A deuterated camptothecin derivative, and a preparation method and use thereof

Abstract

The present application relates to the technical field of medicine, and mainly relates to a kind of deuterated camptothecin derivatives and preparation method and purposes.The deuterated camptothecin derivative described in the present application is the compound shown in formula I or formula II, its pharmaceutically acceptable salt or solvate, is prepared by directly deuterating camptothecin nucleus, and preparation method is simple and effective, and the deuterated camptothecin derivative provided in the present application has excellent tumor inhibition biological activity and faster elimination half-life, simultaneously has more optimal abdominal cavity and oral bioavailability, has good clinical application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to a deuterated camptothecin derivative, a preparation method and use thereof. BACKGROUND

[0002] Camptothecin (CPT) is originally derived from a natural product in the plant Camptotheca acuminata, which has significant antitumor biological activity. It is a topoisomerase I (TOP I) inhibitor, which exerts antitumor effect by inhibiting the formation of ternary complex of TOP1 and DNA. CPT has a broad spectrum of antitumor effect and can exhibit significant biological activity against various tumor types. CPT derivatives irinotecan and topotecan have been approved by FDA for the treatment of colon cancer and ovarian cancer, respectively. CPT compounds have problems such as high toxicity, poor water solubility, and short half-life.

[0003] In recent years, with the rise of antibody-drug conjugates (ADCs), CPT and its derivatives have once again attracted great attention. ADCs are a chemical linkage between the effector molecule and the antibody, which can precisely transport the effector molecule to the target cell by using the high targeting of the antibody. In this way, on the one hand, the toxicity of the drug molecule can be reduced, and on the other hand, the pharmacokinetics of small molecules can be converted into the pharmacokinetics of large molecules. The emergence of ADCs can improve the problems faced by CPT direct drug, so CPT compounds are a very promising molecule and can be used as an effective payload of ADCs. ADCs strastuzumab deruxtecan (DS8201, Enhertu) and sacituzumab govitecan (IMMU-132, Trodelvy) have been approved for marketing, with CPT derivatives Dxd and SN38 (irinotecan active metabolite) as effective payloads, respectively. CPT derivatives have shown great potential in direct application as small molecule drugs or as payload molecules in ADCs.

[0004] Deuterated CPT derivatives have also received more and more attention in recent years. Deuterium (2H, D) is a stable isotope of hydrogen, and carbon-deuterium bonds are more stable than carbon-hydrogen bonds. Deuterium substitution can slow down drug metabolism, thereby prolonging drug half-life, reducing drug clearance, reducing drug toxicity, etc. There are many patent documents reporting deuterated CPT derivatives. Chinese patent document CN117105948A introduces deuterium into the camptothecin lactone ring by means of total synthesis. This method has complex preparation process and high cost; Chinese patent documents CN113943310A and CN118164994A introduce deuterium into the side chain and combine with the linker to synthesize ADCs. This method only modifies deuterium from the side chain. SUMMARY

[0005] In view of the problems in the prior art, the purpose of the present application is to provide a deuterated camptothecin derivative, a preparation method and use thereof, which directly deuterates camptothecin nucleus, has a simple and effective preparation method, excellent tumor inhibition biological activity, short half-life and fast clearance rate, and better abdominal cavity and oral bioavailability.

[0006] To achieve this purpose, the technical scheme adopted by the present application is as follows:

[0007] The first aspect of the present application provides a deuterated camptothecin derivative, which is a compound represented by formula I or formula II, a pharmaceutically acceptable salt or solvate thereof:

[0008]

[0009] wherein R1 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2) n CH3, -F, -Cl, -Br or -I, n represents an integer of 1-3; R2 is selected from -H, -OH, -NH2, -F, -Cl, -Br, -I, -CH3, -OCH3, -(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -O(CH2) n CH3, -O(CH2) n NH2, -NH(CH2) n NH2, -(OCH2CH2) n NH2 or wherein n represents an integer of 1-3; or R1 and R2 are connected to each other to form -(CH2) p -, -X(CH2) (p-1) -, -(CH2)(p-1) X-, -X(CH2) (q-1) X- or -(CH2) (q-1) X(CH2) (q-1) , p and q represent an integer of 2-4, X represents O, NH or S;

[0010] R3is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2) n CH3, -NHCH3, -NH(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -(CH2) n CH=CH2, -NO2, -F, -Cl, -Br or -I, n represents an integer of 1-3; R4is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -Si(CH3)2C(CH3)3, -(CH2) n NHCH, -(CH2) n Si(CH3)3, -(CH3)2, -CH2NH(CH2) n CH3, -CH2O(CH2) n CH3, -F, -Cl, -Br, -I, wherein, n represents an integer of 1-3; or R3and R4are connected to each other to form a structure as shown in the following formula:

[0011]

[0012] In some embodiments of the present application, R1is selected from -H, -F, -Cl, -Br or -I; R2is selected from -H, -OH, -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3; R3is selected from -H, -CH2N(CH3)2, -(CH2)2CH3, -(CH2)3N(CH3)2, R4is selected from -H, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, or R3and R4are connected to each other to form a structure as shown in the following formula:

[0013]

[0014] In some embodiments of the present application, the deuterated camptothecin derivative is selected from the following chemical structural formula, a pharmaceutically acceptable salt or solvate thereof:

[0015]

[0016] A second aspect of the present invention provides a method for preparing a deuterated camptothecin derivative, comprising the following steps: reacting a camptothecin derivative with a deuterated reagent under alkaline conditions to obtain a deuterated camptothecin derivative.

[0017] In some embodiments of the present invention, the chemical structure of the camptothecin derivative is shown in the following formula III:

[0018]

[0019] Wherein, R1 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2) n CH3, -F, -Cl, -Br or -I, n represents an integer of 1 to 3; R2 is selected from -H, -OH, -NH2, -F, -Cl, -Br, -I, -CH3, -OCH3, -(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -O(CH2) n CH3, -O(CH2) n NH2, -NH(CH2) n NH2, -(OCH2CH2) n NH2 or wherein n represents an integer of 1 to 3; or R1 and R2 are connected to each other to form -(CH2) p -, -X(CH2) (p-1) -、-(CH2) (p-1) X-, -X(CH2) (q-1) X- or -(CH2) (q-1) X(CH2) (q-1) , p and q represent integers from 2 to 4, X represents O, NH or S;

[0020] R3 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2) n CH3, -NHCH3, -NH(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -(CH2) n CH=CH2, -NO2, -F, -Cl, -Br or -I, n represents an integer of 1 to 3; R4 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -Si(CH3)2C(CH3)3, -(CH2) nNHCH, -(CH2) n Si(CH3)3, -(CH3)2, -CH2NH(CH2) n CH3, -CH2O(CH2) n CH3, -F, -Cl, -Br, -I, wherein, n represents an integer of 1-3; or R3 and R4 are connected to each other to form a structure as shown in the following formula:

[0021]

[0022] In some embodiments of the present application, the reaction is carried out in the presence of an organic solvent.

[0023] In some embodiments of the present application, the basic condition is in the presence of a strong base.

[0024] In some embodiments of the present application, the temperature of the reaction is -78-40℃.

[0025] In some embodiments of the present application, the reaction time is 0.5-72h.

[0026] In some embodiments of the present application, the deuterium reagent is selected from D2O, CD3OD or CD3COOD.

[0027] In some embodiments of the present application, the reaction post-treatment includes acidification, filtration, purification.

[0028] The third aspect of the present application provides the use of the above-mentioned deuterated camptothecin derivative in the preparation of a medicament for treating tumors.

[0029] The fourth aspect of the present application provides a pharmaceutical composition comprising the above-mentioned deuterated camptothecin derivative and a pharmaceutically acceptable excipient.

[0030] Compared with the prior art, the present application has the following beneficial effects:

[0031] The present application provides a method for preparing a deuterated camptothecin derivative by directly deuterating camptothecin nucleus, which is simple and effective. The deuterated camptothecin derivative provided by the present application has excellent tumor inhibition biological activity, shorter in vivo half-life and faster in vivo clearance rate, and has better abdominal cavity and oral bioavailability, and has good in vivo anti-tumor biological activity, and has good clinical application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0032] Figure 1Growth inhibition of compound 1, 2 on cell lines HL-60, PC-3, CFPAC-1, SY-5Y, Raji, MDA-MB-231, Panc.08.13 and NCI-H1975.

[0033] Figure 2 Growth inhibition of compound 3, 4 on cell lines HL-60, Hela, MDA-MB-231 and SY-5Y.

[0034] Figure 3 Growth inhibition of compound 5-10 on cell lines NCI-H1975, U87-MG, U251 and A172.

[0035] Figure 4 Plasma concentration-time profiles of compound 3 or 4 in mice: (A) 2 mg / kg intravenous injection (IV); (B) 10 mg / kg intraperitoneal injection (IP); (C) and 10 mg / kg oral (PO); (D) 2 mg / kg intravenous injection (IV) plus formic acid to weakly acidic; (E) 10 mg / kg intraperitoneal injection (IP) plus formic acid to weakly acidic; (F) and 10 mg / kg oral (PO) plus formic acid to weakly acidic.

[0036] Figure 5 Tumor volume mean values of human non-small cell lung cancer NCI-H1975 nude mice transplanted tumors versus time (A, B) and mouse weight mean values versus time (C) after administration of compound 3 (TOP) and 4 (TOP-D2). DETAILED DESCRIPTION

[0037] The present application provides a deuterated camptothecin derivative, a preparation method and use thereof.

[0038] The inventors of the present application have found, through a large number of practice researches, that the deuterated camptothecin derivative can be prepared by directly deuterating the camptothecin nucleus, which is simple and effective, and the prepared deuterated camptothecin derivative has excellent tumor inhibition biological activity and a faster elimination half-life, and has better intraperitoneal and oral bioavailability, on the basis of which the present application is completed.

[0039] The present application provides a deuterated camptothecin derivative, a preparation method and use thereof.

[0040]

[0041] wherein R1 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2)n CH3, -F, -Cl, -Br or -I, n represents an integer of 1 to 3; R2 is selected from -H, -OH, -NH2, -F, -Cl, -Br, -I, -CH3, -OCH3, -(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -O(CH2) n CH3, -O(CH2) n NH2, -NH(CH2) n NH2, -(OCH2CH2) n NH2or wherein, n represents an integer of 1 to 3; or R1, R2 are connected to each other to form -(CH2) p -, -X(CH2) (p-1) -, -(CH2) (p-1) X-, -X(CH2) (q-1) X- or -(CH2) (q-1) X(CH2) (q-1) , p and q represent an integer of 2 to 4, X represents O, NH or S;

[0042] R3 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -O(CH2) n CH3, -NHCH3, -NH(CH2) n CH3, -(CH2) n Si(CH3)3, -(CH2) n N(CH3)2, -(CH2) n CH=CH2, -NO2, -F, -Cl, -Br or -I, n represents an integer of 1 to 3; R4 is selected from -H, -CH3, -(CH2) n CH3, -OCH3, -Si(CH3)2C(CH3)3, -(CH2) n NHCH, -(CH2) n Si(CH3)3, -(CH3)2, -CH2NH(CH2) n CH3, -CH2O(CH2) n CH3, -F, -Cl, -Br, -I, wherein, n represents an integer of 1 to 3; or R3 and R4 are connected to each other to form a structure as shown in the following formula:

[0043]

[0044] In some embodiments of the present application, R1is selected from -H, -F, -Cl, -Br, or -I; R2is selected from -H, -OH, -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3; R3is selected from -H, -CH2N(CH3)2, -(CH2)2CH3, -(CH2)3N(CH3)2, R4is selected from -H, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, or R3and R4are linked to each other to form a structure as shown in the following formula:

[0045]

[0046] The term "pharmaceutically acceptable salt" refers to pharmaceutically acceptable organic or inorganic salts of a compound (e.g., a drug, a drug-linker, or an antibody-linker drug conjugate). The compound can contain at least one amino, imino, hydroxy, or carboxyl group and thus be capable of forming a pharmaceutically acceptable addition salt with a corresponding acid or base. Exemplary salts include, but are not limited to, sulfate, trifluoroacetate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate (-H2PO4), phosphite, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, potassium, sodium, ammonium, calcium, and the like. Additionally, pharmaceutically acceptable salts have more than one charged atom in the structure. Examples where multiple charged atoms are part of a pharmaceutically acceptable salt can have multiple counterions. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counterions.

[0047] The term "solvate," "solvate," means the physical association of a compound of this application with one or more solvent molecules, either organic or inorganic. This physical association can include hydrogen bonding. In certain instances, the solvate will be capable of isolation, for example, when one or more solvent molecules are incorporated in the crystal lattice of the crystalline solid. The solvent molecules can be present in a defined and constant ratio. "Solvate" encompasses both solution-phase and isolatable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Methods of solvation are well known in the art.

[0048] In some embodiments of the present application, the deuterated camptothecin derivative is selected from the following chemical structural formula:

[0049]

[0050] The second aspect of the present application provides a method for preparing a deuterated camptothecin derivative, comprising the following steps: reacting a camptothecin derivative with a deuterated reagent under alkaline conditions to obtain a deuterated camptothecin derivative.

[0051] In the above reaction, the reaction is carried out in the presence of an organic solvent, which can generally be an aprotic solvent and is generally a good solvent for the reaction system. For example, the organic solvent used can be a halogenated alkane solvent, an ester solvent, a nitrile solvent, an aromatic solvent, etc. In a specific embodiment of the present application, the organic solvent used can be one or a combination of chloroform, deuterated chloroform, carbon tetrachloride, dichloromethane, acetonitrile, benzene, etc. For another example, the amount of solvent used in the reaction can be determined according to the concentration of the camptothecin derivative in the system. For example, the concentration of the camptothecin derivative in the reaction system can be 0.005-0.3 mmol / mL, specifically 0.005-0.01 mmol / mL, 0.01-0.02 mmol / mL, 0.02-0.03 mmol / mL, 0.03-0.05 mmol / mL, 0.05-0.1 mmol / mL, 0.1-0.2 mmol / mL, 0.2-0.3 mmol / mL. In some preferred embodiments of the present application, the concentration of the camptothecin derivative in the reaction system can be 0.05-0.1 mmol / mL.

[0052] In the above reaction, the alkaline conditions are in the presence of a strong base; the strong base can be an alkali metal hydride, an alkali metal hydroxide, sodium or potassium alcoholate, an organolithium compound, a bicyclic amino base, a guanidine base, etc., for example, one or a combination of sodium hydride, lithium hydride, potassium hydride, rubidium hydride, cesium hydride, sodium hydroxide, potassium hydroxide, 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU), 1,4-diazabicyclooctane (DABCO), sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, butyllithium, lithium diisopropylamide (LDA), sodium hexamethyldisilazide (NaHMDS), potassium hexamethyldisilazide (KHMDS), etc., preferably sodium hydride. The molar ratio of the strong base to the camptothecin derivative is 5:0.2-0.3. In some specific embodiments of the present application, the molar ratio of the strong base to the camptothecin derivative is 5:0.22, 5:0.23, 5:0.26, 5:0.27, 5:0.29.

[0053] In the above reaction, it can specifically include adding the strong alkali metal hydride to the organic solvent containing the camptothecin derivative under an ice water bath, then moving to room temperature for reaction, and then adding the deuterated reagent for quenching. Preferably, the reaction at room temperature is overnight.

[0054] In the above reaction, the reaction is usually carried out at room temperature or low temperature. The temperature of the reaction is -78-40℃, which can be -78--60℃, -60--40℃, -40--20℃, -20-0℃, 0-20℃, or 20-40℃. In some preferred embodiments of the present application, the reaction temperature is room temperature. The reaction time can be adjusted by those skilled in the art according to the reaction progress, which can be judged by methods such as TLC, chromatography, etc. In some preferred embodiments of the present application, the reaction time can be 0.5-72h.

[0055] In the above reaction, the deuterated reagent is selected from D2O, CD3OD or CD3COOD.

[0056] In the above reaction, those skilled in the art can choose a suitable method for post-treatment of the product of the reaction, for example, the post-treatment of the reaction can include acidification, filtration, purification, to provide a deuterated camptothecin derivative. The purpose of the acidification is to ensure that the reaction is completely terminated, remove excess basic substances, and facilitate the extraction of the product. The acidification reagent is selected from any one or several of hydrochloric acid, deuterated acetic acid, acetic acid, formic acid, etc.; in some specific embodiments of the present application, the acidification reagent is hydrochloric acid, and the concentration of the hydrochloric acid is preferably 1M. Suitable filtration methods should be known to those skilled in the art, for example, can be suction filtration, centrifugation. Suitable purification methods should be known to those skilled in the art, which can be high performance liquid chromatography (HPLC) and the like.

[0057] The third aspect of the present application provides the use of the above-mentioned deuterated camptothecin derivative in the preparation of a medicament for treating tumors. The tumors include but are not limited to liver cancer, kidney cancer, lung cancer, gastric cancer, esophageal cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, hematological tumor or glioblastoma, lymphoma, cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer.

[0058] The fourth aspect of the present application provides a pharmaceutical composition comprising the above-mentioned deuterated camptothecin derivative and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient can be one or more.

[0059] The term "pharmaceutically acceptable excipient" shall mean compatible with the effective ingredient, i.e. blendable therewith without substantially diminishing the effect of the drug under normal conditions. Specific examples of some of the substances which can be used as pharmaceutically acceptable excipients can be alcohols such as ethanol, propylene glycol, glycerol, sorbitol, mannitol and polyethylene glycol, etc.; alginic acid; emulsifiers such as Tween, etc.; wetting agents such as sodium lauryl sulfate, etc.; surfactants; lyophilization protecting agents; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic salt solutions; buffers; etc., and combinations thereof. These substances are used as necessary to improve the stability of the formulation or to help improve the activity or its bioavailability.

[0060] The specific embodiments of the present application are further described in the following working examples. When numerical ranges are given, it should be understood that every numerical value within the range is contemplated, unless otherwise indicated. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Except in the Examples, or where otherwise explicitly indicated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, exemplary methods and materials are described herein.

[0061] Example 1: Preparation of deuterated topotecan 2

[0062]

[0063] Topotecan hydrochloride (1, 100 mg, 0.22 mmol) was dissolved in 3 ml of chloroform, NaH (200 mg, 5.0 mmol) was added under ice water bath, and the reaction was allowed to proceed overnight at room temperature. The reaction was quenched with D2O or CD3OD (0.3 ml) and acidified with 1 M hydrochloric acid or deuterated acetic acid. The crude product was filtered and separated by HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O = 10-100%) and lyophilized to give yellow to light yellow solid deuterated topotecan 280.1 mg, yield 79%. ESI-HRMS Calcd for C 23 H 21 D2N3O5[M+H] + : 424.1763, Found: 424.1670. 1H NMR (500 MHz, DMSO) δ 11.58 (s, 1H), 10.08 (s, 1H), 9.01 (s, 1H), 8.10 (d, J = 9.1 Hz, 1H), 7.77 (d, J = 9.2 Hz, 1H), 7.25 (s, 1H), 5.40 (s, 2H), 4.72 (s, 2H), 2.85 (s, 6H), 1.89 (ddt, J = 9.4, 17.9 Hz, 2H), 0.90 (d, J = 7.6 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 172.97, 157.57, 157.25, 150.50, 149.79, 145.92, 143.74, 133.22, 130.47, 129.26, 127.42, 122.70, 118.91, 108.74, 96.67, 72.85, 65.69, 50.71, 42.82, 30.75, 8.24.

[0064] Example 2: Preparation of deuterated camptothecin 4

[0065]

[0066] Camptothecin (100 mg, 0.29 mmol) was dissolved in 3 ml chloroform, NaH (200 mg, 5 mmol) was added under ice water bath, and the mixture was moved to room temperature and stirred overnight. Then, D2O or CD3OD (0.3 ml) was added to quench the reaction, and 1 M HCl aqueous solution was added to acidify the mixture. The crude product was obtained by filtration, and separated by HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O = 10-100%) to obtain the crude product. The yellow to light yellow solid deuterated camptothecin 4 (85.4 mg, 0.24 mmol) was obtained by freeze-drying, with a yield of 85%. ESI-HRMS Calculated for C 23 H 21 D2N3O5[M+H] + : 350.1236, Found 351.1091. 1 H NMR (500 MHz, DMSO) δ 8.69 (s, 1H), 8.17 (d, J = 8.5 Hz, 1H), 8.12 (dd, J = 8.3, 1.4 Hz, 1H), 7.87 (ddd, J = 8.4, 6.7, 1.5 Hz, 1H), 7.71 (ddd, J = 8.1, 6.7, 1.2 Hz, 1H), 7.35 (s, 1H), 5.43 (s, 2H), 1.88 (ddt, J = 17.3, 14.1, 7.1 Hz, 2H), 0.89 (t, J = 7.3 Hz, 3H). 13C NMR (126 MHz, DMSO) δ 172.98, 157.31, 153.04, 150.48, 148.33, 145.95, 132.11, 130.88, 130.18, 129.45, 128.97, 128.39, 128.13, 119.55, 97.21, 72.85, 65.71, 30.70, 8.25.

[0067] Example 3: Preparation of deuterated 7-ethyl-10-hydroxy camptothecin 6

[0068]

[0069] Deuterated 7-ethyl-10-hydroxy camptothecin 6 (85.4 mg, 0.24 mmol), yield 85%, was obtained as a yellow to light yellow solid by dissolving 7-ethyl-10-hydroxy camptothecin (100 mg, 0.26 mmol) in 3 ml chloroform, adding NaH (200 mg, 5 mmol) under ice water bath, moving to room temperature for overnight, then quenching with 1 ml D2O or CD3OD (0.3 ml), dropping 1 M hydrochloric acid aqueous solution for acidification, filtering to obtain the crude product, separating the product by preparative HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O = 10-100%), and freeze-drying. ESI-HRMS Calculated for C 22 H 18 D2N2O5[M+H] + : 394.1498, Found 395.1318:. 1 HNMR (500 MHz, DMSO) δ 8.02 (d, J = 8.9 Hz, 1H), 7.46 - 7.36 (m, 2H), 7.24 (s, 1H), 5.42 (s, 2H), 3.08 (q, J = 7.6 Hz, 2H), 1.86 (dh, J = 21.3, 7.2 Hz, 2H), 1.30 (t, J = 7.6 Hz, 3H), 0.88 (t, J = 7.3 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 173.07, 157.21, 150.57, 149.35, 146.92, 144.06, 143.31132.01, 128.66, 128.39, 122.87, 118.50, 105.25, 96.29, 72.88, 65.71, 40.18, 30.65, 22.77, 13.85, 8.24.

[0070] Example 4: Preparation of deuterated 10-hydroxy camptothecin 8

[0071]

[0072] To a solution of 10-hydroxy camptothecin (100 mg, 0.27 mmol) in 3 ml of chloroform, NaH (200 mg, 5 mmol) was added under ice-water bath, and the mixture was allowed to react overnight at room temperature. Then, 1 ml of D2O or CD3OD was added to quench the reaction (0.3 ml), 1 M hydrochloric acid was added dropwise to acidify the solution, and the crude product was obtained by filtration. The product was separated by HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O = 10-100%) and freeze-dried to obtain deuterated 10-hydroxy camptothecin 8 (84.5 mg, 0.24 mmol) as a yellow to light yellow solid, with a yield of 86%. 1 HNMR (500 MHz, DMSO) δ 10.37 (s, 1H), 8.44 (s, 1H), 8.01 (d, J = 9.1 Hz, 1H), 7.42 (dd, J = 9.1, 2.7 Hz, 1H), 7.27 (d, J = 2.7 Hz, 1H), 7.26 (s, 1H), 6.52 (s, 1H), 5.41 (s, 2H), 1.99 - 1.70 (m, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13 C NMR (126 MHz, DMSO) δ 173.05, 157.35, 157.12, 150.56, 149.92, 146.40, 143.66, 131.10, 130.27, 130.12, 129.78, 123.50, 118.61, 109.25, 96.33, 72.88, 65.69, 30.66, 8.25. ESI-HRMS Calculated for C 20 H 14 D2N2O5[M+H] + : 366.1185, Found 367.1025.

[0073] Example 5: Preparation of deuterated exatecan 10

[0074]

[0075] To a solution of exatecan (100 mg, 0.23 mmol) in 3 ml of chloroform, NaH (200 mg, 5 mmol) was added under ice-water bath, and the mixture was allowed to react overnight at room temperature. Then, 1 ml of D2O or CD3OD was added to quench the reaction (0.3 ml), 1 M hydrochloric acid was added dropwise to acidify the solution, and the crude product was obtained by filtration. The product was separated by preparative HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O = 10-100%) and freeze-dried to obtain deuterated 1 exatecan 10 (82.4 mg, 0.24 mmol) as a yellow to light yellow solid, with a yield of 82%. 1H NMR (600 MHz, DMSO) δ 8.86 (s, 3H), 7.85 (d, J = 10.7 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.44 (d, J = 2.0 Hz, 2H), 5.08 (t, J = 3.2 Hz, 1H), 3.30 - 3.14 (m, 2H), 2.60 (ddt, J = 14.5, 5.3, 2.6 Hz, 1H), 2.41 (d, J = 1.8 Hz, 3H), 2.23 - 2.14 (m, 1H), 1.88 (dp, J = 21.4, 7.2 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). 13 C NMR (151 MHz, DMSO) δ 172.86, 163.03, 161.38, 157.21, 152.94, 150.49, 148.34, 145.50, 135.85, 134.94, 128.11, 125.24, 121.10, 119.99, 110.80, 97.44, 72.82, 65.72, 44.90, 30.76, 25.22, 21.40, 11.53, 8.26. ESI-HRMS Calculated for C 24 H 20 D2FN3O4[M+H] + :437, 1720, Found 438.1517.

[0076] Example 6: In vitro cell growth inhibition activity test

[0077] 1) Cell plating: PC3 (3000 cells / well), Panc 08.13 (3000 cells / well), Hela (3000 cells / well), CFPAC-1 (3000 cells / well), MDA-MB-231 (3000 cells / well), U87-MG (3000 cells / well), NCI-H1975 (3000 cells / well), U251 (3000 cells / well), A172 (3000 cells / well), SY-5Y (5000 cells / well), HL-60 (10000 cells / well), Raji (10000 cells / well), various cells were plated in 96-well plates and incubated in an incubator overnight for adherent growth.

[0078] 2) Drug incubation: Test compounds 1-10 were diluted in eight-row centrifuge tubes using DMSO in a 5-fold concentration gradient, with an initial test concentration of 50 μM, for a total of 8 concentration points. The compounds were diluted in eight-row centrifuge tubes using PBS solution in a 5-fold concentration gradient, with a blank control group (complete medium) and a solvent control group (adding an equal volume of solvent to dissolve the drug) set up. After the drug solution was added to the cells, it was shaken and placed in a cell incubator for 72 h.

[0079] 3) CCK8 detection: After 72 h, 10 μL of CCK8 color developing solution was added to each well, shaken evenly, and incubated in a 37°C incubator for 1-4 h. The absorbance at 450 nm was detected using an enzyme marker, and different cell incubation times were used for data analysis when the OD value reached 1.0-1.2. The calculation formula is as follows:

[0080] Cell growth inhibition rate (%) = (OD 溶媒对照 -OD 给药 ) / (OD 溶媒对照 -OD 空白 ) x 100%

[0081] 4) Data analysis: The experimental results were processed using Graphpadprism 8 software to draw a cell growth inhibition rate curve.

[0082] Figure 1 Growth inhibition of compounds 1, 2 on cell lines HL-60, PC-3, CFPAC-1, SY-5Y, Raji, MDA-MB-231, Panc.08.13, and NCI-H1975. Figure 2 Growth inhibition of compounds 3, 4 on cell lines HL-60, Hela, MDA-MB-231, and SY-5Y. Figure 3 Growth inhibition of compounds 5-10 on cell lines NCI-H1975, U87-MG, U251, and A172.

[0083] From the above results, it can be seen that the IC 50 values of the deuterated camptothecin derivatives (compounds 2, 4, 6, 8, 10) provided by embodiments 1-5 of the present application are similar to those of the non-deuterated camptothecin derivatives (compounds 1, 3, 5, 7, 9), and the inhibition of cell lines can be maintained.

[0084] Example 7: In vivo pharmacokinetic determination in rats

[0085] Rats were randomly divided into groups and pharmacokinetic studies were conducted using single oral, intravenous, and intraperitoneal administration. Plasma samples were precipitated with acetonitrile, and the supernatant of each sample was passed through the LC-MS method to detect the sample concentration in plasma. Pharmacokinetic analysis was performed and the following parameters were calculated: Tmax: time to maximum blood drug concentration; Cmax: maximum blood drug concentration; T 1 / 2 : elimination half-life; AUC: area under the curve, indicating the total exposure of the drug in the body; CL: clearance; Vd: apparent volume of distribution.

[0086] Figure 4 The plasma concentration-time curves of compound 3 (TOP) or 4 (TOP-2D) in mice after administration of the following doses: (A) 2 mg / kg intravenous (IV); (B) 10 mg / kg intraperitoneal (IP); (C) 10 mg / kg oral (PO); (D) 2 mg / kg intravenous (IV) with formic acid adjusted to a weak acidity; (E) 10 mg / kg intraperitoneal (IP) with formic acid adjusted to a weak acidity; (F) 10 mg / kg oral (PO) with formic acid adjusted to a weak acidity. The pharmacokinetics of compounds 3 and 4 in rats (2 mg / kg IV, 10 mg / kg intraperitoneal, and 10 mg / kg oral) are shown in Tables 1-3 below.

[0087] Table 1 Pharmacokinetics of compounds 3 and 4 in rats (2 mg / kg intravenous injection)

[0088]

[0089] Table 2 Pharmacokinetics of compounds 3 and 4 in rats (10 mg / kg intraperitoneal injection)

[0090]

[0091]

[0092] Table 3 Pharmacokinetics of Compounds 3 and 4 in rats (10 mg / kg oral administration)

[0093]

[0094] Depend on Figure 4 As shown in the results of Tables 1 to 3, compared with the non-deuterated camptothecin derivative (Compound 3), the deuterated camptothecin derivative (Compound 4) provided by the present invention has a shorter in vivo half-life and a faster in vivo clearance rate, and has better peritoneal and oral bioavailability.

[0095] Example 8: Antitumor bioactivity assay in mice

[0096] 1) Nanjing Jiquy Pharmaceutical Kang purchased SPF level 5-week-old BALB / c male mice, placed in the national protein center SPF level animal room, checked the gender and body of the mice, and measured the body weight of the mice, and confirmed that the mice were in good condition after 5-7 days of adaptation.

[0097] 2) Large-scale culture of NCI-H1975 using T125 cell culture bottles, when the number of cells reaches the budget, trypsin digestion is collected in a 50 mL centrifuge tube, and the cells are washed once with PBS. Take out the frozen ABW matrix glue in advance and melt it in the 4°C refrigerator overnight, dilute the ABW matrix glue with PBS according to the ratio of 1:2. Mix the diluted matrix glue solution with the collected cell precipitate gently, and place it on ice, taking care to avoid solidification at room temperature.

[0098] 3) Alcohol cotton ball disinfection of the mouse inoculation site, 2*10^6 mycoplasma-free NCI-H1975 cells were subcutaneously injected into the right axillary of the mouse using a 1 mL sterile syringe, and the average tumor volume reached 150mm 3 on the 10th day after inoculation, and mice with similar tumor volume were selected for experimental grouping and drug treatment.

[0099] 4) Experimental design grouping and drug administration (n=5) PBS control group, 1 (po.), 2 (po.), 1 (ip.), 2 (ip.) (po. Drug concentration 1 mg / kg; ip. Drug concentration 0.6 mg / kg).

[0100] 5) Drug treatment of mice by gavage and intraperitoneal injection, once a day for 15 consecutive days, vernier caliper measurement of tumor volume, body weight measurement of mice, daily detection, and continuous detection until the end of the experiment. Tumor volume calculation method: tumor volume = length * width * width / 2.

[0101] 6) When the tumor volume of the control group reached 1500mm 3 , the experimental mice were uniformly sacrificed, the tumors of the mice in each group were dissected, the tumor weight was recorded, and the tumor size comparison was photographed and saved.

[0102] Figure 5 The average tumor volume of human non-small cell lung cancer NCI-H1975 nude mouse transplanted tumor after administration of compounds 3 (TOP) and 4 (TOP-D2) versus time (A, B), and the average mouse body weight versus time (C).

[0103] From Figure 5The results show that the compounds 3 and 4 have good in-vivo anti-tumor biological activity; the deuterated camptothecin derivative (compound 4) provided by the application can keep similar in-vivo anti-tumor biological activity compared with the non-deuterated camptothecin derivative (compound 3), and importantly, can significantly reduce toxic side effects, greatly reduce the influence of the drug on the weight of mice.

[0104] In summary, the present application effectively overcomes the shortcomings in the prior art and has high industrial utilization value.

[0105] The above examples only exemplarily illustrate the principles and effects of the present application, and are not used to limit the present application. Any person skilled in the art can modify or change the above examples without departing from the spirit and scope of the present application. Therefore, all equivalent modifications or changes completed by those skilled in the art without departing from the spirit and technical thought disclosed by the present application should be covered by the claims of the present application.

Claims

1. A deuterated camptothecin derivative, characterized by, The deuterated camptothecin derivative is a compound shown in formula 4, a pharmaceutically acceptable salt thereof: 。 2. A method for preparing a deuterated camptothecin derivative, characterized by, The method comprises the following steps: reacting a camptothecin derivative with a deuterated reagent under alkaline conditions to obtain a deuterated camptothecin derivative, wherein the deuterated camptothecin derivative has a structural formula shown in formula 4 。 3. The production method according to claim 2, wherein One or more of the following features are included: (1) the reaction is carried out in the presence of an organic solvent; (2) the alkaline condition is in the presence of a strong base; (3) the reaction temperature is -78-40 ℃; (4) the reaction time is 0.5-72 h; (5) the deuterated reagent is selected from D2O, CD3OD or CD3COOD; (6) the post-reaction treatment comprises acidification, filtration and purification.

4. The production method according to claim 3, wherein One or more of the following features are included: (1a) the organic solvent is selected from any one or more of chloroform, deuterated chloroform, carbon tetrachloride, dichloromethane, acetonitrile, benzene; (2a) the strong base is selected from any one or more of sodium hydride, lithium hydride, potassium hydride, rubidium hydride, cesium hydride, sodium hydroxide, potassium hydroxide, 1,8-diazabicyclo[5.4.0]undec-7-ene, 1,4-diazabicyclooctane, sodium methoxide, potassium tert-butoxide, sodium tert-butoxide, butyllithium, lithium diisopropylamide, sodium hexamethyldisilylamide, potassium hexamethyldisilylamide; (2b) the molar ratio of the strong base to the camptothecin derivative is 5:0.2-0.3; (2c) the strong base is added to the organic solvent in which the camptothecin derivative is dissolved under an ice-water bath, and then the reaction is carried out at room temperature, followed by the addition of the deuterated reagent for quenching; (6a) the acidification reagent is selected from any one or more of hydrochloric acid, deuterated acetic acid, acetic acid, formic acid; (6b) the purification method is high performance liquid chromatography.

5. Use of the deuterated camptothecin derivative of claim 1 or the deuterated camptothecin derivative prepared by the method of any one of claims 2-4 in the preparation of a medicament for treating tumors; the tumors are breast cancer, lymphoma, cervical cancer, glioma, neuroblastoma, leukemia.

6. A pharmaceutical composition comprising the deuterated camptothecin derivative of claim 1 or the deuterated camptothecin derivative prepared by the method of any one of claims 2-4, a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

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