E3 enzyme ligand-connecting chain compound and preparation method thereof

Abstract

The invention belongs to the technical field of medical intermediates, and particularly relates to an E3 enzyme ligand-connecting chain compound and a preparation method thereof.According to the E3 enzyme ligand-connecting chain compound and the preparation method thereof, an E3 enzyme ligand-connecting chain molecule is synthesized by selecting a CRBN ligand which is widely applied and a flexible polymethylene connecting chain, and the structure is confirmed through mass spectrum and hydrogen spectrum. Functional groups with reaction activity, such as alkynyl, azido, amino and the like, are reserved at the tail ends of one sides of the compounds, and the compounds can be connected with target protein molecules through amide condensation and click chemistry reaction to form complete PROTAC molecules. According to the invention, nucleophilic substitution reaction conditions applied in molecular synthesis of E3 enzyme ligand-linking chain are optimized, the reaction yield is finally optimized from 23% to 69.3%, the preparation process of the product is improved, the reaction yield and the product quality are improved, and the production cost of the product is reduced.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical intermediates, and particularly relates to an E3 enzyme ligand-connector chain complex and a preparation method thereof. Background Art

[0002] CRBN ligand is the most commonly used E3 ubiquitin ligase ligand in PROTAC molecules. To date, CRBN-based PROTACs have been widely studied worldwide and have shown great potential in the treatment of cancer, neurodegenerative diseases, cardiovascular diseases, and immune diseases. CRBN-based PROTAC drugs (such as ARV-110 and ARV-471) have entered the clinical research stage and are expected to play an important role in the treatment of future diseases. Since the design of PROTAC molecules needs to consider factors such as the target protein ligand type, E3 ligase ligand type, connection chain type, and connection chain length, PROTAC molecules have multiple combination possibilities, so a considerable amount of work is required in chemical synthesis.

[0003] In order to reduce the complexity of chemical synthesis of PROTAC molecules, it is of great significance to synthesize a series of "E3 enzyme ligand-linker chain" compounds. Summary of the invention

[0004] The purpose of the present invention is to overcome the above problems existing in the traditional technology and provide an E3 enzyme ligand-connector chain complex and a preparation method thereof.

[0005] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0006] An E3 enzyme ligand-linker chain complex, the structural formula of the E3 enzyme ligand-linker chain complex is as follows:

[0007]

[0008] Where n is 2 to 6; R 1 Azide (-N 3 ), amino groups and their amine salts (-NH 2 or -NH 2 ·HCl) or alkynyl (-C≡CH).

[0009] The present invention also provides a method for preparing an E3 enzyme ligand-connector chain complex, characterized in that the method for preparing the E3 enzyme ligand-connector chain complex is as shown in the following formula:

[0010]

[0011] Furthermore, in the above preparation method, R 1For alkynyl, the preparation of the connecting chain is as follows:

[0012]

[0013] Furthermore, in the above preparation method, the specific reaction steps are:

[0014] Step 1: Take a reaction bottle, add the raw material E3 enzyme ligand, TEA and DCM, slowly add EsCl under ice bath conditions, remove the ice bath after the addition is complete, and react at room temperature; after TLC detection shows that the raw material is basically reacted, pour the reaction solution into water, extract, stand, separate the liquids, wash the aqueous phase with DCM, combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a crude compound as a reddish brown oily liquid, which is directly used in the next step reaction;

[0015] Step 2: Take a reaction bottle, dissolve the reddish brown oily liquid obtained in the previous step in DMF, add LiBr, and react at 80°C overnight; after TLC detection shows that the reaction is complete, stop stirring, remove the oil bath, and wait for the reaction liquid to cool naturally to room temperature; add water and MTBE, extract, stand, separate, wash the aqueous phase with MTBE, combine the organic phases, wash the organic phase with a saturated ammonium chloride solution, and then wash with a saturated sodium chloride solution; dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a reddish brown oily liquid, and purify and separate the compound with flash column chromatography to obtain a light yellow oily liquid;

[0016] Step 3: Take a reaction bottle, dissolve the product of the previous step in DMF, add Potassium phthalimide, and react at 60°C; TLC detection shows that the raw materials are basically reacted, stop stirring, remove the oil bath, and wait for the reaction solution to cool naturally to room temperature; add water and MTBE, extract, stand, separate, wash the aqueous phase with MTBE, combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution; dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow solid compound, which is directly used in the next step reaction;

[0017] Step 4: Take the reaction bottle, add the product of the previous step, MeOH and 80% N 2 H 4 ·H 2 O, react under the condition of internal temperature of 60°C; after TLC detection shows that the reaction is complete, the reaction solution is concentrated under reduced pressure at low temperature, the solvent is removed to obtain a white solid, 200 mL of water is added to dissolve it, 20 mL of ammonia water is added, and then 2DCM is added, extracted, allowed to stand, separated, the aqueous phase is washed with DCM, the organic phases are combined, and the organic phases are washed with saturated NaCl solution; then the organic phases are separated, the organic phases are dried over anhydrous magnesium sulfate, concentrated under reduced pressure at low temperature, and distilled at atmospheric pressure to obtain the compound as a colorless transparent oily liquid.

[0018] Furthermore, in the above preparation method, R 1 For an azido group or an amino group, the preparation of the connecting chain is as follows:

[0019]

[0020] Furthermore, in the above preparation method, R 1 For an azido group, the specific reaction steps are:

[0021] Step 1: Take the reaction bottle, add the raw material compound and EtOH, cool the ice bath to 5°C, and add (Boc) dropwise 2 O, after addition, react at room temperature overnight; TLC detection showed that new spots were generated, and the raw materials reacted completely; directly concentrated and spun off the solvent to obtain the intermediate as a colorless oily liquid, which was directly used in the next step reaction;

[0022] Step 2: Take a reaction bottle, add the intermediate obtained in the previous step, DMF and DBU, slowly add DPPA with a constant pressure dropping funnel under ice bath conditions, remove the ice bath, transfer to an oil bath pot, stir at an internal temperature of 80°C, and react overnight; after the reaction is completed by TLC detection, add water and EA, extract, stand, separate, and wash the aqueous phase with EA; combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a crude product as a light yellow oily liquid, which is purified by flash column chromatography to obtain an intermediate compound as a colorless and transparent oily liquid;

[0023] Step 3: Take a reaction bottle, add the intermediate obtained in the previous step and DCM, slowly add 4M HCl in dioxane, a large amount of white solid precipitates, stir at room temperature, filter, and the filter cake is the target compound.

[0024] Furthermore, in the above preparation method, R 1 The specific steps for the amino reaction are:

[0025] Take the reaction bottle, add the raw material compound, THF and H 2 O, slowly add PPh 3 , react overnight at room temperature, TLC detection shows that the raw material has been basically reacted, the reaction solution is poured into water, DCM is added, extraction is carried out, the liquid is separated, and the aqueous phase is washed with DCM; the organic phases are combined, the organic phase is washed with a saturated ammonium chloride solution, and then with a saturated sodium chloride solution, the organic phase is dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure. The compound separated by flash column chromatography is a colorless transparent liquid.

[0026] Furthermore, in the above preparation method, when synthesizing the nitrogen-substituted E3 enzyme ligand-connector chain series compounds, the general synthesis method used is an aromatic nucleophilic substitution reaction using 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and an amino series connecting chain as raw materials. The reaction formula of the template is as follows:

[0027]

[0028] The solvent is DMSO with a concentration of 8 to 12 M; the equivalent of the connecting chain is 1.2 to 1.5 eq, the equivalent of DIPEA is 1.2 to 1.5 eq, and the reaction temperature is 80 to 100°C.

[0029] The beneficial effects of the present invention are:

[0030] The present invention synthesized the "E3 enzyme ligand-connector chain" molecule by selecting the widely used CRBN ligand and the flexible polymethylene connecting chain, and the structure was confirmed by mass spectrometry and hydrogen spectrum. These compounds have reactive functional groups such as alkynyl, azido, and amino groups at one end, which can be connected to the target protein molecule through amide condensation and click chemistry reaction to form a complete PROTAC molecule. The present invention optimizes the nucleophilic substitution reaction conditions used in the synthesis of the "E3 enzyme ligand-connector chain" molecule, and finally optimizes the reaction yield from 23% to 69.3%, improves the preparation process of the product, improves the reaction yield and product quality, and reduces the production cost of the product.

[0031] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0033] Figure 1 This is a schematic diagram of the detection of the product in Example 5;

[0034] Figure 2 This is a schematic diagram of the detection of the product in Example 6;

[0035] Figure 3 This is a schematic diagram of the detection of the product in Example 7;

[0036] Figure 4 This is a schematic diagram of the detection of the product in Example 8;

[0037] Figure 5This is a schematic diagram of the detection of the product in Example 9;

[0038] Figure 6 This is a schematic diagram of the detection of the product in Example 10;

[0039] Figure 7 This is a schematic diagram of the detection of the product in Example 11;

[0040] Figure 8 This is a schematic diagram of the detection of the product in Example 12;

[0041] Fig. 9 This is a schematic diagram of the detection of the product in Example 13;

[0042] Fig.10 This is a schematic diagram of the detection of the product in Example 14;

[0043] Fig.11 This is a schematic diagram of the detection of the product in Example 15;

[0044] Fig.12 This is a schematic diagram of the detection of the product in Example 16. DETAILED DESCRIPTION

[0045] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] The specific designations of the abbreviations in this embodiment are as follows:

[0047] TEA: triethylamine

[0048] EsCl: ethylsulfonyl chloride

[0049] DCM: dichloromethane

[0050] DMF: N,N-dimethylformamide

[0051] LiBr: lithium bromide

[0052] MeOH: Methanol

[0053] NaCl: Sodium Chloride

[0054] EtOH: ethanol

[0055] (Boc) 2 O: Di-tert-butyl dicarbonate

[0056] DBU: 1,8-diazabicyclo[5.4.0]undec-7-ene

[0057] EA: Ethyl acrylate

[0058] THF: Tetrahydrofuran

[0059] PPh 3 :Triphenylphosphine oxide

[0060] DMSO: dimethyl sulfoxide

[0061] DIPEA: N,N-diisopropylethylamine

[0062] The specific embodiments of the present invention are:

[0063] Example 1

[0064] This type of compound can be divided into two parts: E3 enzyme ligand (mother core) and connecting chain, which are combined through aromatic nucleophilic substitution reaction:

[0065]

[0066] The preparation of the connecting chain can be prepared by the following methods:

[0067] R1 is an alkynyl group:

[0068]

[0069] R 1 is an amino group or an azido group:

[0070]

[0071] Synthesis of alkynyl side chain

[0072]

[0073] operate:

[0074] Step 1: Take a 500mL reaction bottle, add the raw material (20.0g, 203.7mmol), TEA (30.9g, 305.6mmol), DCM 200mL, slowly add EsCl (26.2g, 203.7mol) under ice bath condition, after the addition, remove the ice bath, and react at room temperature for 4h. TLC detection (PE:EA=2:1, R f =0.3) showed that the raw materials were basically reacted, the reaction solution was poured into 200 mL of water, extracted, allowed to stand, separated, the aqueous phase was washed with DCM (200 mL×3), the organic phases were combined, washed with saturated ammonium chloride solution (300 mL×3), and then washed with saturated sodium chloride solution (300 mL×3), the organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude compound of 36.8 g of a reddish brown oily liquid, which was directly used in the next step reaction.

[0075] Step 2: Take a 1L reaction bottle, dissolve the reddish brown oily liquid (36.8 g, 193.4 mmol) obtained in the previous step in 300 mL DMF, add LiBr (20.0 g, 232.1 mmol), and react at 80°C overnight. TLC detection (PE:EA=4:1, R f =0.4) after the reaction was completed, the stirring was stopped, the oil bath was removed, and the reaction solution was cooled to room temperature naturally. 500mL of water and 500mL of MTBE were added, extracted, allowed to stand, and separated. The aqueous phase was washed with MTBE (300mL×3), the organic phases were combined, the organic phases were washed with saturated ammonium chloride solution (500mL×3), and then with saturated sodium chloride solution (500mL×2), the organic phases were dried with anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a reddish brown oily liquid. The compound was purified and separated by flash column chromatography to obtain a light yellow oily liquid (23.6g, 75.8%).

[0076] Step 3: Take a 500mL reaction bottle, dissolve the product of the previous step (10.0g, 62.1mmol) in 100mL DMF, add Potassiumphthalimide (13.8g, 74.5mmol), and react at 60℃ for 4h. TLC detection (PE:EA=10:1, R f =0.3) showed that the raw materials were basically reacted completely, the stirring was stopped, the oil bath was removed, and the reaction solution was cooled to room temperature naturally. 200mL of water and 200mL of MTBE were added, extracted, allowed to stand, and separated. The aqueous phase was washed with MTBE (200mL×3), the organic phases were combined, washed with saturated ammonium chloride solution (500mL×3), and then washed with saturated sodium chloride solution (500mL×2), the organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain 12.2g of the compound as a light yellow solid, which was directly used in the next step reaction.

[0077] Step 4: Take a 500mL reaction bottle, add the product of the previous step (12.2g, 53.6mmol), MeOH 150mL, 80% N 2 H 4 ·H 2 O (6.7 g, 107.4 mmol), reacted at 60°C for 4 hours. TLC detection (PE:EA=1:1, R f=0.1) showed that the reaction was complete, the reaction solution was concentrated under reduced pressure at low temperature, the solvent was removed to obtain a white solid, 200 mL of water was added to dissolve it, 20 mL of ammonia water was added, and then 200 mL of DCM was added, extracted, allowed to stand, separated, the aqueous phase was washed with DCM (100 mL×3), the organic phases were combined, the organic phases were washed with saturated NaCl solution (200 mL×2), then separated, the organic phase was dried over anhydrous magnesium sulfate, concentrated under reduced pressure at low temperature, and distilled at atmospheric pressure to obtain the compound as a colorless transparent oily liquid (3.2 g, 61.4%).

[0078] The preparation process of the remaining alkynyl side chain compounds is the same as above.

[0079] Example 2

[0080] R 1 For the synthesis of azide side chains

[0081]

[0082] operate:

[0083] Step 1: Take a 500mL reaction bottle, add the raw material compound (25.0g, 332.8mmol), EtOH 250mL, cool the ice bath to about 5℃, and add (Boc) dropwise 2 O (72.5 g, 332.8 mmol), after addition, react at room temperature overnight. TLC detection (PE: EA = 1: 1, R f =0.4) showed that new spots were generated, and the raw materials were completely reacted. The solvent was directly concentrated and removed to obtain 58.3 g of the intermediate as a colorless oily liquid, which was directly used in the next step reaction.

[0084] Step 2: Take a 1L reaction bottle, add the intermediate obtained in the previous step (58.3g, 332.8mmol), DMF 500L, DBU (101.0g, 432.6mmol), slowly add DPPA (91.6g, 332.8mmol) using a constant pressure dropping funnel under ice bath conditions, remove the ice bath, transfer to an oil bath pot, stir at an internal temperature of 80℃, and react overnight. TLC detection (PE:EA=10:1, R f =0.3) After the reaction is completed, 800 mL of water and 800 mL of EA are added, extracted, allowed to stand, and separated. The aqueous phase is washed with EA (800 mL×3), the organic phases are combined, washed with saturated ammonium chloride solution (1 L×3), and then washed with saturated sodium chloride solution (1 L×3), the organic phase is dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a crude product as a light yellow oily liquid, which is purified by flash column chromatography (PE:EA=1:0) to obtain the intermediate compound (48.0 g, 72.0%) as a colorless transparent oily liquid.

[0085] Step 3: Take a 500mL reaction bottle, add the intermediate obtained in the step (20.0g, 99.9mmol), DCM 200mL, slowly add 4M HCl in dioxane 40mL, a large amount of white solid precipitated, after stirring at room temperature for 4 hours, filter, the filter cake is the target compound (12.2g, 89.7%) as a white solid.

[0086] The preparation process of the remaining azide side chains is the same as above.

[0087] Example 3

[0088] R 1 For the synthesis of amino side chains

[0089]

[0090] operate:

[0091] Take a 500 mL reaction bottle, add the corresponding product in Example 2 (20.0 g, 99.9 mmol), THF 100 mL, H 2 O100mL, slowly add PPh 3 (39.3 g, 149.8 mmol), react overnight at room temperature, TLC detection shows that the raw material has been basically reacted, the reaction solution is poured into 200 mL of water, DCM 200 mL is added, extraction is carried out, and the liquid is separated. The aqueous phase is washed with DCM (200 mL × 3), the organic phases are combined, the organic phases are washed with saturated ammonium chloride solution (300 mL × 3), and then with saturated sodium chloride solution (300 mL × 2), the organic phase is dried over anhydrous magnesium sulfate, filtered, concentrated under reduced pressure, and purified and separated by flash column chromatography to obtain the compound (7.6 g, 43.6%) as a colorless transparent liquid.

[0092] The preparation process of the remaining amino side chain compounds is the same as above.

[0093] Example 4

[0094] Optimization of process conditions for preparing target products by aromatic nucleophilic substitution reaction between side chain and parent core

[0095]

[0096] When synthesizing nitrogen-substituted E3 enzyme ligand-linker series compounds, the general synthesis method used is an aromatic nucleophilic substitution reaction using 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and an amino series linker as raw materials. The synthesis conditions reported in the literature (×ing Qiu, Ning Sun, Ying Kong, et al. Chemoselective Synthesis of Lenalidomide-based PROTACs Library Using Alkylation Reaction [J]. Organic Letters., 2019, 21 (10): 3838-3841.) were used. It was found that the reaction was complicated, the yield was low (23%), and there were multiple by-products that were difficult to separate and purify. The structural formula of the by-product is as follows:

[0097]

[0098] The inventors used the following reaction formula as a template to screen the solvent, reaction system concentration, raw material equivalent, base type, feed ratio and reaction temperature.

[0099]

[0100] The inventors determined reaction condition 16 as the optimal reaction condition, and finally optimized the reaction yield to 69.3%, as shown in Table 1:

[0101] Table 1

[0102]

[0103]

[0104] The synthetic reactions in the following examples were all carried out with reference to the process parameters optimized in Example 4.

[0105] Example 5

[0106] 2-(2,6-Dioxopiperidin-3-yl)-4-(prop-2-yn-1-ylamino)isoindoline-1,3-dione

[0107]

[0108] The mother core material (5.0 g, 18.1 mmol) was dissolved in 50 mL DMSO, and then DIPEA (3.5 g, 27.2 mmol) and side chain material (1.5 g, 27.2 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw material was complete, the oil bath was removed, and it was cooled to room temperature naturally. The reaction solution was transferred to a separatory funnel, 100 mL of water was added, and EA was extracted (100 mL×3). The organic phases were combined, washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.3) was separated and purified to give the product (3.5 g, 62.1%) as a yellow-green solid.

[0109] 1 H NMR (400 MHz, DMSO-d 6 )δ11.09(s,1H),7.66(dd,J=8.4,7.3Hz,1H),7.15(dd,J=18.4,7.8Hz,2H),6.92(t,J=6.1Hz,1H),5.07(dd,J=12.8,5.4Hz,1H),4.18(d d,J=6.1,2.3Hz,2H),3.17(t,J=2.3Hz,1H),2.89(ddd,J=17.3,13.9,5.4Hz,1H),2.71–2.52(m,2H),2.04(ddd,J=20.4,9.0,6.1Hz,1H). MS(ESI)m / z calcd for C 16 H 13 N 3 O 4 [MH] + :310.09; found,310.03.

[0110] Example 6

[0111] 2-(2,6-Dioxopiperidin-3-yl)-4-(prop-2-yn-1-ylamino)isoindoline-1,3-dione

[0112]

[0113] The mother core material (0.5 g, 1.8 mmol) was dissolved in 2.5 mL DMSO, and then DIPEA (0.4 g, 2.7 mmol) and side chain material (3-alkyne-1-butylamine) (0.3 g, 2.71 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw materials was complete, the oil bath was removed, and the reaction solution was allowed to cool naturally to room temperature. The reaction solution was transferred to a separatory funnel, 10 mL of water was added, and EA was used for extraction (10 mL × 3). The organic phases were combined, and washed with saturated ammonium chloride solution (20 mL × 3) and saturated sodium chloride solution (20 mL × 3) in sequence, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The reaction mixture was analyzed by flash column chromatography (PE:EA=3:1, R f =0.3) was separated and purified to give the product (0.3 g, 50.8%) as a yellow-green solid.

[0114] 1 H NMR (400 MHz, DMSO-d 6 )δ11.08(s,1H),7.60(dd,J=8.3,7.3Hz,1H),7.11(dd,J=42.5,7.8Hz,2H),6.71(t,J=6.2Hz,1H),5.06(dd,J= 12.8, 5.4Hz, 1H), 3.48 (q, J = 6.6Hz, 2H), 2.99–2.80 (m, 2H), 2.69–2.51 (m, 4H), 2.09–1.99 (m, 1H). Purity: 93%. MS(ESI)m / z calcd for C 17 H 15 N 3 O 4 [MH] + :324.11; found,324.22.

[0115] Example 7

[0116] 4-(But-3-yn-1-ylamino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0117]

[0118] The mother core material (5.0 g, 18.1 mmol) was dissolved in 50 mL DMSO, and then DIPEA (7.0 g, 54.3 mmol) and the side chain material (4-pentyn-1-amine) (3.3 g, 27.2 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw materials was complete, the oil bath was removed, and the reaction solution was allowed to cool naturally to room temperature. The reaction solution was transferred to a separatory funnel, 100 mL of water was added, and EA was used for extraction (100 mL×3). The organic phases were combined, and washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.3) was separated and purified to give the product (2.6 g, 42.3%) as a yellow-green solid.

[0119] 1 H NMR (400 MHz, DMSO-d 6 )δ11.06(s,1H),7.63–7.55(m,1H),7.08(dd,J=33.9,7.8Hz,2H),6.62(t,J=6.0Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),3.39(dd, J=13.5,6.6Hz,2H),2.99–2.79(m,2H),2.67–2.52(m,2H),2.26(td,J=7.0,2.5Hz,2H),2.10–2.01(m,1H),1.76(p,J=7.1Hz,2H). MS(ESI)m / z calcd for C 18 H 17 N 3 O 4 [MH] + :338.12; found,338.20.

[0120] Example 8

[0121] 2-(2,6-Dioxopiperidin-3-yl)-4-(hex-5-yn-1-ylamino)isoindoline-1,3-dione

[0122]

[0123] The mother core material (10.0 g, 36.2 mmol) was dissolved in 100 mL DMSO, and then DIPEA (7.0 g, 54.3 mmol) and side chain material (5.3 g, 27.2 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw material was complete, the oil bath was removed, and it was cooled to room temperature naturally. The reaction solution was transferred to a separatory funnel, 200 mL of water was added, and EA was extracted (100 mL×3). The organic phases were combined, washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.4) was separated and purified to give the product (6.4 g, 59.5%) as a yellow-green solid.

[0124] 1 H NMR (400 MHz, DMSO-d 6 )δ11.08(s,1H),7.59(dd,J=8.4,7.2Hz,1H),7.07(dd,J=35.3,7.8Hz,2H),6.57(t,J =6.0Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),2.96–2.82(m,1H),2.76(t,J=2.6Hz,1H),2 .57(dd,J=20.3,10.6Hz,2H),2.22(td,J=7.0,2.6Hz,2H),2.07–1.99(m,1H),1.66(d d, J=14.6, 7.5Hz, 2H), 1.54 (dd, J=15.2, 7.0Hz, 2H), 1.42–1.21 (m, 2H). Purity: 97%. MS(ESI)m / z calcd for C 19 H 19 N 3 O 4 [M+H] + :354.14; found,354.20.

[0125] Example 9

[0126] 2-(2,6-Dioxopiperidin-3-yl)-4-(hept-6-yn-1-ylamino)isoindoline-1,3-dione

[0127]

[0128] The mother core material (10.0 g, 36.2 mmol) was dissolved in 100 mL DMSO, and then DIPEA (7.0 g, 54.3 mmol) and side chain material (6.0 g, 54.3 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw material was complete, the oil bath was removed, and it was cooled to room temperature naturally. The reaction solution was transferred to a separatory funnel, 200 mL of water was added, and EA was extracted (100 mL×3). The organic phases were combined, washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.4) was separated and purified to give the product (8.2 g, 61.6%) as a yellow-green solid.

[0129] 1 H NMR (400 MHz, DMSO-d 6 )δ11.09(s,1H),7.59(dd,J=8.4,7.3Hz,1H),7.06(dd,J=31.0,7.8Hz,2H),6.55(t,J=5.9Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),3.32–3.2 7(m,2H),2.96–2.83(m,1H),2.74(t,J=2.6Hz,1H),2.63–2.56(m,1H),2.17(td,J=6.7,2.6Hz,2H),2.09–1.97(m,1H),1.68–1.30(m,7H). MS(ESI)m / z calcd for C 20 H 21 N 3 O 4 [M+H] + :368.15; found,368.12.

[0130] Example 10

[0131] 2-(2,6-Dioxopiperidin-3-yl)-4-(oct-7-yn-1-ylamino)isoindoline-1,3-dione

[0132]

[0133] The mother core material (10.0 g, 36.2 mmol) was dissolved in 100 mL DMSO, and then DIPEA (7.0 g, 54.3 mmol) and side chain material (6.8 g, 54.3 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw material was complete, the oil bath was removed, and it was cooled to room temperature naturally. The reaction solution was transferred to a separatory funnel, 200 mL of water was added, and EA was extracted (100 mL×3). The organic phases were combined, washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.4) was separated and purified to give the product (7.5 g, 54.3%) as a yellow-green solid.

[0134] 1 H NMR (400 MHz, DMSO-d 6 )δ11.09(s,1H),7.58(dd,J=8.4,7.3Hz,1H),7.05(dd,J=24.7,7.8Hz,2H), 6.52(t,J=5.9Hz,1H),5.05(dd,J=12.9,5.4Hz,1H),3.29(dd,J=13.3,6.6Hz ,2H),2.97–2.81(m,1H),2.72(t,J=2.6Hz,1H),2.65–2.51(m,2H),2.15(td, J=6.7,2.6Hz,2H),2.09–1.99(m,1H),1.62–1.52(m,2H),1.51–1.32(m,6H). MS(ESI)m / zcalcd for C 21 H 23 N 3 O 4 [M+H] + :382.17; found,382.19.

[0135] Embodiment 11

[0136] 4-((4-Azidobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0137]

[0138] The preparation method was the same as that of Example 4 to obtain the product (6.2 g, 46.2%) as a yellow-green solid.

[0139] 1H NMR (400 MHz, DMSO-d 6 )δ11.10(s,1H),7.59(dd,J=8.4,7.2Hz,1H),7.08(dd,J=36.6,7.8Hz,2H),6.62(t,J=6.1Hz,1H),5.06(dd,J=12.8,5.4Hz,1H ),3.39(t,J=6.4Hz,2H),3.35(s,2H),3.04–2.76(m,1H),2.66–2.52(m,2H),2.03(dd,J=14.0,8.8Hz,1H),1.78–1.46(m,4H). MS(ESI)m / z calcd for C 17 H 18 N 6 O 4 [M+H] + :370.14; found,3370.22.

[0140] Example 12

[0141] 4-((5-Azidopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0142]

[0143] The preparation method was the same as that of Example 4, and the product (3.6 g, 25.9%) was obtained as a cyan solid.

[0144] 1 H NMR (400 MHz, DMSO-d 6 )δ11.09(s,1H),7.65–7.53(m,1H),7.07(dd,J=32.7,7.8Hz,2H),6.56(t ,J=5.9Hz,1H),5.05(dd,J=12.8,5.4Hz,1H),3.35(d,J=6.9Hz,2H),3.30( d,J=6.8Hz,2H),2.96–2.82(m,1H),2.57(dd,J=20.2,10.6Hz,2H),2.10–1 .96(m,1H),1.60(dq,J=14.7,7.4Hz,4H),1.48–1.32(m,2H).Purity:96%. MS(ESI)m / z calcdfor C 18 H 20 N 6 O 4 [MH] +:383.15; found,383.21.

[0145] Embodiment 13

[0146] 4-((6-Azidohexyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione

[0147]

[0148] The preparation method was the same as that of Example 4, and the product (5.8 g, 40.2%) was obtained as a cyan solid.

[0149] 1 H NMR (400 MHz, DMSO-d 6 )δ11.09(s,1H),7.58(dd,J=8.3,7.3Hz,1H),7.06(dd,J=27.6,7.8Hz,2H),6.53(t,J=5.9Hz,1H),5.05(dd,J=12.9,5.4Hz,1H),3.30(dd,J=1 2.2,6.7Hz,4H),2.89(ddd,J=17.5,14.1,5.3Hz,1H),2.66–2.51(m,2H),2.09–1.98(m,1H),1.56(dt,J=20.7,6.7Hz,4H),1.43–1.30(m,4H). MS(ESI)m / z calcd forC 19 H 22 N 6 O 4 [M+H] + :399.17; found,399.23.

[0150] Embodiment 14

[0151] 4-((3-Aminopropyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride

[0152]

[0153] The mother core material (10.0 g, 36.2 mmol) was dissolved in 100 mL DMSO, and then DIPEA (7.0 g, 108.6 mmol) and side chain material (9.5 g, 54.3 mmol) were added, and the reaction was carried out at an internal temperature of 90°C for 4 h. After TLC monitoring showed that the reaction of the raw material was complete, the oil bath was removed, and it was cooled to room temperature naturally. The reaction solution was transferred to a separatory funnel, 200 mL of water was added, and EA was used for extraction (100 mL×3). The organic phases were combined, and washed with saturated ammonium chloride solution (200 mL×3) and saturated sodium chloride solution (200 mL×3) in turn, dried over anhydrous magnesium sulfate, filtered, and the filtrate was concentrated under reduced pressure. The filtrate was purified by flash column chromatography (PE:EA=3:1, R f =0.4) to separate and purify the intermediate 6.5 g, which was then dissolved in 20 mL DCM, and 10 mL 4 M HCl indioxane solution was added, solid precipitated, and after half an hour, TLC monitoring showed that the intermediate had basically reacted completely, and the product (5.3 g, 39.9%) was obtained by suction filtration as a yellow-green solid.

[0154] 1 H NMR (400 MHz, DMSO-d 6 )δ11.11(s,1H),7.92(s,3H),7.67–7.52(m,1H),7.16(d,J=8.6Hz,1H),7.05(d,J=7.0Hz,1H),6.76(t,J=6.3Hz,1H),5.06(dd,J =12.8,5.4Hz,1H),3.42(q,J=6.6Hz,2H),2.87(dd,J=13.2,4.9Hz,3H),2.58(t,J=14.4Hz,2H),2.04(s,1H),1.94–1.73(m,2H). MS(ESI)m / z calcd for C 16 H 19 C1N 4 O 4 [M-Cl] - :330.11; found,330.14.

[0155] Embodiment 15

[0156] 4-((4-Aminobutyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride

[0157]

[0158] The preparation method was the same as that of Example 14, and the product (6.4 g, 46.4%) was obtained as a cyan solid.

[0159] 1 H NMR (400 MHz, DMSO-d 6 )δ11.10(s,1H),8.13(s,3H),7.57(t,J=7.6Hz,1H),7.07(dd,J=40.0,7.7Hz,2H),5.05(dd,J=12.6,5.0H z,1H),3.59(d,J=24.4Hz,5H),3.02–2.73(m,3H),2.59(d,J=17.9Hz,1H),2.13–1.92(m,1H),1.64(s,4H). MS(ESI)m / z calcdfor C 17 H 21 C1N 4 O 4 [M-Cl] - :345.13; found,345.20.

[0160] Example 16

[0161] 4-((5-Aminopentyl)amino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione hydrochloride

[0162]

[0163] The preparation method was the same as that of Example 14 to obtain the product (5.3 g, 37.1%) as a yellow-green solid.

[0164] 1 H NMR (400 MHz, DMSO-d 6 )δ11.08(s,1H),7.99(s,3H),7.59(t,J=7.8Hz,1H),7.07(dd,J=32.3,7.8Hz,2 H),6.55(s,1H),5.05(dd,J=12.7,5.3Hz,1H),3.57(s,1H),3.31(dd,J=12.8,6 .6Hz,2H),2.96–2.83(m,1H),2.77(dd,J=13.0,6.6Hz,2H),2.57(dd,J=21.4,1 0.9Hz, 2H), 2.10–1.97 (m, 1H), 1.62 (dd, J=13.9, 6.8Hz, 4H), 1.49–1.35 (m, 2H). MS (ESI) m / z calcd for C18 H 23 C1N 4 O 4 [M-Cl] - :359.14; found,359.13.

[0165] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. An E3 enzyme ligand-linker complex, characterized in that The structural formula of the E3 enzyme ligand-linker complex is as follows: Wherein, n is 2 to 6; R1 is an azido group, an amino group and its amine salt or an alkynyl group.

2. The method for preparing the E3 enzyme ligand-linker complex according to claim 1, characterized in that: The preparation method of the E3 enzyme ligand-connector chain complex is shown in the following formula:

3. The preparation method according to claim 2, characterized in that: R1 is an alkynyl group, and the preparation of the connecting chain is as follows:

4. The preparation method according to claim 3, characterized in that: The specific steps of the reaction are: Step 1: Take a reaction bottle, add the raw material E3 enzyme ligand, TEA and DCM, slowly add EsCl under ice bath conditions, remove the ice bath after the addition is complete, and react at room temperature; after TLC detection shows that the raw material is basically reacted, pour the reaction solution into water, extract, stand, separate the liquids, wash the aqueous phase with DCM, combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a crude compound as a reddish brown oily liquid, which is directly used in the next step reaction; Step 2: Take a reaction bottle, dissolve the reddish brown oily liquid obtained in the previous step in DMF, add LiBr, and react at 80°C overnight; after TLC detection shows that the reaction is complete, stop stirring, remove the oil bath, and wait for the reaction liquid to cool naturally to room temperature; add water and MTBE, extract, stand, separate, wash the aqueous phase with MTBE, combine the organic phases, wash the organic phases with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution; The organic phase was dried over anhydrous magnesium sulfate, filtered, and concentrated under reduced pressure to obtain a reddish brown oily liquid, which was purified and separated by flash column chromatography to obtain a light yellow oily liquid compound; Step 3: Take a reaction bottle, dissolve the product of the previous step in DMF, add Potassium phthalimide, and react at 60°C; TLC detection shows that the raw materials are basically reacted, stop stirring, remove the oil bath, and wait for the reaction solution to cool naturally to room temperature; add water and MTBE, extract, stand, separate, wash the aqueous phase with MTBE, combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution; dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a light yellow solid compound, which is directly used in the next step reaction; Step 4: Take a reaction bottle, add the product MeOH from the previous step and 80% N2H4·H2O, and react at an internal temperature of 60°C; after TLC detection shows that the reaction is complete, concentrate the reaction solution under reduced pressure at low temperature, remove the solvent, and obtain a white solid. Add 200mL of water to dissolve it, add 20mL of ammonia water, and then add 2DCM, extract, let stand, separate the liquids, wash the aqueous phase with DCM, combine the organic phases, and wash the organic phases with saturated NaCl solution; then separate the liquids, dry the organic phase with anhydrous magnesium sulfate, concentrate under reduced pressure at low temperature, and distill at atmospheric pressure to obtain the compound as a colorless, transparent, oily liquid.

5. The preparation method according to claim 2, characterized in that: R1 is an azido group or an amino group, and the preparation of the connecting chain is as follows:

6. The preparation method according to claim 5, characterized in that: R1 is an azido group, and the specific steps of the reaction are: Step 1: Take a reaction bottle, add the raw material compound and EtOH, cool the ice bath to 5°C, add (Boc)2O dropwise, and react at room temperature overnight; TLC detection shows that new spots are generated and the raw material is completely reacted; directly concentrate and remove the solvent to obtain the intermediate as a colorless oily liquid, which is directly used in the next step; Step 2: Take a reaction bottle, add the intermediate obtained in the previous step, DMF and DBU, slowly add DPPA with a constant pressure dropping funnel under ice bath conditions, remove the ice bath, transfer to an oil bath pot, stir at an internal temperature of 80°C, and react overnight; after the reaction is completed by TLC detection, add water and EA, extract, stand, separate, and wash the aqueous phase with EA; combine the organic phases, wash with saturated ammonium chloride solution, and then wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter, and concentrate under reduced pressure to obtain a crude product as a light yellow oily liquid, which is purified by flash column chromatography to obtain an intermediate compound as a colorless and transparent oily liquid; Step 3: Take a reaction bottle, add the intermediate obtained in the previous step and DCM, slowly add 4M HCl in dioxane, a large amount of white solid precipitates, stir at room temperature, filter, and the filter cake is the target compound.

7. The preparation method according to claim 5, characterized in that: R1 is an amino group. The specific steps of the reaction are: Take a reaction bottle, add the raw material compound, THF and H2O, slowly add PPh3, react overnight at room temperature, TLC detection shows that the raw material has been basically reacted, pour the reaction solution into water, add DCM, extract, separate the liquids, wash the aqueous phase with DCM; combine the organic phases, wash the organic phase with saturated ammonium chloride solution, then wash with saturated sodium chloride solution, dry the organic phase with anhydrous magnesium sulfate, filter, concentrate under reduced pressure, and purify and separate the compound with flash column chromatography as a colorless transparent liquid.

8. The preparation method according to claim 2, characterized in that: When synthesizing nitrogen-substituted E3 enzyme ligand-linker series compounds, the general synthesis method used is an aromatic nucleophilic substitution reaction using 2-(2,6-dioxo-piperidin-3-yl)-4-fluoro-isoindole-1,3-dione and an amino series linker as raw materials. The reaction formula of the template is as follows: The solvent is DMSO with a concentration of 8 to 12 M; the equivalent of the connecting chain is 1.2 to 1.5 eq, the equivalent of DIPEA is 1.2 to 1.5 eq, and the reaction temperature is 80 to 100°C.

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