Hyt compound targeting degradation of dapk1 protein and application thereof in neurodegenerative diseases

Abstract

A HyT compound targeting degradation of DAPK1 protein as shown in X-Y-Z, X represents a ligand of DAPK1 protein, Z represents a hydrophobic functional group, Y represents a chain connecting X and Z. The compound can be used for treating or preventing Alzheimer's disease.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of drug preparation for neurodegenerative diseases, and in particular to a HyT compound for targeted degradation of DAPK1 protein, and a preparation method and application thereof. BACKGROUND

[0002] Death-associated protein kinase 1 (DAPK1) is an important serine / threonine kinases (STK) that plays a key role in regulating apoptosis and autophagy. That is, it can play a cancer-inhibiting role by mediating tumor cell growth and metastasis; it can also be involved in the inflammatory process and regulate the immune function of the body; in addition, it also plays a core regulatory role in neural cell death. Studies have found that DAPK1 is highly expressed in the brain and is closely related to diseases such as stroke, epilepsy and Alzheimer disease (AD), and many studies have shown that DAPK1 is related to the pathogenesis of AD. AD is a chronic central nervous system degenerative disease, and its main physiological manifestations are cognitive and memory impairment (dementia), which is the most common form of dementia worldwide and has caused millions of deaths worldwide. The two main pathological features of AD are amyloid beta protein (Aβ) senile plaques and tau neurofibrillary tangles. The formation of Aβ senile plaques is mainly due to the deposition of Aβ protein outside the cell caused by the metabolism of amyloid precursor protein (APP). Tau is a microtubule-associated protein that is over-phosphorylated under pathological conditions to form tau aggregates. DAPK1 is mainly involved in the pathogenesis of AD by over-processing APP, triggering the over-phosphorylation and stabilization of tau aggregates, and is a potential therapeutic target for AD.

[0003] Currently reported DAPK1 inhibitors include TC-DAPK6 and HS-38 and dozens of other inhibitors, but due to problems such as activity, selectivity and drugability, none of them have entered clinical trials.

[0004] Hydrophobic Tagging (HyT) technology is to introduce hydrophobic groups such as adamantyl, fluorene or multiple high hydrophobic aryl groups into the target protein (POI), which leads to the misfolding of the target protein, so that it is recognized and degraded by the proteasome. In 2011, the Crews research group successfully degraded the HaloTag fusion protein using HyT technology, and later the research group also successfully degraded the pseudokinase Her3 and androgen receptor using HyT technology. In 2017, HyT technology was successfully applied to the degradation of Tau associated with Alzheimer's disease, and it was found that the polypeptide combined with HyT could effectively reduce the content of Tau protein in the brain of the mouse model of Alzheimer's disease. HyT technology has the advantages of small molecular weight, good drug properties and good BBB permeability compared with PROTAC.

[0005] The above studies show that DAPK1 protein can mediate the pathogenesis of AD, and inhibiting or reducing the content of DAPK1 may interfere with this process, thereby preventing the onset of AD. Therefore, using HyT technology to reduce the level of DAPK1 protein may play a role in treating or preventing AD. SUMMARY

[0006] In order to overcome the shortcomings of the existing DAPK1 inhibitors, the present application provides a HyT compound for targeted degradation of DAPK1 protein and its preparation method and application. The compound can induce DAPK1 protein degradation by using the ubiquitin-proteasome system through the Hydrophobic Tagging (HyT) technology, so as to achieve the effect of treating or preventing AD. One end of the molecule is targeted to bind to the target protein, and the other end is coupled with a large hydrophobic functional group to induce the ubiquitin-proteasome system to produce specific degradation of the target protein.

[0007] The HyT compound provided by the present application has obvious degradation effect on DAPK1 protein, and can be used for preparing a drug for treating or preventing Alzheimer's disease.

[0008] A HyT compound for targeted degradation of DAPK1 protein, as shown in formula I-1 (X-Y-Z): wherein X represents the ligand of DAPK1 protein, Z represents the hydrophobic functional group, and Y represents the chain connecting X and Z.

[0009] The X is a structure shown in formula II-1, the Z is a structure shown in formula II-2 or II-3, and the Y is a structure shown in formula II-4.

[0010]

[0011]

[0012] Each n is independently an integer between 1 and 6

[0013] In particular, the HyT compounds described above are:

[0014]

[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 = 1

[0028]

[0029] Formula 1-8, Z is Formula II-3, n = 2

[0030]

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

[0032]

[0033] Formula 1-10, Z is Formula II-3, n = 4

[0034] Methods of making HyT compounds that target degradation of DAPK1 protein are as follows:

[0035]

[0036] BRIEF DESCRIPTION OF DRAWINGS

[0037] Figure 1 is to test the degradation of DAPK1 of the CW compound synthesized by the application by taking the HEK293T cell line as the test object;

[0038] Figure 2 is the degradation effect diagram of DAPK1 of the CW compound at different times;

[0039] Figure 3 is the DAPK1 protein degradation of the CJ series compound on N2a cells;

[0040] Figure 4 is an immunofluorescence diagram; wherein (A) immunofluorescence detects the level of hTau in the CA3 region of mice; (B) immunofluorescence detects the phosphorylation level of the Thr231 site of Tau protein in the CA3 region of mice. DETAILED DESCRIPTION

[0041] The embodiments of the application will be described in further detail below, which are exemplary and intended to explain the application, and cannot be understood as a limitation of the application.

[0042] Example 1

[0043] 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)

[0044]

[0045] Step 1: Synthesis of 3-(5-amino-4-(ethoxycarbonyl)-1H-pyrazol-1-yl)benzoic acid (1)

[0046] Under stirring, 2-cyano-3-ethoxyacrylate (2447 mg, 14.5 mmol) was added to a solution of 3-hydrazinylbenzoic acid hydrochloride (3000 mg, 15.9 mmol) and DIPEA (5038 μL, 28.9 mmol) in ethanol (10 mL), and the mixture was heated to reflux for 12 h. The ethanol was removed by concentration under reduced pressure, diluted with water (40 mL), adjusted to pH = 2 with 1M hydrochloric acid, extracted with ethyl acetate (75 mL x 3), washed with saturated brine (30 mL x 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, yield 93.7%. ESI-MS (m / z): 276.20 [M+H] + . 1H NMR (600 MHz, DMSO-d6) δ 13.24 (s, 1H), 8.04 (s, 1H), 7.92 (d, J = 7.6 Hz, 1H), 7.77 (d, J = 7.5 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J = 6.9 Hz, 1H), 6.42 (s, 2H), 4.24-4.13 (m, 2H), 1.27-1.21 (m, 3H).

[0047] Step 2: Synthesis of 3-(4-hydroxy-6-mercapto-lH-pyrazolo[3,4-d]pyrimidin-l- yl)benzoic acid (2)

[0048] Compound 1 (3740 mg, 13.6 mmol) was dissolved in dry tetrahydrofuran (40 mL) with benzoyl isothiocyanate (3995 mg, 24.5 mmol) under nitrogen protection and refluxed for 16 h. The intermediate thiourea was obtained by concentration under reduced pressure. The obtained thiourea was added dropwise to a refluxing solution of ethanol (70 mL) and sodium ethoxide (21% ethanol, 17.6 mL, 54.4 mmol). The stirring was continued for 30 min. Concentration to dryness under reduced pressure, dilution with water (50 mL), adjustment of pH = 2 with 1 M hydrochloric acid, filtration of the solid, washing with water, drying and purification by silica gel column chromatography (dichloromethane: methanol 20: 1) gave the title compound 2 as a yellow solid, 2638 mg, 9.15 mmol, 67.3% yield. ESI-MS (m / z): 289.10 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 13.29 (s, 1H), 12.25 (s, 1H), 8.14 (s, 1H), 8.07-8.00 (m, 2H), 7.80 (s, 1H), 7.66 (d, J = 8.3 Hz, 1H).

[0049] Step 3: Synthesis of 3-{[6-(l-methoxy-l-oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH- pyrazolo[3,4-d]pyrimidin-l-yl}benzoic acid (3)

[0050] Compound 2 (2638 mg, 9.15 mmol) and DIPEA (4.8 mL, 27.5 mmol) were dissolved in anhydrous DMF (4.0 mL). 2-bromopropionic acid methyl ester (1681 mg, 10.1 mol) was added with stirring. The reaction was stirred at rt for 12 h, diluted with water (40 mL), adjusted to pH = 2 with 1 M hydrochloric acid, extracted with ethyl acetate (75 mL x 3), washed with saturated brine (30 mL x 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, 40.5% yield. m.p.: 210.5-212.0 °C. ESI-MS (m / z): 375.10 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 12.87 (s, 1H), 8.60 (s, 1H), 8.30-8.23 (m, 2H), 7.91 (d, J = 7.7 Hz, 1H), 7.64 (t, J = 8.0 Hz, 1H), 4.60 (q, J = 7.3 Hz, 1H), 3.48 (s, 3H), 1.57 (d, J = 7.2 Hz, 3H).

[0051] 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)

[0052] Compound 3 (750 mg, 2 mmol) was dissolved in ammonium methylate solution (7 M, 45 mL) and heated to 65 °C in a reaction kettle for 12 h. It 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. m.p.: 228.6-230.2 °C HRMS (ESI) m / z: [M+H] + calcd for C 15 H 14 N5O4S: 360.07665, found: 360.07608. 1 H NMR (600 MHz, DMSO-d6) δ 13.05 (s, 1H), 8.85-8.69 (m, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.23 (s, 1H), 7.89 (d, J = 7.6 Hz, 1H), 7.70 (s, 1H), 7.64 (t, J = 7.9 Hz, 1H), 7.25 (s, 1H), 4.51 (q, J = 6.6 Hz, 1H), 1.56 (d, J = 7.0 Hz, 3H). 13CNMR (151 MHz, DMSO-d6) δ 172.84, 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.

[0053] Example 2

[0054] Synthesis of 2-(adamantane-l-yl)-N-(2-aminoethyl)acetamide (Jl)

[0055]

[0056] Step 1: Synthesis of tert-butyl 2-{[2-(adamantane-l-yl)acetamido]ethyl}carbamate (Il)

[0057] To a stirred solution of 1-adamantaneacetic acid (129 mg, 0.67 mmol), tert-butyl N-(2- aminoethyl)carbamate (107 mg, 0.67 mmol) and triethylamine (465 μL, 3.35 mmol) in DMSO (2 mL) was added HBTU (254 mg, 0.67 mmol) at rt and the reaction was allowed to proceed overnight. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 3), washed with brine (10 mL x 2), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1) to give the title compound II as a white solid, 152 mg, 0.45 mmol, 67.5% yield, ESI-MS (m / z): 337.25 [M+H] + , 359.20 [M+Na] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.64 (t, J = 5.9 Hz, 1H), 6.68 (t, J = 5.8 Hz, 1H), 3.01 (q, J = 6.3 Hz, 2H), 2.92 (q, J = 6.5 Hz, 2H), 1.86 (d, J = 4.6 Hz, 3H), 1.79 - 1.74 (m, 2H), 1.61 (d, J = 12.2 Hz, 3H), 1.53 (d, J = 12.0 Hz, 3H), 1.50 (s, 6H), 1.33 (s, 9H).

[0058] Step 2: Synthesis of 2-(adamantane-l-yl)-N-(2-aminoethyl)acetamide (Jl)

[0059] Compound I1 was dissolved in dichloromethane (6 mL), ice bath for 30 min, dropwise addition of TFA (2 mL), stirring at room temperature for 2 h, concentrated under reduced pressure, the residue was diluted with dichloromethane, saturated sodium bicarbonate was used to adjust pH = 8, dichloromethane (75 mL x 2) was used for extraction, dried over anhydrous sodium sulfate, and dried under reduced pressure. The title compound J1 was obtained as a white solid, 99 mg, 0.42 mmol, yield 93.3%, ESI-MS (m / z): 237.25 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.69 (d, J = 21.8 Hz, 1H), 2.99 (q, J = 6.4 Hz, 2H), 2.54-2.49 (m, 2H), 1.87 (s, 3H), 1.78 (s, 2H), 1.66-1.53 (m, 6H), 1.51 (s, 8H).

[0060] Example 3

[0061] Synthesis of 2-(adamantan-1-yl)-N-(3-aminopropyl)acetamide (J2)

[0062]

[0063] Compound I2 was prepared as a white solid using the method described for compound I1 in Example 2. 178 mg, 0.51 mmol, yield 75.9%, ESI-MS (m / z): 351.25 [M+H] + ,373.25 [M+Na] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.61 (t, J = 5.6 Hz, 1H), 6.73 (t, J = 5.5 Hz, 1H), 2.96 (q, J = 6.5 Hz, 2H), 2.87 (q, J = 6.7 Hz, 2H), 1.86 (d, J = 5.2 Hz, 3H), 1.76 (d, J = 2.5 Hz, 2H), 1.61 (d, J = 12.0 Hz, 3H), 1.53 (d, J = 11.7 Hz, 3H), 1.50 (s, 6H), 1.47-1.39 (m, 2H), 1.33 (s, 9H).

[0064] Compound J2 was prepared as a white solid using the method described for compound J1 in Example 2. 124 mg, 0.49 mmol, yield 96.7%, ESI-MS (m / z): 251.30 [M+H] + . 1H NMR (600 MHz, DMSO-d6) δ 7.73 (s, 1H), 3.02 (q, J = 6.5 Hz, 2H), 2.57-2.50 (m, 2H), 1.86 (s, 3H), 1.77 (s, 2H), 1.60 (d, J = 12.1 Hz, 3H), 1.53 (s, 3H), 1.50 (s, 8H), 1.45 (t, J = 6.7 Hz, 2H).

[0065] Example 4

[0066] Synthesis of 2-(adamantan-l-yl)-N-(4-aminobutyl)acetamide (J3)

[0067]

[0068] Compound J3 was prepared as a white solid using the method described for compound Jl in Example 2. 134 mg, 0.50 mmol, 97.1% yield, ESI-MS (m / z): 265.30 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.73 (s, 1H), 3.02 (q, J = 6.5 Hz, 2H), 2.57-2.50 (m, 2H), 1.86 (s, 3H), 1.77 (s, 2H), 1.60 (d, J = 12.1 Hz, 3H), 1.53 (s, 3H), 1.50 (s, 8H), 1.45 (t, J = 6.7 Hz, 2H).

[0069] Compound J3 was prepared as a white solid using the method described for compound Jl in Example 2. 134 mg, 0.50 mmol, 97.1% yield, ESI-MS (m / z): 265.30 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.73 (s, 1H), 3.02 (q, J = 6.5 Hz, 2H), 2.57-2.50 (m, 2H), 1.86 (s, 3H), 1.77 (s, 2H), 1.60 (d, J = 12.1 Hz, 3H), 1.53 (s, 3H), 1.50 (s, 8H), 1.45 (t, J = 6.7 Hz, 2H).

[0070] Example 5

[0071] Synthesis of 2-(adamantan-l-yl)-N-(5-aminopentyl)acetamide (J4)

[0072]

[0073] Compound I4 was prepared as a white solid using the method described for compound II in Example 2. 198 mg, 0.52 mmol, 78.1% yield, ESI-MS (m / z): 379.35 [M+H] + , 401.35 [M+Na] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.59 (t, J = 5.5 Hz, 1H), 6.72 (t, J = 5.3 Hz, 1H), 2.94 (q, J = 6.4 Hz, 2H), 2.84 (q, J = 6.6 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 12.0 Hz, 3H), 1.52 (d, J = 12.2 Hz, 3H), 1.50 (s, 6H), 1.33 (s, 11H), 1.31 - 1.29 (m, 2H), 1.18 (p, J = 7.3 Hz, 2H).

[0074] Compound J4 was prepared as a white solid using the method described for compound Jl in Example 2. 132 mg, 0.48 mmol, 91.4% yield, ESI-MS (m / z): 279.35 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.61 (d, J = 7.1 Hz, 1H), 2.95 (d, J = 6.5 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 12.1 Hz, 3H), 1.53 (s, 3H), 1.50 (s, 6H), 1.36 - 1.25 (m, 5H), 1.25 - 1.17 (m, 3H).

[0075] Example 6

[0076] Synthesis of 2-(adamantan-l-yl)-N-(6-aminohexyl)acetamide (J5)

[0077]

[0078] Compound I5 was prepared as a white solid using the method described for compound II in Example 2. 190 mg, 0.48 mmol, 72.1% yield, ESI-MS (m / z): 393.50 [M+H] + , 415.50 [M+Na] + . 1H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 6.0 Hz, 1H), 2.96 (q, J = 6.3 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 12.2 Hz, 3H), 1.55-1.51 (m, 3H), 1.50 (s, 6H), 1.47-1.39 (m, 2H), 1.36-1.30 (m, 2H), 1.29-1.14 (m, 6H).

[0079] Compound J5 was prepared as a white solid using the method described for compound J1 in Example 2. 130 mg, 0.44 mmol, 92.5% yield, ESI-MS (m / z): 293.35 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 6.0 Hz, 1H), 2.96 (q, J = 6.3 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 12.2 Hz, 3H), 1.55-1.51 (m, 3H), 1.50 (s, 6H), 1.47-1.39 (m, 2H), 1.36-1.30 (m, 2H), 1.29-1.14 (m, 6H).

[0080] Example 7

[0081] Synthesis of 2-(adamantan-l-yl)-N-(7-aminopentyl)acetamide (J6)

[0082]

[0083] Compound I6 was prepared as a white solid using the method described for compound I1 in Example 2. 203 mg, 0.50 mmol, 74.4% yield, ESI-MS (m / z): 407.45 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 6.0 Hz, 1H), 2.96 (q, J = 6.3 Hz, 2H), 2.67 (t, J = 7.6 Hz, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 12.2 Hz, 3H), 1.55-1.51 (m, 3H), 1.50 (s, 6H), 1.47-1.39 (m, 2H), 1.36-1.30 (m, 2H), 1.29-1.14 (m, 6H).

[0084] Compound J6 was prepared as a white solid using the method described for compound J1 in Example 2. 145 mg, 0.47 mmol, 94.5% yield, ESI-MS (m / z): 307.40 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.61 (t, J = 5.6 Hz, 1H), 2.96 (t, J = 6.5 Hz, 2H), 2.45 (s, 2H), 1.86 (s, 3H), 1.76 (s, 2H), 1.61 (d, J = 11.7 Hz, 3H), 1.53 (s, 3H), 1.50 (s, 8H), 1.36-1.30 (m, 2H), 1.30-1.25 (m, 2H), 1.22-1.18 (m, 6H).

[0085] Example 8

[0086] Synthesis of N-{2-[2-(adamantan-l-yl)acetylamino]ethyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l-yl}benzamide (CJ1)

[0087]

[0088] To a stirred solution of C9S (571 mg, 0.42 mmol), J1 (99 mg, 0.42 mmol) and triethylamine (292 μL, 2.1 mmol) in DMSO (2 mL) was added HBTU (159 mg, 0.42 mmol) at rt and the reaction was stirred overnight. The reaction mixture was diluted with water (20 mL) and extracted with ethyl acetate (75 mL x 3), washed with brine (10 mL x 2), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane:methanol 10:1) to give the title compound CJ1 as a white solid, 87 mg, 0.15 mmol, 35.9% yield, m.p.: 208.7-209.1 °C. HRMS (ESI) m / z: [M+H] + calcd for C 29 H 36 N7O4S: 578.2549, found: 578.2554. 1H NMR (600 MHz, DMSO-d6) δ 8.67 (d, J = 6.3 Hz, 1H), 8.60 (d, J = 6.4 Hz, 1H), 8.29 - 8.17 (m, 2H), 7.84 - 7.76 (m, 2H), 7.70 (s, 1H), 7.64 - 7.58 (m, 1H), 7.25 (s, 1H), 4.50 (q, J = 7.0 Hz, 1H), 3.31 - 3.28 (m, 2H), 3.25 - 3.19 (m, 2H), 1.78 (d, J = 5.8 Hz, 5H), 1.57 - 1.52 (m, 6H), 1.47 (s, 9H). 13 C NMR (151 MHz, DMSO-d6) δ 172.74, 170.87, 166.31, 161.16, 157.93, 152.10, 138.84, 136.82, 135.99, 129.80, 125.62, 123.93, 120.39, 105.36, 50.61, 45.14, 42.54, 38.42, 36.84, 32.57, 28.48, 18.98.

[0089] Example 9

[0090] Synthesis of N-{3-[2-(adamantan-l-yl)acetylamino]propyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l-yl}benzamide (CJ2)

[0091]

[0092] The title compound CJ2 was prepared as a white solid, 56 mg, 0.09 mmol, yield 22.5%, m.p.: 213.7-214.6 °C, using the method described for compound CJ1 in Example 8. HRMS (ESI) m / z: [M+H] + calcd for C 30 H 38 N7O4S: 592.2706, found: 592.2704; [M+Na] + calcd for C 30 H 37 N7NaO4S: 614.2525, found: 614.2523. 1H NMR (600 MHz, DMSO-d6) δ 8.68 (t, J = 5.6 Hz, 1H), 8.61 (s, 1H), 8.25 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 7.7 Hz, 1H), 7.70 (d, J = 4.7 Hz, 2H), 7.62 (t, J = 7.9 Hz, 1H), 7.25 (s, 1H), 4.50 (q, J = 7.1 Hz, 1H), 3.27 (q, J = 6.6 Hz, 2H), 3.05 (q, J = 6.5 Hz, 2H), 1.85 (s, 3H), 1.78 (s, 2H), 1.65 - 1.57 (m, 5H), 1.54 (d, J = 7.6 Hz, 5H), 1.50 (s, 7H). 13 CNMR (151 MHz, DMSO-d6) δ 172.90, 170.53, 166.25, 162.85, 161.58, 152.35, 139.00, 136.86, 136.11, 129.90, 125.58, 123.86, 120.36, 105.48, 50.68, 45.09, 42.66, 37.88, 36.97, 32.68, 29.86, 28.58, 19.03.

[0093] Example 10

[0094] Synthesis of N-{4-[2-(adamantan-l-yl)acetamido]butyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l- yl}benzamide (CJ3)

[0095]

[0096] The title compound CJ3 was prepared as a white solid, 25 mg, 0.04 mmol, yield 9.8%, m.p.: 217.5-218.0 °C using the method described for compound CJ1 in Example 8. HRMS (ESI) m / z: [M+H] + calcd for C 31 H 40 N7O4S: 606.2862, found: 606.2867; [M+Na] + calcd for C 31 H 39 N7NaO4S: 628.2682, found: 628.2691. 1H NMR (600 MHz, DMSO-d6) δ 8.68 (d, J = 6.0 Hz, 1H), 8.62 (s, 1H), 8.24 (d, J = 8.1 Hz, 1H), 8.15 (s, 1H), 7.78 - 7.72 (m, 2H), 7.66 (d, J = 5.9 Hz, 1H), 7.58 (t, J = 8.0 Hz, 1H), 7.18 (s, 1H), 4.47 (q, J = 7.3 Hz, 1H), 3.26 - 3.23 (m, 2H), 3.00 (q, J = 6.7, 5.6 Hz, 2H), 1.83 (s, 3H), 1.75 (s, 2H), 1.56 (d, J = 12.2 Hz, 3H), 1.49 (d, J = 10.5 Hz, 14H), 1.39 (p, J = 7.5 Hz, 2H). 13 CNMR (151 MHz, DMSO-d6) δ 172.79, 170.35, 166.20, 161.11, 157.90, 152.12, 138.90, 136.89, 136.21, 129.88, 125.67, 123.85, 120.42, 105.43, 50.61, 45.29, 42.65, 38.63, 36.98, 32.66, 28.57, 27.41, 27.18, 19.12.

[0097] Example 11

[0098] Synthesis of N-{5-[2-(adamantan-l-yl)acetylamino]pentyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l-yl}benzamide (CJ4)

[0099]

[0100] The title compound CJ4 was prepared as a white solid, 48 mg, 0.08 mmol, yield 18.4%, m.p.: 222.6-222.7 °C, using the method described for compound CJ1 in Example 8. HRMS (ESI) m / z: [M+H] + calcd for C 32 H 42 N7O4S: 620.3019, found: 620.3014; [M+Na] + calcd for C 32 H 41 N7NaO4S: 642.2838, found: 642.2844. 1H NMR (600 MHz, DMSO-d6) δ 12.78 (s, 1H), 8.67 (d, J = 5.5 Hz, 1H), 8.62 (s, 1H), 8.24 (d, J = 2.1 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.71 (s, 1H), 7.64 - 7.59 (m, 2H), 7.26 (s, 1H), 4.51 (q, J = 7.2 Hz, 1H), 3.26 - 3.22 (m, 2H), 2.97 (q, J = 6.5 Hz, 2H), 1.82 (s, 3H), 1.74 (s, 2H), 1.57 (d, J = 12.2 Hz, 3H), 1.53 (d, J = 6.8 Hz, 3H), 1.50 (s, 3H), 1.47 (s, 8H), 1.40 - 1.34 (m, 2H), 1.29 - 1.25 (m, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 172.81, 170.33, 166.19, 162.86, 157.92, 152.12, 138.90, 136.89, 136.20, 129.87, 125.68, 123.83, 120.41, 105.43, 50.61, 45.29, 42.65, 38.74, 36.98, 32.64, 29.42, 29.23, 28.57, 24.43, 19.10.

[0101] Example 12

[0102] Synthesis of N-{6-[2-(adamantan-l-yl)acetylamino]hexyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l- yl}benzamide (CJ5)

[0103]

[0104] The title compound CJ5 was prepared as a white solid, 45 mg, 0.07 mmol, yield 16.9%, m.p.: 224.2-224.7 °C using the method described for compound CJ1 in Example 8. HRMS (ESI) m / z: [M+H] + calcd for C 33 H 44 N7O4S: 634.3175, found: 634.3179; [M+Na] + calcd for C 33 H 43N7NaO4S: 656.2995, found: 656.2998. 1 H NMR (600 MHz, DMSO-d6) δ 12.76 (s, 1H), 8.67 (d, J = 5.7 Hz, 1H), 8.63 (s, 1H), 8.24 (d, J = 2.4 Hz, 1H), 8.21 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.6 Hz, 1H), 7.70 (s, 1H), 7.63-7.59 (m, 2H), 7.27 (s, 1H), 4.52 (q, J = 6.9 Hz, 1H), 3.24 (q, J = 6.6 Hz, 2H), 2.96 (q, J = 6.3 Hz, 2H), 1.84 (s, 3H), 1.75 (s, 2H), 1.58 (d, J = 12.1 Hz, 3H), 1.55-1.50 (m, 5H), 1.49 (s, 9H), 1.37-1.31 (m, 2H), 1.29-1.24 (m, 4H). 13 C NMR (151 MHz, DMSO-d6) δ 172.78, 170.28, 166.16, 162.85, 161.10, 152.11, 138.89, 136.89, 136.24, 129.88, 125.67, 123.82, 120.41, 105.43, 50.63, 45.31, 42.67, 38.73, 36.99, 36.30, 32.66, 29.71, 29.59, 28.58, 26.73, 19.12.

[0105] Example 13

[0106] Synthesis of N-{7-[2-(adamantan-l-yl)acetylamino]heptyl}-3-{[6-(l-amino-l- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-lH-pyrazolo[3,4-d]pyrimidin-l-yl}benzamide (CJ6)

[0107]

[0108] The title compound CJ6 was prepared as a white solid, 65 mg, 0.10 mmol, yield 23.9%, m.p.: 229.1-229.4 °C, using the method described for compound CJ1 in Example 8. HRMS (ESI) m / z: [M+H] + calcd for C 34 H 46 N7O4S: 648.3332, found: 648.3338; [M+Na] + calcd for C 34H 45 N7NaO4S: 670.3151, found: 670.3155. 1 H NMR (600 MHz, DMSO-d6) δ 12.77 (s, 1H), 8.66 (t, J = 5.5 Hz, 1H), 8.62 (s, 1H), 8.25 (d, J = 2.3 Hz, 1H), 8.22 (d, J = 8.0 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.70 (s, 1H), 7.64 - 7.56 (m, 2H), 7.27 (s, 1H), 4.52 (q, J = 7.2 Hz, 1H), 3.24 (q, J = 6.8 Hz, 2H), 2.96 (q, J = 6.4 Hz, 2H), 1.83 (s, 3H), 1.74 (s, 2H), 1.58 - 1.52 (m, 7H), 1.49 (d, J = 11.3 Hz, 14H), 1.35 - 1.31 (m, 2H), 1.19 (s, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 172.69, 170.13, 166.05, 161.09, 157.87, 152.07, 138.83, 136.82, 136.18, 129.79, 125.59, 123.73, 120.33, 105.37, 50.58, 45.21, 42.61, 38.66, 36.93, 32.58, 29.62, 29.47, 28.95, 28.52, 26.98, 26.87, 19.06.

[0109] Example 14

[0110] Synthesis of N-(2-aminoethyl)-2-(9H-fluoren-9-yl)acetamide (W1)

[0111]

[0112] Step 1: Synthesis of tert-butyl 2-{[2-(9H-fluoren-9-yl)acetamido]ethyl}carbamate (V1)

[0113] To a stirred solution of 9-fluoreneacetic acid (150 mg, 0.67 mmol), N-(2- aminoethyl)carbamic acid tert-butyl ester (107 mg, 0.67 mmol) and triethylamine (465 μL, 3.35 mmol) in DMSO (2 mL) was added HBTU (254 mg, 0.67 mmol) at rt and stirred overnight. The reaction was diluted with water (20 mL) and extracted with ethyl acetate (50 mL x 3), washed with brine (10 mL x 2), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate 2: 1) to give the title compound V1 as a white solid, 185 mg, 0.50 mmol, 75.3% yield, ESI-MS (m / z): 389.25 [M+Na] + . 1 H NMR (600 MHz, DMSO-d6) δ 8.00 (t, J = 5.5 Hz, 1H), 7.83 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 7.4 Hz, 2H), 7.34 (t, J = 7.5 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 6.80 (t, J = 5.7 Hz, 1H), 4.31 (t, J = 7.7 Hz, 1H), 3.17 (q, J = 6.3 Hz, 2H), 3.01 (q, J = 6.6 Hz, 2H), 2.46 (d, J = 4.3 Hz, 2H), 1.34 (s, 9H).

[0114] Step 2: Synthesis of N-(2-aminoethyl)-2-(9H-fluoren-9-yl)acetamide (W1)

[0115] V1 was dissolved in dichloromethane (6 mL), trifluoroacetic acid (3 mL) was added dropwise under ice bath for 30 min, the ice bath was removed, stirred at rt for 2 h, concentrated under reduced pressure. The title compound W1 was obtained as a white solid, 125 mg, 0.47 mmol, 94.0% yield, ESI-MS (m / z): 267.30 [M+H] + . 1 H NMR (600 MHz, Methanol-d4) δ 7.77 (d, J = 7.5 Hz, 2H), 7.48 (d, J = 7.5 Hz, 2H), 7.35 (t, J = 7.5 Hz, 2H), 7.27 (t, J = 7.5 Hz, 2H), 4.38 (t, J = 7.7 Hz, 1H), 3.52 (t, J = 6.2 Hz, 2H), 3.06 (t, J = 6.2 Hz, 2H), 2.63 (d, J = 7.6 Hz, 2H).

[0116] Example 15

[0117] Synthesis of N-(3-aminopropyl)-2-(9H-fluoren-9-yl)acetamide (W2)

[0118]

[0119] Compound W2 was prepared as a white solid using the method described for compound W1 in Example 14. 136 mg, 0.49 mmol, yield 93.4%, ESI-MS (m / z): 281.25 [M+H] + . 1 H NMR (400 MHz, Methanol-d4) δ 7.81 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 6.5 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.32 (td, J = 7.5, 1.2 Hz, 2H), 4.41 (t, J = 7.4 Hz, 1H), 3.37 (t, J = 6.7 Hz, 2H), 2.90 (t, J = 7.4 Hz, 2H), 2.67 (d, J = 7.4 Hz, 2H), 1.93 - 1.81 (m, 2H).

[0120] Compound W2 was prepared as a white solid using the method described for compound W1 in Example 14. 136 mg, 0.49 mmol, yield 93.4%, ESI-MS (m / z): 281.25 [M+H] + . 1 H NMR (400 MHz, Methanol-d4) δ 7.81 (d, J = 7.5 Hz, 2H), 7.53 (d, J = 6.5 Hz, 2H), 7.39 (t, J = 7.3 Hz, 2H), 7.32 (td, J = 7.5, 1.2 Hz, 2H), 4.41 (t, J = 7.4 Hz, 1H), 3.37 (t, J = 6.7 Hz, 2H), 2.90 (t, J = 7.4 Hz, 2H), 2.67 (d, J = 7.4 Hz, 2H), 1.93 - 1.81 (m, 2H).

[0121] Example 16

[0122] Synthesis of N-(4-aminobutyl)-2-(9H-fluoren-9-yl)acetamide (W3)

[0123]

[0124] Compound V3 was prepared as a white solid using the method described for compound V1 in Example 14. 239 mg, 0.60 mmol, 90.1% yield, ESI-MS (m / z): 417.35 [M+Na] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.92 (t, J = 5.6 Hz, 1H), 7.83 (d, J = 7.5 Hz, 2H), 7.47 (d, J = 7.5 Hz, 2H), 7.34 (t, J = 7.4 Hz, 2H), 7.26 (t, J = 7.5 Hz, 2H), 6.81 (t, J = 5.7 Hz, 1H), 4.31 (t, J = 7.8 Hz, 1H), 3.12 (q, J = 6.1 Hz, 2H), 2.90 (q, J = 6.2 Hz, 2H), 2.45 (d, J = 7.2 Hz, 2H), 1.42 - 1.36 (m, 4H), 1.34 (s, 9H).

[0125] Compound W3 was prepared as a white solid using the method described for compound W1 in Example 14. 166 mg, 0.57 mmol, 94.2% yield, ESI-MS (m / z): 295.25 [M+H] + . 1 H NMR (400 MHz, Methanol-d4) δ 7.81 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 8.5 Hz, 2H), 7.39 (t, J = 7.4 Hz, 2H), 7.31 (td, J = 7.5, 1.2 Hz, 2H), 4.41 (t, J = 7.6 Hz, 1H), 3.35 - 3.33 (m, 2H), 2.98 (t, J = 7.3 Hz, 2H), 2.61 (d, J = 7.7 Hz, 2H), 1.77 - 1.59 (m, 4H).

[0126] Example 17

[0127] Synthesis of N-(5-aminopentyl)-2-(9H-fluoren-9-yl)acetamide (W4)

[0128]

[0129] Compound V4 was prepared as a white solid using the method described for compound V1 in Example 14. 170 mg, 0.42 mmol, 62.0% yield, ESI-MS (m / z): 431.35 [M+Na] + . 1H NMR (400 MHz, DMSO-d6) δ 7.95 (t, J = 5.6 Hz, 1H), 7.88 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.38 (t, J = 7.4 Hz, 2H), 7.31 (t, J = 6.8 Hz, 2H), 6.82 (t, J = 5.1 Hz, 1H), 4.36 (t, J = 7.7 Hz, 1H), 3.16 (q, J = 6.5 Hz, 2H), 2.92 (q, J = 6.6 Hz, 2H), 2.51 (d, J = 1.6 Hz, 2H), 2.50 (d, J = 5.2 Hz, 2H), 1.49 - 1.43 (m, 2H), 1.38 (s, 9H), 1.33 - 1.21 (m, 3H).

[0130] The title compound W4 was prepared as a white solid using the method described for compound W1 in Example 14. 122 mg, 0.40 mmol, 94.5% yield, ESI-MS (m / z): 309.25 [M+H] + . 1 H NMR (400 MHz, Methanol-d4) δ 7.81 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.39 (t, J = 7.5 Hz, 2H), 7.30 (td, J = 7.5, 1.2 Hz, 2H), 4.41 (t, J = 7.6 Hz, 1H), 3.29 (d, J = 7.1 Hz, 2H), 2.94 (t, J = 7.7 Hz, 2H), 2.61 (d, J = 7.6 Hz, 2H), 1.77 - 1.67 (m, 2H), 1.64 - 1.56 (m, 2H), 1.47 - 1.38 (m, 2H).

[0131] Example 18

[0132] Synthesis of N-{2-[2-(9H-fluoren-9-yl)acetamido]ethyl}-3-{[6-(1-amino-1- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzamide (CW1)

[0133]

[0134] The title compound CW1 was prepared as a white solid using the method described for compound CJ1 in Example 8. 20 mg, 0.03 mmol, 7.8% yield, m.p.: 166.5-167.4 °C. HRMS (ESI) m / z: [M+H] + calcd for C 32 H 30N7O4S: 608.20800, found: 608.20728. 1 H NMR (600 MHz, DMSO-d6) δ 8.79 (s, 1H), 8.62 (s, 1H), 8.25 (s, 2H), 8.13 (s, 1H), 7.84-7.80 (m, 3H), 7.71 (s, 1H), 7.64 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.31 (t, J = 7.6 Hz, 2H), 7.26 (s, 1H), 7.22 (d, J = 7.8 Hz, 2H), 4.53-4.44 (m, 1H), 4.37-4.30 (m, 1H), 3.38 (s, 4H), 3.26 (s, 2H), 1.53 (d, J = 7.0 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 172.83, 171.73, 166.53, 161.12, 157.90, 152.13, 147.19, 140.60, 138.91, 136.94, 136.04, 129.97, 127.76, 127.61, 125.77, 125.06, 124.07, 120.49, 105.45, 45.22, 44.02, 38.81, 29.59, 18.99.

[0135] Example 19

[0136] Synthesis of N-{3-[2-(9H-fluoren-9-yl)acetylamino]propyl}-3-{[6-(1-amino-1- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzamide (CW2)

[0137]

[0138] The title compound CW2 was prepared as a white solid using the method described for compound CJ1 in Example 8. 22 mg, 0.04 mmol, yield 8.4%, m.p.: 172.3-173.1 °C. HRMS (ESI) m / z: [M+H] + calcd for C 33 H 32 N7O4S: 622.22365, found: 622.22290. 1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.73 (t, J = 5.5 Hz, 1H), 8.62 (s, 1H), 8.25 (d, J = 10.4 Hz, 2H), 8.01 (t, J = 5.4 Hz, 1H), 7.83 (t, J = 6.4 Hz, 3H), 7.73 (s, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.49 (d, J = 7.3 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 7.29 - 7.24 (m, 3H), 4.51 (q, J = 6.7 Hz, 1H), 4.32 (t, J = 6.2 Hz, 1H), 3.31 (d, J = 6.1 Hz, 4H), 3.25 - 3.20 (m, 2H), 1.76 - 1.68 (m, 2H), 1.54 (d, J = 6.9 Hz, 3H). 13 C NMR (151 MHz, DMSO-d6) δ 172.79, 171.33, 166.33, 161.16, 157.92, 152.15, 147.23, 140.62, 138.93, 136.93, 136.16, 129.96, 127.77, 127.63, 125.74, 125.07, 123.98, 120.50, 105.46, 45.26, 44.11, 37.96, 37.26, 29.66, 19.07.

[0139] Example 20

[0140] Synthesis of N-{4-[2-(9H-fluoren-9-yl)acetamido]butyl}-3-{[6-(1-amino-1- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzamide (CW3)

[0141]

[0142] The title compound CW3 was prepared as a white solid using the method described for compound CJ1 in Example 8. 25 mg, 0.04 mmol, 9.4% yield, m.p.: 182.1-183.1 °C. HRMS (ESI) m / z: [M+H] + calcd for C 34 H 34 N7O4S: 636.23930, found: 636.23883. 1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.74 (s, 1H), 8.64 (s, 1H), 8.28-8.23 (m, 2H), 7.96 (s, 1H), 7.82 (d, J = 8.6 Hz, 3H), 7.72 (s, 1H), 7.63 (t, J = 8.2 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.34-7.28 (m, 3H), 7.26-7.21 (m, 2H), 4.51 (d, J = 7.5 Hz, 1H), 4.30 (d, J = 8.0 Hz, 1H), 3.30 (d, J = 6.9 Hz, 4H), 3.17 (d, J = 6.5 Hz, 2H), 1.54 (d, J = 7.2 Hz, 5H), 1.48 (d, J = 9.6 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 172.76, 171.12, 166.19, 161.14, 157.90, 152.15, 147.26, 140.61, 138.92, 136.94, 136.23, 129.93, 127.74, 127.59, 125.72, 125.07, 123.90, 120.50, 105.46, 45.27, 44.11, 38.97, 29.55, 27.22, 27.19, 19.11.

[0143] Example 21

[0144] Synthesis of N-{5-[2-(9H-fluoren-9-yl)acetamido]pentyl}-3-{[6-(1-amino-1- oxopropan-2-yl)thio]-4-oxo-4,5-dihydro-1H-pyrazolo[3,4-d]pyrimidin-1-yl}benzamide (CW4)

[0145]

[0146] The title compound CW4 was prepared as a white solid using the method described for compound CJ1 in Example 8. 46 mg, 0.07 mmol, yield 16.9%, m.p.: 193.0-194.8 °C. HRMS (ESI) m / z: [M+H] + calcd for C 35 H 36 N7O4S: 650.25495, found: 650.25439. 1H NMR (600 MHz, DMSO-d6) δ 12.79 (s, 1H), 8.74 (s, 1H), 8.64 (s, 1H), 8.28-8.23 (m, 2H), 7.96 (s, 1H), 7.82 (d, J = 8.6 Hz, 3H), 7.72 (s, 1H), 7.63 (t, J = 8.2 Hz, 1H), 7.47 (d, J = 7.4 Hz, 2H), 7.34-7.28 (m, 3H), 7.26-7.21 (m, 2H), 4.51 (d, J = 7.5 Hz, 1H), 4.30 (d, J = 8.0 Hz, 1H), 3.30 (d, J = 6.9 Hz, 4H), 3.17 (d, J = 6.5 Hz, 2H), 1.54 (d, J = 7.2 Hz, 5H), 1.48 (d, J = 9.6 Hz, 2H). 13 C NMR (151 MHz, DMSO-d6) δ 172.78, 171.11, 166.19, 161.13, 157.91, 152.13, 147.26, 140.62, 138.92, 136.92, 136.24, 129.90, 127.75, 127.57, 125.73, 125.06, 123.85, 120.50, 105.46, 45.28, 44.13, 40.12, 39.11, 29.36, 29.34, 24.50, 19.13.

[0147] DAPK1 protein degradation of Example 22 compounds on HEK293T cells

[0148] To verify whether the target compounds have degradation effect on DAPK1 protein, the degradation of DAPK1 by the compounds was detected using HEK293T cell line as the experimental object, and the results are shown in Figure 1 CJ3 significantly reduced the DAPK1 protein level after 24 h of action at a concentration of 5 μM, while the other compounds with longer and shorter Linker had no obvious effect on the DAPK1 protein level, which indicated that HyT has strong dependence on Linker, and when the length of Linker is appropriate, HyT can cause degradation of DAPK1 protein.

[0149] CW1 significantly down-regulated the DAPK1 protein level after 24 h of action at a concentration of 5 μM, while the compounds CW2-CW4 with longer Linker had no obvious effect on the DAPK1 protein level. This suggests that when fluorene group is used as a hydrophobic group, a shorter Linker can produce better degradation effect.

[0150] CJ3 and CW1 were further selected for their better degradation effect on DAPK1. The degradation effect of DAPK1 was observed at 10 μΜ concentration after different time of action. The results are shown in Figure 2 CJ3 and CW1 significantly reduced the level of DAPK1 protein after 12 h of action at 10 μΜ concentration. When the action time was prolonged to 24 h, there was no obvious change in the degradation effect of DAPK1 by CJ3, while the level of DAPK1 protein gradually rose after 24 h of action by CW1, which might be due to the speed of compensatory synthesis of DAPK1 protein being greater than the degradation speed of CW1.

[0151] Example 23 Degradation of DAPK1 protein in N2a cells by compounds of Example 23

[0152] Total protein was extracted from N2a 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 PVDF membranes by semi-dry transfer. Then, the non-specific sites were blocked with 5% milk-TBST or 5% BSA-TBST at room temperature for 1 h, and the primary antibody was added to the samples and then incubated at 4°C overnight. The membrane was washed with TBST three times to remove the excess primary antibody, and then the HRP-conjugated secondary antibody was added, and the samples were incubated at room temperature for 1 h. Then, all the membranes were coated with ECL chemiluminescent enzyme substrate, and imaged using an imaging system. The results are shown in Figure 3 CJ1, CJ4, CJ5, and CJ6 in the CJ series can significantly reduce the level of DAPK1 protein.

[0153] Example 24 Effect of CJ1 on tau protein in the CA3 region of mouse hippocampus

[0154] (1) The 2-month-old male mice used in the experiment were purchased from Shanghai Wushi Animal Experiment Center, and were maintained in the standard specific pathogen-free area of Fujian Medical University Experimental Animal Center with a 12-hour light cycle, and were free to eat and drink water.

[0155] (2) Brain stereotactic operation

[0156] AAV-CMV-Tau(P301L)-EGFP-WPRE was purchased from Wuhan Shumi Brain Science Technology Co., Ltd. Mice were anesthetized by isoflurane inhalation and fixed in a stereotactic apparatus. After disinfection with povidone-iodine, the skin was incised along the anteroposterior axis to expose the skull. Injection holes were made using a hand drill at 2.2 mm posterior, 2.7 mm right, and 2.3 mm inferior to the anterior fontanelle. Tau-P301L virus or the vector virus was injected into the CA3 region at a rate of 125 nL / min using a microinfusion pump. The needle was held in place for 5 minutes before withdrawal. The mice were then sutured and placed on a heating pad for resuscitation.

[0157] (3) Immunohistofluorescence

[0158] (3-1) Coronal sections of 5 μm were prepared from paraffin-embedded mouse brain tissue and subjected to immunohistofluorescence staining. After deparaffining, rehydration, and antigen retrieval, the sections were blocked at nonspecific sites with goat serum for 1 hour at room temperature. Primary antibody was added to the sections and incubated overnight at 4°C. After washing three times with PBST, Alexa Fluor 488 or 546 conjugated secondary antibody was added to the sections under light-protected conditions and incubated at room temperature for 1 hour. After washing three times with PBST, the sections were stained with Hoechst 33342 for 10 minutes, washed three times with PBST, and all samples were mounted with an anti-quenching agent and then observed and photographed under a fluorescence microscope.

[0159] (3-2) To investigate the effect of CJ1 on Tau protein lesions in the mouse hippocampus, we measured the levels of total hTau protein and phosphorylation sites of Tau protein associated with Alzheimer's disease. First, we used an HT7 antibody to detect the total hTau protein level in the brain tissue of three groups of mice using immunofluorescence. The results showed that compared with the AAV-eGFP group, the HT7 fluorescence signal was significantly increased in the AAV-P301L group; compared with the AAV-P301L group, the HT7 fluorescence signal was significantly decreased in the AAV-P301L+CJ1 group. Figure 4 (A) shows the results. Simultaneously, we used pT231-tau antibody to detect the phosphorylation level of Tau protein in the brain tissue of three groups of mice. The results showed that compared with the AAV-eGFP group, the pT231-tau fluorescence signal was significantly increased in the AAV-P301L group; compared with the AAV-P301L group, the pT231-tau fluorescence signal was significantly decreased in the AAV-P301L+CJ1 group. Figure 4 (B) shows that the above results indicate that CJ1 treatment can reduce abnormal aggregation and phosphorylation of Tau protein in the mouse hippocampus.

Claims

1. A HyT compound targeting degradation of DAPK1 protein, characterized in that, As shown in formula I-1 X-Y-Z Formula I-1 Wherein, X represents the ligand of DAPK1 protein, which is the structure shown in formula II-1; Z represents the hydrophobic functional group, which is the structure shown in formula II-2 or II-3; Y represents the chain connecting X and Z, which is the structure shown in formula II-4; Ⅱ-1 Ⅱ-2 Ⅱ-3 Ⅱ-4 Each n is independently an integer between 1-6.

2. The HyT compound of claim 1, wherein the HyT compound targets degradation of a DAPK1 protein. 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=1 ； Formula 1-8, Z is formula II-3, n=2 ； Formula 1-9, Z is formula II-3, n=3 ； Formula 1-10, Z is formula II-3, n=4.

3. The method of producing a HyT compound targeted to degrade DAPK1 protein according to claim 1, characterized by, The process is as follows: ； ； 。 4. Use of the HyT compound for targeted degradation of DAPK1 protein according to claim 1 for the preparation of a medicament for the treatment or prevention of a neurodegenerative disease.

5. Use according to claim 4, characterized in that, The neurodegenerative disease is Alzheimer's disease.

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