Deuterated camptothecin derivative as well as preparation method and application thereof

Abstract

The invention relates to the technical field of medicines, and mainly relates to a deuterated camptothecin derivative as well as a preparation method and application thereof. The deuterated camptothecin derivative is a compound as shown in a formula I or a formula II, and pharmaceutically acceptable salt or solvate of the deuterated camptothecin derivative is prepared by directly performing deuteration on a camptothecin mother nucleus, and the preparation method is simple, convenient and effective; and the deuterated camptothecin derivative provided by the invention has excellent tumor inhibition biological activity and relatively fast elimination half-life period, has better abdominal cavity and oral bioavailability, and has a good clinical application prospect.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, in particular to a deuterated camptothecin derivative and a preparation method and use thereof. Background Art

[0002] Camptothecin (CPT) is a natural product originally derived from the plant Camptotheca acuminata, and has significant anti-tumor biological activity. It is a topoisomerase I (TOP I) inhibitor that exerts its anti-tumor effect by inhibiting the formation of a ternary complex between TOP1 and DNA. CPT has a broad-spectrum anti-tumor effect and can show significant biological activity against a variety of tumor types. CPT derivatives irinotecan and topotecan have been approved by the 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 received great attention. ADCs connect pharmacodynamic molecules to antibodies through a chemical link, and use the high targeting of antibodies to accurately transport pharmacodynamic molecules to target cells. In this way, on the one hand, the toxicity of drug molecules 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 just improve the problems faced by CPT direct drug development, so CPT compounds are a very promising class of molecules that can be used as payloads for ADCs. ADCs strastuzumab deruxtecan (DS8201, Enhertu) and sacituzumab govitecan (IMMU-132, Trodelvy) use the CPT compound irinotecan derivatives Dxd and SN38 (irinotecan active metabolite) as payloads, respectively, and have been approved for marketing. CPT derivatives have demonstrated their great potential for direct use as small molecule drugs or as payload molecules in ADCs.

[0004] Deuterium-substituted CPT derivatives have also received increasing attention in recent years. Deuterium (2H, D) is a stable isotope of hydrogen, and the carbon-deuterium bond is more stable than the carbon-hydrogen bond. Deuterium substitution can make the metabolism of drugs slower, thereby extending the half-life of drugs, reducing the clearance rate of drugs, and reducing the toxicity of drugs. Deuterium-substituted CPT derivatives have been reported in many patent documents. Chinese patent document CN117105948A introduces deuterium on the lactone ring of camptothecin by means of total synthesis. This method has a complex preparation process and high cost. Chinese patent documents CN113943310A and CN118164994A introduce deuterium on the side chain and combine it with the linker to synthesize ADCs. This method only performs deuterium modification on the side chain. Summary of the invention

[0005] In view of the problems in the prior art, the purpose of the present invention is to provide a deuterated camptothecin derivative and its preparation method and use, wherein deuteration is directly carried out on the camptothecin parent nucleus, the preparation method is simple and effective, and it has excellent tumor inhibitory biological activity, a shorter half-life and a faster clearance rate, and at the same time has better peritoneal and oral bioavailability.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] The first aspect of the present invention provides a deuterated camptothecin derivative, wherein the deuterated camptothecin derivative is a compound represented by Formula I or Formula II, or 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 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 of 2 to 4, and X represents O, NH or S;

[0010] 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:

[0011]

[0012] In some embodiments of the present invention, R1 is selected from -H, -F, -Cl, -Br or -I; R2 is selected from -H, -OH, -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3; R3 is selected from -H, -CH2N(CH3)2, -(CH2)2CH3, -(CH2)3N(CH3)2, R4 is selected from -H, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, or R3 and R4 are connected to each other to form a structure as shown in the following formula:

[0013]

[0014] In some embodiments of the present invention, the deuterated camptothecin derivative is selected from the compounds represented by the following chemical formulas, and pharmaceutically acceptable salts or solvates thereof:

[0015]

[0016] The 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 of 2 to 4, and 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 to 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 invention, the reaction is carried out in the presence of an organic solvent.

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

[0024] In some embodiments of the present invention, the reaction temperature is -78 to 40°C.

[0025] In some embodiments of the present invention, the reaction time is 0.5 to 72 hours.

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

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

[0028] The third aspect of the present invention provides the use of the above-mentioned deuterated camptothecin derivatives in the preparation of drugs for treating tumors.

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

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

[0031] The present invention provides a method for preparing deuterated camptothecin derivatives by directly performing deuteration on a camptothecin parent nucleus, which is simple and effective. The deuterated camptothecin derivatives provided by the present invention have excellent tumor suppressive biological activity, a shorter in vivo half-life and a faster in vivo clearance rate, and at the same time have better peritoneal and oral bioavailability, and have good in vivo anti-tumor biological activity, and have good clinical application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1The growth inhibitory effects of compounds 1 and 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 The growth inhibitory effects of compounds 3 and 4 on cell lines HL-60, Hela, MDA-MB-231 and SY-5Y.

[0034] Figure 3 The growth inhibitory effect of compound 5-10 on cell lines NCI-H1975, U87-MG, U251 and A172.

[0035] Figure 4 The plasma concentration-time curves after administration 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 administration (PO); (D) 2 mg / kg intravenous injection (IV) plus formic acid adjusted to weak acidity; (E) 10 mg / kg intraperitoneal injection (IP) plus formic acid adjusted to weak acidity; (F) and 10 mg / kg oral administration (PO) plus formic acid adjusted to weak acidity.

[0036] Figure 5 The graphs of mean tumor volume changes over time in nude mice transplanted with human non-small cell lung cancer NCI-H1975 after administration of compounds 3 (TOP) and 4 (TOP-D2) (A, B) and mean mouse body weight changes over time (C). DETAILED DESCRIPTION

[0037] The deuterated camptothecin derivatives of the present invention and their preparation methods and uses are described in detail below.

[0038] After extensive practical research, the inventors of the present invention have discovered that deuterated camptothecin derivatives can be prepared by directly performing deuteration on the camptothecin parent nucleus, which is simple and effective. Experiments have also verified that the prepared deuterated camptothecin derivatives have excellent tumor inhibitory biological activity and a faster elimination half-life, as well as better peritoneal and oral bioavailability. The present invention was completed on this basis.

[0039] The first aspect of the present invention provides a deuterated camptothecin derivative, wherein the deuterated camptothecin derivative is a compound represented by Formula I or Formula II, or a pharmaceutically acceptable salt or solvate 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 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 of 2 to 4, and 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 invention, R1 is selected from -H, -F, -Cl, -Br or -I; R2 is selected from -H, -OH, -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3; R3 is selected from -H, -CH2N(CH3)2, -(CH2)2CH3, -(CH2)3N(CH3)2, R4 is selected from -H, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, or R3 and R4 are connected to each other to form a structure as shown in the following formula:

[0045]

[0046] The term "pharmaceutically acceptable salt" refers to a pharmaceutically acceptable organic or inorganic salt of a compound (e.g., a drug, a drug-linker or an antibody-linker drug conjugate). The compound may contain at least one amino, imino, hydroxyl or carboxyl group, and thus may form an addition salt with the corresponding acid or base. Exemplary salts include, but are not limited to, sulfates, trifluoroacetates, citrates, acetates, oxalates, hydrochlorides, hydrobromides, hydroiodides, nitrates, bisulfates, phosphates, acid phosphates (-H2PO4) salts, phosphites, isonicotinates, lactates, salicylates, acid citrates, tartrates, oleates, tannates, pantothenates, bitartrates, ascorbates, formates, benzoates, glutamates, methanesulfonates, ethanesulfonates, benzenesulfonates, p-toluenesulfonates, potassium salts, sodium salts, ammonium salts, calcium salts, etc. In addition, 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 counter atoms. For example, a pharmaceutically acceptable salt has one or more charged atoms and / or one or more counter atoms.

[0047] The terms "solvate" and "solvate" mean a physical association of a compound of the invention with one or more solvent molecules, whether organic or inorganic. The physical association includes hydrogen bonding. In certain cases, for example, when one or more solvent molecules are incorporated into the crystal lattice of a crystalline solid, the solvate will be able to be separated. The solvent molecules in the solvate may exist in a regular arrangement and / or a disordered arrangement. The solvate may contain stoichiometric or non-stoichiometric amounts of solvent molecules. "Solvate" encompasses solution phase and separable solvates. Exemplary solvates include, but are not limited to, hydrates, ethanolates, methanolates, and isopropanolates. Solvation methods are well known in the art.

[0048] In some embodiments of the present invention, the deuterated camptothecin derivative is selected from the compounds represented by the following chemical formulas, or pharmaceutically acceptable salts or solvates thereof:

[0049]

[0050] The 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.

[0051] In the above reaction, the reaction is carried out in the presence of an organic solvent, and the organic solvent used can generally be an aprotic solvent, and is generally a good solvent for the reaction system. For example, the organic solvent used can specifically be a halogenated alkane solvent, an ester solvent, a nitrile solvent, an aromatic solvent, etc. In a specific embodiment of the present invention, the organic solvent used can be a combination of one or more of chloroform, deuterated chloroform, carbon tetrachloride, dichloromethane, acetonitrile, benzene, etc. For another example, the amount of solvent used in the reaction can refer 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 invention, 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 condition is 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 organic lithium compound, a bicyclic amino base, a guanidine base, etc., for example, 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, butyl lithium, 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 invention, the molar ratio of the strong base to the camptothecin derivative is 5:0.22, 5:0.23, 5:0.26, 5:0.27, or 5:0.29.

[0053] In the above reaction, the strong alkali metal hydride may be added to an organic solvent containing a camptothecin derivative under an ice-water bath, and then the reaction is carried out at room temperature, followed by quenching by adding a deuterated reagent. Preferably, the reaction is carried out at room temperature overnight.

[0054] In the above reaction, the reaction is usually carried out at room temperature or low temperature. The reaction temperature is -78 to 40°C, and can be -78 to -60°C, -60 to -40°C, -40 to -20°C, -20 to 0°C, 0 to 20°C, or 20 to 40°C. In some preferred embodiments of the present invention, the reaction temperature is room temperature. Those skilled in the art can adjust the reaction time according to the progress of the reaction. For example, the progress of the reaction can be determined by TLC, chromatography and the like. In some preferred embodiments of the present invention, the reaction time of the reaction can be 0.5 to 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 to post-treat the product of the reaction. For example, the post-treatment of the reaction may 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 alkaline substances, and facilitate the extraction of products. The acidified reagent is selected from any one or more of hydrochloric acid, deuterated acetic acid, acetic acid, formic acid, etc.; in some specific embodiments of the present invention, the acidified reagent is hydrochloric acid, and the concentration of hydrochloric acid is preferably 1M. Suitable filtering methods should be known to those skilled in the art, for example, it can be suction filtration, centrifugation. Suitable purification methods should be known to those skilled in the art, preferably, it can be methods such as high performance liquid chromatography (HPLC).

[0057] The third aspect of the present invention provides the use of the above-mentioned deuterated camptothecin derivatives in the preparation of drugs 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, blood tumors or multiform glioma, 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 invention provides a pharmaceutical composition, comprising the above-mentioned deuterated camptothecin derivative and a pharmaceutically acceptable excipient. The pharmaceutically acceptable excipient may be one or more.

[0059] The term "pharmaceutically acceptable excipients" should be compatible with the active ingredient, that is, they can be mixed with it without significantly reducing the effect of the drug under normal circumstances. Specific examples of some substances that 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; freeze-drying protectants; stabilizers; diluents; excipients; antioxidants; preservatives; pyrogen-free water; isotonic saline solution; buffer solutions, etc., and combinations thereof. These substances are used to improve the stability of the formulation or help improve the activity or its biological effectiveness as needed.

[0060] The specific embodiments of the present invention are further described in detail below in conjunction with preferred embodiments. When the embodiments provide numerical ranges, it should be understood that, unless otherwise specified in the present invention, the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as those generally understood by those skilled in the art. In addition to the specific methods, equipment, and materials used in the embodiments, as those skilled in the art grasp the prior art and record the present invention, any methods, equipment, and materials of the prior art similar or equivalent to the methods, equipment, and materials described in the embodiments of the present invention can also be used to realize the present invention.

[0061] Example 1: Preparation of deuterated topotecan 2

[0062]

[0063] Dissolve topotecan hydrochloride (1,100 mg, 0.22 mmol) in 3 ml chloroform, add NaH (200 mg, 5.0 mmol) in an ice-water bath, move to room temperature for overnight reaction, then quench with D2O or CD3OD (0.3 ml), drop 1M hydrochloric acid or deuterated acetic acid for acidification, filter to obtain the crude product, separate by HPLC (stationary phase is C-18 silica gel column, mobile phase is CH3CN / H2O=10~100%), freeze-dry to obtain yellow to light yellow solid deuterated topotecan 280.1 mg, yield 79%. ESI-HRMSCalculated for C 23 H 21 D2N3O5[M+H] + :424.1763,Found:424.1670. 1H NMR (500MHz, DMSO) δ11.58(s,1H),10.08(s,1H),9.01(s,1H),8.10(d,J=9.1Hz,1H),7.77(d,J=9.2Hz,1H) ,7.25(s,1H),5.40(s,2H),4.72(s,2H),2.85(s,6H),1.89(dd,J=9.4,17.9Hz,2H),0.90(d,J=7.6Hz,3H). 13 C NMR (126MHz, DMSO) δ172.97,157.57,157.25,150.50,149.79,145.92,143.74,133.22,130.4 7,129.26,127.42,122.70,118.91,108.74,96.67,72.85,65.69,50.71,42.82,30.75,8.24.

[0064] Example 2: Preparation of deuterated camptothecin 4

[0065]

[0066] Camptothecin (100 mg, 0.29 mmol) was dissolved in 3 ml of chloroform, and NaH (200 mg, 5 mmol) was added under an ice-water bath, and the mixture was moved to room temperature for overnight reaction, and then quenched with D2O or CD3OD (0.3 ml), and 1 M hydrochloric acid was added dropwise for acidification, and the crude product was filtered to obtain a crude product, which was separated by HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O=10-100%), and freeze-dried to obtain yellow to light yellow solid deuterated camptothecin 4 (85.4 mg, 0.24 mmol), with a yield of 85%. ESI-HRMS Calculated for C 23 H 21 D2N3O5[M+H] + :350.1236,Found 351.1091. 1 H NMR (500MHz, DMSO) δ8.69(s,1H),8.17(d,J=8.5Hz,1H),8.12(dd,J=8.3,1.4Hz,1H),7.87(ddd,J=8.4,6.7,1.5Hz,1H), 7.71(ddd,J=8.1,6.7,1.2Hz,1H),7.35(s,1H),5.43(s,2H),1.88(ddt,J=17.3,14.1,7.1Hz,2H),0.89(t,J=7.3Hz,3H). 13C NMR (126MHz, DMSO) δ172.98,157.31,153.04,150.48,148.33,145.95,132.11,130.88 ,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-hydroxycamptothecin 6

[0068]

[0069] 7-Ethyl-10-hydroxycamptothecin (100 mg, 0.26 mmol) was dissolved in 3 ml of chloroform, and NaH (200 mg, 5 mmol) was added under an ice-water bath, and the mixture was moved to room temperature for reaction overnight, and then quenched with 1 ml of D2O or CD3OD (0.3 ml), and 1 M hydrochloric acid was added dropwise for acidification, and the crude product was filtered to obtain the product, which was separated by preparative HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O=10-100%), and freeze-dried to obtain yellow to light yellow solid deuterated camptothecin 6 (85.4 mg, 0.24 mmol), with a yield of 85%. ESI-HRMS Calculated for C 22 H 18 D2N2O5[M+H] + :394.1498,Found 395.1318:. 1 HNMR(500MHz,DMSO)δ8.02(d,J=8.9Hz,1H),7.46–7.36(m,2H),7.24(s,1H),5.42(s,2H),3.0 8(q,J=7.6Hz,2H), 1.86(dh,J=21.3,7.2Hz,2H), 1.30(t,J=7.6Hz,3H), 0.88(t,J=7.3Hz,3H). 13 C NMR (126MHz, DMSO) δ173.07,157.21,150.57,149.35,146.92,144.06,143.31132.01,128.6 6,128.39,122.87,118.50,105.25,96.29,72.88,65.71,40.18,30.65,22.77,13.85,8.24.

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

[0071]

[0072] 10-Hydroxycamptothecin (100 mg, 0.27 mmol) was dissolved in 3 ml of chloroform, NaH (200 mg, 5 mmol) was added under an ice-water bath, and the mixture was moved to room temperature for reaction overnight, followed by quenching with 1 ml of D2O or CD3OD (0.3 ml), acidification by dropping 1 M hydrochloric acid water, and the crude product was filtered, and the product was separated by HPLC (the stationary phase was a C-18 silica gel column, and the mobile phase was CH3CN / H2O=10-100%), and freeze-dried to obtain yellow to light yellow solid deuterated 10-hydroxycamptothecin 8 (84.5 mg, 0.24 mmol), with a yield of 86%. 1 HNMR(500MHz,DMSO)δ10.37(s,1H),8.44(s,1H),8.01(d,J=9.1Hz,1H),7.42(dd,J=9.1,2.7Hz,1H),7 .27(d,J=2.7Hz,1H),7.26(s,1H),6.52(s,1H),5.41(s,2H),1.99–1.70(m,2H),0.88(t,J=7.3Hz,3H). 13 C NMR (126MHz, DMSO) δ173.05,157.35,157.12,150.56,149.92,146.40,143.66,131.10,130 .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,Found367.1025.

[0073] Example 5: Preparation of deuterated exitecan 10

[0074]

[0075] Dissolve exitecan (100 mg, 0.23 mmol) in 3 ml of chloroform, add NaH (200 mg, 5 mmol) under an ice-water bath, move to room temperature to react overnight, then quench with 1 ml of D2O or CD3OD (0.3 ml), add 1 M hydrochloric acid water to acidify, filter to obtain the crude product, and separate the product by preparative HPLC (stationary phase: C-18 silica gel column, mobile phase: CH3CN / H2O=10-100%), and freeze-dry to obtain yellow to light yellow solid deuterated 1-exitecan 10 (82.4 mg, 0.24 mmol), with a yield of 82%. 1H NMR (600MHz, DMSO) δ8.86(s,3H),7.85(d,J=10.7Hz,1H),7.33(s,1H),6.55(s,1H),5.44(d,J=2.0Hz,2H),5.08(t,J=3.2Hz,1H),3.30–3.1 4(m,2H),2.60(ddt,J=14.5,5.3,2.6Hz,1H),2.41(d,J=1.8Hz,3H),2.23–2.14(m,1H),1.88(dp,J=21.4,7.2Hz,2H),0.88(t,J=7.3Hz,3H). 13 C NMR (151MHz, DMSO) δ172.86,163.03,161.38,157.21,152.94,150.49,148.34,145.50,135.85,134.94,128. 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,Found438.1517.

[0076] Example 6: In vitro cell growth inhibitory 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, cultured in an incubator overnight, and grown by adherence.

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

[0079] 3) CCK8 detection: After 72 hours, add 10 μL CCK8 colorimetric solution to each well, shake evenly, incubate at 37°C incubator for 1-4 hours, and use a microplate reader to detect the absorbance at 450nm. Different cells have different incubation times, and data analysis is performed when the OD value reaches 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 )×100%

[0081] 4) Data analysis: Graphpadprism 8 software was used to process the experimental results and draw the cell growth inhibition rate curve.

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

[0083] From the above results, it can be seen that compared with the non-deuterated camptothecin derivatives (compounds 1, 3, 5, 7, 9), the IC 50 The values ​​are similar and can maintain the inhibitory effect on the cell lines.

[0084] Example 7: Pharmacokinetics in rats

[0085] Rats were randomly divided into groups and pharmacokinetic studies were conducted by single oral administration, single intravenous injection, and single intraperitoneal injection. 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 for pharmacokinetic analysis, and the following parameters were calculated: Tmax: time to reach 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 rate; Vd: apparent volume of distribution.

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

[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 biological activity assay in mice

[0096] 1) Nanjing Jicui Pharmaceutical purchased 5-week-old SPF-grade BALB / c male mice and placed them in the SPF-grade animal room of the National Protein Center. The gender and physical health of the mice were checked and the weight of the mice was measured. After confirming that the mice were in good condition, they were allowed to acclimate to the environment for 5-7 days.

[0097] 2) Use T125 cell culture flasks to culture NCI-H1975 in large quantities. When the number of cells reaches the budget, digest with trypsin and collect in 50mL centrifuge tubes. Wash the cells once with PBS. Take out the frozen ABW matrix gel in advance and thaw it in a 4℃ refrigerator overnight. Dilute the ABW matrix gel and PBS in a ratio of 1:2. Gently mix the diluted matrix gel solution with the collected cell pellet and place on ice. Be careful to avoid solidification at room temperature.

[0098] 3) Wipe the inoculation site of the mouse with an alcohol cotton ball for disinfection, and use a 1 mL sterile syringe to subcutaneously inoculate 2*10^6 NCI-H1975 cells without mycoplasma contamination under the right armpit of the mouse. The average tumor volume reaches 150mm on the 10th day after inoculation. 3 , select mice with similar tumor sizes, design experimental groups and perform drug treatment.

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

[0100] 5) The mice were treated with drugs by oral gavage and intraperitoneal injection, once a day for 15 consecutive days, and the tumor volume was measured with a vernier caliper and the weight of the mice was measured with a body weight scale, and the test was performed once a day 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 At the same time, the experimental mice were uniformly killed, and the tumors of mice in each group were dissected out and the tumor weights were weighed and recorded using a balance. The tumor sizes were compared and photographed for preservation.

[0102] Figure 5 The graphs of mean tumor volume changes over time in nude mice transplanted with human non-small cell lung cancer NCI-H1975 after administration of compounds 3 (TOP) and 4 (TOP-D2) (A, B) and mean mouse body weight changes over time (C).

[0103] Depend on Figure 5The results show that compounds 3 and 4 have good in vivo anti-tumor biological activity; compared with the non-deuterated camptothecin derivative (compound 3), the deuterated camptothecin derivative (compound 4) provided by the present invention can maintain similar in vivo anti-tumor biological activity, and more importantly, can significantly reduce toxic side effects and greatly reduce the effect of the drug on mouse body weight.

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

[0105] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. A deuterated camptothecin derivative, characterized in that: The deuterated camptothecin derivative is a compound represented by Formula I or Formula II, or a pharmaceutically acceptable salt or solvate thereof: 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 of 2 to 4, X represents O, NH or S; 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:

2. The deuterated camptothecin derivative according to claim 1, characterized in that R1 is selected from -H, -F, -Cl, -Br or -I; R2 is selected from -H, -OH, -CH3, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3; R3 is selected from -H, -CH2N(CH3)2, -(CH2)2CH3, -(CH2)3N(CH3)2, R4 is selected from -H, -CH2CH3, -(CH2)2CH3, -(CH2)3CH3, or R3 and R4 are connected to each other to form a structure shown in the following formula:

3. The deuterated camptothecin derivative according to claim 1 or 2, characterized in that The deuterated camptothecin derivative is selected from the compounds represented by the following chemical formulas, and pharmaceutically acceptable salts or solvates thereof:

4. A method for preparing a deuterated camptothecin derivative, characterized in that: The method comprises the following steps: reacting a camptothecin derivative with a deuterated reagent under alkaline conditions to obtain a deuterated camptothecin derivative.

5. The preparation method according to claim 4, characterized in that: The chemical structure of the camptothecin derivative is shown in the following formula III: 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 of 2 to 4, and X represents O, NH or S; 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:

6. The preparation method according to claim 4, characterized in that: Includes one or more of the following features: (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 to 40°C; (4) The reaction time is 0.5 to 72 hours; (5) The deuterated reagent is selected from D2O, CD3OD or CD3COOD; (6) Post-reaction treatment includes acidification, filtration and purification.

7. The preparation method according to claim 6, characterized in that: Includes one or more of the following features: (1a) The organic solvent is selected from any one or more of chloroform, deuterated chloroform, carbon tetrachloride, dichloromethane, acetonitrile and 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, butyl lithium, lithium diisopropylamide, sodium hexamethyldisilazide, and potassium hexamethyldisilazide; (2b) The molar ratio of the strong base to the camptothecin derivative is 5:0.2-0.3; (2c) adding the strong base to an organic solvent in which a camptothecin derivative is dissolved in an ice-water bath, then moving the mixture to room temperature for reaction, and then adding a deuterated reagent for quenching; (6a) The acidifying agent is selected from any one or more of hydrochloric acid, deuterated acetic acid, acetic acid, and formic acid; (6b) The purification method is high performance liquid chromatography.

8. Use of the deuterated camptothecin derivative according to any one of claims 1 to 3 or the deuterated camptothecin derivative prepared by the preparation method according to any one of claims 4 to 7 in the preparation of a drug for treating tumors.

9. The use according to claim 8, characterized in that The tumors include liver cancer, kidney cancer, lung cancer, stomach cancer, esophageal cancer, urethral cancer, bladder cancer, colon cancer, rectal cancer, prostate cancer, breast cancer, ovarian cancer, pancreatic cancer, melanoma, blood tumors or multiforme glioma, lymphoma, cervical cancer, uterine cancer, endometrial cancer, salivary gland cancer, glioma, neuroblastoma, sarcoma, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer.

10. A pharmaceutical composition comprising the deuterated camptothecin derivative according to any one of claims 1 to 3 or the deuterated camptothecin derivative prepared by the preparation method according to any one of claims 4 to 7, a pharmaceutically acceptable salt or solvate thereof, and a pharmaceutically acceptable excipient.

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