A disulfide-bridged paclitaxel-bms-202 prodrug and its preparation method and application

Abstract

The application discloses a kind of disulfide bond bridged paclitaxel-BMS-202 prodrug and its preparation method and application.The prodrug structure is;Wherein: n is 1,2 or 3;A is modified BMS-202 residue, which retains the PD-1 / PD-L1 blocking activity of BMS-202 pharmacophore and has primary amino group as connection site at the end;B is paclitaxel residue.Preparation: according to the stability difference of different position disulfide bond, alpha position and beta position prodrug are synthesized by step method, and gamma position prodrug is innovatively synthesized by one-pot method at low temperature and special purification strategy.The prodrug of the application can be specifically broken in response to high-concentration glutathione environment in tumor cells, simultaneously release paclitaxel and BMS-202, realize the synergistic treatment of chemotherapy-induced immunogenic cell death and immune checkpoint blocking, solve the problem of pharmacokinetic mismatch and large toxic side effect of two-drug combination, and have wide application prospect in antitumor drug preparation.

Description

Technical Field

[0001] This invention belongs to the field of medicinal chemistry technology, specifically a disulfide bond-bridged paclitaxel-BMS-202 prodrug, its preparation method, and its application. Background Technology

[0002] Paclitaxel (PTX) is a commonly used broad-spectrum chemotherapy drug in clinical practice. It works by promoting microtubule polymerization and inhibiting depolymerization, thereby arresting the cell cycle and inducing apoptosis. Recent studies have found that PTX can also trigger immunogenic cell death (ICD), releasing signaling molecules such as calreticulin (CRT) and HMGB-1, transforming "cold tumors" into "hot tumors" and activating anti-tumor immunity. However, its high hydrophobicity necessitates the use of solubilizers (which can easily trigger hypersensitivity reactions); it also lacks tumor selectivity and is highly toxic to normal proliferating tissues; its pharmacokinetics are nonlinear, making it prone to drug resistance.

[0003] BMS-202 is an oral small molecule antagonist targeting the PD-1 / PD-L1 interaction, inducing PD-L1 dimerization to block immunosuppressive signals. It has strong tissue penetration, can be taken orally, and is inexpensive. However, its short half-life, rapid metabolism, and requirement for high doses lead to increased toxic side effects.

[0004] Chemotherapy and immunotherapy have synergistic potential, but current simple clinical combinations face many challenges: 1) Pharmacokinetic mismatch: PTX is cleared quickly, while antibody drugs accumulate slowly, making it difficult to expose them spatiotemporally at the tumor site; 2) Cumulative toxicity: The dose-limiting toxicity of chemotherapy may overlap with immune-related adverse events; 3) Chemotherapy has a biphasic regulatory effect on the immune system, and high doses may suppress immunity and weaken the efficacy of immunotherapy.

[0005] Therefore, there is an urgent need to develop a novel drug system that can simultaneously deliver chemotherapy drugs and small molecule immunotherapies to the tumor site, achieving precise and controllable release while reducing systemic toxicity. Summary of the Invention

[0006] The purpose of this invention is to address the shortcomings of existing technologies by providing a disulfide-bridged paclitaxel-BMS-202 prodrug, its preparation method, and its application, in order to solve the problems of pharmacokinetic mismatch, significant toxic side effects, and inability to achieve synchronous targeted delivery when paclitaxel and BMS-202 are used in combination.

[0007] This invention involves a rational modification of BMS-202, retaining its key pharmacophores and introducing linker sites to obtain modified BMS-202 (ggBMS-202). Utilizing the high concentration of glutathione (GSH)-sensitive disulfide bonds in the tumor microenvironment as linker arms, ggBMS-202 was covalently linked to PTX, resulting in the design and synthesis of three prodrug molecules with disulfide bonds located at the α, β, and γ positions of the ester bond.

[0008] To achieve the above objectives, the present invention provides the following technical solution:

[0009] In a first aspect, the present invention provides a disulfide-bridged paclitaxel-BMS-202 prodrug having the following structure:

[0010]

[0011] Where: n is 1, 2 or 3, n=1 is the α position, n=2 is the β position, and n=3 is the γ position;

[0012] A represents a modified BMS-202 residue, wherein the modified BMS-202 retains the PD-1 / PD-L1 blocking pharmacophore of BMS-202 and has a primary amino group at the end as a linker site;

[0013] B is a paclitaxel residue, and it forms an ester bond with a carbonyl group through its 2'-hydroxyl group.

[0014] Preferably, the modified BMS-202 residue A is derived from ggBMS-202 prepared by condensation, amination, reduction and deprotection reactions of 2-methyl-3-phenylbenzyl alcohol and 6-chloro-2-methoxypyridine-3-carboxaldehyde.

[0015] Preferably, the specific structure of the prodrug is as follows:

[0016] The prodrug α-ggBMS-202-SS-PTX, with the disulfide bond located at the α-position, is formed by connecting paclitaxel to an intermediate consisting of modified BMS-202 and 2,2'-dithiodiacetic acid.

[0017] The prodrug β-ggBMS-202-SS-PTX, with the disulfide bond located at the β-position, is formed by connecting paclitaxel to an intermediate consisting of modified BMS-202 and 3,3'-dithiodipropionic acid.

[0018] The prodrug γ-ggBMS-202-SS-PTX, with the disulfide bond located at the γ position, is formed by connecting paclitaxel to an intermediate formed by modified BMS-202 and 4,4'-dithiodibutyric acid.

[0019] Secondly, the present invention provides a method for preparing the disulfide-bridged paclitaxel-BMS-202 prodrug described above, comprising the following steps:

[0020] (1) Synthesis of modified BMS-202: Modified BMS-202 was prepared by condensation, amination, reduction and deprotection reaction of 2-methyl-3-phenylbenzyl alcohol and 6-chloro-2-methoxypyridine-3-carboxaldehyde.

[0021] (2) Synthesis of prodrug: The modified BMS-202 was reacted with dithiocarboxylic acid compounds under the action of condensing agent and catalyst, and then esterified with paclitaxel under the action of condensing agent and catalyst to obtain the target prodrug.

[0022] Preferably, in step (2), for the prodrug with the disulfide bond located at the α or β position, a stepwise synthesis method is adopted: first, the condensation intermediate of modified BMS-202 and dithiocarboxylic acid is separated, purified, and then coupled with paclitaxel.

[0023] Preferably: In step (2), for the prodrug with the disulfide bond located at the γ position, a one-pot synthesis is adopted: after the modified BMS-202 and 4,4'-dithiodibutyric acid have been reacted at room temperature, the intermediate is not separated, and paclitaxel is directly added under ice bath conditions for coupling reaction, and the coupling reaction time is strictly controlled within 2 hours.

[0024] Preferably, the purification steps after the one-pot synthesis of the γ-position prodrug include: after the reaction solution is extracted and washed with a pre-cooled reagent, column chromatography is performed using silica gel activated at 60°C, the eluent is prepared fresh for use, a rapid elution method is adopted, and the collected components are immediately concentrated under reduced pressure.

[0025] In a second aspect, the present invention provides the application of the aforementioned prodrug in the preparation of antitumor drugs.

[0026] Preferably, the prodrug is used to prepare an antitumor drug that releases drugs in response to glutathione in the tumor microenvironment and has the synergistic effect of inducing immunogenic cell death and blocking the PD-1 / PD-L1 pathway.

[0027] Compared with the prior art, the beneficial effects of the present invention are:

[0028] 1. Successful Covalent Bridging of Small Molecule Immunosuppressants and Chemotherapy Drugs: Addressing the challenge of directly coupling the original chemical structure of BMS-202 with paclitaxel, this invention employs a rational molecular modification strategy. While retaining the key pharmacophore (methoxy-1-pyridine structure) of BMS-202, it successfully introduces functional groups that facilitate assembly, resulting in modified BMS-202 (ggBMS-202). This design not only ensures the preservation of immunoblocking activity but also enables two drugs that were previously unable to connect directly to achieve a precise 1:1 covalent bond via disulfide bonds, providing a solid material basis for the simultaneous delivery of chemotherapy and immunotherapy drugs.

[0029] 2. A series of prodrug molecules with tunable disulfide bond positions were provided: This invention successfully synthesized three prodrugs with disulfide bonds located at the α, β, and γ positions of adjacent ester bonds by selecting dithiodic acid compounds of different chain lengths (2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, and 4,4'-dithiodibutyric acid). This structural design enriches the diversity of prodrug molecules, making the relative position of the disulfide bonds a tunable factor, and providing a structural basis for subsequent investigation of the effects of different steric hindrances and carbon chain lengths on prodrug assembly behavior and drug release kinetics.

[0030] 3. Overcoming the synthetic challenge of unstable γ-configuration intermediates, an innovative one-pot process was developed: During the research and development process, it was discovered that the intermediate for the γ-configuration prodrug (ggBMS-202-4,4'-dithiodibutyrate) has poor stability and is prone to disulfide bond breakage or rearrangement during separation and purification. To address this technical bottleneck, this invention innovatively employs a one-pot continuous reaction strategy, where the intermediate is directly coupled with paclitaxel in an ice bath (0-5℃) without separation. This process cleverly avoids the separation step of the unstable intermediate, fundamentally solving its easy degradation problem.

[0031] 4. Optimized purification conditions ensured the yield and quality of unstable products: Addressing the temperature and time sensitivity of the γ-configuration prodrug, this invention systematically optimized the post-reaction processing and column chromatography purification steps. By employing special techniques such as rapid washing with pre-cooled reagents, silica gel activation, freshly prepared eluent, rapid elution (flow rate 2-3 mL / min), and immediate vacuum concentration of collected fractions, the residence time of the product in solution and its exposure to heat were minimized. This effectively suppressed the formation of byproducts and the degradation of the target product, successfully obtaining a high-purity γ-configuration prodrug with a yield of 58%. Attached Figure Description

[0032] Figure 1 The synthetic route for the modified BMS-202 (ggBMS-202) of Example 1 is as follows;

[0033] Figure 2 The synthetic routes for the three prodrugs obtained in Examples 2 to 4 are as follows;

[0034] Figure 3 (A) Proton spectrum and (B) high-resolution mass spectrum of α-ggBMS-202-SS-PTX;

[0035] Figure 4 (A) Proton spectrum and (B) high-resolution mass spectrum of β-ggBMS-202-SS-PTX;

[0036] Figure 5 (A) is the proton spectrum and (B) is the high-resolution mass spectrum of γ-ggBMS-202-SS-PTX. Detailed Implementation

[0037] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention. It should be noted that, unless otherwise specified, the following embodiments and features described therein can be combined with each other.

[0038] Example 1: Synthesis of modified BMS-202 (ggBMS-202)

[0039] The synthetic route for modified BMS-202 (ggBMS-202) is as follows: Figure 1 As shown in the figure, the reaction reagents and conditions are as follows: a) 6-chloro-2-methoxypyridine-3-carboxaldehyde, potassium tert-butoxide, N,N-dimethylformamide (DMF), tetrahydrofuran (THF), 80 ℃; b) tert-butyl 2-(methylamino)ethylcarbamate, acetic acid, DMF, 80 ℃; c) sodium cyanoborohydride, acetic acid, ethanol, 90 ℃; d) hydrochloric acid, dichloromethane (DCM), room temperature. The specific synthesis method is as follows:

[0040] 1. Dissolve 1.0 mmol of 2-methyl-3-phenylbenzyl alcohol in 13 mL of a mixed solvent of DMF and THF (volume ratio 10:3). Place the solution in a round-bottom flask and add 1.5 mmol of 6-chloro-2-methoxypyridine-3-carboxaldehyde under magnetic stirring. After stirring until homogeneous, add 1.5 mmol of potassium tert-butoxide in portions. Reflux the reaction mixture at 80 °C for 10 h. Monitor the reaction progress using thin-layer chromatography (TLC) (petroleum ether:ethyl acetate = 5:1). Stop the reaction when the starting material spot of 2-methyl-3-phenylbenzyl alcohol completely disappears. Cool the reaction solution to room temperature and dilute with 30 mL of ethyl acetate. Wash successively with 20 mL of distilled water and 20 mL of saturated sodium chloride solution. Dry the solution on anhydrous sodium sulfate, filter, and concentrate under reduced pressure. Purify the crude product by silica gel column chromatography using petroleum ether:ethyl acetate (20:1→5:1) as eluent. Collect the target fractions, combine them, and concentrate under reduced pressure to obtain intermediate 1a.

[0041] 2. Intermediate 1a (1.0 mmol) was dissolved in DCM (10 mL) in a round-bottom flask, and 2-(methylamino)ethylcarbamate tert-butyl ester (2.5 mmol) was added. Acetic acid (0.5 mmol) was slowly added dropwise under magnetic stirring. The reaction was stirred at room temperature for 12 h, and the reaction progress was monitored by TLC. After the reaction was complete, the reaction solution was diluted with DCM (30 mL), washed successively with saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 2a, which was directly used in the next reaction.

[0042] 3. Intermediate 2a (1.0 mmol) was dissolved in anhydrous ethanol (15 mL) in a round-bottom flask. Sodium cyanoborohydride (3.0 mmol) was added, and acetic acid (1.0 mmol) was added dropwise under magnetic stirring. The reaction system was heated to 90 °C and refluxed for 24 h. The reaction progress was monitored by TLC. After the reaction was complete, it was cooled to room temperature, concentrated under reduced pressure to remove ethanol, and extracted with DCM (30 mL) and distilled water (20 mL). The aqueous phase was back-extracted with DCM (20 mL), and the organic phases were combined. The organic phase was washed successively with saturated sodium bicarbonate solution (20 mL) and saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain intermediate 3a.

[0043] 4. Intermediate 3a (1.0 mmol) was dissolved in DCM (10 mL) in a round-bottom flask. Concentrated hydrochloric acid (0.1 mmol) was added with magnetic stirring. The reaction was carried out at room temperature, and the reaction progress was monitored by TLC. After the reaction was complete, DCM (20 mL) and distilled water (20 mL) were added for extraction and separation. The organic layer was collected. The organic phase was washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The residue was dissolved in methanol (5 mL), and the pH was adjusted to below 3.0 by adding dilute hydrochloric acid. A pale yellow oily solid precipitated. This solid was filtered under reduced pressure and dried to obtain the target compound ggBMS-202.

[0044] Example 2: Synthesis of α-ggBMS-202-SS-PTX (stepwise method)

[0045] The synthetic route of the α-ggBMS-202-SS-PTX prodrug is as follows: Figure 2 As shown in the figure, the reaction reagents and conditions are: a) 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, 4,4'-dithiodibutyric acid, EDCI, DMAP, DCM, room temperature; b) EDCI, DMAP, DCM, room temperature (an ice bath is required for the γ-position). The specific synthesis method is as follows:

[0046] 1. ggBMS-202 (1.0 mmol, based on actual weight) and 2,2'-dithiodiacetic acid (1.2 mmol) were placed in a round-bottom flask containing DCM (15 mL). 1-Ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 1.2 mmol) and 4-dimethylaminopyridine (DMAP, 0.2 mmol) were added. The reaction system was stirred at room temperature (25 °C), and the reaction progress was monitored by TLC (dichloromethane:methanol = 15:1). The reaction was complete after approximately 4 h. The reaction solution was diluted with DCM (30 mL), transferred to a separatory funnel, and washed successively with saturated ammonium chloride solution (20 mL) and saturated sodium chloride solution (20 mL). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure (water bath temperature 35 °C). The crude product was purified by silica gel chromatography using dichloromethane:methanol (60:1) as the eluent. The purified product was then concentrated under reduced pressure to obtain a yellow oily compound (ggBMS-202-2,2'-dithiodiacetic acid ester).

[0047] 2. Paclitaxel (1.0 mmol) and DMAP (0.1 mmol) were added to a round-bottom flask containing DCM (10 mL) and stirred for 10 min to dissolve completely. Then, ggBMS-202-2,2'-dithiodiacetic acid ester (2.0 mmol) obtained in the previous step was added. Finally, EDCI (1.0 mmol) was added to the reaction flask. The reaction system was purged with nitrogen three times and stirred at room temperature (25 °C) under nitrogen protection. The reaction was monitored by TLC. After about 3 h, the reaction was complete. After the reaction was complete, DCM (30 mL) and distilled water (20 mL) were added for extraction and separation. The aqueous phase was back-extracted with DCM (20 mL), and the organic phases were combined. The organic phase was washed successively with saturated ammonium chloride solution (20 mL) and saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure (water bath temperature 35 °C). The crude product was purified by silica gel chromatography using dichloromethane:methanol (50:1) as the eluent to obtain the target compound α-ggBMS-202-SS-PTX, a white solid with a yield of 85%.

[0048] Example 3: Synthesis of β-ggBMS-202-SS-PTX (stepwise method)

[0049] The synthetic route of the β-ggBMS-202-SS-PTX prodrug is as follows: Figure 2 As shown in the figure, the reaction reagents and conditions are: a) 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, 4,4'-dithiodibutyric acid, EDCI, DMAP, DCM, room temperature; b) EDCI, DMAP, DCM, room temperature (an ice bath is required for the γ-position). The specific synthesis method is as follows:

[0050] 1. ggBMS-202 (1.0 mmol) and 3,3'-dithiodipropionic acid (1.2 mmol) were placed in a round-bottom flask containing DCM (15 mL). EDCI (1.2 mmol) and DMAP (0.2 mmol) were added under magnetic stirring. The reaction mixture was stirred at room temperature (25 °C), and the reaction progress was monitored by TLC (dichloromethane:methanol = 15:1). The reaction was complete after approximately 5 h. The reaction solution was diluted with DCM (30 mL), washed successively with saturated ammonium chloride solution (20 mL), saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure (water bath temperature 35 °C). The crude product was purified by silica gel chromatography using dichloromethane:methanol (60:1) as the eluent to obtain a pale yellow oily compound (ggBMS-202-3,3'-dithiodipropionic acid ester).

[0051] 2. Paclitaxel (1.0 mmol) and DMAP (0.1 mmol) were added to a round-bottom flask containing DCM (10 mL) and stirred thoroughly. Then, ggBMS-202-3,3'-dithiodipropionate (2.0 mmol) obtained in the previous step was added. Finally, EDCI (1.0 mmol) was added to the round-bottom flask. Under nitrogen protection (N2), the reaction was stirred at room temperature and monitored by TLC. After the reaction was completed in about 4.5 h, DCM (30 mL) and distilled water (20 mL) were added for extraction and separation. The aqueous phase was back-extracted with DCM (20 mL), and the organic phases were combined. The organic phase was washed successively with saturated ammonium chloride solution (20 mL) and saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure (water bath temperature 35℃). The crude product was purified by silica gel chromatography using dichloromethane:methanol (50:1) as the eluent to obtain the target compound (β-ggBMS-202-SS-PTX), a white solid with a yield of 80%.

[0052] Example 4: Synthesis of γ-ggBMS-202-SS-PTX (one-pot method)

[0053] Because the γ-configuration intermediate was found to have poor stability during the experiment, and disulfide bond breaking or rearrangement easily occurred during the separation and purification process, a one-pot continuous reaction was adopted, and the intermediate was used directly in the next coupling reaction without separation. The synthetic route of the γ-ggBMS-202-SS-PTX prodrug is as follows: Figure 2 As shown in the figure, the reaction reagents and conditions are: a) 2,2'-dithiodiacetic acid, 3,3'-dithiodipropionic acid, 4,4'-dithiodibutyric acid, EDCI, DMAP, DCM, room temperature; b) EDCI, DMAP, DCM, room temperature (an ice bath is required for the γ-position). The specific synthesis method is as follows:

[0054] 1. ggBMS-202 (1.0 mmol) and 4,4'-dithiodibutyric acid (1.2 mmol) were placed in a round-bottom flask containing DCM (15 mL). EDCI (1.2 mmol) and DMAP (0.2 mmol) were added with magnetic stirring. The reaction system was stirred at room temperature (25 °C), and the reaction progress was monitored by TLC (dichloromethane:methanol = 15:1). The reaction was completed after approximately 6 h, at which point the ggBMS-202-4,4'-dithiodibutyrate intermediate was formed in the reaction system.

[0055] 2. The reaction system was placed directly in an ice-water bath (0-5℃) for 10 min without any pretreatment. Paclitaxel (1.0 mmol) and DMAP (0.1 mmol) were dissolved in anhydrous DCM (10 mL) and then slowly added dropwise to the above reaction solution over a period of 15 min. Subsequently, EDCI (1.0 mmol) was added in batches, and the reaction was stirred while maintaining the ice-water bath conditions. The reaction progress was monitored by TLC. The reaction must be strictly controlled to be completed within 2 h (the total reaction time starts from the first step; after 2 h, the byproducts increase significantly).

[0056] 3. After the reaction is complete, immediately add pre-cooled DCM (30 mL) and pre-cooled distilled water (20 mL) for extraction and separation. Back-extract the aqueous phase with pre-cooled DCM (20 mL) and combine the organic phases. Quickly wash the organic phase with pre-cooled saturated ammonium chloride solution (20 mL) and pre-cooled saturated sodium chloride solution (20 mL), dry with anhydrous sodium sulfate, filter, and concentrate under reduced pressure (water bath temperature ≤25℃).

[0057] 4. Special Purification: Silica gel was activated at 60℃ for 2 h before use; the eluent, dichloromethane:methanol (50:1), was prepared fresh before use; after column packing, the column was equilibrated with the eluent for 15 min; after sample loading, a rapid elution method was used (flow rate controlled at 2-3 mL / min); the collected fraction should be concentrated under reduced pressure immediately to avoid prolonged standing in solution. After purification under the above conditions, the target compound (γ-ggBMS-202-SS-PTX), a white solid, was obtained with a yield of 58%.

[0058] Example 5: Structural confirmation of the target compound

[0059] Take an appropriate amount of prodrug powder (α-ggBMS-202-SS-PTX, β-ggBMS-202-SS-PTX or γ-ggBMS-202-SS-PTX) and dissolve it in DMSO. Use high-resolution mass spectrometry to determine the molecular weight of the sample.

[0060] Weigh approximately 5 mg of the prodrug powder (α-ggBMS-202-SS-PTX, β-ggBMS-202-SS-PTX, or γ-ggBMS-202-SS-PTX) and dissolve it in deuterated chloroform. 1 H-NMR was used to analyze the structure of the sample. The NMR and mass spectrometry results are shown below. Figure 3 (α-ggBMS-202-SS-PTX) Figure 4 (β-ggBMS-202-SS-PTX). Figure 5 (γ-ggBMS-202-SS-PTX).

[0061] High-resolution mass spectrometry (HRMS) and proton nuclear magnetic resonance (NMR) spectrometry were performed on the three prodrugs mentioned above. 1 Characterization by H-NMR, the results are as follows:

[0062] like Figure 3 As shown:

[0063] α-ggBMS-202-SS-PTX:(2aR,4R,4aS,6S,9S,11S,12S,12bS)-9-(((R)-13-((S)-benzamido(phenyl)methyl)-1-(2- methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)-2-methyl-6,11-dioxo-12-oxa-8,9-di thia-2,5-diazatetradecan-14-oyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo- 3,4,4a,5,6,9,10,11,12,12a-decahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxete-6,12b(2aH)-diyl diacetate

[0064] HRMS (ESI) m / z calcd for C 75 H 82 N4O 18 S2([M+H)) + ):1391.5138; found:1391.5144.

[0065] The NMR spectrum analysis is as follows: 1H NMR (300 MHz, Chloroform-d) δ 8.14 (d, J = 1.3Hz, 2H), 8.11 (d, J = 1.7 Hz, 1H), 7.90 – 7.84 (m, 2H), 7.65 – 7.60 (m, 1H),7.57 – 7.52 (m, 2H), 7.50 (dt, J = 4.9, 1.8 Hz, 2H), 7.48 – 7.45 (m, 2H),7.44 (q, J = 1.5 Hz, 2H), 7.41 (d, J = 1.6 Hz, 2H), 7.39 (s, 2H), 7.37 (dd, J= 3.3, 1.5 Hz, 2H), 7.35 (d, J = 2.6 Hz, 1H), 7.32 (dd, J = 3.3, 1.5 Hz, 2H),7.29 (q, J = 1.9, 1.5 Hz, 1H), 7.25 (d, J = 1.6 Hz, 2H), 7.23 (d, J = 2.6 Hz,2H), 6.36 (dd, J = 7.9, 2.6 Hz, 1H), 6.27 (s, 1H), 6.15 (t, J = 9.0 Hz, 1H),5.92 (dd, J = 8.5, 5.8 Hz, 1H), 5.64 (d, J = 7.2 Hz, 1H), 5.49 (d, J = 5.8Hz, 1H), 5.42 (s, 2H), 4.95 (dd, J = 9.7, 2.3 Hz, 1H), 4.42 (dd, J = 10.9,6.5 Hz, 1H), 4.31 – 4.26 (m, 1H), 4.18 (d, J = 8.4 Hz, 1H), 3.98 (d, J = 7.7Hz, 1H), 3.96 (s, 2H), 3.76 – 3.72 (m, 1H), 3.56 – 3.50 (m, 2H), 3.46 (d, J =4.4 Hz, 2H), 3.43 (s, 1H), 3.30 (d, J = 3.2 Hz, 2H), 2.56 (d, J = 6.2 Hz,1H), 2.52 (d, J = 5.6 Hz, 2H), 2.39 (s, 2H), 2.27 (s, 2H), 2.21 (s, 2H), 2.20(s, 1H), 2.12 (dd, J = 15.4, 9.2 Hz, 2H), 1.89 (d, J = 1.3 Hz, 2H), 1.85 (d,J = 7.0 Hz, 1H), 1.75 (s, 1H), 1.66 (s, 2H), 1.42 (s, 2H), 1.30 – 1.25 (m,2H), 1.21 (s, 3H), 1.11 (s, 3H), 0.96 (dd, J = 6.9, 5.2 Hz, 1H), 0.86 (td, J= 6.4, 2.3 Hz, 3H).

[0066] α-ggBMS-202-SS-PTX 1 The H NMR data are highly consistent with the expected structure.

[0067] like Figure 4 As shown:

[0068] β-ggBMS-202-SS-PTX:(2aR,4R,4aS,6S,9S,11S,12S,12bS)-9-(((R)-15-((S)-benzamido(phenyl)methyl)-1-(2- methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)-2-methyl-6,13-dioxo-14-oxa-9,10-d ithia-2,5-diazahexadecan-16-oyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo- 3,4,4a,5,6,9,10,11,12,12a-decahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxete-6,12b(2aH)-diyl diacetate

[0069] HRMS (ESI) m / z calcd for C 77 H 86 N4O 18 S2([M+H)) + ):1419.5451; found:1419.5453.

[0070] The NMR spectrum analysis is as follows:

[0071] 1H NMR (300 MHz, Chloroform-d) δ 8.09 – 8.03 (m, 2H), 7.73 – 7.69 (m,2H), 7.55 (dd, J = 8.6, 6.4 Hz, 2H), 7.46 (d, J = 7.8 Hz, 2H), 7.41 (d, J =9.3 Hz, 2H), 7.38 – 7.37 (m, 2H), 7.35 (s, 2H), 7.32 (d, J = 1.5 Hz, 2H),7.30 (s, 2H), 7.27 (s, 1H), 7.23 (dd, J = 6.6, 1.7 Hz, 2H), 7.20 (d, J = 4.0Hz, 2H), 7.18 – 7.14 (m, 2H), 6.31 (d, J = 7.9 Hz, 1H), 6.21 (s, 1H), 6.10(t, J = 9.0 Hz, 1H), 5.88 (dd, J = 9.1, 4.7 Hz, 1H), 5.59 (d, J = 7.1 Hz,1H), 5.46 (d, J = 4.7 Hz, 1H), 5.35 (s, 2H), 4.89 (d, J = 9.3 Hz, 1H), 4.36(dd, J = 10.9, 6.6 Hz, 1H), 4.23 (d, J = 8.5 Hz, 1H), 4.12 (d, J = 8.4 Hz,1H), 3.90 (s, 2H), 3.71 (d, J = 7.0 Hz, 1H), 3.43 (s, 2H), 3.28 (s, 2H), 2.82(d, J = 6.3 Hz, 2H), 2.80 (s, 2H), 2.76 (d, J = 11.5 Hz, 2H), 2.49 (s, 2H),2.48 – 2.41 (m, 2H), 2.39 (s, 1H), 2.35 (s, 2H), 2.20 (s, 2H), 2.17 (s, 2H),2.14 (s, 2H), 1.94 (d, J = 10.6 Hz, 2H), 1.84 (d, J = 1.4 Hz, 2H), 1.81 (s,1H), 1.74 (d, J = 10.0 Hz, 2H), 1.60 (s, 2H), 1.35 (s, 2H), 1.18 (s, 3H),1.14 (s, 3H), 1.05 (s, 2H), 0.92 – 0.86 (m, 1H), 0.78 (ddd, J = 7.0, 5.0, 2.0Hz, 3H).

[0072] β-ggBMS-202-SS-PTX 1 The H NMR data are highly consistent with the expected structure.

[0073] like Figure 5 As shown:

[0074] γ-ggBMS-202-SS-PTX:(2aR,4R,4aS,6S,9S,11S,12S,12bS)-9-(((R)-17-((S)-benzamido(phenyl)methyl)-1-(2- methoxy-6-((2-methyl-[1,1'-biphenyl]-3-yl)methoxy)pyridin-3-yl)-2-methyl-6,15-dioxo-16-oxa-10,11-d ithia-2,5-diazaoctadecan-18-oyl)oxy)-12-(benzoyloxy)-4,11-dihydroxy-4a,8,13,13-tetramethyl-5-oxo- 3,4,4a,5,6,9,10,11,12,12a-decahydro-1H-7,11-methanocyclodeca[3,4]benzo[1,2-b]oxete-6,12b(2aH)-diyl diacetate

[0075] HRMS (ESI) m / z calcd for C 79 H 90 N4O 18 S2([M+H)) + ):1447.5764; found:1447.5757.

[0076] The NMR spectrum analysis is as follows: 1H NMR (300 MHz, Chloroform-d) δ 8.11 – 8.03 (m,2H), 7.72 – 7.65 (m, 2H), 7.53 (d, J = 7.2 Hz, 1H), 7.47 (d, J = 2.5 Hz, 1H),7.45 (d, J = 1.8 Hz, 1H), 7.42 (d, J = 2.4 Hz, 1H), 7.41 – 7.36 (m, 2H), 7.35(d, J = 2.1 Hz, 2H), 7.34 (s, 2H), 7.32 (d, J = 3.1 Hz, 2H), 7.31 (s, 1H),7.31 – 7.27 (m, 2H), 7.25 (d, J = 1.8 Hz, 2H), 7.22 (t, J = 1.5 Hz, 1H), 7.18– 7.15 (m, 2H), 7.09 (d, J = 9.1 Hz, 1H), 6.31 (d, J = 7.9 Hz, 1H), 6.20 (d,J = 17.5 Hz, 2H), 5.90 (dd, J = 9.1, 3.6 Hz, 1H), 5.61 (d, J = 7.0 Hz, 1H),5.45 (d, J = 3.6 Hz, 1H), 5.35 (s, 2H), 4.90 (d, J = 9.1 Hz, 1H), 4.41 – 4.34(m, 1H), 4.26 – 4.21 (m, 2H), 4.14 (d, J = 8.4 Hz, 1H), 3.90 (s, 2H), 3.74(d, J = 6.8 Hz, 1H), 3.28 (s, 2H), 2.61 (d, J = 6.3 Hz, 2H), 2.57 (d, J = 5.8Hz, 2H), 2.48 (d, J = 6.5 Hz, 2H), 2.45 (s, 1H), 2.39 (s, 2H), 2.27 (d, J =6.2 Hz, 1H), 2.20 (s, 2H), 2.18 (s, 2H), 2.15 (s, 2H), 2.11 – 2.01 (m, 2H),1.96 (s, 2H), 1.90 (s, 2H), 1.87 (s, 2H), 1.83 (s, 2H), 1.77 (s, 1H), 1.66(d, J = 7.2 Hz, 1H), 1.61 (s, 3H), 1.41 (s, 1H), 1.36 (s, 2H), 1.18 (s, 3H), 1.16 (s, 3H), 1.06 (s, 2H), 0.89 (t, J = 7.4 Hz, 2H), 0.80 (d, J = 7.7 Hz, 3H).

[0077] γ-ggBMS-202-SS-PTX 1 The H NMR data are highly consistent with the expected structure.

[0078] The successful synthesis of three target prodrugs has been confirmed.

[0079] The embodiments described above are merely illustrative of specific implementations of the present invention, and while the descriptions are detailed, they should not be construed as limiting the scope of protection of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. A disulfide-bridged paclitaxel-BMS-202 prodrug, characterized in that, It has the following structure: Where: n is 1, 2 or 3, n=1 is the α position, n=2 is the β position, and n=3 is the γ position; A represents a modified BMS-202 residue, wherein the modified BMS-202 retains the PD-1 / PD-L1 blocking pharmacophore of BMS-202 and has a primary amino group at the end as a linker site; B is a paclitaxel residue, and it forms an ester bond with a carbonyl group through its 2'-hydroxyl group.

2. The prodrug according to claim 1, characterized in that, The modified BMS-202 residue A is derived from ggBMS-202, which is prepared by condensation, amination, reduction, and deprotection reactions using 2-methyl-3-phenylbenzyl alcohol and 6-chloro-2-methoxypyridine-3-carboxaldehyde as raw materials.

3. The prodrug according to claim 2, characterized in that, The specific structure of the prodrug is as follows: The prodrug α-ggBMS-202-SS-PTX, with the disulfide bond located at the α-position, is formed by connecting paclitaxel to an intermediate consisting of modified BMS-202 and 2,2'-dithiodiacetic acid. The prodrug β-ggBMS-202-SS-PTX, with the disulfide bond located at the β-position, is formed by connecting paclitaxel to an intermediate consisting of modified BMS-202 and 3,3'-dithiodipropionic acid. The prodrug γ-ggBMS-202-SS-PTX, with the disulfide bond located at the γ position, is formed by connecting paclitaxel to an intermediate consisting of modified BMS-202 and 4,4'-dithiodibutyric acid.

4. A method for preparing a disulfide-bridged paclitaxel-BMS-202 prodrug according to any one of claims 1-3, characterized in that, Includes the following steps: (1) Synthesis of modified BMS-202: Modified BMS-202 was prepared by condensation, amination, reduction and deprotection reaction of 2-methyl-3-phenylbenzyl alcohol and 6-chloro-2-methoxypyridine-3-carboxaldehyde. (2) Synthesis of prodrug: The modified BMS-202 was reacted with dithiocarboxylic acid compounds under the action of condensing agent and catalyst, and then esterified with paclitaxel under the action of condensing agent and catalyst to obtain the target prodrug.

5. The preparation method according to claim 4, characterized in that, In step (2), for prodrugs with disulfide bonds located at the α or β positions, a stepwise synthesis method is adopted: first, the condensation intermediate of modified BMS-202 and dithiocarboxylic acid is separated, purified, and then coupled with paclitaxel.

6. The preparation method according to claim 4, characterized in that, In step (2), for the prodrug with the disulfide bond located at the γ position, a one-pot synthesis method is adopted: after the modified BMS-202 and 4,4'-dithiodibutyric acid are reacted at room temperature, the intermediate is not separated, and paclitaxel is directly added under ice bath conditions for coupling reaction, and the coupling reaction time is strictly controlled within 2 hours.

7. The preparation method according to claim 6, characterized in that, The purification steps after the one-pot synthesis of the γ-position prodrug include: after the reaction solution is extracted and washed with a pre-cooled reagent, column chromatography is performed using silica gel activated at 60°C. The eluent is prepared fresh for use, and a rapid elution method is adopted. The collected components are immediately concentrated under reduced pressure.

8. The use of the prodrug according to any one of claims 1-3 in the preparation of an antitumor drug.

9. The application according to claim 8, characterized in that, The prodrug is used to prepare an antitumor drug that releases drugs in response to glutathione in the tumor microenvironment and has the synergistic effect of inducing immunogenic cell death and blocking the PD-1 / PD-L1 pathway.

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