Proteolysis-targeting chimera (protac) small molecule preparation method and application

Abstract

The application relates to the technical field of medicine synthesis, in particular to synthesis and application of a PROTAC small molecule. The application designs a PROTAC small molecule capable of specifically degrading CBX3. Test results show that the PROTAC small molecule with the structure of QTARK(Me3)T-(NH-PEG4-CH2COOH-) -Pomalidomide can reduce the CBX3 protein expression level in prostate cancer, and has the effect of preventing cancer.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of medicine synthesis, in particular to a PROTAC small molecule preparation method and application. BACKGROUND

[0002] Chromobox protein homolog 3 (CBX3) is an important part of the core protein complex (Polycomb complex), which regulates the expression of downstream target genes by recognizing and binding to histone H3K9me3. Currently, CBX3 is found to be highly expressed in lung cancer, reproductive system tumors and digestive system tumors and other tumors and participates in promoting tumor progression. Therefore, taking CBX3 as a treatment target can significantly inhibit the progression of prostate cancer and other tumors. However, there is no specific inhibitor targeting CBX3.

[0003] Proteolysis-targeting chimera (PROTAC) technology is to bring E3 ligase and target protein close in space and combine together, so that the target protein is ubiquitinated and degraded to play a role. PROTAC small molecules can target "undruggable" proteins, and have the characteristics of strong blocking of target proteins, high stability and not easy to be degraded. PROTAC small molecules are composed of a POI ligand that binds to a target protein, a ligand that binds to an E3 ligase, and a linker that connects them.

[0004] Since there is no CBX3 specific drug at present, it is of great practical significance to develop a specific PROTAC small molecule targeting CBX3 using ROTAC technology. SUMMARY

[0005] Therefore, the technical problem to be solved by the present application is to provide a PROTAC small molecule preparation method and application.

[0006] The present application provides a PROTAC small molecule, which comprises a target protein ligand, a linker and an E3 ubiquitin ligase ligand connected in sequence;

[0007] The target protein ligand comprises X1X2AX3K(Me3)X4, wherein X1 is selected from Q or N; X2 is selected from T or V; X3 is selected from R, I, F, W or V; and X4 is selected from S or T;

[0008] The type of the linker is PEG, Alkyl / ether and / or Alkyl-Chain; the linker has an active end group, and the active end group comprises an amino group, a carboxyl group and / or a mercapto group;

[0009] The E3 ubiquitin ligase ligand is selected from at least one of MDM2, MDMX, cIAP, VH032, VHL, BRD4, CRBN, VH032 or Pomalidomide.

[0010] Further,

[0011] The target protein ligand is QTARK(Me3)T. Experiments have shown that QTARK(Me3)T can specifically bind to CBX3.

[0012] In the present application, the type of linker is PEG; specifically, (PEG)n with active end groups, the active end groups at both ends of (PEG)n include amino NH2, carboxyl COOH, azide N3, thiol SH, aldehyde CHO and / or hydroxyl, etc. n in (PEG)n is 1-5, the active end groups at both ends of (PEG)n can be the same or two independent kinds; in the present application, the active end groups are amino NH2, carboxyl COOH and / or thiol SH, which are located at both ends of (PEG)n; specifically, the linker in the present application can be any one of -NH-PEG4-COOH-, -NH-PEG4-acp- and / or -COOH-PEG4-COOH-, acp is a phosphopantetheinyl group with a thiol SH active group at the end. Further, either end or both ends of the linker can be connected to a protecting group, the protecting group includes FMOC, BOC and CBZ, the protecting group blocks the functional group from reacting with other groups, and when the protecting group is removed, the functional group regains activity.

[0013] In the present application, the E3 ubiquitin ligase ligand includes but is not limited to MDM2, MDMX, cIAP, VH032, VHL, BRD4, CRBN, VH032 or Pomalidomide. The present application independently uses VH032 or Pomalidomide as the ubiquitin ligase ligand for the synthesis of PROTAC small molecules. The results show that the PROTAC small molecule composed of Boc-NH-PEG4-CH2COOH as a linker, Pamolidomide as an E3 ligand, and integrated QTARK(Me3)T can significantly down-regulate the protein level of CBX3 in cells, and can play a role in preventing and treating cancer.

[0014] Further, the PROTAC small molecule of the present application has a structure as shown in at least one of formula I-a to formula I-f:

[0015]

[0016]

[0017] The application provides a preparation process of a PROTAC small molecule, a linker is coupled with an E3 ubiquitin ligase ligand, and then coupled with a target protein ligand.

[0018] Or the linker is coupled with the target protein ligand, and then coupled with the E3 ubiquitin ligase ligand.

[0019] Further, the application further discloses the use of the PROTAC small molecule in the preparation of a cancer prevention and treatment drug.

[0020] The coupling conditions comprise that EDCl is used as a condensing agent, and DMAP and HOBt are used as catalysts.

[0021] When the linker is coupled with the E3 ubiquitin ligase ligand, the N terminal of the linker is protected by a Boc group.

[0022] The application provides the use of the following I) to II) in the preparation of a cancer prevention and treatment drug:

[0023] I) the PROTAC small molecule provided by the application;

[0024] II) the mixture containing the PROTAC small molecule prepared by the preparation method.

[0025] Further, the cancer provided by the application comprises at least one of prostate cancer, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, large intestine cancer, sigmoid colon cancer, rectal cancer, liver cancer, pancreatic cancer, periampullar cancer, biliary tract cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penis cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic cancer, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma or multiple myeloma.

[0026] The prostate cancer comprises but is not limited to one or more of ductal adenocarcinoma, urothelial carcinoma, squamous cell carcinoma and adenosquamous carcinoma.

[0027] The application takes prostate cancer cells C4-2 as a test object, and performs a prostate cancer prevention effect test of the PROTAC small molecule with the structure of QTARK (Me3) T- (-NH-PEG4-CH2COOH-) -Pomalidomide, and results show that the QTARK (Me3) T- (-NH-PEG4-CH2COOH-) -Pomalidomide can significantly reduce the protein level of CBX3 in cells, and can play a role in preventing prostate cancer.

[0028] Further, the tumor prevention provided by the application comprises any one of inhibiting tumor growth, reducing tumor volume or delaying the growth rate of the tumor, and the application does not make any limitation.

[0029] The application provides a medicine for preventing and treating tumors, which is prepared from a raw material including a PROTAC small molecule and / or a mixture containing the PROTAC small molecule prepared by the preparation method.

[0030] The application provides a medicine including the compound or derivative of the formula I.

[0031] The medicine is in the form of a tablet, capsule, granule, oral liquid, sustained-release preparation, controlled-release preparation, nano-preparation or injection.

[0032] The application provides a medicine for preventing and treating tumors, which is prepared from a raw material including a PROTAC small molecule and / or a mixture containing the PROTAC small molecule prepared by the preparation method.

[0033] Further, the medicine also includes a pharmaceutically acceptable excipient or carrier.

[0034] Further, the application provides a method for preventing and treating tumors, which is to administer the medicine.

[0035] The application designs a PROTAC small molecule capable of specifically degrading CBX3. Test results show that the PROTAC small molecule with the structure of QTARK(Me3)T-(-NH-PEG4-CH2COOH-)-Pomalidomide can reduce the expression level of CBX3 protein in prostate cancer, and has the effect of preventing cancer. BRIEF DESCRIPTION OF DRAWINGS

[0036] Figure 1 A structural diagram showing the specific binding of CBX3 and H3K9me3 to QTAR M3L;

[0037] Figure 2 A structural formula of a PROTAC small molecule;

[0038] Figure 3 A condensation reaction of an amide connecting a linker and a CRBN ligand Pomalidomide;

[0039] Figure 4 A synthesis of a short peptide QTARK(Me3)T from the C-terminal end by Fmoc method;

[0040] Figure 5 A condensation reaction of a short peptide QTARK(Me3)T and an E3 ligand;

[0041] Figure 6 An HPLC purification diagram of a PROTAC small molecule;

[0042] Figure 7 Mass spectrometry identification of PROTAC small molecules;

[0043] Figure 8 Western blot detection of intracellular CBX3 protein levels. DETAILED DESCRIPTION

[0044] The present application provides a PROTAC small molecule preparation method and application. Those skilled in the art can refer to the content herein, and appropriately improve the process parameters to achieve. It is particularly pointed out that all similar substitutions and changes are obvious to those skilled in the art, and they are considered to be included in the present application. The method and application of the present application have been described by the preferred embodiments, and the relevant personnel can obviously modify or appropriately change and combine the method and application herein without departing from the content, spirit and scope of the present application, to realize and apply the present application technology.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art. For the purposes of the present application, the reader is directed to Current Protocols in Molecular Biology (Ausubel) for definitions and terms of the art. Abbreviations for amino acid residues are the standard three letter and / or one letter codes used in the art to designate one of the 20 commonly occurring L-amino acids.

[0046] The test materials used in the present application are all ordinary commercially available products, which can be purchased in the market.

[0047] The present application is further illustrated below in conjunction with examples:

[0048] Example 1 Preparation and effect detection of PROTAC small molecule peptide degrading CBX3

[0049] 1. Using the Protein Data Bank (Protein Data Bank) database (https: / / www.rcsb.org / ), we found that the QTARM3L ((Q / N)(T / V)A(R / I / F / W / V)Kme3(S / T)) peptide segment from H3K9me3 can specifically bind to CBX3. Figure 1 ).

[0050] 2. We choose QTARK(Me3)T sequence as the Protein of interest (POI) ligand in its structure, according to the characteristics of nuclear proteins and small molecule drugability, we select von Hippel-Lindau protein (VHL) ligand VH032 (CAS NO: 1448188-62-2) and Cereblon ligand Pomalidomide (CAS NO: 19171-19-8) as E3 ligase ligand part, based on the above scheme we designed a number of PROTAC small molecules, such as Figure 2 The small molecules used for synthesis were purchased from MedChemExpress company, and the synthesis of POI polypeptide ligand was supported by Nanjing Peptide Valley company.

[0051] Take QTARK(Me3)T-PEG-Pomalidomide( Figure 2 ) as an example, introduce the synthesis process:

[0052] (1) Link E3 ligand and linker: as shown in Figure 3 , E3 ligase ligand Pamolidomide and Boc-NH-PEG4-COOH were condensed under the catalysis of DMAP (4-dimethylaminopyridine), HOBt (1-hydroxybenzotriazole) and EDCl (carbodiimide) condensing agent to obtain Boc-NH-PEG4-Pamolidomide. After purification, the Boc protecting group was removed to obtain NH2-PEG4-Pamolidomide.

[0053] (2) Solid-phase synthesis of short peptide QTARK(Me3)T: Fmoc method was used for polypeptide synthesis( Figure 4 ), starting with 2-Chlorotrityl Chloride Resin, sequentially connecting condensing T, K(Me3), R, A, T, Q from the C-terminal to obtain the fully protected QTARK(Me3)T polypeptide.

[0054] (3) Condensation reaction to connect short peptide QTARK(Me3)T and E3 ligand: the synthesized short peptide was purified and condensed with NH2-PEG4-Pamolidomide from the previous step, then the N-terminal protecting group of the polypeptide was removed and purified to obtain the designed PROTAC molecule QTARK(Me3)T-PEG-Pomalidomide( Figure 5 ). The obtained small molecule was further purified by HPLC (HPLC analysis results are shown in Figure 6 ), the highest red peak is the PROTAC molecule peak, the retention time is 8.227 min; mass spectrometric identification analysis is as followsFigure 7 The peak chart of the corresponding group is shown.

[0055] (4) In the culture medium of prostate cancer cells C4-2, we added CBX3 PROTAC small molecules. After 24 hours, the cells were collected for Western blot to detect the protein level of CBX3 in the cells. It was found that CBX3-PROTAC small molecules could down-regulate the protein level of CBX3 in the cells (as shown in Figure 8

[0056] The above is only a preferred embodiment of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the scope of protection of the present application.​

Claims

1. A PROTAC small molecule characterized in that, The structure is at least one of formula I-a~formula I-c:

2. The method of producing a PROTAC small molecule according to claim 1, characterized by, It is: After coupling the linker with the E3 ubiquitin ligase ligand, the target protein ligand is coupled again. Or the linker is coupled with the target protein ligand, and then the E3 ubiquitin ligase ligand is coupled.

3. The production method according to claim 2, characterized by, The coupling conditions include: EDCl as condensing agent, DMAP and HOBt as catalyst.

4. The production method according to claim 3, characterized by, When the linker is coupled with the E3 ubiquitin ligase ligand, the N-terminal of the linker is protected by Boc group.

5. The use of a)~b) in the preparation of a drug for preventing and treating tumors: a), the PROTAC small molecule of claim 1; b), the mixture containing PROTAC small molecules prepared by the preparation method of any one of claims 2~4.

6. Use according to claim 5, characterized in that, The tumor includes at least one of prostate cancer, thyroid cancer, tongue cancer, lung cancer, esophageal cancer, breast cancer, gastric cancer, large intestine cancer, sigmoid colon cancer, rectal cancer, liver cancer, pancreatic cancer, periampullar cancer, biliary cancer, kidney cancer, prostate cancer, bladder cancer, testicular malignant tumor, penis cancer, cervical cancer, endometrial cancer, ovarian cancer, fibrous histiocytic cancer, rhabdomyosarcoma, synovial sarcoma, melanoma, osteosarcoma, Ewing's sarcoma, lymphoma or multiple myeloma.

7. Use according to claim 5 or 6, characterized in that, The tumor prevention includes inhibiting and / or degrading CBX3 in cells.

8. A drug for preventing and treating tumors, which raw materials include the PROTAC small molecule of claim 1, and / or the mixture containing PROTAC small molecules prepared by the preparation method of any one of claims 2~4.

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