CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug, albumin nanoparticles thereof, and preparation methods and applications of CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug and albumin nanoparticles

Abstract

The invention relates to a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug and albumin nanoparticles thereof as well as a preparation method and application thereof, and belongs to the technical field of medicines. The CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug is a prodrug as shown in a formula (I) or a pharmaceutically acceptable salt of the prodrug. The invention also relates to a combined albumin nanoparticle prepared from the camptothecin-fatty alcohol prodrug entrapped with CES1 / CES2 cascade activation. The albumin nanoparticles are small in particle size and uniform in form, have good placement stability and colloidal stability, can stably exist in systemic circulation and normal tissues, can release an intermediate through CES1-mediated primary hydrolysis after being taken by tumor cells, and then the intermediate is subjected to secondary hydrolysis under the action of CES2 to release a parent drug camptothecin, so that the curative effect of the camptothecin on tumor cells is improved, and the curative effect of the camptothecin on tumor cells is improved. Therefore, specific killing of tumor cells is realized without generation of serious toxic and side effects, and good clinical development prospects are achieved. Formula (I)

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of medicine, and relates to a camptothecin-fatty alcohol prodrug activated by CES1 / CES2 cascade, an albumin nanoparticle thereof, and a preparation method and application thereof, in particular to synthesis of camptothecin-fatty alcohol prodrugs with different chain lengths activated by CES1 / CES2 cascade, preparation of albumin nanoparticles thereof, and application of the albumin nanoparticles in tumor drug delivery. BACKGROUND

[0002] Camptothecin (CPT) and its derivatives are one of the most important families of traditional antitumor drugs, which can inhibit the rapid proliferation of tumor cells by inhibiting the activity of topoisomerase I, thereby achieving the purpose of treating tumors. However, the water solubility and fat solubility of camptothecin are too poor, resulting in poor drugability of camptothecin, so there is no camptothecin drug on the market. Currently, the camptothecin drugs on the market are mostly water-soluble derivatives of camptothecin. Therefore, the modification of camptothecin prodrugs needs to consider the release efficiency, stability and improvement of physicochemical properties.

[0003] Albumin is the most abundant protein in plasma (about 35-50 g / L in human serum), with a molecular weight of 66.5 kDa. Many poorly water-soluble substances (such as long-chain fatty acids) can be transported by binding to it. Albumin is a large molecule that naturally exists in the body, and has the following advantages as a drug carrier: (1) low immunogenicity, non-toxicity, biodegradability, etc.; (2) long half-life in the body, which can significantly prolong the circulation time of the drug in the body; (3) after the drug is combined with albumin, the protective effect of the large molecule of albumin can be utilized to improve the stability of the drug. In the past few decades, human serum albumin (HSA) has become one of the most important multifunctional drug carriers, which has been used to deliver various therapeutic drugs and diagnostic reagents. Some HSA-based drug formulations have been approved by the FDA for clinical use. Abraxane is the first HSA-based chemotherapy drug approved by the FDA in 2005 for the treatment of cancer. The formulation is prepared into nanoparticles by Nab technology with HSA and paclitaxel for the treatment of breast cancer, non-small cell lung cancer and pancreatic cancer. Since then, HSA-based formulations for the treatment of diabetes and hemophilia and imaging have been successfully approved for listing. Recently, the second drug Fararrotm TM based on Nab technology has also been listed in 2021 for the treatment of locally advanced unresectable or metastatic malignant perivascular epithelioid cell tumor. In addition, a large number of preclinical and clinical trials of HSA formulations are ongoing. The research on HSA-based formulations is still continuing, and it is expected that in the near future, more new HSA-based therapeutic and diagnostic products will be approved for clinical use to benefit patients.

[0004] Whether for prodrugs or nanodelivery systems, selective drug release at the target site is crucial for the efficacy and safety of formulations, and enzymatic reactions are widely used in this process. Enzymes are involved in almost all physiological and metabolic processes in the body, and their high selectivity and substrate specificity give them a significant advantage in stimulus-responsive prodrugs. Studies have shown that the expression levels of carboxylesterases (CES) in human colon cancer tumor cells are higher than those in normal cells. The main human carboxylesterases are carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2), with CES1 showing the highest expression level in the liver and CES2 in the intestine. These two enzymes have different substrate preferences: CES1 tends to hydrolyze ester substrates with "large acids and small alcohols," while CES2 tends to hydrolyze ester substrates with "small acids and large alcohols." This difference is mainly due to the different three-dimensional structures of the two enzymes. To address this phenomenon, a camptothecin prodrug capable of CES1 / CES2 cascade activation was developed. This prodrug can release the parent drug in tumor cells that highly express carboxylesterase while only undergoing primary hydrolysis in normal tissues, thereby reducing toxicity to normal tissues and achieving targeted drug delivery. Summary of the Invention

[0005] Based on this, the present invention provides a CES1 / CES2 cascade-activated camptothecin-fatty alcohol prodrug, its albumin nanoparticles, its preparation method, and its applications. A series of camptothecin-fatty alcohol prodrugs with different chain lengths are designed and synthesized, and succinic acid is introduced as a motif for CES1 / CES2 cascade activation, forming CES1 / CES2 cascade-activated camptothecin-fatty alcohol prodrugs. The present invention further uses human or bovine serum albumin as a carrier to encapsulate CES1 / CES2 cascade-activated camptothecin-succinic acid-fatty alcohol prodrugs of different chain lengths, preparing bound albumin nanoparticles. These albumin nanoparticles have a small particle size and uniform morphology, exhibiting good placement and colloidal stability. They remain stable in systemic circulation and normal tissues. However, after being taken up by tumor cells, they release the parent drug camptothecin through CES1 / CES2 cascade activation, thereby achieving specific killing of tumor cells without producing serious toxic side effects, showing good prospects for clinical development.

[0006] The purpose of this invention is to design and synthesize a series of camptothecin-succinic acid-fatty alcohol prodrugs activated by CES1 / CES2 cascades with different fatty alcohol chain lengths, and to prepare albumin nanoparticles by encapsulation with human or bovine serum albumin. The effects of fatty alcohols with different chain lengths on the properties of the prodrugs and albumin nanoparticles were verified, providing a new approach for developing tumor-stimulation-responsive albumin nanodelivery systems.

[0007] The present invention achieves the above objectives through the following technical solutions: This invention provides a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug, which is a prodrug having the structure shown in formula (I), its geometric isomers, and its pharmaceutically acceptable salts, hydrates, and solvates. Formula (I) Where n = 6-14, preferably n = 10-14.

[0008] The aforementioned is a CES1 / CES2 cascade activated camptothecin-succinic acid-fatty alcohol prodrug, wherein the carbon chain length of the fatty alcohol is 2-30 carbon atoms.

[0009] This invention provides a method for preparing a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug, comprising the following steps: Step 1: Weigh succinic anhydride, add fatty alcohol and 4-dimethylaminopyridine (DMAP), and react with dichloromethane (DCM) as solvent to obtain fatty alcohol monoester of succinic acid. Step 2: Weigh camptothecin (CPT), add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), and use dichloromethane (DCM) as solvent. Add the mixture directly to the reaction vessel from Step 1 to obtain the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug. The specific synthesis route is as follows: .

[0010] Depending on the type of fatty alcohol, the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug of the present invention is a compound represented by the following structural formula (I), with a preferred structure being one of the following: (A) camptothecin-hexanol prodrug (CPT-SA-C6), (B) camptothecin-octanol prodrug (CPT-SA-C8), (C) camptothecin-decylol prodrug (CPT-SA-C10), (D) camptothecin-dodecylol prodrug (CPT-SA-C12), or (E) camptothecin-tetradecylol prodrug (CPT-SA-C14). .

[0011] This invention provides albumin nanoparticles of camptothecin-fatty alcohol prodrugs activated by CES1 / CES2 cascade. These are a series of camptothecin-fatty alcohol prodrugs of succinic acid, and are obtained by encapsulation with albumin as the carrier of the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrugs.

[0012] The CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles have a diameter of 135-150 nm, a polydispersity index (PDI) of 0.102-0.197, a zeta potential of -8.18 to -9.41 mV, an encapsulation efficiency of ≥95%, and a sphericity of ≥25%.

[0013] This invention provides a method for preparing CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles, comprising the following steps: Step 1: Dissolve the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug in an organic solvent to obtain a prodrug solution; wherein, the mass concentration of the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug in the prodrug solution is 1 mg / mL-20 mg / mL. In step 1, the organic solvent is selected from one or a mixture of several of the following: chloroform, dichloromethane, ethyl acetate, methanol, ethanol, acetone, acetonitrile, and dimethyl sulfoxide.

[0014] Step 2: Dissolve albumin in water for injection until it is fully dissolved to obtain an albumin aqueous solution; In step 2, the albumin used is human serum albumin or bovine serum albumin; the albumin concentration in the aqueous solution is 0.5 mg / mL-2 mg / mL.

[0015] Step 3: Mix the prodrug solution and albumin aqueous solution, and homogenize by ultrasonication or high pressure at 0-20℃. The CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug is loaded with albumin. Remove the organic solvent to obtain CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles. The mass ratio of CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug to albumin is (1-5):(1-50). In step 3, the ultrasonic power is 50-650W, the ultrasonic time is 1-20min; the pressure of the high-pressure homogenizer is 5000-50000psi, and the number of high-pressure homogenization cycles is 3-20; the method for removing organic solvents is selected from one or a combination of rotary evaporation, vacuum drying, ultrafiltration, and dialysis.

[0016] The present invention also provides a pharmaceutical composition comprising a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug having the structure shown in formula (I), its geometric isomer, or a pharmaceutically acceptable salt, hydrate, or solvate thereof, and further comprising a pharmaceutically acceptable carrier or excipient.

[0017] The present invention also provides the use of a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug having the structure shown in formula (I), its geometric isomer, or its pharmaceutically acceptable salt, hydrate or solvate, or its CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles, or the pharmaceutical composition thereof, in the preparation of an antitumor drug.

[0018] The antitumor drug is selected from one of the following: oral administration, injection administration, or topical administration.

[0019] Compared with the prior art, the present invention has the following advantages: (1) Design and synthesize a CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug and prepare it as an albumin nanodelivery system. Utilize the high permeability and retention (EPR) effect and active tumor targeting of albumin nanoparticles to promote the accumulation of nanoparticles at the tumor site. (2) This nanodelivery system can reduce the systemic toxicity of camptothecin and rapidly release the parent drug when tumor cells highly express carboxylesterase (CES), thereby achieving the purpose of reducing toxicity and increasing efficacy. (3) It has a high drug loading capacity, avoiding the immunotoxicity caused by using a large amount of carrier material; (4) The preparation process is simple and easy to scale up for production. Attached Figure Description

[0020] Figure 1 The mass spectrum of the camptothecin-succinic acid-hexanol prodrug (CPT-SA-C6) activated by the CES1 / CES2 cascade in Example 1 is shown. Figure 2 The mass spectrum of the camptothecin-succinic acid-octanol prodrug (CPT-SA-C8) activated by the CES1 / CES2 cascade in Example 2 is shown. Figure 3 The mass spectrum of the camptothecin-succinic acid-decyl alcohol prodrug (CPT-SA-C10) activated by the CES1 / CES2 cascade in Example 3 is shown. Figure 4 The mass spectrum of the camptothecin-succinic acid-dodecyl alcohol prodrug (CPT-SA-C12) activated by the CES1 / CES2 cascade in Example 4 is shown. Figure 5 The mass spectrum of the camptothecin-succinic acid-tetradecyl alcohol prodrug (CPT-SA-C14) activated by the CES1 / CES2 cascade in Example 5 is shown. Figure 6 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 6 are shown. Figure 7 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 7 are shown. Figure 8 The particle size distribution diagram (A) and electron microscope image (B) of albumin nanoparticles in Example 8 are shown. Figure 9 The image shows the particle size versus storage time of albumin nanoparticles in Examples 6-8 under storage conditions at 4°C. Figure 10 The following are particle size-time graphs of albumin nanoparticles in 10% FBS in Examples 6-8; Figure 11 The figures below show the in vitro release assays of albumin nanoparticles from Examples 6-8. Specifically, A shows the release assay of albumin nanoparticles from Examples 6-8 in phosphate buffer; B, C, and D show the release assays of albumin nanoparticles from Examples 6-8 in phosphate buffer (pH=7.4) containing 10 U / mL CES1 and 10 U / mL CES2; E shows the release assay of albumin nanoparticles from Examples 6-8 in phosphate buffer (pH=7.4) containing 10 U / mL CES1; and F shows the release assay of intermediate albumin nanoparticles in phosphate buffer (pH=7.4) containing 10 U / mL CES2. Figure 12 The images show the blood concentration-time curves of albumin nanoparticles from Examples 6-8. Specifically, A represents the blood concentration-time curve of camptothecin solution and the blood concentration-time curves of the prodrugs from Examples 3-5 corresponding to the albumin nanoparticles from Examples 6-8; B represents the blood concentration-time curve of camptothecin released from the albumin nanoparticles from Examples 6-8. Figure 13 The graphs shown are from the in vivo antitumor assays of albumin nanoparticles in Examples 6-8. In the graphs, A represents tumor growth curves; B represents body weight changes. Detailed Implementation

[0021] The present invention will be further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein.

[0022] In the following embodiments, unless otherwise specified, "%" refers to mass percentage.

[0023] In the following embodiments, unless otherwise specified, all raw materials and equipment used are commercially available.

[0024] Example 1: Synthesis of a CES1 / CES2 cascade-activated succinic acid-bridged camptothecin-hexanol prodrug (CPT-SA-C6) Step 1: Weigh succinic anhydride (2 g, 20 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add hexanol (2.76 mL, 22 mmol) and 4-dimethylaminopyridine (DMAP, 0.73 g, 6 mmol) to the reaction system and react at room temperature for 2 hours.

[0025] Step 2: Weigh camptothecin (CPT, 3.48 g, 10 mmol) and add it to the reaction vessel from Step 1. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 5.75 mg, 30 mmol) and 4-dimethylaminopyridine (DMAP, 6 g, 50 mmol). React overnight at room temperature. Wash the reaction solution three times each with water and saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude CPT-SA-C6. Purify the crude product by column chromatography to obtain pure CPT-SA-C6.

[0026] Analysis of the obtained product revealed that the prodrug contained [M+H] atoms. + =533.30. Mass spectrum as follows Figure 1 As shown. The results of the proton NMR spectrum analysis are as follows: 1 H-NMR (400 MHz, CDCl3) δ = 8.39 (d, J=1.0,1H), 8.23 ​​(dd, J=8.5, 0.9, 1H), 7.94 (dd, J=8.2, 1.7, 1H), 7.83 (ddd, J=8.6,6.9, 1.5, 1H), 7.66 (ddd, J=8.1, 6.8, 1.2, 1H), 7.28 (s, 1H), 5.68 (d, J=17.1, 1H), 5.39 (d, J=17.1, 1H), 5.28 (dd, J=2.8, 1.3, 2H), 4.12 – 3.97 (m,2H), 2.97 – 2.75 (m,2H), 2.73 – 2.57 (m, 2H), 2.35 – 2.08 (m, 2H), 1.53 (dq, J=8.4, 6.4, 2H), 1.29 – 1.10 (m, 6H), 0.99 (t, J=7.5, 3H), 0.82 (t, 3H); After verification, its structural formula is: .

[0027] Example 2: Synthesis of CES1 / CES2 cascade-activated succinate-bridged camptothecin-octanol prodrug (CPT-SA-C8) Step 1: Weigh succinic anhydride (2 g, 20 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add octanol (3.46 mL, 22 mmol) and 4-dimethylaminopyridine (DMAP, 0.73 g, 6 mmol) to the reaction system and react at room temperature for 2 hours.

[0028] Step 2: Weigh camptothecin (CPT, 3.48 g, 10 mmol) and add it to the reaction vessel from Step 1. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 5.75 mg, 30 mmol) and 4-dimethylaminopyridine (DMAP, 6 g, 50 mmol). React overnight at room temperature. Wash the reaction solution three times each with water and saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude CPT-SA-C6. Purify the crude product by column chromatography to obtain pure CPT-SA-C8.

[0029] Analysis of the obtained product revealed that the prodrug contained [M+H] atoms. + =561.35. Mass spectrum as follows. Figure 2 As shown. The results of the proton NMR spectrum analysis are as follows: 1 H-NMR (400 MHz, CDCl3) δ = 8.38 (s, 1H),8.23 (d, J=8.6, 1H), 7.93 (dd, J=8.3, 1.5, 1H), 7.83 (ddd, J=8.6, 6.8, 1.6,1H), 7.66 (ddd, J=8.1, 6.8, 1.2, 1H), 7.32 – 7.26 (m, 1H), 5.68 (d, J=17.1,1H), 5.40 (d, J=17.2, 1H), 5.34 – 5.24 (m, 2H), 4.88 (dh, J=12.2, 6.2, 1H),2.84 (tdd, J=6.7, 4.1, 2.2, 2H), 2.62 (td, J=7.5, 2.9, 2H), 2.21 (ddq, J=46.0, 14.9, 7.4, 2H), 1.95 (s, 1H), 1.28 – 1.05 (m, 12H), 1.00 (t, J=7.5, 3H), 0.84 (dt, J=16.6, 6.9, 3H); After verification, its structural formula is: .

[0030] Example 3: Synthesis of a CES1 / CES2 cascade-activated succinic acid-bridged camptothecin-decanol prodrug (CPT-SA-C10) Step 1: Weigh succinic anhydride (2 g, 20 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add decanol (4.20 mL, 22 mmol) and 4-dimethylaminopyridine (DMAP, 0.73 g, 6 mmol) to the reaction system and react at room temperature for 2 hours.

[0031] Step 2: Weigh camptothecin (CPT, 3.48 g, 10 mmol) and add it to the reaction vessel from Step 1. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 5.75 mg, 30 mmol) and 4-dimethylaminopyridine (DMAP, 6 g, 50 mmol). React overnight at room temperature. Wash the reaction solution three times each with water and saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude CPT-SA-C6. Purify the crude product by column chromatography to obtain pure CPT-SA-C10.

[0032] Analysis of the obtained product revealed that the prodrug contained [M+H] atoms. + =589.37. Mass spectrum as follows Figure 3 As shown. The results of the proton NMR spectrum analysis are as follows: 1 H-NMR (400 MHz, CDCl3) δ = 8.38 (s, 1H),8.22 (d, J=8.4, 1H), 7.93 (dd, J=8.3, 1.6, 1H), 7.82 (ddd, J=8.4, 6.9, 1.5,1H), 7.66 (ddd, J=8.2, 6.8, 1.3, 1H), 7.27 (s, 1H), 5.68 (d, J=17.2, 1H), 5.39 (d, J=17.2, 1H), 5.27 (dd, J=3.0, 1.3, 2H), 4.04 (d, J=8.4, 2H), 2.84(d, J=6.1, 2H), 2.64 (d, J=2.2, 2H), 2.21 (ddd, J=51.8, 13.9, 7.5, 2H), 1.27– 1.16 (m, 16H), 0.99 (t, J=7.5, 3H), 0.87 (t, J=7.0, 3H); After verification, its structural formula is: .

[0033] Example 4: Synthesis of a CES1 / CES2 cascade-activated succinic acid-bridged camptothecin-dodecyl prodrug (CPT-SA-C12) Step 1: Weigh succinic anhydride (2 g, 20 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add dodecanol (4.10 mg, 22 mmol) and 4-dimethylaminopyridine (DMAP, 0.73 g, 6 mmol) to the reaction system and react at room temperature for 2 hours.

[0034] Step 2: Weigh camptothecin (CPT, 3.48 g, 10 mmol) and add it to the reaction vessel from Step 1. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 5.75 mg, 30 mmol) and 4-dimethylaminopyridine (DMAP, 6 g, 50 mmol). React overnight at room temperature. Wash the reaction solution three times each with water and saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude CPT-SA-C6. Purify the crude product by column chromatography to obtain pure CPT-SA-C6.

[0035] Analysis of the obtained product revealed that the prodrug contained [M+Na]. + = 617.35. Mass spectrum as follows Figure 4 As shown. The results of the proton NMR spectrum analysis are as follows: 1 H-NMR (400 MHz, CDCl3) δ = 8.37 (s, 1H),8.22 (d, J=8.6, 1H), 7.92 (dd, J=8.2, 1.5, 1H), 7.82 (ddd, J=8.6, 6.8, 1.6,1H), 7.65 (ddd, J=8.1, 6.8, 1.2, 1H), 7.27 (s, 1H), 5.67 (d, J=17.1, 1H), 5.38 (d, J=17.2, 1H), 5.31 – 5.23 (m, 2H), 4.03 (qt, J=10.8, 6.9, 2H), 2.84(q, J=7.0, 2H), 2.69 – 2.59 (m, 2H), 2.21 (ddd, J=51.5, 13.9, 7.5, 2H), 1.53 (t, J=7.2, 2H), 1.32 – 1.15 (m, 18H), 0.98 (t, J=7.5, 3H), 0.86 (t, J=6.9, 3H); After verification, its structural formula is: .

[0036] Example 5: Synthesis of a CES1 / CES2 cascade-activated succinic acid-bridged camptothecin-tetradecyl alcohol prodrug (CPT-SA-C14) Step 1: Weigh succinic anhydride (2 g, 20 mmol) and dissolve it in 60 mL of dichloromethane (DCM). Add tetradecanol (4.72 mg, 22 mmol) and 4-dimethylaminopyridine (DMAP, 0.73 g, 6 mmol) to the reaction system and react at room temperature for 2 hours.

[0037] Step 2: Weigh camptothecin (CPT, 3.48 g, 10 mmol) and add it to the reaction vessel from Step 1. Then add 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 5.75 mg, 30 mmol) and 4-dimethylaminopyridine (DMAP, 6 g, 50 mmol). React overnight at room temperature. Wash the reaction solution three times each with water and saturated brine. Collect the dichloromethane (DCM) phase and filter under reduced pressure to obtain crude CPT-SA-C6. Purify the crude product by column chromatography to obtain pure CPT-SA-C14.

[0038] Analysis of the obtained product showed that the [M+Na]+ concentration of the prodrug was 645.37, according to high-resolution mass spectrometry. The mass spectrum is shown below. Figure 5 As shown. The results of the proton NMR spectrum analysis are as follows: 1 H-NMR (400 MHz, CDCl3) δ = 8.36 (s, 1H), 8.21 (d, J=8.6, 1H), 7.91 (d, J=8.2, 1H), 7.81 (t, J=7.6, 1H), 7.64 (t, J=7.5, 1H), 5.66 (d, J=17.1, 1H), 5.24 (d, J=4.0, 2H), 4.10 – 3.95 (m, 2H), 2.83 (q, J=6.9, 2H), 2.70 – 2.59 (m, 2H), 2.20 (dp, J=43.6, 7.3, 2H), 1.51(q, J=7.0, 2H), 1.20 (d, J=24.8, 24H), 0.98 (t, J=7.5, 3H), 0.86 (t, J=6.8, 3H); After verification, its structural formula is: .

[0039] Example 6: Formulation and preparation process of the prodrug albumin nanoparticles from Example 3 Table 1: Formulation of prodrug albumin nanoparticles in Example 3 Preparation process: (1) Accurately weigh 50 mg of the prodrug from Example 3, dissolve it thoroughly in 5 mL of chloroform, and set aside; (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 50 mL of water for injection, and set aside. (3) Add (1) to (2) and sonicate at 105W for 3 minutes in an ice bath. The resulting emulsion is then subjected to rotary evaporation to remove the organic solvent and obtain albumin nanoparticles.

[0040] like Figure 6 A and Figure 6 As shown in B, the albumin nanoparticles in Example 6 are spherical with uniform particle size, with a particle size of 142.5±4.850 nm, a PDI of 0.134±0.011, a Zeta potential of -8.41±0.968 mV, an encapsulation efficiency of 97.56±1.19%, and a sphericity of 31.21±3.41%.

[0041] Example 7: Formulation and preparation process of the prodrug albumin nanoparticles from Example 4 Table 2: Formulation of prodrug albumin nanoparticles in Example 4 Preparation process: (1) Accurately weigh 50 mg of the prodrug from Example 4, dissolve it thoroughly in 5 mL of chloroform, and set aside; (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 50 mL of water for injection, and set aside. (3) Add (1) to (2) and sonicate at 105W for 3 minutes in an ice bath. The resulting emulsion is then subjected to rotary evaporation to remove the organic solvent and obtain albumin nanoparticles.

[0042] like Figure 7 A and Figure 7 As shown in B, the albumin nanoparticles in Example 7 were spherical with uniform particle size, with a particle size of 135.3±7.267 nm, a PDI of 0.102±0.013, a Zeta potential of -8.23±0.780 mV, an encapsulation efficiency of 95.60±1.54%, and a sphericity of 28.43±3.40%.

[0043] Example 8: Formulation and preparation process of the prodrug albumin nanoparticles from Example 5 Table 3: Formulation of the prodrug albumin nanoparticles in Example 5 Preparation process: (1) Accurately weigh 50 mg of the prodrug from Example 5, dissolve it thoroughly in 5 mL of chloroform, and set aside; (2) Accurately weigh 50 mg of human serum albumin, dissolve it thoroughly in 50 mL of water for injection, and set aside. (3) Add (1) to (2) and sonicate at 105W for 3 minutes in an ice bath. The resulting emulsion is then subjected to rotary evaporation to remove the organic solvent and obtain albumin nanoparticles.

[0044] like Figure 8 A and Figure 8 As shown in B, the albumin nanoparticles in Example 8 are spherical with uniform particle size, with a particle size of 135.7±3.092 nm, a PDI of 0.197±0.015, a Zeta potential of -8.18±0.853 mV, an encapsulation efficiency of 98.69±0.77%, and a sphericity of 28.17±5.20%.

[0045] Example 9: Storage stability test of albumin nanoparticles from Examples 6-8 at 4°C The albumin nanoparticles from Examples 6-8 were stored at 4°C. During this period, particle size changes were measured at set time points using dynamic light scattering. The results are as follows: Figure 9 As shown, the particle size of albumin nanoparticles in Examples 6-8 showed no significant change over 30 days, indicating that the nanoparticles have good storage stability.

[0046] Example 10: Colloidal stability test of albumin nanoparticles from Examples 5-8 The albumin nanoparticles from Examples 6-8 were diluted to 0.1 mg / mL with 10% FBS and placed in a shaker at 37 °C. The particle size change was measured at set time points using dynamic light scattering. The results are as follows: Figure 10 As shown, the particle size of albumin nanoparticles in Examples 6-8 did not change significantly within 24 hours, indicating that the nanoparticles have good colloidal stability.

[0047] Example 11: In vitro release test of albumin nanoparticles from Examples 5-8 The in vitro release of albumin nanoparticles from Examples 6-8 was investigated using phosphate-buffered saline (pH=7.4) and phosphate-buffered saline (pH=7.4) containing 10 U / mL CES1 and 10 U / mL CES2 as release media. The albumin nanoparticles from Examples 6-8 were added to the release media and incubated in a shaker at 37 °C. Samples were taken at predetermined time points, and acetonitrile (3 times the volume) was added to precipitate the protein. The mixture was vortexed, centrifuged, and the supernatant was collected. The concentrations of the released intermediates and camptothecin were determined by high-performance liquid chromatography (HPLC) to investigate the release of albumin nanoparticles from Examples 6-8 under different conditions.

[0048] Depend onFigure 11 It is known that the albumin nanoparticles in Examples 6-8 can only be released in a phosphate buffer (pH=7.4) containing 10 U / mL CES1 and 10 U / mL CES2. Figure 11 Camptothecin can only be released in A), while in a neutral physiological environment ( Figure 11 Under C), the albumin nanoparticles in Examples 6-8 released almost no camptothecin or intermediates.

[0049] Subsequently, the in vitro release of albumin nanoparticles from Examples 6-8 was investigated using phosphate buffer (pH=7.4) containing 10 U / mL CES1 as the release medium; the in vitro release of the parent drug from the intermediate was investigated using phosphate buffer (pH=7.4) containing 10 U / mL CES2 as the medium. The albumin nanoparticles and intermediates from Examples 6-8 were added to the stiffest release medium and incubated in a shaker at 37 °C. Samples were taken at set time points, and three volumes of acetonitrile were added to precipitate the protein. The mixture was vortexed, centrifuged, and the supernatant was collected. The concentrations of the released intermediate and camptothecin were determined by high-performance liquid chromatography (HPLC) to investigate the release of albumin nanoparticles from Examples 6-8 under different conditions.

[0050] Depend on Figure 11 It can be seen that the albumin nanoparticles in Examples 6-8 were released in a phosphate buffer (pH=7.4) containing 10 U / mL CES1. Figure 11 In A), a large amount of intermediate is released, with almost no camptothecin released. However, the intermediate successfully released camptothecin in a release medium of 10 U / mLCES2.

[0051] This demonstrates that the albumin nanoparticles of Examples 6-8 possess the characteristic of CES1 / CES2 cascade activation. Compared with the albumin nanoparticles of Examples 6, 8, and 9, the albumin nanoparticles of Example 7 achieved a release rate of 96.08% within 48 hours, suggesting the potential to release the parent drug within tumor cells highly expressing carboxylesterase 1 (CES1) and carboxylesterase 2 (CES2), thereby achieving a tumor-killing effect.

[0052] Example 12: Pharmacokinetic Study of Albumin Nanoparticles from Examples 6-8 SD rats (weight: 180-220g) were used as the research subjects and randomly divided into groups of 5 rats each. They were fasted for 12 hours before administration but had free access to water. Camptothecin solution and albumin nanoparticles (2 mg / kg based on camptothecin) from Examples 6-8 were administered via tail vein, respectively. The plasma concentrations of the prodrug camptothecin from Examples 3-5 were then measured.

[0053] Experimental results are as follows Figure 12 A, Figure 12As shown in B and Table 4, camptothecin solution is rapidly cleared from the blood, and the peak concentration of CPT in the blood (C max The C value was lower, while the albumin nanoparticles in Examples 6-8 showed significantly higher C values. max Furthermore, the different groups of nanoparticles exhibited significant differences due to the varying lengths of their fatty acid chains.

[0054] Table 4: Key Pharmacokinetic Parameters of Albumin Nanoparticles in Examples 6-8 Example 13: In vivo antitumor experiment of albumin nanoparticles from Examples 6-8 CT26 cell suspension (5×10) 6 150 μL of cells per cell were subcutaneously injected into the dorsal side of male BALB / c mice. The tumors were allowed to grow to 100 mm in size. 3 Around 10:00 AM, tumor-bearing mice were randomly divided into groups of five. Each group received a tail vein injection of saline, irinotecan hydrochloride for injection (10 mg / kg based on SN-38), camptothecin (3 mg / kg), and albumin nanoparticles from Examples 6-8 (10 mg / kg based on camptothecin). The drugs were administered every two days for four consecutive days. Mice were observed for survival, weighed, and tumor volume measured every two days.

[0055] In vivo anti-tumor effects such as Figure 13 As shown, the camptothecin solution had no significant antitumor effect, tumor volume increased rapidly, and it was highly toxic, indicating that the instability of the lactone ring in the camptothecin structure led to its poor antitumor effect and severe toxicity. The commercially available irinotecan hydrochloride injection group showed good antitumor effects; compared to the saline group, tumor volume growth in mice was effectively controlled, but it also exhibited some toxicity, manifested as a decrease in mouse body weight. The albumin nanoparticles in Examples 6-8 also showed excellent antitumor effects; compared to the saline group, tumor volume growth in mice was effectively controlled. Furthermore, the antitumor effects varied among different albumin nanoparticles, with the order of antitumor effect being: Example 7 albumin nanoparticles > Example 6 albumin nanoparticles ≈ Irinotecan hydrochloride injection > Example 8 albumin nanoparticles. This indicates that the albumin nanoparticle group in Example 7 had the best antitumor activity, and compared to the saline group, the body weight of the albumin nanoparticle group in Example 7 did not decrease significantly, demonstrating good safety.

[0056] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope of the present invention.

Claims

1. A CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14, which is a CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14, respectively, as shown in the following structural formula (I), a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, characterized in that, Formula (I) wherein n = 6 - 14.

2. The CES1 / CES2 cascade-activated camptothecin-fatty alcohol prodrug, geometric isomer thereof, or pharmaceutically acceptable salt, hydrate, or solvate thereof according to claim 1, characterized in that, According to the type of fatty alcohol, the specific structure is one of (A) CPT-SA-C6, (B) CPT-SA-C8, (C) CPT-SA-C10, (D) CPT-SA-C12 or (E) CPT-SA-C14 as shown in the following structural formula: 。 3. A process for the preparation of a CESl / CES2 cascade-activated camptothecin- fatty alcohol prodrug, a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof according to claim 1 or claim 2, characterized in that, The preparation method of the CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14 includes the following steps: Step 1: Weigh succinic anhydride, add fatty alcohol and 4-dimethylaminopyridine (DMAP), and use dichloromethane (DCM) as the solvent to obtain a fatty alcohol monosuccinate; Step 2: Weigh CPT, add 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) and 4-dimethylaminopyridine (DMAP), and use dichloromethane (DCM) as the solvent to directly add to the reaction container in step 1 to obtain a CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14; The specific synthesis route is as follows: 。 4. A process for the preparation of a camptothecin-fatty alcohol prodrug of CES1 / CES2 cascade activation, a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate, or solvate thereof according to claim 3, characterized in that, In step 1, the molar ratio of succinic anhydride, fatty alcohol and DMAP is 1:1.1:(0-1); and / or in step 2, the molar ratio of CPT, 4-dimethylaminopyridine and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride is (0-1):(1-10):(1-10).

5. A camptothecin-fatty alcohol prodrug albumin nanoparticle with CES1 / CES2 cascade activation, characterized in that, The CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14 is encapsulated by using albumin as a carrier of the CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14 to obtain an albumin nanoparticle.

6. The CES1 / CES2 cascade-activated camptothecin-fatty alcohol prodrug albumin nanoparticle according to claim 5, characterized in that, The CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14 albumin nanoparticle has a particle size of 135-150 nm, a polydispersity index (PDI) of 0.102-0.197, a Zeta potential of -8.18 to -9.41 mV, an encapsulation efficiency of ≥95%, and a balling rate of ≥25%.

7. A method of preparing a CES1 / CES2 cascade-activated camptothecin-fatty alcohol prodrug albumin nanoparticle of claim 5 or claim 6, characterized in that, The method includes the following steps: Step 1: Dissolve the CPT-SA-C6, CPT-SA-C8, CPT-SA-C10, CPT-SA-C12 or CPT-SA-C14 in an organic solvent to obtain a prodrug solution; Step 2: Dissolve albumin in water for injection to obtain an albumin aqueous solution; Step 3: mixing the prodrug solution and the albumin aqueous solution, ultrasonic or high pressure homogenization at 0-20℃, the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug is encapsulated by albumin, removing the organic solvent to obtain the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles; wherein, the mass ratio of CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug: albumin is (1-5):(1-50).

8. The process for the preparation of a CES1 / CES2 cascade-activated camptothecin- fatty alcohol prodrug albumin nanoparticle according to claim 7, characterized in that, In the step 1, the mass concentration of the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug in the prodrug solution is 0.5 mg / mL-20 mg / mL; The organic solvent is selected from one or a mixture of several of trichloromethane, dichloromethane, ethyl acetate, methanol, ethanol, acetone, acetonitrile, dimethyl sulfoxide; And / or, in the step 2, the albumin is selected from human serum albumin or bovine serum albumin; the mass concentration of the albumin in the albumin aqueous solution is 0.1 mg / mL-2 mg / mL; And / or, in the step 3, the ultrasonic power is 50-650W, the ultrasonic time is 1~20 min; the high pressure homogenizer pressure is 5000-50000 psi, the high pressure homogenization cycle number is 3-20 times; the method for removing the organic solvent is selected from one or a combination of several of spin evaporation, vacuum drying, ultrafiltration, dialysis.

9. A pharmaceutical composition, characterized by, It comprises the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug having the formula (I) as claimed in claim 1 or claim 2, a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, and further comprises a pharmaceutically acceptable carrier or excipient.

10. The use of the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug as claimed in claim 1 or claim 2, a geometric isomer thereof, or a pharmaceutically acceptable salt, hydrate or solvate thereof, or the CES1 / CES2 cascade activated camptothecin-fatty alcohol prodrug albumin nanoparticles as claimed in claim 5 or claim 6, or the pharmaceutical composition as claimed in claim 9 in the preparation of an antitumor drug.

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