A protac compound targeting degradation of dapk1 protein and preparation method and application thereof

Abstract

The application discloses a PROTAC compound for targeted degradation of DAPK1 protein, and a preparation method and application thereof, and has a structural formula of X-Y-Z; wherein X represents a ligand of the DAPK1 protein, Z represents a ligand of an E3 ligase, and Y represents a chain connecting X and Z. Alternatively, X and Z can also be directly connected through a piperazine ring. The compound can be used for treating or preventing Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to the field of preparation of drugs for neurodegenerative diseases, and specifically to a PROTAC compound that targets and degrades DAPK1 protein, and a preparation method and application thereof. Background Art

[0002] With the aging of the population, neurodegenerative diseases are becoming a major threat to human health. Alzheimer's disease (AD) is the most common form of senile dementia and one of the most complex diseases. It is a chronic neurodegenerative disorder caused by degeneration of the central nervous system. The primary neuropathological hallmarks of AD are hyperphosphorylation of the tau protein and deposition of amyloid beta (Aβ). However, the pathogenesis of AD is not fully understood, and effective treatments are lacking. Currently, there is a significant unmet need for AD treatments, but technological advances are insufficient to combat the disease, creating a situation where there is no drug available. This presents a hot area for new drug development, but also carries significant risks. Between 1993 and 2003, only five drugs were approved by the FDA to alleviate AD symptoms: tacrine, donepezil, rivastigmine, galantamine, and memantine. None of these drugs has been able to halt or slow the progression of AD. In the nearly two decades since then, leading global pharmaceutical companies such as Pfizer, Roche, Eli Lilly, Merck, and Johnson & Johnson have all struggled to develop anti-AD drugs. From 2021 to 2023, the FDA granted accelerated approvals to Aduhelm (adunatumab) and Lecanemab (lencanelumab), jointly developed by Japan's Eisai Pharmaceuticals and the US company Biogen, due to their effective clearance of β-amyloid protein. However, these monoclonal antibodies face challenges such as excessive pricing, questionable efficacy, and safety. Therefore, new approaches to developing small molecule drugs for the prevention and treatment of AD are urgently needed.

[0003] Death-associated protein kinase 1 (DAPK1) is a calcium / calmodulin-regulated serine / threonine protein kinase. DAPK1 has multiple activities, including regulating apoptosis and autophagy, contributing to the pathogenesis of AD, inhibiting tumor metastasis as a tumor suppressor, participating in the regulation of inflammation and immune function, mediating the body's response to viral infection, and regulating synaptic plasticity.

[0004] DAPK1 plays a central role in neuronal cell death. Studies have found that DAPK1 is expressed in large quantities in the brain and is closely associated with diseases of neuronal damage such as ischemic stroke, depression, epilepsy, and AD. Many studies have shown that DAPK1 is related to the onset of AD. The two main pathological characteristics of AD are amyloid β protein (Aβ) senile plaques and tau neurofibrillary tangles. The formation of Aβ senile plaques is mainly due to the metabolism of amyloid precursor protein (APP), which causes Aβ protein to deposit outside the cell. Tau is a microtubule-associated protein that is hyperphosphorylated under pathological conditions to form tau aggregates. DAPK1 mainly participates in the onset of AD by over-processing APP, triggering the hyperphosphorylation and stabilization of tau aggregates, and is a potential target for the treatment of AD.

[0005] Currently, dozens of DAPK1 inhibitors have been reported, including TC-DAPK6 and HS-38, but none of them have entered clinical trials due to issues with activity, selectivity, and drugability.

[0006] PROteolysis TArgeting Chimeras (PROTAC) is a technology that uses E3 ligase ubiquitination to tag target proteins (POI) to specifically induce their degradation by the proteasome pathway in cells. From a chemical structure perspective, PROTAC is a hybrid bifunctional molecule that uses a linker to couple the target protein ligand (POI) and the E3 ubiquitin ligase ligand (E3). After the PROTAC molecule ubiquitinates and tags the POI and promotes the degradation of the target protein, it can dissociate from the POI and be recycled within the cell. PROTAC is an event-driven mode of action. In theory, a catalytic dose is all that is needed to degrade almost all target proteins in the cell. Therefore, it has a high safety profile and is not prone to drug resistance. It can also regulate some "undruggable proteins". Currently, more than one hundred target proteins have been successfully degraded using PROTAC, the largest of which are kinase families and regulatory proteins.

[0007] DAPK1 is highly expressed in the brains of 75% of AD patients; it can increase tau hyperphosphorylation and Aβ secretion. DAPK1 knockout in mice reduces tau phosphorylation and Aβ production; mice with systemic DAPK1 knockout exhibit no overt phenotypic defects. DAPK1 mediates the pathogenesis of AD, and inhibiting or degrading DAPK1 levels may disrupt this process, thereby preventing the onset of AD. DAPK1 degraders offer the advantages of greater efficacy and lower toxicity compared to inhibitors. Using PROTAC technology to reduce DAPK1 protein levels may have therapeutic or preventive effects on AD. SUMMARY

[0008] To overcome the shortcomings of the existing DAPK1 inhibitors, the present application provides a PROTAC compound targeting degradation of DAPK1 protein and a preparation method and application thereof. The compound is targeted at the ubiquitin-proteasome system to induce degradation of DAPK1 protein by proteolysis-targeting chimeras (PROTACs) technology, so as to achieve the effect of treating or preventing AD. One end of the molecule is targeted to bind E3 ligase CRBN, and the other end is targeted to bind DAPK1 protein, and a stable ternary complex is formed through the linker. The compound ubiquinates the target protein by bridging the E3 ligase, so that the target protein is recognized by the proteasome degradation system, and then specific degradation of the target protein is achieved.

[0009] The PROTAC compound provided by the present application has obvious degradation effect on DAPK1 protein.

[0010] A PROTAC compound targeting degradation of DAPK1 protein, as shown in formula I-1 (X-Y-Z): wherein X represents a ligand of DAPK1 protein, Z represents a ligand of E3 ligase, and Y represents a chain connecting X and Z; or X and Z are directly connected through a piperazine ring.

[0011] The PROTAC compound targeting degradation of DAPK1 protein, wherein X is a compound shown in formula II-1, Z is a compound shown in formula II-2 or II-3, and Y is a compound shown in formula II-4.

[0012]

[0013] Each n is independently an integer between 1 and 6.

[0014] The PROTAC compound targeting degradation of DAPK1 protein, specifically, the compound is:

[0015] Formula 1-1, Z is formula II-2, n=1

[0016]

[0017] Formula 1-2, Z is formula II-2, n=2

[0018]

[0019] Formula 1-3, Z is formula II-2, n=3

[0020]

[0021] Formula 1-4, Z is Formula II-2, n=4

[0022]

[0023] Formula 1-5, Z is Formula II-2, n=5

[0024]

[0025] Formula 1-6, Z is Formula II-2, n=6

[0026]

[0027] Formula 1-7, Z is Formula II-3, n=2

[0028]

[0029] Formula 1-8, Z is Formula II-3, n=3

[0030]

[0031] Formula 1-9, Z is Formula II-3, n=4

[0032]

[0033] Formula 1-10, Z is Formula II-3, n=5

[0034]

[0035] Formula 1-11, Z is Formula II-3, n=6

[0036]

[0037] MG1, Z is formula II-2, X and Z are directly connected through the piperazine ring

[0038]

[0039] MG2, Z is of formula II-2, and X and Z are directly connected through the piperazine ring.

[0040] The preparation method of the PROTAC compound targeting the degradation of DAPK1 protein is as follows:

[0041]

[0042]

[0043] The process flow chart of direct connection between X and Z through the piperazine ring is as follows:

[0044]

[0045] The above-mentioned PROTAC compound targeting the degradation of DAPK1 protein is used to prepare a drug for treating or preventing Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0046] Figure 1 This is a diagram showing the degradation of DAPK1 by the compounds obtained in the present invention, using the HEK293T cell line as the experimental subject;

[0047] Figure 2 The figure shows the degradation effect of compounds at different concentrations on DAPK1;

[0048] Figure 3 This is a graph showing the degradation effect of CL2 compound on DAPK1 over time;

[0049] Figure 4 The degradation of DAPK1 protein in N2a cells by CP compounds;

[0050] Figure 5 These are immunohistofluorescence images, in which: A. The hTau level in the CA3 region of the three groups of mice was detected by immunofluorescence using the HT7 antibody; B. The phosphorylation level of the Tau protein Thr231 site in the CA3 region of the hippocampus of the three groups of mice was detected by immunofluorescence using the pT231-tau antibody. DETAILED DESCRIPTION

[0051] The following will further describe the embodiments of the present invention in detail. The embodiments are exemplary and intended to be used to explain the present invention, but should not be construed as limiting the present invention.

[0052] Example 1

[0053] Synthesis of 3-{[6-(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzoic acid (C9S)

[0054]

[0055] Step 1: Synthesis of 3-(5-amino-4-(ethoxycarbonyl)-1H-pyrazol-1-yl)benzoic acid (1)

[0056] To a solution of 3-hydrazinobenzoic acid hydrochloride (3000 mg, 15.9 mmol) and DIPEA (5038 μL, 28.9 mmol) in ethanol (10 mL) was added ethyl 2-cyano-3-ethoxyacrylate (2447 mg, 14.5 mmol) with stirring, and the mixture was heated at reflux for 12 h. The ethanol was removed by concentration under reduced pressure, and the mixture was diluted with water (40 mL). The pH was adjusted to 2 with 1 M hydrochloric acid, and the mixture was extracted with ethyl acetate (75 mL × 3), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol 50:1). The title compound 1 was obtained as a yellow solid, 3740 mg, 13.6 mmol, with a yield of 93.7%. ESI-MS (m / z): 276.20 [M+H]. + . 1 H NMR (600MHz, DMSO-d6) δ13.24(s,1H),8.04(s,1H),7.92(d,J=7.6Hz,1H),7.77(d,J=7.5Hz, 1H), 7.71 (s, 1H), 7.63 (t, J = 6.9Hz, 1H), 6.42 (s, 2H), 4.24-4.13 (m, 2H), 1.27-1.21 (m, 3H).

[0057] Step 2: Synthesis of 3-(4-hydroxy-6-mercapto-1H-pyrazolo[3,4-d]pyrimidin-1-yl)benzoic acid (2)

[0058] Compound 1 (3740 mg, 13.6 mmol) and benzoyl isothiocyanate (3995 mg, 24.5 mmol) were dissolved in anhydrous tetrahydrofuran (40 mL) and refluxed under nitrogen for 16 h. The mixture was concentrated under reduced pressure to obtain the intermediate thiourea. The resulting thiourea was added dropwise to a refluxing solution of ethanol (70 mL) and sodium ethoxide (21% ethanol, 17.6 mL, 54.4 mmol), and stirring was continued for 30 min. The mixture was concentrated to dryness under reduced pressure, diluted with water (50 mL), and adjusted to pH 2 with 1 M hydrochloric acid. The solid was filtered, washed with water, dried, and purified by silica gel column chromatography (dichloromethane:methanol 20:1). The title compound 2 was obtained as a yellow solid, 2638 mg, 9.15 mmol, in a 67.3% yield. ESI-MS (m / z): 289.10 [M+H]. + . 1 H NMR (600MHz, DMSO-d6) δ13.29 (s, 1H), 12.25 (s, 1H), 8.14 (s, 1H), 8.07-8.00 (m, 2H), 7.80 (s, 1H), 7.66 (d, J = 8.3Hz, 1H).

[0059] Step 3: Synthesis of 3-{[6-(1-methoxy-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzoic acid (3)

[0060] Compound 2 (2638 mg, 9.15 mmol) and DIPEA (4.8 mL, 27.5 mmol) were dissolved in anhydrous DMF (4.0 mL). Methyl 2-bromopropionate (1681 mg, 10.1 mol) was added with stirring. The reaction was stirred at rt for 12 h, diluted with water (40 mL), and the pH was adjusted to 2 with 1 M hydrochloric acid. The mixture was extracted with ethyl acetate (75 mL × 3), washed with saturated brine (30 mL × 2), dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol 50:1). The title compound 3 was obtained as a yellow solid, 1387 mg, 3.70 mmol, in a 40.5% yield. mp: 210.5-212.0°C. ESI-MS (m / z): 375.10 [M+H] + . 1 H NMR(600MHz,DMSO-d6)δ12.87(s,1H),8.60(s,1H),8.30-8.23(m,2H),7.91(d,J=7.7Hz ,1H),7.64(t,J=8.0Hz,1H),4.60(q,J=7.3Hz,1H),3.48(s,3H),1.57(d,J=7.2Hz,3H).

[0061] Step 4: Synthesis of 3-{[6-(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzoic acid (C9S)

[0062] Compound 3 (750 mg, 2 mmol) was dissolved in a 7 M ammonia-methanol solution (45 mL) and heated to 65°C in a reactor for 12 h. The mixture was concentrated under reduced pressure and purified by silica gel column chromatography (dichloromethane:methanol 7.5:1). The title compound C9S was obtained as a white solid (474 ​​mg, 1.32 mmol, 65.9% yield). mp: 228.6-230.2°C. HRMS (ESI) m / z: [M+H] + calcdfor C 15 H 14 N5O4S:360.07665,found:360.07608. 1H NMR (600MHz, DMSO-d6) δ13.05(s,1H),8.85-8.69(m,1H),8.32(d,J=7.7Hz,1H),8.23(s,1H),7.89(d,J=7 .6Hz,1H),7.70(s,1H),7.64(t,J=7.9Hz,1H),7.25(s,1H),4.51(q,J=6.6Hz,1H),1.56(d,J=7.0Hz,3H). 13 CNMR(151MHz,DMSO-d6)δ172.84,168.30,162.87,161.70,158.62,152.39 ,138.98,136.82,129.84,127.62,124.03,121.77,105.50,45.09,19.10.

[0063] Example 2

[0064] Synthesis of 2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}ethan-1-amine trifluoroacetate (P1)

[0065]

[0066] Step 1: Synthesis of tert-butyl 2-({[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}ethyl)carbamate (O1)

[0067] To a stirred solution of 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione (500 mg, 1.81 mmol) in DMSO (4 mL) were added tert-butyl N-(2-aminoethyl)carbamate (377 mg, 2.35 mmol) and DIPEA (1576 μL, 9.1 mmol). The mixture was stirred at 145°C for 2 h, diluted with water (30 mL), extracted with ethyl acetate (50 mL × 3), and washed with saturated brine (20 mL × 3). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (petroleum ether:ethyl acetate 2:1). The title compound O1 was obtained as a yellow solid (146 mg, 0.35 mmol, 19.4% yield), mp: 196.3-197.8°C. ESI-MS (m / z): 417.30 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ11.05(s,1H),7.54(dd,J=8.6,7.0Hz,1H),7.10(d,J=8.6H z,1H),6.99(d,J=7.1Hz,1H),6.97(d,J=5.7Hz,1H),6.67(t,J=6.3Hz,1H),5.01(dd ,J=12.9,5.5Hz,1H),3.33(q,J=6.6Hz,2H),3.08(q,J=6.1Hz,2H),2.89-2.81(m,1H ),2.59-2.53(m,1H),2.50(dd,J=13.0,4.4Hz,1H),2.01-1.94(m,1H),1.33(s,9H).

[0068] Step 2: Synthesis of 2-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}ethan-1-amine trifluoroacetate

[0069] Compound O1 was dissolved in TFA (2 mL), heated to 50°C and stirred for 30 min. The reaction was stopped, and dichloromethane (30 mL) was added and concentrated under reduced pressure to obtain the title compound P1 as a yellow oily liquid. ESI-MS (m / z): 317.25 [M+H] + . 1 H NMR(600MHz, Methanol-d4)δ7.60(t,J=7.8Hz,1H),7.16-7.09(m,2H),5.06(dd,J=11.7,4.9Hz,1H ),3.70-3.61(m,2H),3.19-3.14(m,2H),2.86-2.80(m,1H),2.76-2.67(m,2H),2.12-2.05(m,1H).

[0070] Example 3

[0071] Synthesis of 3-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}propan-1-amine trifluoroacetate (P2)

[0072]

[0073] Compound O2 was prepared as a yellow solid using the method described for compound O1 in Example 2. 186 mg, 0.43 mmol, 23.9% yield, mp: 96.3-97.0°C. ESI-MS (m / z): 453.25 [M+Na] + . 1H NMR (600 MHz, DMSO-d6) δ 11.05 (s, 1H), 7.57 - 7.50 (m, 1H), 7.05 (d, J = 8.6 Hz, 1H), 6.98 (d, J = 7.1 Hz, 1H), 6.87 (t, J = 5.8 Hz, 1H), 6.62 (t, J = 6.0 Hz, 1H), 5.01 (dd, J = 12.8, 5.4 Hz, 1H), 3.29 - 3.26 (m, 2H), 2.96 (q, J = 6.4 Hz, 2H), 2.89 - 2.80 (m, 1H), 2.59 - 2.49 (m, 2H), 2.03 - 1.95 (m, 1H), 1.62 (p, J = 6.9 Hz, 2H), 1.34 (s, 9H).

[0074] Compound P2 was prepared as a yellow oil liquid using the method described for compound P1 in Example 2. ESI-MS (m / z): 331.15 [M+H] + . 1 H NMR (600 MHz, Methanol-d4) δ 7.56 (t, J = 7.9 Hz, 1H), 7.10 - 7.04 (m, 2H), 5.04 (dd, J = 12.9, 5.1 Hz, 1H), 3.48 - 3.41 (m, 2H), 3.07 - 3.00 (m, 2H), 2.89 - 2.79 (m, 1H), 2.76 - 2.65 (m, 2H), 2.12 - 2.05 (m, 1H), 1.98 (p, J = 7.1 Hz, 2H).

[0075] Example 4

[0076] Synthesis of 4-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4- yl]amino}butan-1-amine trifluoroacetate salt (P3)

[0077]

[0078] Compound O3 was prepared as a yellow solid using the method described for compound O1 in Example 2. 335 mg, 0.75 mmol, 41.6% yield, m.p.: 86.9-88.7 °C. ESI-MS (m / z): 445.30 [M+H] + . 1HNMR(600MHz,DMSO-d6)δ11.07(s,1H),7.53(t,J=7.8Hz,1H),7.06(d,J=8.5Hz,1H),6.98 (d,J=6.9Hz,1H),6.81(t,J=5.6Hz,1H),6.52(t,J=6.0Hz,1H),5.01(dd,J=12.9,5.3Hz,1 H),3.26(q,J=6.6Hz,2H),2.91(q,J=6.5Hz,2H),2.88-2.82(m,1H),2.60-2.52(m,1H),2. 05-1.94(m,1H),1.50(p,J=7.4Hz,2H),1.44-1.38(m,2H),1.37-1.34(m,1H),1.33(s,9H).

[0079] Compound P3 was prepared as a yellow oily liquid using the method described for compound P1 in Example 2. ESI-MS (m / z): 345.25 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.53(t,J=7.8Hz,1H),7.04(t,J=8.7Hz,2H),5.03(dd,J=12.9,5.3Hz,1H),3.44 -3.33(m,2H),2.99-2.92(m,2H),2.90-2.79(m,1H),2.76-2.64(m,2H),2.13-2.04(m,1H),1.80-1.66(m,4H).

[0080] Example 5

[0081] Synthesis of 5-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}pentan-1-amine trifluoroacetate (P4)

[0082]

[0083] Compound O4 was prepared as a yellow solid using the method described for compound O1 in Example 2. 352 mg, 0.77 mmol, 42.4% yield, mp: 75.9-76.7°C. ESI-MS (m / z): 481.25 [M+Na] + . 1HNMR(600MHz,DMSO-d6)δ11.04(s,1H),7.59-7.49(m,1H),7.05(d,J=8.6Hz,1H),6.98(d,J= 7.0Hz,1H),6.73(t,J=5.7Hz,1H),6.49(d,J=6.0Hz,1H),5.01(dd,J=12.9,5.5Hz,1H),3.24( q,J=6.8Hz,2H),2.92-2.86(m,2H),2.86-2.81(m,1H),2.58-2.53(m,1H),2.50-2.47(m,1H), 2.02-1.96(m,1H),1.53(p,J=7.3Hz,2H),1.40-1.35(m,2H),1.32(s,9H),1.31-1.26(m,2H).

[0084] Compound P4 was prepared as a yellow oily liquid using the method described for compound P1 in Example 2. ESI-MS (m / z): 359.15 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ7.57-7.52(m,1H),7.06(d,J=8.7Hz,1H),6.99(d,J=7.0Hz, 1H), 6.50 (t, J=5.9Hz, 1H), 5.01 (dd, J=12.9, 5.5Hz, 1H), 3.26 (q, J=6.8Hz, 2H), 2.8 8-2.81(m,1H),2.61(q,J=6.6,6.1Hz,2H),2.58-2.53(m,1H),2.03-1.92(m,2H),1. 54(p,J=7.4Hz,2H), 1.43(q,J=7.5Hz,2H), 1.36-1.32(m,2H), 1.19(t,J=2.3Hz,2H).

[0085] Example 6

[0086] Synthesis of 6-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}hexane-1-amine trifluoroacetate (P5)

[0087]

[0088] Yellow solid compound O5 was prepared using the method described for compound O1 in Example 2. 293 mg, 0.62 mmol, 34.3% yield, ESI-MS (m / z): 473.50 [M+H] + . 1H NMR (600 MHz, DMSO-d6) δ 11.08 (s, 1H), 7.54 (t, J = 7.9 Hz, 1H), 7.05 (d, J = 8.7 Hz, 1H), 6.98 (d, J = 7.1 Hz, 1H), 6.75 (d, J = 5.8 Hz, 1H), 6.50 (t, J = 5.8 Hz, 1H), 5.01 (dd, J = 12.9, 5.3 Hz, 1H), 3.24 (q, J = 6.8 Hz, 2H), 2.86 (q, J = 6.5 Hz, 3H), 2.57 - 2.53 (m, 1H), 2.49 (d, J = 3.3 Hz, 1H), 2.02 - 1.95 (m, 1H), 1.52 (p, J = 7.6, 7.1 Hz, 2H), 1.32 (s, 11H), 1.31 - 1.26 (m, 2H), 1.26 - 1.21 (m, 2H).

[0089] Compound P5 was prepared as a yellow oil liquid using the method described for compound P1 in Example 2. ESI-MS (m / z): 373.30 [M+H] + . 1 H NMR (600 MHz, Methanol-d4) δ 7.53 (t, J = 7.8 Hz, 1H), 7.06 - 6.98 (m, 2H), 5.03 (dd, J = 12.8, 5.3 Hz, 1H), 3.36 - 3.31 (m, 2H), 2.90 (t, J = 7.7 Hz, 2H), 2.85 - 2.80 (m, 1H), 2.76 - 2.70 (m, 2H), 2.12 - 2.05 (m, 1H), 1.72 - 1.61 (m, 4H), 1.51 - 1.41 (m, 4H).

[0090] Example 7

[0091] 7-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]amino}heptan-1-amine trifluoroacetate salt (P6)

[0092]

[0093] Compound O6 was prepared as a yellow solid using the method described for compound O1 in Example 2. 276 mg, 0.57 mmol, 31.3% yield, ESI-MS (m / z): 487.30 [M+H] + ,509.25 [M+Na] + . 1H NMR (600MHz, DMSO-d6) δ11.07(s,1H),7.54(t,J=7.8Hz,1H),7.05(d,J=8.6Hz,1 H),6.98(d,J=7.0Hz,1H),6.73(t,J=5.6Hz,1H),6.49(t,J=5.9Hz,1H),5.01(dd, J=12.8,5.4Hz,1H),3.25(q,J=6.8Hz,2H),2.87-2.80(m,4H),2.59-2.53(m,1H) ,2.02-1.96(m,1H),1.52(p,J=7.5,7.0Hz,2H),1.32(s,13H),1.29-1.23(m,4H).

[0094] Compound P6 was prepared as a yellow oily liquid using the method described for compound P1 in Example 2. ESI-MS (m / z): 387.25 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.52(t,J=7.8Hz,1H),7.01(d,J=7.8Hz,2H),5.03(dd,J=12.9,5.3Hz,1H),3.33-3.29(m,2H),2.91 -2.86(m,2H),2.85-2.79(m,1H),2.75-2.67(m,2H),2.11-2.05(m,1H),1.68-1.60(m,4H),1.47-1.42(m,2H),1.42-1.37(m,4H).

[0095] Example 8

[0096] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({2-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}ethyl)benzamide (CP1)

[0097]

[0098] To a solution of C9S (571 mg, 0.42 mmol), P1 (181 mg, 0.42 mmol), and triethylamine (292 μL, 2.1 mmol) in DMSO (2 mL) was added HBTU (159 mg, 0.42 mmol) with stirring. The mixture was allowed to react overnight at rt, diluted with water (20 mL), extracted with ethyl acetate (75 mL × 3), washed with brine (10 mL × 2), dried over anhydrous sodium sulfate, and concentrated under reduced pressure. Purification was performed by silica gel column chromatography (dichloromethane:methanol 15:1). The title compound CP1 was obtained as a yellow solid (35 mg, 0.05 mmol, 12.8% yield), mp: 223.8-224.4°C. HPLC: 95.932%. HRMS (ESI) m / z: [M+H] + calcd for C 30 H 28 N9O7S:658.18324,found:658.18335. 1 H NMR (600MHz, DMSO-d6) δ12.79(s,1H),11.07(s,1H),8.92(t,J=5.2Hz,1H),8.61(s,1H),8.26(d,J=3.8Hz, 2H),7.80(d,J=7.7Hz,1H),7.71(s,1H),7.62(t,J=8.1Hz,1H),7.54(t,J=7.8Hz,1H),7.26(s,1H),7.23(d ,J=8.5Hz,1H),6.99(d,J=7.0Hz,1H),6.84(t,J=5.6Hz,1H),5.01(dd,J=13.0,5.4Hz,1H),4.49(q,J=7.2H z,1H),3.54-3.43(m,4H),2.88-2.79(m,1H),2.58-2.52(m,1H),2.01-1.93(m,1H),1.54(d,J=7.0Hz,3H). 13 C NMR (151MHz, DMSO-d6) δ173.34,172.81,170.63,169.26,167.84,166.85,162.85,161.29,152.20,146.88,138.95,136.94,136. 76,135.89,132.79,129.98,125.71,124.01,120.37,117.71,111.13,109.88,105.48,49.07,45.17,41.78,31.51,22.69,19.02.

[0099] Example 9

[0100] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({3-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}propyl)benzamide (CP2)

[0101]

[0102] The title compound CP2 was prepared using the method described for compound CP1 in Example 8 as a yellow solid, 46 mg, 0.07 mmol, 16.1% yield, mp: 209.9-210.0°C. HPLC: 97.911%. HRMS (ESI) m / z: [M+H] + calcd for C 31 H 30 N9O7S:672.19889,found:672.19910. 1 H NMR (600MHz, DMSO-d6) δ11.07(s,1H),8.79(d,J=5.8Hz,1H),8.63(s,1H),8.29(d,J=8.1Hz,1H),8.10(s,1H),7. 77(d,J=7.4Hz,2H),7.59(t,J=7.9Hz,1H),7.54(t,J=7.9Hz,1H),7.13(s,1H),7.08(d,J=8.6Hz,1H),6.98(d,J=6 .9Hz,1H),6.74(t,J=6.0Hz,1H),5.01(dd,J=12.9,5.4Hz,1H),4.44(q,J=7.2Hz,1H),3.37-3.34(m,4H),2.89-2. 78(m,1H),2.59-2.51(m,1H),2.50-2.47(m,1H),2.02-1.95(m,1H),1.79(p,J=6.7Hz,2H),1.47(d,J=7.0Hz,3H). 13C NMR(151MHz,DMSO-d6)δ173.37,172.82,170.66,169.38,167.85,166.51,161.19,157.96,152.15,146.80,138.92,136.92,136.77, 136.10,132.80,129.93,125.72,123.92,120.38,117.66,110.95,109.75,105.47,49.09,45.23,37.43,31.53,29.15,22.71,19.02.

[0103] Example 10

[0104] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({4-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}butyl)benzamide (CP3)

[0105]

[0106] The title compound CP3 was prepared using the method described for compound CP1 in Example 8 as a yellow solid, 67 mg, 0.10 mmol, 23.3% yield, mp: 206.9-207.5°C. HPLC: 99.951%. HRMS (ESI) m / z: [M+H] + calcd for C 32 H 32 N9O7S:686.2145,found:686.2137;[M+Na] + calcd for C 32 H 31 N9NaO7S:708.1965,found:708.1982. 1H NMR (600MHz, DMSO-d6) δ12.77(s,1H),11.06(s,1H),8.71(t,J=5.6Hz,1H),8.62(s,1H),8.25(d,J=2.0Hz,1H),8. 23(d,J=8.0Hz,1H),7.81(d,J=7.7Hz,1H),7.70(s,1H),7.61(t,J=8.2Hz,1H),7.52(t,J=7.8Hz,1H),7.26(s,1H) ,7.08(d,J=8.5Hz,1H),6.97(d,J=6.9Hz,1H),6.55(t,J=5.9Hz,1H),5.01(dd,J=13.2,5.4Hz,1H),4.51(q,J=7.2 Hz,1H),3.31(s,4H),2.89-2.79(m,1H),2.58-2.52(m,1H),2.03-1.91(m,2H),1.59(s,4H),1.53(d,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ173.38,172.82,170.66,169.47,167.84,166.27,161.16,157.98,152.14,146.93,138.91,136.91,136.78,136 .16,132.72,129.92,125.69,123.88,120.39,117.72,110.93,109.57,105.45,49.08,45.24,42.09,31.51,26.96,26.79,22.69,19.07.

[0107] Example 11

[0108] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({5-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}pentyl)benzamide (CP4)

[0109]

[0110] The title compound CP4 was prepared using the method described for compound CP1 in Example 8 as a yellow solid, 40 mg, 0.06 mmol, 13.6% yield, mp: 187.9-188.5°C. HPLC: 99.764%. HRMS (ESI) m / z: [M+H] + calcd for C33 H 34 N9O7S:700.23019,found:700.23016. 1 H NMR (600MHz, DMSO-d6) δ11.08(s,1H),8.76-8.70(m,1H),8.64(s,1H),8.25(d,J=8.1Hz,1H),8.15(s,1H),7.7 7(d,J=5.9Hz,2H),7.59(t,J=7.9Hz,1H),7.53(t,J=7.8Hz,1H),7.20(s,1H),7.06(d,J=8.6Hz,1H),6.97(d,J= 6.9Hz,1H),6.52(t,J=5.8Hz,1H),5.01(dd,J=12.9,5.5Hz,1H),4.56-4.43(m,1H),3.29-3.23(m,5H),2.89-2 .80(m,1H),2.59-2.51(m,1H),2.02-1.94(m,1H),1.61-1.53(m,4H),1.49(d,J=7.1Hz,3H),1.40-1.35(m,2H). 13 C NMR(151MHz,DMSO-d6)δ173.36,172.80,170.65,169.49,167.84,166.20,161.13,157.92,152.12,146.95,138.90,136.91,136.79,136.20 ,132.70,129.89,125.69,123.82,120.38,117.69,110.92,109.55,10 5.45,49.08,45.27,42.34,31.52,29.28,28.98,24.35,22.69,19.12.

[0111] Example 12

[0112] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({6-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}hexyl)benzamide (CP5)

[0113]

[0114] The title compound CP5 was prepared using the method described for compound CP1 in Example 8 as a yellow solid, 202 mg, 0.28 mmol, 67.4% yield, mp: 164.9-165.9°C. HPLC: 98.260%. HRMS (ESI) m / z: [M+H] + calcd for C 34 H 36 N9O7S:714.24584,found:714.24506. 1 H NMR (600MHz, DMSO-d6) δ11.08(s,1H),8.68(d,J=6.7Hz,1H),8.63(s,1H),8.25(s,1H),8.22(d,J=8.0Hz, 1H),7.81(d,J=7.7Hz,1H),7.71(s,1H),7.61(t,J=7.9Hz,1H),7.52(t,J=7.9Hz,1H),7.28(s,1H),7.04( d,J=8.6Hz,1H),6.97(d,J=7.0Hz,1H),6.51(s,1H),5.01(dd,J=13.2,5.6Hz,1H),4.57-4.46(m,1H),3.2 8-3.22(m,5H),2.89-2.80(m,1H),2.59-2.52(m,1H),2.03-1.95(m,1H),1.57-1.48(m,7H),1.34(s,4H). 13 C NMR(151MHz,DMSO-d6)δ173.25,172.70,170.55,169.43,167.76,166.10,161.05,157.83,152.05,146.89,138.83,136.82,136.70,136.17,13 2.65,129.81,125.61,123.75,120.33,117.58,110.83,109.51,105.37 ,49.03,45.22,42.29,31.46,29.44,29.13,26.69,26.56,22.64,19.06.

[0115] Example 13

[0116] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({7-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-4-yl]amino}heptyl)benzamide (CP6)

[0117]

[0118] The title compound CP6 was prepared using the method described for compound CP1 in Example 8 as a yellow solid, 124 mg, 0.17 mmol, 40.5% yield, mp: 158.7-159.1°C. HPLC: 98.465%. HRMS (ESI) m / z: [M+H] + calcd for C 35 H 38 N9O7S:728.26149,found:728.26123. 1 H NMR (600MHz, DMSO-d6) δ12.78(s,1H),11.07(s,1H),8.67(d,J=6.7Hz,1H),8.62(s,1H),8.25(s,1H),8.21(d,J=8 .0Hz,1H),7.80(d,J=7.7Hz,1H),7.71(s,1H),7.61(t,J=8.0Hz,1H),7.52(t,J=7.8Hz,1H),7.28(s,1H),7.04(d,J =8.7Hz,1H),6.97(d,J=7.1Hz,1H),6.50(s,1H),5.01(dd,J=13.1,5.3Hz,1H),4.51(q,J=7.2Hz,1H),3.29-3.19(m ,5H),2.89-2.80(m,1H),2.60-2.51(m,1H),2.02-1.95(m,1H),1.57-1.52(m,5H),1.51-1.47(m,2H),1.31(s,6H). 13 C NMR(151MHz,DMSO-d6)δ173.26,172.70,170.55,169.42,167.76,166.08 ,161.04,157.83,152.05,146.89,138.83,136.82,136.70,136.17,132.6 4,129.81,125.60,123.73,120.32,117.58,110.82,109.48,105.37,49.0 3,45.22,42.32,31.46,29.45,29.14,28.97,26.93,26.78,22.63,19.07.

[0119] Example 14

[0120] Synthesis of 3-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}propan-1-amine trifluoroacetate (L2)

[0121]

[0122] Step 1: Synthesis of tert-butyl 3-({[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}propyl)carbamate (K2)

[0123] To a stirred solution of lenalidomide (771 mg, 2.97 mmol) in NMP (4 mL) were added tert-butyl N-(3-bromopropyl)carbamate (848 mg, 3.56 mmol) and DIPEA (2590 μL, 14.9 mmol). The mixture was stirred at 110°C for 12 h, diluted with water (30 mL), extracted with ethyl acetate (75 mL × 3), and washed with saturated brine (20 mL × 5). The mixture was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by silica gel column chromatography (dichloromethane:methanol 200:3). Compound K2 was obtained as a yellow solid, 176 mg, 0.42 mmol, with a yield of 14.2%. ESI-MS (m / z): 417.25 [M+H]. + ,439.20[M+Na] + . 1 H NMR (600MHz, DMSO-d6) δ10.98(s,1H),7.24(t,J=7.7Hz,1H),6.89(d,J=7.3Hz,1H),6.86(d,J=5. 9Hz,1H),6.70(d,J=8.0Hz,1H),5.54(t,J=5.5Hz,1H),5.08(dd,J=13.4,4.9Hz,1H),4.19(d,J=1 7.0Hz,1H),4.09(d,J=17.1Hz,1H),3.09(q,J=6.6Hz,2H),2.98(q,J=6.6Hz,2H),2.94-2.84(m,1 H),2.62-2.55(m,1H),2.32-2.21(m,1H),2.03-1.97(m,1H),1.65(p,J=7.2Hz,2H),1.34(s,9H).

[0124] Step 2: Synthesis of 3-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}propan-1-amine trifluoroacetate (L2)

[0125] K2 was dissolved in dichloromethane (6 mL), ice-bathed for 30 min, trifluoroacetic acid (3 mL) was added dropwise, the ice-bath was removed, stirred at rt for 2 h, and concentrated under reduced pressure to obtain compound L2 as a yellow oily liquid. ESI-MS (m / z): 317.25 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.33(t,J=7.8Hz,1H),7.09(d,J=7.4Hz,1H),6.86(d,J=8.0Hz,1H),5.14(dd,J=13.4,5.0Hz,1H),4.26(q,J=16.8 Hz,2H),3.39-3.31(m,2H),3.09-3.01(m,2H),2.95-2.84(m,1H),2.81 -2.72(m,1H),2.49-2.38(m,1H),2.20-2.11(m,1H),2.04-1.94(m,2H).

[0126] Example 15

[0127] Synthesis of 4-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}butan-1-amine trifluoroacetate (L3)

[0128]

[0129] Yellow solid compound K3.76 mg, 0.18 mmol, 5.9% yield, ESI-MS (m / z): 431.30 [M+H] + ,453.25[M+Na] + . 1 H NMR (400MHz, DMSO-d6) δ11.03(s,1H),7.28(t,J=7.7Hz,1H),6.93(d,J=7.4Hz,1H),6.83(t,J=5.6H z,1H),6.75(d,J=8.1Hz,1H),5.60(t,J=5.6Hz,1H),5.12(dd,J=13.3,5.1Hz,1H),4.23(d,J=17.1H z,1H),4.12(d,J=17.1Hz,1H),3.12(q,J=6.4Hz,2H),3.00-2.93(m,2H),2.932.86(m,1H),2.672.5 7(m,1H),2.36-2.22(m,1H),2.09-1.98(m,1H),1.61-1.52(m,2H),1.51-1.42(m,2H),1.37(s,9H).

[0130] Compound L3 was prepared as a yellow oily liquid using the method described for compound L2 in Example 14. ESI-MS (m / z): 331.15 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.31(t,J=7.9Hz,1H),7.07(d,J=7.4Hz,1H),6.83(d,J=8.0Hz,1H),5.14(dd,J=13.4,5.2Hz,1H),4.31-4.18(m, 2H),3.28-3.26(m,2H),2.94(t,J=7.3Hz,2H),2.92-2.87(m,1H),2.81 -2.73(m,1H),2.48-2.39(m,1H),2.19-2.11(m,1H),1.81-1.68(m,4H).

[0131] Example 16

[0132] Synthesis of 5-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}pentan-1-amine trifluoroacetate (L4)

[0133]

[0134] Yellow solid compound K4 was prepared using the method described for compound K2 in Example 14. 345 mg, 0.78 mmol, yield 26.1%, ESI-MS (m / z): 467.30 [M+Na] + . 1 H NMR (600MHz, DMSO-d6) δ10.98(s,1H),7.24(t,J=7.6Hz,1H),6.88(d,J=7.5Hz,1H),6.75(t,J=5.9Hz ,1H),6.70(d,J=7.9Hz,1H),5.53(t,J=5.2Hz,1H),5.08(dd,J=13.4,5.0Hz,1H),4.19(d,J=17.0Hz, 1H),4.09(d,J=17.0Hz,1H),3.06(q,J=6.4Hz,2H),2.88(q,J=6.3Hz,3H),2.63-2.55(m,1H),2.32-2 .21(m,1H),2.04-1.97(m,1H),1.58-1.47(m,2H),1.40-1.35(m,2H),1.33(s,9H),1.31-1.27(m,2H).

[0135] Compound L4 was prepared as a yellow oily liquid using the method described for compound L2 in Example 14. ESI-MS (m / z): 345.30 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.30(t,J=7.7Hz,1H),7.05(d,J=7.4Hz,1H),6.80(d,J=8.0Hz,1H),5.12(dd,J=13.6,5.0Hz,1H),4.25(q,J=16.8Hz,2H),3.24 -3.19(m,2H),2.91(t,J=7.6Hz,2H),2.88-2.83(m,1H),2.79-2.72(m,1H),2. 47-2.37(m,1H),2.17-2.11(m,1H),1.72-1.64(m,4H),1.49(p,J=7.7Hz,2H).

[0136] Example 17

[0137] Synthesis of 6-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}hexane-1-amine trifluoroacetate (L5)

[0138]

[0139] Yellow solid compound K5 was prepared using the method described for compound K2 in Example 14. 378 mg, 0.82 mmol, yield 27.7%, ESI-MS (m / z): 459.40 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.98(s,1H),7.24(t,J=7.8Hz,1H),6.88(d,J=7.3Hz,1H),6.75( d,J=5.9Hz,1H),6.70(d,J=8.0Hz,1H),5.53(t,J=5.6Hz,1H),5.08(dd,J=13.4,5.0Hz,1H) ,4.19(d,J=17.1Hz,1H),4.09(d,J=17.1Hz,1H),3.06(q,J=6.5Hz,2H),2.93-2.84(m,3H), 2.64-2.55(m,1H),2.33-2.22(m,1H),2.04-1.95(m,1H),1.59-1.49(m,2H),1.33(s,15H).

[0140] Compound L5 was prepared as a yellow oil liquid using the method described for compound L2 in Example 14. ESI-MS (m / z): 359.30 [M+H] + . 1 H NMR (600 MHz, Methanol-d4) δ 7.30 (t, J = 7.9 Hz, 1H), 7.05 (d, J = 7.5 Hz, 1H), 6.80 (d, J = 8.0 Hz, 1H), 5.13 (dd, J = 13.2, 4.9 Hz, 1H), 4.25 (q, J = 16.8 Hz, 2H), 3.25 - 3.19 (m, 2H), 2.92 - 2.85 (m, 3H), 2.80 - 2.76 (m, 1H), 2.49 - 2.41 (m, 1H), 2.18 - 2.13 (m, 1H), 1.71 - 1.61 (m, 4H), 1.51 - 1.39 (m, 4H).

[0141] Example 18

[0142] 7-{[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl]amino}heptan-1-amine trifluoroacetic acid salt (L6)

[0143]

[0144] Compound K6 was prepared as a yellow solid using the method described for compound K2 in Example 14. 428 mg, 0.91 mmol, 30.5% yield, ESI-MS (m / z): 473.35 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 7.24 (t, J = 7.7 Hz, 1H), 6.88 (d, J = 7.4 Hz, 1H), 6.74 (t, J = 5.6 Hz, 1H), 6.69 (d, J = 8.1 Hz, 1H), 5.54 (d, J = 5.5 Hz, 1H), 5.08 (dd, J = 13.3, 5.0 Hz, 1H), 4.18 (d, J = 17.1 Hz, 1H), 4.08 (d, J = 17.1 Hz, 1H), 3.06 (q, J = 6.5 Hz, 2H), 2.93 - 2.88 (m, 1H), 2.85 (q, J = 6.5 Hz, 2H), 2.61 - 2.55 (m, 1H), 2.30 - 2.21 (m, 1H), 2.02 - 1.96 (m, 1H), 1.53 (p, J = 7.1 Hz, 2H), 1.32 (s, 13H), 1.28 - 1.23 (m, 2H), 1.23 - 1.18 (m, 2H).

[0145] Compound L6 was prepared as a yellow oily liquid using the method described for compound L2 in Example 14. ESI-MS (m / z): 373.35 [M+H] + . 1 H NMR (600MHz, Methanol-d4) δ7.30(t,J=7.8Hz,1H),7.06(d,J=7.5Hz,1H),6.81(d,J=8.1Hz,1H),5.13(dd,J=13.4,5.0Hz,1H),4.31-4.19(m,2H),3.2 5-3.19(m,2H),2.91-2.85(m,3H),2.80-2.75(m,1H),2.50-2.38(m,1H),2 .19-2.12(m,1H),1.71-1.59(m,4H),1.47-1.42(m,2H),1.41-1.36(m,4H).

[0146] Example 19

[0147] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({3-[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}propyl)benzamide (CL2)

[0148]

[0149] The title compound CL2 was prepared using the method described for compound CP1 in Example 8 as a white solid, 23 mg, 0.03 mmol, 8.33% yield, mp: 158.7-159.1°C. HPLC: 95.715%. HRMS (ESI) m / z: [M+H] + calcd for C 31 H 32 N9O6S:658.21963,found:658.21930. 1H NMR (600MHz, DMSO-d6) δ12.79(s,1H),10.99(s,1H),8.76(d,J=5.6Hz,1H),8.61(s,1H),8.24(d,J=14.4Hz,2H),7.81(d,J=7.7Hz,1H ),7.72(s,1H),7.62(t,J=7.9Hz,1H),7.25(dd,J=15.6,8.0Hz,2H),6.90(d,J=7.4Hz,1H),6.74(d,J=8.1Hz,1H),5.62(t,J=5.5Hz,1H ),5.07(dd,J=13.4,5.1Hz,1H),4.52-4.48(m,1H),4.19(d,J=17.0Hz,1H),4.10(d,J=17.1Hz,1H),3.38(q,J=6.6Hz,2H),3.18(q,J= 6.6Hz,2H),2.93-2.83(m,1H),2.60-2.54(m,1H),2.31-2.21(m,1H),2.02-1.93(m,1H),1.83(p,J=6.8Hz,2H),1.53(d,J=7.1Hz,3H). 13 C NMR(151MHz,DMSO-d6)δ173.45,172.79,171.79,169.44,166.43,161.12,157.89,152.13,144.15,138.91,136.94,136.16,132.62, 129.95,129.78,127.14,125.73,123.93,120.41,112.41,110.66,105.46,52.06,46.23,45.26,40.99,31.77,28.91,23.33,19.06.

[0150] Example 20

[0151] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({4-[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}butyl)benzamide (CL3)

[0152]

[0153] The title compound CL3 was prepared as a white solid, 62 mg, 0.09 mmol, yield 22.0%, m.p.: 206.4-207.1 °C. HPLC: 95.747%. HRMS (ESI) m / z: [M+H] + calcd for C 32 H 34 N9O6S: 672.23528, found: 672.23462. 1 H NMR (600 MHz, DMSO-d6) δ 10.99 (s, 1H), 8.71 (t, J = 5.2 Hz, 1H), 8.62 (s, 1H), 8.26 (s, 1H), 8.23 (d, J = 7.8 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.61 (t, J = 7.8 Hz, 1H), 7.28 (s, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.88 (d, J = 7.3 Hz, 1H), 6.72 (d, J = 8.0 Hz, 1H), 5.62-5.56 (m, 1H), 5.07 (dd, J = 13.6, 5.0 Hz, 1H), 4.50 (q, J = 6.9 Hz, 1H), 4.18 (d, J = 17.1 Hz, 1H), 4.08 (d, J = 17.1 Hz, 1H), 3.31-3.26 (m, 2H), 3.17-3.10 (m, 2H), 2.922.83 (m, 1H), 2.61-2.53 (m, 1H), 2.30-2.20 (m, 1H), 2.02-1.95 (m, 1H), 1.61 (s, 4H), 1.53 (d, J = 6.9 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 173.46, 172.82, 171.80, 169.48, 166.25, 161.11, 157.91, 152.12, 144.26, 138.90, 136.92, 136.20, 132.58, 129.92, 129.75, 127.03, 125.70, 123.89, 120.40, 112.31, 110.51, 105.45, 52.04, 46.28, 45.26, 42.96, 31.77, 27.25, 26.50, 23.35, 19.06.

[0154] Example 21

[0155] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({5-[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}pentyl)benzamide (CL4)

[0156]

[0157] The title compound CL4 was prepared using the method described for compound CP1 in Example 8 as a white solid, 49 mg, 0.07 mmol, 17.0% yield, mp: 249.6-250.3°C. HPLC: 96.664%. HRMS (ESI) m / z: [M+H] + calcd for C 33 H 36 N9O6S:686.25093,found:686.25018. 1 H NMR (600MHz, DMSO-d6) δ10.99(s,1H),8.70(d,J=5.7Hz,1H),8.62(s,1H),8.25(s,1H),8.22(d,J=8.2Hz,1H),7.81(d,J=7.7Hz,1H),7.71(s ,1H),7.61(t,J=7.9Hz,1H),7.28(s,1H),7.24(t,J=7.7Hz,1H),6.88(d,J=7.4Hz,1H),6.71(d,J=8.0Hz,1H),5.55(t,J=5.4Hz,1H),5.07(d d,J=13.4,5.1Hz,1H),4.51(q,J=7.1Hz,1H),4.18(d,J=17.1Hz,1H),4.08(d,J=17.0Hz,1H),3.27(q,J=7.0Hz,2H),3.09(q,J=6.5Hz,2H),2 .93-2.82(m,1H),2.62-2.54(m,1H),2.29-2.19(m,1H),2.03-1.93(m,1H),1.62-1.55(m,4H),1.54(d,J=7.0Hz,3H),1.39(p,J=7.7Hz,2H). 13C NMR(151MHz,DMSO-d6)δ173.47,172.82,171.80,169.49,166.23,161.09,157.89,152.11,144.31,138.89,136.91,136.23,132.56,129.91 ,129.76,126.99,125.71,123.86,120.40,112.27,110.48,105.45,5 2.05,46.29,45.27,43.24,31.77,29.44,28.81,24.67,23.34,19.09.

[0158] Example 22

[0159] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({6-[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}hexyl)benzamide (CL5)

[0160]

[0161] The title compound CL5 was prepared using the method described for compound CP1 in Example 8 as a white solid, 27 mg, 0.04 mmol, 9.19% yield, mp: 259.4-259.7°C. HPLC: 98.693%. HRMS (ESI) m / z: [M+H] + calcd for C 34 H 38 N9O6S:700.26658,found:700.26599. 1H NMR (600MHz, DMSO-d6) δ12.79(s,1H),10.99(s,1H),8.69(d,J=5.6Hz,1H),8.64-8.60(m,1H),8.26(d,J=1.9Hz,1H),8.22(d,J=8.0Hz,1H), 7.81(d,J=7.7Hz,1H),7.72(s,1H),7.61(t,J=8.1Hz,1H),7.29(s,1H),7.23(t,J=7.8Hz,1H),6.87(d,J=7.3Hz,1H),6.69(d,J=8.0Hz,1H),5 .54(t,J=5.3Hz,1H),5.07(dd,J=13.5,5.0Hz,1H),4.51(q,J=6.8Hz,1H),4.18(d,J=17.1Hz,1H),4.08(d,J=17.1Hz,1H),3.26(q,J=6.6Hz,2 H),3.07(q,J=6.6Hz,2H),2.93-2.84(m,1H),2.61-2.54(m,1H),2.30- 2.21(m,1H),2.03-1.96(m,1H),1.60-1.48(m,7H),1.41-1.29(m,4H). 13 C NMR(151MHz,DMSO-d6)δ173.45,172.74,171.81,169.45,166.15,161.10,157.87,152.12,144.31,138.89,136.94,136.26,132.58,129.92,12 9.75,127.00,125.71,123.86,120.41,112.24,110.43,105.44,52.02, 46.27,45.28,43.22,31.77,29.59,29.04,26.93,26.91,23.36,19.13.

[0162] Example 23

[0163] Synthesis of 3-{6-[(1-amino-1-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}-N-({7-[2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindol-4-yl]amino}heptyl)benzamide (CL6)

[0164]

[0165] The title compound CL6 was prepared using the method described for compound CP1 in Example 8 as a white solid, 35 mg, 0.05 mmol, 11.7% yield, mp: 266.5-267.0°C. HPLC: 97.859%. HRMS (ESI) m / z: [M+H] + calcd for C 35 H 40 N9O6S:714.28223,found:714.28131. 1 H NMR(400MHz,DMSO-d6)δ12.82(s,1H),11.03(s,1H),8.89-8.54(m,2H),8.43-8.13(m,2H),7.92-7.58( m,3H),7.41-7.17(m,2H),6.99-6.86(m,1H),6.73(d,J=8.0Hz,1H),5.69-5.47(m,1H),5.24-5.02(m,1 H),4.56(q,J=15.2,12.6Hz,1H),4.31-4.03(m,2H),3.29-3.23(m,2H),3.16-3.06(m,2H),2.95-2.85( m,1H),2.71-2.59(m,1H),2.37-2.22(m,1H),2.10-1.96(m,1H),1.68-1.46(m,7H),1.44-1.26(m,6H). 13 C NMR(151MHz,DMSO-d6)δ173.46,172.79,171.81,169.47,166.16,161.10 ,157.89,152.12,144.31,138.89,136.92,136.24,132.56,129.92,129.7 4,126.99,125.69,123.85,120.40,112.24,110.43,105.44,52.04,46.2 8,45.28,43.27,31.77,29.56,29.21,29.06,27.16,27.06,23.35,19.11.

[0166] Example 24

[0167] MG1 and MG2 are prepared by the following process flow chart:

[0168]

[0169] Example 25: Degradation of DAPK1 protein in HEK293T cells by the compound

[0170] Total protein was extracted from HEK293T cells using RIPA lysis buffer containing protease and phosphatase inhibitors. The protein concentration was then determined using BCA protein assay reagent. Protein samples (15-30 μg) were separated by SDS / PAGE and transferred to a PVDF membrane by semi-dry transfer. Non-specific sites were then blocked with 5% milk-TBST or 5% BSA-TBST at room temperature for 1 hour, and the primary antibody was added to the sample and incubated overnight at 4°C. The membrane was washed three times with TBST to remove additional primary antibodies, and then HRP-conjugated secondary antibodies were added, and the samples were incubated at room temperature for 1 hour. All membranes were then coated with ECL chemiluminescent enzyme-linked substrate and imaged using an imaging system. To determine whether the target compound has a degradation effect on DAPK1 protein, the HEK293T cell line was used as the experimental subject to test the degradation of DAPK1 by the compound. The degradation results are shown in the figure. Figure 1 As shown in Figure 3, after 24 h of treatment at a concentration of 5 μM, CL2 and CP1 were able to significantly degrade DAPK1 protein.

[0171] CL2 and CP1 with better activity were selected to test the degradation effect of the compounds on DAPK1 with increasing concentrations over a 24-h period. The results are as follows: Figure 2 As shown. CL2 and CP1 significantly downregulated DAPK1 protein levels after 24 hours at a concentration of 5μM, and the degradation effect increased with increasing concentration. When the concentration reached 10μM, DAPK1 protein was almost completely degraded. We then selected CL2 and continued to test the degradation effect of DAPK1 protein at different exposure times at a concentration of 10μM. The results are shown in Figure 2. Figure 3 As shown in Figure 2, CL2 at a concentration of 10 μM significantly reduced DAPK1 protein levels after 6 hours of treatment, reaching maximum degradation at 12 hours. After 12 hours, the degradation effect no longer varied with the duration of treatment. This may be due to the compensatory synthesis of new DAPK1 protein in the cells following the decrease in DAPK1 protein levels.

[0172] Example 26: Degradation of DAPK1 protein in N2a cells by the compound

[0173] Total protein was extracted from N2a cells using RIPA lysis buffer containing protease and phosphatase inhibitors. Protein concentration was then determined using BCA protein assay reagent. Protein samples (15-30 μg) were separated by SDS / PAGE and transferred to a PVDF membrane by semi-dry transfer. Non-specific sites were then blocked with 5% milk-TBST or 5% BSA-TBST at room temperature for 1 hour, and the primary antibody was added to the sample and then incubated overnight at 4°C. The membrane was washed three times with TBST to remove additional primary antibody, and then HRP-conjugated secondary antibody was added, and the sample was incubated at room temperature for 1 hour. All membranes were then coated with ECL chemiluminescent enzyme-linked substrate and imaged using an imaging system, see. Figure 4 shown.

[0174] Example 27 Effect of CP1 on tau protein in the CA3 region of the mouse hippocampus

[0175] Two-month-old male mice were purchased from the Shanghai Wu Animal Experimental Center and maintained under a 12-h light cycle in a standard specific pathogen-free area of ​​the Experimental Animal Center of Fujian Medical University with free access to food and water.

[0176] Stereotaxic surgery: AAV-CMV-Tau(P301L)-EGFP-WPRE, AAV-CMV-EGFP-WPRE was purchased from Wuhan Shumi Brain Science Technology Co., Ltd. Mice were anesthetized by isoflurane inhalation and fixed in a stereotaxic apparatus. After disinfection with iodine, the skin was incised along the anterior-posterior axis to expose the skull. Injection holes were made 2.2 mm posterior, 2.7 mm right, and 2.3 mm inferior to the bregma using a handheld drill. Tau-P301L virus or vector virus was injected into the CA3 region at a rate of 125 nL / min using a microinjection pump. The needle remained in place for 5 minutes before being withdrawn. The mouse was then sutured and placed on a heating pad for recovery.

[0177] Immunohistofluorescence: Paraffin-embedded mouse brain tissue specimens were used to prepare 5 μm coronal sections and perform immunohistofluorescence staining. After deparaffinization, rehydration, and antigen retrieval, sections were blocked with goat serum for 1 hour at room temperature to remove nonspecific sites. Primary antibodies were applied to the sections and incubated overnight at 4°C. Sections were washed three times with PBST, and Alexa Fluor 488- or 546-conjugated secondary antibodies were added to the sections in the dark and incubated for 1 hour at room temperature. After washing three times with PBST, sections were stained with Hoechst 33342 for 10 minutes and washed three times with PBST. All samples were mounted with antifade reagent and observed and photographed under a fluorescence microscope.

[0178] To investigate the effect of CP1 on Tau pathology in the hippocampus of mice, we examined the levels of total hTau protein and phosphorylation sites of Tau protein associated with Alzheimer's disease. First, we used HT7 antibody to detect the levels of total hTau protein in the brain tissues of the three groups of mice by immunofluorescence. Figure 5 , we found that compared with the AAV-eGFP group, the HT7 fluorescence signal in the AAV-P301L group was significantly increased; compared with the AAV-P301L group, the HT7 fluorescence signal in the AAV-P301L+CP1 group was significantly decreased. Figure 5 (A) Simultaneously, we used a pT231-tau antibody to detect Tau phosphorylation in the brain tissues of the three mouse groups. The results showed that compared with the AAV-eGFP group, the pT231-tau fluorescence signal in the AAV-P301L group was significantly increased; compared with the AAV-P301L group, the pT231-tau fluorescence signal in the AAV-P301L+CP1 group was significantly decreased. These results indicate that CP1 treatment can alleviate abnormal Tau protein aggregation and phosphorylation in the mouse hippocampus.

Claims

1. A PROTAC compound targeting the degradation of DAPK1 protein, characterized in that As shown in formula I-1: XYZ formula I-1; Wherein, X represents a ligand of the DAPK1 protein, whose structure is the compound shown in Formula II-1; Z represents a ligand of the E3 ligase, whose structure is the compound shown in Formula II-2 or II-3; Y represents a chain connecting X and Z, whose structure is the compound shown in Formula II-4, wherein the N in Y is connected to X: Ⅱ-1 Ⅱ-2 Ⅱ-3 Ⅱ-4 Each n is independently an integer between 1 and 6.

2. The PROTAC compound targeting the degradation of DAPK1 protein according to claim 1, characterized in that The compound is: ； Formula 1-1, Z is Formula II-2, n=1; ； Formula 1-2, Z is Formula II-2, n=2; ； Formula 1-3, Z is Formula II-2, n=3; ； Formula 1-4, Z is Formula II-2, n=4; ； Formula 1-5, Z is Formula II-2, n=5; ； Formula 1-6, Z is Formula II-2, n=6; ； Formula 1-7, Z is Formula II-3, n=2; ； Formula 1-8, Z is Formula II-3, n=3; ； Formula 1-9, Z is Formula II-3, n=4; ； Formula 1-10, Z is Formula II-3, n=5; ； Formula 1-11, Z is Formula II-3, n=6.

3. The method for preparing a PROTAC compound targeting the degradation of DAPK1 protein according to any one of claims 1 to 2, characterized in that: The process is as follows: ； ； ； 。 4. Use of the PROTAC compound targeting the degradation of DAPK1 protein according to claim 1 for the preparation of a drug for treating or preventing neurodegenerative diseases.

5. The use according to claim 4, characterized in that The neurodegenerative disease is Alzheimer's disease, Parkinson's disease or Huntington's disease.

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