HyT compound for targeted degradation of DAPK1 protein, preparation method of HyT compound and application of HyT compound in neurodegenerative diseases

Abstract

The invention relates to a HyT compound for targeted degradation of DAPK1 protein as well as a preparation method and application of the HyT compound in neurodegenerative diseases, the structural formula is X-Y-Z, X represents a ligand of the DAPK1 protein, Z represents a hydrophobic functional group, and Y represents a chain connecting X and Z. The compounds are useful in the treatment or prevention of neurodegenerative diseases such as 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 kinase (STK) that plays a key role in regulating apoptosis and autophagy. It can inhibit cancer by mediating tumor cell growth and metastasis, and can also participate in the inflammatory process and regulate immune function. In addition, it also plays a key regulatory role in neural cell death. DAPK1 plays a key role in the process of neural cell death. Related studies have shown that DAPK1 is highly expressed in the brain and is closely related to neuronal damage diseases such as stroke and epilepsy. Moreover, several studies have shown that DAPK1 is related to the pathogenesis of AD. AD has two major pathological features: senile plaques composed of amyloid β protein (Aβ) and tau neurofibrillary tangles. Tau is a microtubule-associated protein that forms tau aggregates in pathological conditions. The formation of Aβ senile plaques is mainly due to the metabolism of amyloid precursor protein (APP), which can lead to extracellular deposition of Aβ protein under these conditions. DAPK1 can trigger excessive phosphorylation and stabilization of tau aggregates by overprocessing APP, and is a potential therapeutic target for AD.

[0003] Currently reported DAPK1 inhibitors include TC-DAPK6 (compound A3) and HS-38 and dozens of other inhibitors. However, due to poor selectivity and low activity, there have been no reports of FDA-approved DAPK1 inhibitors or clinical candidate drugs.

[0004] Hydrophobic Tagging (HyT) technology is to introduce hydrophobic groups such as adamantyl, fluorene or multiple highly 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 inhibition or reduction of 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 a preparation method and application thereof in neurodegenerative diseases. The compound induces 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 specifically degrade 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 is shown in formula I-1:

[0009] X-Y-Z formula I-1

[0010] Wherein, X represents the ligand of DAPK1 protein, Z represents the hydrophobic functional group, and Y represents the chain connecting X and Z.

[0011] 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, wherein the N terminal of Y is connected with Z.

[0012]

[0013] In Formula II-4, each n is independently an integer between 1 and 7

[0014] Specifically, the HyT compound described above is:

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

[0016] Z is Formula II-2, n = 2

[0017] Z is Formula II-2, n = 3

[0018] Z is Formula II-2, n = 4

[0019] Z is Formula II-2, n = 5

[0020] Z is Formula II-2, n = 6

[0021] Z is Formula II-2, n = 7

[0022] The above Z is Formula II-2, each n is independently an integer between 1 and 7, resulting in compounds of Formula 1-1 to Formula 1-7, which correspond to compounds AJ1~AJ7 of Example 1 to Example 14, respectively.

[0023] Z is Formula II-3, n = 1

[0024] Z is Formula II-3, n = 2

[0025] Z is Formula II-3, n = 3

[0026] Z is Formula II-3, n = 4

[0027] Z is Formula II-3, n = 5

[0028] Z is Formula II-3, n = 6

[0029] Z is Formula II-3, n = 7.

[0030] The above Z is Formula II-3, each n is independently an integer between 1 and 7, resulting in compounds of Formula 1-8 to Formula 1-14, which correspond to compounds AW1~AW7 of Example 15 to Example 28, respectively.

[0031] The preparation method of the HyT compound targeting degradation of DAPK1 protein is as follows:

[0032]

[0033] BRIEF DESCRIPTION OF DRAWINGS

[0034] Figure 1 The degradation of the AJ and AW compounds synthesized in the application on DAPK1 is tested by taking mouse primary neurons as the test object. Among them, Figure 1 A and B are diagrams of the degradation of the compounds AJ1-AJ7 obtained in the embodiments of the application on DAPK1 in mouse primary neurons. Figure 1 C and D are diagrams of the degradation of the compounds AW1-AW7 obtained in the embodiments of the application on DAPK1 in mouse primary neurons. DETAILED DESCRIPTION

[0035] The embodiments of the application will be further described in detail below, which are exemplary and are intended to explain the application, and cannot be understood as a limitation of the application.

[0036] Preparation of N-{[2-(adamantane-1-yl)acetyl]amino}acetic acid (Jc1)

[0037]

[0038] Step 1: Preparation of tert-butyl N-{[2-(adamantane-1-yl)acetyl]amino}acetate (Jb1)

[0039] In a 100 mL single-neck flask, (adamantane-1-yl)acetic acid (388.2 mg, 2 mmol), tert-butyl 2-aminoacetate (262.3 mg, 2 mmol), DMSO (5 mL) and Et3N (202.3 mg, 4 mmol) were sequentially added, and finally HBTU (1137.7 mg, 3 mmol) was added. The reaction was carried out under magnetic stirring at room temperature overnight, and TLC was used to monitor the completion of the reaction. Deionized water (20 mL) was added to quench the reaction, and then transferred to a separatory funnel, extracted with ethyl acetate (50 mL x 3), the organic layer was reserved, washed with saturated brine (50 mL x 2), and dried over anhydrous sodium sulfate. The dried reaction liquid was transferred to a 250 mL single-neck flask and concentrated under vacuum and reduced pressure. The residue was purified by chromatography (petroleum ether: ethyl acetate = 5:1) to obtain compound Jb1 white solid powder 557.2 mg, with a yield of 90.7%. ESI-MS (m / z): 308.15 [M+H] + . 1H NMR (600 MHz, DMSO-d6) δ 8.02 (t, J = 5.9 Hz, 1H), 3.66 (d, J = 5.9 Hz, 2H), 1.92 (d, J = 21.7 Hz, 3H), 1.86 (s, 2H), 1.65 (d, J = 11.8 Hz, 3H), 1.57 (s, 9H), 1.39 (s, 9H).

[0040] Step 2: Preparation of N-{[2-(adamantan-l-yl)acetyl]amino}acetic acid (Jcl)

[0041] Compound Jbl was dissolved in dichloromethane (10 mL), ice bath for 30 min, dropwise addition of TFA (3 mL), remove the ice bath, transfer to an oil bath pot and stir at 55 °C for 1 h, concentrated under reduced pressure to give compound Jcl white solid powder 422.3 mg, yield 92.7%. ESI-MS (m / z): 252.05 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ

[0042] 12.35 (s, 1H), 7.98 (t, J = 6.4 Hz, 1H), 3.70 (d, J = 3.3 Hz, 2H), 1.92 (d, J = 22.9 Hz, 3H), 1.87 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.57 (s, 9H).

[0043] Example 2 Preparation of N-{{3-[2-(adamantan-l-yl)acetyl]}amino}propanoic acid (Jc2)

[0044]

[0045] Compound Jb2 was prepared as a white solid using the method described for compound Jbl in Example 1. 591.7 mg, yield 92.1%. ESI-MS (m / z): 322.15 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.76 (t, J = 5.6 Hz, 1H), 3.19 (q, J = 6.4 Hz, 2H), 2.34 (t, J = 6.7 Hz, 2H), 1.89 (s, 3H), 1.79 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.56 (d, J = 12.1 Hz, 3H), 1.52 (s, 6H), 1.39 (s, 9H).

[0046] Compound Jc2 was prepared as a white solid using the method described for compound Jc1 in Example 1. 457.6 mg, 93.7% yield. ESI-MS (m / z): 266.05 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 12.16 (s, 1H), 7.75 (t, J = 5.7 Hz, 1H), 3.20 (q, J = 6.5 Hz, 2H), 2.36 (t, J = 6.9 Hz, 2H), 1.89 (s, 3H), 1.79 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.56 (d, J = 12.1 Hz, 3H), 1.53 (d, J = 2.9 Hz, 6H).

[0047] Example 3 Preparation of N-{{4-[2-(adamantan-l-yl)acetyl]}amino}butanoic acid (Jc3)

[0048]

[0049] Compound Jb3 was prepared as a white solid using the method described for compound Jb1 in Example 1. 614.8 mg, 91.7% yield. ESI-MS (m / z): 336.15 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.69 (t, J = 5.6 Hz, 1H), 3.00 (q, J = 6.5 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 1.90 (s, 3H), 1.80 (s, 2H), 1.65 (d, J = 12.2 Hz, 3H), 1.58 (d, J = 12.1 Hz, 3H), 1.56 (p, J = 7.9 Hz, 2H), 1.54 (s, 6H), 1.39 (s, 9H).

[0050] Compound Jc3 was prepared as a white solid using the method described for compound Jc1 in Example 1. 473.62 mg, 92.5% yield. ESI-MS (m / z): 280.05 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 12.05 (s, 1H), 7.70 (t, J = 5.6 Hz, 1H), 3.01 (q, J = 6.5 Hz, 2H), 2.21 (t, J = 7.4 Hz, 2H), 1.90 (s, 3H), 1.80 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.62 - 1.58 (m, 2H), 1.54 (s, 9H).

[0051] Preparation of N-{{5-[2-(adamantan-l-yl)acetyl]}amino}pentanoic acid (Jc4)

[0052]

[0053] Compound Jb4 was prepared as a white solid using the method described for compound Jb1 in Example 1. 651.0 mg, 93.2% yield. ESI-MS (m / z): 350.15 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.66 (t, J = 5.8 Hz, 1H), 2.99 (q, J = 6.5 Hz, 2H), 2.17 (t, J = 7.4 Hz, 2H), 1.89 (s, 3H), 1.79 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.55 (d, J = 18.9 Hz, 9H), 1.47 (p, J = 7.4 Hz, 2H), 1.38 (s, 9H), 1.37 - 1.33 (m, 2H).

[0054] Compound Jc4 was prepared as a white solid using the method described for compound Jc1 in Example 1. 508.8 mg, 93.1% yield. ESI-MS (m / z): 294.05 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.67 (t, J = 5.6 Hz, 1H), 3.00 (q, J = 6.5 Hz, 2H), 2.19 (t, J = 7.4 Hz, 2H), 1.90 (s, 3H), 1.79 (s, 2H), 1.64 (d, J = 12.1 Hz, 3H), 1.55 (d, J = 22.5 Hz, 9H), 1.48 (p, J = 7.4 Hz, 2H), 1.37 (p, J = 6.8, 2H).

[0055] Preparation of N-{{6-[2-(adamantan-l-yl)acetyl]}amino}hexanoic acid (Jc5)

[0056]

[0057] Compound Jb5 was prepared as a white solid using the method described for compound Jb1 in Example 1. 653.1 mg, 89.9% yield. ESI-MS (m / z): 364.15 [M+H] + . 1H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 5.7 Hz, 1H), 2.99 (q, J = 6.6 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.90 (s, 3H), 1.79 (s, 2H), 1.65 (d, J = 12.1 Hz, 3H), 1.56 (d, J = 19.5 Hz, 9H), 1.47 (p, J = 7.4 Hz, 2H), 1.39 (s, 9H), 1.35 (p, J = 7.3 Hz, 2H), 1.25 (p, J = 8.6 Hz, 2H).

[0058] Compound Jc5 was prepared as a white solid using the method described for compound Jc1 in Example 1. 501.0 mg, 90.7% yield. ESI-MS (m / z): 308.15 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 5.7 Hz, 1H), 2.99 (q, J = 6.6 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.90 (s, 3H), 1.79 (s, 2H), 1.65 (d, J = 12.1 Hz, 3H), 1.56 (d, J = 19.5 Hz, 9H), 1.47 (p, J = 7.4 Hz, 2H), 1.39 (s, 9H), 1.35 (p, J = 7.3 Hz, 2H), 1.25 (p, J = 8.6 Hz, 2H).

[0059] Example 6 Preparation of N-{{7-[2-(adamantan-l-yl)acetyl]}amino}heptanoic acid (Jc6)

[0060]

[0061] Compound Jb6 was prepared as a white solid using the method described for compound Jb1 in Example 1. 690.4 mg, 91.5% yield ESI-MS (m / z): 378.20 [M+H] + . 1 H NMR (600 MHz, DMSO-d6) δ 7.65 (t, J = 5.7 Hz, 1H), 2.99 (q, J = 6.6 Hz, 2H), 2.16 (t, J = 7.3 Hz, 2H), 1.90 (s, 3H), 1.79 (s, 2H), 1.65 (d, J = 12.1 Hz, 3H), 1.56 (d, J = 19.5 Hz, 9H), 1.47 (p, J = 7.4 Hz, 2H), 1.39 (s, 9H), 1.35 (p, J = 7.3 Hz, 2H), 1.25 (p, J = 8.6 Hz, 2H).

[0062] The white solid compound Jc6 was prepared using the method described for compound Jc1 in Example 1. 536.1 mg, yield 91.2%. ESI-MS (m / z): 322.15 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ7.64(t,J=5.7Hz,1H),2.99(q,J=6.7Hz,2H),2.18(t,J=7.4Hz,2H),1.90(s,3H),1.79(s,2 H), 1.64 (d, J = 12.1Hz, 3H), 1.55 (d, J = 17.2Hz, 9H), 1.47 (p, J = 7.2Hz, 2H), 1.35 (p, J = 6.9Hz, 2H), 1.28–1.19 (m, 4H).

[0063] Example 7: Preparation of N-{{8-[2-(adamantane-1-yl)acetyl]}amino}octanoic acid (Jc7)

[0064]

[0065] The white solid compound Jb7 was prepared using the method described for compound Jb1 in Example 1. 722.3 mg, yield 92.3%. ESI-MS (m / z): 392.20 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ7.64(t,J=5.7Hz,1H),2.99(q,J=6.6Hz,2H),2.16(t,J=7.3Hz,2H),1.90(s,3H),1.79(s,2H),1.6 4(d,J=12.0Hz,3H),1.56(d,J=17.2Hz,9H),1.47(p,J=7.2Hz,2H),1.38(s,9H),1.35(p,J=6.8Hz,2H),1.26–1.20(m,6H).

[0066] The white solid compound Jc7 was prepared using the method described for compound Jc1 in Example 1. 574.9 mg, yield 92.9%. ESI-MS (m / z): 336.20 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ7.64(t,J=5.7Hz,1H),2.99(q,J=6.6Hz,2H),2.17(t,J=7.4Hz,2H),1.89(s,3H),1.79(s,2 H), 1.64 (d, J = 12.1Hz, 3H), 1.55 (d, J = 17.0Hz, 9H), 1.47 (p, J = 7.1Hz, 2H), 1.35 (p, J = 7.5Hz, 2H), 1.26–1.20 (m, 6H).

[0067] Example 8: Preparation of (Z)-2-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}acetamide (AJ1) Step 1: Preparation of tert-butyl 2-(4-aminobenzoylamino)acetate (F4)

[0068] In a 100 mL single-necked flask, 4-aminobenzoic acid (137.1 mg, 1 mmol), tert-butyl 2-aminoacetate (131.1 mg, 1 mmol), DMSO (5 mL), and Et3N (202.3 mg, 2 mmol) were added sequentially, followed by HBTU (568.8 mg, 1.5 mmol). The reaction was carried out overnight at room temperature with magnetic stirring, and the reaction was monitored by TLC until completion. After quenching with 20 mL of deionized water, the mixture was transferred to a separatory funnel and extracted with ethyl acetate (50 mL × 3). The organic layer was retained, washed with saturated brine (50 mL × 2), and dried over anhydrous sodium sulfate. The dried reaction solution was transferred to a 250 mL single-necked flask and concentrated under vacuum. The residue was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 1:1) to obtain 227.1 mg of a yellow oily liquid, with a yield of 90.7%. ESI-MS (m / z): 273.00 [M + Na] + . 1 H NMR (600MHz, DMSO-d6) δ8.32 (t, J = 6.0 Hz, 1H), 7.57 (d, J = 8.6 Hz, 2H), 6.54 (d, J = 8.6 Hz, 2H), 5.63 (s, 2H), 3.82 (d, J = 5.9 Hz, 2H), 1.41 (s, 9H).

[0069] Step 2: Preparation of N-{{4-N-{2-[2-(adamantane-1-yl)acetamido]acetamido}benzoyl}amino}tert-butyl acetate (Jd1)

[0070] In a 100 mL round-bottom flask, HATU (570.3 mg, 1.5 mmol), N-{[2-(adamantane-1-yl)acetyl]amino}acetic acid (Jc1) (251.15 mg, 1 mmol), and DMF (5 mL) were added sequentially and stirred for 1 h. Then, tert-butyl 2-(4-aminobenzoylamino)acetate (F4) (250.1 mg, 1 mmol) was added and stirred for 30 min. DIPEA (522 μL, 5 mmol) was then added, and the reaction was carried out overnight at room temperature with magnetic stirring. The reaction was monitored by TLC until completion. After quenching with deionized water (20 mL), the mixture was transferred to a separatory funnel and extracted with ethyl acetate (50 mL × 3), retaining the organic layer. The mixture was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, and then transferred to a 250 mL single-necked flask for vacuum concentration. The residue was purified by column chromatography (dichloromethane:methanol = 50:1) to give 261.45 mg of compound Jd1 as a yellow solid powder, with a yield of 54.1%. ESI-MS (m / z): 484.25 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.18(s,1H),8.71(t,J=5.9Hz,1H),8.05(t,J=5.8Hz,1H),7.83(d,J=8.8Hz,2H),7.66(d,J=8 .7Hz, 2H), 3.87 (d, J = 6.0Hz, 4H), 1.92 (d, J = 1.4Hz, 3H), 1.91 (s, 2H), 1.66 (d, J = 12.2Hz, 3H), 1.59 (s, 9H), 1.42 (s, 9H).

[0071] Step 3: Preparation of N-{{4-N-{2-[2-(adamantane-1-yl)acetamido]acetamido}benzoyl}amino}acetic acid (Je1)

[0072] Jd1 was dissolved in 10 mL of dichloromethane in a 100 mL single-necked flask, placed in an ice bath for 30 min, and TFA (3 mL) was added dropwise. The ice bath was removed, and the mixture was transferred to an oil bath and stirred at 55 °C for 1 h. The solution was then concentrated under reduced pressure to obtain 214.2 mg of a yellow solid powder, with a yield of 92.7%. ESI-MS (m / z): 426.20 [MH] - . 1H NMR (600MHz, DMSO-d6) δ12.54(s,1H),10.19(s,1H),8.70(t,J=4.5Hz,1H),8.05(t,J=5.8Hz,1H),7.83(d,J=8.7Hz,2H) ,7.66(d,J=8.7Hz,2H),3.89(d,J=17.5Hz,4H),1.92(d,J=3.0Hz,3H),1.91(s,2H),1.65(d,J=13.4Hz,3H),1.59(s,9H).

[0073] Step 4: Preparation of (Z)-2-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}acetamide (AJ1)

[0074] In a 32 mL pressure-resistant flask, N-{{4-N-{2-[2-(adamantane-1-yl)acetamido]acetamido}benzoyl}amino}acetic acid (Je1) (213.6 mg, 0.5 mmol), pyridine-3-carboxaldehyde (69.5 mg, 0.65 mmol), NaOAc (41.0 mg, 0.5 mmol), and Ac2O (1 mL) were added sequentially. The flask was then placed in an oil bath. Under stirring, the reaction temperature was raised to 100 °C, and heating continued for 30 min before the reaction was stopped. The pressure-resistant flask was removed and cooled to room temperature. After the pressure-resistant flask was placed in a refrigerator overnight, ethyl acetate was added, and the mixture was filtered. The filter cake was subjected to column chromatography (dichloromethane:ethyl acetate = 2:1), followed by preparative column separation and purification. The purified product was then dried under vacuum at 25 °C to obtain 145.7 mg of compound AJ1 as a yellow solid powder, with a yield of 58.5%. ESI-MS (m / z): 499.25 [M+H] + . 1 H NMR(600MHz,DMSO-d6)δ10.23(s,1H),9.27(s,1H),8.73(d,J=7.9Hz,1H),8 .63(d,J=4.7Hz,1H),8.11(d,J=9.4Hz,2H),7.85(d,J=9.5Hz,2H),7.82(t, J=7.0Hz,1H),7.54(dd,J=8.0,4.7Hz,1H),7.31(s,1H),3.93(d,J=5.7Hz,2 H), 1.96 (s, 3H), 1.93 (s, 2H), 1.68 (d, J = 12.0Hz, 3H), 1.63 (d, J = 7.0Hz, 9H). 13C NMR (151MHz, DMSO-d6) δ171.15,169.25,166.92,164.20,153.14,151.37,144.68,138.71,135. 56,130.26,130.03,126.59,124.44,119.83,119.66,50.41,43.66,42.74,37.12,32.88,28.78.

[0075] Example 9: Preparation of (Z)-3-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}propionamide (AJ2)

[0076]

[0077] The pale yellow solid compound Jd2 was prepared using the method described for compound Jd1 in Example 8. 257.1 mg, yield 51.7%. ESI-MS (m / z): 498.35 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.18(s,1H),8.71(t,J=5.9Hz,1H),7.82(t,J=6.3Hz,1H),7.80(d,J=8.1Hz,2H),7.68(d,J=8.4Hz,2H),3.87(d,J= 5.8Hz, 2H), 3.30 (q, J = 6.2Hz, 2H), 2.53 (t, J = 6.8Hz, 2H), 1.81 (d, J = 15.9Hz, 5H), 1.59 (d, J = 12.2Hz, 3H), 1.52 (d, J = 7.5Hz, 9H), 1.42 (s, 9H).

[0078] The pale yellow solid compound Je2 was prepared using the method described for compound Je1 in Example 8. 205.9 mg, yield 90.3%. ESI-MS (m / z): 440.25 [MH] - . 1H NMR (600MHz, DMSO-d6) δ10.18(s,1H),8.70(t,J=5.9Hz,1H),7.82(t,J=5.8Hz,1H),7.81(d,J=8.7Hz,2H),7.68(d,J=8.4Hz,2H), 3.89(d,J=5.9Hz,2H), 3.31(q,J=6.1Hz,2H), 2.53(t,J=6.8Hz,2H), 1.81(d,J=18.3Hz,5H), 1.59(d,J=12.2Hz,3H), 1.52(s,9H).

[0079] The yellow solid compound AJ2 was prepared using the method described for compound AJ1 in Example 8. 144.7 mg, yield 56.5%. ESI-MS (m / z): 513.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.23 (s, 1H), 9.91 (s, 1H), 8.66 (d, J = 2.2Hz, 1H), 8.44 (d,J=4.8Hz,1H),7.91(d,J=8.4Hz,2H),7.85(d,J=9.5Hz,2H),7.83(t,J=7.0Hz ,1H),7.67(s,1H),7.35(dd,J=8.0,4.8Hz,1H),3.32–3.31(m,2H),1.83(s,3H) ,1.80(s,2H),1.60(d,J=12.3Hz,3H),1.52(d,J=3.0Hz,9H),1.23–1.22(m,2H). 13 C NMR(151MHz,DMSO-d6)δ170.61,170.55,165.86,165.70,150.61,149.48,142.93,136.32,132.72, 130.99,129.36,128.17,125.51,124.06,118.62,50.52,42.58,40.93,36.92,35.41,32.66,28.56.

[0080] Example 10: Preparation of (Z)-4-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}butyramide (AJ3)

[0081]

[0082] The pale yellow solid compound Jd3 was prepared using the method described for compound Jd1 in Example 8. 266.9 mg, yield 52.2%. ESI-MS (m / z): 512.35 [M+H] + . 1 H NMR(600MHz,DMSO-d6)δ10.16(s,1H),8.71(t,J=6.0Hz,1H),7.80(d,J=8.8H z,2H),7.74(t,J=5.7Hz,1H),7.67(d,J=8.7Hz,2H),3.86(d,J=5.9Hz,2H),3 .06(q,J=6.6Hz,2H),2.34(t,J=7.5Hz,2H),1.90(s,3H),1.81(s,2H),1.70( p, J = 7.4Hz, 2H), 1.64 (d, J = 12.2Hz, 3H), 1.56 (d, J = 12.8Hz, 9H), 1.41 (s, 9H).

[0083] The pale yellow solid compound Je3 was prepared using the method described for compound Je1 in Example 8. 221.2 mg, yield 93.1%. ESI-MS (m / z): 454.25 [MH] - . 1 H NMR(600MHz,DMSO-d6)δ12.45(s,1H),10.16(s,1H),8.70(t,J=5.9Hz,1H),7 .81(d,J=8.7Hz,2H),7.74(t,J=5.7Hz,1H),7.67(d,J=8.5Hz,2H),3.89(d,J= 5.8Hz,2H),3.06(q,J=6.7Hz,2H),2.34(t,J=7.5Hz,2H),1.90(s,3H),1.81( s, 2H), 1.70 (p, J = 7.3Hz, 2H), 1.64 (d, J = 12.1Hz, 3H), 1.56 (d, J = 12.5Hz, 9H).

[0084] The yellow solid compound AJ3 was prepared using the method described for compound AJ1 in Example 8. 136.0 mg, yield 51.7%. ESI-MS (m / z): 527.30 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.18(s,1H),9.22(s,1H),8.68(d,J=8.1Hz,1H),8.58(d,J=4 .9Hz,1H),8.05(d,J=8.5Hz,2H),7.82(d,J=8.4Hz,2H),7.50(dd,J=8.0,4.9Hz,1H),7 .46(t,J=7.0Hz,1H),7.26(s,1H),2.47–2.46(m,2H),2.37(t,J=7.4Hz,2H),1.88(s,3 H), 1.81 (s, 2H), 1.73 (p, J = 7.6Hz, 2H), 1.63 (d, J = 12.3Hz, 3H), 1.56 (d, J = 10.0Hz, 9H). 13 C NMR(151MHz,DMSO-d6)δ172.28,170.59,166.92,164.22,153.10,151.33,145.07,138.69,135.55,130 .25,129.94,126.48,124.43,119.76,119.36,50.74,42.81,38.63,37.11,34.77,32.73,28.75,25.72.

[0085] Example 11 Preparation of (Z)-5-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}pentanamide (AJ4)

[0086]

[0087] The pale yellow solid compound Jd4 was prepared using the method described for compound Jd1 in Example 8. 280.5 mg, yield 53.4%. ESI-MS (m / z): 526.35 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.12(s,1H),8.71(t,J=6.0Hz,1H),7.80(d,J=8.7Hz,2H) ,7.70(t,J=5.6Hz,1H),7.67(d,J=8.7Hz,2H),3.87(d,J=5.9Hz,2H),3.03(q,J=6. 6Hz,2H),2.34(t,J=7.4Hz,2H),1.89(s,3H),1.80(s,2H),1.63(d,J=12.2Hz,3H), 1.59(p,J=7.7Hz,2H), 1.55(d,J=13.6Hz,9H), 1.41(s,9H), 1.40(p,J=5.2Hz,2H).

[0088] The pale yellow solid compound Je4 was prepared using the method described for compound Je1 in Example 8. 235.0 mg, yield 93.8%. ESI-MS (m / z): 468.25 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ12.50(s,1H),10.12(s,1H),8.71(t,J=5.9Hz,1H),7.81(d ,J=8.3Hz,2H),7.70(t,J=5.8Hz,1H),7.67(d,J=8.6Hz,2H),3.89(d,J=5.8Hz,2H) ,3.03(q,J=6.5Hz,2H),2.34(t,J=7.4Hz,2H),1.89(s,3H),1.80(s,2H),1.63(d,J =12.3Hz, 3H), 1.59 (p, J = 7.5Hz, 2H), 1.55 (d, J = 13.7Hz, 9H), 1.41 (p, J = 7.1Hz, 2H).

[0089] The yellow solid compound AJ4 was prepared using the method described for compound AJ1 in Example 8. 141.2 mg, yield 52.3%. ESI-MS (m / z): 541.35 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.18(s,1H),9.92(s,1H),8.67(d,J=2.2Hz,1H),8.44(d,J=6.4Hz ,1H),7.92(d,J=8.5Hz,2H),7.70(,J=9.5Hz,2H),7.67(t,J=7.0Hz,1H),7.35(dd,J=8.0,4. 8Hz,1H),7.15(s,1H),3.07–3.04(m,2H),2.35(t,J=7.4Hz,2H),1.89(s,3H),1.81(s,2H), 1.63(d,J=12.5Hz,3H), 1.60(p,J=7.6Hz,2H), 1.54(d,J=2.9Hz,9H), 1.42(p,J=7.1Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ172.16,170.27,165.85,165.69,150.60,149.48,143.04,136.33,130.99 ,129.42,125.52,124.08,118.55,50.62,42.67,38.52,36.98,36.57,32.68,29.37,28.56,23.03.

[0090] Example 12 Preparation of (Z)-6-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}hexanoamide (AJ5)

[0091]

[0092] The pale yellow solid compound Jd5 was prepared using the method described for compound Jd1 in Example 8. 295.0 mg, yield 54.7%. ESI-MS (m / z): 540.40 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.11(s,1H),8.71(t,J=6.0Hz,1H),7.80(d,J=8.5Hz,2H),7.6 7(d,J=8.5Hz,2H),7.65(t,J=5.5Hz,1H),3.87(d,J=5.9Hz,2H),3.01(q,J=6.5Hz,2H), 2.32(t,J=7.4Hz,2H),1.89(s,3H),1.79(s,2H),1.63(d,J=12.0Hz,3H),1.58(p,J=7.5 Hz, 2H), 1.55 (d, J = 14.9 Hz, 9H), 1.41 (s, 9H), 1.30 (p, J = 9.1, 2H), 1.26 (p, J = 7.9 Hz, 2H).

[0093] The pale yellow solid compound Je5 was prepared using the method described for compound Je1 in Example 8. 241.8 mg, yield 91.5%. ESI-MS (m / z): 484.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.11(s,1H),8.71(t,J=5.9Hz,1H),7.81(d,J=8.7Hz,2H),7. 67(d,J=8.7Hz,2H),7.65(t,J=5.8Hz,1H),3.89(d,J=5.8Hz,2H),3.01(q,J=6.5Hz,2H ),2.32(t,J=7.4Hz,2H),1.89(s,3H),1.79(s,2H),1.63(d,J=12.0Hz,3H),1.58(p,J= 7.5Hz, 2H), 1.55 (d, J = 15.1Hz, 9H), 1.40 (p, J = 7.0, 6.6Hz, 2H), 1.30 (p, J = 8.6Hz, 2H).

[0094] The yellow solid compound AJ5 was prepared using the method described for compound AJ1 in Example 8. 147.1 mg, yield 53.1%. ESI-MS (m / z): 555.40 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.34(s,1H),9.22(s,1H),8.74(d,J=8.0Hz,1H),8.59(d,J=4.7Hz,1H ),8.05(d,J=8.0Hz,2H),7.82(d,J=8.4Hz,2H),7.62(t,J=5.6Hz,1H),7.52(dd,J=8.1,4.9Hz,1 H),7.28(s,1H),2.97(q,J=6.6Hz,2H),2.32(t,J=7.4Hz,2H),1.85(s,3H),1.75(s,2H),1.59( d,J=12.6Hz,2H),1.56(p,J=7.3Hz,2H),1.49(s,9H),1.36(p,J=7.3Hz,2H),1.27–1.25(m,2H). 13 C NMR(151MHz,DMSO-d6)δ172.56,170.24,167.09,164.05,153.19,151.40,145.03,138.69,135.62,130.28,130.0 4,126.18,124.59,119.48,119.16,50.61,42.67,40.94,38.70,37.03,37.00,32.66,29.56,28.56,26.69,25.15.

[0095] Example 13 Preparation of (Z)-7-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}heptamide (AJ6)

[0096]

[0097] The pale yellow solid compound Jd6 was prepared using the method described for compound Jd1 in Example 8. 289.4 mg, yield 52.3%. ESI-MS (m / z): 554.40 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.11(s,1H),8.71(t,J=6.0Hz,1H),7.80(d,J=8.7Hz,2H),7 .67(d,J=8.8Hz,2H),7.64(t,J=5.7Hz,1H),3.87(d,J=5.9Hz,2H),3.00(q,J=6.5Hz,2 H),2.32(t,J=7.5Hz,2H),1.89(s,3H),1.79(s,2H),1.63(d,J=12.2Hz,3H),1.55(d,J =14.5Hz,9H),1.42(s,9H),1.38(p,J=7.0,2H),1.31–1.27(m,4H),1.26–1.22(m,2H).

[0098] The pale yellow solid compound Je6 was prepared using the method described for compound Je1 in Example 8. 234.8 mg, yield 90.3%. ESI-MS (m / z): 496.30 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ12.54(s,1H),10.11(s,1H),8.70(t,J=6.0Hz,1H),7.81(d,J= 8.6Hz,2H),7.67(d,J=8.5Hz,2H),7.64(t,J=5.6Hz,1H),3.90(d,J=5.9Hz,2H),3.00(q ,J=6.5Hz,2H),2.32(t,J=7.5Hz,2H),1.89(s,3H),1.79(s,2H),1.63(d,J=12.1Hz,3H ),1.55(d,J=14.5Hz,9H),1.37(p,J=7.0Hz,2H),1.31–1.27(m,4H),1.26–1.22(m,2H).

[0099] The yellow solid compound AJ6 was prepared using the method described for compound AJ1 in Example 8. 144.6 mg, yield 50.9%. ESI-MS (m / z): 569.40 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.34(s,1H),9.21(s,1H),8.73(d,J=8.2Hz,1H),8.59(d,J=4.7Hz,1H) ,8.04(d,J=8.4Hz,2H),7.81(d,J=8.3Hz,2H),7.61(t,J=5.6Hz,1H),7.52(dd,J=8.1,4.9Hz,1H ),7.28(s,1H),2.97(q,J=6.6Hz,2H),2.32(t,J=7.4Hz,2H),1.85(s,3H),1.75(s,2H),1.59(d, J=12.3Hz,2H),1.51(d,J=15.3Hz,9H),1.37–1.31(m,2H),1.29–1.22(m,4H),1.23–1.11(m,2H). 13 C NMR(151MHz,DMSO-d6)δ172.65,170.26,167.09,164.06,153.18,151.40,145.03,138.70,135.61,130.28,130.0 5,126.19,124.59,119.50,119.17,50.63,42.67,40.93,38.75,37.01,32.68,29.59,28.89,28.58,26.79,25.46.

[0100] Example 14 Preparation of (Z)-8-{N-[(adamantane-1-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}octamide (AJ7)

[0101]

[0102] The pale yellow solid compound Jd7 was prepared using the method described for compound Jd1 in Example 8. 293.9 mg, yield 51.8%. ESI-MS (m / z): 568.45 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.11(s,1H),8.71(t,J=6.0Hz,1H),7.80(d,J=8.8Hz,2H),7.67 (d,J=8.7Hz,2H),7.63(t,J=5.7Hz,1H),3.86(d,J=5.9Hz,2H),2.99(q,J=6.5Hz,2H),2. 31(t,J=7.4Hz,2H),1.88(s,3H),1.79(s,2H),1.62(d,J=12.2Hz,3H),1.57(p,J=6.8Hz, 2H), 1.54 (d, J=12.6Hz, 9H), 1.41 (s, 9H), 1.36 (p, J=5.3, 3.7Hz, 2H), 1.31–1.26 (m, 6H).

[0103] The pale yellow solid compound Je7 was prepared using the method described for compound Je1 in Example 8. 246.0 mg, yield 92.9%. ESI-MS (m / z): 512.35 [M+H] + . 1 H NMR (600MHz, DMSO-d6) δ10.12(s,1H),8.71(t,J=5.9Hz,1H),7.81(d,J=8.3Hz,2H), 7.67(d,J=8.5Hz,2H),7.64(t,J=5.9Hz,1H),3.90(d,J=5.8Hz,2H),3.00(q,J=6.5H z,2H),2.32(t,J=7.4Hz,2H),1.88(s,3H),1.79(s,2H),1.62(d,J=12.3Hz,3H),1.5 7(p,J=6.7Hz,2H),1.54(d,J=13.3Hz,9H),1.36(p,J=6.9Hz,2H),1.26–1.24(m,6H).

[0104] The yellow solid compound AJ7 was prepared using the method described for compound AJ1 in Example 8. 172.6 mg, yield 59.3%. ESI-MS (m / z): 583.45 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.39(s,1H),9.27(s,1H),8.79(d,J=8.0Hz,1H),8.63(d,J=6.4Hz,1H), 8.10(d,J=8.8Hz,2H),7.86(d,J=8.8Hz,2H),7.64(t,J=5.7Hz,1H),7.56(dd,J=8.1,4.8Hz,1H),7 .33(s,1H),3.00(q,J=6.5Hz,2H),2.36(t,J=7.4Hz,2H),1.88(s,3H),1.79(s,2H),1.62(d,J=12. 6Hz, 3H), 1.59 (p, J = 6.8Hz, 2H), 1.54 (d, J = 12.5Hz, 9H), 1.36 (p, J = 6.5Hz, 2H), 1.31–1.26 (m, 6H). 13 CNMR(151MHz,DMSO-d6)δ172.66,170.20,167.11,164.06,153.20,151.42,145.03,138.71,135.65,130.29,130.07,1 26.17,124.61,119.48,119.17,50.63,42.67,38.71,37.08,36.99,32.66,29.69,29.16,29.03,28.55,26.84,25.41.

[0105] Example 15: Preparation of N-{[2-(9H-fluorene-9-yl)acetyl]amino}acetic acid (Wcl)

[0106]

[0107] Step 1: Preparation of N-{[2-(9H-fluorene-9-yl)acetyl]amino}tert-butyl acetate (Wb1)

[0108] In a 100 mL single-necked flask, 9-fluoreneacetic acid (448.5 mg, 2 mmol), tert-butyl 2-aminoacetate (262.3 mg, 2 mmol), DMSO (5 mL), and Et3N (202.3 mg, 4 mmol) were added sequentially, followed by HBTU (1137.7 mg, 3 mmol). The reaction was carried out overnight at room temperature with magnetic stirring, and the reaction was monitored by TLC until completion. After quenching with deionized water (20 mL), the mixture was transferred to a separatory funnel and extracted with ethyl acetate (50 mL × 3). The organic layer was retained, washed with saturated brine (50 mL × 2), and dried over anhydrous sodium sulfate. The dried reaction solution was transferred to a 250 mL single-necked flask and concentrated under vacuum. The residue was purified by chromatography (petroleum ether: ethyl acetate = 5:1) to obtain 617.0 mg of compound Wb1 as a white solid powder, with a yield of 91.5%. ESI-MS (m / z): 360.10 [M + Na] + . 1 H NMR (600MHz, DMSO-d6) δ8.42(t,J=5.9Hz,1H),7.87(d,J=7.5Hz,2H),7.59(d,J=7.6Hz,2H),7.38(t,J=7.4Hz ,2H),7.29(t,J=7.4Hz,2H),4.36(t,J=7.6Hz,1H),3.85(d,J=5.9Hz,2H),2.58(d,J=7.6Hz,2H),1.46(s,9H).

[0109] Step 2: Preparation of N-{[2-(9H-fluorene-9-yl)acetyl]amino}acetic acid (Wcl)

[0110] Compound Wb1 was dissolved in dichloromethane (10 mL), incubated in an ice bath for 30 min, and TFA (3 mL) was added dropwise. The ice bath was removed, and the mixture was transferred to an oil bath and stirred at 55 °C for 1 h. The solution was then concentrated under reduced pressure to obtain 458.8 mg of a white solid powder, with a yield of 89.2%. ESI-MS (m / z): 280.05 [MH] - . 1 H NMR (600MHz, DMSO-d6)δ

[0111] 12.64(s,1H),8.41(t,J=5.9Hz,1H),7.87(d,J=7.5Hz,2H),7.60(d,J=7.5Hz,2H),7.38(t,J=7.4H z, 2H), 7.29 (t, J = 7.5Hz, 2H), 4.36 (t, J = 7.6Hz, 1H), 3.88 (d, J = 5.8Hz, 2H), 2.58 (d, J = 7.6Hz, 2H).

[0112] Example 16 Preparation of N-{{3-[2-(9H-fluorene-9-yl)acetyl]}amino}propionic acid (Wc2)

[0113]

[0114] The white solid compound Wb2 was prepared using the method described for compound Wb1 in Example 15. 654.6 mg, yield 93.2%. ESI-MS (m / z): 374.10 [M+Na] + . 1 H NMR (600MHz, DMSO-d6) δ8.09(t,J=5.7Hz,1H),7.86(d,J=7.4Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.29( t,J=7.5Hz,2H), 4.34(t,J=7.7Hz,1H), 3.36(q,J=6.5Hz,2H), 2.49(d,J=2.2Hz,2H), 2.45(t,J=6.8Hz,2H), 1.41(s,9H).

[0115] The white solid compound Wc2 was prepared using the method described for compound Wc1 in Example 15. 501.6 mg, yield 91.2%. ESI-MS (m / z): 294.05 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ8.08(t,J=5.6Hz,1H),7.86(d,J=7.5Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t,J=7.4Hz ,2H),7.29(t,J=7.4Hz,2H),4.34(t,J=7.7Hz,1H),3.37(q,J=6.5Hz,2H),2.48(s,2H),2.44(t,J=6.9Hz,2H).

[0116] Example 17 Preparation of N-{{4-[2-(9H-fluorene-9-yl)acetyl]}amino}butyric acid (Wc3)

[0117]

[0118] The white solid compound Wb3 was prepared using the method described for compound Wb1 in Example 15. 687.3 mg, yield 94.1%. ESI-MS (m / z): 388.15 [M+Na] + . 1H NMR (600MHz, DMSO-d6) δ7.97(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.52(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.29(t,J=7.5Hz ,2H),4.35(t,J=7.8Hz,1H),3.18(q,J=6.5Hz,2H),2.49(d,J=2.2Hz,2H),2.24(t,J=7.5Hz,2H),1.67(p,J=7.2Hz,2H),1.42(s,9H).

[0119] The white solid compound Wc3 was prepared using the method described for compound Wc1 in Example 15. 527.6 mg, yield 90.7%. ESI-MS (m / z): 308.10 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ8.02(t,J=5.7Hz,1H),7.86(d,J=7.3Hz,2H),7.51(d,J=7.5Hz,2H),7.38(d,J=7.4Hz,2H),7.28(d,J= 7.4Hz, 2H), 4.35 (t, J = 7.7Hz, 1H), 3.18 (q, J = 6.5Hz, 2H), 2.49 (d, J = 1.5Hz, 2H), 2.24 (t, J = 7.4Hz, 2H), 1.69 (p, J = 7.2Hz, 2H).

[0120] Example 18 Preparation of N-{{5-[2-(9H-fluorene-9-yl)acetyl]}amino}valeric acid (Wc4)

[0121]

[0122] The white solid compound Wb4 was prepared using the method described for compound Wb1 in Example 15. 703.0 mg, yield 92.7%. ESI-MS (m / z): 402.20 [M+Na] + . 1H NMR (600MHz, DMSO-d6) δ7.96(t,J=5.7Hz,1H),7.87(d,J=7.5Hz,2H),7.52(d,J=7.5Hz,2H),7.37(t,J=7.5Hz,2H),7.29(t,J=7.5Hz,2H),4.35( t,J=7.7Hz,1H),3.17(q,J=6.4Hz,2H),2.49(d,J=4.5Hz,2H),2.23(t,J=7.2Hz,2H),1.52(p,J=14.3Hz,2H),1.46(p,J=7.0Hz,2H),1.41(s,9H).

[0123] The white solid compound Wc4 was prepared using the method described for compound Wc1 in Example 15. 522.4 mg, yield 87.2%. ESI-MS (m / z): 322.10 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ12.08(s,1H),7.96(t,J=5.7Hz,1H),7.87(d,J=7.5Hz,2H),7.52(d,J=7.5Hz,2H),7.37(t,J=7.5Hz,2H),7 .30(t,J=7.5Hz,2H),4.35(t,J=7.8Hz,1H),3.17(q,J=6.5Hz,2H),2.49(d,J=4.0Hz,2H),2.25(t,J=7.3Hz,2H),1.64–1.43(m,4H).

[0124] Example 19: Preparation of N-{{6-[2-(9H-fluorene-9-yl)acetyl]}amino}hexanoic acid (Wc5)

[0125]

[0126] The white solid compound Wb5 was prepared using the method described for compound Wb1 in Example 15. 712.5 mg, yield 90.6%. ESI-MS (m / z): 416.20 [M+Na] + . 1H NMR (600MHz, DMSO-d6) δ7.94(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t,J=7.5Hz,2H),7.29(t ,J=7.4Hz,2H),4.35(t,J=7.7Hz,1H),3.15(q,J=6.6Hz,2H),2.49(d,J=5.2Hz,2H),2.19(t,J=7.4Hz,2H),1.51(p,J=14.3 2H), 1.45 (p, J = 7.1Hz, 2H), 1.40 (s, 9H), 1.29 (p, J = 8.3Hz, 2H).

[0127] The white solid compound Wc5 was prepared using the method described for compound Wc1 in Example 15. 544.3 mg, yield 89.1%. ESI-MS (m / z): 336.10 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ12.03(s,1H),7.94(t,J=5.7Hz,1H),7.87(d,J=7.5Hz,2H) ,7.51(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.29(t,J=7.5Hz,2H),4.35(t,J=7. 7Hz,1H),3.16(q,J=6.1Hz,2H),2.49(d,J=2.6Hz,2H),2.31(t,J=7.4Hz,1H),2.21 (t,J=7.4Hz,1H),1.54(p,J=15.3Hz,2H),1.45(p,J=7.1Hz,2H),1.36–1.24(m,2H).

[0128] Example 20: Preparation of N-{{7-[2-(9H-fluorene-9-yl)acetyl]}amino}heptanoic acid (Wc6)

[0129]

[0130] The white solid compound Wb6 was prepared using the method described for compound Wb1 in Example 15. 722.4 mg, yield 88.7%. ESI-MS (m / z): 430.20 [M+Na] + . 1H NMR(600MHz,DMSO-d6)δ7.92(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.51(d ,J=7.4Hz,2H),7.37(t,J=7.5Hz,2H),7.29(t,J=7.4Hz,2H),4.35(t,J=7.7H z,1H),3.15(q,J=6.6Hz,2H),2.49(d,J=4.2Hz,2H),2.19(t,J=7.4Hz,2H),1 .50(p,J=6.9Hz,2H),1.43(p,J=6.8Hz,2H),1.40(s,9H),1.31–1.26(m,4H).

[0131] The white solid compound Wc6 was prepared using the method described for compound Wc1 in Example 15. 571.2 mg, yield 91.7%. ESI-MS (m / z): 350.10 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ12.01(s,1H),7.93(t,J=5.6Hz,1H),7.87(d,J=7.5H z,2H),7.52(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.29(t,J=7.4Hz,2H),4 .35(t,J=7.8Hz,1H),3.16(q,J=6.6Hz,2H),2.49(d,J=4.5Hz,2H),2.21(t,J =7.4Hz,2H),1.50(p,J=7.3Hz,2H),1.45(p,J=7.0Hz,2H),1.36–1.24(m,4H).

[0132] Example 21: Preparation of N-{{8-[2-(9H-fluorene-9-yl)acetyl]}amino}octanoic acid (Wc7)

[0133]

[0134] The white solid compound Wb7 was prepared using the method described for compound Wb1 in Example 15. 765.8 mg, yield 90.9%. ESI-MS (m / z): 444.20 [M+Na] + . 1H NMR(600MHz,DMSO-d6)δ7.93(t,J=5.7Hz,1H),7.87(d,J=7.5Hz,2H),7.52(d ,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.29(t,J=7.4Hz,2H),4.35(t,J=7.7H z,1H),3.16(q,J=6.5Hz,2H),2.49(d,J=4.6Hz,2H),2.18(t,J=7.3Hz,2H),1 .50(p,J=7.2Hz,2H),1.44(p,J=6.8Hz,2H),1.39(s,9H),1.30–1.25(m,6H).

[0135] The white solid compound Wc7 was prepared using the method described for compound Wc1 in Example 15. 605.5 mg, yield 91.2%. ESI-MS (m / z): 364.15 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ11.99(s,1H),7.93(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.51(d,J=7.5Hz,2H),7.38(t,J=7.4Hz,2H),7.29(t,J=7.4Hz,2 H),4.35(t,J=7.7Hz,1H),3.16(q,J=6.5Hz,2H),2.49(s,2H),2.21(t,J=7 .4Hz,2H),1.50(p,J=7.0Hz,2H),1.44(p,J=7.0Hz,2H),1.36–1.24(m,6H).

[0136] Example 22 Preparation of (Z)-2-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}acetamide (AW1)

[0137]

[0138] Step 1: Preparation of N-{{4-N-{2-[2-(9H-fluoren-9-yl)acetamido]acetamido}benzoyl}amino}tert-butyl acetate (Wd1)

[0139] In a 100 mL round-bottom flask, HATU (570.3 mg, 1.5 mmol), N-{[2-(9H-fluorene-9-yl)acetyl]amino}acetic acid (Wc1) (281.1 mg, 1 mmol), and DMF (5 mL) were added sequentially and stirred for 1 h. Then, tert-butyl 2-(4-aminobenzoylamino)acetate (250.1 mg, 1 mmol) was added and stirred for 30 min. DIPEA (522 μL, 5 mmol) was then added, and the reaction was carried out overnight at room temperature with magnetic stirring. The reaction was monitored by TLC until completion. After quenching with deionized water (20 mL), the mixture was transferred to a separatory funnel and extracted with ethyl acetate (50 mL × 3), retaining the organic layer. The mixture was washed with saturated brine (50 mL × 2), dried over anhydrous sodium sulfate, and then transferred to a 250 mL single-necked flask for vacuum concentration. The residue was purified by column chromatography (dichloromethane:methanol = 50:1) to give 301.2 mg of compound Wd1 as a pale yellow solid powder, with a yield of 58.7%. ESI-MS (m / z): 512.25 [MH] - . 1 H NMR (600MHz, DMSO-d6) δ10.33(s,1H),8.75(t,J=6.0Hz,1H),8.47(t,J=5.8Hz,1 H),7.87(d,J=7.7Hz,2H),7.85(d,J=1.9Hz,2H),7.72(d,J=8.7Hz,2H),7.61(d,J =7.5Hz,2H),7.38(t,J=7.4Hz,2H),7.30(t,J=7.4Hz,2H),4.38(t,J=7.6Hz,1H), 4.05(d,J=5.7Hz,2H), 3.89(d,J=5.9Hz,2H), 2.63(d,J=7.6Hz,2H), 1.42(s,9H).

[0140] Step 2: Preparation of N-{{4-N-{2-[2-(9H-fluoren-9-yl)acetamido]acetamido}benzoyl}amino}acetic acid (We1)

[0141] Compound Wd1 was dissolved in dichloromethane (10 mL), incubated in an ice bath for 30 min, and TFA (3 mL) was added dropwise. The ice bath was removed, and the mixture was transferred to an oil bath and stirred at 55 °C for 1 h. The solution was then concentrated under reduced pressure to obtain 247.1 mg of a pale yellow solid powder, yielding 92.1%. ESI-MS (m / z): 458.20 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ12.47(s,1H),10.34(s,1H),8.75(t,J=5.9Hz,1H),8.47( t,J=5.8Hz,1H),7.88(d,J=3.1Hz,2H),7.86(d,J=4.4Hz,2H),7.73(d,J=8.4Hz,2 H),7.61(d,J=7.5Hz,2H),7.38(t,J=7.4Hz,2H),7.30(t,J=7.4Hz,2H),4.38(t,J =7.6Hz, 1H), 4.05 (d, J = 5.7Hz, 2H), 3.92 (d, J = 5.8Hz, 2H), 2.63 (d, J = 7.6Hz, 2H).

[0142] Step 3: Preparation of (Z)-2-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}acetamide (AW1)

[0143] In a 32 mL pressure-resistant flask, N-{{4-N-{2-[2-(9H-fluoren-9-yl)acetamido]acetamido}benzoyl}amino}acetic acid (We1) (228.5 mg, 0.5 mmol), pyridine-3-carboxaldehyde (69.5 mg, 0.65 mmol), NaOAc (41.0 mg, 0.5 mmol), and Ac2O (1 mL) were added sequentially. The flask was then placed in an oil bath. Under stirring, the reaction temperature was raised to 100 °C, and heating continued for 30 min before the reaction was stopped. The pressure-resistant flask was removed and cooled to room temperature. After the pressure-resistant flask was placed in a refrigerator overnight, ethyl acetate was added, and the mixture was filtered. The filter cake was subjected to column chromatography (dichloromethane:ethyl acetate = 2:1), followed by preparative column separation and purification. The purified product was then dried under vacuum at 25 °C to obtain 142.8 mg of compound AW1 as a yellow solid powder, with a yield of 54.1%. ESI-MS (m / z): 529.15 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.33(s,1H),9.23(s,1H),8.70(d,J=7.9Hz,1H),8.59(d,J=4.9Hz ,1H),8.23(t,J=5.9Hz,1H),8.10(d,J=8.8Hz,2H),7.86(d,J=5.8Hz,2H),7.81(d,J=4.6Hz, 2H),7.58(dd,J=8.1,4.8Hz,1H),7.51(d,J=8.3Hz,1H),7.34(t,J=7.6Hz,2H),7.28(t,J=3. 1Hz, 2H), 7.26 (s, 1H), 4.37 (d, J = 7.8Hz, 1H), 4.06 (t, J = 4.4Hz, 2H), 2.64 (d, J = 10.2Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ172.07,169.08,166.92,164.19,153.11,151.36,147.31,144.64,140.71,138.74,13 5.54,130.25,130.04,127.72,127.58,126.63,125.21,124.45,120.35,119.94,119.75,44.21,43.89,41.27.

[0144] Example 23 Preparation of (Z)-3-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}propionamide (AW2)

[0145]

[0146] The pale yellow solid compound Wd2 was prepared using the method described for compound Wd1 in Example 22. 293.6 mg, yield 55.7%. ESI-MS (m / z): 528.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0147] 10.28(s,1H),8.73(t,J=6.0Hz,1H),8.16(t,J=5.7Hz,1H),7.85(d,J=7.2Hz,2 H),7.83(d,J=1.8Hz,2H),7.73(d,J=8.7Hz,2H),7.52(d,J=7.5Hz,2H),7.34(t, J=7.4Hz,2H),7.24(t,J=7.4Hz,2H),4.35(t,J=7.7Hz,1H),3.88(d,J=5.9Hz,2H ),3.48(q,J=6.3Hz,2H),2.66(t,J=6.6Hz,2H),2.55–2.48(m,2H),1.42(s,9H).

[0148] The pale yellow solid compound We2 was prepared using the method described for compound We1 in Example 22. 238.5 mg, yield 90.9%. ESI-MS (m / z): 472.20 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0149] 12.38(s,1H),10.28(s,1H),8.73(t,J=5.9Hz,1H),8.16(t,J=5.6Hz,1H),7.85( d,J=3.1Hz,2H),7.84(d,J=2.8Hz,2H),7.73(d,J=8.4Hz,2H),7.52(d,J=7.5Hz,2 H),7.34(t,J=7.5Hz,2H),7.24(t,J=7.4Hz,2H),4.36(t,J=7.7Hz,1H),3.91(d,J =5.8Hz, 2H), 3.48 (q, J = 6.4Hz, 2H), 2.66 (t, J = 6.6Hz, 2H), 1.34 (d, J = 8.8Hz, 2H).

[0150] The yellow solid compound AW2 was prepared using the method described for compound AW1 in Example 22. 149.9 mg, yield 55.3%. ESI-MS (m / z): 543.25 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.28(s,1H),9.23(s,1H),8.69(d,J=8.2Hz,1H),8.59(d,J=7.0Hz,1H) ,8.07(d,J=8.4Hz,2H),7.89(t,J=6.3Hz,1H),7.86(d,J=8.5Hz,2H),7.78(d,J=7.6Hz,2H),7.51 (dd,J=8.1,4.8Hz,1H),7.49(d,J=8.3Hz,1H),7.31(t,J=7.6Hz,2H),7.27(s,1H),7.23(t,J=3. 1Hz, 2H), 4.34 (t, J = 7.6Hz, 1H), 3.49 (q, J = 6.4Hz, 2H), 2.66 (d, J = 6.8Hz, 2H), 2.53–2.51 (m, 2H). 13 CNMR(151MHz,DMSO-d6)δ171.48,170.79,166.91,164.21,153.11,151.33,147.37,145.00,140.69,138.70,135.5 5,130.25,129.95,127.66,127.53,126.52,125.08,124.42,120.34,119.83,119.49,44.27,41.33,37.18,35.73.

[0151] Example 24 Preparation of (Z)-4-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}butyramide (AW3)

[0152]

[0153] The pale yellow solid compound Wd3 was prepared using the method described for compound Wd1 in Example 22. 304.7 mg, yield 56.3%. ESI-MS (m / z): 542.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0154] 10.19(s,1H),8.72(t,J=6.0Hz,1H),8.05(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.8 2(d,J=8.6Hz,2H),7.70(d,J=8.6Hz,2H),7.52(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H), 7.29(t,J=7.4Hz,2H),4.36(t,J=7.7Hz,1H),3.88(d,J=5.9Hz,2H),3.24(q,J=6.6Hz, 2H), 2.53 (d, J = 14.8Hz, 2H), 2.40 (t, J = 7.5Hz, 2H), 1.80 (p, J = 7.3Hz, 2H), 1.42 (s, 9H).

[0155] The pale yellow solid compound We3 was prepared using the method described for compound We1 in Example 22. 239.8 mg, yield 87.8%. ESI-MS (m / z): 486.20 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0156] 12.25(s,1H),10.19(s,1H),8.72(t,J=5.9Hz,1H),8.05(t,J=5.6Hz,1H),7.87(d,J=7 .6Hz,2H),7.83(d,J=8.4Hz,2H),7.70(d,J=8.4Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t, J=7.5Hz,2H),7.29(t,J=7.4Hz,2H),4.36(t,J=7.7Hz,1H),3.90(d,J=5.8Hz,2H),3.24 (q, J=6.6Hz, 2H), 2.53 (d, J=15.3Hz, 2H), 2.40 (t, J=7.5Hz, 2H), 1.80 (p, J=7.3Hz, 2H).

[0157] The yellow solid compound AW3 was prepared using the method described for compound AW1 in Example 22. 138.2 mg, yield 49.7%. ESI-MS (m / z): 557.25 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.24(s,1H),9.27(s,1H),8.73(d,J=8.1Hz,1H),8.63(d,J=4.3Hz,1H),8.10( d,J=8.8Hz,2H),7.88(d,J=9.2Hz,2H),7.84(d,J=7.6Hz,2H),7.82(t,J=6.3Hz,1H),7.55(dd,J=8.1,4 .8Hz,1H),7.54(d,J=8.3Hz,1H),7.37(t,J=7.6Hz,2H),7.31(s,1H),7.29(t,J=7.5Hz,2H),4.38(t,J= 7.5Hz, 1H), 3.28 (q, J = 6.6Hz, 2H), 2.56 (d, J = 10.2Hz, 2H), 2.46 (t, J = 7.4Hz, 2H), 1.85 (p, J = 7.2Hz, 2H). 13 C NMR(151MHz,DMSO-d6)δ172.22,171.28,166.92,164.23,153.12,151.34,147.40,145.08,140.72,138.69,135.56,1 30.26,129.96,127.68,127.53,126.49,125.06,124.43,120.37,119.78,119.39,44.27,41.33,38.95,34.72,25.58.

[0158] Example 25 Preparation of (Z)-5-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}pentanamide (AW4)

[0159]

[0160] The pale yellow solid compound Wd4 was prepared using the method described for compound Wd1 in Example 22. 303.7 mg, yield 54.7%. ESI-MS (m / z): 556.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0161] 10.17(s,1H),8.72(t,J=6.0Hz,1H),7.98(t,J=5.7Hz,1H),7.86(d,J=7.5Hz,2H),7.82(d,J =8.6Hz,2H),7.70(d,J=8.7Hz,2H),7.52(d,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.28(t,J= 7.4Hz,2H),4.35(t,J=7.8Hz,1H),3.87(d,J=5.9Hz,2H),3.21(q,J=6.6Hz,2H),2.49(d,J=2 .8Hz, 2H), 2.39 (t, J = 7.4Hz, 2H), 1.64 (p, J = 7.2Hz, 2H), 1.51 (p, J = 7.1Hz, 2H), 1.42 (s, 9H).

[0162] The pale yellow solid compound We4 was prepared using the method described for compound We1 in Example 22. 243.3 mg, yield 89.1%. ESI-MS (m / z): 500.20 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0163] 10.17(s,1H),8.72(t,J=5.9Hz,1H),7.98(t,J=5.7Hz,1H),7.86(d,J=7.5Hz,2H),7.83( d,J=8.4Hz,2H),7.70(d,J=8.3Hz,2H),7.52(d,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.28 (t,J=7.5Hz,2H),4.35(t,J=7.8Hz,1H),3.90(d,J=5.8Hz,2H),3.21(q,J=6.6Hz,2H),2. 49(d,J=15.3Hz,2H), 2.39(t,J=7.4Hz,2H), 1.64(p,J=7.5Hz,2H), 1.51(p,J=7.4Hz,2H).

[0164] The yellow solid compound AW4 was prepared using the method described for compound AW1 in Example 22. 142.8 mg, yield 50.1%. ESI-MS (m / z): 571.35 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.43(s,1H),9.26(s,1H),8.78(d,J=8.1Hz,1H),8.63(d,J=4.7Hz,1H),8.11(d,J=8 .8Hz,2H),7.99(t,J=5.7Hz,1H),7.88(d,J=8.8Hz,2H),7.85(d,J=7.5Hz,2H),7.56(dd,J=8.1,4.8Hz,1H),7 .52(d,J=7.5Hz,2H),7.35(t,J=7.5Hz,2H),7.33(s,1H),7.28(t,J=7.4Hz,2H),4.35(t,J=7.7Hz,1H),3.21( q, J=6.6Hz, 2H), 2.49 (d, J=10.2Hz, 2H), 2.43 (t, J=7.3Hz, 2H), 1.65 (p, J=7.2Hz, 2H), 1.52 (p, J=7.6Hz, 2H). 13 C NMR (151MHz, DMSO-d6) δ172.55,171.16,167.10,164.07,153.18,151.41,147.24,145.01,140.61,138.72,135.63,130. 29,130.10,127.77,127.60,126.21,125.08,124.61,120.52,119.52,119.24,44.11,38.88,36.71,29.20,28.27,22.98.

[0165] Example 26 Preparation of (Z)-6-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}hexanoamide (AW5)

[0166]

[0167] The pale yellow solid compound Wd5 was prepared using the method described for compound Wd1 in Example 22. 300.5 mg, yield 52.8%. ESI-MS (m / z): 570.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0168] 10.14(s,1H),8.71(t,J=6.0Hz,1H),7.96(t,J=5.7Hz,1H),7.86(d,J=7.5Hz,2H),7.81(d,J=8.8 Hz,2H),7.69(d,J=8.7Hz,2H),7.51(d,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.29(t,J=7.4Hz,2H ),4.35(t,J=7.7Hz,1H),3.87(d,J=5.9Hz,2H),3.18(q,J=6.6Hz,2H),2.49(d,J=6.3Hz,2H),2.3 6(t,J=7.4Hz,2H), 1.63(p,J=7.5Hz,2H), 1.49(p,J=7.5Hz,2H), 1.42(s,9H), 1.34(p,J=8.4,2H).

[0169] The pale yellow solid compound We5 was prepared using the method described for compound We1 in Example 22. 247.1 mg, yield 91.2%. ESI-MS (m / z): 514.20 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0170] 10.15(s,1H),8.72(t,J=5.9Hz,1H),7.97(t,J=5.6Hz,1H),7.87(d,J=7.5Hz,2H),7.83(d,J=8 .5Hz,2H),7.69(d,J=8.5Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t,J=7.4Hz,2H),7.30(t,J=7.4H z,2H),4.35(t,J=7.7Hz,1H),3.90(d,J=5.8Hz,2H),3.18(q,J=6.2Hz,2H),2.49(d,J=6.0Hz,2 H), 2.36 (t, J = 7.4Hz, 2H), 1.64 (p, J = 7.5Hz, 2H), 1.50 (p, J = 7.2Hz, 2H), 1.34 (p, J = 4.9Hz, 2H).

[0171] The yellow solid compound AW5 was prepared using the method described for compound AW1 in Example 22. 104.5 mg, yield 48.1%. ESI-MS (m / z): 585.35 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.37(s,1H),9.21(s,1H),8.72(d,J=7.7Hz,1H),8.59(d,J=2.8Hz,1H),8.04(d,J=8.4 Hz,2H),7.94(t,J=5.6Hz,1H),7.83(d,J=9.2Hz,2H),7.80(s,2H),7.51(dd,J=8.1,4.8Hz,1H),7.47(d,J=7.5Hz ,2H),7.32(t,J=7.5Hz,2H),7.27(s,1H),7.25(t,J=7.4Hz,2H),4.31(t,J=7.7Hz,1H),3.15(q,J=6.5Hz,2H),2. 45(d,J=10.2Hz,2H), 2.36(t,J=7.4Hz,2H), 1.61(p,J=7.5Hz,2H), 1.47(p,J=7.1Hz,2H), 1.31(p,J=6.8Hz,2H). 13 C NMR(151MHz,DMSO-d6)δ172.58,171.11,167.08,164.03,153.18,151.38,147.26,145.03,140.62,138.69,135.59,130.27, 130.04,127.75,127.58,126.19,125.06,124.57,120.52,119.49,119.16,44.13,40.93,39.08,37.05,29.45,26.71,25.19.

[0172] Example 27 Preparation of (Z)-7-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}heptanamide (AW6)

[0173]

[0174] The pale yellow solid compound Wd6 was prepared using the method described for compound Wd1 in Example 22. 302.7 mg, yield 51.9%. ESI-MS (m / z): 606.25 [M+Na] + . 1 H NMR (600MHz, DMSO-d6)δ

[0175] 10.13(s,1H),8.71(t,J=6.0Hz,1H),7.93(t,J=5.7Hz,1H),7.86(d,J=7.5Hz,2H),7.81(d,J=8. 7Hz,2H),7.68(d,J=8.7Hz,2H),7.51(d,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.29(t,J=7.4Hz, 2H),4.35(t,J=7.7Hz,1H),3.87(d,J=5.9Hz,2H),3.19–3.15(m,2H),2.49(d,J=2.8Hz,2H),2.3 5(t,J=7.4Hz,2H),1.61(p,J=7.5Hz,2H),1.46(p,J=7.1Hz,2H),1.41(s,9H),1.36–1.24(m,4H).

[0176] The pale yellow solid compound We6 was prepared using the method described for compound We1 in Example 22. 252.3 mg, yield 92.2%. ESI-MS (m / z): 550.20 [M+Na] + . 1 H NMR (600MHz, DMSO-d6)δ

[0177] 10.17(s,1H),8.72(t,J=6.1Hz,1H),7.93(d,J=7.8Hz,1H),7.86(d,J=7.5Hz,2H),7.82(d,J= 8.2Hz,2H),7.69(d,J=8.3Hz,2H),7.51(d,J=7.5Hz,2H),7.37(t,J=7.3Hz,2H),7.29(t,J=7. 4Hz,2H),4.35(t,J=7.8Hz,1H),3.90(d,J=5.8Hz,2H),3.17(q,J=6.6Hz,2H),2.54(d,J=6.0H z, 2H), 2.36 (t, J = 7.4Hz, 2H), 1.61 (p, J = 7.5Hz, 2H), 1.46 (p, J = 7.3Hz, 2H), 1.36–1.30 (m, 4H).

[0178] The yellow solid compound AW6 was prepared using the method described for compound AW1 in Example 22. 153.4 mg, yield 51.3%. ESI-MS (m / z): 599.30 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.16(s,1H),8.81(s,1H),8.11(d,J=8.9Hz,1H),7.95(t,J=5.7Hz,1H),7.88(s,2H), 7.86(d,J=7.7Hz,1H),7.81(d,J=8.5Hz,2H),7.69(d,J=8.4Hz,2H),7.52(dd,J=8.1,4.8Hz,1H),7.50(d,J=7.5 Hz,2H),7.37(t,J=7.5Hz,2H),7.34(s,1H),7.29(t,J=7.5Hz,2H),4.35(t,J=7.7Hz,1H),4.11(q,J=7.1Hz,2H) ,3.96(d,J=5.8Hz,2H),2.35(t,J=7.5Hz,2H),1.60(p,J=7.5Hz,2H),1.47(p,J=6.8Hz,2H),1.35–1.31(m,4H). 13 C NMR(151MHz,DMSO-d6)δ172.28,171.06,165.88,165.71,151.42,150.60,149.47,147.27,143.06,140.63,136.35,130.09,129 .44,127.77,127.57,125.55,125.07,124.08,120.54,119.52,118.60,44.12,40.94,39.10,37.01,29.50,28.98,26.83,25.54.

[0179] Example 28 Preparation of (Z)-8-{N-[(9H-fluorene-9-yl)acetyl]amino}-N-{4-[5-oxo-4-(pyridin-3-yl)methylene-4,5-dihydrooxazol-2-yl]phenyl}octamide (AW7)

[0180]

[0181] The pale yellow solid compound Wd7 was prepared using the method described for compound Wd1 in Example 22. 311.2 mg, yield 52.1%. ESI-MS (m / z): 598.30 [M+H] + . 1 H NMR (600MHz, DMSO-d6)δ

[0182] 10.14(s,1H),8.72(t,J=6.0Hz,1H),7.93(t,J=5.6Hz,1H),7.86(d,J=7.5Hz,2H),7.82(d,J=8.8Hz,2 H),7.69(d,J=8.7Hz,2H),7.51(d,J=7.6Hz,2H),7.36(t,J=7.0Hz,2H),7.28(t,J=7.4Hz,2H),4.35(t ,J=7.7Hz,1H),3.87(d,J=5.9Hz,2H),3.17(q,J=6.5Hz,2H),2.49(d,J=5.4Hz,2H),2.35(t,J=7.4Hz, 2H), 1.62 (p, J = 7.3Hz, 2H), 1.46 (p, J = 7.0Hz, 2H), 1.42 (s, 9H), 1.36–1.30 (m, 4H), 1.25–1.22 (m, 2H).

[0183] The pale yellow solid compound We7 was prepared using the method described for compound We1 in Example 22. 256.9 mg, yield 91.1%. ESI-MS (m / z): 564.25 [M+Na] + . 1 H NMR (600MHz, DMSO-d6)δ

[0184] 10.15(s,1H),8.57(t,J=5.8Hz,1H),7.94(t,J=5.7Hz,1H),7.86(d,J=7.5Hz,2H),7.81(d, J=8.5Hz,2H),7.69(d,J=8.4Hz,2H),7.51(d,J=7.5Hz,2H),7.36(t,J=7.4Hz,2H),7.28(t, J=7.4Hz,2H),4.35(t,J=7.8Hz,1H),3.84(d,J=5.7Hz,2H),3.16(q,J=6.5Hz,2H),2.49(s, 2H), 2.35(t,J=7.4Hz,2H), 1.62(p,J=7.3Hz,2H), 1.46(p,J=7.0Hz,2H), 1.36–1.30(m,6H).

[0185] The yellow solid compound AW7 was prepared using the method described for compound AW1 in Example 22. 165.0 mg, yield 53.9%. ESI-MS (m / z): 613.30 [M+H] + . 1H NMR (600MHz, DMSO-d6) δ10.41(s,1H),9.27(s,1H),8.78(d,J=8.0Hz,1H),8.63(d,J=6.5Hz,1H),8.10(d,J=8. 9Hz,2H),7.94(t,J=5.3Hz,1H),7.86(d,J=4.9Hz,2H),7.85(s,2H),7.56(dd,J=8.0,4.8Hz,1H),7.50(d,J=7.5 Hz,2H),7.36(t,J=7.5Hz,2H),7.33(s,1H),7.29(t,J=7.4Hz,2H),4.35(t,J=7.7Hz,1H),3.17(q,J=6.3Hz,2H) ,2.39(d,J=7.4Hz,2H),2.36(t,J=6.5Hz,2H),1.62(p,J=7.3Hz,2H),1.45(p,J=7.1Hz,2H),1.33–1.30(m,6H). 13 C NMR(151MHz,DMSO-d6)δ172.68,171.04,167.11,164.06,153.20,151.41,147.26,145.04,140.61,138.70,135.64,130.29,130.09 ,127.76,127.56,126.17,125.05,124.60,120.53,119.49,119.18,44.11,40.93,39.10,37.09,29.60,29.20,29.13,26.92,25.45.

[0186] Example 29: Degradation of DAPK1 protein in mouse primary neurons by the compound

[0187] To verify whether the target compound has a degradation effect on DAPK1 protein, primary mouse neurons were used as experimental subjects to detect the degradation effect of the compound on DAPK1. The results are as follows: Figure 1 As shown, compounds AJ2, AJ3, and AJ4 from the AJ series, and AW5 from the AW series, significantly reduced DAPK1 protein levels after treatment at a concentration of 3 μM for 24 h. This experimental result indicates that HyT is highly dependent on the linker; HyT can only induce DAPK1 protein degradation when the linker length is within a suitable range.

Claims

1. A HyT compound that targets and degrades DAPK1 protein, characterized in that, As shown in Equation I-1: XYZ type I-1; Where X represents the ligand of the DAPK1 protein, Z represents the hydrophobic functional group, and Y represents the chain connecting X and Z.

2. The HyT compound for targeted degradation of DAPK1 protein according to claim 1, characterized in that, X is the structure shown in Formula II-1, Z is the structure shown in Formula II-2 or II-3 or a hydrophobic compound of norbornene, norbornane, D-cyclopentaenoic acid, or carborane, and Y is the structure shown in Formula II-4, wherein the N-terminus of Y is connected to Z. Each n is an independent integer between 1 and 7.

3. The HyT compound for targeted degradation of DAPK1 protein according to claim 2, characterized in that, The compound is: Equation 1-1, Z is Equation II-2, n=1 Equation 1-2, Z is Equation II-2, n=2 Equations 1-3, Z is Equation II-2, n=3 Equations 1-4, Z is Equation II-2, n=4 Equations 1-5, Z is Equation II-2, n = 5 Equations 1-6, Z is Equation II-2, n=6 Equations 1-7, Z is Equation II-2, n = 7 Equations 1-8, Z is Equation II-3, n=1 Equations 1-9, Z is Equation II-3, n=2 Equation 1-10, Z is Equation II-3, n=3 Equation 1-11, Z is Equation II-3, n=4 Equation 1-12, Z is Equation II-3, n=5 Equation 1-13, Z is Equation II-3, n=6 Equation 1-14, Z is Equation II-3, n=7.

4. The method for preparing the HyT compound for targeted degradation of DAPK1 protein according to claim 1, characterized in that, The process is as follows:

5. Use of the HyT compound of claim 1, which targets and degrades DAPK1 protein, in the preparation of a medicament for the treatment or prevention of neurodegenerative diseases.

6. The use according to claim 5, characterized in that, The neurodegenerative disease mentioned is Alzheimer's disease.

Images