receptor mouse 14 days after in vitro treatment of CD45.2 mouse hematopoietic stem cells with DMSO and Compound I-5;
FIG. 6 shows the proportion of B cells, myeloid cells, erythrocytes, T cells in total mononuclear cells in mice receiving lethal irradiation, as measured by flow cytometry, after 14 days of in vitro treatment of CD45.2 murine hematopoietic stem cells with DMSO and Compound I-5, transplanted into the mice receiving the lethal irradiation.
Detailed Description
The present application will be described in detail with reference to specific examples.
Example 1: methyl (3- (5-nitro-1H-benzimidazol-2-yl) propyl) terephthalate (I-1)
The synthetic route is as follows:
。
the synthesis method comprises the following steps:
step one, 4-nitroo-phenylenediamine (3.0 g,19.59 mmol) and 4-chlorobutyric acid (3.02 g,24.68 mmol) were taken into a round bottom flask and 4N HCl (50 mL) was added and the temperature was raised to 100deg.C and the reaction was refluxed for 24 h. After the reaction was completed, the aqueous phase was adjusted to basic ph=8 with a concentrated NaOH solution, concentrated under reduced pressure, and purified by silica gel column chromatography (DCM: meoh=20:1) to give compound A1 (3.0 g, yield 69%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.94 (s, 1H), 8.38 (s, 1H), 8.07 (dd,J= 8.8, 2.3 Hz, 1H), 7.64 (d,J= 8.9 Hz, 1H), 4.63 (s, 1H), 3.49 (t,J= 6.3 Hz, 2H), 2.93 (t,J= 7.5 Hz, 1H), 2.02–1.86 (m, 2H). HPLC,t R = 7.580 min, purity 97.7%。
Step two, A1 (3.0 g,13.56 mmol) and methyl 4- (chloroformyl) benzoate (4.04 g,20.34 mmol) were taken in a base flask, dichloromethane 80 mL was added, then triethylamine 3 mL was slowly added dropwise, a large amount of solid was precipitated, after stirring 3 h the reaction was completed, TLC monitored the reaction was complete, triethylamine hydrochloride solid was removed by suction filtration, the organic phase was washed twice with water, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (PE: ea=1: 1) to give I-1 (3.5 g, yield 67%) as a white solid. 1 H NMR (400 MHz, DMSO-d 6 )δ 12.97 (s, 1H), 8.39–8.20 (m, 1H), 8.03 (d,J= 8.9 Hz, 1H), 7.97–7.87 (m, 4H), 7.68–7.50 (m, 1H), 4.43 (t,J= 6.0 Hz, 2H), 3.88 (s, 3H), 3.08 (t,J= 7.2 Hz, 2H), 2.35–2.24 (m, 2H). 13 C NMR (101 MHz, DMSO-d 6 ) δ 165.85, 165.30, 133.84, 133.74, 129.72, 129.66, 65.18,52.94, 26.60, 26.17. HPLC,t R = 9.407 min, purity 99.7%。
Example 2: n- (2- (1H-benzoimidazol-2-yl) ethyl) -2, 3-dihydrobenzodioxane-6-carboxamide (I-2)
The synthetic route is as follows:
。
the synthesis method comprises the following steps:
step one, compound B1 (0.5 g,2.78 mmol) was dissolved in 10 mL thionyl chloride, heated to 80 ℃, stirred under reflux for reaction 2 h, and concentrated under reduced pressure to give 500 mg as a white solid product B2.
Step two, compound B3 (0.49 g,2.09 mmol) was dissolved in 10 mL dichloromethane, triethylamine 2 mL was added thereto, a dichloromethane solution (10 mL) in which 500 mg white solid product B2 was slowly added dropwise, the reaction solution was washed twice with water, concentrated under reduced pressure, and purified by a silica gel column chromatography to give a white solid product I-2 (0.5 g, yield 74%). 1 H NMR (400 MHz, DMSO-d 6 )δ12.26 (s, 1H), 8.51 (t,J= 5.6 Hz, 1H), 7.58–7.47 (m, 1H), 7.45–7.30 (m, 3H), 7.16–7.05 (m, 2H), 6.90 (d,J= 8.2 Hz, 1H), 4.32–4.18 (m, 4H), 3.67 (q,J= 6.0 Hz, 2H), 3.06 (t,J= 7.3 Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ165.89, 153.35, 146.40, 143.32, 127.90, 121.09, 117.19, 116.68, 64.76, 64.44, 38.48, 29.37. HPLC,t R = 6.632 min, purity 99.3%。
Example 3: n- (2- (1- (2, 3-dihydrobenzodioxane-6-carbonyl) -1H-benzoimidazol-2-yl) ethyl) -2, 3-dihydrobenzodioxane-6-carboxamide (I-3)
The synthetic route is as follows:
。
the synthesis method comprises the following steps:
compound B2 (1.27, g) and compound B3 (0.5 g,2.14 mmol) prepared in example 2 were taken and dissolved in 20 mL dichloromethane, 5 mL triethylamine was slowly added dropwise thereto, followed by stirring at room temperature for 6 h,the reaction solution was washed twice with water, concentrated under reduced pressure, and purified by silica gel chromatography to give the product I-3 (0.79. 0.79 g, yield 76%) as a white solid. 1 H NMR (400 MHz, CDCl 3 )δ7.75 (dt,J= 8.1, 0.9 Hz, 1H), 7.53–7.43 (m, 1H), 7.36 (d,J= 2.1 Hz, 2H), 7.32–7.27 (m, 2H), 7.26–7.23 (m, 1H), 7.19–7.13 (m, 1H), 6.99–6.82 (m, 3H), 4.42–4.21 (m, 8H), 4.00 (q,J= 5.8 Hz, 2H), 3.28 (t,J= 5.3 Hz, 2H). HPLC,t R = 9.536 min, purity 95.9%.
Example 4: n- (2- (1H-benzimidazol-2-yl) ethyl) -4-bromobenzamide (I-4)
The synthesis method of example 2 was followed by substituting p-bromobenzoic acid for the starting material B1 to give compound I-4. The yield thereof was found to be 80%. 1 H NMR (400 MHz, DMSO-d 6 )δ12.40 (s, 1H), 8.78 (t,J= 5.6 Hz, 1H), 7.85–7.73 (m, 2H), 7.72–7.61 (m, 2H), 7.55–7.41 (m, 2H), 7.18–7.08 (m, 2H), 3.74–3.65 (m, 2H), 3.09 (t,J= 7.3 Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ165.83, 153.26, 133.97, 131.76, 129.80, 125.38, 38.58, 29.22. HPLC,t R = 7.337 min, purity 98.7%.
Example 5: 4-bromo-N- (2- (1- (4-bromophenyl) -1H-benzimidazol-2-yl) ethyl) benzamide (I-5)
The synthesis method of example 3 was followed by substituting p-bromobenzoyl chloride for the starting material B2 to give compound I-5. The yield thereof was found to be 74%. 1 H NMR (400 MHz, DMSO-d 6 )δ8.70 (t,J= 5.5 Hz, 1H), 7.83–7.55 (m, 9H), 7.31–7.23 (m,1H), 7.19–7.09 (m,1H), 6.70–6.63 (d,J= 8.2 Hz, 1H), 3.74 (q,J= 6.5 Hz, 2H), 3.30 (t,J= 6.9 Hz, 2H). 13 C NMR (101 MHz, DMSO-d 6 )δ 168.03, 165.88, 154.78, 142.58, 133.87, 133.84, 132.54, 132.52, 132.36, 131.66, 129.67, 128.64, 125.32, 124.20, 124.09, 119.77, 113.81, 38.28, 29.96. HPLC,t R = 10.874 min, purity 95.5%.
EXAMPLE 6 biological evaluation experiment
(1) Micro-thermophoresis assay (MST), an activity test of interaction between small molecules and YTDDF 2 protein
The experiment was performed using the Lance Ultra method of Perkinelmer company.
Firstly, 100 mu L of 10 mu MYTHDF2 protein (amino acid sequence is shown as SEQ ID NO. 1: SEPHPVLEKLRSINNYNPKDFDWNLKHGRVFIIKSYSE DDIHRSIKYNIWCSTEHGNKRLDAAYRSMNGKGPVYLLFSVNGSGHFCGV AEMKSAVDYNTCAGVWSQDKWKGRFDVRWIFVKDVPNSQLRHIRLENNEN KPVTNSRDTQEVPLEKAKQVLKIIASYKHT TSI) is taken, a 30K ultrafiltration tube is adopted for concentration and liquid exchange to prepare PBS buffer solution, and the protein is marked according to a test scheme of a protein marking kit RED-NHS. The compounds used in the MST experiments were diluted with PBS buffer to a DMSO content of 5%. The labeled proteins were mixed with diluted compounds (500. Mu.M, 250. Mu.M, 125. Mu.M, 62.5. Mu.M, 31.25. Mu.M, 15.63. Mu.M, 7.81. Mu.M, 3.91. Mu.M, 1.95. Mu.M, 0.98. Mu.M, 0.49. Mu.M, 0.24. Mu.M, 0.12. Mu.M, 0. Mu.M) at various concentrations in equal volumes (5. Mu.L: 5. Mu.L) and incubated at 37℃for 30 minutes in the absence of light. The binding of the compound and protein was measured by detecting thermophoresis under a 20% LED light source, and finally the measured data was analyzed using mo.affinity Analysis v2.2.4, all experiments being repeated at least three times.
Dissociation constants calculated using mo.affinity Analysis v2.2.4 software based on data from different concentration testsK D The values are shown in Table 1 below, and it can be seen from the experimental results that the compounds of the present application have binding activity to YTDDF 2 protein.
TABLE 1 compounds of the application against YTHDF2 proteinK D Measurement value
(2) In vitro amplification of small molecules hematopoietic stem cell Activity test
Selecting 7-week-old C57BL/6J mice (Shanghai Laek laboratory animal Co., ltd.), separating bone marrow cells from femur and tibia under aseptic conditions, and measuring 2×10 per well 5 Individual bone marrow cells at 100 μlstemspan (Stemcell, 02691) medium (containing 100 ng/mL mouse Stem cell factor (Sinobiologic, 50487-M08B) and 100 ng/mL mouse thrombopoietin (Sinobiologic, 50146-M08H)) was seeded into 96-well U-plates at 37℃with 5% CO 2 Cell culture is performed under the conditions of (2). After 24 hours, the small molecule inhibitors prepared in examples 1 to 5 (final concentration 20. Mu.M) were added to 100. Mu.L of stemspan medium (containing 100 ng/mL mouse stem cell factor and 100 ng/mL mouse thrombopoietin), respectively, and half the liquid change was performed every 3 days and the corresponding small molecule inhibitors (final concentration 20. Mu.M) were added, respectively. After 14 days of culture, the cells were collected and counted, and flow cytometry was performed to examine the proportion and number of hematopoietic stem cells. The results are shown in FIG. 1, and it can be seen from the experimental results that the compounds I-1, I-2, I-3, I-4 and I-5 prepared by the application have remarkable amplification activity on hematopoietic stem cells. Wherein the statistical analysis uses a unpaired Student's t two-tailed test,Pvalues less than 0.05 are considered statistically different,P<0.01; ***，P<0.001；
(3) In vivo transplantation hematopoietic stem cell Activity test of small molecules
One day prior to bone marrow transplantation, 7 weeks of CD45.1 mice (Shanghai Laek laboratory animal Co., ltd.) were subjected to lethal dose of whole body irradiation at 4.5 gray twice, 3 h intermediate intervals, and total dose of 9 gray. One week before and one week after the transplantation, 1 mL bayer was added to the drinking water (bayer, germany). 2X 10 mice treated with small molecule inhibitors the next day after irradiation 5 Bone marrow cells (from CD45.2 mice, shanghai Srile laboratory animal Co., ltd.) plus 2X 10 5 The individual CD45.1 protective cells were tail vein injected into lethally irradiated CD45.1 recipient mice at a liquid volume of 200. Mu.L each. For the second generation, 1×10 mice from the recipient of the first generation were sacrificed 6 The tail vein of each bone marrow cell was transplanted into a lethal dose irradiated CD45.1 receptor mouse, and 4 weeks, 8 weeks, 12 weeks and 16 weeks after transplantation, and peripheral blood was used for flow cytometry analysis experiments to detect the CD45.2 chimerism rate and the differentiation of the stranguria and medullary lines. The measured results are shown in fig. 2-6, and the experimental results show that the applicationAfter the hematopoietic stem cells are treated in vitro, the compound I-5 can obviously improve the ability of the hematopoietic stem cells of the CD45.2 donor mouse to reconstruct the hematopoietic system of the lethal irradiated CD45.1 acceptor mouse, namely improve the chimeric proportion of various blood cells of the CD45.2 mouse in the CD45.1 mouse; meanwhile, I-5 expands only hematopoietic stem cells, and does not expand various hematopoietic progenitor cells (lymphoblastic progenitor cell CLP, myelogenous progenitor cell CMP, granulocyte macrophage progenitor GMP, megakaryocyte-erythrocyte progenitor cell MEP) and mature B cells (Bcell), myelogenous cells (Myeioid), erythrocytes (Erythro) and T cells (T cells), namely does not cause leukemia tendency, and the safety of the compound I-5 is shown. Wherein the statistical analysis uses unpaired Student' stThe two-tailed test is carried out,Pvalues less than 0.05, with statistical differences,P<0.05; **，P<0.01; ***，P<0.001；N.S, Not significant。
Claims (7)
1. a compound of formula (I) or a pharmaceutically acceptable salt thereof:
，
wherein R is 1 Selected from the following groups:
。
2. a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the compounds are shown below:
。
3. a compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein: the pharmaceutically acceptable salt is an acid addition salt of a compound of formula (I), wherein the acid used for salt formation is an inorganic acid, which is hydrochloric acid, sulfuric acid or phosphoric acid, or an organic acid, which is acetic acid, trichloroacetic acid, propionic acid, butyric acid, maleic acid, p-toluenesulfonic acid, malic acid, malonic acid, cinnamic acid, citric acid, fumaric acid, camphoric acid, digluconic acid, aspartic acid and tartaric acid or methanesulfonic acid.
4. A process for the preparation of a compound according to claim 2, or a pharmaceutically acceptable salt thereof, wherein when the compound is selected from the group consisting of I-3, the synthesis of the compound comprises the steps of:
；
dissolving the compound B2 and the compound B3 in dichloromethane, and dropwise adding triethylamine to obtain a compound I-3;
when the compound is selected from I-5, the synthesis of the compound comprises the steps of:
；
dissolving the compound C2 and the compound B3 in dichloromethane, dropwise adding triethylamine, and stirring to obtain a compound I-5.
5. A pharmaceutical composition characterized by: a compound of general formula (I) or a pharmaceutically acceptable salt thereof as claimed in claim 1, and a pharmaceutically acceptable carrier.
6. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a composition according to claim 5 in the manufacture of a medicament for an inhibitor of the YTHDF2 target.
7. Use of a compound according to claim 1 or a pharmaceutically acceptable salt thereof or a composition according to claim 5 in the manufacture of a medicament for expanding hematopoietic stem cells.
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Non-Patent Citations (4)
| Title |
|---|
| Barlin, Gordon B.，等.Imidazo[1,2-b]pyridazines. XVI. Synthesis and central nervous system activities of some 6-(chloro,alkylthio, phenylthio, benzylthio or benzoyl)imidazo[1,2-b]pyridazines.《Australian Journal of Chemistry》.1994,第47卷(第11期),第1989-1999页. * |
| Barlin, Gordon B..Imidazo[1,2-b]pyridazines: syntheses and interaction with central and peripheral-type (mitochondrial) benzodiazepine receptors.《Journal of Heterocyclic Chemistry》.1998,第35卷(第5期),第1205-1217页. * |
| Fragment Ligands of the m6A‑RNA Reader YTHDF2;Francesco Nai，等;《ACS Medicinal Chemistry Letters》;第13卷;1500−1509 * |
| Transient regulation of RNA methylation in human hematopoietic stem cells promotes their homing and engraftment;Xuepeng Wang，等;Leukemia;第37卷;453-464 * |
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