CN116425828A - Small molecule compound for degrading HDAC7 protein, and preparation and application thereof - Google Patents
Small molecule compound for degrading HDAC7 protein, and preparation and application thereof Download PDFInfo
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- CN116425828A CN116425828A CN202310345461.1A CN202310345461A CN116425828A CN 116425828 A CN116425828 A CN 116425828A CN 202310345461 A CN202310345461 A CN 202310345461A CN 116425828 A CN116425828 A CN 116425828A
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- 102100038719 Histone deacetylase 7 Human genes 0.000 title claims abstract description 29
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Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06017—Dipeptides with the first amino acid being neutral and aliphatic
- C07K5/06034—Dipeptides with the first amino acid being neutral and aliphatic the side chain containing 2 to 4 carbon atoms
-
- 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
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
- C07B2200/00—Indexing scheme relating to specific properties of organic compounds
- C07B2200/07—Optical isomers
-
- 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
Abstract
The invention discloses a small molecule compound for degrading HDAC7 protein, and preparation and application thereof. The small molecule compounds include pharmaceutically acceptable salts thereof. The small molecular compound provided by the invention is a PROTAC compound I-1 for targeted degradation of HDAC7 protein, and the small molecular compound I-1 has an obvious degradation effect on HDAC7 in NB4 cells, provides a novel treatment means for treating acute myeloid lymphoblastic leukemia, and can be applied to preparation of drugs for treating acute myeloid lymphoblastic leukemia. The structural formula of the small molecule compound is shown as I-1:
Description
Technical Field
The invention belongs to the technical field of medicines, and relates to a small molecular compound for degrading HDAC7 protein, and preparation and application thereof.
Background
HDAC7 (Histone Deacetylase 7) full-name histone deacetylase 7 belongs to class IIa HDAC. Numerous documents indicate that HDAC7 protein has important regulatory role in hematological tumors, solid tumors and related autoimmune diseases, and is a potential therapeutic target. At present, only 5 HDAC drugs are marketed and are all pan-HDAC inhibitors, the indications are narrow, and the drugs are only approved to be suitable for skin T cell lymphomas and peripheral T cell lymphomas, and have the defects of large toxicity, poor drug substitution, poor selectivity and the like. With the progress of drug development, selective small molecule inhibitors of IIa HDAC are developed successively, and representative drugs include TMP269, TMP195, CHDI-390576 and NVS-HD1, and although the drugs have a certain degree of improvement in selectivity compared with the former drugs, the proliferation inhibition effect at the cell level is generally weak, an ideal therapeutic effect is difficult to achieve, and further pharmaceutical research is impossible. The literature shows that the enzyme activity of IIa HDAC is more than 1000 times lower than that of I, and the IIa HDAC has related non-enzyme functions, which is a main reason that small molecule inhibitors are difficult to effectively interfere. PROTAC can target and degrade target protein, can eliminate enzyme activity and non-enzyme function, and has advantages compared with small molecule inhibitor. Therefore, there is a need to develop a compound that targets degradation of HDAC7.
Disclosure of Invention
The invention aims to provide a small molecular compound for degrading HDAC7 protein, which is a PROTAC compound I-1 for degrading the HDAC7 protein in a targeting way, wherein the PROTAC compound I-1 for degrading the HDAC7 protein in a targeting way can obviously degrade the HDAC7 in NB4 cells, and has a wide application prospect.
The PROTAC compound I-1 for targeted degradation of HDAC7 protein provided by the invention is (4R) -1- (S) -2-tertiary butyl-4, 16-dioxo-17- (4-phenylthiazole-2-yl) -4- (3- (5-trifluoromethyl) -1,2, 4-oxadiazole-3-yl) benzamide methyl) piperidine-1-yl) -6,9, 12-trioxa-3, 15-diazaheptadecanoyl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide, and has a structural formula shown in the following I-1:
further, the HDAC7-PROTAC compound I-1 provided by the invention is a stereoisomer, a geometric isomer, a tautomer, a nitrogen oxide, a hydrate, a solvate, a metabolite, a pharmaceutically acceptable salt or a prodrug thereof.
It is a second object of the present invention to provide a method for preparing the protoc compound I-1 targeted to degrade HDAC7 protein,
(1) Compound 1 (methods of synthesis refer to Xiong Y, donovan KA, eleuteri NA, et al chemo-proteomics exploration of HDAC degradability by small molecule degraders.cell Chem biol.2021;28 (10): 1514-1527.) is dissolved in Dichloromethane (DCM), trifluoroacetic acid is added to remove the Boc protecting group, stirring is carried out at room temperature for 1h, the solvent is dried by spinning, and then dissolved in trifluoroacetic acid (DMF), followed by the addition of tert-butyl 2-bromoacetate in K 2 CO 3 Stirring for 2h at room temperature under the condition to carry out nucleophilic substitution to obtain a compound 2;
(2) Dissolving a compound 3 (the synthesis method is referred to patent WO 2020/093370 A1) in DCM, adding trifluoroacetic acid, stirring for 1h at room temperature, spin-drying the solvent, dissolving in DMF, sequentially adding 2, 2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-azahexadecane-16-acid, 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethylurea Hexafluorophosphate (HATU) and N, N-Diisopropylethylamine (DIPEA), stirring for 2h at room temperature, and carrying out acid-amine condensation to obtain a compound 4;
(3) Compound 2 and compound 4 are respectively dissolved in DCM, trifluoroacetic acid is added, stirring is carried out for 1h at room temperature, solvent is dried by spin, then the mixture is dissolved in DMF, HATU and DIPEA are sequentially added, stirring is carried out for 2h at room temperature, TLC monitoring reaction is completed, water is added into the reaction, dichloromethane extraction is carried out, organic solvent is dried by spin, and the target compound I-1 is obtained through silica gel column chromatography purification.
The reaction formula is as follows:
it is a further object of the present invention to provide the use of said compound I-1, including pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of acute myeloid lymphoblastic leukemia, wherein said compound I-1 is useful for the treatment of acute myeloid lymphoblastic leukemia based on the mechanism of protein degradation of HDAC7.
The invention synthesizes and screens a novel HDAC7-PROTAC degradation agent. The western blot experiment proves that the novel HDAC7-PROTAC degradation agent (compound I-1) provided by the invention has obvious degradation effect on HDAC7 in NB4 cells, and the degradation agent is suggested to have potential treatment effect in acute myeloid lymphoblastic leukemia.
Drawings
Fig. 1: of the compounds of the invention 1 H NMR chart.
Fig. 2: of the compounds of the invention 13 C NMR chart.
Fig. 3: WB plots of the compounds of the invention on NB4 cells.
Detailed Description
The following detailed description of some embodiments of the invention is provided merely as an example and is intended to provide a detailed illustration of the invention in order to facilitate an understanding of the invention and is not to be construed as limiting the invention.
Throughout the description of the examples, the materials, methods, and terms of art used are those which are recognized in the art and unless otherwise indicated, the description of the invention will take place in the presence of an ambiguity. The various raw materials, reagents, instruments, equipment, etc. used in the present invention are commercially available or available by existing methods unless otherwise specified.
EXAMPLE 1 Synthesis of Compound I-1
Step one: preparation of tert-butyl 2- (4- (4-phenylthiazol-2-yl) -4- (3- (5-trifluoromethyl) -1,2, 4-oxadiazol-3-yl) benzamido) methyl) piperidin-1-yl) acetate (Compound 2)
4- (4-phenylthiazol-2-yl) -4- (3- (5-trifluoromethyl) -1,2, 4-oxadiazol-3-yl) benzamido) methyl) piper-dinePyridine-1-carboxylic acid tert-butyl ester (Compound 1,614mg,1.0 mmol) was dissolved in 35mL of DCM solution, 2mL of trifluoroacetic acid was added, stirring was performed at room temperature for 1h, the solvent was dried by spinning, 35mL of DMF solution was added, and K was added sequentially 2 CO 3 (276 mg,2.0 mmol), tert-butyl bromoacetate (234 mg,1.2 mmol), stirring at room temperature for 2h, diluting with water, extracting with DCM, anhydrous Na 2 SO 4 Washing, spin-drying the solvent, and purifying by silica gel column chromatography to obtain white solid 324mg, YIeld:50%; ESI-MS 628.2[ M+H ]] + ； 1 H NMR(500MHz,DMSO-d 6 )δ8.79(t,J＝6.5Hz,1H),8.44(d,J＝1.8Hz,1H),8.19(dt,J＝7.8,1.6Hz,1H),8.07(d,J＝5.8Hz,2H),7.94–7.90(m,2H),7.69(t,J＝7.8Hz,1H),7.38(t,J＝7.7Hz,2H),7.29(t,J＝7.5Hz,1H),3.53(d,J＝6.4Hz,2H),3.06(s,2H),2.77(q,J＝5.1,4.5Hz,2H),2.39(t,J＝10.9Hz,2H),2.30(d,J＝13.3Hz,2H),2.03–1.89(m,2H),1.31(s,9H).
Step two: preparation of tert-butyl-13- ((2 s,4 r) -4-hydroxy-2- (4- (4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidine-1-carbonyl) -14, 14-dimethyl-11-oxo-3, 6, 9-trioxa-12-aza-pentadecyl) carbamate (Compound 4)
(2S) -1- ((4R) -4-hydroxy-2- (4- (4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidin-1-yl) -3, 3-dimethyl-1-oxobutan-2-ylcarbamic acid tert-butyl ester (compound 3,530mg,1.0 mmol) was dissolved in 35mL of DCM, 2mL of trifluoroacetic acid was added, stirring was carried out at room temperature for 1h, the spin-dry solvent was dissolved in 35mL of DMF solution, 2-dimethyl-4-oxo-3, 8,11, 14-tetraoxa-5-azahexadecane-16-oic acid (365 mg,1.2 mmol), HATU (560 mg,1.5 mmol), DIPEA (0.5 mL,3.0 mmol) was added, stirring was carried out at room temperature for 2h, water was added for dilution, the organic layer was combined, silica gel was stirred, the spin-dry solvent was purified by passing through DCM=1:50 column to give white solid 0.4g, YIE:56; ESI-MS 720.4[ M+H ]] + .
Step three: preparation of (4R) -1- (S) -2-tert-butyl-4, 16-dioxo-17- (4-phenylthiazol-2-yl) -4- (3- (5-trifluoromethyl) -1,2, 4-oxadiazol-3-yl) benzoylaminomethyl) piperidin-1-yl) -6,9, 12-trioxa-3, 15-diazaheptadecanoyl) -4-hydroxy-N- (4- (4-methylthiazol-5-yl) benzyl) pyrrolidine-2-carboxamide (Compound I-1)
Tert-butyl 2- (4- (4-phenylthiazol-2-yl) -4- (3- (5-trifluoromethyl) -1,2, 4-oxadiazol-3-yl) benzamido) methyl) piperidin-1-yl acetate (compound 2,627mg,1.0 mmol), tert-butyl-13- ((2 s,4 r) -4-hydroxy-2- (4- (4-methylthiazol-5-yl) benzyl) carbamoyl) pyrrolidine-1-carbonyl) -14, 14-dimethyl-11-oxo-3, 6, 9-trioxa-12-aza pentadecyl) carbamate (compound 4,791mg,1.1 mmol) was dissolved in 30mL of DCM, 3mL of trifluoroacetic acid was added, stirred at room temperature for 1h, spin-drying solvents were dissolved in 20mL of DMF solution, respectively, the reaction solutions were combined, HATU (560 mg,1.5 mmol), DIPEA (0.5 mL,3.0 mmol) were added in sequence, diluted with water, and the combined, the organic layers were combined, and dried in vacuo to obtain a white solid by chromatography column=50:35:35:; ESI-MS 1173.4[ M+H ]] + The method comprises the steps of carrying out a first treatment on the surface of the Concrete embodiments 1 H、 13 The spectrogram C is shown in fig. 1 and 2, and the data are as follows: 1 H NMR(500MHz,DMSO-d 6 )δ8.98(d,J＝5.4Hz,1H),8.73(t,J＝6.4Hz,1H),8.61(t,J＝6.1Hz,1H),8.44(t,J＝1.8Hz,1H),8.18(dt,J＝7.8,1.4Hz,1H),8.11–7.99(m,2H),7.97–7.90(m,2H),7.74(t,J＝5.9Hz,1H),7.68(t,J＝7.8Hz,1H),7.46–7.35(m,7H),7.32–7.25(m,1H),5.17(d,J＝3.5Hz,1H),4.57(dd,J＝9.6,2.8Hz,1H),4.45(t,J＝8.1Hz,1H),4.42–4.34(m,2H),4.26(dt,J＝15.8,5.3Hz,1H),3.97(d,J＝6.5Hz,2H),3.75–3.43(m,14H),3.39(t,J＝6.0Hz,2H),3.33(d,J＝5.8Hz,1H),3.22(q,J＝5.8Hz,2H),2.77(d,J＝53.7Hz,3H),2.44(d,J＝4.8Hz,3H),2.34–2.29(m,1H),2.20(d,J＝11.5Hz,1H),2.05(dt,J＝15.8,10.7Hz,3H),1.91(m,1H),0.97–0.93(m,9H). 13 C NMR(126MHz,DMSO-d 6 )δ174.89,172.23,169.84,169.60,169.06(d,J＝3.4Hz),168.56,166.12,154.18,151.90,148.21,139.91,136.25,134.86,131.73,131.60,130.31,130.17,130.06,129.15,129.06,128.21,127.93,126.80,126.44,125.02,114.74,70.92,70.31,70.21,70.09(d,J＝3.5Hz),70.00,69.47,69.35,68.30,61.85,59.21,57.04,56.16,50.32,44.70,42.16,38.51,38.39,36.19,34.17,26.64,16.38.
EXAMPLE 2 in vitro enzyme Activity of Compounds of the invention
1. Experimental method
Instrument: microplate reader TECAN SPARK (TECAN, switzerland);
materials: HDAC7 protein purified from SF9 cells; the substrate Ac-Leu-Lys (TFAc) -AMC;
sample processing: the sample is dissolved by DMSO, stored at low temperature and diluted in a gradient way, and the concentration of the DMSO in a final system is controlled within a range which does not influence the enzyme activity detection. The positive compound used in the experiment was TMP269.
2. The experimental procedure is as follows: 100nM HDAC7 protein and 50. Mu.M substrate were dissolved in kinase reaction buffer (500mM NaCl,50mM Tris,PH8.0). 0.3. Mu.M, 3. Mu.M of compound was added to the reaction system (100. Mu.L) and the non-dosing group, positive compound (TMP 269, 0.3. Mu.M and 3. Mu.M) and blank group were set simultaneously, and 2 sub-wells were set for each concentration of each sample. After sufficient lysis, the system was transferred to a 96-well plate and 100. Mu.l of 10mg/ml of trypsin was added and incubated at 37 ℃. Fluorescence values (absorption light 460nm, excitation light 390 nm) were detected by an enzyme-labeled instrument to indicate the release of AMC. And calculating the enzyme activity inhibition rate of the sample according to the sample reading, wherein the calculation formula is as follows: (RFU non-dosing-RFU compound)/RFU non-dosing x100%. The results are shown in Table 1.
3. Experimental results: compound I-1 can bind to HDAC7, with the potential to form a stable ternary complex.
TABLE 1 inhibition of enzyme activity of the compounds of the invention at concentrations of 0.3. Mu.M and 3. Mu.M
"A" represents >75%; "B" represents 50% to 75%; "C" represents 25% to 50%; "D" represents <25%. The results show that: the PROTAC molecule I-1 after the structure modification has the capability of combining with HDAC7 protein and does not have strong inhibition.
EXAMPLE 3 degradation of HDAC7 in NB4 cells by the Compounds of the present invention
1. The experimental method comprises the following steps:
cell culture and administration: NB4 cells were placed in 5% CO 2 Is cultured in a constant temperature incubator at 37 ℃ under the condition of RPMI-1640+10% Gibco serum. Front three of resuscitationInstead of antimycotic agents. The cell suspension was added to a 15mL centrifuge tube, centrifuged at 1500rpm for 4min, the supernatant was discarded, resuspended in 2mL of culture medium and counted. The cell suspension was seeded in 6-well plates, 50 ten thousand cells per well, and the test compound was added at the appropriate concentration. And collecting samples after 12-24 hours.
Sample preparation: at the end of the time of action, the cells are collected and washed once with PBS; adding 4% SDS according to the cell quantity to lyse the cells, and performing ultrasonic treatment until the cells are not sticky; centrifuging at room temperature for 30min at 12,000 g; the supernatant was transferred to a new EP tube for protein quantification.
The degradation of HDAC7 in NB4 cells was tested by Western Blot at concentrations of 0.3. Mu.M, 1. Mu.M, and 3. Mu.M, respectively, and the degradation data are shown in Table 2, and the experimental results are shown in FIG. 3.
2. Experimental results: compound I-1 has the ability to significantly degrade HDAC7 in NB4 cells.
TABLE 2 degradation screening test of the inventive Compounds in NB4 cells
"A" represents >75%; "B" represents 50% to 75%; "C" represents 25% to 50%; "D" represents <25%. The results show that: compound I-1 exerts degradation of PROTAC in NB4 cells, significantly degrading HDAC7 in the range of 0.3-3 μm.
Claims (4)
1. A small molecule compound for degrading an HDAC7 protein, wherein the small molecule compound has a structural formula shown in I-1:
or a pharmaceutically acceptable salt thereof.
2. The method for preparing a small molecule compound according to claim 1, characterized by comprising the steps of:
(1) Dissolving compound 1 in dichloromethane, adding trifluoroacetic acid to remove Boc protecting group, and adding tert-butyl 2-bromoacetate in K 2 CO 3 Nucleophilic substitution is carried out under the condition to obtain a compound 2;
(2) Removing Boc from the compound 3 under the condition of trifluoroacetic acid, and then carrying out acid-amine condensation on the compound and 2, 2-dimethyl-4-oxo-3,8,11,14-tetraoxa-5-aza-hexadecane-16-acid to obtain a compound 4;
(3) Removing Boc from the compound 2 and the compound 4 under the condition of trifluoroacetic acid, and performing acid-amine condensation to obtain the compound I-1.
3. Use of compound I-1 according to claim 1, including pharmaceutically acceptable salts thereof, for the manufacture of a medicament for the treatment of acute myeloid lymphoblastic leukemia.
4. The use according to claim 3, wherein said compound I-1 is based on a mechanism for degradation of HDAC7 protein for the treatment of acute myeloid lymphoblastic leukemia.
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