CN113512035B - Dihydropyrimidine-pomalidomide conjugate, and preparation method and application thereof - Google Patents
Dihydropyrimidine-pomalidomide conjugate, and preparation method and application thereof Download PDFInfo
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- CN113512035B CN113512035B CN202110452224.6A CN202110452224A CN113512035B CN 113512035 B CN113512035 B CN 113512035B CN 202110452224 A CN202110452224 A CN 202110452224A CN 113512035 B CN113512035 B CN 113512035B
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- Prior art keywords
- pomalidomide
- dihydropyrimidine
- room temperature
- boc
- acid
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- 229960000688 pomalidomide Drugs 0.000 title claims abstract description 33
- 238000002360 preparation method Methods 0.000 title claims abstract description 20
- 150000001875 compounds Chemical class 0.000 claims abstract description 34
- 239000003814 drug Substances 0.000 claims abstract description 15
- UVSMNLNDYGZFPF-UHFFFAOYSA-N pomalidomide Chemical compound O=C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O UVSMNLNDYGZFPF-UHFFFAOYSA-N 0.000 claims abstract description 4
- 239000008194 pharmaceutical composition Substances 0.000 claims abstract description 3
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 claims description 99
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 claims description 35
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 claims description 28
- BWHMMNNQKKPAPP-UHFFFAOYSA-L potassium carbonate Chemical compound [K+].[K+].[O-]C([O-])=O BWHMMNNQKKPAPP-UHFFFAOYSA-L 0.000 claims description 28
- JGFZNNIVVJXRND-UHFFFAOYSA-N N,N-diisopropylethylamine Substances CCN(C(C)C)C(C)C JGFZNNIVVJXRND-UHFFFAOYSA-N 0.000 claims description 27
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 24
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 21
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 claims description 21
- 238000003756 stirring Methods 0.000 claims description 20
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 17
- PCLIMKBDDGJMGD-UHFFFAOYSA-N N-bromosuccinimide Chemical compound BrN1C(=O)CCC1=O PCLIMKBDDGJMGD-UHFFFAOYSA-N 0.000 claims description 14
- 229910000027 potassium carbonate Inorganic materials 0.000 claims description 14
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 12
- 239000001632 sodium acetate Substances 0.000 claims description 12
- 235000017281 sodium acetate Nutrition 0.000 claims description 12
- 239000002904 solvent Substances 0.000 claims description 11
- OPZDXMCOWFPQPE-UHFFFAOYSA-N 2-bromo-4-fluorobenzaldehyde Chemical compound FC1=CC=C(C=O)C(Br)=C1 OPZDXMCOWFPQPE-UHFFFAOYSA-N 0.000 claims description 7
- YCPULGHBTPQLRH-UHFFFAOYSA-N 3-aminopiperidine-2,6-dione;hydron;chloride Chemical compound Cl.NC1CCC(=O)NC1=O YCPULGHBTPQLRH-UHFFFAOYSA-N 0.000 claims description 7
- XYIBRDXRRQCHLP-UHFFFAOYSA-N ethyl acetoacetate Chemical compound CCOC(=O)CC(C)=O XYIBRDXRRQCHLP-UHFFFAOYSA-N 0.000 claims description 7
- INQOMBQAUSQDDS-UHFFFAOYSA-N iodomethane Chemical compound IC INQOMBQAUSQDDS-UHFFFAOYSA-N 0.000 claims description 7
- 238000000034 method Methods 0.000 claims description 7
- CWXPZXBSDSIRCS-UHFFFAOYSA-N tert-butyl piperazine-1-carboxylate Chemical compound CC(C)(C)OC(=O)N1CCNCC1 CWXPZXBSDSIRCS-UHFFFAOYSA-N 0.000 claims description 7
- -1 1-methyllenalidomide Chemical compound 0.000 claims description 6
- 239000007821 HATU Substances 0.000 claims description 6
- 238000010992 reflux Methods 0.000 claims description 6
- WWJAZKZLSDRAIV-UHFFFAOYSA-N 4-fluoro-2-benzofuran-1,3-dione Chemical compound FC1=CC=CC2=C1C(=O)OC2=O WWJAZKZLSDRAIV-UHFFFAOYSA-N 0.000 claims description 4
- 239000012467 final product Substances 0.000 claims description 4
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims description 4
- 239000002994 raw material Substances 0.000 claims description 4
- UPBQMAHYLWJGDW-UHFFFAOYSA-N 2-[2-[(2-methylpropan-2-yl)oxycarbonylamino]ethoxy]acetic acid Chemical compound CC(C)(C)OC(=O)NCCOCC(O)=O UPBQMAHYLWJGDW-UHFFFAOYSA-N 0.000 claims description 3
- HIDJWBGOQFTDLU-UHFFFAOYSA-N 4-[(2-methylpropan-2-yl)oxycarbonylamino]butanoic acid Chemical compound CC(C)(C)OC(=O)NCCCC(O)=O HIDJWBGOQFTDLU-UHFFFAOYSA-N 0.000 claims description 3
- RUFDYIJGNPVTAY-UHFFFAOYSA-N 6-[(2-methylpropan-2-yl)oxycarbonylamino]hexanoic acid Chemical compound CC(C)(C)OC(=O)NCCCCCC(O)=O RUFDYIJGNPVTAY-UHFFFAOYSA-N 0.000 claims description 3
- FPRZYWCRQHFPSX-UHFFFAOYSA-N 8-[(2-methylpropan-2-yl)oxycarbonylamino]octanoic acid Chemical compound CC(C)(C)OC(=O)NCCCCCCCC(O)=O FPRZYWCRQHFPSX-UHFFFAOYSA-N 0.000 claims description 3
- 239000003513 alkali Substances 0.000 claims description 3
- NMVVJCLUYUWBSZ-UHFFFAOYSA-N aminomethylideneazanium;chloride Chemical compound Cl.NC=N NMVVJCLUYUWBSZ-UHFFFAOYSA-N 0.000 claims description 3
- 239000003153 chemical reaction reagent Substances 0.000 claims description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 3
- UEJJHQNACJXSKW-UHFFFAOYSA-N 2-(2,6-dioxopiperidin-3-yl)-1H-isoindole-1,3(2H)-dione Chemical compound O=C1C2=CC=CC=C2C(=O)N1C1CCC(=O)NC1=O UEJJHQNACJXSKW-UHFFFAOYSA-N 0.000 claims description 2
- RUVRGYVESPRHSZ-UHFFFAOYSA-N 2-[2-(2-azaniumylethoxy)ethoxy]acetate Chemical compound NCCOCCOCC(O)=O RUVRGYVESPRHSZ-UHFFFAOYSA-N 0.000 claims description 2
- SLXKOJJOQWFEFD-UHFFFAOYSA-N 6-aminohexanoic acid Chemical compound NCCCCCC(O)=O SLXKOJJOQWFEFD-UHFFFAOYSA-N 0.000 claims description 2
- UQXNEWQGGVUVQA-UHFFFAOYSA-N 8-aminooctanoic acid Chemical group NCCCCCCCC(O)=O UQXNEWQGGVUVQA-UHFFFAOYSA-N 0.000 claims description 2
- GNRLUBOJIGSVNT-UHFFFAOYSA-N Aminoethoxyacetic acid Chemical compound NCCOCC(O)=O GNRLUBOJIGSVNT-UHFFFAOYSA-N 0.000 claims description 2
- 238000005761 Biginelli synthesis reaction Methods 0.000 claims description 2
- 229960002684 aminocaproic acid Drugs 0.000 claims description 2
- 238000005893 bromination reaction Methods 0.000 claims description 2
- 239000003937 drug carrier Substances 0.000 claims description 2
- 239000012634 fragment Substances 0.000 claims description 2
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims description 2
- 239000005457 ice water Substances 0.000 claims description 2
- 229960004942 lenalidomide Drugs 0.000 claims description 2
- GOTYRUGSSMKFNF-UHFFFAOYSA-N lenalidomide Chemical compound C1C=2C(N)=CC=CC=2C(=O)N1C1CCC(=O)NC1=O GOTYRUGSSMKFNF-UHFFFAOYSA-N 0.000 claims description 2
- 239000000546 pharmaceutical excipient Substances 0.000 claims description 2
- 239000007858 starting material Substances 0.000 claims description 2
- 229960003433 thalidomide Drugs 0.000 claims description 2
- VQNDBXJTIJKJPV-UHFFFAOYSA-N 2h-triazolo[4,5-b]pyridine Chemical compound C1=CC=NC2=NNN=C21 VQNDBXJTIJKJPV-UHFFFAOYSA-N 0.000 claims 1
- 239000003054 catalyst Substances 0.000 claims 1
- 229940079593 drug Drugs 0.000 abstract description 13
- WCFAPJDPAPDDAQ-UHFFFAOYSA-N 1,2-dihydropyrimidine Chemical compound C1NC=CC=N1 WCFAPJDPAPDDAQ-UHFFFAOYSA-N 0.000 abstract description 3
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 71
- GVNVAWHJIKLAGL-UHFFFAOYSA-N 2-(cyclohexen-1-yl)cyclohexan-1-one Chemical compound O=C1CCCCC1C1=CCCCC1 GVNVAWHJIKLAGL-UHFFFAOYSA-N 0.000 description 66
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- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical class [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 9
- 238000004440 column chromatography Methods 0.000 description 9
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 8
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- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
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- OMBVJVWVXRNDSL-UHFFFAOYSA-N 2-[2-[2-[(2-methylpropan-2-yl)oxycarbonylamino]ethoxy]ethoxy]acetic acid Chemical compound CC(C)(C)OC(=O)NCCOCCOCC(O)=O OMBVJVWVXRNDSL-UHFFFAOYSA-N 0.000 description 2
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- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 description 2
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- 229930182555 Penicillin Natural products 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing three or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/12—Antivirals
- A61P31/20—Antivirals for DNA viruses
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
- C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
- C07D417/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
-
- 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 provides a dihydropyrimidine pomalidomide conjugate, a preparation method and application thereof. The compound has a structure shown in a formula I. The invention also relates to a preparation method of the compound containing the structure shown in the formula I, a pharmaceutical composition and application of the compound in preparing anti-HBV medicines.
Description
Technical Field
The invention belongs to the technical field of medicines, and particularly relates to a dihydropyrimidine-pomalidomide conjugate, a preparation method thereof and an anti-HBV (hepatitis B Virus) drug application.
Background
Viral Hepatitis B (viral Hepatitis type B), abbreviated as Hepatitis B (HBV), is a major infectious disease caused by the Hepatitis B Virus (HBV) and can lead to acute and chronic viral Hepatitis, severe Hepatitis, cirrhosis and primary hepatocellular carcinoma (hepatocellular carcinoma, HCC) after long-term development. The current drugs for preventing and treating chronic hepatitis B mainly include vaccines, interferons, immunomodulators and DNA polymerase inhibitors. However, the traditional Chinese medicine has the defects of drug resistance, side effect, rebound after stopping drug, incomplete removal of hepatitis B virus and the like, so that the research and development of a new generation of safe, efficient, low-toxic and drug-resistant non-nucleoside hepatitis B virus inhibitor has important scientific significance. The core protein is the main structural protein composed of HBV nucleocapsids, is relatively conserved in the virus evolution process, and the assembly of the core protein plays an important role in the life cycle of hepatitis B virus. However, no related target drugs are currently marketed.
Protein degradation targeting chimera (PROteolysis TArgeting Chimeria, PROTAC) is a technology that utilizes the intracellular ubiquitin-protease degradation system to target degradation of a target protein. The PROTAC molecule is a bifunctional molecule consisting of three parts, the target protein receptor, the intermediate link chain and the E3 ubiquitin protease ligand, respectively. It can recognize both the target protein and the E3 ligase, forming a triple complex with them in space, and then mediating ubiquitination and degradation of the target protein. The procac technology is blooming in a variety of target applications due to its multiple advantages. The PROTAC strategy is used for HBV core protein, so that the virus capsid can be degraded in a targeted way, and the multifunctional property of the virus protein is utilized to exert greater antiviral potential.
Among the HBV core protein assembly modulators that have been reported to date, dihydropyrimidines can induce HBV capsid misinduction to exert antiviral effects. Through research on the crystal complex structure of core protein and dihydropyrimidine ligand, the 6-position of dihydropyrimidine is located in the solvent opening region, and the modification method is suitable for modification of PROTAC analogues. Position 6 was selected as a ligation site for ligation of a "Linker arm" (Linker); the use of different hydrophilicities and different lengths of "linker arms" for linking the core protein ligand and the E3 ligase ligand; using an E3 ligase ligand for recognition of E3 ligase, a total of 15 dihydropyrimidine-pomalidomide conjugates were designed and synthesized, and no related report is found in the prior art.
Disclosure of Invention
The invention provides a dihydropyrimidine-pomalidomide conjugate and a preparation method thereof, and also provides an activity screening result of the compound serving as a non-nucleoside HBV inhibitor and pharmaceutical application thereof.
The technical scheme of the invention is as follows:
1. dihydropyrimidine-pomalidomide conjugates
The dihydropyrimidine-pomalidomide conjugate has a structure shown in the following general formula I:
wherein,
the connecting arm (Linker) is a fatty amino acid chain with the number of main chain atoms of 3-20, an ethoxyamino acid chain with the number of main chain atoms of 4-20 or an amino acid chain with the number of atoms of 3-20 and containing O, S, N atoms;
r is a hydrogen atom, pomalidomide, 1-methylppomalidomide, lenalidomide, 1-methyllenalidomide, thalidomide or 1-methylthalidomide.
According to the invention, the connecting arm is preferably a fatty amino acid chain with the main chain atom number of 4-8, an ethoxyamino acid chain with the main chain atom number of 5-8 or an amino acid chain with the atom number of 4-8 and containing O, S, N atoms;
r is a hydrogen atom, pomalidomide or 1-methylppomalidomide.
Further preferred, the linker arm is 8-aminocaprylic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2- (2- (2-aminoethoxy) ethoxy) acetic acid, 2- (2-aminoethoxy) acetic acid;
still further preferred, the dihydropyrimidine-pomalidomide conjugate is one of the compounds having the following structure:
TABLE 1 Structure of dihydropyrimidine-pomalidomide conjugates of target compounds
2. Process for preparing dihydropyrimidine-pomalidomide conjugates
A method for preparing a dihydropyrimidine-pomalidomide conjugate, comprising the steps of: 3-fluorophthalic anhydride II-1 is taken as a raw material, and is refluxed with 3-amino-2, 6-piperidinedione hydrochloride and sodium acetate in an acetic acid solvent at 120 ℃ for 10 hours to obtain II-2; dissolving II-2 in N, N-dimethylformamide solution, adding methyl iodide under stirring at room temperature by taking potassium carbonate as alkali, and reacting at room temperature for 24 hours to obtain an intermediate II-3; 2-thiazole formamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and ethyl acetoacetate are taken as starting materials, and are cyclized through a Biginelli reaction to obtain a key intermediate 2; in dichloromethane solution, the intermediate 2 and N-bromosuccinimide undergo bromination reaction to obtain an important intermediate 3; taking the intermediate 3 as a raw material, adding potassium carbonate, potassium iodide and 1-Boc piperazine, and refluxing in acetonitrile solution at 75 ℃ for 1h to obtain an intermediate 4; dissolving the 4 in dichloromethane solution of trifluoroacetic acid, stirring at room temperature for 10 hours, and removing Boc to obtain an intermediate 5; activating the N-Boc- "connecting arm" fragment and 2- (7-azabenzotriazol) -N, N, N ', N' -tetramethyluronium Hexafluorophosphate (HATU) in an ice-water bath for half an hour, adding intermediate 5 and N, N-Diisopropylethylamine (DIPEA) and stirring overnight at room temperature to obtain intermediate 6; dissolving 6 in dichloromethane solution of trifluoroacetic acid, stirring at room temperature for 10h, and removing Boc to obtain a final product 7 (a-e); 7 (a-e) and II-2 or II-3 are refluxed for 10 hours in N, N-dimethylformamide solution by taking DIPEA as a base at 90 ℃ to obtain a final product 8 (a-j).
The synthetic route is as follows:
reagents and conditions: (i) 3-amino-2, 6-piperidinedione hydrochloride, acetic acid, sodium acetate, 120 ℃ for 10h; (ii) Potassium carbonate, methyl iodide, N, N-dimethylformamide, at room temperature for 24 hours; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80 ℃; the method comprises the steps of carrying out a first treatment on the surface of the (iv) N-bromosuccinimide, dichloromethane, 40 ℃; (v) 1-tert-butoxycarbonyl-piperazine, potassium carbonate, potassium iodide, acetonitrile, 75 ℃ for 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N-Boc-Linker, HATU, DIPEA, dichloromethane, 0deg.C, 30min, room temperature, 6h; (viii) trifluoroacetic acid, dichloromethane, room temperature, 10h; (ix) II-2 or II-3, N-dimethylformamide, DIPEA,90℃for 10h.
Wherein, N-tert-butoxycarbonyl-Linker includes: N-Boc-8-aminocaprylic acid, N-Boc-6-aminocaproic acid, N-Boc-4-aminobutyric acid, N-Boc-2- (2- (2-aminoethoxy) ethoxy) acetic acid, N-Boc-2- (2-aminoethoxy) acetic acid.
According to the preferred preparation method of the dihydropyrimidine-pomalidomide conjugate, the preparation method comprises the following specific preparation steps:
(1) Dissolving 3-fluorophthalic anhydride, 3-amino-2, 6-piperidinedione hydrochloride and sodium acetate in acetic acid, and refluxing at 120 ℃ for 10 hours; after the reaction is finished, performing rapid column chromatography separation to obtain II-2;
(2) Adding the solvent N, N-dimethylformamide into the II-2 and potassium carbonate, dropwise adding methyl iodide under stirring at room temperature, and stirring at room temperature for 24 hours. After the reaction is finished, extracting and separating by a rapid column chromatography to obtain II-3;
(3) Dissolving 2-thiazole formamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and sodium acetate in absolute ethyl alcohol, adding ethyl acetoacetate under stirring at room temperature, and refluxing with ethanol at 80 ℃ for 8 hours; after the reaction is finished, extracting, separating by a rapid column chromatography, and recrystallizing to obtain 2;
(4) Dissolving the intermediate 2 in dichloromethane, adding a small amount of NBS for many times under stirring at room temperature, and refluxing the dichloromethane for 1.5 hours; after the reaction is finished, extracting, separating by using a rapid column chromatography, and recrystallizing to obtain a compound 3;
(5) Intermediate 3 and 1-Boc-piperazine, potassium carbonate and potassium iodide were taken, acetonitrile was added, and the mixture was refluxed at 75℃for 1 hour. After the reaction is finished, extracting, separating by using a rapid column chromatography, and recrystallizing to obtain a compound 4;
(6) Intermediate 4 was taken and dissolved in dichloromethane, trifluoroacetic acid was added and stirred at room temperature for 10h. After the reaction is finished, adding saturated sodium carbonate solution and dichloromethane for extraction, spin-drying to obtain a crude product 5, and directly carrying out the next reaction;
(7) The different N-Boc-Linker "arms" and HATU were dissolved in dichloromethane, ice-bath, added 5 and DIPEA and stirred at room temperature for 10h. After the reaction is finished, extracting, separating by a rapid column chromatography, and recrystallizing to obtain 6 (a-e);
(8) Intermediate 6 (a-e) was taken and dissolved in methylene chloride, trifluoroacetic acid was added thereto, and the mixture was stirred at room temperature for 10 hours. After the reaction, adding saturated sodium carbonate solution and dichloromethane for extraction, separating by using preparative TLC, and recrystallizing to obtain the target compound 7 (a-e);
(9) 7 (a-e) and 2 or 3 are dissolved in N, N-dimethylformamide, DIPEA is added, and the mixture is reacted for 10 hours at 90 ℃. After the reaction, the target compound 8 (a-j) is obtained by extraction, flash column chromatography and recrystallization.
The room temperature of the invention is 20-30 ℃.
3. Use of dihydropyrimidine-pomalidomide conjugates
The invention discloses a dihydropyrimidine-pomalidomide conjugate anti-HBV activity screening result and application thereof as an anti-HBV inhibitor. Experiments prove that the dihydropyrimidine-pomalidomide conjugate can be applied as a classical HBV non-nucleoside inhibitor.
As shown in Table 2, the in vitro anti-HBV activity evaluation and cytotoxicity evaluation were performed on the synthesized target compounds 7 (a-e) and 8 (a-j). HBV DNA inhibition activity was determined by PCR and cytotoxicity was determined by MTS, while the marketed drug lamivudine (3 TC) and the clinical drug candidate moxadine mesylate (GLS 4) were selected as positive controls.
The novel synthetic dihydropyrimidine-pomalidomide conjugates of the present invention exhibit significant anti-HBV activity. All dihydropyrimidine-PROTAC analogs exhibit anti-HBV activity at low micromolar concentration levels, EC 50 The value was in the range of 0.43-3.77. Mu.M, which is weaker than the GLS4 activity (EC 50 =0.046 μm), but there are three compounds whose activity is close to that of the positive drug 3TC (EC 50 =0.40 μm), in particular 8c (EC 50 ＝0.48μM)、8i(EC 50 =0.46 μm) and 8j (EC 50 =0.43 μm), with value for further investigation.
The dihydropyrimidine-pomalidomide conjugate is a non-nucleoside HBV inhibitor with a novel structure and can be used as a lead compound for resisting HBV.
The dihydropyrimidine-pomalidomide conjugate of the present invention can be used as a non-nucleoside HBV inhibitor. In particular, the HBV inhibitor is used for preparing anti-hepatitis B medicines.
An anti-HBV pharmaceutical composition comprising a dihydropyrimidine-pomalidomide conjugate of the invention and one or more pharmaceutically acceptable carriers or excipients.
The invention discloses a dihydropyrimidine-pomalidomide conjugate, a preparation method thereof, an anti-HBV activity screening result and first application of the dihydropyrimidine-pomalidomide conjugate as an anti-HBV inhibitor. Experiments prove that the dihydropyrimidine-pomalidomide conjugate can be used as an HBV inhibitor for preparing anti-HBV medicines.
Detailed Description
The present invention will be further understood by the following examples, in which all target compounds are numbered the same as in table 1, but the content of the present invention is not limited thereto.
The synthetic route is as follows:
reagents and conditions: (i) 3-amino-2, 6-piperidinedione hydrochloride, acetic acid, sodium acetate, 120 ℃ for 10h; (ii) Potassium carbonate, methyl iodide, N, N-dimethylformamide, at room temperature for 24 hours; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80 ℃; the method comprises the steps of carrying out a first treatment on the surface of the (iv) N-bromosuccinimide, dichloromethane, 40 ℃; (v) 1-tert-butoxycarbonyl-piperazine, potassium carbonate, potassium iodide, acetonitrile, 75 ℃ for 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N-Boc-Linker, HATU, DIPEA, dichloromethane, 0deg.C, 30min, room temperature, 6h; (viii) trifluoroacetic acid, dichloromethane, room temperature, 10h; (ix) II-2 or II-3, N-dimethylformamide, DIPEA,90℃for 10h.
EXAMPLE 1 preparation of Compound II-2
3-fluorophthalic anhydride (200 mg,1.2 mmol), 3-amino-2, 6-piperidinedione hydrochloride (319 mg,1.2 mmol) sodium acetate (118 mg,1.44 mmol) was dissolved in 20ml acetic acid and refluxed at 120℃for 10h. And after the reaction is finished, spin-drying the solvent to obtain a large amount of black solid, adding a large amount of methanol for dissolution, adding silica gel for sample mixing and loading on a column, and performing rapid column chromatography separation to obtain II-2.
White solid product, 58% yield; 1 H NMR(400MHz,DMSO-d 6 )δ11.15(s,1H,CONHCO),7.95(q,J＝7.4Hz,1H,Ph-H),7.83–7.66(m,2H,Ph-H),5.16(dd,J＝12.8,5.1Hz,1H,COCHN),2.97–2.82(m,1H,CH 2 ),2.68–2.52(m,1H,CH 2 ),2.18–1.96(m,2H,CH 2 )；EI-MS:275.06[M-H] - ；C 13 H 9 FN 2 O 4 [276.05].
EXAMPLE 2 preparation of Compound II-3
II-2 (100.00 mg,0.36 mmol) and potassium carbonate (50 mg,0.36 mmol) were added to 5ml of N, N-dimethylformamide as solvents, and methyl iodide was added dropwise with stirring at room temperature, followed by stirring at room temperature for 24 hours. After the reaction, the solvent was spun-dried, extracted with water (20 ml) and dichloromethane (20 ml x 2), the organic phases were combined, extracted once with saturated sodium chloride, dried over anhydrous magnesium sulfate and filtered. The organic phase is added to be stirred with silica gel, and is separated by a rapid column chromatography to obtain II-3.
White solid product, 46% yield; 1 H NMR(400MHz,DMSO-d 6 )δ7.96(tdt,J＝7.4,4.7,2.3Hz,1H,Ph-H),7.83–7.70(m,2H,Ph-H),5.30–5.12(m,1H,COCHN),3.08–2.85(m,3H,CH 3 ),2.85–2.73(m,1H,CH 2 ),2.66–2.51(m,1H,CH 2 ),2.08(tdd,J＝12.7,7.5,4.2Hz,1H,CH 2 )；EI-MS:290.25[M-H] - ；C 14 H 11 FN 2 O 4 [290.07].
EXAMPLE 3 preparation of Compound 2
2-Thiazolidine hydrochloride (1.0 g,6.11 mmol), 2-bromo-4-fluorobenzaldehyde (1.86 g,9.16 mmol) and sodium acetate (1.0 g,1.22 mmol) were weighed into absolute ethanol (100 mL), ethyl acetoacetate (1.2 mL,9.20 mmol) was added with stirring at room temperature, and ethanol was refluxed for 8h at 80 ℃; after the reaction, the solution was filtered to remove salts. The mother liquor was cooled to room temperature, and yellow crystals (I-2) were precipitated. The remaining mother liquor was freed from absolute ethanol under reduced pressure, water (60 mL) was added, extracted with ethyl acetate (25 mL. Times.3), the organic phases were collected and combined, extracted once with saturated sodium chloride (25 mL) and the organic phase was dried over anhydrous magnesium sulfate. Filtering, adding 200 mesh silica gel, stirring, separating by flash column chromatography, and recrystallizing to obtain compound 2. 0.75g of yellow powder was obtained, yield: 58%; melting point 153-156 ℃.
1 H NMR(400MHz,DMSO-d6)δ9.92(s,1H),7.97(d,J＝2.8Hz,1H),7.89(s,1H),7.59–7.50(m,1H),7.42–7.31(m,1H),7.23(t,J＝8.3Hz,1H),5.98(s,1H),3.94(q,J＝6.9Hz,2H),2.48(s,3H),1.03(t,J＝7.0Hz,3H)； 13 C NMR(100MHz,DMSO-d6)δ166.07,163.13,159.93,147.99,144.78,143.69,141.19,131.14(d,J＝8.7Hz),124.85,122.94(d,J＝9.6Hz),119.98(d,J＝24.2Hz),115.85(d,J＝21.0Hz),97.33,59.57,58.14,17.86,14.46；EI-MS:426.04[M+2+H] + ,C 17 H 15 BrFN 3 O 2 S[423.01].
EXAMPLE 4 preparation of Compound 3
Intermediate 2 (1.86 g,4.39 mmol) was dissolved in dichloromethane (50 mL) and NBS (1.95 g,1.10 mmol) was added in small portions with stirring at room temperature and the dichloromethane was refluxed for 1.5h; after the reaction was completed, dichloromethane was removed under reduced pressure, water (50 mL) was added, extraction was performed with ethyl acetate (20 ml×3), and the organic phases were collected and combined, extracted once with saturated sodium chloride (25 mL), and dried over anhydrous magnesium sulfate. Filtering, separating by flash column chromatography, and recrystallizing to obtain the compound 3. Yellow solid 1.30g, yield 59%; melting point 123-128 ℃.
1 H NMR(400MHz,CDCl 3 )δ7.84(d,J＝3.1Hz,1H),7.52(s,2H),7.44–7.35(m,1H),7.32(dd,J＝8.1,2.6Hz,1H),7.02(t,J＝8.0Hz,1H),6.09(s,1H),4.94(d,J＝8.9Hz,1H),4.61(s,1H),4.09(d,J＝7.0Hz,2H),1.16(t,J＝7.1Hz,3H)；EI-MS:502.2[M+H] + ； 13 C NMR(100MHz,CDCl 3 )δ164.73,163.27,160.76,155.66,150.28,143.87,143.01,137.84,130.60(d,J＝8.6Hz),124.62,123.45,122.10(d,J＝9.2Hz),120.26(d,J＝24.8Hz),115.72(d,J＝20.9Hz),106.39,60.72,51.61,31.79,14.03；EI-MS:499.90[M-H] - ,501.94[M+2-H] - ,503.91[M+4-H] - ,C 17 H 14 Br 2 FN 3 O 2 S[500.92].
EXAMPLE 5 preparation of Compound 4
Intermediate 3 (100 mg,0.2 mmol) and 1-Boc-piperazine (37 mg,0.2 mmol), potassium carbonate (41 mg,0.3 mmol), potassium iodide (50 mg,0.3 mmol) were taken, 10mL acetonitrile was added to give a yellow suspension, and the mixture was refluxed at 75℃for 1h. After the completion of the reaction, acetonitrile was removed under reduced pressure, water (20 mL) was added, extraction was performed with ethyl acetate (20 ml×3), and the organic phases were collected and combined, extracted once with saturated sodium chloride (25 mL), dried over anhydrous magnesium sulfate, and filtered. Flash column chromatography and recrystallization gave compound 4. 76.2mg of the product was obtained as a yellow solid in 63% yield.
1 H NMR(400MHz,DMSO-d 6 )δ9.69(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.93(s,1H,thiazole-H),7.56(d,J＝8.1Hz,1H,Ph-H),7.37(m,1H,Ph-H),7.22(s,1H,Ph-H),6.02(s,1H,CH),4.08–3.79(m,4H,dihydropyrimidine-CH 2 , 2 CHCH 3 ),3.31(s,4H,CH 2 NCH 2 ),2.50(s,4H,2×BocNCH 2 ),1.39(s,9H,Boc),1.05(t,J＝6.9Hz,3H,CH 2 CH 3 )；EI-MS:610.01[M+H] + .
EXAMPLE 6 preparation of Compound 5
Intermediate 4 (100 mg,0.16 mmol) was dissolved in 5mL dichloromethane, and 0.5mL trifluoroacetic acid was added dropwise with stirring at room temperature and stirred overnight at room temperature. After the reaction was completed, a saturated sodium carbonate solution (20 mL) was added to generate a large amount of bubbles. The organic phases were collected and combined, extracted once with saturated sodium chloride (25 mL), dried over anhydrous magnesium sulfate and filtered. And spin-drying to obtain a crude product 5, and directly carrying out the next reaction.
EXAMPLE 7 preparation of Compound 6
The different N-Boc-Linker "Linker arms" (68 mg,0.26 mmol) and HATU (112 mg,0.26 mmol) were dissolved in dichloromethane, ice-cooled for 30min, 5 (100 mg,0.20 mmol) and DIPEA (60. Mu.L, 0.59 mmol) were added and stirred overnight at room temperature. After the reaction, the solvent was dried, extracted with water (20 mL) and dichloromethane (20 ml×2), and the organic phases were combined, extracted once with saturated sodium chloride, dried over anhydrous magnesium sulfate, and filtered. Flash column chromatography and recrystallization gave 6 (a-e).
N-Boc-L usedThe anker is N-tert-butyloxycarbonyl-8-aminocaprylic acid, the product 6a is yellow solid with the yield of 51.7% and the melting point of 73-75 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.01(d,J＝3.0Hz,1H,thiazole-H),7.95(d,J＝2.8Hz,1H,thiazole-H),7.57(dd,J＝8.6,2.8Hz,1H,Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t,J＝8.4Hz,1H,Ph-H),6.75(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),3.95(q,J＝6.7,5.8Hz,2H, 2 CHCH 3 ),3.89(d,J＝17.2Hz,2H,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),2.89(q,J＝6.8Hz,2H,COCH 2 CH 2 CH 2 CH 2 CH 2 CH 2 2 CHNH),2.69(d,J＝2.1Hz,2H,CO 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH),2.55(s,4H, 2 CHN 2 CH),2.31(t,J＝7.5Hz,2H,COCH 2 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 NH),1.52-1.44(m,2H,COCH 2 CH 2 CH 2 CH 2 CH 2 2 CHCH 2 NH),1.36(s,9H,Boc),1.25(d,J＝2.2Hz,6H,COCH 2 CH 2 2 2 2 CHCHCHCH 2 CH 2 NH),1.05(td,J＝7.1,2.1Hz,3H,CH 2 3 CH)；EI-MS:848.09[M+H] + ；C 34 H 46 BrFN 6 O 5 S[848.24].
the N-Boc-Linker is N-t-butoxycarbonyl-6-aminocaproic acid, the product 6b is yellow solid with 76% yield and 53-55deg.C melting point; 1 H NMR(400MHz,DMSO-d 6 )δ9.66(s,1H,dihydropyrimidine-H),8.02(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(d,J＝8.5Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.24(s,1H,Ph-H),6.77(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),3.99–3.93(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.55–3.50(m,4H, 2 CHNCO 2 CH),2.89(q,J＝6.9Hz,2H,COCH 2 CH 2 CH 2 CH 2 2 CHNH),2.69(d,J＝2.0Hz,2H,CO 2 CHCH 2 CH 2 CH 2 CH 2 NH),2.57(s,4H, 2 CHN 2 CH),2.32(s,2H,COCH 2 2 CHCH 2 CH 2 CH 2 NH),1.53–1.45(m,2H,COCH 2 CH 2 CH 2 2 CHCH 2 NH),1.37(s,9H,Boc),1.26(q,J＝7.2Hz,2H,COCH 2 CH 2 2 CHCH 2 CH 2 NH),1.09–1.02(m,3H,CH 2 3 CH)；EI-MS:721.19[M+H] + ；C 32 H 42 BrFN 6 O 5 S[720.21].
the N-Boc-Linker is N-t-butoxycarbonyl-4-aminobutyric acid, the product 6c is yellow solid, the yield is 88.7%, and the melting point is 100-108 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.23(s,1H,Ph-H),6.81(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),3.96(s,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),2.93(q,J＝6.7Hz,2H,COCH 2 CH 2 2 CHNH),2.69(d,J＝1.9Hz,4H, 2 CHN 2 CH),2.32(s,2H,CO 2 CHCH 2 CH 2 NH),1.62(q,J＝7.2Hz,2H,COCH 2 2 CHCH 2 NH),1.37(s,9H,Boc),1.05(t,J＝7.3Hz,3H,CH 2 3 CH)；EI-MS:693.25[M+H] + ；C 30 H 38 BrFN 6 O 5 S[692.18].
the N-Boc-Linker is N-t-butoxycarbonyl-2- (2- (2-aminoethoxy) ethoxy) acetic acid, the product 6d is a yellow solid with a yield of 48% and a melting point of 73-75 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.22(s,1H,Ph-H),6.76(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.17(s,2H,COCH 2 O),4.04–3.86(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.54(d,J＝10.3Hz,8H, 2 CHNCO 2 CH,OCH 2 CH 2 O 2 2 CHCHN),3.38(d,J＝6.4Hz,2H,O 2 CHCH 2 OCH 2 CH 2 N),3.07(q,J＝6.3Hz,2H,OCH 2 2 CHOCH 2 CH 2 N),1.36(s,9H,Boc),1.05(t,J＝7.3Hz,3H,CH 2 3 CH)；EI-MS:755.18[M+2+H] + ；C 32 H 42 BrFN 6 O 7 S[752.20].
the N-Boc-linker N-tert-butoxycarbonyl-2- (2-aminoethoxy) acetic acid used, product 6e was a yellow solid with a yield of 93%, melting point of 53-55deg.C; 1 H NMR(400MHz,DMSO-d 6 )δ9.66(s,1H,dihydropyrimidine-H),8.04–7.99(m,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.5Hz,1H,Ph-H),7.40(s,1H,Ph-H),7.23(s,1H,Ph-H),6.82(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.15(s,2H,COCH 2 O),3.98–3.92(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.49-3.43(m,6H, 2 CHNCO 2 CH,O 2 CHCH 2 N),3.09(q,J＝6.3,5.8Hz,2H,OCH 2 2 CHN),2.69(d,J＝1.9Hz,2H),2.55(s,4H, 2 CHN 2 CH),1.35(s,9H,Boc),1.05(t,J＝7.1Hz,3H,CH 2 3 CH)；EI-MS:708.92[M+H] + ；C 30 H 38 BrFN 6 O 6 S[708.17].
EXAMPLE 8 preparation of Compound 7 (a-e)
Intermediate 6 (a-e) (400 mg,0.56 mmol) was dissolved in 20mL dichloromethane, 2mL trifluoroacetic acid was added dropwise with stirring at room temperature, and stirring overnight at room temperature. After the reaction was completed, a saturated sodium carbonate solution (20 mL) was added to generate a large amount of bubbles. The organic phases were collected and combined, extracted once with saturated sodium chloride (25 mL), dried over anhydrous magnesium sulfate and filtered. The excess solvent was evaporated under reduced pressure leaving 2mL of solvent, which was separated by preparative TLC and recrystallized to give the target compound 7 (a-e).
7a is yellow solid, the yield is 61.7%, and the melting point is 98-100 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ8.00(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.55(d,J＝8.2Hz,1H,Ph-H),7.41(t,J＝6.9Hz,1H,Ph-H),7.22(t,J＝7.8Hz,1H,Ph-H),6.75(s,1H,NH),6.04(s,1H,dihydropyrimidine-CH),4.05–3.83(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.53(s,4H, 2 CHNCO 2 CH),2.80–2.70(m,2H,COCH 2 CH 2 CH 2 CH 2 CH 2 CH 2 2 CHNH),2.53(d,J＝18.8Hz,4H, 2 CHN 2 CH),2.31(s,2H,CO 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 CH 2 NH),1.53(d,J＝24.3Hz,4H,COCH 2 2 CHCH 2 CH 2 CH 2 2 CHCH 2 NH),1.28(s,6H,COCH 2 CH 2 2 2 2 CHCHCHCH 2 CH 2 NH),1.04(t,J＝6.5Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ171.20,165.64,162.50,160.04,146.77,144.40,144.08,140.64,131.43,125.24,123.00,120.20,119.96,116.07,115.86,97.73,59.88,58.66,55.84,53.46,53.04,45.63,41.66,32.71,29.10,28.91,28.00,26.32,25.15,14.45；EI-MS:649.10[M+H] + ；C 29 H 38 BrFN 6 O 3 S[648.19].
7b is yellow solid, the yield is 76%, and the melting point is 95-100 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ8.01(d,J＝2.8Hz,1H,thiazole-H),7.95(d,J＝3.5Hz,1H,thiazole-H),7.57(d,J＝8.6Hz,1H,Ph-H),7.39(t,J＝7.1Hz,1H,Ph-H),7.22(t,J＝8.7Hz,1H,Ph-H),6.03(s,1H,dihydropyrimidine-CH),3.95(q,J＝9.0,8.4Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),2.69–2.51(m,6H,COCH 2 CH 2 CH 2 CH 2 2 CHNH, 2 CHN 2 CH),2.32(t,J＝7.0Hz,2H,COCH 2 2 CHCH 2 CH 2 CH 2 NH),1.47(dt,J＝24.4,8.0Hz,4H,COCH 2 2 CHCH 2 2 CHCH 2 NH),1.28(dd,J＝20.1,12.1Hz,2H,COCH 2 CH 2 2 CHCH 2 CH 2 NH),1.04(t,J＝7.1Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ171.06,165.64,162.50,160.04,146.78,144.40,144.08,140.64,125.25,120.20,119.96,116.07,115.86,97.72,59.88,58.66,55.84,53.43,53.02,45.62,41.69,32.50,28.05,26.13,24.72,14.45；EI-MS:622.92[M+2+H] + ；C 27 H 34 BrFN 6 O 3 S[620.16].
7c is yellow solid, the yield is 52.0%, and the melting point is 128-135 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ7.99(d,J＝24.0Hz,2H,thiazole-H),7.58(d,J＝8.6Hz,1H,Ph-H),7.40(t,J＝7.5Hz,1H,Ph-H),7.23(t,J＝8.7Hz,1H,Ph-H),6.04(s,1H,dihydropyrimidine-CH),3.96(q,J＝8.9Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.53(s,4H, 2 CHNCO 2 CH),2.72–2.52(m,6H,COCH 2 CH 2 2 CHNH, 2 CHN 2 CH),2.39(t,J＝7.5Hz,2H,CO 2 CHCH 2 CH 2 NH),1.64(t,J＝7.3Hz,2H,COCH 2 2 CHCH 2 NH),1.05(t,J＝7.2Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ170.40,167.42,165.64,162.51,160.04,146.75,144.40,144.06,140.65,132.18,131.98,129.13,125.23,123.11,123.01,120.20,119.95,116.05,115.84,97.73,65.49,59.87,58.68,55.82,53.29,52.96,45.47,41.77,30.48,29.79,23.24,19.12,14.45,14.00；EI-MS:592.97[M+H] + ；C 25 H 30 BrFN 6 O 3 S[592.13].
7d is yellow solid, the yield is 81.7 percent, and the melting point is 60-68 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ8.07(d,J＝22.0Hz,2H,thiazole-H),7.66(d,J＝8.6Hz,1H,Ph-H),7.48(t,J＝7.4Hz,1H,Ph-H),7.34–7.26(m,1H,Ph-H),6.11(s,1H,dihydropyrimidine-CH),4.29(s,2H,COCH 2 O),4.03(q,J＝7.7Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.64(d,J＝27.7Hz,10H, 2 CHNCO 2 CH,O 2 2 CHCHO 2 CHCH 2 N),2.98(s,2H,OCH 2 CH 2 OCH 2 2 CHN),2.64(s,4H, 2 CHN 2 CH),1.13(t,J＝7.0Hz,3H,CH 2 3 CH)；13C NMR(100MHz,DMSO-d 6 )δ167.99,165.65,162.51,160.04,146.74,144.39,144.07,140.65,140.61,131.44,131.36,125.26,120.20,119.96,116.08,115.87,97.73,70.25,69.90,69.62,69.19,59.89,58.66,55.82,53.23,52.91,44.90,41.80,14.45；EI-MS:653.18[M+H] + ；C 27 H 34 BrFN 6 O 5 S[652.15].
7e is a yellow solid with a yield of 58.8% and a melting point of 121-129 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ8.03–7.94(m,2H,thiazole-H),7.58(d,J＝8.5Hz,1H,Ph-H),7.40(t,J＝7.4Hz,1H,Ph-H),7.23(t,J＝8.7Hz,1H,Ph-H),6.04(s,1H,dihydropyrimidine-CH),4.25(s,2H,COCH 2 O),3.96(q,J＝7.3,6.7Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.61–3.41(m,6H, 2 CHNCO 2 CH,O 2 CHCH 2 N),2.86(t,J＝5.4Hz,2H,OCH 2 2 CHN),2.69(s,2H,NH 2 ),2.56(d,J＝13.5Hz,4H, 2 CHN 2 CH),1.05(t,J＝7.1Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ168.38,165.64,162.52,160.04,146.69,144.39,144.04,140.64,131.34,125.24,123.11,120.20,119.96,116.05,115.84,97.81,69.08,67.89,67.78,63.28,59.88,58.69,55.80,53.10,44.67,14.45；EI-MS:608.92[M+H] + ；C 25 H 30 BrFN 6 O 4 S[608.12].
EXAMPLE 9 preparation of Compound 8 (a-j)
7 (a-e) (123 mg,0.16 mmol) and 2 (57 mg,0.20 mmol) or 3 (60 mg,0.20 mmol) were dissolved in N, N-dimethylformamide, DIPEA (38. Mu.l, 0.29 mmol) was added with stirring, and the reaction was carried out at 90℃for 10 hours. After the reaction, the solvent was spun-dried, extracted with water (20 mL) and EA (20 mL x 2), the organic phases were combined, extracted once with saturated sodium chloride, dried over anhydrous magnesium sulfate, and filtered. The organic phase is added to silica gel for sample mixing, the mixture is subjected to rapid column chromatography and recrystallized to obtain the target compound 8 (a-j).
8a is yellow solid, the yield is 41.7 percent, and the melting point is 119-120 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ11.10(s,1H,CONHCO),9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(t,J＝7.8Hz,2H,Ph-H,Pomalidomide-Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t,J＝8.4Hz,1H,Ph-H),7.10(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.53(s,1H,NH),6.03(s,2H,dihydropyrimidine-CH),5.05(dd,J＝12.7,4.8Hz,1H,Pomalidomide-CH),3.95(q,J＝9.4,8.4Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),3.30(d,J＝6.4Hz,2H,Pomalidomide-CH 2 ),2.95–2.84(m,1H,Pomalidomide-CH 2 ),2.64–2.53(m,4H, 2 CHNCO 2 CH),2.31(t,J＝7.1Hz,2H,COCH 2 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 NH),2.07–1.99(m,1H,Pomalidomide-CH 2 ),1.58(s,2H,COCH 2 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 NH),1.50(s,2H,COCH 2 CH 2 CH 2 CH 2 CH 2 2 CHCH 2 NH),1.28(d,J＝34.5Hz,6H,COCH 2 CH 2 2 2 2 CHCHCHCH 2 CH 2 NH),1.04(t,J＝6.9Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,30.12,29.30,28.98,26.77,26.02,24.30,14.46；EI-MS:906.97[M+2+H] + ；C 42 H 46 BrFN 8 O 7 S[904.24].
8b is yellow solid, the yield is 20.6%, and the melting point is 114-117 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ11.11(s,1H,CONHCO),9.73(s,1H,dihydropyrimidine-H),8.09(s,2H,thiazole-H),7.66–7.55(m,2H,Ph-H,Pomalidomide-Ph-H),7.51(s,1H,Ph-H),7.29(s,1H,Ph-H),7.11(d,J＝8.4Hz,1H,Pomalidomide-Ph-H),7.04(d,J＝6.9Hz,1H,Ph-H),6.55(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.0,5.0Hz,1H,Pomalidomide-CH),4.23(t,J＝6.4Hz,2H,dihydropyrimidine-CH 2 ),4.02(d,J＝6.7Hz,2H, 2 CHCH 3 ),3.32(d,J＝5.7Hz,4H, 2 CHNCO 2 CH),2.89(t,J＝13.0Hz,1H,Pomalidomide-CH 2 ),2.60(d,J＝17.6Hz,2H,COCH 2 CH 2 CH 2 CH 2 2 CHNH),2.39(s,2H,CO 2 CHCH 2 CH 2 CH 2 CH 2 NH),2.03(d,J＝7.7Hz,1H,Pomalidomide-CH 2 ),1.69–1.52(m,4H,COCH 2 2 CHCH 2 2 CHCH 2 NH),1.39(s,2H,Pomalidomide-CH 2 ),1.24(s,2H,COCH 2 CH 2 2 CHCH 2 CH 2 NH),1.10(d,J＝6.3Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,28.98,26.02,24.80,24.30,14.46；EI-MS:878.95[M+2+H] + ；C 40 H 42 BrFN 8 O 7 S[876.21].
8c is yellow solid, the yield is 26.6 percent, and the melting point is 76-82 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ11.11(s,1H,CONHCO),9.69(s,1H,dihydropyrimidine-H),8.08(s,2H,thiazole-H),7.61(t,J＝7.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.50(s,1H,Ph-H),7.29(s,1H,Ph-H),7.19(d,J＝8.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.04(d,J＝6.8Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.1,5.0Hz,1H,Pomalidomide-CH),4.58(s,2H),4.23(t,J＝6.4Hz,4H,dihydropyrimidine-CH 2 ),4.01(d,J＝6.3Hz,2H, 2 CHCH 3 ),3.35(d,J＝6.1Hz,4H, 2 CHNCO 2 CH),2.96–2.57(m,3H,Pomalidomide-CH 2 ,COCH 2 CH 2 2 CHNH),2.48(s,2H,CO 2 CHCH 2 CH 2 NH),2.07–2.00(m,1H,Pomalidomide-CH 2 ),1.88–1.76(m,2H,COCH 2 2 CHCH 2 NH),1.09(t,J＝6.4Hz,3H,CH 2 3 CH)；13C NMR(100MHz,DMSO-d 6 )δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.56,34.75,30.69,25.15,24.30,14.46；EI-MS:848.93[M+H] + ；C 38 H 38 BrFN 8 O 7 S[848.18].
8d is yellow solid, the yield is 13.0 percent, and the melting point is 122-129 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ11.10(s,1H,CONHCO),9.65(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.93(t,J＝2.8Hz,1H,thiazole-H),7.57(ddd,J＝8.7,5.9,2.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.39(dd,J＝8.8,6.3Hz,1H,Ph-H),7.25–7.18(m,1H,Ph-H),7.13(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.03(dd,J＝7.1,2.1Hz,1H,Pomalidomide-Ph-H),6.62(d,J＝6.3Hz,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.05(dd,J＝13.0,5.4Hz,1H,Pomalidomide-CH),4.17(s,2H,COCH 2 O),4.00–3.88(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.65–3.44(m,12H, 2 CHNCO 2 CH,O 2 2 CHCHO 2 2 CHCHN),2.93–2.82(m,1H,Pomalidomide-CH 2 ),2.69–2.52(m,6H,Pomalidomide-CH 2 , 2 CHN 2 CH),2.08–1.90(m,1H,Pomalidomide-CH 2 ),1.04(td,J＝7.1,2.1Hz,3H)； 13 C NMR(100MHz,DMSO-d 6 )δ173.20,170.49,169.42,167.82,167.74,165.62,162.52,160.03,146.89,146.68,144.37,144.03,140.64,136.68,132.58,131.43,125.17,123.10,123.01,120.19,119.95,117.87,116.03,115.82,111.15,109.78,97.75,70.31,70.05,69.96,69.33,59.86,58.69,55.83,49.07,42.24,41.80,31.47,22.63,14.44；EI-MS:909.10[M+H] + ；C 40 H 42 BrFN 8 O 9 S[908.20].
8e is yellow solid, the yield is 4.3 percent, and the melting point is 62-68 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ11.10(s,1H,CONHCO),9.66(s,1H,dihydropyrimidine-H),8.05–7.92(m,2H,thiazole-H),7.66–7.50(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.6Hz,1H,Ph-H),7.23(d,J＝8.5Hz,1H,Ph-H),7.18(t,J＝9.5Hz,1H,Pomalidomide-Ph-H),7.04(d,J＝7.1Hz,1H,Pomalidomide-Ph-H),6.70(d,J＝6.2Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.05(dd,J＝12.9,5.5Hz,1H,Pomalidomide-CH),4.24(s,2H,COCH 2 O),4.00–3.81(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.72–3.63(m,2H,O 2 CHCH 2 N),3.56–3.46(m,4H, 2 CHNCO 2 CH),2.92–2.81(m,1H,Pomalidomide-CH 2 ),2.56(d,J＝17.2Hz,6H, 2 CHN 2 CH,OCH 2 2 CHN),2.01(d,J＝11.7Hz,1H,Pomalidomide-CH 2 ),1.12–0.97(m,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ173.22,170.51,169.36,167.79,167.75,165.63,162.50,160.04,146.87,146.73,144.39,144.05,140.65,136.68,132.61,131.36,125.20,123.11,123.01,120.20,119.96,117.90,116.05,115.84,111.15,109.75,97.72,69.66,69.54,67.77,63.27,59.87,58.68,55.82,53.23,49.05,44.91,42.24,41.80,41.02,31.46,22.64,14.44；EI-MS:865.10[M+H] + ；C 38 H 38 BrFN 8 O 8 S[864.17].
8f is yellow solid, the yield is 8 percent, and the melting point is 85-90 ℃; 1 HNMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.94(d,J＝2.7Hz,1H,thiazole-H),7.64–7.49(m,2H,Ph-H,Pomalidomide-Ph-H),7.39(t,J＝7.3Hz,1H,Ph-H),7.22(t,J＝8.7Hz,1H,Ph-H),7.10(d,J＝8.5Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.54(d,J＝5.6Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.32(dd,J＝12.7,4.8Hz,1H,Pomalidomide-CH),4.03–3.84(m,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),3.30(d,J＝6.4Hz,2H,Pomalidomide-CH 2 ),3.01(d,J＝2.1Hz,3H,Pomalidomide-CH 3 ),2.93–2.63(m,2H,Pomalidomide-CH 2 ),2.53(d,J＝13.6Hz,4H, 2 CHN 2 CH),2.31(t,J＝7.5Hz,2H,COCH 2 2 CHCH 2 CH 2 CH 2 CH 2 CH 2 NH),2.01(dt,J＝13.9,9.6Hz,2H,COCH 2 2 CHCH 2 CH 2 CH 2 2 CHCH 2 NH),1.53(d,J＝34.6Hz,4H,COCH 2 2 CHCH 2 CH 2 CH 2 2 CHCH 2 NH),1.30(d,J＝19.0Hz,6H,COCH 2 CH 2 2 2 2 CHCHCHCH 2 CH 2 NH),1.09–1.00(m,3H,CH 2 3 CH)； 13 CNMR(100MHz,DMSO-d 6 )δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.58,36.13,30.50,30.12,29.30,28.98,28.93,26.77,26.02,24.21,14.46；EI-MS:920.99[M+2+H] + ；C 43 H 48 BrFN 8 O 7 S[918.25].
8g of the solid is yellow, the yield is 14.5 percent, and the melting point is 128-130 ℃; 1 HNMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),7.98(d,J＝24.6Hz,2H,thiazole-H),7.58(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.11(s,1H,Ph-H),7.03(s,1H,Ph-H),6.55(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(s,1H,Pomalidomide-CH),3.94(s,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.53(s,4H, 2 CHNCO 2 CH),3.02(s,3H,Pomalidomide-CH 3 ),2.95(s,1H,Pomalidomide-CH 2 ),2.76(d,J＝16.1Hz,1H,Pomalidomide-CH 2 ),2.54(s,4H, 2 CHN 2 CH),2.34(s,2H,COCH 2 CH 2 CH 2 CH 2 2 CHNH),2.05(s,2H,CO 2 CHCH 2 CH 2 CH 2 CH 2 NH),1.58(s,4H,Pomalidomide-CH 2 ,COCH 2 2 CHCH 2 2 CHCH 2 NH),1.38(s,2H,COCH 2 CH 2 CH 2 2 CHCH 2 NH),1.24(s,2H,COCH 2 CH 2 2 CHCH 2 CH 2 NH),1.05(s,3H,CH 2 3 CH)； 13 CNMR(100MHz,DMSO-d 6 )δ172.26,171.11,170.30,169.40,167.74,165.63,162.50,160.03,146.93,146.79,144.38,144.07,140.66,140.62,136.77,132.66,131.43,125.21,123.11,123.01,120.20,119.96,117.68,116.05,115.84,110.86,109.44,97.69,59.87,58.66,55.85,53.43,53.03,49.58,45.61,42.26,41.67,32.63,31.60,29.03,27.06,26.58,24.98,21.86,14.44；EI-MS:890.94[M+H] + ；C 41 H 44 BrFN 8 O 7 S[890.22].
8h is yellow solid, the yield is 26.8 percent, and the melting point is 108-114 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.69–7.48(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.2Hz,1H,Ph-H),7.29–7.12(m,2H,Ph-H,Pomalidomide-Ph-H),7.03(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(dd,J＝12.7,4.7Hz,1H,Pomalidomide-CH),3.95(q,J＝7.2Hz,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.52(s,4H, 2 CHNCO 2 CH),3.01(s,3H,Pomalidomide-CH 3 ),2.94–2.62(m,2H,Pomalidomide-CH 2 ),2.55(s,4H, 2 CHNCO 2 CH),2.40(d,J＝23.7Hz,2H,COCH 2 CH 2 2 CHNH),2.04(d,J＝11.7Hz,2H,Pomalidomide-CH 2 ),1.77(d,J＝32.1Hz,2H,CO 2 CHCH 2 CH 2 NH),1.24(s,2H,COCH 2 2 CHCH 2 NH),1.05(t,J＝6.9Hz,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.56,34.75,30.50,28.93,25.15,24.21,14.46；EI-MS:862.90[M+H] + ；C 39 H 40 BrFN 8 O 7 S[862.19].
8i is yellow solid, the yield is 4.8 percent, and the melting point is 90-104 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.66(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.94(s,1H,thiazole-H),7.57(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.14(s,1H,Pomalidomide-Ph-H),7.04(s,1H,Pomalidomide-Ph-H),6.62(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(s,1H,Pomalidomide-CH),4.17(s,1H,COCH 2 O),3.94(s,4H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.64(s,8H,OCH 2 CH 2 OCH 2 CH 2 N)3.34(s,4H, 2 CHNCO 2 CH),3.01(s,3H,Pomalidomide-CH 3 ),2.76(s,1H,Pomalidomide-CH 2 ),2.02(s,2H,Pomalidomide-CH 2 ),1.04(s,3H,CH 2 3 CH)； 13 C NMR(100MHz,DMSO-d 6 )δ172.23,170.26,169.41,167.82,167.71,165.62,162.51,160.03,146.90,146.68,144.36,144.02,140.67,136.71,132.55,131.43,125.18,123.10,120.18,119.94,117.89,116.02,115.82,111.17,109.72,97.72,70.30,70.06,69.96,69.32,59.86,58.68,55.83,53.23,52.92,49.62,44.99,42.23,41.79,31.61,27.05,21.84,14.43；EI-MS:924.95[M+2+H] + ；C 41 H 44 BrFN 8 O 9 S[922.21].
8j is yellow solid, the yield is 19.8%, and the melting point is 78-80 ℃; 1 H NMR(400MHz,DMSO-d 6 )δ9.67(s,1H,dihydropyrimidine-H),8.00(s,2H,thiazole-H),7.94(s,1H,Ph-H),7.67–7.52(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝6.9Hz,1H,Ph-H),7.28–7.13(m,1H,Pomalidomide-Ph-H),7.05(d,J＝6.7Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(dd,J＝12.7,4.6Hz,3H,Pomalidomide-CH),4.24(s,2H,COCH 2 O),3.93(dd,J＝15.1,11.3Hz,5H, 2 CHCH 3 ,dihydropyrimidine-CH 2 ),3.70(d,J＝17.1Hz,2H,O 2 CHCH 2 N),3.50(d,J＝20.7Hz,6H,OCH 2 2 CHN, 2 CHNCO 2 CH),3.00(s,2H,Pomalidomide-CH 3 ),2.73(d,J＝16.8Hz,1H,Pomalidomide-CH 2 ),2.53(d,J＝8.3Hz,4H, 2 CHN 2 CH),2.02(s,1H,Pomalidomide-CH 2 ),1.05(t,J＝6.8Hz,3H,CH 2 3 CH)；13C NMR(100MHz,DMSO-d 6 )δ172.22,170.27,169.34,167.75,167.71,165.62,162.50,160.04,146.88,146.76,144.38,144.05,140.66,136.71,132.59,131.43,131.34,125.22,123.11,123.01,120.20,119.96,117.94,116.05,115.84,111.17,109.69,97.65,69.72,69.53,67.77,63.27,59.86,58.66,55.82,53.22,52.87,49.60,44.94,42.21,41.81,41.01,31.60,27.05,21.84,14.44；EI-MS:878.91[M+H] + ；C 39 H 40 BrFN 8 O 8 S[878.19].
example 10 in vitro anti-HBV Activity assay of Compounds of interest (HepDES 19 cells)
The test principle is as follows: hepDES19 cells are derivatives of the HepG2 (human hepatoblastoma) cell line stably transfected with HBV D-type genome gene under the control of tetracycline (repressible promoter) 1 . Inducing HBV replication in HepDES19 cells in the absence of tetracycline, adding the compound, and incubating the cells for 3 days with a change in HBV DNA content expressed by the cells and survival of the cells 2 . Analysis of HBV DNA content by quantitative polymerase chain reaction (qPCR)The amount, i.e., the effectiveness of the compound in inhibiting HBV replication, gives a half-Effective Concentration (EC) of the compound required for HBV DNA to be reduced to half 50 ) Representing the anti-HBV activity of the compound. Testing toxicity of the compound to cells by MTS method to obtain half-killing concentration (CC) 50 ) Indicating cytotoxicity of the compound. ( Guo H, jiang D, zhou T, et al journal of virology 2007;81 (22) 12472-12484; edwards TC, lomonosova E, patel JA, et al anti-viral research 2017; (143):205-217. )
The experimental method comprises the following steps:
(1) And (5) culturing the cells. Cells were kept in Dulbecco 'S modified Eagle' S medium (DMEM)/F12 medium supplemented with 10% Fetal Bovine Serum (FBS) and 1% penicillin/streptomycin (P/S) and 1. Mu.g/mL tetracycline. Synchronous expression of HBV pgRNA was induced by removal of tetracycline from the medium.
(2) And (5) culturing cells with drugs. HepDES19 cells were plated at 4X 10 per well in the absence of tetracycline 4 The density of individual cells was seeded in 96-well plates. 48 hours after HBV replication was induced, a final DMSO concentration of 1% compound solution was added and incubated with the cells for 3 days.
(3) Cell Activity (EC) 50 ) Test method. Cells were washed in 200 μl Phosphate Buffered Saline (PBS) and lysed in 150 μl core lysis buffer (10mM Tris pH 7.4,1%Tween20, 150mM NaCl). Cells were incubated at 350rpm for 40 minutes on an orbital shaker at 20-23 ℃. Cell lysates were transferred to 96-well PCR plates and centrifuged at 3300×g for 5 min. Transfer 50. Mu.L of supernatant to another 96-well PCR plate and mix with 20 units micrococcus nuclease and 100. Mu.M CaCl 2 Mixing. The lysate was incubated at 37℃for 1 hour, and then the nuclease was inactivated at 70℃for 10 minutes. Qiagen protease (0.005 Anson unit) was added to the lysate and the mixture incubated overnight, then the protease was inactivated at 95℃for 10 min.
Lysates were used as templates for chain-preferential quantitative polymerase chain reaction (qPCR) analysis. The reaction was carried out at 95℃for 15s with 40 cycles, atQuantitative PCR was performed at 60℃for 1 min. Kappa Probe Force universal PCR premix was used. Primers and probes for the positive polarity DNA strand were 5' CATGAACAAGAGAGAGATGTAGGTAGGCAGGAG3 ',5' GGAGGCTGTAGGAAATTGG 3' and 5'/56-FAM/CTGCGCACC/ZEN/AGCACCATGCA/3IABkFQ. Primers and probes for the negative polarity DNA strand were 5' GCAGATGAGAAGGCACAG3 ',5' CTTCTCCGTCCGTT3 ' and 5'/56-FAM/AGTCCGCGT/ZEN/AAAGAGAGGTGCG/3IABkFQ. EC of forward DNA was calculated using GraphPad Prism and three-parameter variable response log (inhibitor) -response algorithm (variable-response log (inhibitor) -versus-response algorithm) with the minimum set to zero 50 Values.
(4) Cytotoxicity (CC) 50 ) Test method. Using CellTiter 96 TM AQueuus non-radioactive cell proliferation assay (MTS) cell viability in the presence of compounds was measured in HepDES19 cells. In the absence of tetracycline, cells were grown at 1X 10 per well 4 The density of individual cells was seeded into 96-well plates, compounds were used two days later, and cells were incubated for 3 days. Calculation of 50% Cytotoxicity Concentration (CC) using GraphPad Prism and three-parameter variable response log (inhibitor) -response algorithm (floor set to zero) 50 ) Values.
TABLE 2 evaluation of Activity of target Compounds (dihydropyrimidine-pomalidomide conjugates) to inhibit HBV DNA replication and cytotoxicity
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The results show that the novel synthetic dihydropyrimidine-pomalidomide conjugates of the present invention exhibit significant anti-HBV activity. All dihydropyrimidine-PROTAC analogs exhibit anti-HBV activity at low micromolar concentration levels, EC 50 The values are in the range of 0.43-3.77. Mu.M. The activity of the three compounds was even lower than that of GLS4 (EC 50 =0.046 μm), but close to the positive drug 3TC (EC 50 =0.40 μm), in particular 8c (EC 50 ＝0.48μM)、8i(EC 50 =0.46 μm) and 8j (EC 50 =0.43 μm), with value for further investigation.
Claims (5)
1. The dihydropyrimidine-pomalidomide conjugate is characterized by having a structure shown in the following general formula I:
I
wherein,
the Linker arm Linker is 8-aminocaprylic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2- (2- (2-aminoethoxy) ethoxy) acetic acid, 2- (2-aminoethoxy) acetic acid;
r is a hydrogen atom, pomalidomide, 1-methylppomalidomide, lenalidomide, 1-methyllenalidomide, thalidomide or 1-methylthalidomide.
2. The dihydropyrimidine-pomalidomide conjugate of claim 1, which is one of the compounds having the following structure:
。
3. the method of preparing the dihydropyrimidine-pomalidomide conjugate of claim 2, comprising the steps of: 3-fluorophthalic anhydride II-1 is taken as a raw material, and is refluxed with 3-amino-2, 6-piperidinedione hydrochloride and sodium acetate in an acetic acid solvent at 120 ℃ for 10 hours to obtain II-2; dissolving II-2 inN,N-dimethylformamideAdding methyl iodide into the solution by taking potassium carbonate as alkali and stirring at room temperature, and reacting for 24 hours at room temperature to obtain an intermediate II-3; 2-thiazole formamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and ethyl acetoacetate are taken as starting materials, and are cyclized through a Biginelli reaction to obtain a key intermediate 2; in dichloromethane solution, the intermediate 2 and N-bromosuccinimide undergo bromination reaction to obtain an important intermediate 3; taking the intermediate 3 as a raw material, adding potassium carbonate, potassium iodide and 1-Boc piperazine, and refluxing in acetonitrile solution at 75 ℃ for 1h to obtain an intermediate 4; dissolving the 4 in dichloromethane solution of trifluoroacetic acid, stirring at room temperature for 10 hours, and removing Boc to obtain an intermediate 5; will beNBoc- "linker arm" fragment and 2- (7-azabenzotriazol) o-N,N,N', N'-tetramethyl urea hexafluorophosphate, activated in an ice-water bath for half an hour, added intermediate 5 andN,Ndiisopropylethylamine, stirred at room temperature overnight to afford intermediate 6; dissolving 6 in dichloromethane solution of trifluoroacetic acid, stirring at room temperature for 10h, and removing Boc to obtain a final product 7 (a-e); 7 (a-e) and II-2 or II-3 in the presence of a catalystN,N-In dimethylformamide solution, DIPEA is taken as alkali, and reflux is carried out for 10 hours at 90 ℃ to obtain a final product 8 (a-j);
the synthetic route is as follows:
reagents and conditions: (i) 3-amino-2, 6-piperidinedione hydrochloride, acetic acid, sodium acetate, 120 ℃ for 10h; (ii) Potassium carbonate, methyl iodide, and the like,N,N-dimethylformamide, room temperature, 24h; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80 ℃; the method comprises the steps of carrying out a first treatment on the surface of the (iv) N-bromosuccinimide, dichloromethane, 40 ℃; (v) 1-tert-butoxycarbonyl-piperazine, potassium carbonate, potassium iodide, acetonitrile, 75 ℃ for 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N-t-butoxycarbonyl-Linker, HATU, DIPEA, dichloromethane, 0 ℃,30min, room temperature, 6h; (viii) trifluoroacetic acid, dichloromethane, room temperature, 10h; (ix) II-2 or II-3,N,N-dimethylformamide, DIPEA,90 ℃,10h;
wherein the saidA kind of electronic deviceN-t-butoxycarbonyl-Linker comprising:N-Boc-8-amino octanoic acid,N-Boc-6-aminocaproic acid,N-Boc-4-aminobutyric acid,N-Boc-2- (2- (2-aminoethoxy) ethoxy) acetic acid,N-t-butoxycarbonyl-2- (2-aminoethoxy) acetic acid.
4. Use of a dihydropyrimidine-pomalidomide conjugate according to any one of claims 1-2 in the preparation of an anti-HBV medicament.
5. An anti-HBV pharmaceutical composition comprising the dihydropyrimidine-pomalidomide conjugate of any of claims 1-2 and one or more pharmaceutically acceptable carriers or excipients.
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| US6503913B1 (en) * | 1998-04-18 | 2003-01-07 | Bayer Aktiengesellschaft | 2-heterocyclically substituted dihydropyrimidines |
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