Compound as well as preparation method and application thereof

Abstract

The invention discloses a compound as well as a preparation method and application thereof, and belongs to the technical field of drug delivery systems and tumor targeted therapy. The compound as shown in the formula 1 is novel and unique in structure, can be used as an anti-melanoma prodrug compound, realizes targeted release and collaborative treatment of drugs (Devistat and TPEDCH) under laser irradiation, has a fluorescence imaging function, is used for monitoring the distribution and activation process of the drugs in tumor cells in real time, and has a good application prospect. The traditional Chinese medicine composition has excellent anti-melanoma effect and low toxic and side effects. Formula 1.

Description

Technical Field

[0001] This invention relates to the fields of drug delivery systems and tumor targeted therapy, and particularly to compounds, their preparation methods, and applications. Background Technology

[0002] Melanoma is a highly malignant skin tumor originating from melanocytes. It is highly aggressive, prone to metastasis, and easily develops resistance to traditional chemotherapy drugs, posing significant challenges to clinical treatment. Current main treatment methods include surgical resection, chemotherapy, targeted therapy, and immunotherapy; however, these methods generally suffer from poor selectivity, high systemic toxicity, high relapse rates, or limited response rates. Therefore, developing novel drug delivery systems that can precisely target tumor tissue, reduce toxic side effects, and achieve synergistic treatment through multiple mechanisms is of great significance.

[0003] In recent years, prodrug strategies have become an effective way to improve the physicochemical properties of drugs, enhance targeting, and reduce toxicity. Among these, stimulus-responsive prodrugs can release active drugs in response to specific signals in the tumor microenvironment (such as pH, enzymes, and reducing substances), thereby achieving targeted and controlled drug release. Glutathione (GSH) concentrations in tumor cells are significantly higher than in normal cells, providing ideal endogenous triggering conditions for the design of reduction-responsive prodrugs based on disulfide bonds (-SS-). Disulfide bonds can break under the influence of high intracellular GSH concentrations, achieving selective drug release.

[0004] Meanwhile, peptide-mediated active targeting is widely used to enhance drug accumulation at tumor sites. For example, the melanoma-targeting peptide YRWPKPVRA has been shown to specifically recognize and bind to receptors overexpressed on the surface of melanoma cells, enhancing drug endocytosis efficiency. Furthermore, photodynamic therapy (PDT), as a local treatment, uses photosensitizers to generate reactive oxygen species (ROS) under light irradiation to kill tumor cells, offering advantages such as spatiotemporal controllability and low invasiveness. Aggregation-induced emission (AIE) photosensitizers (such as TPEDCH) exhibit enhanced fluorescence and efficient ROS generation in the aggregated state, overcoming the fluorescence quenching drawback of traditional photosensitizers caused by aggregation, making them more suitable for tumor cell imaging and treatment.

[0005] However, the existing technology still has the following limitations: (1) single-mechanism therapeutic drugs are prone to insufficient efficacy due to tumor heterogeneity or drug resistance pathways; (2) most delivery systems only achieve targeted release of a single drug, making it difficult to deliver multiple functional molecules (such as chemotherapy drugs and photosensitizers) simultaneously and achieve synergistic therapy; (3) there is a lack of an integrated system that can simultaneously achieve real-time tracking of drug release and efficacy monitoring; (4) simple physical encapsulation or non-selective bonding methods may lead to premature drug leakage or uncontrollable release.

[0006] Therefore, there is an urgent need in this field to develop a smart prodrug molecule that can integrate active targeting, reduction-triggered drug release, multi-mechanism synergistic therapy, and real-time fluorescence tracing to improve the precision, efficacy, and safety of melanoma treatment. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a compound, its preparation method, and its application. The compound has a novel and unique structure and exhibits excellent anti-melanoma efficacy.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] This invention provides a compound represented by Formula 1:

[0010]

[0011] Formula 1.

[0012] The compound represented by Formula 1 of this invention has a novel and unique structure (which can be represented as YRW-SS-Dev-SS-TPEDCH), which has a three-arm structure. One arm is the parent nucleus (i.e., the melanoma-targeting peptide YRWPKPVRA), and the other two arms are connected to functional groups (Devimistat and TPEDCH) by disulfide bonds, respectively.

[0013] The compounds described in this invention can act precisely on melanoma cells as prodrugs and break disulfide bonds in the high glutathione (GSH) microenvironment of melanoma cells, thereby achieving targeted release and synergistic treatment of drugs (Devimistat and TPEDCH). They also have fluorescence imaging capabilities to monitor their distribution and activation process in tumor cells in real time.

[0014] The present invention also provides a method for preparing the above-mentioned compound, comprising the following steps:

[0015] (1) The polypeptide shown in Formula 2, 2-hydroxymethyl-1,3-propanediol, the first condensing agent, the first organic base and the organic solvent are mixed and reacted to obtain the intermediate shown in Formula 3;

[0016] (2) The intermediate, the compound shown in Formula 4, the second condensing agent, the second organic base and the organic solvent are mixed and reacted to obtain reaction system S1;

[0017] (3) The reaction system S1 and the compound shown in Formula 5 are mixed and reacted to obtain the compound;

[0018]

[0019] Formula 2;

[0020]

[0021] Formula 3;

[0022]

[0023] Equation 4;

[0024]

[0025] Formula 5.

[0026] The above preparation method involves a condensation reaction between the carboxyl group of the polypeptide shown in Formula 2 and the hydroxyl group of 2-hydroxymethyl-1,3-propanediol to prepare the intermediate shown in Formula 3 (denoted as YRW-pro); then YRW-pro is sequentially condensed with the compound shown in Formula 4 (denoted as SS-Dev) and the compound shown in Formula 5 (denoted as SS-TPEDCH) to prepare the compound shown in Formula 1 of the present invention.

[0027] Preferably, the first condensing agent and the second condensing agent are independently selected from 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxyhexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI), dicyclohexylcarbodiimide (DCC), or diisopropylcarbodiimide (DIC); more preferably, HATU.

[0028] Preferably, the first organic base and the second organic base are independently selected from N,N-diisopropylethylamine (DIPEA), triethylamine (TFA) or 4-dimethylaminopyridine (DMAP); more preferably, DIPEA.

[0029] Preferably, the organic solvent is selected from anhydrous N,N-dimethylformamide, anhydrous tetrahydrofuran, anhydrous dichloromethane, or anhydrous dimethyl sulfoxide; more preferably, it is anhydrous N,N-dimethylformamide.

[0030] Preferably, the first condensing agent is selected from 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-hexafluorophosphate;

[0031] Preferably, the second condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0032] Preferably, both the first organic base and the second organic base are selected from N,N-diisopropylethylamine.

[0033] Preferably, in step (1), the molar ratio of the polypeptide represented by Formula 2 to 2-hydroxymethyl-1,3-propanediol is 1:(1-1.5); more preferably, it is 1:(1-1.2); and even more preferably, it is 1:1.

[0034] Preferably, the molar ratio of the intermediate and the compound shown in Formula 4 in step (2) is 1:(1-1.3); in some specific embodiments of the present invention, it is preferably 1:1.

[0035] Preferably, the molar ratio of the intermediate to the compound shown in Formula 5 is 1:(1-1.6); more preferably, it is 1:(1-1.3). In some specific embodiments of the present invention, a ratio of 1:1 is preferred.

[0036] Preferably, the molar ratio of the first condensing agent to 2-hydroxymethyl-1,3-propanediol is (1-1.5):1; more preferably (1-1.2):1. In some specific embodiments of the present invention, a ratio of 1.2:1 is preferred.

[0037] Preferably, the molar ratio of the first organic base to 2-hydroxymethyl-1,3-propanediol is (2-3):1; more preferably (2.2-3.6):1; in some specific embodiments of the present invention, it is preferably 2.5:1.

[0038] Preferably, the molar ratio of the second condensing agent to the intermediate is (1-1.5):0.5; more preferably, it is 1.2:0.5.

[0039] Preferably, the molar ratio of the second organic base to the intermediate is (2-3):0.5; more preferably (2.2-3.6):0.5; in some specific embodiments of the present invention, it is preferably 2.5:0.5.

[0040] In the above preparation method, the specific synthesis methods of the compounds of formula 4 and formula 5 are as follows:

[0041] (a) The method for synthesizing the compound shown in Formula 4 includes the following steps:

[0042] Devimistat, 2-[(2-aminoethyl)dithio]-acetic acid, a third condensing agent, a third organic base, and an organic solvent were mixed and reacted under stirring to obtain the compound shown in Formula 4, which is denoted as SS-Dev.

[0043] The synthetic method prepares SS-Dev through the condensation reaction of Devimistat and 2-[(2-aminoethyl)dithio]-acetic acid.

[0044] The molar ratio of Devimistat and 2-[(2-aminoethyl)dithio]acetic acid is preferably 1:(1-1.3); more preferably 1:1.

[0045] The third condensing agent includes, but is not limited to, EDCI, DCC, or DIC;

[0046] The third organic base includes, but is not limited to, EDCI, TFA, or DMAP.

[0047] In some specific embodiments of the present invention, the third condensing agent is selected from EDCI, and the third organic base is selected from DMAP.

[0048] The organic solvent includes, but is not limited to, anhydrous N,N-dimethylformamide, anhydrous dichloromethane, or anhydrous dimethyl sulfoxide; more preferably, anhydrous N,N-dimethylformamide.

[0049] (ii) The method for synthesizing the compound shown in Formula 5 includes the following steps:

[0050] TPEDCH, dithiodipropionic acid, a fourth condensing agent, a fourth organic base, and an organic solvent are mixed and reacted to obtain the compound shown in Formula 5, which is represented as SS-TPEDCH.

[0051] The synthesis method prepares SS-TPEDCH through the condensation reaction of TPEDCH and dithiodipropionic acid.

[0052] The molar ratio of TPEDCH to dithiodipropionic acid is preferably 0.5:(0.8-1.2); more preferably 1:2.

[0053] The fourth condensing agent includes, but is not limited to, EDCI, DCC, or DIC.

[0054] The fourth organic base includes, but is not limited to, EDCI, TFA, or DMAP.

[0055] In some specific embodiments of the present invention, the fourth condensing agent is selected from EDCI, and the fourth organic base is selected from DMAP.

[0056] The organic solvent includes, but is not limited to, anhydrous N,N-dimethylformamide, anhydrous dichloromethane, or anhydrous dimethyl sulfoxide; more preferably, anhydrous dichloromethane.

[0057] The compound shown in Formula 1 or the compound prepared by the above preparation method can be used alone as an anti-melanoma prodrug or in combination with other pharmaceutical adjuvants to prepare anti-melanoma drugs.

[0058] The compound described in this invention (denoted as YRW-SS-Dev-SS-TPEDCH) serves as a prodrug molecule. Through its YRW targeting peptide structure, it can efficiently and specifically accumulate in melanoma A375 cells via a specific recognition-mediated active transport mechanism, thereby achieving subsequent intracellular reduction-responsive drug release and synergistic therapy.

[0059] The YRW-SS-Dev-SS-TPEDCH of this invention can efficiently initiate photodynamic therapy after being taken up by tumor cells through a cascade process of "reduction-release-laser excitation", generating a large amount of ROS, thereby achieving synergy with the chemotherapy effect of Devimistat and enhancing the killing effect on melanoma cells.

[0060] The compounds described in this invention can be used to develop intelligent anti-tumor drugs that combine targeted delivery, synergistic therapy, and visual monitoring functions.

[0061] The present invention also provides a composition comprising the above-described compound or the compound prepared by the above-described preparation method, and other pharmaceutical adjuvants.

[0062] The pharmaceutical adjuvants include one or more of the following: buffers, solubilizers, antioxidants, and preservatives.

[0063] The buffers include, but are not limited to, buffers composed of sodium dihydrogen phosphate and disodium hydrogen phosphate, buffers composed of potassium dihydrogen phosphate and dipotassium hydrogen phosphate, etc.

[0064] The cosolvents include, but are not limited to, ethanol, propylene glycol, glycerin, etc.

[0065] The antioxidants include, but are not limited to, vitamin C (ascorbic acid), sodium sulfite, sodium bisulfite, etc.

[0066] The preservatives include, but are not limited to, benzyl alcohol, ethanol, phenol, etc.

[0067] The present invention also provides the use of the above-described composition in the preparation of an anti-melanoma drug.

[0068] Melanoma can be divided into cutaneous malignant melanoma and non-cutaneous malignant melanoma.

[0069] Preferably, the anti-melanoma drug of the present invention is an anti-malignant melanoma drug for the skin.

[0070] Preferably, the anti-melanoma drug is used under laser irradiation conditions.

[0071] Under laser irradiation, the composition of the present invention achieves a significant synergistic effect between chemotherapy and photodynamic therapy through highly efficient intracellular delivery mediated by targeted peptides and simultaneous release of Devimistat and TPEDCH in response to GSH, resulting in excellent in vitro growth inhibition of melanoma cells.

[0072] Compared with the prior art, the compound structure shown in Formula 1 provided by the present invention is novel and unique, and can be used as a prodrug compound for anti-melanoma. Under laser irradiation, it can achieve targeted release and synergistic treatment of drugs (Devimistat and TPEDCH). At the same time, it also has the function of fluorescence imaging to monitor the distribution and activation process of drugs in tumor cells in real time, and has excellent anti-melanoma effect and low toxic side effects. Attached Figure Description

[0073] Figure 1 The image shows the 1H NMR spectrum (DMSO, 500 MHz) of SS-TPEDCH.

[0074] Figure 2 The image shows the 1H NMR spectrum (DMSO, 500 MHz) of the SS-Dev.

[0075] Figure 3 The 1H NMR spectrum (DMSO, 500 MHz) of compound YRW-SS-Dev-SS-TPEDCH is shown.

[0076] Figure 4 The target uptake verification diagram of YRW-SS-Dev-SS-TPEDCH in A375 cells (scale bar 50 μm) is shown. Group 1 is the SS-Dev-SS-TPEDCH group, Group 2 is the YRW-SS-Dev-SS-TPEDCH group, and Group 3 is the YRW-SS-Dev-SS-TPEDCH + YRW group.

[0077] Figure 5 A diagram validating the selective release of YRW-SS-Dev-SS-TPEDCH in the A375 and HUVEC co-culture model (scale bar: 100 μm).

[0078] Figure 6 To verify the effect of YRW-SS-Dev-SS-TPEDCH on inducing ROS generation in A375 cells, group 1 was TPEDCH + laser, group 2 was YRW-SS-Dev-TPEDCH + laser, group 3 was YRW-SS-TPEDCH + laser, group 4 was YRW-SS-Dev-SS-TPEDCH, and group 5 was YRW-SS-Dev-SS-TPEDCH + laser.

[0079] Figure 7 To verify the in vitro killing effect of YRW-SS-Dev-SS-TPEDCH on A375 cells (data are expressed as mean ± standard deviation, n=3). Detailed Implementation

[0080] To further illustrate the present invention, the compounds, their preparation methods, and applications provided by the present invention are described in detail below with reference to embodiments.

[0081] Example 1

[0082] 1. Synthesis and characterization of the melanoma-targeting prodrug YRW-SS-Dev-SS-TPEDCH

[0083] 1.1 Synthesis of the reactant SS-TPEDCH

[0084] In a dry round-bottom flask, 0.5 mmol (299 mg) of TPEDCH and 1 mmol of dithiodipropionic acid were dissolved in anhydrous dichloromethane. The reaction mixture was cooled to 0–5°C in an ice-water bath. While stirring, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.6 mmol, 115 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 1.2 mmol, 155 mg) were added sequentially. After the addition was complete, the ice bath was removed, the reaction mixture was brought to room temperature, and stirring continued for 24 hours. Figure 1 The reaction process was monitored by thin-layer chromatography. After the reaction was completed, saturated ammonium chloride aqueous solution was added to the reaction solution to quench the reaction, followed by extraction three times with dichloromethane. The combined organic phases were washed successively with 1M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The crude product was purified by silica gel column chromatography to obtain 302 mg of the target product SS-TPEDCH, with a yield of 77%.

[0085] Characterizing the structure of compounds using nuclear magnetic resonance (NMR) 1H NMR (e.g.) Figure 1 (As shown). Specific data is as follows:

[0086] 1H NMR (500 MHz, DMSO-d6) δ 11.98 (s, 1H), 8.39 (s, 1H), 7.84 – 7.75 (m, 4H), 7.69 – 7.64 (m, 2H), 7.64 – 7.54 (m, 4H), 7.47 – 7.35 (m, 5H), 7.33– 7.27 (m, 2H), 7.25 – 7.19 (m, 4H), 7.15 (t, J = 7.6 Hz, 1H), 6.86 – 6.81(m, 2H), 3.12 – 2.97 (m, 4H), 2.72 (dt, J = 13.1, 7.1 Hz, 4H).

[0087]

[0088] 1.2 Synthesis of the reactant SS-Dev

[0089] In a dry round-bottom flask, 1 mmol (388 mg) of Devimistat and 1 mmol (167 mg) of 2-[(2-aminoethyl)dithio]acetic acid were dissolved in anhydrous DMF. The reaction flask was cooled to 0–5°C in an ice-water bath, and the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 1.2 mmol, 230 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 2.5 mmol, 323 mg) were added sequentially with stirring. After the addition was complete, the ice bath was removed, and the reaction was continued to be stirred at room temperature for 18 hours. Figure 3 The reaction was monitored by thin-layer chromatography. After the reaction was complete, the reaction solution was poured into ice water and extracted three times with ethyl acetate. The combined organic phases were washed successively with 1M dilute hydrochloric acid, saturated sodium bicarbonate solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent. The crude product was purified by silica gel column chromatography to give 452 mg of the target product SS-Dev, with a yield of 82%.

[0090] Characterizing the structure of compounds using nuclear magnetic resonance (NMR) 1H NMR (e.g.) Figure 2 (As shown). Specific data is as follows:

[0091] 1H NMR (500 MHz, DMSO-d6) δ 11.97 (s, 1H), 7.66 (t, J = 6.5 Hz, 1H), 7.38 – 7.26 (m, 8H), 7.26 – 7.19 (m, 2H), 3.72 (dt, J = 12.2, 1.0 Hz, 1H),3.63 (dt, J = 12.4, 1.1 Hz, 1H), 3.60 – 3.54 (m, 2H), 3.43 – 3.26 (m, 2H),3.11 (dt, J = 12.2, 7.1 Hz, 1H), 3.02 (dt, J = 12.5, 7.1 Hz, 1H), 2.88 – 2.79(m, 2H), 2.74 (dt, J = 12.5, 7.1 Hz, 1H), 2.70 – 2.57 (m, 4H), 2.22 – 2.13(m, 2H), 1.92 (dq, J = 12.2, 7.0 Hz, 1H), 1.83 (dq, J = 12.2, 7.0 Hz, 1H),1.77 – 1.68 (m, 1H), 1.65 – 1.56 (m, 3H), 1.56 – 1.47 (m, 1H), 1.47 – 1.40(m, 1H).

[0092]

[0093] 1.3 Synthesis of the polypeptide YRWPKPVRA

[0094] A solid-phase synthesis strategy using Fmoc / tBu was employed, with rink amide MBHA resin (C-terminal amidation) or Wang resin (C-terminal carboxylation) of 0.3-0.8 mmol / g as the support. Amino acid residues Ala, Arg, Val, Pro, Lys, Pro, Trp, Arg, and Tyr were sequentially coupled from C-terminus to N-terminus. The specific steps were as follows: 0.1 mmol of resin was weighed and placed in a solid-phase synthesis tube, 10 mL of N,N-dimethylformamide (DMF) was added, and the mixture was shaken at room temperature for 30 min to swell. After filtration, 10 mL of 20% piperidine / DMF solution was added, and the mixture was shaken at room temperature for 10 min and then 5 min to remove Fmoc residues from the resin. After washing the protecting group six times with DMF, the free amino group was confirmed by ninhydrin assay. Then, an activation system for Fmoc-Ala-OH was prepared according to the molar ratio of resin amino: amino acid: HBTU: HOBt: DIEA = 1:3:3:3:6. Specifically, 0.3 mmol Fmoc-Ala-OH, 0.3 mmol HBTU, and 0.3 mmol HOBt were dissolved in 5 mL DMF, and 0.6 mmol DIEA was added. Activation was performed at room temperature for 5 min. The activated solution was added to the synthesis tube and coupled at room temperature with shaking for 2 h. For amino acids with large steric hindrance, such as Arg and Trp, the coupling time was extended to 4 h, or a double coupling strategy was used. After coupling, the amino group was washed six times with DMF, and the complete coupling was confirmed by ninhydrin assay. The deprotection-coupling operation was repeated until all amino acids were coupled. Finally, the N-terminal Fmoc protecting group was removed, and the amino group was washed six times each with DMF and dichloromethane (DCM), then vacuum dried for 30°C. For 2–3 h of cleavage at room temperature with shaking, 1 g of resin was added to 10 mL of cleavage buffer (TFA / TIS / H2O = 95:2.5:2.5, v / v / v). The cleavage buffer was collected by filtration, and 50 mL of anhydrous diethyl ether pre-cooled to -20°C was slowly added dropwise to the filtrate. The mixture was stirred to precipitate the peptide, allowed to stand for 30 min, and then centrifuged at 4000 rpm for 10 min. The supernatant was discarded, and the precipitate was washed three times with pre-cooled diethyl ether and dried under vacuum to obtain crude peptide powder. The powder was purified by reversed-phase high-performance liquid chromatography (RP-HPLC) using a C18 column (250 mm × 10 mm, 5 μm). The mobile phase consisted of 0.1% TFA aqueous solution (phase A) and 0.1% TFA acetonitrile solution (phase B). The gradient elution conditions were 10%–60% elution in phase B for 30 min, with a detection wavelength of 220 nm. The eluent of the target peak was collected, concentrated under nitrogen, and freeze-dried for 24 h to obtain a purity ≥95%. The total yield of the pure peptide YRWPKPVRA obtained by this method was 65%. The 1H NMR spectral data of the pure peptide YRWPKPVRA are shown below:

[0095] 1 H NMR (500 MHz, DMSO-d6) δ 12.19 (s, 1H), 10.71 (d, J = 8.3 Hz, 1H),8.37 – 8.31 (m, 2H), 8.09 – 8.02 (m, 3H), 7.99 (d, J = 10.5 Hz, 1H), 7.75 (d,J = 11.0 Hz, 1H), 7.64 – 7.58 (m, 3H), 7.33 (dd, J = 7.3, 1.7 Hz, 1H), 7.19 –6.99 (m, 7H), 6.71 – 6.65 (m, 2H), 6.22 (t, J = 7.9 Hz, 1H), 6.05 (d, J = 6.5Hz, 2H), 5.91 (t, J = 8.1 Hz, 1H), 5.67 (d, J = 6.6 Hz, 2H), 4.79 (dt, J =12.0, 7.0 Hz, 1H), 4.45 – 4.32 (m, 2H), 4.26 (dt, J = 11.7, 7.1 Hz, 1H), 4.23– 4.14 (m, 3H), 3.89 (tt, J = 7.8, 6.8 Hz, 1H), 3.54 – 3.35 (m, 4H), 3.19 –2.92 (m, 8H), 2.70 (dp, J = 12.5, 7.3 Hz, 1H), 2.56 (dp, J = 12.2, 7.0 Hz,1H), 2.13 – 1.95 (m, 5H), 1.83 – 1.70 (m, 9H), 1.70 – 1.54 (m, 3H), 1.54 –1.48 (m, 2H), 1.48 – 1.42 (m, 2H), 1.42 – 1.38 (m, 2H), 1.38 – 1.29 (m, 4H),0.86 (d, J = 6.8 Hz, 3H), 0.81 (d, J = 6.8 Hz, 3H). 13C NMR (125 MHz, Chloroform-d) δ 179.23, 173.67, 173.48, 173.29, 173.17, 172.23, 172.14,171.44, 171.00, 158.07, 156.03, 136.65, 130.47, 129.02, 127.55, 123.58,120.80, 119.29, 118.14, 116.32, 111.97, 110.30, 61.75, 60.61, 58.87, 54.30,53.49, 53.37, 52.21, 52.15, 52.12, 47.71, 47.24, 41.30, 41.25, 40.30, 39.17,31.00, 30.69, 30.22, 29.93, 29.87, 29.70, 28.34, 28.00, 25.25, 25.22, 24.57,24.54, 24.18, 19.15, 17.99. HR-MS (ESI): [M+3H] 3+ m / z calcd 391.5611, found391.5617.

[0096] 1.4 Synthesis of YRW-SS-Dev-SS-TPEDCH

[0097] (1) In a dry 50 mL round-bottom flask, the polypeptide YRWPKPVRA (0.1 mmol, 117 mg) was dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF) and stirred until completely dissolved. The reaction flask was placed in an ice-water bath and cooled to 0-5°C. 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxyhexafluorophosphate (HATU, 0.12 mmol, 46 mg) and N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 33 mg) were added to the reaction solution in sequence. After stirring for 30 minutes, 2-hydroxymethyl-1,3-propanediol (0.1 mmol, 11 mg) was added dropwise. After the addition was complete, the ice-water bath was removed, and the reaction solution was allowed to rise to room temperature naturally. Stirring was continued for 3 hours. After the reaction was complete, the reaction solution was slowly added dropwise to 50 mL of ice-cold diethyl ether under vigorous stirring, and a solid precipitated out. The solid was collected by vacuum filtration and washed with a small amount of cold diethyl ether to obtain the white solid product YRW-pro, which was directly used in the next reaction.

[0098] (2) In a dry 50 mL round-bottom flask, the intermediate YRW-pro (0.05 mmol) and SS-Dev (0.05 mmol, 27 mg) obtained in the previous step were dissolved together in 8 mL of anhydrous DMF. The reaction flask was placed in an ice-water bath and cooled to 0-5°C. At this temperature, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.12 mmol, 23 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 32 mg) were added to the reaction solution. After the addition was complete, the ice-water bath was removed, and the reaction solution was stirred at room temperature for 6 hours.

[0099] (3) Then, SS-TPEDCH (0.05 mmol, 40 mg) was added to the above reaction flask, and the reaction was continued to be stirred at room temperature for 24 hours. After the reaction was completed by HPLC monitoring, the reaction solution was diluted with 50 mL of ethyl acetate and washed three times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding the crude product ( Figure 5 The crude product was purified by preparative high-performance liquid chromatography, the target fraction was collected, and after lyophilization, the final product was 65 mg of YRW-SS-Dev-SS-TPEDCH, with an overall yield of 26% for both steps.

[0100] The structure of compound YRW-SS-Dev-SS-TPEDCH was characterized by 1H NMR spectroscopy (e.g., Figure 3 (As shown), the specific data is as follows:

[0101] 1H NMR (500 MHz, DMSO-d6) δ 10.74 (s, 1H), 9.42 (s, 1H), 9.03 (s,1H), 8.15 – 8.09 (m, 5H), 7.97 (s, 1H), 7.70 – 7.63 (m, 5H), 7.63 – 7.54 (m,3H), 7.54 – 7.47 (m, 3H), 7.45 – 7.02 (m, 33H), 6.87 – 6.79 (m, 4H), 6.71 –6.65 (m, 2H), 6.50 (s, 2H), 6.15 (s, 2H), 4.75 (t, J = 7.0 Hz, 1H), 4.44 –4.27 (m, 3H), 4.26 – 4.09 (m, 7H), 4.08 – 3.96 (m, 3H), 3.87 – 3.76 (m, 2H), 3.69 (q, J = 1.0 Hz, 2H), 3.52 – 3.42 (m, 3H), 3.42 – 3.32 (m, 3H), 3.32 –3.22 (m, 4H), 3.22 – 2.97 (m, 11H), 2.85 – 2.63 (m, 11H), 2.55 (ddt, J =22.7, 12.5, 7.1 Hz, 2H), 2.18 – 2.05 (m, 4H), 2.05 – 1.99 (m, 4H), 1.96 (s,2H), 1.89 – 1.24 (m, 28H), 0.85 (d, J = 6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz,3H).

[0102]

[0103] Comparative Example 1

[0104] Preparation of compound (prodrug) SS-Dev-SS-TPEDCH

[0105]

[0106] (1) In a dry 50 mL round-bottom flask, 2-hydroxymethyl-1,3-propanediol (0.1 mmol, 11 mg) was dissolved in 10 mL of anhydrous N,N-dimethylformamide (DMF) and stirred until completely dissolved. The reaction flask was placed in an ice-water bath and cooled to 0–5°C. At this temperature, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.12 mmol, 23 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 32 mg), as well as the intermediate SS-Dev (0.05 mmol, 27 mg) obtained above, were added to the reaction solution. After the addition was complete, the ice-water bath was removed, and the reaction solution was stirred at room temperature for 8 hours.

[0107] (2) Then, SS-TPEDCH (0.05 mmol, 40 mg) was added to the above reaction flask, and the reaction was continued to be stirred at room temperature for 24 hours. After the reaction was completed by HPLC monitoring, the reaction solution was diluted with 50 mL of ethyl acetate and washed three times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by preparative high performance liquid chromatography, the target fraction was collected, and lyophilized to obtain the final product of 24 mg of SS-Dev-SS-TPEDCH, with an overall yield of 34% for both steps.

[0108] The structure of compound SS-Dev-SS-TPEDCH was characterized by 1H NMR spectroscopy, and the specific data are as follows:

[0109] 1H NMR (500 MHz, DMSO-d6) δ 8.39 (s, 1H), 7.84 – 7.75 (m, 4H), 7.66 – 7.57 (m, 4H), 7.59 – 7.53 (m, 3H), 7.43 – 7.36 (m, 5H), 7.39 – 7.23 (m, 11H),7.27 – 7.18 (m, 6H), 7.15 (t, J = 7.4 Hz, 1H), 6.87 – 6.82 (m, 2H), 4.19 –4.06 (m, 4H), 3.75 – 3.64 (m, 2H), 3.67 – 3.57 (m, 3H), 3.60 – 3.54 (m, 1H),3.41 (dtd, J = 12.2, 7.1, 6.2 Hz, 1H), 3.38 – 3.28 (m, 1H), 3.12 – 2.95 (m,8H), 2.88 – 2.81 (m, 1H), 2.84 – 2.78 (m, 1H), 2.80 – 2.73 (m, 2H), 2.72 (dd,J = 5.1, 1.1 Hz, 1H), 2.73 – 2.68 (m, 3H), 2.70 – 2.57 (m, 4H), 2.54 (hept, J= 7.0 Hz, 1H), 2.22 – 2.13 (m, 2H), 1.95 (dq, J = 12.5, 7.0 Hz, 1H), 1.83(dq, J = 12.2, 7.0 Hz, 1H), 1.78 – 1.51 (m, 6H), 1.53 – 1.41 (m, 1H).

[0110] Comparative Example 2

[0111] Preparation of compound (prodrug) YRW-SS-Dev-TPEDCH

[0112]

[0113] (1) In a dry 50 mL round-bottom flask, the intermediates YRW-pro (0.05 mmol, 63 mg) and SS-Dev (0.05 mmol, 27 mg) obtained above were dissolved together in 12 mL of anhydrous DMF. The reaction flask was placed in an ice-water bath and cooled to 0-5°C. At this temperature, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.12 mmol, 23 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 32 mg) were added to the reaction solution. After the addition was complete, the ice-water bath was removed, and the reaction solution was stirred at room temperature for 6 hours.

[0114] (2) Triphosgene (0.02 mmol, 6 mg) was then added to the above reaction flask. After stirring at room temperature for 2 hours, compound TPEDCH (0.05 mmol, 30 mg) was added. After the reaction was completed by HPLC monitoring, the reaction solution was diluted with 50 mL of ethyl acetate and washed three times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated saline solution. The organic phase was dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by preparative high-performance liquid chromatography, and the target fraction was collected, lyophilized, and the final product was 30 mg of YRW-SS-Dev-TPEDCH, with an overall yield of 25% for both steps.

[0115] The structure of compound YRW-SS-Dev-TPEDCH was characterized by 1H NMR spectroscopy, and the specific data are as follows:

[0116] 1H NMR (500 MHz, DMSO-d6) δ 8.15 – 8.09 (m, 2H), 7.70 – 7.60 (m, 2H), 7.63 – 7.54 (m, 1H), 7.56 – 7.47 (m, 1H), 7.45 – 7.02 (m, 13H), 6.87 – 6.79(m, 1H), 6.71 – 6.65 (m, 1H), 6.50 (s, 1H), 6.15 (s, 1H), 4.44 – 4.29 (m,1H), 4.26 – 4.09 (m, 3H), 4.02 (ddd, J = 17.8, 12.5, 7.1 Hz, 1H), 3.87 – 3.76(m, 1H), 3.69 (q, J = 1.0 Hz, 1H), 3.52 – 3.26 (m, 3H), 3.22 – 2.97 (m, 4H), 2.85 – 2.63 (m, 3H), 2.55 (ddt, J = 22.7, 12.5, 7.1 Hz, 1H), 2.18 – 2.05 (m,1H), 2.08 – 1.99 (m, 2H), 1.96 (s, 1H), 1.89 – 1.24 (m, 12H), 0.85 (d, J =6.8 Hz, 1H), 0.80 (d, J = 6.8 Hz, 1H).

[0117] Comparative Example 3

[0118] Preparation of compound (prodrug) YRW-SS-TPEDCH

[0119]

[0120] In a dry 50 mL round-bottom flask, the intermediates YRW-pro (0.05 mmol, 63 mg) and SS-TPEDCH (0.05 mmol, 40 mg) obtained above were dissolved together in 15 mL of anhydrous DMF. The reaction flask was cooled to 0–5°C in an ice-water bath. At this temperature, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.12 mmol, 23 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 32 mg) were added to the reaction solution. After the addition was complete, the ice-water bath was removed, and the reaction solution was stirred at room temperature for 12 hours. After the reaction was complete as monitored by HPLC, the reaction solution was diluted with 50 mL of ethyl acetate and washed three times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by preparative high performance liquid chromatography, the target fraction was collected, and after freeze-drying, the final product was 44 mg of YRW-SS-TPEDCH, with a yield of 43%.

[0121] The structure of compound YRW-SS-TPEDCH was characterized by 1H NMR spectroscopy, and the specific data are as follows:

[0122] 1H NMR (500 MHz, DMSO-d6) δ 10.74 (s, 1H), 9.42 (s, 1H), 9.03 (s, 1H),8.15 – 8.09 (m, 5H), 7.97 (s, 1H), 7.70 – 7.64 (m, 4H), 7.62 (dd, J = 7.2,1.8 Hz, 1H), 7.59 – 7.54 (m, 2H), 7.53 (dd, J = 7.6, 1.5 Hz, 1H), 7.51 – 7.46(m, 2H), 7.42 (dd, J = 7.4, 1.6 Hz, 1H), 7.41 – 7.34 (m, 4H), 7.34 – 7.26 (m,2H), 7.28 (s, 2H), 7.23 (d, J = 7.6 Hz, 2H), 7.24 – 7.15 (m, 4H), 7.18 – 7.08(m, 6H), 7.12 – 7.02 (m, 3H), 6.87 – 6.79 (m, 4H), 6.71 – 6.65 (m, 2H), 6.50(s, 2H), 6.15 (s, 2H), 4.75 (t, J = 7.0 Hz, 1H), 4.63 (t, J = 5.5 Hz, 1H),4.42 (d, J = 7.1 Hz, 1H), 4.37 (q, J = 6.7 Hz, 1H), 4.30 (dd, J = 12.5, 6.8Hz, 1H), 4.25 – 4.19 (m, 1H), 4.21 – 4.09 (m, 7H), 4.00 (dd, J = 12.5, 7.1Hz, 1H), 3.84 (t, J = 7.0 Hz, 1H), 3.71 (ddd, J = 12.6, 7.0, 5.5 Hz, 1H),3.59 (ddd, J = 12.5, 7.0, 5.5 Hz, 1H), 3.46 (dtd, J = 12.2, 7.1, 2.0 Hz, 2H),3.37 (dt, J = 12.4, 7.1 Hz, 2H), 3.28 (dt, J = 12.2, 7.1 Hz, 1H), 3.21 (td, J= 7.1, 2.1 Hz, 2H), 3.19 – 3.11 (m, 2H), 3.10 (dd, J = 7.7, 1.3 Hz, 2H), 3.11– 2.97 (m, 5H), 2.93 (dt, J = 12.5, 7.1 Hz, 1H), 2.89 – 2.63 (m, 7H), 2.53 (dt, J = 12.5, 7.1 Hz, 1H), 2.11 – 1.98 (m, 5H), 1.96 (s, 2H), 1.84 (dq, J =12.0, 6.9 Hz, 1H), 1.81 – 1.62 (m, 9H), 1.62 – 1.24 (m, 13H), 0.85 (d, J =6.8 Hz, 3H), 0.80 (d, J = 6.8 Hz, 3H).

[0123] Comparative Example 4

[0124] Preparation of compound (prodrug) YRW-SS-Dev

[0125]

[0126] In a dry 50 mL round-bottom flask, the intermediates YRW-pro (0.05 mmol, 63 mg) and SS-Dev (0.05 mmol, 27 mg) obtained above were dissolved together in 15 mL of anhydrous DMF. The reaction flask was cooled to 0–5°C in an ice-water bath. At this temperature, the condensing agent 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDCI, 0.12 mmol, 23 mg) and the organic base N,N-diisopropylethylamine (DIPEA, 0.25 mmol, 32 mg) were added to the reaction solution. After the addition was complete, the ice-water bath was removed, and the reaction solution was stirred at room temperature for 6 hours. After the reaction was complete as monitored by HPLC, the reaction solution was diluted with 50 mL of ethyl acetate and washed three times each with 5% citric acid aqueous solution, saturated sodium bicarbonate aqueous solution, and saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent, yielding the crude product. The crude product was purified by preparative high performance liquid chromatography, the target fraction was collected, and after lyophilization, the final product was 35 mg of YRW-SS-Dev, with an overall yield of 39% for both steps.

[0127] The structure of compound YRW-SS-Dev was characterized by 1H NMR spectroscopy, and the specific data are as follows:

[0128] 1H NMR (500 MHz, DMSO-d6) δ 8.15 – 8.09 (m, 2H), 7.39 – 7.17 (m, 5H), 7.16 – 7.02 (m, 2H), 6.85 (s, 1H), 6.71 – 6.65 (m, 1H), 6.50 (s, 1H), 6.15(s, 1H), 4.44 – 4.26 (m, 1H), 4.25 – 4.13 (m, 2H), 4.16 – 4.09 (m, 1H), 3.87– 3.54 (m, 3H), 3.52 – 3.26 (m, 3H), 3.20 – 2.88 (m, 4H), 2.88 – 2.75 (m,1H), 2.78 – 2.70 (m, 1H), 2.72 – 2.66 (m, 1H), 2.68 – 2.49 (m, 1H), 2.18 –1.94 (m, 4H), 1.89 – 1.24 (m, 12H), 0.85 (d, J = 6.8 Hz, 1H), 0.80 (d, J =6.8 Hz, 1H).

[0129] Application Example 1

[0130] YRW-SS-Dev-SS-TPEDCH's melanoma targeting capability

[0131] Verifying the specific targeting ability of the prodrug molecule on melanoma cells is a crucial step in confirming the effectiveness of its design. This section of the experiment aims to demonstrate that the melanoma-targeting peptide YRWPKPVRA (YRW) attached to the prodrug molecule significantly enhances the compound's accumulation efficiency in target cells, thus laying the foundation for subsequent microenvironment-responsive drug release and synergistic therapy through cellular uptake. By setting up a control molecule without the targeting peptide and a competition experiment with the free targeting peptide, the specificity and dependence of this targeting function can be systematically evaluated, providing direct evidence for elucidating its active targeting mechanism.

[0132] Human melanoma A375 cells were used as an in vitro model. Cells were seeded in confocal culture dishes and cultured to an appropriate density under standard conditions. The cells were then grouped and treated as follows:

[0133] Group 1: Co-incubated with 10 µM YRW-SS-Dev-SS-TPEDCH (prepared in Example 1).

[0134] Group 2: Co-incubated with 10 µM of SS-Dev-SS-TPEDCH (prepared from Comparative Example 1), an analogue without the target peptide YRW.

[0135] Group 3: After pre-incubating with excess free YRW peptide (100 µM) for 1 hour, 10 µM of YRW-SS-Dev-SS-TPEDCH was added for further co-incubation.

[0136] All groups were incubated under the same conditions (37°C, 5% CO2) in the dark for 6 hours. After incubation, the culture medium was discarded, and the cells were gently washed three times with pre-cooled phosphate-buffered saline (PBS) to remove any compounds that had not yet entered the cells. Immediately afterwards, fluorescence images of each group of cells were observed and acquired using a fluorescence microscope in the characteristic fluorescence channel of TPEDCH. The uptake level of the compounds was assessed by comparing the average fluorescence intensity within the cells.

[0137] Fluorescence microscopy observation results show (e.g.) Figure 4 As shown in the figure, strong characteristic fluorescence signals were observed in A375 cells treated with Group 1 (YRW-SS-Dev-SS-TPEDCH), indicating that the compound could be efficiently taken up and accumulated by cells. In contrast, the fluorescence signal intensity in cells treated with Group 2 (SS-Dev-SS-TPEDCH) was significantly weaker than that in the experimental group, confirming that the absence of the targeting peptide YRW would lead to a significant reduction in the efficiency of the compound entering the cell. In Group 3 (free YRW peptide competition group), the fluorescence signal intensity in the cells was significantly weaker than that in the experimental group, and similar to that in Group 2. This result indicates that excessive free targeting peptides competitively block the relevant receptors on the cell surface, thereby effectively inhibiting the entry of YRW-SS-Dev-SS-TPEDCH into cells via the receptor-mediated pathway. The above results collectively demonstrate that the YRW-SS-Dev-SS-TPEDCH prodrug molecule described in this invention, by virtue of its linked YRW targeting peptide, can efficiently and specifically target and accumulate in melanoma A375 cells through a specific recognition-mediated active transport mechanism. This targeting capability is an important prerequisite for achieving subsequent intracellular reduction-responsive drug release and synergistic therapy.

[0138] Application Example 2

[0139] Selective release of YRW-SS-Dev-SS-TPEDCH in tumor cells

[0140] Demonstrating that the prodrug molecule can selectively release drugs against tumor cells in an environment where tumor cells and normal cells coexist is a core step in evaluating its precision therapeutic potential and safety. This part of the experiment aims to simulate a heterogeneous in vivo cellular environment using a co-culture model of tumor cells (A375) and normal cells (HUVECs) to directly verify whether YRW-SS-Dev-SS-TPEDCH can rely on the highly reducing microenvironment (high concentration of GSH) specific to tumor cells to specifically break disulfide bonds and release its loaded TPEDCH fluorophore. This verification is directly related to the core design concepts of this invention: "tumor microenvironment response" and "reducing off-target toxicity to normal cells."

[0141] A co-culture system of A375 melanoma cells and human umbilical vein endothelial cells (HUVECs) was established. A375 cells were pre-labeled with the cell membrane green fluorescent dye PKH67 to facilitate differentiation from unlabeled HUVEC cells under a fluorescence microscope. Labeled A375 cells and HUVEC cells were co-cultured in a confocal culture dish at an appropriate ratio. After cell attachment, YRW-SS-Dev-SS-TPEDCH (10 µM) was added to the co-culture system for 6 hours of co-incubation. After incubation, the culture medium was discarded, and the cells were thoroughly washed with pre-cooled PBS to remove any unadulterated compounds. Immediate observation was performed using a fluorescence microscope. Images were acquired under the green fluorescence channel (corresponding to PKH67-labeled A375 cells) and the orange fluorescence channel (corresponding to the characteristic fluorescence of TPEDCH), and the cell localization of fluorescence signals was analyzed by image overlay.

[0142] Fluorescence microscopy observation results show (e.g.) Figure 5 As shown in the image, where DAPI represents the cell nucleus and indicates the location of all cells; merge represents the images of each fluorescence channel combined together;), a strong orange fluorescence signal characteristic of TPEDCH can be clearly observed in PKH67-labeled A375 cells (exhibiting green fluorescence), and the two fluorescence signals are highly co-localized, indicating that the prodrug molecule was successfully activated and released TPEDCH in A375 tumor cells. In stark contrast, the characteristic orange fluorescence signal of TPEDCH is almost undetectable in the unlabeled green fluorescence region of HUVEC normal cells. This result fully demonstrates that YRW-SS-Dev-SS-TPEDCH can distinguish between tumor cells and normal cells, and selectively trigger disulfide bond breakage and release the active payload (TPEDCH) in the highly reducing microenvironment within tumor cells (A375); while in normal cells (HUVEC), it basically remains in the prodrug state without significant release. This selective release characteristic is an important foundation for the precise and low-toxicity treatment achieved by this invention.

[0143] Application Example 3

[0144] Photodynamic therapy effects of YRW-SS-Dev-SS-TPEDCH

[0145] Verifying whether YRW-SS-Dev-SS-TPEDCH can effectively exert photodynamic therapy within tumor cells is one of the key functional evaluations of this invention. Its core mechanism lies in the fact that the released TPEDCH, acting as an aggregation-induced emission (AIE) photosensitizer, can convert intracellular oxygen into cytotoxic reactive oxygen species (ROS) under specific laser irradiation, thereby inducing tumor cell death.

[0146] This experiment, by detecting and comparing intracellular ROS generation levels under different treatment conditions, aims to directly demonstrate that: 1) the prodrug system can successfully deliver and release a functional photosensitizer; 2) the released photosensitizer possesses a high ROS generation capacity under laser excitation; and 3) a complete "drug release-laser irradiation" process is a necessary condition for initiating the PDT effect. This provides an important mechanistic basis for evaluating its synergistic therapeutic potential.

[0147] A375 melanoma cells were used as a model. Cells were seeded in 96-well plates or confocal culture dishes and cultured to an appropriate density. They were then randomly divided into the following five groups for treatment: Group 1 (TPEDCH + laser group): cells were incubated with free TPEDCH molecules only and then irradiated with laser.

[0148] Group 2 (YRW-SS-Dev-TPEDCH + laser group): Incubated with the control compound YRW-SS-Dev-TPEDCH (prepared from Comparative Example 2) which does not have key disulfide bonds (or has a different connection mode) and subjected to laser irradiation.

[0149] Group 3 (YRW-SS-TPEDCH + laser group): Incubated with the control compound YRW-SS-TPEDCH (prepared from Comparative Example 3) without the Devimistat module and subjected to laser irradiation.

[0150] Group 4 (YRW-SS-Dev-SS-TPEDCH group): Incubated with the target prodrug YRW-SS-Dev-SS-TPEDCH, without laser irradiation.

[0151] Group 5 (YRW-SS-Dev-SS-TPEDCH + laser group): Incubated with the target prodrug YRW-SS-Dev-SS-TPEDCH and irradiated with laser.

[0152] All compound concentrations were kept consistent. After incubation for an appropriate time (6 hours), cells in each group were washed. Groups 2, 3, and 5 were irradiated with a 530 nm laser for a predetermined time to elicit the PDT effect; Group 4 was treated in the dark. Immediately after irradiation, cells from all groups were loaded with a reactive oxygen species-sensitive fluorescent probe (DCFH-DA). After incubation, intracellular fluorescence intensity was quantitatively detected by a fluorescence microplate reader or observed by a fluorescence microscope; this intensity was proportional to the ROS level.

[0153] Intracellular ROS level detection results showed that the fluorescence signal intensity produced by group 5 (YRW-SS-Dev-SS-TPEDCH + laser) was significantly higher than that of all other groups, indicating that this group had the highest intracellular ROS production level (e.g., Figure 6 (As shown). In contrast, although Group 1 (TPEDCH + laser group) had a certain ROS background, it was much lower than that of Group 5, confirming the difference in efficiency between a simple free photosensitizer and a complete targeted delivery system. The ROS signals of Groups 2 and 3 were significantly lower than those of Group 5, demonstrating that the integrity of the prodrug structure (including disulfide bond connections and the presence of the Devimistat module) may have an important impact on achieving optimal intracellular drug release and subsequent PDT effects. Group 4 (YRW-SS-Dev-SS-TPEDCH, no laser) produced only a very weak background fluorescence signal, similar to the negative control group. This clearly shows that laser irradiation is an indispensable exogenous condition for triggering ROS generation in TPEDCH and initiating photodynamic therapy. These results strongly demonstrate that the YRW-SS-Dev-SS-TPEDCH prodrug system constructed in this invention can efficiently initiate photodynamic therapy and generate a large amount of ROS after being taken up by tumor cells through a cascade process of "reduction release-laser excitation". This provides direct mechanistic support for its ability to synergize with the chemotherapy effects of Devimistat, thereby enhancing its killing effect on melanoma cells.

[0154] Application Example 4

[0155] In vitro antitumor activity of YRW-SS-Dev-SS-TPEDCH

[0156] Evaluating the direct killing effect of YRW-SS-Dev-SS-TPEDCH on melanoma cells is a core step in verifying its ultimate effectiveness as an anti-tumor prodrug. This experiment aims to systematically compare the efficacy of the target compound and its functional modules individually or in combination, through cell viability assays, thereby clarifying: 1) the enhancing effect of the targeting peptide YRW; 2) the respective contributions of Devimistat and TPEDCH-mediated photodynamic therapy (PDT); and 3) most importantly, whether the integration of the two through this patented design can produce a significant synergistic effect. These results provide the most direct pharmacodynamic evidence for elucidating the therapeutic advantages of this invention and clarifying its "targeted-chemotherapy-phototherapy" triple synergistic mechanism.

[0157] Human melanoma A375 cells were used as a model. Cells were uniformly seeded in 96-well plates and allowed to adhere overnight under standard culture conditions. Subsequently, the cells were randomly divided into seven groups for intervention treatment:

[0158] Group 1 (PBS group): An equal volume of PBS was added as a negative control.

[0159] Group 2 (Devimistat group): Add free Devimistat.

[0160] Group 3 (TPEDCH + laser group): Free TPEDCH was added and the mixture was irradiated with laser after incubation.

[0161] Group 4 (SS-Dev-SS-TPEDCH + laser group): The control prodrug SS-Dev-SS-TPEDCH without the target peptide was added and then subjected to laser irradiation after incubation.

[0162] Group 5 (YRW-SS-TPEDCH + laser group): YRW-SS-TPEDCH (prepared in Comparative Example 3) without Devimistat was added and then subjected to laser irradiation after incubation.

[0163] Group 6 (YRW-SS-Dev group): The control prodrug YRW-SS-Dev (prepared in Comparative Example 4) without TPEDCH and phototherapy module was added.

[0164] Group 7 (YRW-SS-Dev-SS-TPEDCH + laser group): The target prodrug YRW-SS-Dev-SS-TPEDCH was added and the product was irradiated with laser after incubation.

[0165] All drug treatment groups maintained a consistent concentration (10 µM). Incubation continued for 24 hours after drug addition. For groups requiring light (groups 3, 4, 5, and 7), laser irradiation for a preset time was used at the end of incubation to induce the PDT effect. After irradiation, all groups continued incubation for 24 hours. Subsequently, CCK-8 reagent was added to each well, and after incubation in the dark for 1 hour, the absorbance of each well was measured at 450 nm using a microplate reader. Cell viability was expressed as a percentage relative to the PBS control group (group 1). Each group had three replicates, and the experiment was independently repeated three times.

[0166] Cell viability results as determined by the CCK-8 assay are as follows: Figure 7 As shown, the cell survival rate of group 1 (PBS) was 100%. When group 2 (Devimistat group) and group 3 (TPEDCH + laser group) were treated alone, the cell survival rates decreased to approximately 66% and 74%-83%, respectively, indicating that single therapy had a certain inhibitory effect. Compared with the non-targeted group 4 (SS-Dev-SS-TPEDCH + laser), group 5 (YRW-SS-TPEDCH + laser group), which contained the targeting peptide but lacked Devimistat, had a lower survival rate (group 4 approximately 53%-57%, group 5 approximately 30%-39%), initially showing the enhancing effect of the targeting peptide on PDT. Similarly, group 6 (YRW-SS-Dev group), which contained only the targeting peptide and Devimistat, also showed strong cytotoxicity (survival rate approximately 28%-36%). Group 7 (YRW-SS-Dev-SS-TPEDCH + laser group) exhibited the strongest cell-killing effect, with a cell survival rate of only 5.6%-9.5%, significantly lower than any single-therapy group (groups 2 and 3) or partial functional combination group (groups 4, 5, and 6). This result clearly demonstrates that the complete prodrug system YRW-SS-Dev-SS-TPEDCH constructed in this invention, under laser irradiation, achieves a significant synergistic effect between chemotherapy and photodynamic therapy through highly efficient intracellular delivery mediated by targeted peptides and GSH-responsive simultaneous release of Devimistat and TPEDCH, ultimately producing the strongest in vitro growth inhibition effect on melanoma cells.

[0167] The above description of the embodiments is only for the purpose of helping to understand the method and core ideas of the present invention. It should be noted that those skilled in the art can make several improvements and modifications to the present invention without departing from the principles of the present invention, and these improvements and modifications also fall within the protection scope of the claims of the present invention.

Claims

1. The compound shown in Formula 1: Formula 1.

2. The method for preparing the compound according to claim 1, characterized in that, Includes the following steps: (1) The polypeptide shown in Formula 2, 2-hydroxymethyl-1,3-propanediol, the first condensing agent, the first organic base and the organic solvent are mixed and reacted to obtain the intermediate shown in Formula 3; (2) The intermediate, the compound shown in Formula 4, the second condensing agent, the second organic base and the organic solvent are mixed and reacted to obtain reaction system S1; (3) The reaction system S1 and the compound shown in Formula 5 are mixed and reacted to obtain the compound; Formula 2; Formula 3; Equation 4; Formula 5.

3. The preparation method according to claim 2, characterized in that, The first and second condensing agents are independently selected from 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxyhexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, dicyclohexylcarbodiimide, or diisopropylcarbodiimide; The first organic base and the second organic base are independently selected from N,N-diisopropylethylamine (DIPEA), triethylamine, or 4-dimethylaminopyridine; The organic solvent is selected from anhydrous N,N-dimethylformamide, anhydrous tetrahydrofuran, anhydrous dichloromethane, or anhydrous dimethyl sulfoxide.

4. The preparation method according to claim 3, characterized in that, The first condensing agent is selected from 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-hexafluorophosphate; The second condensing agent is selected from 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; Both the first organic base and the second organic base are selected from N,N-diisopropylethylamine.

5. The preparation method according to any one of claims 2-4, characterized in that, The molar ratio of the polypeptide shown in Formula 2 to 2-hydroxymethyl-1,3-propanediol is 1:(1-1.5). The molar ratio of the intermediate and the compound shown in Formula 4 in step (2) is 1:(1-1.3). The molar ratio of the intermediate to the compound shown in Formula 5 is 1:(1-1.6).

6. The preparation method according to claim 5, characterized in that, The molar ratio of the first condensing agent to 2-hydroxymethyl-1,3-propanediol is (1-1.5):1; The molar ratio of the first organic base to 2-hydroxymethyl-1,3-propanediol is (2-3):1; The molar ratio of the second condensing agent to the intermediate is (1-1.5):0.5; The molar ratio of the second organic base to the intermediate is (2-3):0.

5.

7. A composition, characterized in that, This includes the compound described in claim 1 or the compound prepared by the preparation method described in any one of claims 2-6, as well as other pharmaceutical adjuvants.

8. Use of the composition of claim 7 in the preparation of an anti-melanoma drug.

9. The application according to claim 8, characterized in that, The aforementioned anti-melanoma drug is an anti-malignant melanoma drug for the skin.

10. The application according to claim 8 or 9, characterized in that, The anti-melanoma drug is used under laser irradiation conditions.

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