Compound for targeted degradation of p300 / CBP protein as well as synthesis method and application of compound

Abstract

The invention discloses a compound for targeted degradation of p300 / CBP protein as well as a synthesis method and application thereof, and relates to the technical field of medicinal chemistry. The invention provides a method for constructing a proteolysis targeting chimera and other bifunctional molecules by utilizing a SuFEx reaction for the first time; according to the invention, fluorosulfonate and a sulfonyl fluoride analogue of a compound CPI644 are used as p300 / CBP protein ligands, and are connected with E3 ubiquitin ligase ligands 5-OH and 4-OH thalidomide which are introduced in advance and take alkanes with different lengths as connecting arms through SuFEx reaction to prepare two series of 12 PROTACs molecules; the PROTACs molecule designed by the invention has the capability of degrading p300 / CBP protein, and the PROTACs molecule can degrade the p300 / CBP protein in human breast cancer cells in a concentration-dependent manner; the PROTACs molecules show a relatively good anti-tumor effect, and have a wide application prospect in the aspects of diagnosis and treatment of tumors.

Description

Technical Field

[0001] The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a compound for targeted degradation of p300 / CBP protein, a synthesis method and application thereof. Background Art

[0002] Currently, p300 / CBP is an important target for disease research, and the design and discovery of high-quality small molecule inhibitors targeting p300 / CBP is also one of the hot topics for researchers. However, since small molecule inhibitors targeting p300 / CBP still have potential problems in terms of specificity, selectivity, and safety, protein degradation targeting chimeras (PROTACs) targeting p300 / CBP have aroused great interest, and the new p300 / CBP PROTACs degrader is expected to become a potential cancer therapy targeting p300 / CBP.

[0003] Unlike traditional small molecule inhibitors, PROTACs have a degradative effect on non-drugable proteins; in addition, because PROTACs molecules are event-driven, they also have the advantages of reducing drug dosage, improving drug resistance and off-target toxicity. PROTACs is a bifunctional molecule, one end of which is a ligand connected to the E3 ubiquitin ligase, and the other end is a ligand connected to the target protein, and the two are connected by a linker of appropriate length. PROTACs molecules form a ternary complex with E3 ubiquitin ligase and target protein. The formation of the ternary complex allows the poly-Ub chain to bind to the substrate protein, ultimately leading to the degradation of the protein through UPS.

[0004] It should be pointed out that the linker between the two ligands plays a very important role. If the linker is too short, the two ligands will hinder each other from binding to the target protein or ubiquitin enzyme; if the linker is too long, it will be difficult for the PROTACs molecule to bring the E3 ubiquitin ligase and the target protein closer, resulting in ubiquitination failure.

[0005] SuFEx reaction is a new type of "click chemistry" proposed by Sharpless et al., which is based on the reaction of arylsulfonyl fluoride or arylfluorosulfonate with silyl ether or with amino compounds. SuFEx reaction has the following potential advantages, such as: mild reaction conditions and easy operation; strong substrate tolerance and good biocompatibility; its reaction substrate sulfur (VI) fluoride has very high chemical stability compared with the more common S-Cl, which provides convenience for the preparation and storage of raw materials; when arylsulfonyl fluoride or arylfluorosulfonate reacts with amino compounds, it will generate "sulfonic acid amine" or "sulfuric acid amine" connection mode, which are the dominant skeletons in drugs and may bring new properties to PROTACs molecules.

[0006] Therefore, based on the above research background, the inventors believe that how to design a new PROTACs molecule based on the SuFEx reaction and construct a synthetic method for PROTACs molecules are technical problems that technicians in this field urgently need to solve.

[0007] The information disclosed in this background technology section is only intended to enhance the understanding of the overall background of the invention and should not be regarded as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the invention

[0008] In view of the above technical problems, the embodiments of the present invention provide a compound for targeted degradation of p300 / CBP protein and a synthesis method and application thereof, so as to solve the problems raised in the above background technology.

[0009] A compound for targeted degradation of p300 / CBP protein, with a general structural formula as follows Figure 4 As shown in formula (A) and formula (B).

[0010] Preferably, the compound targeting the degradation of p300 / CBP protein has the following structural formula: Figure 5 One of the formulas Ⅰ 1-Ⅰ 6 and Ⅱ 1-Ⅱ 6.

[0011] A method for synthesizing a compound for targeted degradation of p300 / CBP protein as described above, the reaction route is as follows Figure 3 and Figure 4 .

[0012] Preferably, the method for synthesizing a compound that targets the degradation of p300 / CBP protein comprises the following steps: Mixing 2-fluoro-3-bromonitrobenzene shown in Formula 1 and (R)-3-aminobutyric acid shown in Formula 2, and subjecting the two to a substitution reaction to obtain an intermediate 3; Under the conditions of iron powder and acetic acid, the nitro group on intermediate 3 was reduced to amino group and then self-cyclized to obtain intermediate 4; Intermediate 4 underwent Suzuki-Miyaura cross-coupling with boron reagent 5 to give olefin 6; Olefin 6 is subjected to Heck coupling with bromophenylsulfonyl fluoride shown in formula 7 to give sulfonyl fluoride IV; Intermediate 4 was subjected to Heck coupling with styrene 8 to give compound 9; Compound 9 is acylated with sulfuryl fluoride gas under alkaline conditions to obtain fluorosulfonate III; Fluorosulfonate III and sulfonyl fluoride IV are respectively subjected to SuFEx reaction with thalidomide containing alkane with different lengths as connecting arms to obtain target compounds represented by formula (A) and formula (B).

[0013] A use of the compound for targeted degradation of p300 / CBP protein as described above in the preparation of a drug for degrading p300 / CBP protein.

[0014] A use of the compound for targeted degradation of p300 / CBP protein as described above in the preparation of drugs for treating tumors.

[0015] Preferably, the compound that targets and degrades p300 / CBP protein is used as an active ingredient in the preparation of a tumor treatment drug.

[0016] Preferably, the dosage form of the drug is an oral preparation, a mucosal administration preparation or an injection.

[0017] It should be noted that p300 / CBP proteins play an important role in tumorigenesis and development. They promote the growth and survival of tumor cells by regulating key processes such as gene transcription, cell cycle, and apoptosis. Overexpression or abnormal activity of p300 / CBP is one of the characteristics of many cancers, so they are also potential targets in tumor treatment.

[0018] The compound for targeted degradation of p300 / CBP protein provided in the embodiment of the present invention and its synthesis method and application have the following beneficial effects: 1. The present invention provides for the first time a method for constructing proteolysis targeting chimeras and other bifunctional molecules using the SuFEx reaction; the present invention uses the fluorinated sulfonate and sulfonyl fluoride analogs of the compound CPI644 as p300 / CBP protein ligands, and connects them with the E3 ubiquitin ligase ligands 5-OH and 4-OH thalidomide pre-introduced with alkanes of different lengths as connecting arms through the SuFEx reaction, thereby preparing two series of 12 PROTACs molecules; 2. The PROTACs molecules designed in the present invention have the ability to degrade p300 / CBP protein, and PROTACs molecules can degrade p300 / CBP protein in a concentration-dependent manner in human breast cancer cells; these PROTACs molecules show good anti-tumor effects and have broad application prospects in the diagnosis and treatment of tumors. BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 The predicted map of compound CPI644 binding to p300 / CBP protein bromodomain (PDB: 5DBM); Figure 2 A design roadmap for the synthesis of PROTACs based on the SuFEx reaction of compound CPI644 fluorosulfonate III with sulfonyl fluoride IV; Figure 3 The synthetic route of fluorosulfonate III and sulfonyl fluoride IV is shown in FIG. Figure 4 A synthetic roadmap for the target PROTACs molecules; Figure 5 The structural formulas of 12 target PROTACs molecules; Figure 6 The degradation result of p300 protein by PROTACs molecules; Figure 7 This is the mechanism by which compounds I-4 and II-4 induce p300 protein degradation. DETAILED DESCRIPTION

[0020] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.

[0021] In view of the above technical problems, the embodiments of the present invention provide a compound for targeted degradation of p300 / CBP protein and a synthesis method and application thereof, so as to solve the problems raised in the above background technology.

[0022] 1. Design idea of ​​the present invention Compared with p300 / CBP HAT inhibitors, the development of p300 / CBP bromodomain inhibitors has progressed faster, and p300 / CBP bromodomain inhibitors with various structural types have been reported. Among them, the compound CPI644 reported by Genentech has good affinity for CBP bromodomain. The relevant research data of compound CPI644 are as follows: ITC (Isothermal titration calorimetry) experiments showed that K d = 0.084 uM; NanoBRETCellular Assays indicate that the EC of CPI644 to CBP 50 = 0.53 uM, EC for EP300 50 = 0.36 uM (reference: S. Ghosh, A. Taylor, M. Chin, et al., J. Biol. Chem. 291 (2016) 13014-13027). The above results suggest that compound CPI644 can be used as a ligand targeting the p300 / CBP bromodomain.

[0023] Molecular docking experiment of compound CPI644 molecule and p300 / CBP protein bromodomain (PDB: 5DBM) (see Figure 1) showed that CPI644 binds to the acetyllysine recognition site of p300 / CBP protein: its benzodiazepine core forms a key hydrogen bond with ASN1168 and forms a π-π stack with TRP1125. It should be noted that the sulfonamide group at the end of CPI644 points to the solvent region. The above results suggest that the terminal sulfonamide of CPI644 can be rationally coupled with E3 ubiquitin ligase ligands to prepare the corresponding PROTACs molecules.

[0024] Based on the results of the molecular docking experiment between CPI644 and the p300 / CBP protein bromodomain, we designed the modification of the sulfonamide group at the end of CPI644 to fluorinated sulfonate III and sulfonyl fluoride IV (see Figure 2 ). The fluorinated sulfonate group in compound III and the sulfonyl fluoride group in compound IV can both be extended by SuFEx reaction, that is, reacting with the E3 ubiquitin ligase fragment containing an amino group to generate PROTACs molecules connected with sulfate amine or sulfonamide, respectively.

[0025] 2. Specific synthesis method of compounds targeting the degradation of p300 / CBP protein The synthesis method of fluorinated sulfonate III and sulfonyl fluoride IV is as follows Figure 3 As shown. Commercial raw materials 1 (2-fluoro-3-bromonitrobenzene) and 2 ((R)-3-aminobutyric acid) were used for substitution reaction to obtain intermediate 3, and then the nitro group was reduced to amino group under the conditions of iron powder and acetic acid and self-cyclized to obtain intermediate 4; intermediate 4 was subjected to Suzuki-Miyaura cross coupling with boron reagent 5 to obtain olefin 6 (olefin 6 was prepared according to the synthetic route of the literature: J. Biol. Chem. 2016, 291, (25), 13014), and then Heck coupling with bromophenylsulfonyl fluoride 7 to obtain sulfonyl fluoride IV. Intermediate 4 was subjected to Heck coupling with styrene 8 to obtain 9, and then acylated with sulfuryl fluoride gas under alkaline conditions to obtain fluorosulfonate III.

[0026] After obtaining fluorosulfonate III and sulfonyl fluoride IV, fluorosulfonate III and sulfonyl fluoride IV were subjected to SuFEx reaction with thalidomide fragments (which are known compounds) containing alkane with different lengths as connecting arms (see Figure 4 ), 12 target PROTACs molecules were prepared; (Table 1).

[0027] Table 1. Synthesis information of target PROTACs molecules Linking position to thalidomide n Sulfonamide series compounds (yield) Sulfate ammonium compounds (yield) 4 n = 1 I-1 (79%) II-1 (76%) 4 n = 2 I-3 (88%) II-3 (78%) 4 n = 3 I-5 (72%) II-5 (81%) 5 n = 1 I-2 (82%) II-2 (72%) 5 n = 2 I-4 (85%) II-4 (84%) 5 n = 3 I-6 (78%) II-6 (82%) Reaction conditions: Fluorosulfonate III or sulfonyl fluoride IV (1.0 equiv.), thalidomide with different linker lengths (2.0 equiv.), HOBt (5 mol%), DIPEA (1.0 equiv.), and TMDS (2.0 equiv.) were reacted in anhydrous DMSO.

[0028] 3. Preparation Examples of Specific Key Compounds Example 1: Synthesis of Compound 5 Add compound 4 (100 mg, 0.39 mmol, 1.0 eq), boron reagent 5 (98 uL, 0.57mmol, 1.5 eq), PdCl2(dppf) (27 mg, 0.04 mmol, 0.1 eq), Cs2CO3 (260 mg, 0.80 mmol, 2.0eq), dioxane (1 mL), and H2O (0.3 mL) to a microwave reaction tube. Bubble nitrogen into the system for 5 min to create an oxygen-free environment. Set the temperature to 100°C and microwave for 45 min. After cooling to room temperature, TLC detection shows that the reaction is complete. The product was extracted from the aqueous phase into the organic phase with ethyl acetate (3×30 mL), and the aqueous phase and the organic phase were monitored by TLC respectively to ensure that all the products entered the organic phase. After there was no product in the aqueous phase, the organic phases were combined, washed with saturated brine to absorb excess water, and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered out, and the organic phase was concentrated under reduced pressure on a rotary evaporator and separated using silica gel column chromatography (DCM: MeOH = 10: 1) to obtain compound 5 as a brown solid (72 mg, 80%). 1H NMR (400 MHz, DMSO-d6) d 8.92 (s, 1H), 7.32 (dd, J = 7.9, 1.2 Hz, 1H), 7.17 (dd, J = 6.6, 1.2 Hz, 1H), 7.00 (dd, J =7.9, 6.6 Hz, 1H), 6.86 (dd, J = 16.3, 10.8 Hz, 1H), 6.26 (d, J = 8.5 Hz, 1H), 5.62 (dd, J = 16.2, 2.4 Hz, 1H), 5.44 (dd, J = 10.8, 2.4 Hz, 1H), 4.09 (m,1H), 2.68 (dd, J = 16.4, 6.4 Hz, 1H), 2.47- 2.39 (m, 1H), 1.29 (d, J = 6.6Hz, 3H). ESI-MS: m / z calcd. for C 12 H 15 N2O＋ [M+H] ＋ 203.1, found 203.0. Example 2: Synthesis of Compound IV Dissolve 5 (50 mg, 0.24 mmol, 1.0 eq) and 7 (88 mg, 0.37 mmol, 1.5 eq) in DMF (10 mL), add TEA (0.6 mL, 4.80 mmol, 20.0 eq) and tri(o-methylphenyl)phosphine (15 mg, 0.05 mmol, 0.2 eq) to the system, replace nitrogen three times, add Pd(OAc)2 (11 mg, 0.05 mmol, 0.2 eq), and replace nitrogen three times. Heat to 150 ° C and react overnight. TLC detection of the reaction is complete. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3×30 mL). The aqueous phase and the organic phase were monitored by TLC respectively to ensure that all the products entered the organic phase. After there was no product in the aqueous phase, the organic phases were combined and washed with saturated brine to absorb excess water, and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered out, and the organic phase was concentrated under reduced pressure on a rotary evaporator, and separated by silica gel column chromatography (PE: EA = 1:1) to obtain compound IV as a brown solid (62 mg, 47%). 1 H NMR (400 MHz, cdcl3) δ 8.09(ddd, J = 14.3, 8.1, 1.3 Hz, 1H), 8.01 (s, 1H), 7.86 (d, J = 8.0 Hz, 2H),7.81 – 7.66 (m, 1H), 7.56 – 7.40 (m, 1H), 7.37 – 7.19 (m, 3H), 5.30 (s, 1H), 4.18 (s, 1H), 2.66 (ddd, J = 17.5, 13.3, 4.7 Hz, 1H), 2.42 (dt, J = 13.3, 8.0Hz, 1H), 1.40 (dd, J = 6.3, 1.2 Hz, 3H). ESI-MS: m / z calcd. for C 18 H 18 FN2O3S ＋ [M+H] ＋ 361.1, found 361.1. Example 3: Synthesis of Compound 9 4 (50 mg, 0.2 mmol, 1.0 eq) and 8 (35 mg, 0.3 mmol, 1.5 eq) were dissolved in DMF (10 mL). TEA (277 μL, 2.0 mmol, 10.0 eq) and tri(o-methylphenyl)phosphine (12 mg, 0.04 mmol, 0.2 eq) were added to the system. After replacing nitrogen three times, Pd(OAc)2 (4 mg, 0.04 mmol, 0.2 eq) was added and nitrogen was replaced three times. The mixture was heated to 150°C and reacted overnight. The reaction was completed by TLC. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3×30 mL). The aqueous phase and the organic phase were monitored by TLC respectively to ensure that all the products entered the organic phase. After there was no product in the aqueous phase, the organic phases were combined and washed with saturated brine to absorb excess water, and dried over anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered out, and the organic phase was concentrated under reduced pressure on a rotary evaporator, and separated by silica gel column chromatography (DCM: MeOH = 10:1) to obtain compound 9 as a yellow solid (30 mg, 51%). 1 H NMR (400 MHz, DMSO-d6) δ 8.99 (s,1H), 8.78 (s, 1H), 7.49 (dd, J = 8.0, 1.3 Hz, 1H), 7.36 – 7.21 (m, 3H), 7.21– 7.08 (m, 4H), 6.79 (dd, J 1.29 (d, J = 6.6 Hz, 3H). ESI-MS: m / z calcd. for C 18 H 19 N2O2 ＋ [M+H] ＋ 295.1, found 295.2. Example 4: Synthesis of Compound III 9 (30 mg, 0.1 mmol, 1.0 eq) was dissolved in DCM (5 mL), and TEA (41 μL, 0.3 mmol, 3.0 eq) was added to replace the SO2F2 gas, and the reaction was carried out for 2 h. The reaction was completed by TLC. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3×30 mL). The aqueous phase and the organic phase were monitored by TLC respectively to ensure that all the products entered the organic phase and there was no product in the aqueous phase. The organic phases were combined, washed with saturated brine to absorb excess water, and dried with anhydrous sodium sulfate. The anhydrous sodium sulfate was filtered out, and the organic phase was concentrated under reduced pressure by a rotary evaporator without further purification and directly put into the next step.

[0029] Example 5: General method for the synthesis of target PROTACs Compound III or IV (0.1 mmol, 1.0 eq) and thalidomide with different lengths of connecting arms (0.1 mmol, 1.0 eq) were dissolved in DMSO (2 mL), and HOBT (1.0 mg, 0.005 mmol, 0.05 eq), TMDS (35 μL, 0.2 mmol, 2.0 eq), and DIPEA (17 μL, 0.1 mmol, 1.0 eq) were added to the system. After the addition was completed, the reaction was allowed to proceed at room temperature for 24 h. The reaction was detected by TLC. The reaction solution was diluted with water, and the product was extracted from the aqueous phase into the organic phase with ethyl acetate (3×30 mL). The aqueous phase and the organic phase were monitored by TLC respectively to ensure that all the products entered the organic phase and there was no product in the aqueous phase. The organic phases were combined, washed with saturated brine, and excess water was absorbed. The anhydrous sodium sulfate was fully dried, and the anhydrous sodium sulfate was filtered out. The organic phase was concentrated under reduced pressure by a rotary evaporator and separated using a silica gel preparation plate.

[0030] I-1: light yellow powder, 57.5 mg, 79%. 1H NMR (400 MHz, DMSO) δ 11.16 (s, 1H),9.54 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 8.31 (t, J = 5.8 Hz, 1H), 8.10 – 8.01(m, 1H), 7.78 (dd, J = 8.5, 7.3 Hz, 1H), 7.59 (d, J = 16.1 Hz, 1H), 7.49 (d,J = 7.3 Hz, 1H), 7.44 – 7.32 (m, 6H), 7.27 (d, J = 16.1 Hz, 1H), 6.96 – 6.79(m, 2H), 5.14 (dd, J = 12.9, 5.4 Hz, 2H), 4.81 (s, 2H), 4.05 (dq, J = 11.6,6.6 Hz, 2H), 3.34 (q, J = 6.4 Hz, 2H), 3.25 (q, J = 6.3 Hz, 3H), 2.91 (ddd, J= 17.4, 13.9, 5.5 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.52 – 2.41 (m, 2H), 2.15(dd, J = 13.0, 7.2 Hz, 1H), 2.10 – 2.01 (m, 1H), 2.00 (s, 1H), 1.28 (d, J =6.1 Hz, 3H), 1.19 (t, J = 7.1 Hz, 1H). 13C NMR (101 MHz, DMSO) δ 173.34,172.32, 172.26, 170.90, 170.42, 167.81, 167.25, 165.90, 155.60, 147.67,137.91, 137.43, 133.52, 132.85, 131.33, 131.04, 129.92, 129.12, 127.48,127.34, 127.27, 126.89, 125.13, 124.13, 123.01, 122.81, 122.51, 122.27,122.11, 121.38, 120.85, 119.63, 117.20, 116.53, 116.25, 67.96, 60.29, 56.23,56.13, 49.29, 43.14, 40.73, 38.86, 31.45, 23.76, 23.68, 22.51, 21.28, 14.59.ESI-MS: m / z calcd. for C 35 H 35 N6O 10 S ＋ [M+H] ＋ 731.2, found 731.2. I-2: pale yellow powder, 60.8 mg, 82%. 1H NMR (400 MHz, DMSO) δ 11.15 (s, 1H),9.52 (s, 1H), 8.82 (t, J = 5.6 Hz, 1H), 8.45 (t, J = 5.7 Hz, 1H), 8.08 – 8.01(m, 1H), 7.91 – 7.83 (m, 1H), 7.58 (d, J = 16.1 Hz, 1H), 7.50 – 7.41 (m, 2H),7.41 – 7.35 (m, 5H), 7.28 (t, J = 14.8 Hz, 2H), 6.95 – 6.86 (m, 2H), 6.86 –6.79 (m, 1H), 5.18 – 5.07 (m, 2H), 4.72 (s, 2H), 4.04 (q, J = 7.1 Hz, 2H),3.32 (t, J = 6.1 Hz, 3H), 3.25 (t, J = 6.1 Hz, 2H), 3.23 – 3.15 (m, 1H), 2.97– 2.84 (m, 1H), 2.65 – 2.52 (m, 2H), 2.52 – 2.38 (m, 2H), 2.19 – 2.02 (m,2H), 2.00 (s, 2H), 1.26 (t, J = 6.9 Hz, 4H), 1.18 (t, J = 7.1 Hz, 3H). 13C NMR(101 MHz, DMSO) δ 173.31, 172.27, 172.21, 171.96, 170.87, 170.44, 167.67,167.28, 167.23, 163.48, 163.44, 155.33, 147.67, 137.91, 137.38, 134.20,132.85, 131.33, 131.01, 129.91, 129.37, 129.11, 127.49, 127.33, 127.26,126.87, 125.83, 124.78, 124.06, 122.99, 122.80, 122.49, 122.25, 122.08,121.34, 121.26, 119.61, 116.25, 110.08, 67.75, 60.28, 56.20, 56.10, 49.48,43.33, 43.08, 40.73, 38.95, 38.77, 31.45, 24.14, 23.67, 23.08, 22.55, 21.60,21.27, 14.59.ESI-MS: m / z calcd. for C 35 H 35 N6O 10 S ＋ [M+H] ＋ 731.2, found 731.2. I-3: yellow powder, 66.8 mg, 88%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H), 9.49(s, 1H), 8.53 (t, J = 5.5 Hz, 1H), 8.04 – 7.95 (m, 2H), 7.85 – 7.77 (m, 1H),7.57 – 7.46 (m, 2H), 7.43 – 7.31 (m, 5H), 7.30 – 7.21 (m, 1H), 6.94 – 6.89(m, 1H), 6.89 – 6.82 (m, 1H), 5.15 – 5.08 (m, 1H), 4.76 (d, J = 5.1 Hz, 2H),4.03 (qd, J = 6.7, 5.3 Hz, 1H), 3.18 – 3.07 (m, 4H), 2.93 – 2.84 (m, 1H),2.69 (d, J = 1.3 Hz, 4H), 2.63 – 2.56 (m, 1H), 2.53 (d, J = 16.0 Hz, 8H),2.43 (dd, J = 13.0, 5.0 Hz, 1H), 2.21 – 2.10 (m, 1H), 2.06 – 1.97 (m, 2H),1.53 – 1.48 (m, 2H), 1.48 (d, J = 7.9 Hz, 2H), 1.37 – 1.28 (m, 2H), 1.24 (dd,J = 14.5, 5.6 Hz, 6H), 1.17 (td, J = 7.1, 1.3 Hz, 1H), 0.90 – 0.83 (m, 1H). 13C NMR (101 MHz, DMSO) δ 173.48, 172.61, 170.46, 167.42, 167.28, 166.06,155.51, 147.71, 137.88, 137.58, 133.43, 132.75, 131.20, 130.02, 129.20,127.32, 127.26, 123.07, 122.98, 122.53, 122.18, 121.47, 120.81, 117.13,116.60, 82.28, 67.96, 56.22, 49.27, 43.72, 40.63, 40.59, 38.36, 34.84, 31.39,27.15, 26.87, 26.54, 23.62, 22.46. ESI-MS: m / z calcd. for C 37 H 39 N6O 10 S ＋ [M+H] ＋ 759.2, found 759.2. I-4: pale yellow powder, 64.5 mg, 85%. 11H NMR (400 MHz, DMSO) δ 11.15 (s, 1H), 9.52 (s, 1H), 8.30 (q, J = 5.1 Hz, 1H), 8.06 – 7.99 (m, 1H), 7.88 (d, J = 8.3 Hz, 1H), 7.57 (d, J = 16.1 Hz, 1H), 7.45 (d, J = 2.3 Hz, 1H), 7.43 – 7.35 (m, 4H), 7.35 – 7.22 (m, 2H), 6.95 – 6.71 (m, 2H), 5.18 – 5.05 (m, 2H), 4.72 (d, J = 2.8 Hz, 2H), 4.10 – 3.99 (m, 1H), 3.13 (dt, J = 6.7, 3.6 Hz, 5H), 2.90 (ddd, J = 17.0, 14.0, 5.4 Hz, 1H), 2.59 (dd, J = 15.5, 11.9 Hz, 3H), 2.51 – 2.36 (m, 2H), 2.19 – 1.98 (m, 3H), 1.56 – 1.46 (m, 4H), 1.26 (t, J = 6.0 Hz, 4H), 1.23 – 1.15 (m, 1H). 13 13C NMR (101 MHz, DMSO) δ 173.30, 172.18, 170.44, 167.30, 167.24, 167.17, 163.57, 147.84, 137.89, 134.20, 132.86, 131.30, 129.96, 129.08, 127.37, 127.22, 127.15, 125.81, 124.01, 123.00, 122.39, 122.07, 121.32, 109.97, 67.83, 60.27, 56.09, 49.48, 43.79, 40.74, 38.34, 31.45, 27.02, 26.69, 23.69, 22.54, 21.28, 14.60. ESI-MS: m / z calcd. for C 37 H 39 N6O 10 S ＋ [M+H] ＋ 759.2,found 759.2. I-5: Yellow powder, 6.1 mg, 72%. 1H NMR (600 MHz, DMSO) δ 11.13 (s, 1H), 9.51(s, 1H), 8.56 (q, J = 4.0 Hz, 1H), 8.06 – 8.00 (m, 2H), 7.84 – 7.77 (m, 1H),7.57 (d, J = 16.1 Hz, 1H), 7.49 (d, J = 7.3 Hz, 1H), 7.45 – 7.31 (m, 5H),7.26 (d, J = 16.1 Hz, 1H), 6.90 – 6.84 (m, 2H), 5.76 (s, 1H), 5.16 – 5.09 (m,1H), 5.09 – 5.05 (m, 1H), 4.78 (s, 2H), 4.16 (s, 1H), 4.05 (tt, J = 7.3, 3.6Hz, 1H), 3.18 (d, J = 3.1 Hz, 3H), 3.16 – 3.11 (m, 2H), 3.11 – 3.02 (m, 2H),2.90 (ddd, J = 17.0, 13.9, 5.4 Hz, 1H), 2.64 – 2.51 (m, 3H), 2.47 – 2.38 (m,1H), 2.14 (dd, J = 12.9, 7.2 Hz, 1H), 2.04 (dtd, J = 12.5, 4.9, 2.0 Hz, 1H),1.55 – 1.37 (m, 4H), 1.25 (dd, J = 14.7, 8.9 Hz, 7H). 13C NMR (151 MHz, DMSO)δ 173.25, 172.16, 171.72, 170.35, 167.21, 167.11, 165.97, 155.54, 147.77,137.87, 137.37, 133.50, 132.80, 131.25, 129.93, 129.04, 127.44, 127.26,127.16, 127.05, 122.95, 122.89, 122.37, 122.06, 121.23, 120.81, 117.25,116.47, 68.06, 56.01, 55.38, 55.35, 49.28, 49.05, 43.98, 43.77, 40.73, 40.50,38.72, 31.41, 29.44, 29.37, 26.34, 26.31, 26.15, 23.63, 22.47 . ESI-MS: m / zcalcd. for C 39 H 43 N6O 10 S ＋ [M+H] ＋ 787.2, found 787.3. I-6: yellow powder, 60.7 mg, 78%. 1H NMR (400 MHz, DMSO) δ 9.53 (s, 1H), 8.31(t, J = 5.8 Hz, 1H), 8.04 (dd, J = 6.1, 3.3 Hz, 1H), 7.87 (dd, J = 8.3, 2.2Hz, 1H), 7.58 (d, J = 16.1 Hz, 1H), 7.44 (d, J = 2.4 Hz, 1H), 7.43 – 7.30 (m,6H), 7.30 – 7.22 (m, 2H), 6.93 – 6.84 (m, 2H), 5.14 (dt, J = 12.8, 3.4 Hz,1H), 5.08 (s, 1H), 4.73 (s, 2H), 4.04 (ddt, J = 14.2, 7.1, 4.0 Hz, 1H), 3.10(t, J = 6.9 Hz, 4H), 2.90 (tt, J = 13.6, 5.4 Hz, 2H), 2.65 – 2.54 (m, 2H),2.44 (dd, J = 13.1, 4.9 Hz, 2H), 2.19 – 1.95 (m, 4H), 1.54 – 1.34 (m, 6H),1.26 (d, J = 5.8 Hz, 5H), 0.85 (s, 1H). 13 C NMR (151 MHz, DMSO) δ 173.23,171.97, 171.70, 170.37, 167.27, 167.20, 167.05, 163.57, 159.19, 158.94,158.69, 158.44, 147.78, 134.17, 133.02, 131.19, 130.20, 129.13, 127.23,127.20, 127.18, 125.74, 123.99, 123.26, 122.92, 122.44, 122.14, 121.87,121.37, 116.72, 114.80, 109.86, 67.83, 60.22, 56.23, 55.34, 49.47, 43.98,40.58, 40.47, 38.74, 31.41, 29.42, 29.35, 26.35, 26.15, 23.25, 22.88, 22.52,14.52. ESI-MS: m / z calcd. for C 39 H 43 N6O10 S ＋ [M+H] ＋ 787.2, found 787.3. II-1: Yellow powder, 53.9 mg, 76%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.08 (d, J = 7.9 Hz, 1H), 8.04 (dt, J = 6.1, 3.1 Hz, 1H), 7.98 (t, J = 6.0 Hz, 1H), 7.85 (dd, J = 8.0, 1.4 Hz, 1H), 7.79 – 7.71 (m, 2H), 7.64 (t, J = 7.6 Hz, 1H), 7.50 – 7.43 (m, 3H), 7.41 (dd, J = 6.4, 3.0 Hz, 1H), 7.35 (d, J = 8.6 Hz, 1H), 6.89 (q, J = 4.2 Hz, 2H), 5.18 – 5.01 (m, 2H), 4.71 (d, J = 1.6 Hz, 2H), 4.04 (q, J = 7.0 Hz, 1H), 3.20 (q, J = 6.6 Hz, 2H), 3.01 – 2.78 (m, 3H), 2.47 – 2.38 (m, 2H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.03 (dtd, J = 12.8, 5.3, 2.2 Hz, 1H), 1.37 – 1.29 (m, 1H), 1.24 (dd, J = 13.5, 4.9 Hz, 5H). 13 C NMR (101 MHz, DMSO) δ 173.28, 172.16, 170.85, 170.38, 167.64, 167.23, 165.88, 155.57, 137.41, 133.54, 126.16, 123.29, 122.20, 120.89, 117.26, 116.52, 67.97, 60.27, 56.05, 55.42, 49.29, 49.09, 48.97, 40.92, 31.45, 23.70, 22.51, 21.27, 14.59. ESI-MS: m / z calcd. for C 35 H 35 N6O9S＋ [M+H] ＋ 715.2, found 715.2. II-2: Yellow powder, 51.1 mg, 72%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.22 (t, J = 5.9 Hz, 1H), 8.09 (dd, J = 8.0, 1.2 Hz, 1H), 7.99 (t, J = 6.0 Hz, 1H), 7.90 – 7.82 (m, 2H), 7.75 (d, J = 15.8 Hz, 1H), 7.69 – 7.57 (m, 1H), 7.50 – 7.40 (m, 4H), 7.40 – 7.32 (m, 1H), 6.93 – 6.86 (m, 2H), 5.15 – 5.08 (m, 2H), 4.64 (s, 2H), 4.11 (q, J = 5.2 Hz, 2H), 4.03 (q, J = 7.1 Hz, 2H), 3.19 (dd, J = 17.0, 5.9 Hz, 6H), 2.95 – 2.90 (m, 2H), 2.90 – 2.85 (m, 1H), 2.64 – 2.59 (m, 1H), 2.59 – 2.50 (m, 4H), 2.43 (dd, J = 12.9, 5.0 Hz, 1H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.05 (dtd, J = 13.0, 5.4, 2.4 Hz, 1H), 1.99 (s, 2H), 1.25 (d, J = 6.2 Hz, 3H), 1.18 (t, J = 7.1 Hz, 2H). 13C NMR(151 MHz, DMSO) δ 173.24, 172.13, 170.82, 170.37, 167.49, 167.23, 167.18,163.35, 138.00, 137.55, 136.58, 134.17, 133.01, 132.73, 129.85, 129.13,129.07, 128.94, 128.57, 127.83, 126.12, 125.76, 124.04, 123.26, 122.18,121.24, 121.12, 109.98, 67.69, 60.22, 56.00, 55.37, 49.46, 49.07, 42.00,40.70, 40.51, 38.97, 31.42, 23.65, 22.52, 21.22, 14.55. ESI-MS: m / z calcd.for C 35 H 35 N6O9S ＋ [M+H] ＋ 715.2, found 715.3. II-3: yellow powder, 57.9 mg, 78%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H),9.49 (s, 1H), 8.09 (dd, J = 8.0, 1.3 Hz, 1H), 7.92 (t, J = 5.8 Hz, 1H), 7.87– 7.80 (m, 2H), 7.79 (dd, J = 7.2, 1.4 Hz, 1H), 7.76 (s, 1H), 7.67 – 7.62 (m,1H), 7.51 – 7.43 (m, 3H), 7.42 (dd, J = 7.2, 2.3 Hz, 1H), 7.36 (d, J = 8.6Hz, 1H), 6.93 – 6.86 (m, 2H), 5.16 – 5.05 (m, 2H), 4.74 (s, 2H), 4.04 (q, J =6.6 Hz, 1H), 3.06 (q, J = 6.4 Hz, 2H), 2.89 (ddd, J = 17.1, 13.9, 5.5 Hz,1H), 2.82 (q, J = 6.3 Hz, 2H), 2.60 (ddd, J = 17.0, 4.2, 2.5 Hz, 1H), 2.47 –2.41 (m, 1H), 2.13 (dd, J = 13.0, 7.2 Hz, 1H), 2.03 (dtd, J = 12.8, 5.2, 2.3Hz, 1H), 1.39 (dd, J = 6.3, 3.1 Hz, 3H), 1.26 – 1.24 (m, 3H). 13 C NMR (151MHz, DMSO) δ 173.24, 172.13, 170.35, 167.20, 167.14, 165.95, 155.53, 137.99,137.40, 133.50, 132.87, 129.00, 128.78, 128.47, 127.79, 126.27, 123.23,122.17, 121.13, 120.85, 117.26, 116.51, 68.06, 56.01, 49.27, 42.48, 40.89,40.71, 38.27, 31.42, 27.05, 26.63, 23.65, 22.46. ESI-MS: m / z calcd. forC 37 H 39 N6O9S ＋ [M+H]＋ 743.2, found 743.2. II-4: Yellow powder, 62.1 mg, 84%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.50 (s, 1H), 8.17 (t, J = 5.9 Hz, 1H), 8.10 (dd, J = 7.9, 1.2 Hz, 1H), 7.88–7.85 (m, 2H), 7.82 (t, J = 5.8 Hz, 1H), 7.78 (d, J = 15.8 Hz, 1H), 7.65 (td, J = 7.6, 1.4 Hz, 1H), 7.51 – 7.40 (m, 4H), 7.37 (dd, J = 8.3, 2.3 Hz, 1H), 6.94 – 6.87 (m, 2H), 5.15 – 5.08 (m, 2H), 4.68 (s, 2H), 4.14 – 4.02 (m, 1H), 3.18 (d, J = 5.0 Hz, 1H), 3.05 (q, J = 6.3 Hz, 2H), 2.83 (q, J = 6.3 Hz, 2H), 2.52 – 2.41 (m, 7H), 2.14 (dd, J = 12.9, 7.2 Hz, 1H), 2.10 – 2.00 (m, 1H), 1.43 – 1.33 (m, 4H), 1.26 (d, J = 6.2 Hz, 4H). 13 C NMR (101 MHz, DMSO) δ 172.31, 170.85, 170.19, 169.67, 167.20, 167.03, 161.43, 138.09, 136.53, 133.91, 133.40, 131.42, 130.37, 128.63, 128.54, 128.31, 127.87, 127.53, 127.38, 126.20, 125.54, 124.06, 124.02, 122.82, 121.65, 108.38, 67.77, 52.50, 49.98, 43.97, 43.52, 40.15, 29.91, 26.43, 26.33, 24.73, 20.89. ESI-MS: m / z calcd. for C 37 H 39 N6O9S ＋[M+H] ＋ 743.2, found 743.2. II-5: Yellow powder, 62.4 mg, 81%. 1 H NMR (600 MHz, DMSO) δ 11.12 (s, 1H), 9.49 (s, 1H), 8.10 (dd, J = 7.8, 1.2 Hz, 1H), 7.92 – 7.84 (m, 2H), 7.84 – 7.75 (m, 3H), 7.68 – 7.62 (m, 1H), 7.51 – 7.40 (m, 4H), 7.38 (d, J = 8.6 Hz, 1H), 6.94 – 6.86 (m, 2H), 5.15 – 5.10 (m, 1H), 5.10 – 5.07 (m, 1H), 4.75 (s, 2H), 4.04 (q, J = 6.2 Hz, 1H), 3.07 (p, J = 6.3 Hz, 2H), 2.94 – 2.84 (m, 1H), 2.81 (q, J = 6.7 Hz, 2H), 2.48 – 2.40 (m, 1H), 2.13 (dd, J = 12.9, 7.3 Hz, 1H), 2.04 (dtd, J = 13.0, 5.4, 2.4 Hz, 1H), 1.33 (dt, J = 14.7, 7.1 Hz, 5H), 1.28 – 1.21 (m, 6H), 1.21 – 1.17 (m, 2H), 1.17 – 1.14 (m, 1H), 1.14 – 1.08 (m, 2H). 13C NMR (151 MHz, DMSO) δ 173.24, 172.13, 170.34, 167.20, 167.06, 165.97, 155.51, 137.97, 137.38, 136.54, 133.50, 132.86, 132.77, 129.90,129.06, 128.74, 128.44, 127.77, 126.28, 123.23, 122.17, 121.14, 120.83,117.28, 116.51, 68.08, 56.03, 49.27, 42.67, 40.90, 40.71, 40.52, 38.70,31.41, 29.53, 29.34, 26.25, 26.11, 23.65, 22.46. ESI-MS: m / z calcd. forC 39 H 43 N6O9S ＋ [M+H] ＋ 771.2, found 771.1. II-6: yellow powder, 63.2 mg, 82%. 1H NMR (600 MHz, DMSO) δ 11.11 (s, 1H),9.49 (s, 1H), 8.15 (t, J = 5.8 Hz, 1H), 8.10 (dd, J = 8.0, 1.2 Hz, 1H), 7.86(dt, J = 6.6, 2.4 Hz, 2H), 7.85 – 7.75 (m, 2H), 7.68 – 7.62 (m, 1H), 7.51 –7.31 (m, 5H), 6.94 – 6.86 (m, 2H), 5.15 – 5.07 (m, 2H), 4.69 (s, 2H), 4.04(q, J = 6.6 Hz, 1H), 3.06 (dq, J = 13.2, 6.6 Hz, 2H), 2.93 – 2.84 (m, 2H),2.80 (q, J = 6.6 Hz, 2H), 2.69 (s, 1H), 2.63 – 2.57 (m, 1H), 2.43 (dd, J =12.9, 5.0 Hz, 1H), 2.13 (dd, J = 13.0, 7.3 Hz, 1H), 2.05 (dtd, J = 12.7, 5.3,2.2 Hz, 1H), 1.42 – 1.33 (m, 3H), 1.31 (dd, J = 13.3, 6.1 Hz, 3H), 1.26 –1.18 (m, 8H), 1.17 (d, J = 8.3 Hz, 1H), 1.11 (dt, J = 14.4, 8.6 Hz, 3H), 0.85(t, J = 6.8 Hz, 1H). 13C NMR (151 MHz, DMSO) δ 173.24, 172.12, 170.37, 167.27,167.20, 167.00, 163.57, 137.97, 136.54, 134.17, 132.86, 132.77, 129.90,129.06, 128.74, 128.44, 127.77, 126.28, 125.76, 123.98, 123.23, 122.17,121.38, 121.14, 109.86, 67.83, 56.03, 49.46, 42.69, 40.90, 40.72, 40.52,38.71, 32.01, 31.42, 30.30, 29.90, 29.52, 29.45, 29.40, 26.31, 26.13, 23.65,22.56, 22.52, 14.42. ESI-MS: m / z calcd. for C 39 H 43 N6O9S ＋ [M+H] ＋ 771.2, found 771.4. IV. Active pharmacological testing experiments of PROTACs molecules The obtained target compound was used to perform a p300 protein degradation experiment in human breast cancer MDA-MB-468 cells. The Western Blot experimental method includes: discarding the culture medium in the plate and rinsing with PBS. Add 100 uL of pre-prepared 2% SDS solution to each well to ensure that the solution evenly covers all cells. Stir with a pipette until the cells at the bottom are completely digested and form a viscous liquid, transfer it to a 1.5 mL EP tube, heat it in a 100℃ metal bath for 15 min, and the viscous liquid in the EP tube becomes clear and transparent, which is the cell protein solution. The BCA kit detects the protein concentration of each sample. Add 1 / 4 volume of 5× loading buffer to the protein solution and mix well. Add the sample to the SDS-PAGE well (total protein is 40 ug / well). Set the electrophoresis instrument to an 80 V constant voltage power supply, wait for the sample to leave the concentrated gel, and then complete the protein separation with a 150 V constant voltage current to transfer the protein sample on the separation gel to the PVDF membrane. Set the electrophoresis instrument to a 400 mA constant current, and the transfer time is 60 min. The PVDF membrane was placed in a TBST solution containing 5% BSA and blocked at room temperature for 1 h. After that, it was washed three times with TBST, each time for 5 min. The PVDF strip containing the target protein was completely immersed in the primary antibody solution, so that it was fully in contact and slowly shaken at 4°C, and incubated overnight. The PVDF membrane was washed with TBST 5 times, each time for 6 min. After that, the PVDF strip was completely immersed in the primary antibody solution, so that it was fully in contact, and slowly shaken at room temperature for 2 h. After that, it was washed with TBST 5 times, each time for 6 min. The ECL luminescent liquid evenly covered the PVDF strips, and the protein bands were detected using a Bio-rad gel imager.

[0031] Western Blot results showed that (see Figure 6 ), the 12 PROTACs molecules designed and synthesized by the present invention can degrade p300 protein in MDA-MB-468 cells in a concentration-dependent manner. Among them, I-4 and II-4 showed better protein degradation ability in their respective series, and their DC 50 0.18 uM and 1.04 uM respectively.

[0032] Next, using I-4 and II-4 as examples, the mechanism verification experiment of competitive inhibition confirmed that I-4 and II-4 induced the degradation of p300 protein through the ubiquitin-proteasome pathway (see Figure 7). First, I-4 and II-4 significantly degraded p300 protein. Next, MDA-MB-468 cells were pretreated with proteasome inhibitor MG132, and then treated with compounds I-4 and II-4. After treatment, the degradation of p300 activity by both compounds was significantly weakened, suggesting that PROTACs molecules degrade the target protein through the proteasome pathway. Then, MDA-MB-468 cells were pretreated with CRBN ligand thalidomide and p300 protein ligand CPI644, respectively, and then compounds I-4 and II-4 were added. After treatment, the degradation of p300 activity by both compounds was also significantly weakened, suggesting that PROTACs molecules degrade the target protein by forming a p300-PROTACs-CRBN ternary complex.

[0033] The embodiments described above are only descriptions of the preferred implementation modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the claims of the present invention.

Claims

1. A compound for targeted degradation of p300 / CBP protein, characterized in that: The general structural formula is shown in formula (A) and formula (B): .

2. The compound for targeted degradation of p300 / CBP protein according to claim 1, characterized in that: The structural formula is one of formula Ⅰ1-Ⅰ6 and Ⅱ1-Ⅱ6: 。 3. A method for synthesizing a compound for targeted degradation of p300 / CBP protein as claimed in claim 1 or 2, characterized in that: The synthetic reaction route is as follows: ； 。 4. The method for synthesizing a compound for targeted degradation of p300 / CBP protein according to claim 3, characterized in that: The synthesis method of the compound targeting the degradation of p300 / CBP protein comprises the following steps: Mixing 2-fluoro-3-bromonitrobenzene shown in Formula 1 and (R)-3-aminobutyric acid shown in Formula 2, and subjecting the two to a substitution reaction to obtain an intermediate 3; Under the conditions of iron powder and acetic acid, the nitro group on intermediate 3 was reduced to amino group and then self-cyclized to obtain intermediate 4; Intermediate 4 underwent Suzuki-Miyaura cross-coupling with boron reagent 5 to give olefin 6; Olefin 6 is subjected to Heck coupling with bromophenylsulfonyl fluoride shown in formula 7 to give sulfonyl fluoride IV; Intermediate 4 was subjected to Heck coupling with styrene 8 to give compound 9; Compound 9 is acylated with sulfuryl fluoride gas under alkaline conditions to obtain fluorosulfonate III; Fluorosulfonate III and sulfonyl fluoride IV are respectively subjected to SuFEx reaction with thalidomide containing alkane with different lengths as connecting arms to obtain target compounds represented by formula (A) and formula (B).

5. Use of the compound for targeted degradation of p300 / CBP protein as claimed in any one of claims 1 to 4 in the preparation of a drug for degrading p300 / CBP protein.

6. Use of the compound for targeted degradation of p300 / CBP protein as claimed in any one of claims 1 to 4 in the preparation of a drug for treating tumors.

7. Use of the compound for targeted degradation of p300 / CBP protein according to claim 6 in the preparation of a drug for treating tumors, characterized in that: The invention relates to the use of a compound that targets and degrades p300 / CBP protein as an active ingredient in the preparation of a tumor treatment drug.

8. Use of the compound for targeted degradation of p300 / CBP protein according to claim 7 in the preparation of a drug for treating tumors, characterized in that: The drug is in the form of oral preparations, transmucosal preparations or injections.

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