CDK9 protein degradation targeting chimera

Abstract

The invention relates to the technical field of medicines, in particular to a CDK9 protein degradation targeting chimera as well as a preparation method and application thereof. The CDK9 protein degradation targeting chimera compound disclosed by the invention shows an obvious CDK9 inhibition capability and inhibits the proliferation of MDA-MB-231 cells.

Description

Technical Field

[0001] The present disclosure relates to the field of medical technology, and in particular to a CDK9 protein degradation targeted chimera and a preparation method and use thereof. Background Art

[0002] The proliferation and division of eukaryotic cells is a precise and complex regulatory process. The proliferation process is completed through the cell cycle, and the orderly progress of the cell cycle is through its strict molecular regulatory mechanism. At present, it has been found that there are three main types of molecules involved in cell cycle regulation: cyclin-dependent kinases (CDK), cyclins, and cyclin-dependent kinase inhibitors (CKI), among which CDK is at the center. 13 members of the CDK family have been found (CDK1-CDK13), which are divided into two categories according to their different intracellular functions: CDK that controls the cell cycle and CDK that controls cell transcription. CDK9 belongs to the serine kinase class. The complex formed by its combination with the corresponding cell cycle protein (cyclin) is called positive transcription elongation factor b (P-TEFb). This complex can phosphorylate RNA polymerase II (RNA polymerase II) and some negative transcription elongation factors (NELF and N-TEF) so that transcription can be extended from the starting site. It is the core molecule for transcription extension (Sims RJ 3rd et al. Genes Dev, 2004, 18: 2437-68; Yamaguchi Y et al. Mol Cell Biol, 2002, 22: 2918-27). Studies have found that abnormalities in the expression level or / and kinase activity of CDK9 can cause abnormal expression of multiple proteins in cells or / and their mRNA levels. It has been confirmed that anti-apoptotic proteins (such as Bcl-2), cell cycle-related regulatory proteins (such as cyclinD1), p53 pathway-related proteins, certain proteins of the NF-κB pathway, and proteins related to the tumor microenvironment (such as VEGF) are closely related to tumors. It can be seen that CDK9 is one of the most critical molecules in the development of tumors.

[0003] Therefore, the development of drugs to regulate CDK9 is crucial for the prevention and treatment of CDK9-related diseases. Summary of the invention

[0004] In view of this, one aspect of the present disclosure provides a targeted chimera for inhibiting CDK9 protein degradation, wherein the targeted chimera for CDK9 protein degradation is shown in the following formula I:

[0005]

[0006] In the formula,

[0007] R 1 Halogen or C 1-3 Alkoxy;

[0008] R 2 is hydrogen or C 1-3 alkyl;

[0009] C is a ligand of CDK9 protein, which has a structure represented by Formula IA or Formula IB:

[0010]

[0011] In formula IA, R A1 is halogen; R A2 Halogen or C 1-3 Alkoxy; R A3 is hydrogen or C 1-3 Alkyl; R A4 C 1-3 Alkyl; in Formula IB, R B1 C 4-6 Cycloalkyl; R B2 , R B3 Each independently is C 1-3 alkyl.

[0012] In some embodiments, the protein degradation targeting chimera has the structure shown in the following formula I-1:

[0013]

[0014] Among them, R 1 , R 2 , R A1 , R A2 , R A3 , R A4 Each defines a compound of formula I.

[0015] In some embodiments, the protein degradation targeting chimera has the structure shown in the following formula I-2:

[0016]

[0017] Among them, R 1 , R 2 , R B1 , R B2 , R B3 Each defines a compound of formula I.

[0018] In some embodiments, R 1 Halogen or C 1-3 Alkoxy.

[0019] In some embodiments, R 1 It is a halogen.

[0020] In some embodiments, R 1 C 1-3 Alkoxy.

[0021] In some embodiments, R 1 is fluorine, chlorine or bromine.

[0022] In some embodiments, R 1 It is methoxy or ethoxy.

[0023] In some embodiments, R 1 is fluorine or methoxy.

[0024] In some embodiments, R 2 For hydrogen.

[0025] In some embodiments, R A1 It is a halogen.

[0026] In some embodiments, R A1 For fluorine.

[0027] In some embodiments, R A2 Halogen or C 1-3 Alkoxy.

[0028] In some embodiments, R A2 is fluorine or methoxy.

[0029] In some embodiments, R A3 is hydrogen or C 1-3 alkyl.

[0030] In some embodiments, R A3 It is isopropyl.

[0031] In some embodiments, R A4 It is methyl.

[0032] In some embodiments, R A1 is fluorine, R A2 is fluorine; R A3 is isopropyl, R A4 It is methyl.

[0033] In some embodiments, R B1 is cyclobutyl, cyclopentyl or cyclohexyl.

[0034] In some embodiments, R B1 It is cyclopentyl.

[0035] In some embodiments, R B2 , RB3 Each is independently methyl or ethyl.

[0036] In some embodiments, R B2 It is methyl.

[0037] In some embodiments, R B3 It is methyl.

[0038] In some embodiments, R B1 is cyclopentyl; R B2 is methyl, R B3 It is methyl.

[0039] In some embodiments, the preparation method of the compound of Formula I-1 or Formula I-2 is selected from the following Synthesis Route 1 or Synthesis Route 2:

[0040] Synthetic route 1:

[0041]

[0042] The compound of formula 1 is subjected to condensation reaction with the compound of formula 2 to obtain a compound of formula I-2;

[0043] Among them, R 1 , R 2 , R A1 , R A2 , R A3 , R A4 The respective definitions are the same as before;

[0044] Synthesis route 2:

[0045]

[0046] The compound of formula 1 is reacted with the compound of formula 3 to obtain a compound of formula I-2;

[0047] Among them, R 1 , R 2 , R B1 , R B2 , R B3 The definitions are the same as before.

[0048] In some embodiments, the compound of Formula 1 is or a pharmaceutically acceptable salt thereof.

[0049] In some embodiments, the compound of Formula 1 is or its trifluoroacetate salt.

[0050] In some embodiments, the compound of formula 2 is or a pharmaceutically acceptable salt thereof;

[0051] In some embodiments, the compound of formula 3 is or a pharmaceutically acceptable salt thereof.

[0052] Another aspect of the present disclosure provides the following intermediate compound or a pharmaceutically acceptable salt thereof:

[0053]

[0054] The present disclosure provides the following protein degradation targeting chimeras:

[0055]

[0056]

[0057] Another aspect of the present disclosure provides a CDK9 degrader prepared by the protein degradation targeting chimera described above.

[0058] Another aspect of the present disclosure provides a pharmaceutical composition prepared by the protein degradation targeting chimera described above.

[0059] The pharmaceutical composition optionally further comprises a pharmaceutically acceptable carrier.

[0060] Another aspect of the present disclosure provides use of the protein degradation targeting chimera described above or the pharmaceutical composition described above in the preparation of a CDK9 degrader.

[0061] Another aspect of the present disclosure provides use of the aforementioned protein degradation targeting chimera or the aforementioned pharmaceutical composition in the preparation of a drug for treating cancer, preferably breast cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] Figure 1 The degradation activity of the compound on CDK9 in MDA-MB-231 cells at different concentrations is shown, with an action time of 16 hours. DETAILED DESCRIPTION

[0063] I. Definitions

[0064] As used herein and unless otherwise specified, the terms "comprising", "including", "having", "containing", including grammatical equivalents thereof, should generally be understood as open and non-limiting, for example, not excluding other unlisted elements or steps.

[0065] Unless otherwise specified, the following terms used in the specification and claims have the following meanings:

[0066] The "protein degradation targeting chimera" or "compound" of the present invention may be asymmetric, for example, having one or more chiral centers. Unless otherwise indicated, the "compound" or "protein degradation targeting chimera" of the present invention may be any one isomer or a mixture of two or more isomers. The "compound" of the present invention includes isomers (such as stereoisomers), enantiomers, diastereomers, racemates or mixtures of two or more isomers of the compound, and pharmaceutically acceptable salts thereof.

[0067] The "protein degradation targeting chimera" of the present invention also includes tautomer forms. Tautomer forms are derived from the exchange of a single bond with an adjacent double bond and the migration of a proton. The term "tautomer" or "tautomer form" means that at room temperature, isomers with different functional groups are in dynamic equilibrium and can quickly convert into each other.

[0068] The term "optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes both occurring and not occurring of said event or circumstance.

[0069] In this disclosure, It refers to the position where a substituent is bonded.

[0070] The term "halogen" refers to fluorine, chlorine, bromine and iodine.

[0071] The term "substituted" means that any one or more (e.g., 1, 2, 3, or 4, etc.) hydrogen atoms on a specific group are replaced by a substituent, which may be the same or different, as long as the valence state of the specific group is normal and the substituted compound is stable. For example, "substituted by halogen" means that any one or more (e.g., 1, 2, 3, or 4, etc.) hydrogen atoms on a specific group are replaced by the same or different halogens, as long as the valence state of the specific group is normal and the substituted compound is stable.

[0072] Numeric ranges herein refer to each integer in the given range. For example, "C1-C3" means that the group can have 1 carbon atom, 2 carbon atoms, or 3 carbon atoms.

[0073] The term "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight-chain or branched saturated hydrocarbon groups, having the indicated number of carbon atoms. 1-3 The term "alkyl" includes C1 alkyl, C2 alkyl, C3 alkyl, and examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, and the like.

[0074] The term "alkoxy" may be linear or branched. Specific examples thereof include methoxy, ethoxy, n-propoxy, isopropoxy, isopropyloxy, n-butoxy, isobutoxy, tert-butoxy, sec-butoxy, n-pentoxy, neopentoxy, isopentyloxy, etc., but are not limited thereto.

[0075] The term "cycloalkyl" refers to a monocyclic saturated hydrocarbon system, without heteroatoms and without double bonds. 4-6 Examples of "cycloalkyl" include, but are not limited to, cyclobutyl, cyclopentyl, and cyclohexyl.

[0076] The term "pharmaceutically acceptable" means that a carrier, vehicle, excipient, diluent, and / or formed salt is generally chemically or irrationally compatible with the other ingredients constituting a pharmaceutical dosage form and physiologically compatible with the receptor.

[0077] The term "pharmaceutically acceptable carrier" refers to those carriers that have no significant irritation to the body and do not impair the biological activity and performance of the active compound, including but not limited to any diluents, disintegrants, binders, glidants, and wetting agents commonly used in the art that can be used for humans or animals.

[0078] The term "pharmaceutically acceptable salt" refers to a salt that retains the biological effectiveness of the free acid and base of a particular compound without biological adverse effects. For example, acid (including organic acid and inorganic acid) addition salts or base addition salts (including organic bases and inorganic bases), zwitterionic salts, and quaternary ammonium salts, such as alkylammonium salts. The pharmaceutically acceptable salts of the present invention can be synthesized by conventional chemical methods from parent compounds containing acid radicals or bases. In general, the preparation method of such salts is: in water or an organic solvent or a mixture of the two, these compounds in the form of free acid or base are reacted with a stoichiometric amount of an appropriate base or acid to prepare.

[0079] The term "effective amount" or "therapeutically effective amount" refers to a non-toxic but sufficient amount of a drug or pharmaceutical agent to achieve the desired effect. The exact dosage will vary depending on factors such as subject dependent variables (e.g., age, immune system health, etc.), the disease or illness, and the treatment being administered.

[0080] The term "pharmaceutical composition" means a composition comprising the compound described in the present disclosure or a pharmaceutically acceptable salt thereof, and at least one pharmaceutically acceptable ingredient selected from the following depending on the mode of administration and the nature of the dosage form, including but not limited to: carriers, diluents, adjuvants, excipients, preservatives, fillers, disintegrants, wetting agents, emulsifiers, suspending agents, sweeteners, flavoring agents, fragrances, antibacterial agents, antifungal agents, lubricants, dispersants, temperature-sensitive materials, temperature regulators, adhesives, stabilizers, suspending agents, etc.

[0081] The drugs or pharmaceutical compositions of the present disclosure can be administered orally, topically, parenterally or mucosally (e.g., buccally, by inhalation or rectally) in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers. It is generally desirable to use an oral route. The active agent can be administered orally in the form of capsules, tablets, etc.

[0082] The medicine or pharmaceutical composition of the present disclosure can be delivered parenterally, that is, administered intravenously (iv), intracerebroventricularly (icv), subcutaneously (sc), intraperitoneally (ip), intramuscularly (im), subcutaneously (sd) or intradermally (id), by direct injection, for example, by bolus injection or continuous infusion. The formulation for injection can be presented in unit dosage form, for example, in an ampoule or multi-dose container with an added preservative. The composition can be in the shape of an excipient, in the form of a suspension, solution or emulsion in an oil or aqueous vehicle, and can include formulation agents such as anti-settling agents, stabilizers and / or dispersants. Alternatively, the active ingredient can be reconstituted with a suitable carrier (e.g., sterile pyrogen-free water) before use in powder form.

[0083] The medicaments or pharmaceutical compositions of the present disclosure may also be formulated for rectal administration, for example, as suppositories or retention enemas (eg, containing conventional suppository bases such as cocoa butter or other glycerides).

[0084] The term "treating" includes inhibiting, alleviating, preventing or eliminating one or more symptoms or side effects associated with the disease, condition or disorder being treated.

[0085] II. Embodiment

[0086] The preparation method of compound of the present invention is described in more detail below, but these specific preparation methods do not constitute any restriction to the scope of the present invention.In addition, reaction conditions such as reactant, solvent, alkali, the amount of compound used, reaction temperature, reaction time etc. are not limited to the following examples.

[0087] The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in the specification or known in the art. Such a combination can be easily performed by those skilled in the art.

[0088] Unless otherwise specified, the raw materials and equipment used in the specific embodiments of the present invention are all known products and can be obtained by purchasing commercially available products.

[0089] Preparation Example

[0090] Synthesis of intermediate 1

[0091]

[0092] Step 1: Synthesis of tert-butyl (4-((7-cyclopentyl-6-(dimethylcarbamoyl)-7H-pyrrolo[2,3-d]pyrimidin-2-yl)amino)phenyl)carbamate

[0093] 2-Chloro-7-cyclopentyl-N, N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide (5 g, 17.08 mmol), tert-butyl (4-aminophenyl)carbamate (3.55 g, 17.08 mmol), palladium acetate (384 mg, 1.71 mmol), Xantphos (1.98 g, 3.42 mmol) and cesium carbonate (16.65 g, 51.24 mmol) were added to 1,4-dioxane (100 mL). Nitrogen was replaced and the reaction solution was stirred at room temperature for 18 hours. LCMS showed that the reaction was complete. The reaction solution was poured into water and extracted with ethyl acetate. The organic phase was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was separated and purified by silica gel column (PE / EA, 3 / 1-2 / 1-1 / 2) to obtain 4.12 g of the target compound as a yellow solid with a yield of 51.9%.

[0094] LC-MS (ESI) m / z: 465.3 [M+H] + .

[0095] 1 H NMR (400MHz, MeOD) δ: 1.52 (s, 9H), 1.65-1.67 (m, 2H), 2.01-2.05 (m, 4H), 2.54-2.57 (m, 2H), 3.14 ( s,6H),4.76(s,1H),6.40(s,1H),6.65(s,1H),7.30-7.33(m,2H),7.61-7.64(s,3H),8.62(s,1H).

[0096] Step 2: Synthesis of 2-((4-aminophenyl)amino)-7-cyclopentyl-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide hydrochloride

[0097] The product of the previous step (4.12 g) was dissolved in anhydrous dichloromethane (40 mL), 20 ml of hydrochloric acid (4 M dioxane solution) was added, and stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The mixture was concentrated under reduced pressure to obtain 3.5 g of the target compound, with a yield of 100%.

[0098] LC-MS (ESI) m / z: 365.2 [M+H] + .

[0099] Synthesis of intermediate 2

[0100]

[0101] Step 1: Synthesis of tert-butyl (4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzimidazol-6-yl)pyrimidin-2-yl)amino)phenyl)carbamate

[0102] 6-(2-chloro-5-fluoropyrimidin-4-yl)-4-fluoro-1-isopropyl-2-methyl-1H-benzimidazole (15.0 g, 46.56 mmol) was dissolved in 1,4-dioxane (200 mL), and tert-butyl (4-aminophenyl)carbamate (19.37 g, 93.12 mmol), XantPhos (2.69 g, 4.6 mmol), cesium carbonate (30.26 g, 93.12 mmol) and palladium acetate (527 mg, 2.3 mmol) were added, and stirred at 110°C under nitrogen protection for 16 hours. LCMS showed that the reaction was complete. The reaction solution was directly filtered, the filtrate was concentrated under reduced pressure, and the residue was separated and purified by silica gel column (DCM / MeOH, 80 / 1) to obtain 15 g of the target compound as a yellow solid with a yield of 63%.

[0103] LC-MS (ESI) m / z: 495.2 [M+H] + .

[0104] Step 2: Synthesis of N1-(5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)benzene-1,4-diamine hydrochloride

[0105] Tert-butyl (4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzimidazol-6-yl)pyrimidin-2-yl)amino)phenyl)carbamate (15 g, 30.4 mmol, 1.0 eq) was dissolved in DCM (300 mL) and added to hydrogen chloride-1,4-dioxane (200 mL). The reaction solution was stirred at room temperature for 16 hours. LCMS showed that the reaction was complete. The reaction solution was concentrated under reduced pressure to obtain 15 g of the target compound as a light yellow solid.

[0106] LC-MS(ESI)m / z:395.1[M+H]+,RT=0.993min

[0107] 1 H NMR (400MHz, DMSO) δ: 10.38 (br, 3H), 10.09 (s, 1H), 8.74 (d, J = 3.6Hz, 1H), 8.41 (s, 1H), 7.92 -7.89(m,3H),7.38(d,J=9.2Hz,2H),5.04-4.97(m,1H),2.86(s,3H),1.69(d,J=6.8Hz,6H).

[0108] Synthesis of intermediate 3

[0109]

[0110] Step 1: Synthesis of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoate

[0111] A mixture of 4-(dimethoxymethyl)piperidine (5.5 g, 34.9 mmol), methyl 2,4-difluorobenzoate (4 g, 23.3 mmol) and potassium carbonate (6.4 g, 46.6 mmol) in DMSO (40 mL) was replaced with nitrogen three times, heated to 80°C and stirred for 2 hours. The reaction solution was cooled to room temperature, water (300 mL) was added, and extracted twice with ethyl acetate (200 mL). The organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the residue obtained by vacuum rotary evaporation was separated and purified by flash silica gel column chromatography (petroleum ether-ethyl acetate, 95-90%) to obtain 3.5 g of the title compound as a white solid, with a yield of 49%.

[0112] LCMS: Rt=2.551min, MS(ESI)m / z=312.0[M+H] + .

[0113] 1H NMR(400MHz,DMSO-d6)δ7.86-7.76ppm(m,1H),6.63(dt,J=9.4,2.6Hz,1H),6.51(dt,J=14.8,2.6Hz,1H),4.20-4.01(m,1H),3 .93-3.84(m,5H),3.39(q,J=1.3Hz,6H),3.02-2.71(m,2H),1.86(dt,J=10.7,3.5Hz,3H),1.74-1.55(m,1H),1.52-1.23(m,2H)

[0114] Step 2: Synthesis of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoic acid

[0115] An aqueous solution (60 mL) of lithium hydroxide monohydrate (7.9 g, 329.8 mmol) was slowly added to a methanol solution (60 mL) of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoate (25.4 g, 82.5 mmol), and the system was heated to 50°C and stirred for 1 h. The reaction solution was evaporated under reduced pressure to remove methanol, diluted with water (60 ml), and a dilute aqueous hydrochloric acid solution (1 mol / L) was added dropwise under stirring until a large amount of precipitation was precipitated, and the pH was about 6. The mixture was filtered, and the filter cake was washed with water and dried to obtain 20 g of the title compound as a white solid with a yield of 87.0%.

[0116] LCMS: Rt=2.391min, MS(ESI)m / z=298.0[M+H] + .

[0117] 1H NMR (400MHz, DMSO-d6) δ10.93ppm (s, 1H), 9.82 (d, J = 7.0Hz, 1H), 7.97-7.76 (m ,1H),7.29-7.08(m,1H),6.99(td,J=8.3,2.3Hz,1H),4.85-4.68(m,1H),4.50 (s,1H),3.16(s,2H),2.88-2.62(m,3H),2.24(d,J=12.6Hz,1H),2.12-1.91(m ,1H),1.77(d,J=11.5Hz,2H),1.45(d,J=12.3Hz,3H),1.27(d,J=17.1Hz,1H).

[0118] Step 3: Synthesis of 4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-fluorobenzamide

[0119] 2-(7-Azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (32.3 g, 85.5 mmol) was added to a dimethylformamide solution (1700 mL) of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-fluorobenzoic acid (17 g, 57 mmol), 3-aminopiperidine-2,6-dione hydrochloride (9.35 g, 57 mmol) and diisopropylethylamine (22.1 g, 171 mmol) at once, and the system was stirred at room temperature for 1 hour. Water (1000 mL) was added to the system, washed with ethyl acetate (200 mL), and the filter cake was filtered to obtain a relatively pure target compound. DCM:EA=1:10 was used for slurrying to obtain 17 g of the light yellow solid title compound with a yield of 73.0%.

[0120] LCMS: Rt=1.12min, MS(ESI)m / z=408.3[M+H] + .

[0121] 1H NMR (400MHz, DMSO-d6) δ10.85(s,1H),8.01(t,J=7.3Hz,1H),7.62(t,J=9.1Hz,1H), 6.95-6.67(m,2H),4.83-4.67(m,1H),4.07(d,J=6.9Hz,1H),3.90(dd,J=13.2,3.4Hz ,2H),3.27(s,6H),2.78(tdd,J=13.5,9.6,4.1Hz,3H),2.19-1.93(m,2H),1.83(dtt, J=11.4,7.3,3.6Hz,1H),1.68(dd,J=13.4,3.7Hz,2H),1.26(qd,J=12.4,4.1Hz,2H).

[0122] Step 4: Synthesis of N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(4-formylpiperidin-1-yl)benzamide 2,2,2-trifluoroacetate

[0123] Trifluoroacetic acid (17 mL) was added dropwise to a dichloromethane solution (170 mL) of 4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-fluorobenzamide (17 g, 2.9 mmol), and the system was stirred at room temperature for 4 hours. The reaction solution was evaporated under reduced pressure to obtain a residue, and the pH was adjusted to weak alkalinity with saturated sodium bicarbonate solution, and DCM1.5 L was added to extract once, and the organic phase was washed with water 3 times and dried to obtain 12 g of the title compound as an off-white solid, with a yield of 79%.

[0124] LCMS: Rt=0.967min, MS(ESI)m / z=362.0[M+H] + .

[0125] 1H NMR (400MHz, DMSO-d6) δ = 10.85ppm (s, 1H), 1H NMR (400MHz, DMSO-d6) δ10.85(s,1H),9.62(s,1H),8.03(t,J=7.2Hz,1H),7.63(t,J=9. 1Hz,1H),6.97-6.64(m,2H),4.73(ddd,J=12.7,7.7,5.4Hz,1H),3.02(ddd,J=13.5,10.9 ,3.0Hz,2H),2.78(ddd,J=17.2,13.3,5.6Hz,1H),2.59(dtd,J=12.2,8.2,7.3,4.0Hz,1H ),2.19-1.97(m,2H),1.90(dt,J=12.4,3.8Hz,2H),1.54(dtd,J=14.4,10.8,3.9Hz,2H).

[0126] Synthesis of intermediate 4

[0127]

[0128] Step 1: Preparation of methyl 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoate

[0129] Palladium acetate (640 mg, 2.86 mmol), 1,1'-binaphthyl-2,2'-bisdiphenylphosphine (3810 mg, 6.12 mmol), cesium carbonate (26.59 g, 81.61 mmol) were added to a mixed solution of methyl 4-bromo-2-methoxybenzoate (10.00 g, 40.81 mmol) and 4-(dimethoxymethyl)piperidine (7.15 g, 44.89 mmol) in 1,4-dioxane (150 mL), argon was replaced 3 times, and the mixture was stirred at 100°C for 8 hours under argon protection. After the reaction was completed, diatomaceous earth was used for suction filtration and washed with ethyl acetate. The filtrate was dried and the obtained crude product was separated by a normal silica gel column (ethyl acetate / petroleum ether = 1 / 10 to 3 / 1) to obtain 8.0 g of the crude product of the light yellow solid title compound, with a yield of 60.63%.

[0130] LCMS: MS (ESI) m / z = 324.2 [M+H] + .

[0131] Step 2: Preparation of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid

[0132] Dissolve 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid methyl ester (8.0g, 24.74mmol) in 40mL tetrahydrofuran and 40mL methanol, add 40mL of prepared lithium hydroxide (4.15g, 98.95mmol) aqueous solution, and stir at room temperature for 16 hours. After the reaction is completed, spin dry, add 100mL of water again, extract twice with ethyl acetate (60mL*2), adjust the pH of the aqueous phase to 4-5 with 1N hydrochloric acid, a large amount of solid precipitates, and filter to obtain 6.0g of light yellow solid title compound with a yield of 78.40%.

[0133] LCMS: MS (ESI) m / z = 310.2 [M+H] + .

[0134] Step 3: Preparation of (S)-4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-methoxybenzamide

[0135] Under ice bath, (S)-3-aminopiperidine-2,6-dione hydrochloric acid (3.35 g, 20.37 mmol) and diisopropylethylamine (7.52 g, 58.19 mmol) were added to a mixed solution of 4-(4-(dimethoxymethyl)piperidin-1-yl)-2-methoxybenzoic acid (6.0 g, 19.40 mmol) and O-(7-azabenzotriazol-1-yl)-N,N,N',N'-tetramethyluronium hexafluorophosphate (8.85 g, 23.27 mmol) in N,N-dimethylformamide (30 mL) and dichloromethane (120 mL), and the system was stirred at 25 ° C for 16 hours. The dichloromethane and diisopropylethylamine were removed by rotary evaporation under reduced pressure, and the reaction solution was added dropwise into 200 mL of water with stirring. After stirring for 1 hour, the mixture was filtered and the filter cake was washed with water. After the filter cake was dried, it was rotary evaporated under reduced pressure to obtain 7.8 g of the title compound as a white solid with a yield of 95.87%.

[0136] LCMS: MS (ESI) m / z = 420.2 [M+H] + .

[0137] Step 4: Preparation of (S)-N-(2,6-dicarbonylpiperidin-3-yl)-4-(4-formylpiperidin-1-yl)-2-methoxybenzamide trifluoroacetate

[0138] (S)-4-(4-(dimethoxymethyl)piperidin-1-yl)-N-(2,6-dicarbonylpiperidin-3-yl)-2-methoxybenzamide (7.80 g, 18.59 mmol) was dissolved in 30 mL of dichloromethane, and 30 mL of trifluoroacetic acid was added, and stirred at room temperature for 3 hours. After the reaction was completed, the reaction solution was spin-dried, and the obtained crude product was separated by a normal silica gel column (methanol / dichloromethane = 1 / 20 to 10 / 1) to obtain 3.90 g of the light yellow solid title compound, with a yield of 46.01%.

[0139] LCMS: MS (ESI) m / z = 374.2 [M+H] + .

[0140] Example 1: Preparation of 7-cyclopentyl-2-((4-(((1-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-fluorophenyl)piperidin-4-yl)methyl)amino)phenyl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

[0141]

[0142] The E3 aldehyde compound intermediate (0.1 mmol) was added to a solution (3 mL) of the amine compound intermediate (0.1 mmol) in N,N-dimethylformamide, and stirred at room temperature for 5 minutes. Triethylamine (0.2 mmol) was added dropwise, and stirred at room temperature for 2 hours. Then sodium triacetoxyborohydride (0.25 mmol) was added in two batches, and the system was stirred at room temperature for 1 hour. The reaction was completed under TLC monitoring. 15 ml of water was added to the system, and then extracted with ethyl acetate and n-butanol in sequence. The organic phases were combined, dried over Na2SO4 and filtered. The filtrate was concentrated under reduced pressure and further purified by preparative TLC (DCM:MeOH=10:1) to obtain 57 mg of the target compound in a yield of 80%.

[0143] LC-MS: [M+H] + :710.3

[0144] 1H NMR(400MHz,Chloroform-d)δ8.57(s,1H),7.92(t,J=9.2Hz,1H),7.48(d,J=7.9Hz,2H ),6.72(d,J=9.0Hz,1H),6.64(d,J=8.3Hz,2H),6.53(d,J=16.1Hz,1H),6.41(s,1H),4 .79-4.69(m,2H),3.88(d,J=13.0Hz,2H),3.15(s,6H),3.07(d,J=6.2Hz,2H),2.96-2. 76(m,5H),2.71-2.48(m,4H),2.07-1.94(m,6H),1.44-1.37(m,2H),1.33-1.29(m,2H).

[0145] Example 2: Preparation of (S)-7-cyclopentyl-2-((4-(((1-(4-((2,6-dioxopiperidin-3-yl)carbamoyl)-3-methoxyphenyl)piperidin-4-yl)methyl)amino)phenyl)amino)-N,N-dimethyl-7H-pyrrolo[2,3-d]pyrimidine-6-carboxamide

[0146]

[0147] Referring to the method of Example 1, Example 2 (59 mg, 82%) can be prepared.

[0148] LC-MS: [M+H] + :722.4

[0149] 1 H NMR(400MHz,Chloroform-d)δ8.70(d,J=5.6Hz,1H),8.58(s,1H),8.03(d,J=8.9Hz,1H),7 .49(d,J=8.3Hz,2H),6.64(d,J=8.3Hz,2H),6.59(dd,J=9.0,2.2Hz,1H),6.40(s,2H),4.7 9-4.69(m,2H),3.99(s,3H),3.89(d,J=12.7Hz,2H),3.15(s,6H),3.12-3.02(m,2H),2.93 -2.78(m,4H),2.77-2.69(m,1H),2.60-2.49(m,2H),2.08-1.95(m,8H),1.49-1.32(m,4H).

[0150] Example 3: Preparation of (S)-N-(2,6-dioxopiperidin-3-yl)-4-(4-(((4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)phenyl)amino)methyl)piperidin-1-yl)-2-methoxybenzamide

[0151]

[0152] Referring to the method of Example 1, Example 3 (63 mg, 84%) can be prepared.

[0153] LC-MS: [M+H] + :752.3

[0154] 1 H NMR(400MHz,Chloroform-d)δ8.71(d,J=5.6Hz,1H),8.29(d,J=3.9Hz,1H),8.15(s,1H),8.02(d,J=8.9Hz,1H),7 .78(d,J=11.7Hz,1H),7.44(d,J=8.2Hz,2H),6.66(d,J=8.4Hz,1H),6.59(dd,J=9.1,2.2Hz,1H),6.40(d,J=2.3H z,1H),4.79-4.71(m,2H),3.99(s,3H),3.89(d,J=12.8Hz,2H),3.75(t,J=6.1Hz,1H),3.08(d,J=6.4Hz,2H),2.9 2-2.76(m,4H),2.67(s,3H),2.60(t,J=6.1Hz,1H),1.98-1.88(m,3H),1.70(d,J=7.0Hz,6H),1.49-1.35(m,2H).

[0155] Example 4: Preparation of N-(2,6-dioxopiperidin-3-yl)-2-fluoro-4-(4-(((4-((5-fluoro-4-(4-fluoro-1-isopropyl-2-methyl-1H-benzo[d]imidazol-6-yl)pyrimidin-2-yl)amino)phenyl)amino)methyl)piperidin-1-yl)benzamide

[0156]

[0157] Referring to the method of Example 1, Example 4 (63.4 85%) can be prepared.

[0158] LC-MS: [M+H] + :740.3

[0159] 1 H NMR(400MHz,Chloroform-d)δ8.37-8.31(m,1H),8.20(d,J=20.3Hz,1H),7.78(d,J=11.6Hz,1H) ,7.68(dd,J=14.7,8.6Hz,2H),7.56(d,J=8.8Hz,1H),7.44(d,J=8.3Hz,1H),7.10(d,J=8.5Hz,1H ),6.78-6.70(m,1H),6.55(t,J=16.8Hz,1H),4.80-4.70(m,2H),3.77-3.62(m,2H),2.98-2.75( m,3H),2.68(s,3H),2.27-2.16(m,2H),2.09-1.85(m,6H),1.77-1.70(m,6H),1.33-1.30(m,2H).

[0160] Test Case

[0161] 1. Antiproliferation activity of compounds on tumor cells

[0162] MDA-MB-231 cells in the logarithmic growth phase (purchased from the Cell Bank of the Chinese Academy of Sciences) were inoculated in a 96-well plate (NEST, 701001), with 80 μL per well (2 × 10 cells per well). 3 ), place the culture plate in a 37°C incubator containing 5% carbon dioxide and incubate for 4 hours. The test compound was prepared into a 10mM stock solution using dimethyl sulfoxide (DMSO) (Aladdin, D103277). After gradient dilution, a series of working solutions of different concentrations were prepared using complete culture medium. Add 20uL of the working solution to the corresponding wells of the well plate, place the well plate in a 5% carbon dioxide incubator, and take it out after incubation at 37°C for 72h. Add 10μL Cellcounting kit-8 reagent (Mei5bio, MF128-Pro-02) to each well, incubate at room temperature for 90min, and measure the absorbance at 450nm using an enzyme reader. Calculate the cell proliferation inhibition rate, cell proliferation inhibition rate = [1-(A 样品孔 -A 空白孔 ) / (A 对照孔 -A 空白孔 )] × 100%. The inhibition curve was drawn using the four-parameter equation in GraphPad 8.0 software, and the IC 50 The results of the inhibitory activity of each compound in the examples of the present invention on MDA-MB-231 cell proliferation are shown in Table 1. "++++" represents IC 50 <10nM; "+++" represents 10nM <IC 50 <100nM; “++” represents 100nM <IC50 <500nM.

[0163] Table 1: Inhibitory activity of compounds on MDA-MB-231 cell proliferation

[0164] Serial number <![CDATA[IC 50 (nM)]]> Example 1 ++ Example 2 ++ Example 3 +++ Example 4 ++++ Palbociclib ++

[0165] 2. Western Blot assay to determine the degradation activity of the compound on CDK9 protein:

[0166] Experimental steps:

[0167] 1) After subculturing MDA-MB-231 cells with cell culture medium, cells with good growth status were inoculated into 12-well plates (NEST, 712001), with 1 mL of culture medium per well and 2*10 cells per well. 5 , and cultured overnight in a 37°C, 5% CO2 cell incubator.

[0168] 2) Prepare the test compound with dimethyl sulfoxide (DMSO) into a 10mM stock solution. Dilute with DMSO again before use, add 1μl of the diluted compound to the cell culture well, and gently shake to mix. Also set up a negative control well (add an equal amount of DMSO) and a positive control well.

[0169] 3) After culturing for 16-24 hours, cells were lysed with RIPA cell lysis buffer (Biyuntian, P0013) (containing 1% Cocktail), protein was extracted, and protein concentration was measured with BCA kit (Thermo, 23227). 5-fold concentrated protein loading buffer was added, heated at 100°C for 5 minutes, and then the samples were stored at -20°C.

[0170] 4) SDS-PAGE gel electrophoresis experiment: a. Gel preparation: All 7.5%-15% polyacrylamide gels of the present invention are used to separate proteins; b. Sample loading: After the device is assembled, add Running buffer, add protein markers and samples to the sample loading wells, the sample loading volume of each well is 5-15μL, and the amount of sampled protein is 20-30μg; c. Electrophoresis: Connect the power supply, run the sample at a constant voltage of 80V until the pre-stained protein marker is completely separated, then stop the voltage, discard the electrophoresis well, and rinse the sample tank with water; d. Transfer to membrane: Remove the gel in the glass plate, cut off the required part and place it in Trans buffer, assemble the transfer to membrane device in the order of sponge-filter paper-gel-activated PVDF membrane-filter paper-sponge, then place the electrophoresis tank in an ice water bath, turn on the power supply, and transfer the membrane at a voltage of 300V for 1h. After the transfer to membrane is completed, quickly place the PVDF membrane in TBS buffer.

[0171] 5) Immunoblotting and color development: a. Wash the PVDF membrane with 50 mL 1×TBS for 5 min, then add 50 mL 5% skim milk blocking buffer and shake at room temperature for 1 h; b. Wash the PVDF membrane with 50 mL 1×TBS / T for 3 times, 10 min each time; c. Add primary antibodies of appropriate dilution, CDK4 (Beyotime, AF2515), CDK6 (Beyotime, AF0114), CDK9 (Beyotime, AF6468), α-Actin (abclonal, A2319), and incubate at 4°C overnight; d. Wash the PVDF membrane with 50 mL 1×TBS / T buffer for 3 times, 10 min each time; e. Add secondary antibodies of appropriate dilution, Peroxidase Affinipure (HRP) Goat Anti-Rabbit IgG (Bio-Ray, Cat. BN20601), incubate at room temperature for 2 hours; 6. Wash the membrane three times with 50 mL 1×TBS / T buffer, 10 minutes each time; 7. Develop and expose, using α-actin as an internal reference.

[0172] 6) Result detection: Finally, add ECL color developing solution (Biyuntian, Cat. No. P0018S) for color development, take pictures with an automatic chemiluminescence instrument, collect pictures and analyze them.

[0173] Experimental results: The degradation activity of the compound on CDK9 at the action concentration was measured by Western Blot. Figure 1 ), the degradation effects of the four PROTAC molecules in Examples 1-4 on CDK4, CDK6 and CDK9 in MDA-MB-231 cells were evaluated by Western Blot experiments. After different PROTACs molecules were treated with MDA-MB-231 cells for 16 hours, the whole protein was collected, and the expression of CDK9 protein in the cells was detected by Western Blot experiments, and quantified by ImageJ software. The experimental results show that Examples 3 and 4 have good CDK9 degradation activity. The degradation activity of the compounds is shown in Table 2.

[0174] A represents that the degradation percentage of the target protein is greater than 80%; B represents that the degradation percentage of the target protein is between 50% and 80%; C represents that the degradation percentage of the target protein is less than 25% and 50%; D represents that the degradation percentage of the target protein is less than 25%.

[0175] Table 2: Degradation activity of the example compounds on CDK9 at different concentrations

[0176]

[0177]

[0178] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. These descriptions are not intended to limit the present invention to the precise form disclosed, and it is clear that many changes and variations can be made based on the above teachings. The purpose of selecting and describing the exemplary embodiments is to explain the specific principles of the present invention and its practical application, so that those skilled in the art can realize and utilize various different exemplary embodiments of the present invention and various different selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A CDK9 protein degradation targeting chimera, characterized in that: The CDK9 protein degradation targeting chimera is shown in the following formula I: In the formula, R 1 Halogen or C 1-3 Alkoxy; R 2 is hydrogen or C 1-3 alkyl; C is a ligand of CDK9 protein, which has a structure represented by Formula IA or Formula IB: In formula IA, R A1 is halogen; R A2 Halogen or C 1-3 Alkoxy; R A3 is hydrogen or C 1-3 Alkyl; R A4 C 1-3 alkyl; In formula IB, R B1 C 4-6 Cycloalkyl; R B2 , R B3 Each independently is C 1-3 alkyl.

2. The protein degradation targeting chimera according to claim 1, wherein R 1 is fluorine or methoxy.

3. The protein degradation targeting chimera according to claim 1, wherein R 2 For hydrogen.

4. The protein degradation targeting chimera according to any one of claims 1 to 3, wherein: R A1 is fluorine, R A2 is fluorine; R A3 is isopropyl, R A4 It is methyl.

5. The protein degradation targeting chimera according to any one of claims 1 to 3, wherein: R B1 is cyclopentyl; R B2 is methyl, R B3 It is methyl.

6. The protein degradation targeting chimera according to claim 1, which is a compound of formula I-1 or formula I-2, and its preparation method is selected from the following synthetic route 1 or synthetic route 2: Synthetic route 1: The compound of formula 1 is subjected to condensation reaction with the compound of formula 2 to obtain a compound of formula I-2; Among them, R 1 , R 2 , R A1 , R A2 , R A3 , R A4 Each definition of claim 1; Synthesis route 2: The compound of formula 1 is reacted with the compound of formula 3 to obtain a compound of formula I-2; Among them, R 1 , R 2 , R B1 , R B2 , R B3 Each is defined as claim 1.

7. The protein degradation targeting chimera according to claim 6, wherein: The compound of formula 1 is or a pharmaceutically acceptable salt thereof; the compound of formula 2 is or a pharmaceutically acceptable salt thereof; The compound of formula 3 is or a pharmaceutically acceptable salt thereof.

8. The protein degradation targeting chimera according to claim 1, which is selected from:

9. A CDK9 degrader or pharmaceutical composition prepared using the protein degradation targeting chimera according to any one of claims 1 to 8.

10. Use of the protein degradation targeting chimera according to any one of claims 1 to 8 in the preparation of a CDK9 degrader.

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