CN117024413B - 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof - Google Patents

3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof Download PDF

Info

Publication number
CN117024413B
CN117024413B CN202311279870.2A CN202311279870A CN117024413B CN 117024413 B CN117024413 B CN 117024413B CN 202311279870 A CN202311279870 A CN 202311279870A CN 117024413 B CN117024413 B CN 117024413B
Authority
CN
China
Prior art keywords
compound
aminopyrazine
targeted
atr
proteolytic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202311279870.2A
Other languages
Chinese (zh)
Other versions
CN117024413A (en
Inventor
马岚
杨光
魏明明
张坤
王宇博
李明
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Tianjin Jiangxin Zhicheng Technology Co ltd
Original Assignee
Tianjin Jiangxin Zhicheng Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Tianjin Jiangxin Zhicheng Technology Co ltd filed Critical Tianjin Jiangxin Zhicheng Technology Co ltd
Priority to CN202311279870.2A priority Critical patent/CN117024413B/en
Publication of CN117024413A publication Critical patent/CN117024413A/en
Application granted granted Critical
Publication of CN117024413B publication Critical patent/CN117024413B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom 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
    • A61P35/00—Antineoplastic agents
    • 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

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Nitrogen Condensed Heterocyclic Rings (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

The invention belongs to the field of chemical drugs, and discloses a 3-aminopyrazine-2-formamide targeted proteolytic chimera, a preparation method, a pharmaceutical composition and application thereof. The novel 3-aminopyrazine-2-formamide targeted proteolytic chimeric body is obtained through the eutectic structure of the small molecular inhibitor of ATR and the binding domain thereof, the preparation process is simple and easy, various 3-aminopyrazine-2-formamide targeted proteolytic chimeric body compounds are obtained through different paths, the synthesized proteolytic targeted chimeric body can selectively induce the targeted degradation of ATR protein by utilizing a ubiquitin-proteinase system, inhibit the downstream signal path thereof, obviously induce AML cells to apoptosis, and the obtained compound can inhibit the activation of PI3K-AKT signal path, thereby being suitable for the development of cancer drugs for treating acute myelogenous leukemia and the like.

Description

3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof
Technical Field
The invention belongs to the field of chemical drugs, and particularly relates to a 3-aminopyrazine-2-formamide targeted proteolytic chimera, a preparation method, a pharmaceutical composition and application thereof.
Background
Ataxia telangiectasia and RAD3 associated Protein (ATR) are key proteins for DNA damage response, and their non-kinase activity may play an important role in the development and progression of tumors, which is responsible for the poor efficacy of certain conventional kinase inhibitors. ATR kinase is considered a potential drug target for cancer therapy because it plays an important role in DNA damage repair, and is receiving extensive attention from academia and pharmaceutical companies.
According to the existing data, the ATR kinase inhibitor has good in vivo safety and has advantages in anti-tumor treatment. First, ATR kinase inhibitors show significantly enhanced cytotoxicity in a variety of gene mutations: (1)ATMThe deletion of (2) significantly enhances the antitumor activity of ATR kinase inhibitors. At the position ofATMIn the tumors such as defective chronic lymphocytic leukemia, non-small cell lung cancer and the like, the ATR kinase inhibitor can kill tumor cells, and can inhibit the tumor cellsATMNormal cells have poor antitumor effects and even have no significant toxicity to some tumor cells. (2) Presence ofTP53The mutated tumor cells are sensitive to ATR inhibitors.TP53As an oncogene, the gene is involved in regulating biological processes such as cell cycle, cell senescence, apoptosis and the like by controlling a G1/S phase checkpoint. Tumor cellsTP53Following mutational inactivation, the cells will be more dependent on ATR passing S and G2/M cell cycle checkpoints. And wild typeTP53In comparison, there isTP53Mutated chronic lymphocytic leukemia cells, colorectal cancer cells are sensitive to ATR inhibitors. (3)XRCC1A deletion of (2),ARID1AA deficiency of,CYCLINEHigh expression,APOBEC3BOver-expression of,MYCActivated tumors also show increased sensitivity to ATR kinase inhibitors.
At this stage, at least 8 ATR kinase inhibitors have entered the clinical trial phase, and indications involve solid tumors as well as hematological tumors. However, the existing ATR kinase inhibitors have the problems of poor anti-tumor effect of single use, lack of biomarker research and the like. Although there are a number of ATR kinase inhibitors that enter the clinical trial phase, the development of selective ATR inhibitors with therapeutic effects remains a challenge. First, because ATR has a large protein size and the need to activate downstream proteins by forming complexes with DNA and multiple proteins, it has led to difficulties in developing in vitro structure-based drug designs and high throughput screening, while ATR kinase has structural similarities to other PIKKs family members, inhibition of all PIKKs family members can be potentially toxic, and these have also led to slower development of selective ATR kinase inhibitors.
The protein degradation technology (protein-targeting Chimera, PROTACs) is an emerging protein degradation technology in recent years, and the selective degradation of target proteins is realized by virtue of an in-vivo ubiquitin-proteinase system in a manner different from the event-driven mode of the traditional inhibitor, so that the protein degradation technology has remarkable advantages in basic research and new drug development. After two molecules ARV-110 and ARV-471 with milestone significance were introduced from Arvinas in 2009, the development of PROTAC technology entered the fast lane and many molecules began to rapidly enter clinical transformations, which also brought new promise for the development of traditional inhibitors.
Disclosure of Invention
The invention aims to solve the defects in the prior art and provides a 3-aminopyrazine-2-formamide targeted proteolytic chimera, a preparation method, a pharmaceutical composition and application thereof, and the following technical scheme is adopted:
according to a first aspect of the present invention, there is provided a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera having the structural formula (I):
formula (I);
wherein Linker is any chemically feasible connection structure.
The invention develops a series of small molecules for selectively degrading ATR by virtue of PROTAC technology, and obtains a series of molecules which can exert high-efficiency anti-tumor activity in vivo and in vitro, and the molecules are obviously superior to ATR kinase inhibitors. The non-kinase function of ATR is related to the proliferation of AML, a series of synthesized small molecules can be designed to selectively induce the targeted degradation of ATR protein by using a ubiquitin-proteinase system, inhibit the downstream signal path of the ATR protein, obviously induce AML cells to apoptosis, and the screened compound can inhibit the activation of PI3K-AKT signal path. Not only provides a new strategy for the treatment of AML, but also provides a molecular probe for the study of ATR non-kinase function.
Preferably, linker is a saturated fatty chain, an unsaturated fatty chain or a fatty acid chain. By selecting ideal linkers of different types and different lengths, the aim of not only spatially affecting the binding of two proteins but also maintaining the binding thereof is achieved.
Preferably, the molecular structure of the 3-aminopyrazine-2-carboxamide targeted proteolytic chimera is any one of formula (Ia), formula (Ib), formula (Ic):
formula (Ia)>Formula (Ib),formula (Ic);
wherein in the formula (Ia), n is any positive integer from 4 to 6; in the formula (Ib), n is any positive integer from 2 to 4; formula (Ic), n is any positive integer from 5 to 7.
More preferably, the 3-aminopyrazine-2-carboxamide targeted proteolytic chimera has a molecular structure represented by formula (II):
formula (II).
The compound shown in the formula (II) has the strongest degradation activity on ATR protein in MV-4-11 cells, can effectively induce the degradation of ATR in various cancer cell lines in a dose-dependent and time-dependent manner, has proliferation inhibition effect on common leukemia cells, breast cancer cells and colorectal cancer cells, obviously enhances the antiproliferative activity on acute myeloid leukemia cells (AML), and particularly obviously enhances the killing property on MV-4-11 and MOLM-13. This suggests that the compound may have selectivity in antitumor activity.
According to a second aspect of the present invention, there is also provided a method for preparing the above 3-aminopyrazine-2-carboxamide targeted proteolytic chimera, wherein the preparation route is route one, route two or route three:
route one:
；
route two:
；
route three:
。
the invention utilizes the technology of protein hydrolysis targeting chimeras (PROTACs), obtains a lead with certain inhibitory activity based on modification of a small molecule ATR inhibitor, combines a molecular docking model, selects piperazine of the lead as a connecting part to introduce Linker, connects different types of CRBN ligands, and designs and synthesizes a series of PROTAC molecules of targeting ATR protein.
According to a third aspect of the present invention there is also provided the use of a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera as described above or a pharmaceutically acceptable salt thereof in the preparation of an ATR inhibitor.
Preferably, ATR inhibitors are useful in the manufacture of a medicament for the treatment of cancers associated with ATR abnormalities. More preferably, the cancer of interest comprises acute myelogenous leukemia.
According to a fourth aspect of the present invention there is also provided a pharmaceutical composition comprising a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera as described above or a pharmaceutically acceptable salt thereof.
Preferably, the pharmaceutical composition comprises an excipient, a solvent, and a pharmaceutically acceptable carrier. The excipient comprises at least one of acacia, syrup, lanolin, and starch. The excipient has stable property, no incompatibility with main medicine, no side effect, no influence on curative effect, no deformation, dry crack, mildew and moth damage at normal temperature, no harm to human body, no physiological effect, no chemical or physical effect with main medicine, and no influence on content measurement of main medicine. The solvent comprises water, glycerol or ethanol.
The beneficial effects of the invention are as follows: the novel 3-aminopyrazine-2-formamide targeted proteolytic chimera is obtained by the invention, the preparation process is simple and easy to implement, and various 3-aminopyrazine-2-formamide targeted proteolytic chimeras are obtained through different paths, can be applied to the preparation of ATR inhibitors, can form a pharmaceutical composition, has a certain inhibition effect on proliferation of various tumor cells, and is suitable for the development of medicines for treating cancers such as acute myelogenous leukemia and the like.
Drawings
FIG. 1 is a graph showing the results of a test for the degradation capacity of compound 12b of the present invention for target ATR;
FIG. 2 is a graph showing the results of ATR protein assays of compound 12b of the present invention in various cell lines depending on intracellular proteasome degradation.
Detailed Description
The conception and the technical effects produced by the present invention will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects and effects of the present invention. It should be noted that, in the case of no conflict, the embodiments and features in the embodiments may be combined with each other.
Example 1
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 10 a) having the structure shown below:
compound 10a;
the preparation method comprises the following steps:
；
1) Synthesis of Compound 2: to a solution of compound 1 (2.18 g,10.0 mmol) in N, N-dimethylformamide (65.0 mL) was added aniline (0.930 g,10.0 mmol), benzotriazole-1-tetramethylhexafluorophosphate (4.55 g,12.0 mmol), N, N-diisopropylethylamine (5.95 mL,36.0 mmol) in this order. The mixture was stirred at 25 ℃ for 12 hours. The mixture was then diluted with ethyl acetate (150 mL) and washed with saturated NaCl solution (100 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was purified by column chromatography over a silica gel column (PE: ethyl acetate=9:1) to give compound 2 (2.26 g, 77%) as a yellow gum compound.
Compound 2 was tested as follows: IR (KBr): 3434, 3341, 1661, 1591, 1523, 1437, 752 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.46 (s, 1H), 8.26 (s, 1H), 7.67 (d,J= 8.0 Hz, 2H), 7.38 (t,J= 7.8 Hz, 2H), 7.16 (t,J= 7.4 Hz, 1H). 13 C NMR (100 MHz, Chloroform-d) Delta 162.9, 154.2, 149.4, 137.1, 129.1, 125.6, 124.8, 123.0, 120.1. HRMS (ESI) predicted values: c (C) 11 H 9 BrN 4 NaO + [M+Na] + 314.8952, measured values: 314.9847.
2) Synthesis of Compound 4: to compound 2 (1.17 g,4.00 mmol) in 1, 4-dioxane (20.0/20.0 mL) was added p-bromobenzoic acid (664 mg,4.00 mmol), tetrakis (triphenylphosphine) palladium (462 mg,0.400 mmol) and Na 2 CO 3 (1.02 g,9.60 mmol). The mixture was stirred under argon at 90 ℃ for 12 hours. The mixture was then diluted with ethyl acetate (150 mL) and washed with saturated NaCl solution (120 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate is fed through a silica gel columnPurification by column chromatography (dichloromethane: methanol=50:1) afforded compound 4 (1.23 g, 92%) as a yellow solid.
Compound 4 was tested as follows: IR (KBr) 3409, 3164, 1690, 1357, 1269, 1191, 1122 cm -1 . 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.43 (s, 1H), 9.00 (s, 1H), 8.35 (d,J= 8.1 Hz, 2H), 8.04 (d,J= 7.9 Hz, 2H), 7.83 (d,J= 8.1 Hz, 4H), 7.40 (t,J= 7.8 Hz, 2H), 7.17 (t,J= 7.4 Hz, 1H). 13 C NMR (100 MHz, DMSO-d 6 ) Delta 167.6, 164.4, 154.5, 145.1, 139.4, 137.8, 137.4, 130.0, 129.7, 128.6, 126.9, 125.5, 124.2, 121.2. HRMS (ESI) predicted values: c (C) 18 H 14 N 4 NaO 3 + [M+Na] + 357.0958, measured values: 357.0961.
3) Synthesis of Compound 6: to a solution of compound 3 (200 mg,0.598 mmol) in N, N-dimethylformamide (5.00 mL) was added 1-Boc-piperazine (111 mg,0.598 mmol), 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (172 mg,0.897 mmol), 1-hydroxybenzotriazole (97.0 mg,0.718 mmol) and N, N-diisopropylethylamine (231 mg,1.79 mmol). The mixture was stirred at 25 ℃ for 8 hours. The mixture was then diluted with ethyl acetate (50 mL) and washed with saturated NaCl solution (50 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was purified by column chromatography over a silica gel column (dichloromethane: methanol=80:1) to give compound 6 (255 mg, 85%) as a yellow solid.
Compound 6 was tested as follows: IR (KBr) 3372, 1690, 1634, 1545, 1439 and 1439 cm -1 . 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.43 (s, 1H), 8.98 (s, 1H), 8.31 (d,J= 8.0 Hz, 2H), 7.95 – 7.67 (m, 4H), 7.53 (d,J= 8.0 Hz, 2H), 7.41 (t,J= 7.8 Hz, 2H), 7.17 (t,J= 7.4 Hz, 1H), 3.61 (s, 8H), 1.42 (s, 9H). 13 C NMR (100 MHz, DMSO-d 6 ) δ 169.0, 164.5, 154.4, 153.8, 144.8, 137.9, 137.6, 136.9, 135.2, 128.6, 127.5, 125.6, 124.2124.1, 121.1, 79.2, 28.0. HRMS (ESI) predicted values: c (C) 27 H 30 N 6 NaO 4 + [M+Na] + 525.2221, measured values: 525.2223.
4) Synthesis of compound 8 a: trifluoroacetic acid (2.0 mL) was added to a dichloromethane (3.0 mL) solution of compound 6 (150 mg,0.300 mmol) and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to give the crude intermediate compound as a yellow solid. The above crude intermediate compound was dissolved in N, N-dimethylformamide (3.0. 3.0 mL). The resulting solution was added 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (86.0 mg,0.450 mmol), 1-hydroxybenzotriazole (49.0 mg,0.360 mmol), N, N-diisopropylethylamine (194 mg,1.50 mmol) and 5- ((t-butoxycarbonyl) amino) pentanoic acid (65.0 mg,0.300 mmol). The mixture was stirred at room temperature for 8 hours, then the mixture was diluted with ethyl acetate (80 mL) and washed with saturated NaCl solution (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (dichloromethane: methanol=50:1) to give compound 8a (135 mg, 75%) as a yellow solid.
Compound 8a was tested as follows: IR (KBr): 3421, 1707, 1607, 1517, 1254, 1195 cm -1 . 1 H NMR (400 MHz, DMSO-d 6 ) δ 10.43 (s, 1H), 8.98 (s, 1H), 8.35 – 8.23 (m, 2H), 7.86 – 7.81 (m, 2H), 7.76 (s, 2H), 7.54 (d,J= 8.3 Hz, 2H), 7.41 (t,J= 7.9 Hz, 2H), 7.21 – 7.13 (m, 1H), 6.79 (t,J= 5.8 Hz, 1H), 3.73 – 3.44 (m, 8H), 2.92 (q,J= 5.9, 5.0 Hz, 2H), 2.42 – 2.20 (m, 2H), 1.52 – 1.47 (m, 2H), 1.43 (d,J= 5.6 Hz, 2H), 1.38 (d,J= 8.0 Hz, 9H). 13 C NMR (100 MHz, DMSO-d 6 ) Delta 171.3, 169.5, 165.0, 156.1, 155.0, 145.3, 138.4, 138.1, 137.4, 135.6, 129.1, 128.0, 126.1, 124.7, 124.6, 121.6, 79.6, 77.8, 45.2, 36.2, 32.4, 29.6, 28.7, 22.5. HRMS (ESI) predicted values: c (C) 32 H 39 N 7 NaO 5 + [M+Na] + 624.2905, measured values: 24.2901。
5) Synthesis of Compound 10 a: trifluoroacetic acid (1.0 mL) was added to a dichloromethane (1.5. 1.5 mL) solution of compound 8a (135 mg,0.225 mmol) and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to give the crude intermediate compound as a yellow solid. The above crude intermediate compound was dissolved in N-methylpyrrolidone (4 mL). The resulting solution was added N, N-diisopropylethylamine (87.0 mg,0.630 mmol) and compound 9 (69.0 mg,0.250 mmol). The mixture was stirred at 110 ℃ overnight. The mixture was then diluted with ethyl acetate (50 mL) and washed with saturated NaCl solution (30 mL), with Na 2 SO 4 Dried, filtered and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (dichloromethane: methanol=40:1) to give compound 10a (65.0 mg, 38%) as a yellow solid.
Compound 10a was tested as follows: IR (KBr): 3423, 3344, 1705, 1620, 1532, 1261 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.86 (s, 1H), 8.71 (d,J= 2.7 Hz, 1H), 7.99 – 7.94 (m, 2H), 7.72 (d,J= 8.0 Hz, 2H), 7.56 (d,J= 8.0 Hz, 2H), 7.53 – 7.47 (m, 1H), 7.41 (t,J= 7.9 Hz, 2H), 7.18 (t,J= 7.4 Hz, 1H), 7.10 (d,J= 7.1 Hz, 1H), 6.89 (d,J= 8.5 Hz, 1H), 6.26 (s, 1H), 4.91 (dd,J= 11.9, 5.5 Hz, 1H), 3.60 (d,J= 34.6 Hz, 8H), 3.35 (dd,J= 15.0, 8.8 Hz, 2H), 2.92 – 2.70 (m, 3H), 2.44 (s, 2H), 2.14 (d,J= 11.0 Hz, 1H), 1.78 (d,J= 11.1 Hz, 4H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.5, 170.4, 169.8, 168.9, 167.7, 163.9, 154.5, 146.9, 144.8, 139.1, 137.9, 137.4, 136.3, 132.6, 129.3, 128.2, 125.9, 125.1, 124.8, 120.2, 116.8, 111.7, 110.2, 49.0, 45.6, 42.4, 33.1, 31.6, 28.8, 23.0, 22.7. HRMS (ESI) predicted values: c (C) 40 H 39 N 9 NaO 7 + [M+Na] + 780.2865, measured values: 780.2864.
example 2
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 10 b) having the structure shown below:
compound 10b;
the preparation method comprises the following steps: the synthesis method of the compound 2-compound 6 is the same as that of the example 1, and the synthesis methods of the compound 8b and the compound 10b are as follows:
；
1) Synthesis of compound 8 b: according to the method for synthesizing compound 8a, only reactant 7a was replaced with 7b, to obtain the target compound 8b. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 8b (72%) as a yellow solid.
Compound 8b was tested as follows: IR (KBr): 3423, 1706, 1619, 1528, 1261, 1193 and 1193 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.86 (s, 1H), 8.69 (d,J= 1.8 Hz, 1H), 8.02 – 7.93 (m, 2H), 7.72 (d,J= 8.0 Hz, 2H), 7.60 – 7.52 (m, 2H), 7.40 (t,J= 7.9 Hz, 2H), 7.18 (t,J= 7.4 Hz, 1H), 4.66 (s, 1H), 3.94 – 3.39 (m, 8H), 3.12 (q,J= 7.2, 6.8 Hz, 2H), 2.48 – 2.28 (m, 2H), 2.10 – 2.02 (m, 1H), 1.67 (p,J= 7.5 Hz, 2H), 1.50 (m, 2H), 1.44 (s, 9H), 1.41 – 1.32 (m, 2H). 13 C NMR (100 MHz, Chloroform-d) δ 171.7, 170.2, 163.9, 156.1, 154.5, 144.8, 139.0, 137.8, 137.3, 135.1, 129.2, 128.0, 125.8, 124.9, 124.7, 120.1, 79.1, 45.5, 41.6, 40.4, 33.3, 30.0, 28.5, 26.6, 24.8. HRMS (ESI) predicted values: c (C) 33 H 41 N 7 NaO 5 + [M+Na] + 638.3061, measured values: 638.3062.
2) Synthesis of Compound 10 b: according to the method for synthesizing compound 10a, only reactant 8a was replaced with 8b, to obtain the target compound 10b. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 10b (45%) as a yellow solid.
Compound 10b was tested as follows: IR (KBr): 3421, 3332, 1708, 1617, 1522, 1248 and 1248 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.85 (s, 1H), 8.88 (d,J= 31.8 Hz, 1H), 8.70 (s, 1H), 7.95 (d,J= 8.2 Hz, 2H), 7.73 – 7.69 (m, 2H), 7.57 – 7.54 (m, 2H), 7.48 (dd,J= 8.5, 7.1 Hz, 1H), 7.42 – 7.37 (m, 2H), 7.18 (tt,J= 7.3, 1.2 Hz, 1H), 7.08 (d,J= 7.0 Hz, 1H), 6.87 (d,J= 8.5 Hz, 1H), 6.23 (t,J= 5.7 Hz, 1H), 4.90 (dd,J= 12.0, 5.4 Hz, 1H), 3.56 (s, 8H), 3.29 (q,J= 6.5 Hz, 2H), 2.89 – 2.67 (m, 3H), 2.38 (s, 2H), 2.16 – 2.08 (m, 1H), 1.76 – 1.68 (m, 4H), 1.52 – 1.44 (m, 2H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.3, 168.6, 167.6, 163.8, 154.4, 146.9, 144.6, 139.0, 137.8, 137.2, 136.2, 132.5, 129.2, 128.0, 125.8, 125.0, 124.7, 120.0, 116.6, 111.5, 109.9, 48.9, 42.3, 33.0, 31.4, 29.0, 26.6, 24.8, 22.8. HRMS (ESI) predicted values: c (C) 41 H 41 N 9 NaO 7 + [M+Na] + 794.3021, measured values: 794.3023.
example 3
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 10 c) having the structure shown below:
compound 10c;
the preparation method comprises the following steps: the synthesis method of the compound 2-compound 6 is the same as that of the example 1, and the synthesis methods of the compound 8c and the compound 10c are as follows:
；
1) Synthesis of Compound 8 c: according to the method for synthesizing compound 8a, only reactant 7a was replaced with 7c, to obtain the target compound 8c. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 8c (83%) as a yellow solid.
Compound 8c was tested as follows: IR (KBr): 3424, 1703, 1606, 1523, 1239, 1205 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.85 (s, 1H), 8.68 (s, 1H), 7.95 (d,J= 8.3 Hz, 2H), 7.71 (d,J= 7.7 Hz, 2H), 7.55 (d,J= 8.2 Hz, 2H), 7.39 (t,J= 7.9 Hz, 2H), 7.16 (t,J= 7.4 Hz, 1H), 4.68 (t,J= 6.0 Hz, 1H), 3.82 – 3.47 (m, 8H), 3.10 (q,J= 6.7 Hz, 2H), 2.35 (t,J= 7.4 Hz, 2H), 1.63 (dt,J= 13.5, 6.6 Hz, 2H), 1.44 (m, 11H), 1.39 – 1.31 (m, 4H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.8, 170.1, 163.8, 156.0, 154.4, 144.7, 138.9, 137.7, 137.3, 135.0, 129.1, 128.0, 125.7, 124.8, 124.6, 120.0, 79.0, 45.5, 41.7, 40.4, 33.1, 29.9, 29.0, 28.4, 26.5, 25.0. HRMS (ESI) predicted values: c (C) 34 H 43 N 7 NaO 5 + [M+Na] + 652.3218, measured values: 652.3220.
2) Synthesis of Compound 10 c: according to the method for synthesizing compound 10a, only reactant 8a was replaced with 8c, to obtain the target compound 10c. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 10c (34%) as a yellow solid.
Compound 10c was tested as follows: IR (KBr): 3419, 3315, 1711, 1623, 1509, 1235 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.83 (s, 1H), 9.39 (s, 1H), 8.69 (s, 1H), 7.96 – 7.91 (m, 2H), 7.73 – 7.67 (m, 2H), 7.56 – 7.51 (m, 2H), 7.47 (dd,J= 8.5, 7.1 Hz, 1H), 7.40 – 7.35 (m, 2H), 7.16 (td,J= 7.3, 1.3 Hz, 1H), 7.06 (d,J= 7.1 Hz, 1H), 6.86 (d,J= 8.5 Hz, 1H), 6.21 (t,J= 5.6 Hz, 1H), 5.01 – 4.82 (m, 1H), 3.59 (d,J= 44.4 Hz, 8H), 3.26 (q,J= 6.5 Hz, 2H), 2.89 – 2.68 (m, 3H), 2.35 (s, 2H), 2.10 (ddd,J= 12.4, 5.8, 3.3 Hz, 1H), 1.82 (s, 2H), 1.71 – 1.59 (m, 4H), 1.47 – 1.36 (m, 4H). 13 C NMR (100 MHz, Chloroform-d) δ 171.7, 170.2, 169.6, 168.9, 167.6, 163.8, 154.4, 147.0, 144.5, 139.0, 137.7, 137.2, 136.1, 135.0, 132.5, 129.2, 128.0, 125.8, 125.1, 125.0, 124.7, 120.0, 116.6, 111.4, 109.9, 48.9, 42.4, 33.0, 31.5, 29.0, 26.6, 25.0, 22.8. HRMS (ESI) predicted values: c (C) 42 H 43 N 9 NaO 7 + [M+Na] + 808.3178, measured values: 808.3179.
example 4
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 12 a) having the structure shown below:
compound 12a;
the preparation method comprises the following steps:
；
1) Synthesis of Compound 11 a: to a solution of compound 6 (603 mg,1.20 mmol) in dichloromethane (6.0 mL) was added trifluoroacetic acid (4 mL) and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to give a crude yellow-green solid. The above crude intermediate compound was dissolved in N, N-dimethylformamide (12, mL). To the resulting solution were added 1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride (345 mg,1.80 mmol), 1-hydroxybenzotriazole (195 mg,1.44 mmol), N, N-diisopropylethylamine (775 mg,6.00 mmol) and pent-4-ynoic acid (118 mg,1.20 mmol). The mixture was stirred at room temperature for 8 hours. The mixture was then diluted with ethyl acetate (80 mL) and washed with saturated NaCl solution (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (dichloromethane: methanol=60:1) to give compound 11a (428 mg, 74%) as a yellow solid.
Compound 11a was tested as follows: IR (KBr): 3419, 3302, 1635, 1611, 1523, 1432 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.63 (s, 1H), 7.95 – 7.88 (m, 2H), 7.67 (d,J= 8.0 Hz, 2H), 7.55 – 7.48 (m, 2H), 7.35 (t,J= 7.9 Hz, 2H), 7.13 (t,J= 7.4 Hz, 1H), 3.58 (d,J= 45.1 Hz, 8H), 2.65 – 2.51 (m, 4H), 2.04 – 1.96 (m, 1H). 13 C NMR (100 MHz, Chloroform-d) Delta 170.1, 169.7, 163.8, 154.4, 144.6, 138.8, 137.7, 137.2, 134.9, 129.1, 128.0, 125.6, 124.8, 124.6, 120.0, 83.2, 69.0, 45.3, 41.9, 32.1, 14.5. HRMS (ESI) predicted values: c (C) 27 H 26 N 6 NaO 3 + [M+Na] + 505.1959, measured values: 505.1960.
2) Synthesis of compound 12 a: to a solution of compound 11a (179 mg, 370. Mu. Mol) in N, N-dimethylformamide (2 mL) was added 2- (2, 6-dioxopiperidin-3-yl) -4-iodoisoindoline-1, 3-dione (142 mg, 370. Mu. Mol), pd (PPh 3 ) 2 Cl 2 (26.0 mg, 37.0. Mu. Mol), cuI (7.05 mg, 37.0. Mu. Mol) and N, N-diisopropylethylamine (1 mL). The mixture was stirred under argon for 12 hours at 80 ℃. The mixture was then diluted with ethyl acetate (50 mL) and washed with saturated NaCl solution (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (dichloromethane: methanol=50:1) to give compound 12a (156 mg, 57%) as a yellow solid.
Compound 12a was tested as follows: IR (KBr): 3421, 3298, 1725, 1624, 1513, 1436 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.76 (d,J= 24.8 Hz, 2H), 8.57 (d,J= 4.9 Hz, 1H), 7.82 (d,J= 7.6 Hz, 2H), 7.68 (d,J= 6.4 Hz, 1H), 7.58 (q,J= 8.2 Hz, 4H), 7.43 (d,J= 7.8 Hz, 2H), 7.33 – 7.25 (m, 2H), 7.19 (d,J= 3.7 Hz, 1H), 7.06 (t,J= 7.1 Hz, 1H), 4.96 – 4.80 (m, 1H), 3.65 (d,J= 65.5 Hz, 8H), 2.87 – 2.61 (m, 6H), 2.09 – 1.86 (m, 2H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.6, 170.5, 169.8, 168.8, 166.4, 166.2, 163.8, 154.3, 144.3, 138.8, 138.2, 137.6, 137.3, 135.0, 133.9, 132.2, 130.8, 129.1, 129.1, 128.0, 125.6, 125.0, 124.6, 122.8, 121.2, 120.1, 120.0, 98.0, 49.3, 31.6, 31.4, 22.8, 16.0. HRMS (ESI) predicted values: c (C) 40 H 34 N 8 NaO 7 + [M+Na] + 761.2443, measured values: 761.2445.
example 5
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 12 b) having the structure shown below:
compound 12b;
the preparation method comprises the following steps:
；
1) Synthesis of Compound 11 b: according to the synthesis method of the compound 11a, only pentynoic acid is changed into hexynoic acid to obtain the target compound 11b. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 11b (70%) as a yellow solid.
Compound 11b was tested as follows: IR (KBr): 3421, 3292, 1635, 1596, 1528, 1439, 1004 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.82 (s, 1H), 8.65 (s, 1H), 7.93 (d,J= 7.9 Hz, 2H), 7.68 (d,J= 8.0 Hz, 2H), 7.53 (d,J= 7.9 Hz, 2H), 7.37 (t,J= 7.7 Hz, 2H), 7.15 (t,J= 7.4 Hz, 1H), 3.59 (d,J= 36.4 Hz, 8H), 2.48 (q,J= 8.0, 7.4 Hz, 2H), 2.32 – 2.25 (m, 2H), 2.01 – 1.95 (m, 1H), 1.86 (p,J= 7.1 Hz, 2H). 13 C NMR (100 MHz, Chloroform-d) δ 171.0, 170.1, 163.8, 154.4, 144.6, 138.9, 137.7, 137.2, 135.0, 129.1, 128.0, 125.7, 124.9, 124.7, 120.0, 83.6, 77.4, 77.2 (d,J=20.3 Hz), 76.8, 69.3, 45.4, 41.8, 31.5, 23.7, 17.9. HRMS (ESI) predicted values: c (C) 28 H 28 N 6 NaO 3 + [M+Na] + 519.2115, measured values: 519.2113.
2) Synthesis of compound 12 b: according to the method for synthesizing compound 12a, only 11a was replaced with 11b, to obtain the target compound 12b. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 12b (65%) as a yellow solid.
Compound 12b was tested as follows: IR (KBr): 3417, 3291, 1710, 1601, 1522, 1406 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 10.35 (s, 1H), 9.84 (s, 1H), 8.67 (s, 1H), 7.93 (d,J= 8.3 Hz, 2H), 7.78 (d,J= 6.9 Hz, 1H), 7.72 – 7.62 (m, 4H), 7.57 (d,J= 8.0 Hz, 2H), 7.38 (t,J= 7.9 Hz, 2H), 7.19 – 7.10 (m, 1H), 4.99 (s, 1H), 3.82 – 3.39 (m, 8H), 2.88 – 2.72 (m, 4H), 2.64 (t,J= 6.3 Hz, 2H), 2.17 – 1.94 (m, 4H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.8, 171.5, 170.6, 168.7, 166.4, 166.2, 163.8, 154.3, 144.5, 138.8, 138.5, 137.7, 137.3, 134.8, 133.9, 132.2, 130.6, 129.1, 128.2, 125.6, 124.9, 124.6, 122.8, 121.3, 120.0, 98.3, 53.5, 49.3, 45.5, 42.8, 41.8, 41.7, 31.4, 23.4, 22.9, 19.3. HRMS (ESI) predicted values: c (C) 41 H 36 N 8 NaO 7 + [M+Na] + 775.2599, measured values: 775.2601.
example 6
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 12 c) having the structure shown below:
compound 12c;
the preparation method comprises the following steps:
；/>
1) Synthesis of Compound 11 c: according to the synthesis method of the compound 11a, only hexynoic acid is changed into heptynoic acid, and the target compound 11c is obtained. The resulting compound 11c (82%) was purified by silica gel column chromatography (dichloromethane: methanol=50:1) as a yellow solid.
Compound 11c was tested as follows: IR (KBr): 3426, 3295, 1657, 1602, 1524, 1435 and 1435 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.85 (s, 1H), 8.67 (d,J= 1.8 Hz, 1H), 7.94 (dd,J= 8.3, 1.8 Hz, 2H), 7.71 (d,J= 8.0 Hz, 2H), 7.60 – 7.51 (m, 2H), 7.44 – 7.35 (m, 2H), 7.23 – 7.10 (m, 1H), 3.61 (d,J= 45.5 Hz, 8H), 2.39 (t,J= 8.0 Hz, 2H), 2.26 – 2.20 (m, 2H), 1.97 (d,J= 2.9 Hz, 1H), 1.78 (p,J= 7.5 Hz, 2H), 1.60 (p,J= 7.5, 7.0 Hz, 2H). 13 C NMR (100 MHz, Chloroform-d) Delta 171.4, 170.1, 163.8, 154.4, 144.6, 138.8, 137.7, 137.3, 135.0, 129.1, 128.0, 125.7, 124.8, 124.6, 120.0, 84.0, 68.8, 45.4, 41.7, 32.7, 28.0, 24.2, 18.2, 18.1. HRMS (ESI) predicted values: c (C) 29 H 30 N 6 NaO 3 + [M+Na] + 533.2272, measured values: 533.2271.
2) Synthesis of Compound 12 c: according to the method for synthesizing compound 12a, only 11a was replaced with 11c, to obtain the target compound 12c. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 12c (67%) as a yellow solid.
Compound 12c was tested as follows: IR (KBr): 3403, 3295, 1703, 1611, 1509 and 1509 cm -1 . 1 H NMR (400 MHz, Chloroform-d) δ 9.83 (s, 2H), 8.67 (s, 1H), 7.92 (d,J= 8.0 Hz, 2H), 7.68 (dh,J= 15.0, 7.4 Hz, 5H), 7.52 (d,J= 7.9 Hz, 2H), 7.38 (t,J= 7.9 Hz, 2H), 7.15 (t,J= 7.4 Hz, 1H), 4.99 (s, 1H), 3.92 – 3.37 (m, 8H), 2.93 – 2.72 (m, 3H), 2.63 – 2.41 (m, 4H), 2.11 (d,J= 10.0 Hz, 1H), 1.89 (dq,J= 13.5, 6.9, 6.1 Hz, 2H), 1.75 (q,J= 6.9 Hz, 2H). 13 C NMR (100 MHz, Chloroform-d) δ 171.7, 171.6, 170.3, 168.6, 166.5, 166.1, 163.8, 154.4, 144.4, 138.9, 138.6, 137.6, 137.3, 134.9, 133.9, 132.2, 130.6, 129.1, 128.0, 125.6, 125.0, 124.7, 122.6, 121.5, 120.0, 98.8, 49.3, 45.7, 45.7, 41.7, 33.1, 31.4, 27.8, 24.5, 22.7, 19.5. HRMS (ESI) predicted values: c (C) 42 H 38 N 8 NaO 7 + [M+Na] + 789.2756, measured values: 789.2757.
example 7
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 14 a) having the structure shown below:
compound 14a;
the preparation method comprises the following steps:
；
synthesis of compound 14 a: trifluoroacetic acid (400 μl) was added to a dichloromethane (0.60 mL) solution of compound 6 (60.0 mg,0.12 mmol), and stirred at room temperature for 2 hours. The mixture was concentrated under reduced pressure to give a crude intermediate compound as a yellow-green solid. The above crude intermediate compound was dissolved in N, N-dimethylformamide (1.0. 1.0 mL). N, N-diisopropylethylamine (77.0 mg,0.60 mmol) and tert-butyl (5-bromopentyl) carbamate (32.0 mg,0.120 mmol) were added to the resulting solution. The mixture was stirred at room temperature for 8 hours, then the crude product was obtained. To the crude N, N-dimethylformamide solution were added 2- (2, 6-dioxopiperidin-3-yl) -4-iodoisoindoline-1, 3-dione (73.0 mg, 191. Mu. Mol) and N, N-diisopropylethylamine (550. Mu.L). The mixture was stirred at 80 ℃ for 12 hours. The mixture was then diluted with ethyl acetate (50 mL) and washed with saturated NaCl solution (30 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The concentrate was purified by silica gel column chromatography (dichloromethane: methanol=50:1) to give compound 14a (58 mg, 41%) as a yellow solid.
Compound 14a was tested as follows: 1 H NMR (400 MHz, Chloroform-d) δ 9.81 (s, 1H), 8.66 (d,J= 3.6 Hz, 1H), 8.32 (s, 1H), 7.96 (s, 1H), 7.94 – 7.86 (m, 2H), 7.67 (d,J= 7.8 Hz, 2H), 7.59 (s, 1H), 7.56 – 7.51 (m, 2H), 7.45 – 7.39 (m, 1H), 7.36 (q,J= 8.3, 7.1 Hz, 2H), 7.16 – 7.09 (m, 2H), 4.91 (dd,J= 11.5, 5.7 Hz, 1H), 3.99 (d,J= 31.5 Hz, 2H), 3.78 – 3.60 (m, 2H), 3.45 – 3.21 (m, 4H), 2.90 (t,J= 2.7 Hz, 3H), 2.83 (t,J= 2.7 Hz, 4H), 2.73 (dd,J= 29.7, 16.6 Hz, 2H), 2.37 – 1.84 (m, 2H), 1.73 (s, 2H), 1.43 – 1.22 (m, 2H). 13 C NMR (100 MHz, Chloroform-d) δ 170.9, 170.0, 168.1, 167.1, 166.6, 163.8, 154.4, 149.9, 144.7, 139.2, 137.6, 137.3, 135.9, 135.4, 134.2, 129.2, 128.1, 125.7, 125.0, 124.7, 123.4, 120.0, 118.2, 116.6, 77.2, 49.2, 48.1, 37.3, 36.5, 31.4, 29.7, 29.3, 25.9, 22.7. HRMS (ESI) predicted values: c (C) 40 H 41 N 9 NaO 6 + [M+Na] + 766.3072, measured values: 766.3068.
example 8
A 3-aminopyrazine-2-carboxamide targeted proteolytic chimera (designated compound 14 b) having the structure shown below:
compound 14b;
the preparation method comprises the following steps:
；
synthesis of compound 14 b: according to the method for synthesizing the compound 14a, only 13a was replaced with 13b, thereby obtaining the target compound 14b. Purification by column chromatography on silica gel (dichloromethane: methanol=50:1) afforded compound 14b (37%) as a yellow solid.
Detection of Compound 14bThe detection results are as follows: 1 H NMR (400 MHz, Chloroform-d) δ 9.85 (s, 1H), 9.58 (s, 1H), 8.68 (s, 1H), 7.91 (d,J= 8.0 Hz, 2H), 7.69 (d,J= 8.0 Hz, 2H), 7.52 (d,J= 8.0 Hz, 2H), 7.50 – 7.43 (m, 1H), 7.38 (t,J= 7.8 Hz, 2H), 7.15 (t,J= 7.4 Hz, 1H), 7.06 (d,J= 7.0 Hz, 1H), 6.85 (d,J= 8.5 Hz, 1H), 6.21 (t,J= 5.6 Hz, 1H), 4.89 (dd,J= 12.0, 5.3 Hz, 1H), 3.81 (s, 2H), 3.48 (s, 2H), 3.24 (q,J= 6.6 Hz, 2H), 2.89 – 2.67 (m, 3H), 2.60 (s, 5H), 2.52 (s, 2H), 2.38 (q,J= 8.7, 7.6 Hz, 3H), 2.14 – 2.06 (m, 1H), 2.03 (s, 2H), 1.66 (d,J= 7.2 Hz, 1H), 1.51 (p,J= 7.6 Hz, 2H), 1.39 (dt,J= 21.4, 7.3 Hz, 4H). 13 C NMR (100 MHz, Chloroform-d) δ 171.7, 169.8, 169.6, 169.0, 167.7, 163.8, 154.3, 147.0, 144.5, 139.1, 137.3, 137.2, 136.1, 135.7, 132.5, 129.2, 127.9, 125.6, 124.9, 124.7, 120.0, 116.6, 111.4, 109.9, 77.3, 58.3, 53.5, 52.9, 48.9, 47.8, 42.5, 42.2, 40.9, 31.5, 29.7, 29.1, 27.1, 26.8, 26.5, 22.8. HRMS (ESI) predicted values: c (C) 41 H 43 N 9 NaO 6 + [M+Na] + 780.3229, measured values: 780.3231.
example 9
This example is directed to the 3-aminopyrazine-2-carboxamide targeted proteolytic chimeras prepared in examples 1-8 for evaluation of ATR protein degradation in MV-4-11 cells.
After 24. 24 h incubation of PROTAC molecules at different concentrations (0.1. Mu.M and 0.5. Mu.M) with MV-4-11 cells, whole proteins were harvested, and the expression of ATR protein in the cells was detected by Western Blot assay and quantified using imageJ software, and the results are shown in Table 1.
TABLE 1 determination of the ATR degradation Activity of PROTAC molecules on MV-4-11 cells
The compound 12b for degrading ATR more effectively is selected, and the degradation effect on ATR at a lower concentration is detected by using a Western Blot experiment, and the result is shown in figure 1 that the degradation activity of the compound 12b on ATR protein in MV-4-11 cells is obviously stronger than that of other PROTAC molecules. In addition, it was verified that compound 12b ubiquitin proteasome pathway mediated degradation of the target, pretreatment with MG132, lenalidomide and prodrug molecule (LC-7-20) all inhibited ATR degradation induced by compound 12b, indicating that ATR protein degradation was dependent on the formation of the "target protein-PROTAC-E3 ubiquitin ligase" ternary complex and proteasome activity.
Example 10
This example uses the CCK-8 method to examine the proliferation inhibition of compound 12b on common leukemia cells, breast cancer cells and colorectal cancer cells. After incubating 72 h different concentrations of compound 12b with cells using ATR kinase inhibitor as a control, the absorbance was measured and IC was calculated 50 Values, results are shown in table 2.
TABLE 2 determination of the Effect of Compound 12b on cell proliferation
As shown in Table 2, the antiproliferative activity of compound 12b against acute myeloid leukemia cells (AML), especially the killing of MV-4-11 and MOLM-13, was significantly enhanced. This suggests that the antitumor activity of compound 12b may be selective.
As shown in panel a of fig. 2, degradation of the ATR target by compound 12b can be competitively inhibited by the proteasome inhibitor MG132, lenalidomide; panels B and C of fig. 2 show that the degradation of ATR by a compound can be competitively inhibited by the ribosome inhibitor Cycloheximide (CHX). Thus, it was shown that compound 12b could effect ATR degradation in a ubiquitin dependent pathway.
In summary, the novel ATR-targeted PROTAC degrading agent 12b has a significant degrading effect on ATR protein. As shown by the results of in vitro studies, compound 12b can effectively induce ATR degradation in various cancer cell lines in a dose-dependent and time-dependent manner, and exhibits excellent antitumor cell activity.
While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.

Claims (5)

1. The 3-aminopyrazine-2-formamide targeted proteolytic chimera is characterized in that the structural formula is shown in a formula (I):
formula (I);
wherein, linker is'、/>、/>、Any one of the following.
2. The 3-aminopyrazine-2-carboxamide targeted proteolytic chimera according to claim 1, characterized in that the molecular structure of the 3-aminopyrazine-2-carboxamide targeted proteolytic chimera is represented by formula (II):
formula (II).
3. A method for preparing a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera according to any one of claims 1-2, characterized in that the preparation route is route one or route two:
route one:
；
route two:
。
4. use of a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera according to any of claims 1-2 or a pharmaceutically acceptable salt thereof for the preparation of a medicament for the treatment of leukemia, breast cancer, colorectal cancer.
5. A pharmaceutical composition comprising a 3-aminopyrazine-2-carboxamide targeted proteolytic chimera according to any one of claims 1 to 2 or a pharmaceutically acceptable salt thereof.
CN202311279870.2A 2023-10-07 2023-10-07 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof Active CN117024413B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202311279870.2A CN117024413B (en) 2023-10-07 2023-10-07 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202311279870.2A CN117024413B (en) 2023-10-07 2023-10-07 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof

Publications (2)

Publication Number Publication Date
CN117024413A CN117024413A (en) 2023-11-10
CN117024413B true CN117024413B (en) 2024-01-09

Family

ID=88630224

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202311279870.2A Active CN117024413B (en) 2023-10-07 2023-10-07 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof

Country Status (1)

Country Link
CN (1) CN117024413B (en)

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN117736187B (en) * 2024-02-19 2024-04-30 天津匠新致成科技有限公司 Proteolysis targeting chimeric and preparation method, pharmaceutical composition and application thereof

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102300862A (en) * 2008-12-19 2011-12-28 沃泰克斯药物股份有限公司 Pyrazine Derivatives Useful As Inhibitors Of Atr Kinase
CN103562204A (en) * 2011-04-05 2014-02-05 沃泰克斯药物股份有限公司 Aminopyrazine compounds useful as inhibitors of TRA kinase
CN104582795A (en) * 2012-04-05 2015-04-29 沃泰克斯药物股份有限公司 Compounds useful as inhibitors of ATR kinase and combination therapies thereof
CN104736569A (en) * 2012-01-12 2015-06-24 耶鲁大学 Compounds & methods for the enhanced degradation of targeted proteins & other polypeptides by an e3 ubiquitin ligase
CN113527263A (en) * 2021-07-20 2021-10-22 南开大学 Proteolysis targeting chimera and pharmaceutical composition and application thereof
CN115244053A (en) * 2020-08-05 2022-10-25 上海领泰生物医药科技有限公司 Targeted protein degradation compound and preparation method and application thereof

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102300862A (en) * 2008-12-19 2011-12-28 沃泰克斯药物股份有限公司 Pyrazine Derivatives Useful As Inhibitors Of Atr Kinase
CN103562204A (en) * 2011-04-05 2014-02-05 沃泰克斯药物股份有限公司 Aminopyrazine compounds useful as inhibitors of TRA kinase
CN104736569A (en) * 2012-01-12 2015-06-24 耶鲁大学 Compounds & methods for the enhanced degradation of targeted proteins & other polypeptides by an e3 ubiquitin ligase
CN104582795A (en) * 2012-04-05 2015-04-29 沃泰克斯药物股份有限公司 Compounds useful as inhibitors of ATR kinase and combination therapies thereof
CN115244053A (en) * 2020-08-05 2022-10-25 上海领泰生物医药科技有限公司 Targeted protein degradation compound and preparation method and application thereof
CN113527263A (en) * 2021-07-20 2021-10-22 南开大学 Proteolysis targeting chimera and pharmaceutical composition and application thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Proteolysis-Targeting Chimera (PROTAC) Modification of DovitinibEnhances the Antiproliferative Effect against FLT3-ITD-PositiveAcute Myeloid Leukemia Cells;Sheng Cao等;《J. Med. Chem.》;第64卷;第16497-16511页 *
小分子蛋白降解靶向嵌合体的研究进展;谢妙红等;《中国现代应用药学》;第38卷(第22期);第2891-2899页 *

Also Published As

Publication number Publication date
CN117024413A (en) 2023-11-10

Similar Documents

Publication Publication Date Title
US10793543B2 (en) Selective C-KIT kinase inhibitor
Zhang et al. Quinazoline-1-deoxynojirimycin hybrids as high active dual inhibitors of EGFR and α-glucosidase
CN117024413B (en) 3-aminopyrazine-2-formamide targeted proteolytic chimera, and preparation method, pharmaceutical composition and application thereof
Wei et al. Design, synthesis and biological evaluation of novel 4-anilinoquinazoline derivatives as hypoxia-selective EGFR and VEGFR-2 dual inhibitors
CN113603676B (en) Erlotinib-based EGFR protein targeted degradation small molecule compound and preparation method and application thereof
CN105646454B (en) The 2- aryl amine pyridine derivatives of the fragment containing hydroxamic acid and preparation and application
EP3312180A1 (en) Use of pteridinone derivative serving as egfr inhibitor
CN110845474B (en) Target I-type PRMT compound and preparation method and application thereof
Yin et al. Design, synthesis and biological activities of novel oxazolo [4, 5-g] quinazolin-2 (1H)-one derivatives as EGFR inhibitors
Jing et al. Discovery and optimization of tetrahydropyrido [4, 3-d] pyrimidine derivatives as novel ATX and EGFR dual inhibitors
Elbastawesy et al. Arylidenes of Quinolin-2-one scaffold as Erlotinib analogues with activities against leukemia through inhibition of EGFR TK/STAT-3 pathways
CN107531683A (en) USP7 inhibitor compounds and application method
Yu et al. Design, synthesis and antitumor activity of 4-aminoquinazoline derivatives targeting VEGFR-2 tyrosine kinase
WO2022199547A1 (en) 7,9-dihydropurine derivative and pharmaceutical purpose thereof
Zhao et al. Synthesis and SAR of 4, 5-dihydro-1H-pyrazolo [4, 3-h] quinazoline derivatives as potent and selective CDK4/6 inhibitors
Zhang et al. Design, synthesis and biological evaluation of cinnamamide-quinazoline derivatives as potential EGFR inhibitors to reverse T790M mutation
Gan et al. Discovery of novel 4-arylamino-quinazoline derivatives as EGFRL858R/T790M inhibitors with the potential to inhibit the non-small cell lung cancers
Kaviarasan et al. 1, 3, 4-Thiadiazolo (3, 2-) Pyrimidine-6-Carbonitrile Scaffold as PARP1 Inhibitors
Zhang et al. Design, synthesis and biological evaluation of KRASG12C-PROTACs
Nunes et al. Synthesis, biological evaluation and molecular docking studies of novel 1, 2, 3-triazole-quinazolines as antiproliferative agents displaying ERK inhibitory activity
Feng et al. Synthesis and biological evaluation of 4-(piperid-3-yl) amino substituted 6-pyridylquinazolines as potent PI3Kδ inhibitors
Argyros et al. Design and synthesis of novel 7-aminosubstituted pyrido [2, 3-b] pyrazines exhibiting anti-breast cancer activity
Kayumov et al. Design, synthesis and pharmacological evaluation of new PARP1 inhibitors by merging pharmacophores of olaparib and the natural product alantolactone
Riaz et al. Novel pyridine-2, 4, 6-tricarbohydrazide derivatives: Design, synthesis, characterization and in vitro biological evaluation as α-and β-glucosidase inhibitors
CN102264699A (en) Novel benzylidene-indolinone and their medical and diagnostic uses

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant