CN113512035B - Dihydropyrimidine-pomalidomide conjugate, and preparation method and application thereof - Google Patents

Abstract

The invention provides a dihydropyrimidine pomalidomide conjugate, a preparation method and application thereof. The compound has a structure shown in a formula I. The invention also relates to a preparation method of the compound containing the structure shown in the formula I, a pharmaceutical composition and application of the compound in preparing anti-HBV medicines.

Description

Dihydropyrimidine-pomalidomide conjugate and preparation method and application thereof

Technical Field

The present invention belongs to the field of medical technology, and in particular relates to a dihydropyrimidine-pomalidomide conjugate, a preparation method thereof, and an anti-HBV drug use thereof.

Background Art

Viral hepatitis type B (HBV) is a major infectious disease caused by hepatitis B virus (HBV). Long-term development can lead to acute and chronic viral hepatitis, severe hepatitis, cirrhosis and primary hepatocellular carcinoma (HCC). Currently, the drugs used to prevent and treat chronic hepatitis B are mainly vaccines, interferons, immunomodulatory drugs and DNA polymerase inhibitors. However, they have shortcomings such as drug resistance, side effects, rebound after drug withdrawal and inability to completely eliminate HBV. Therefore, the development of a new generation of safe, efficient, low-toxic and anti-resistance non-nucleoside HBV inhibitors has important scientific significance. Core protein is the main structural protein of HBV nucleocapsid, which is relatively conserved during viral evolution, and the assembly of core protein plays an important role in the life cycle of HBV. However, there are currently no drugs targeting related targets on the market.

PROteolysis TArgeting Chimeria (PROTAC) is a technology that uses the intracellular ubiquitin-protease degradation system to target and degrade the target protein. The PROTAC molecule is a bifunctional molecule composed of three parts: a target protein receptor, an intermediate connecting chain, and an E3 ubiquitin protease ligand. It can simultaneously recognize the target protein and the E3 ligase, spatially forming a tripartite complex with them, and then mediating the ubiquitination and degradation of the target protein. PROTAC technology shines in the application of multiple targets due to its multiple advantages. Using the PROTAC strategy for HBV core protein can target the degradation of the viral capsid and utilize the versatility of viral proteins to exert greater antiviral potential.

Among the HBV core protein assembly regulators that have been reported so far, dihydropyrimidines can induce HBV capsid misinduction and exert antiviral effects. By investigating the crystal complex structure of core protein and dihydropyrimidine ligands, it was found that the 6th position of dihydropyrimidine is in the solvent opening area, which is suitable for modification of PROTAC analogs. The 6th position was selected as the connection site to connect the "linker"; "linkers" with different hydrophilicities and different lengths were used to connect core protein ligands and E3 ligase ligands; E3 ligase ligands were used to identify E3 ligases. A total of 15 dihydropyrimidine-pomalidomide conjugates were designed and synthesized. Such compounds have not been reported in the prior art.

Summary of the invention

The present invention provides a dihydropyrimidine-pomalidomide conjugate and a preparation method thereof. The present invention also provides the activity screening results of the above-mentioned compound as a non-nucleoside HBV inhibitor and its pharmaceutical application.

The technical solution of the present invention is as follows:

1. Dihydropyrimidine-pomalidomide conjugate

The dihydropyrimidine-pomalidomide conjugate of the present invention has a structure shown in the following general formula I:

in,

The linker is a fatty amino acid chain with 3-20 main chain atoms, an ethoxy amino acid chain with 4-20 main chain atoms, or an amino acid chain containing 3-20 O, S, and N atoms;

R is a hydrogen atom, pomalidomide, 1-methylpomalidomide, lenalidomide, 1-methyllenalidomide, thalidomide or 1-methylthalidomide.

According to the present invention, preferably, the connecting arm is a fatty amino acid chain with 4-8 main chain atoms, an ethoxy amino acid chain with 5-8 main chain atoms, or an amino acid chain containing 4-8 O, S, and N atoms;

R is a hydrogen atom, pomalidomide or 1-methylpomalidomide.

More preferably, the linker is 8-aminooctanoic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, 2-(2-aminoethoxy)acetic acid;

More preferably, the dihydropyrimidine-pomalidomide conjugate is one of the compounds having the following structures:

Table 1. Structures of target compounds dihydropyrimidine-pomalidomide conjugates

2. Preparation method of dihydropyrimidine-pomalidomide conjugate

The preparation method of the dihydropyrimidine-pomalidomide conjugate comprises the following steps: using 3-fluorophthalic anhydride II-1 as a raw material and 3-amino-2,6-piperidinedione hydrochloride and sodium acetate in an acetic acid solvent, refluxing at 120°C for 10 hours to obtain II-2; dissolving II-2 in an N,N-dimethylformamide solution, using potassium carbonate as a base, adding iodomethane under stirring at room temperature, and reacting at room temperature for 24 hours to obtain an intermediate II-3; using 2-thiazolecarboxamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and ethyl acetoacetate as starting materials, and obtaining a key intermediate 2 through a "Biginelli" reaction cyclization; in a dichloromethane solution, the intermediate 2 undergoes a bromination reaction with N-bromosuccinimide to obtain an important intermediate 3; using intermediate 3 as a raw material, adding potassium carbonate, potassium iodide and 1-Boc piperazine , refluxed at 75°C in acetonitrile solution for 1h to obtain intermediate 4; 4 was dissolved in a dichloromethane solution of trifluoroacetic acid, stirred at room temperature for 10h, and de-Boc was obtained to obtain intermediate 5; the N-Boc-"linker arm" fragment and 2-(7-azabenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) were activated in an ice-water bath for half an hour, and intermediate 5 and N,N-diisopropylethylamine (DIPEA) were added and stirred at room temperature overnight to obtain intermediate 6; 6 was dissolved in a dichloromethane solution of trifluoroacetic acid, stirred at room temperature for 10h, and de-Boc was obtained to obtain the final product 7(a-e); 7(a-e) and II-2 or II-3 were refluxed at 90°C for 10h in N,N-dimethylformamide solution with DIPEA as a base to obtain the final product 8(a-j).

The synthetic route is as follows:

Reagents and conditions: (i) 3-amino-2,6-piperidindione hydrochloride, acetic acid, sodium acetate, 120°C, 10h; (ii) potassium carbonate, methyl iodide, N,N-dimethylformamide, room temperature, 24h; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80°C; (iv) N-bromosuccinimide, dichloromethane, 40°C; (v) 1-tert-butyloxycarbonyl-piperazine, potassium carbonate, Potassium iodide, acetonitrile, 75°C, 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N-tert-butyloxycarbonyl-Linker, HATU, DIPEA, dichloromethane, 0°C, 30min, room temperature, 6h; (viii) trifluoroacetic acid, dichloromethane, room temperature, 10h; (ix) II-2 or II-3, N,N-dimethylformamide, DIPEA, 90°C, 10h.

Wherein, the N-tert-butoxycarbonyl-Linker includes: N-tert-butoxycarbonyl-8-aminooctanoic acid, N-tert-butoxycarbonyl-6-aminohexanoic acid, N-tert-butoxycarbonyl-4-aminobutyric acid, N-tert-butoxycarbonyl-2-(2-(2-aminoethoxy)ethoxy)acetic acid, and N-tert-butoxycarbonyl-2-(2-aminoethoxy)acetic acid.

According to a preferred embodiment of the present invention, the preparation method of the dihydropyrimidine-pomalidomide conjugate of the present invention comprises the following specific preparation steps:

(1) Dissolve 3-fluorophthalic anhydride, 3-amino-2,6-piperidindione hydrochloride, and sodium acetate in acetic acid and reflux at 120° C. for 10 h. After the reaction is completed, separate by flash column chromatography to obtain II-2;

(2) Add II-2 and potassium carbonate to N,N-dimethylformamide solvent, add iodomethane dropwise under stirring at room temperature, and stir at room temperature for 24 hours. After the reaction is completed, extract and separate by flash column chromatography to obtain II-3;

(3) 2-thiazolecarboxamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and sodium acetate were dissolved in anhydrous ethanol, ethyl acetoacetate was added under stirring at room temperature, and the ethanol was refluxed at 80° C. for 8 h. After the reaction was completed, extraction was performed, rapid column chromatography was performed, and recrystallization was performed to obtain 2;

(4) The intermediate 2 was dissolved in dichloromethane, and NBS was added in small amounts several times under stirring at room temperature, and the dichloromethane was refluxed for 1.5 hours. After the reaction was completed, the mixture was extracted, separated by flash column chromatography, and recrystallized to obtain compound 3;

(5) Take intermediate 3 and 1-Boc-piperazine, potassium carbonate, and potassium iodide, add acetonitrile, and reflux at 75°C for 1 hour. After the reaction is completed, extract, separate by flash column chromatography, and recrystallize to obtain compound 4;

(6) Intermediate 4 was dissolved in dichloromethane, trifluoroacetic acid was added, and the mixture was stirred at room temperature for 10 h. After the reaction was completed, saturated sodium carbonate solution and dichloromethane were added for extraction, and the crude product 5 was obtained by spin drying, and the crude product 5 was directly subjected to the next step of reaction;

(7) Dissolve different N-Boc-Linker "connector arms" and HATU in dichloromethane, place in an ice bath, add 5 and DIPEA, and stir at room temperature for 10 h. After the reaction, extract, separate by flash column chromatography, and recrystallize to obtain 6 (a-e);

(8) Intermediate 6 (a-e) was dissolved in dichloromethane, trifluoroacetic acid was added, and the mixture was stirred at room temperature for 10 h. After the reaction was completed, saturated sodium carbonate solution and dichloromethane were added for extraction, and the mixture was separated by preparative TLC and recrystallized to obtain the target compound 7 (a-e);

(9) Compound 7(a-e) and 2 or 3 were dissolved in N,N-dimethylformamide, and DIPEA was added. The mixture was reacted at 90°C for 10 h. After the reaction, the mixture was extracted, separated by flash column chromatography, and recrystallized to obtain the target compound 8(a-j).

The room temperature described in the present invention is 20-30°C.

3. Application of dihydropyrimidine-pomalidomide conjugate

The present invention discloses the anti-HBV activity screening results of a dihydropyrimidine-pomalidomide conjugate and its application as an anti-HBV inhibitor. Experiments prove that the dihydropyrimidine-pomalidomide conjugate of the present invention can be used as a classic HBV non-nucleoside inhibitor.

As shown in Table 2, the synthesized target compounds 7 (a-e) and 8 (a-j) were evaluated for in vitro anti-HBV activity and cytotoxicity. The HBV DNA inhibitory activity was determined by PCR, and the cytotoxicity was determined by MTS. At the same time, the marketed drug lamivudine (3TC) and the clinical candidate drug mofetil mesylate (GLS4) were selected as positive controls.

The newly synthesized dihydropyrimidine-pomalidomide conjugate of the present invention exhibits significant anti-HBV activity. All dihydropyrimidine-PROTAC analogs showed anti-HBV activity at low micromolar concentration levels, with EC ₅₀ values in the range of 0.43-3.77 μM, which was weaker than the activity of GLS4 (EC ₅₀ = 0.046 μM), but the activity of three compounds was close to that of the positive drug 3TC (EC ₅₀ = 0.40 μM), especially 8c (EC ₅₀ = 0.48 μM), 8i (EC ₅₀ = 0.46 μM) and 8j (EC ₅₀ = 0.43 μM), which are worthy of further study.

The dihydropyrimidine-pomalidomide conjugate of the present invention is a class of non-nucleoside HBV inhibitors with novel structures and can be used as a lead compound for anti-HBV.

The dihydropyrimidine-pomalidomide conjugate of the present invention can be used as a non-nucleoside HBV inhibitor, and specifically, used as a HBV inhibitor to prepare anti-hepatitis B drugs.

An anti-HBV pharmaceutical composition comprises the dihydropyrimidine-pomalidomide conjugate of the present invention and one or more pharmaceutically acceptable carriers or excipients.

The present invention discloses a dihydropyrimidine-pomalidomide conjugate, a preparation method thereof, anti-HBV activity screening results and the first application thereof as an anti-HBV inhibitor. Experiments have shown that the dihydropyrimidine-pomalidomide conjugate of the present invention can be used as a HBV inhibitor for preparing anti-hepatitis B drugs.

DETAILED DESCRIPTION

The following examples are helpful for understanding the present invention, but they cannot limit the content of the present invention. In the following examples, the numbers of all target compounds are the same as those in Table 1.

Synthesis route:

Reagents and conditions: (i) 3-amino-2,6-piperidindione hydrochloride, acetic acid, sodium acetate, 120°C, 10h; (ii) potassium carbonate, methyl iodide, N,N-dimethylformamide, room temperature, 24h; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80°C; (iv) N-bromosuccinimide, dichloromethane, 40°C; (v) 1-tert-butyloxycarbonyl-piperazine, potassium carbonate, Potassium iodide, acetonitrile, 75°C, 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N-tert-butyloxycarbonyl-Linker, HATU, DIPEA, dichloromethane, 0°C, 30min, room temperature, 6h; (viii) trifluoroacetic acid, dichloromethane, room temperature, 10h; (ix) II-2 or II-3, N,N-dimethylformamide, DIPEA, 90°C, 10h.

Example 1. Preparation of Compound II-2

3-Fluorophthalic anhydride (200 mg, 1.2 mmol), 3-amino-2,6-piperidindione hydrochloride (259 mg, 1.2 mmol) and sodium acetate (118 mg, 1.44 mmol) were dissolved in 20 ml of acetic acid and refluxed at 120°C for 10 h. After the reaction was completed, the solvent was dried to obtain a large amount of black solid, which was dissolved in a large amount of methanol, and silica gel was added to mix the sample and loaded on the column for rapid column chromatography to obtain II-2.

White solid product, yield 58%; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 11.15 (s, 1H, CONHCO), 7.95 (q, J = 7.4 Hz, 1H, Ph-H), 7.83-7.66 (m, 2H, Ph-H), 5.16 (dd, J = 12.8, 5.1 Hz, 1H, COCHN), 2.97-2.82 (m, 1H, CH ₂ ), 2.68-2.52 (m, 1H, CH ₂ ), 2.18-1.96 (m, 2H, CH ₂ ); EI-MS: 275.06 [MH] ^- ; C ₁₃ H ₉ FN ₂ O ₄ [276.05].

Example 2. Preparation of Compound II-3

II-2 (100.00 mg, 0.36 mmol) and potassium carbonate (50 mg, 0.36 mmol) were added to 5 ml of N,N-dimethylformamide solvent, and iodomethane was added dropwise under stirring at room temperature, and stirred at room temperature for 24 hours. After the reaction was completed, the solvent was dried, and water (20 ml) and dichloromethane (20 ml*2) were added for extraction, and the organic phases were combined, extracted once with saturated sodium chloride, dried with anhydrous magnesium sulfate, and filtered. Silica gel was added to the organic phase and mixed, and rapid column chromatography was performed to obtain II-3.

White solid product, yield 46%; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ 7.96 (tdt, J = 7.4, 4.7, 2.3 Hz, 1H, Ph-H), 7.83–7.70 (m, 2H, Ph-H), 5.30–5.12 (m, 1H, COCHN), 3.08–2.85 (m, 3H, CH ₃ ), 2.85–2.73 (m, 1H, CH ₂ ), 2.66–2.51 (m, 1H, CH ₂ ), 2.08 (tdd, J = 12.7, 7.5, 4.2 Hz, 1H, CH ₂ ); EI-MS: 290.25 [MH] ^- ; C ₁₄ H ₁₁ FN ₂ O ₄ [290.07].

Example 3. Preparation of Compound 2

Weigh 2-thiazolecarboxamidine hydrochloride (1.0 g, 6.11 mmol), 2-bromo-4-fluorobenzaldehyde (1.86 g, 9.16 mmol) and sodium acetate (1.0 g, 1.22 mmol) and dissolve in anhydrous ethanol (100 mL). Add ethyl acetoacetate (1.2 mL, 9.20 mmol) under stirring at room temperature. Reflux the ethanol at 80°C for 8 h. After the reaction is completed, filter and remove the salts. Cool the mother liquor to room temperature, and yellow crystals (I-2) precipitate. Remove anhydrous ethanol from the remaining mother liquor under reduced pressure, add water (60 mL), extract with ethyl acetate (25 mL×3), collect and combine the organic phases, extract once with saturated sodium chloride (25 mL), and dry the organic phase with anhydrous magnesium sulfate. Filter, add 200 mesh silica gel, mix the sample, separate by flash column chromatography, and recrystallize to obtain compound 2. Obtain 0.75 g of yellow powder, yield: 58%; melting point 153-156°C.

¹ H NMR (400MHz, DMSO-d6) δ9.92 (s, 1H), 7.97 (d, J = 2.8Hz, 1H), 7.89 (s, 1H), 7.59–7.50 (m, 1H), 7.42–7.31 (m,1H),7.23(t,J＝8.3Hz,1H),5.98(s,1H),3.94(q,J＝6.9Hz,2H),2.48(s,3H),1.03(t,J＝ 7.0Hz,3H); ^13C NMR (100MHz, DMSO-d6) δ166.07,163.13,159.93,147.99,144.78,143.69,141.19,131.14(d,J＝8.7Hz),124.85,122.94(d,J＝9.6Hz),119.98(d,J＝ 24.2Hz), 115.85 (d, J＝21.0Hz), 97.33, 59.57, 58.14, 17.86, 14.46; EI-MS: 426.04[M+2+H] ⁺ ,C ₁₇ H ₁₅ BrFN ₃ O ₂ S[423.01] .

Example 4. Preparation of Compound 3

Intermediate 2 (1.86 g, 4.39 mmol) was dissolved in dichloromethane (50 mL), and NBS (1.95 g, 1.10 mmol) was added in small amounts several times under stirring at room temperature. The dichloromethane was refluxed for 1.5 h. After the reaction was completed, the dichloromethane was removed under reduced pressure, water (50 mL) was added, and the mixture was extracted with ethyl acetate (20 mL×3). The organic phases were collected and combined, and extracted once with saturated sodium chloride (25 mL). The organic phases were dried over anhydrous magnesium sulfate. Filtered, separated by rapid column chromatography, and recrystallized to obtain compound 3. Yellow solid 1.30 g, yield 59%; melting point 123-128°C.

¹ H NMR (400MHz, CDCl ₃ ) δ7.84 (d, J＝3.1Hz, 1H), 7.52 (s, 2H), 7.44–7.35 (m, 1H), 7.32 (dd, J＝8.1, 2.6Hz, 1H),7.02(t,J＝8.0Hz,1H),6.09(s,1H),4.94(d,J＝8.9Hz,1H),4.61(s,1H),4.09(d,J＝7.0Hz, 2H), 1.16 (t, J=7.1Hz, 3H); EI-MS: 502.2[M+H] ⁺ ; ¹³ C NMR (100MHz, CDCl ₃ )δ164.73,163.27,160.76,155.66,150.28,143.87,143.01,137.84,130.60(d,J＝8.6Hz),124.62,123.45,122.10(d,J＝9.2Hz),120.26(d,J＝24.8Hz) ,115.72(d,J＝20.9Hz),106.39,60.72,51.61,31.79,14.03;EI-MS:499.90[MH] ^- ,501.94[M+2-H] ^- ,503.91[M+4-H] ^- ,C ₁₇ H ₁₄ Br ₂ FN ₃ O ₂ S[500.92].

Example 5. Preparation of Compound 4

Take intermediate 3 (100 mg, 0.2 mmol) and 1-Boc-piperazine (37 mg, 0.2 mmol), potassium carbonate (41 mg, 0.3 mmol), potassium iodide (50 mg, 0.3 mmol), add 10 mL of acetonitrile to obtain a yellow suspension, and reflux at 75 ° C for 1 h. After the reaction is completed, remove acetonitrile under reduced pressure, add water (20 mL), extract with ethyl acetate (20 mL × 3), collect and combine the organic phases, extract once with saturated sodium chloride (25 mL), and then dry with anhydrous magnesium sulfate and filter. Rapid column chromatography separation and recrystallization give compound 4. Obtain 76.2 mg of yellow solid product with a yield of 63%.

¹ H NMR (400MHz, DMSO-d ₆ ) δ9.69(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.93(s,1H,thiazole-H),7.56(d, J＝8.1Hz,1H,Ph-H),7.37(m,1H,Ph-H),7.22(s,1H,Ph-H),6.02(s,1H,CH),4.08–3.79(m,4H ,dihydropyrimidine-CH ₂ , CH ₂ CH ₃ ),3.31(s,4H,CH ₂ NCH ₂ ),2.50(s,4H,2×BocNCH ₂ ),1.39(s,9H,Boc),1.05(t,J ＝6.9Hz,3H,CH ₂ CH ₃ ); EI-MS: 610.01[M+H] ⁺ .

Example 6. Preparation of Compound 5

Take intermediate 4 (100 mg, 0.16 mmol) and dissolve it in 5 mL of dichloromethane. Add 0.5 mL of trifluoroacetic acid dropwise under stirring at room temperature and stir overnight at room temperature. After the reaction is completed, add saturated sodium carbonate solution (20 mL) to produce a large number of bubbles. Extract with dichloromethane (20 mL × 3), collect and combine the organic phases, extract once with saturated sodium chloride (25 mL), dry with anhydrous magnesium sulfate, and filter. Spin dry to obtain crude product 5, which is directly subjected to the next step of reaction.

Example 7. Preparation of Compound 6

Different N-Boc-Linker "connector arms" (68 mg, 0.26 mmol) and HATU (112 mg, 0.26 mmol) were dissolved in dichloromethane, ice-bathed for 30 min, 5 (100 mg, 0.20 mmol) and DIPEA (60 μL, 0.59 mmol) were added, and stirred at room temperature overnight. After the reaction was completed, the solvent was dried, and water (20 ml) and dichloromethane (20 mL×2) were added for extraction. The organic phases were combined, extracted once with saturated sodium chloride, dried with anhydrous magnesium sulfate, and filtered. Rapid column chromatography was performed and recrystallization was performed to obtain 6 (a-e).

The N-Boc-Linker used was N-tert-butyloxycarbonyl-8-aminooctanoic acid. The product 6a was a yellow solid with a yield of 51.7% and a melting point of 73-75°C. ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.01(d,J＝3.0Hz,1H,thiazole-H),7.95(d,J＝2.8Hz,1H,thiazole-H),7.57(dd,J＝8.6,2.8Hz,1H,Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t ,J＝8.4Hz,1H,Ph-H),6.75(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),3.95(q,J＝6.7,5.8Hz,2H, CH ₂ CH ₃ ),3.89(d,J＝17.2Hz,2H,dihydropyrimidine-CH ₂ ),3.52(s,4H, CH ₂ NCO CH ₂ ),2.89(q,J＝6.8Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.69(d,J＝2.1Hz,2H,CO CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.55(s,4H, CH ₂ N CH ₂ ),2.31(t,J＝7.5Hz,2H ,COCH ₂ CH _{2 CH} 2 CH ₂ CH ₂ CH ₂ CH ₂ NH),1.52-1.44(m,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.36(s,9H,Boc),1.25(d,J＝2.2Hz,6H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.05 (td, J=7.1, 2.1Hz, 3H, CH ₂ CH ₃ ); EI-MS: 848.09[M+H] ⁺ ; C ₃₄ H ₄₆ BrFN ₆ O ₅ S[848.24].

The N-Boc-Linker used was N-tert-butyloxycarbonyl-6-aminohexanoic acid. The product 6b was a yellow solid with a yield of 76% and a melting point of 53-55°C. ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.66(s,1H,dihydropyrimidine-H),8.02(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(d,J＝8.5Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.24(s,1H,Ph-H),6.77(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH ),3.99–3.93(m,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.55–3.50(m,4H, CH ₂ NCO CH ₂ ),2.89(q,J＝6.9Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.69(d,J＝2.0Hz,2H,CO CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.57(s,4H, CH ₂ N CH ₂ ),2.3 2(s,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.53–1.45(m,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.37(s,9H,Boc),1.26(q,J＝7.2Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.09–1.02(m ,3H,CH ₂ CH ₃ ); EI-MS: 721.19[M+H] ⁺ ; C ₃₂ H ₄₂ BrFN ₆ O ₅ S[720.21].

The N-Boc-Linker used was N-tert-butyloxycarbonyl-4-aminobutyric acid. The product 6c was a yellow solid with a yield of 88.7% and a melting point of 100-108°C. ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.23(s,1H,Ph-H),6.81(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH ),3.96(s,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.52(s,4H, CH ₂ NCO CH ₂ ),2.93(q,J＝6.7Hz,2H,COCH ₂ CH ₂ CH ₂ NH),2.69(d,J＝1.9Hz,4H, CH ₂ N CH ₂ ),2.32(s,2H,CO CH ₂ CH ₂ CH ₂ NH),1.62(q,J＝7.2Hz,2H,COCH ₂ CH ₂ CH ₂ NH),1.37 (s,9H,Boc),1.05(t,J=7.3Hz,3H,CH ₂ CH ₃ ); EI-MS: 693.25[M+H] ⁺ ; C ₃₀ H ₃₈ BrFN ₆ O ₅ S[692.18].

The N-Boc-Linker used was N-tert-butyloxycarbonyl-2-(2-(2-aminoethoxy)ethoxy)acetic acid. The product 6d was a yellow solid with a yield of 48% and a melting point of 73-75°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.3Hz,1H,Ph-H),7.41(s,1H,Ph-H),7.22(s,1H,Ph-H),6.76(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.17(s,2H,COCH ₂ O),4.04–3.86(m,4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.54 (d, J = 10.3Hz, 8H, CH ₂ NCO CH ₂ ,OCH ₂ CH ₂ O CH ₂ CH ₂ N), 3.38 (d, J = 6.4Hz, 2H, O CH ₂ CH ₂ OCH ₂ CH ₂ N), 3.07 (q, J = 6.3Hz, 2H, OCH ₂ CH ₂ OCH ₂ CH ₂ N), 1.36 (s, 9H, Boc), 1.05 (t, J＝7.3Hz, 3H, CH ₂ CH ₃ ); EI-MS: 755.18[M+2+H] ⁺ ; C ₃₂ H ₄₂ BrFN ₆ O ₇ S[752.20].

The N-Boc-Linker N-tert-butyloxycarbonyl-2-(2-aminoethoxy)acetic acid used, the product 6e was a yellow solid, the yield was 93%, the melting point was 53-55°C; ¹ H NMR (400MHz, DMSO-d ₆ )δ9.66(s,1H,dihydropyrimidine-H),8.04–7.99(m,1H,thiazole-H),7.95(s,1H,thiazole-H),7.57(d,J＝8.5Hz,1H,Ph-H),7.40(s,1H,Ph-H),7.23(s,1H,Ph-H),6.82(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),4.15(s,2H,COCH ₂ O),3.98–3.92(m,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.49-3.43(m,6H, CH ₂ NCO CH ₂ ,O CH ₂ CH ₂ N),3.09(q,J＝6.3,5.8Hz,2H,OCH ₂ CH ₂ N),2.69(d,J＝1.9Hz,2H),2.55(s,4H, CH ₂ N CH ₂ ),1.35(s ,9H,Boc),1.05(t,J=7.1Hz,3H,CH ₂ CH ₃ ); EI-MS: 708.92[M+H] ⁺ ;C ₃₀ H ₃₈ BrFN ₆ O ₆ S[708.17].

Example 8. Preparation of Compound 7 (a-e)

Take intermediate 6 (a-e) (400mg, 0.56mmol) and dissolve it in 20ml dichloromethane. Add 2mL of trifluoroacetic acid dropwise under stirring at room temperature and stir at room temperature overnight. After the reaction is completed, add saturated sodium carbonate solution (20mL) to produce a large number of bubbles. Extract with dichloromethane (20mL×3), collect and combine the organic phases, extract once with saturated sodium chloride (25mL), dry with anhydrous magnesium sulfate, and filter. Evaporate the excess solvent under reduced pressure, leaving 2mL of solvent, separate using preparative TLC, and recrystallize to obtain the target compound 7 (a-e).

7a is a yellow solid with a yield of 61.7% and a melting point of 98-100°C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.00 (s, 1H, thiazole-H), 7.95 (s, 1H, thiazole-H), 7.55 (d, J＝8.2 Hz, 1H, Ph-H), 7.41 (t, J＝6.9 Hz, 1H, Ph-H), 7.22 (t, J＝7.8 Hz, 1H, Ph-H), 6.75 (s, 1H, NH ), 6.04 (s, 1H, dihydropyrimidine-CH ), 4.05–3.83 (m, 4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.53 (s, 4H, CH ₂ NCO CH ₂ ), 2.80–2.70 (m, 2H, COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.53(d,J＝18.8Hz,4H, CH ₂ N CH ₂ ),2.31(s,2H,CO CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.53(d,J＝24.3Hz,4H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.28 (s, 6H, COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.04 (t, J＝6.5Hz, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ171.20,165.64,162.50,160.04,146.77,144.40,144.08,140.64,131.43,125.24,123.00,120.20,119.96,116.07,115.86,97.73,59.88,58. 66,55.84,53.46,53.04,45.63,41.66,32.71,29.10,28.91,28.00,26.32,25.15,14.45; EI-MS: 649.10[M+H] ⁺ ; C ₂₉ H ₃₈ BrFN ₆ O ₃ S[648.19].

7b is a yellow solid with a yield of 76% and a melting point of 95-100°C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.01 (d, J=2.8 Hz, 1H, thiazole-H), 7.95 (d, J=3.5 Hz, 1H, thiazole-H), 7.57 (d, J=8.6 Hz, 1H, Ph-H), 7.39 (t, J=7.1 Hz, 1H, Ph-H), 7.22 (t, J=8.7 Hz, 1H, Ph-H), 6.03 (s, 1H, dihydropyrimidine-CH ), 3.95 (q, J=9.0, 8.4 Hz, 4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.52 (s, 4H, CH ₂ NCO CH ₂ ),2.69–2.51(m,6H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH, CH ₂ N CH ₂ ),2.32(t,J＝7.0Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.47(dt,J＝24.4,8.0Hz,4H,COCH _{2 CH} 2 CH ₂ CH ₂ CH ₂ NH ), 1.28 (dd, J＝20.1, 12.1Hz, 2H, COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.04 (t, J＝7.1Hz, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ171.06,165.64,162.50,160.04,146.78,144.40,144.08,140.64,125.25,120.20,119.96,116.07,115.86,97.72,59.88,58.66,55.84,53.43 ,53.02,45.62,41.69,32.50,28.05,26.13,24.72,14.45; EI-MS:622.92[M+2+H] ⁺ ; C ₂₇ H ₃₄ BrFN ₆ O ₃ S[620.16].

7c is a yellow solid with a yield of 52.0% and a melting point of 128-135°C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ7.99 (d, J=24.0 Hz, 2H, thiazole-H), 7.58 (d, J=8.6 Hz, 1H, Ph-H), 7.40 (t, J=7.5 Hz, 1H, Ph-H), 7.23 (t, J=8.7 Hz, 1H, Ph-H), 6.04 (s, 1H, dihydropyrimidine-CH ), 3.96 (q, J=8.9 Hz, 4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.53 (s, 4H, CH ₂ NCO CH ₂ ), 2.72–2.52 (m, 6H, COCH ₂ CH ₂ CH ₂ NH, CH ₂ N CH _{2 13 C NMR ​ ​ ​ ​ ​} ^​ _​ )δ170.40,167.42,165.64,162.51,160.04,146.75,144.40,144.06,140.65,132.18,131.98,129.13,125.23,123.11,123.01,120.20,119.95,1 16.05,115.84,97.73,65.49,59.87,58.68,55.82,53.29,52.96,45.47,41.77,30.48,29.79,23.24,19.12,14.45,14.00; EI-MS:592.97[M+H] ⁺ ; C ₂₅ H ₃₀ BrFN ₆ O ₃ S[592.13].

7d is a yellow solid with a yield of 81.7% and a melting point of 60-68°C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.07 (d, J＝22.0 Hz, 2H, thiazole-H), 7.66 (d, J＝8.6 Hz, 1H, Ph-H), 7.48 (t, J＝7.4 Hz, 1H, Ph-H), 7.34–7.26 (m, 1H, Ph-H), 6.11 (s, 1H, dihydropyrimidine-CH ), 4.29 (s, 2H, COCH ₂ O), 4.03 (q, J＝7.7 Hz, 4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.64 (d, J＝27.7 Hz, 10H, CH ₂ NCO CH ₂ , O CH ₂ CH ₂ O CH ₂ CH ₂ N), 2.98 (s, 2H, OCH ₂ CH ₂ OCH ₂ CH ₂ N), 2.64 (s, 4H, CH ₂ N CH ₂ ), 1.13 (t, J＝7.0Hz, 3H, CH ₂ CH ₃ ); 13C NMR (100MHz, DMSO-d ₆ )δ167.99,165.65,162.51,160.04,146.74,144.39,144.07,140.65,140.61,131.44,131.36,125.26,120.20,119.96,116.08,115.87,97.73,70 .25,69.90,69.62,69.19,59.89,58.66,55.82,53.23,52.91,44.90,41.80,14.45; EI-MS: 653.18[M+H] ⁺ ; C ₂₇ H ₃₄ BrFN ₆ O ₅ S[652.15].

7e is a yellow solid with a yield of 58.8% and a melting point of 121-129°C; ¹ H NMR (400 MHz, DMSO-d ₆ ) δ8.03–7.94 (m, 2H, thiazole-H), 7.58 (d, J=8.5 Hz, 1H, Ph-H), 7.40 (t, J=7.4 Hz, 1H, Ph-H), 7.23 (t, J=8.7 Hz, 1H, Ph-H), 6.04 (s, 1H, dihydropyrimidine-CH ), 4.25 (s, 2H, COCH ₂ O), 3.96 (q, J=7.3, 6.7 Hz, 4H, CH ₂ CH ₃ , dihydropyrimidine-CH ₂ ), 3.61–3.41 (m, 6H, CH ₂ NCO CH ₂ , O CH ₂ CH ₂ N), 2.86 (t, J = 5.4Hz, 2H, OCH ₂ CH ₂ N), 2.69 (s, 2H, NH ₂ ), 2.56 (d, J = 13.5Hz, 4H, CH ₂ N CH ₂ ), 1.05 (t, J = 7.1Hz, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ168.38,165.64,162.52,160.04,146.69,144.39,144.04,140.64,131.34,125.24,123.11,120.20,119.96,116.05,115.84,97.81,69.08,67. 89, 67.78, 63.28, 59.88, 58.69, 55.80, 53.10, 44.67, 14.45; EI-MS: 608.92[M+H] ⁺ ; C ₂₅ H ₃₀ BrFN ₆ O ₄ S[608.12].

Example 9. Preparation of Compound 8 (a-j)

Take 7(a-e) (123 mg, 0.16 mmol) and 2 (57 mg, 0.20 mmol) or 3 (60 mg, 0.20 mmol) and dissolve them in N,N-dimethylformamide, add DIPEA (38 μl, 0.29 mmol) under stirring, and react at 90°C for 10 h. After the reaction, spin dry the solvent, add water (20 mL) and EA (20 mL*2) for extraction, combine the organic phases, extract once with saturated sodium chloride, dry with anhydrous magnesium sulfate, and filter. Add silica gel to the organic phase, mix the sample, separate by flash column chromatography, and recrystallize to obtain the target compound 8(a-j).

8a is a yellow solid with a yield of 41.7% and a melting point of 119-120°C; ¹ H NMR (400MHz, DMSO-d ₆ )δ11.10(s,1H,CONHCO),9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.58(t,J＝7.8Hz,2H,Ph-H,Pomalidomide-Ph-H),7.44–7.35(m,1H,Ph-H),7.22(t,J＝8.4Hz,1H,Ph-H), 7.10(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.53(s,1H,NH),6.03(s,2H,dihydropyrimidine-CH),5.05(dd,J＝12.7,4.8Hz,1H,Pomalidomide-CH),3 .95(q,J＝9.4,8.4Hz,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.52(s,4H, CH ₂ NCO CH ₂ ),3.30(d,J＝6.4Hz,2H,Pomalidomide-CH ₂ ),2.95–2.84(m,1H,Pomalidomide-CH ₂ ),2.64–2.53(m,4H, CH ₂ NCO CH ₂ ),2.31 (t,J＝7.1Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.07–1.99(m,1H,Pomalidomide-CH ₂ ),1.58(s,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.50(s,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.28 (d, J＝34.5Hz, 6H, COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.04 (t, J＝6.9Hz, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,1 29.28,126.57,126.51,125.44,124.96,122.52, 121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,30.12,29.30,28.98, 26.77, 26.02, 24.30, 14.46; EI-MS: 906.97[M+2+H] ⁺ ; C ₄₂ H ₄₆ BrFN ₈ O ₇ S[904.24].

8b is a yellow solid with a yield of 20.6% and a melting point of 114-117°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ11.11(s,1H,CONHCO),9.73(s,1H,dihydropyrimidine-H),8.09(s,2H,thiazole-H),7.66–7.55(m,2H,Ph-H,Pomalidomide-Ph-H),7.51(s,1H,Ph-H),7.29(s,1H,Ph-H),7.11(d,J＝8.4Hz,1H,Po malidomide-Ph-H),7.04(d,J＝6.9Hz,1H,Ph-H),6.55(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.0,5.0Hz,1H,Pomalidomide-CH),4.23(t,J＝6.4Hz,2H,dihydropyrimidine-CH) ₂ ),4.02(d,J＝6.7Hz,2H, CH ₂ CH ₃ ),3.32(d,J＝5.7Hz,4H, CH ₂ NCO CH ₂ ),2.89(t,J＝13.0Hz,1H,Pomalidomide-CH ₂ ),2.60(d,J＝17.6Hz,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 2.39(s,2H,CO CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.03(d,J＝7.7Hz,1H,Pomalidomide-CH ₂ ),1.69–1.52(m,4H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.39(s,2H,Pomalidomide-CH ₂ ),1.24(s,2H, COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH), 1.10 (d, J=6.3Hz, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ ) δ 174.13, 173.76, 171.78, 169.15, 166.82, 166.78, 162.39, 160.37, 152. 23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,129.28,126.57,126.51,125.44,124.96,1 22.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.58,36.13,30.69,28.98,26.02, 24.80, 24.30, 14.46; EI-MS: 878.95[M+2+H] ⁺ ; C ₄₀ H ₄₂ BrFN ₈ O ₇ S[876.21].

8c is a yellow solid with a yield of 26.6% and a melting point of 76-82°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ11.11(s,1H,CONHCO),9.69(s,1H,dihydropyrimidine-H),8.08(s,2H,thiazole-H),7.61(t,J＝7.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.50(s,1H,Ph-H),7.29(s,1H,Ph-H),7.19(d,J＝8.5Hz,2H,Ph-H,Pomalidomi de-Ph-H),7.04(d,J＝6.8Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.06(dd,J＝13.1,5.0Hz,1H,Pomalidomide-CH),4.58(s,2H),4.23(t,J＝6.4 Hz,4H,dihydropyrimidine-CH ₂ ),4.01(d,J＝6.3Hz,2H, CH ₂ CH ₃ ),3.35(d,J＝6.1Hz,4H, CH ₂ NCO CH ₂ ),2.96–2.57(m,3H,Pomalidomide-CH ₂ ,COCH ₂ CH ₂ CH ₂ NH),2.48(s,2H,CO CH ₂ CH ₂ CH ₂ NH),2.0 7–2.00(m,1H,Pomalidomide-CH ₂ ),1.88–1.76(m,2H,COCH ₂ CH ₂ CH ₂ NH),1.09(t,J＝6.4Hz,3H,CH ₂ CH ₃ );13C NMR(100MHz,DMSO-d ₆ )δ174.13,173.76,171.78,169.15,166.82,166.78,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,1 29.28,126.57,126.51,125.44,1 24.96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,52.71,45.07,41.56,34.75,30.69,25.15 ,24.30,14.46;EI-MS:848.93[M+H] ⁺ ;C ₃₈ H ₃₈ BrFN ₈ O ₇ S[848.18].

8d is a yellow solid with a yield of 13.0% and a melting point of 122-129°C; ¹ H NMR (400MHz, DMSO-d ₆ )δ11.10(s,1H,CONHCO),9.65(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.93(t,J＝2.8Hz,1H,thiazole-H),7.57(ddd,J＝8.7,5.9,2.5Hz,2H,Ph-H,Pomalidomide-Ph-H),7.39(dd,J＝8.8,6.3Hz,1H,Ph-H),7.25–7.1 8(m,1H,Ph-H),7.13(d,J＝8.6Hz,1H,Pomalidomide-Ph-H),7.03(dd,J＝7.1,2.1Hz,1H,Pomalidomide-Ph-H),6.62(d,J＝6.3Hz,1H,NH),6.02(s,1H,dihydropyrimidine-CH),5.05(dd,J＝ 13.0,5.4Hz,1H,Pomalidomide-CH),4.17(s,2H,COCH ₂ O),4.00–3.88(m,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.65–3.44(m,12H, CH ₂ NCO CH ₂ ,O CH ₂ CH ₂ O CH ₂ CH ₂ N),2.93–2.82(m,1H,Pomalidomide-CH ₂ ),2.69–2.52(m,6H , Pomalidomide-CH ₂ , CH ₂ N CH ₂ ), 2.08–1.90 (m, 1H, Pomalidomide-CH ₂ ), 1.04 (td, J＝7.1, 2.1Hz, 3H); ¹³ C NMR (100MHz, DMSO-d ₆ )δ173.20,170.49,169.42,167.82,167.74,165.62,162.52,160.03,146.89,146.68,144.37,144.03,140.64,136.68,132.58,131.43,125.17,1 23.10,123.01,120.19, 119.95,117.87,116.03,115.82,111.15,109.78,97.75,70.31,70.05,69.96,69.33,59.86,58.69,55.83,49.07,42.24,41.80,31.47,22.63,1 4.44; EI-MS: 909.10[M+H] ⁺ ; C ₄₀ H ₄₂ BrFN ₈ O ₉ S[908.20].

8e is a yellow solid with a yield of 4.3% and a melting point of 62-68°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ11.10(s,1H,CONHCO),9.66(s,1H,dihydropyrimidine-H),8.05–7.92(m,2H,thiazole-H),7.66–7.50(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.6Hz,1H,Ph-H),7.23(d,J＝8.5Hz,1H,Ph-H),7.18(t,J ＝9.5Hz,1H,Pomalidomide-Ph-H),7.04(d,J＝7.1Hz,1H,Pomalidomide-Ph-H),6.70(d,J＝6.2Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.05(dd,J＝12.9,5.5Hz,1H,Pomalidomide-CH),4. 24(s,2H,COCH ₂ O),4.00–3.81(m,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.72–3.63(m,2H,O CH ₂ CH ₂ N),3.56–3.46(m,4H, CH ₂ NCO CH ₂ ),2.92–2.81(m,1H,Pomalidomide-CH ₂ ),2.56(d, J＝17.2Hz, 6H, CH ₂ N CH ₂ ,OCH ₂ CH ₂ N), 2.01 (d, J＝11.7Hz, 1H, Pomalidomide-CH ₂ ), 1.12–0.97 (m, 3H, CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ173.22,170.51,169.36,167.79,167.75,165.63,162.50,160.04,146.87,146.73,144.39,144.05,140.65,136.68,132.61,131.36,125.20,1 23.11,123.01,120.20,119.96,11 7.90,116.05,115.84,111.15,109.75,97.72,69.66,69.54,67.77,63.27,59.87,58.68,55.82,53.23,49.05,44.91,42.24,41.80,41.02,31.4 6,22.64,14.44;EI-MS:865.10[M+H] ⁺ ;C ₃₈ H ₃₈ BrFN ₈ O ₈ S[864.17].

8f is a yellow solid with a yield of 8% and a melting point of 85-90°C; ¹ HNMR (400MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.00(t,J＝2.7Hz,1H,thiazole-H),7.94(d,J＝2.7Hz,1H,thiazole-H),7.64–7.49(m,2H,Ph-H,Pomalidomide-Ph-H),7.39(t,J＝7.3Hz,1H,Ph-H),7.22(t,J＝8.7Hz,1H,Ph-H),7. 10(d,J＝8.5Hz,1H,Pomalidomide-Ph-H),7.02(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.54(d,J＝5.6Hz,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.32(dd,J＝12.7,4.8Hz,1H,Pomalidomide -CH),4.03–3.84(m,4H, _{2 ... ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​ ​} NH),1.53(d,J＝34.6Hz,4H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.30(d,J＝19.0Hz,6H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.09–1.00(m,3H,CH ₂ CH ₃ ); ¹³ CNMR(1 00MHz, DMSO-d ₆ )δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65,131.84,129.35,1 29.28,126.57,126.51,125.44,124.96,122.52,121 .71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.58,36.13,30.50,30.12,29.30,28.98,28. 93, 26.77, 26.02, 24.21, 14.46; EI-MS: 920.99[M+2+H] ⁺ ; C ₄₃ H ₄₈ BrFN ₈ O ₇ S[918.25].

8g as yellow solid, yield 14.5%, melting point 128-130°C; ¹ HNMR (400MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),7.98(d,J＝24.6Hz,2H,thiazole-H),7.58(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.11(s,1H,Ph-H),7.03(s,1H,Ph-H),6.55(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(s,1H,Pomalidomide-CH),3.94(s,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.53(s,4H, CH ₂ NCO CH ₂ ),3.02(s,3H,Pomalidomide-CH ₃ ),2.95(s,1H,Pomalidomide-CH ₂ ),2.76(d,J＝16.1Hz,1H,Pomalidomide-CH ₂ ),2.54(s,4H, CH ₂ N CH ₂ ),2.34(s,2H ,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),2.05(s,2H,CO CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.58(s,4H,Pomalidomide-CH ₂ ,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.38(s,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.24(s,2H,COCH ₂ CH ₂ CH ₂ CH ₂ CH ₂ NH),1.05(s,3H,CH ₂ CH ₃ ); ¹³ CNMR(100MHz,DMSO-d ₆ )δ172.26,171.11,170.30,169.40,167.74,165.63,162.50,16 0.03,146.93,146.79,144.38,144.07,140.66,140.62,136.77,132.66,131.43,125.21,123.11,123.01,120.20,119.96,11 7.68,116.05,115.84,110.86,109.44,97.69,59.87,58.66,55.85,53.43,53.03,49.58,45.61,42.26,41.67,32.63,31.60,29.03,27.06,26.5 8, 24.98, 21.86, 14.44; EI-MS: 890.94[M+H] ⁺ ; C ₄₁ H ₄₄ BrFN ₈ O ₇ S[890.22].

8h is a yellow solid, yield 26.8%, melting point 108-114°C; ¹ H NMR (400MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.01(s,1H,thiazole-H),7.95(s,1H,thiazole-H),7.69–7.48(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝7.2Hz,1H,Ph-H),7.29–7.12(m,2H,Ph-H,Pomalidomide-Ph-H),7.03(d,J＝7.0Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.13(dd,J＝12.7,4.7Hz,1H,Pomalidomide-CH),3.95(q,J＝7.2Hz,4H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.52(s,4H, _{CH 2} NCO _{CH 2} ),3.01(s,3H,Pomalidomide-CH ₃ ),2.94–2.62(m,2H,Pomalidomide-CH ₂ ) _, 2.55(s,4H, CH _{2 NCO CH} ² _{) ,} 2.40(d, _{J ＝} 23.7Hz _, 2H, _{COCH 1 3C} NMR (100MHz, DMSO-d ₆ ) δ172.68,171.78,169.15,166.82,166.78,166.64,162.39,160.37,152.23,146.11,143.15,142.71,138.89,138.87,138.65, 131.84,129.35,129.28,126.57,126.51,125.44,124. 96,122.52,121.71,120.94,120.78,113.30,113.14,112.47,100.34,65.57,61.37,60.21,52.75,51.73,45.07,41.56,34.75,30.50,28.93,25 .15,24.21,14.46;EI-MS:862.90[M+H] ⁺ ;C ₃₉ H ₄₀ BrFN ₈ O ₇ S[862.19].

8i is a yellow solid with a yield of 4.8% and a melting point of 90-104°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.66(s,1H,dihydropyrimidine-H),8.00(s,1H,thiazole-H),7.94(s,1H,thiazole-H),7.57(s,2H,Ph-H,Pomalidomide-Ph-H),7.40(s,1H,Ph-H),7.22(s,1H,Ph-H),7.14(s,1H,P omalidomide-Ph-H),7.04(s,1H,Pomalidomide-Ph-H),6.62(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(s,1H,Pomalidomide-CH),4.17(s,1H,COCH ₂ O),3.94(s,4H, CH ₂ CH ₃ ,dihydropyrimidine -CH ₂ ),3.64(s,8H,OCH ₂ CH ₂ OCH ₂ CH ₂ N)3.34(s,4H, CH ₂ NCO CH ₂ ),3.01(s,3H,Pomalidomide-CH ₃ ),2.76(s,1H,Pomalidomide-CH ₂ ),2.02(s,2H,Pomalidomide-CH ₂ ),1.04(s,3H,CH ₂ CH ₃ ); ¹³ C NMR (100MHz, DMSO-d ₆ )δ172.23,170.26,169.41,167.82,167.71,165.62,162.51,160.03,146.90,146.68,144.36,144.02,140.67,136.71,132.55,131.43,125.18,1 23.10,120.18,119.94,117.89,116 .02,115.82,111.17,109.72,97.72,70.30,70.06,69.96,69.32,59.86,58.68,55.83,53.23,52.92,49.62,44.99,42.23,41.79,31.61,27.05, 21.84,14.43; EI-MS:924.95[M+2+H] ⁺ ; C ₄₁ H ₄₄ BrFN ₈ O ₉ S[922.21].

8j is a yellow solid with a yield of 19.8% and a melting point of 78-80°C; ¹ H NMR (400 MHz, DMSO-d ₆ )δ9.67(s,1H,dihydropyrimidine-H),8.00(s,2H,thiazole-H),7.94(s,1H,Ph-H),7.67–7.52(m,2H,Ph-H,Pomalidomide-Ph-H),7.40(t,J＝6.9Hz,1H,Ph-H),7.28–7.13(m,1H,P omalidomide-Ph-H),7.05(d,J＝6.7Hz,1H,Pomalidomide-Ph-H),6.69(s,1H,NH),6.03(s,1H,dihydropyrimidine-CH),5.12(dd,J＝12.7,4.6Hz,3H,Pomalidomide-CH),4.24(s,2H,COCH ₂ O),3.93(dd,J＝15.1,11.3Hz,5H, CH ₂ CH ₃ ,dihydropyrimidine-CH ₂ ),3.70(d,J＝17.1Hz,2H,O CH ₂ CH ₂ N),3.50(d,J＝20.7Hz,6H,OCH ₂ CH ₂ N, CH ₂ NCO CH ₂ ),3.00(s,2H,Poma lidomide-CH ₃ ), 2.73 (d, J = 16.8Hz, 1H, Pomalidomide-CH ₂ ), 2.53 (d, J = 8.3Hz, 4H, CH ₂ N CH ₂ ), 2.02 (s, 1H, Pomalidomide-CH ₂ ), 1.05 (t, J = 6.8Hz, 3H, CH ₂ CH ₃ ); 13C NMR (100MHz, DMSO- d ₆ )δ172.22,170.27,169.34,167.75,167.71,165.62,162.50,160.04,146.88,146.76,144.38,144.05,140.66,136.71,132.59,131.43,131.3 4,125.22,123.11,123.01,120.20,119.96,117.9 4,116.05,115.84,111.17,109.69,97.65,69.72,69.53,67.77,63.27,59.86,58.66,55.82,53.22,52.87,49.60,44.94,42.21,41.81,41.01,3 1.60, 27.05, 21.84, 14.44; EI-MS: 878.91[M+H] ⁺ ; C ₃₉ H ₄₀ BrFN ₈ O ₈ S[878.19].

Example 10. In vitro anti-HBV activity test of the target compound (HepDES19 cells)

Test principle: HepDES19 cells are HepG2 (human hepatoblastoma) cell line derivatives that are stably transfected with HBV D-type genomic genes under the control of tetracycline (repressible promoter) ¹ . In the absence of tetracycline, HBV replication is induced in HepDES19 cells, compounds are added, and cells are incubated for 3 days. The HBV DNA content expressed by the cells and the survival status of the cells will ^change2 . The HBV DNA content, that is, the effectiveness of the compound in inhibiting HBV replication, is analyzed by quantitative polymerase chain reaction (qPCR). The concentration of the compound required to reduce HBV DNA to half is obtained, which is the half effective concentration (EC ₅₀ ), indicating the anti-HBV activity of the compound. The toxicity of the compound to cells is tested by the MTS method, and the concentration required for the compound to kill half of the cells is obtained, which is the half lethal concentration (CC ₅₀ ), indicating the cytotoxicity of the compound. (Guo H, Jiang D, Zhou T, et al. Journal of virology 2007; 81(22): 12472-12484; Edwards TC, Lomonosova E, Patel JA, et al. Antiviral research 2017; (143): 205-217.)

Experimental methods:

(1) Cell culture. The cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium supplemented with 10% fetal bovine serum (FBS), 1% penicillin/streptomycin (P/S), and 1 μg/mL tetracycline. Synchronous expression of HBV pgRNA was induced by removing tetracycline from the culture medium.

(2) Cell culture with drugs. In the absence of tetracycline, HepDES19 cells were seeded in a 96-well plate at a density of 4 x 10 ⁴ cells per well. After inducing HBV replication for 48 hours, a compound solution with a final DMSO concentration of 1% was added and incubated with the cells for 3 days.

(3) Cell activity (EC ₅₀ ) test method. Cells were washed in 200 μL phosphate buffered saline (PBS) and lysed in 150 μL core lysis buffer (10 mM Tris pH 7.4, 1% Tween 20, 150 mM NaCl). The cells were incubated at 350 rpm for 40 minutes on an orbital shaker at 20-23°C. The cell lysate was transferred to a 96-well PCR plate and centrifuged at 3300×g for 5 minutes. 50 μL of the supernatant was transferred to another 96-well PCR plate and mixed with 20 units of micrococcal nuclease and 100 μM CaCl _2. The lysate was incubated at 37°C for 1 hour, and the nuclease was then inactivated at 70°C for 10 minutes. Qiagen protease (0.005 Anson units) was added to the lysate, and the mixture was incubated overnight, and then the protease was inactivated at 95°C for 10 minutes.

The lysate was used as a template for strand-first quantitative polymerase chain reaction (qPCR) analysis. Quantitative PCR was performed with 40 cycles of 15 s at 95 °C and 1 min at 60 °C. Kappa Probe Force Universal PCR Master Mix was used. The primers and probe for the positive polarity DNA strand were 5′CATGAACAAGAGATGTGTAGTAGGCAGAG3′, 5′GGAGGCTGTAGGCATAAATTGG3′, and 5′/56-FAM/CTGCGCACC/ZEN/AGCACCATGCA/3IABkFQ. The primers and probe for the negative polarity DNA strand were 5'GCAGATGAGAAGGCACAGA3', 5'CTTCTCCGTCTGCCGTT3', and 5'/56-FAM/AGTCCGCGT/ZEN/AAAGAGAGGTGCG/3IABkFQ. _EC50 values for positive-strand DNA were calculated using GraphPad Prism and the three-parameter variable-response log(inhibitor)-versus-response algorithm with the lowest value set to zero.

(4) Cytotoxicity (CC ₅₀ ) test method. Cell viability in the presence of compounds was measured in HepDES19 cells using the CellTiter 96 ^TM AQueous non-radioactive cell proliferation assay (MTS). In the absence of tetracycline, cells were seeded into 96-well plates at a density of 1×10 ⁴ cells per well, compounds were applied two days later, and cells were incubated for 3 days. 50% cytotoxic concentration (CC ₅₀ ) values were calculated using GraphPad Prism and the three-parameter variable response logarithm (inhibitor)-response algorithm (bottom value set to zero).

Table 2. Evaluation of the activity of the target compound (dihydropyrimidine-pomalidomide conjugate) in inhibiting HBV DNA replication and cytotoxicity

The results showed that the newly synthesized dihydropyrimidine-pomalidomide conjugate of the present invention exhibited significant anti-HBV activity. All dihydropyrimidine-PROTAC analogs showed anti-HBV activity at low micromolar concentration levels, with EC ₅₀ values ranging from 0.43 to 3.77 μM. Although the activity of the three compounds was lower than that of GLS4 (EC ₅₀ = 0.046 μM), it was close to the positive drug 3TC (EC ₅₀ = 0.40 μM), especially 8c (EC ₅₀ = 0.48 μM), 8i (EC ₅₀ = 0.46 μM) and 8j (EC ₅₀ = 0.43 μM), which is worthy of further study.

Claims (5)

1. Dihydropyrimidine-pomalidomide conjugate, characterized in that it has a structure represented by the following general formula I: I in, The connecting arm Linker is 8-aminooctanoic acid, 6-aminocaproic acid, 4-aminobutyric acid, 2-(2-(2-aminoethoxy)ethoxy)acetic acid, 2-(2-aminoethoxy) acetic acid; R is a hydrogen atom, pomalidomide, 1-methylpomalidomide, lenalidomide, 1-methyllenalidomide, thalidomide or 1-methylthalidomide. 2. The dihydropyrimidine-pomalidomide conjugate as claimed in claim 1, which is one of the compounds with the following structures: . 3. The preparation method of dihydropyrimidine-pomalidomide conjugate as claimed in claim 2, the steps include: taking 3-fluorophthalanhydride II-1 as raw material and 3-amino-2,6-piperidine di Ketone hydrochloride and sodium acetate were refluxed at 120°C for 10 hours in an acetic acid solvent to obtain II-2; dissolve II-2 in N,N -dimethylformamide solution, use potassium carbonate as the base, and add it with stirring at room temperature. Methyl iodide, react at room temperature for 24 hours to obtain intermediate II-3; using 2-thiazoleformamidine hydrochloride, 2-bromo-4-fluorobenzaldehyde and ethyl acetoacetate as starting materials, obtain it through "Biginelli" reaction cyclization Key intermediate 2; in dichloromethane solution, intermediate 2 reacts with N-bromosuccinimide to obtain important intermediate 3; using intermediate 3 as raw material, add potassium carbonate, potassium iodide and 1- Boc piperazine, reflux at 75°C for 1 hour in an acetonitrile solution to obtain intermediate 4; dissolve 4 in a dichloromethane solution of trifluoroacetic acid, stir at room temperature for 10 hours, remove Boc to obtain intermediate 5; connect N -Boc-"Arm" fragment and 2-(7-azabenzotriazole) -N,N,N', N' -tetramethylurea hexafluorophosphate, activate in an ice water bath for half an hour, add intermediate 5 and N,N -diisopropylethylamine, stir at room temperature overnight to obtain intermediate 6; dissolve 6 in a dichloromethane solution of trifluoroacetic acid, stir at room temperature for 10 hours, and remove Boc to obtain the final product 7 (ae); 7 (ae ) and II-2 or II-3 in N,N -dimethylformamide solution, using DIPEA as the base, refluxed at 90°C for 10 hours to obtain the final product 8 (aj); The synthesis route is as follows: Reagents and conditions: (i) 3-amino-2,6-piperidinedione hydrochloride, acetic acid, sodium acetate, 120°C, 10h; (ii) Potassium carbonate, iodomethane, N,N- dimethylform Amide, room temperature, 24h; (iii) 2-bromo-4-fluorobenzaldehyde, ethyl acetoacetate, sodium acetate, ethanol, 80℃; (iv) N-bromosuccinimide, dichloromethane, 40 ℃; (v) 1-tert-butoxycarbonyl-piperazine, potassium carbonate, potassium iodide, acetonitrile, 75 ℃, 1h; (vi) trifluoroacetic acid, dichloromethane, room temperature, 10h; (vii) N -tert-butyl Oxycarbonyl-Linker, HATU, DIPEA, methylene chloride, 0℃, 30min, room temperature, 6h; (viii) Trifluoroacetic acid, methylene chloride, room temperature, 10h; (ix) II-2 or II-3, N, N -Dimethylformamide, DIPEA, 90℃, 10h; Wherein, the N -tert-butoxycarbonyl-Linker includes: N -tert-butoxycarbonyl-8-aminooctanoic acid, N -tert-butoxycarbonyl-6-aminocaproic acid, N -tert-butoxycarbonyl-4-amino Butyric acid, N -tert-butoxycarbonyl-2-(2-(2-aminoethoxy)ethoxy)acetic acid, N -tert-butoxycarbonyl-2-(2-aminoethoxy)acetic acid. 4. Application of the dihydropyrimidine-pomalidomide conjugate according to any one of claims 1 to 2 in the preparation of anti-HBV drugs. 5. An anti-HBV pharmaceutical composition, comprising the dihydropyrimidine-pomalidomide conjugate according to any one of claims 1-2 and one or more pharmaceutically acceptable carriers or excipients.