WO2024227432A1 - Camptothecin derivative, method for preparing same and use thereof, antibody-drug conjugate, and use thereof - Google Patents

Abstract

The present invention relates to a camptothecin derivative, a method for preparing same and use thereof, an antibody-drug conjugate, and use thereof. The camptothecin derivative is a compound represented by general formula I, or a tautomer, a stereoisomer, a polymorph, a nitrogen oxide or an isotope-labeled form thereof. The camptothecin derivative and the antibody-drug conjugate of the present invention have good in-vitro cell-killing activity and a superior anti-tumor effect.

Description

Camptothecin derivatives, preparation methods and uses thereof, antibody-drug conjugates and applications thereof Technical Field

The present invention relates to a camptothecin derivative with a novel structure, a preparation method and use thereof, an antibody-drug conjugate and application thereof.

Background Art

Camptothecin (CPT) is an alkaloid extracted from the plant Camptotheca acuminata in the Davidiaceae family in the mid-1950s. It is a selective inhibitor of topoisomerase I (TopⅠ), which forms a stable ternary complex with topoisomerase I and cellular DNA through hydrogen bonding, making it impossible for the broken DNA chains to reconnect, thus leading to DNA damage and cell apoptosis (Am J Cancer Res 2017; 7(12): 2350-2394).

Although camptothecin has a wide range of therapeutic applications, its efficacy is limited by its extremely low water solubility. Since its discovery, researchers have synthesized many camptothecin derivatives with stronger water solubility and conducted anti-tumor biological evaluation studies. Currently, three camptothecin drugs have been approved for marketing, including irinotecan for colorectal cancer, topotecan for ovarian cancer, and belotecan for ovarian cancer and small cell lung cancer. In addition, other camptothecin derivatives under development include Exatecan, Rubitecan, Chimmitecan, Karenitecan and Diflomotecan.

Most camptothecin drugs or their derivatives cause blood toxicity, as well as adverse reactions to the digestive system and urinary system. In clinical studies, in addition to improving their pharmacokinetic properties, regulating activity or reducing dosage, methods to improve the safety and effectiveness of camptothecin compounds also include forming antibody-drug conjugates with antibodies. For example, Exatecan has significant gastrointestinal toxicity and bone marrow toxicity, while the derivative DXd based on Exatecan has similar topoisomerase I inhibition, but lower bone marrow toxicity. On this basis, the DXd-based anti-HER2 antibody-drug conjugate DS8201 (Enhertu) was further developed. There is still a high clinical demand for anti-tumor drugs to develop new camptothecin derivatives and corresponding antibody-drug conjugates, and to further improve effectiveness and improve safety.

Summary of the invention

The present invention provides camptothecin derivatives with novel structures. The camptothecin compounds have good anti-tumor activity and antibody-coupled drugs based thereon have the potential to be used for the treatment of tumor diseases.

In one aspect, the present invention provides a camptothecin derivative, characterized in that its structure is a compound shown in general formula I:

_R1 and _R2 are each independently selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy or cycloalkyl;

A is selected from hydrogen, amino, W is selected from cycloalkyl, Y is selected from -C(=O)-, -S(=O) ₂ -, -CHCF ₃ -, -C(=CH ₂ )-, -P(=O)R ₁₀ -, -C(=NR ₁₁ )-, or -C(=O)NH-; n=an integer of 0-4; Z is selected from hydrogen or -OH;

R ₃ in A is selected from hydrogen or alkyl;

_R4 and _R5 in A are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl, cycloalkyl substituted alkyl, heterocyclyl, aryl or heteroaryl;

B is selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkanoyloxy-substituted alkyl, glycosyl-substituted alkyl, mercapto-substituted alkyl, amino-substituted alkyl, carboxyl-substituted alkyl, ester-substituted alkyl, alkynyl, alkenyl, cyano or cyano-substituted alkyl;

R ₆ and R ₇ are the same or different and are independently selected from hydrogen, fluorine or alkyl;

R ₈ and R ₉ are the same or different and are independently selected from hydrogen, fluorine or alkyl;

R ₁₀ and R ₁₁ are the same or different and are independently selected from saturated or unsaturated alkyl groups and aryl groups.

According to one embodiment of the present invention, R ₁ and R ₂ are each independently selected from hydrogen, halogen, hydroxyl, cyano, alkyl, alkoxy or cycloalkyl;

A is selected from hydrogen, amino, Y is selected from -C(=O)-, -S(=O) ₂ -, -CHCF ₃ -, -C(=CH ₂ )-, -P(=O)R ₁₀ -, -C(=NR ₁₁ )-, or -C(=O)NH-; n=an integer of 0 to 4; Z is selected from hydrogen or -OH;

R ₃ in A is selected from hydrogen or alkyl;

_R4 and _R5 in A are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl, cycloalkyl substituted alkyl, heterocyclyl, aryl or heteroaryl;

B is selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkanoyloxy-substituted alkyl, glycosyl-substituted alkyl, mercapto-substituted alkyl, amino-substituted alkyl, carboxyl-substituted alkyl, ester-substituted alkyl, alkynyl, alkenyl, cyano or cyano-substituted alkyl;

R ₆ and R ₇ are the same or different and are independently selected from hydrogen, fluorine or alkyl;

R ₈ and R ₉ are the same or different and are independently selected from hydrogen, fluorine or alkyl;

R ₁₀ and R ₁₁ are the same or different and are independently selected from saturated or unsaturated alkyl or aryl groups.

According to one embodiment of the present invention, R ₁ is selected from fluorine; R ₂ is selected from methyl;

A is selected from hydrogen, amino, W is selected from cyclobutyl, Y is selected from -C(=O)-, -S(=O) ₂ - or -CHCF ₃ -; n=an integer from 0 to 3; Z is selected from hydrogen or -OH; R ₃ in A is selected from hydrogen or methyl;

_R4 and _R5 in A are the same or different and are independently selected from hydrogen, methyl, trifluoromethyl,

B is selected from hydrogen, _-CH2OH , _-CH2OAc , _-CH2SH , _-CH2NH2 , _-CH2C ( ₌ O)OH, _-CH2C (=O)OMe, ethynyl, vinyl, cyano or _-CH2CN ;

R ₆ and R ₇ are the same or different and are independently selected from hydrogen or fluorine;

_R8 and _R9 are the same or different and are independently selected from hydrogen or fluorine.

According to an alternative embodiment of the present invention, R ₄ , R _{5 ,} together with the carbon atom to which they are connected, form a cyclopropyl group.

According to an optional embodiment of the present invention, R ₄ and R ₅ are not hydrogen at the same time.

According to an optional embodiment of the present invention, when B is hydrogen, A is also hydrogen; when A is amino, B is not hydrogen.

According to an optional solution of the present invention, in said A, Y is selected from -C(=O)-; n=0 or 1.

According to an optional scheme of the present invention, R ₃ , B, and the carbon atom to which A and B are commonly connected and/or the nitrogen atom to which R ₃ is connected in A form an aziridine group or an azetidine group, and/or R ₁ and R ₂ and their respective adjacent carbon atoms form a cycloalkyl group or a heterocyclic group, and/or R ₄ and R ₅ together with the carbon atom to which they are commonly connected form a cycloalkyl group or a heterocyclic group.

According to a preferred embodiment of the present invention, in the general formula I:

A is selected from hydrogen, amino or Y is selected from -C(=O)-; n=0-4; Z is selected from hydrogen or -OH;

B is selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkanoyloxy-substituted alkyl, glycosyl-substituted alkyl, mercapto-substituted alkyl, amino-substituted alkyl, carboxyl-substituted alkyl, ester-substituted alkyl, alkynyl, alkenyl, cyano or cyano-substituted alkyl;

_R1 and _R2 are each independently selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy, cycloalkyl;

R ₃ in A is selected from hydrogen or alkyl;

_R4 and _R5 in A are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl, cycloalkyl-substituted alkyl, Heterocyclyl, aryl or heteroaryl; _R4 and _R5 are not hydrogen at the same time;

R ₆ and R ₇ are the same or different and are independently selected from hydrogen, fluorine or alkyl;

_R8 and _R9 are the same or different and are independently selected from hydrogen, fluorine or alkyl.

According to an optional scheme of the present invention, B and R ₃ in A and the carbon atom and nitrogen atom to which A and B are commonly connected constitute aziridine or azetidine, and/or R ₁ and R ₂ and their respective adjacent carbon atoms constitute cycloalkyl or heterocyclic group, and/or R ₄ , R ₅ and the carbon atom to which they are commonly connected constitute cycloalkyl or heterocyclic group.

Further preferably, the compound of general formula I is selected from at least one of the structures shown in the following formula:

According to one embodiment of the present invention, it also includes prodrugs of the compound of formula I, or pharmaceutically acceptable salts, esters, solvates, tautomers, stereoisomers, polymorphs, nitrogen oxides or isotope labels thereof.

Another aspect of the present invention provides use of the aforementioned camptothecin derivatives in preparing drugs for treating cancer.

Another aspect of the present invention provides a compound of formula II:

Among them, M is selected from and/or,

L ₁ is selected from n ₁ = an integer from 1 to 10, n ₂ = an integer from 1 to 12, n ₃ = an integer from 1 to 10, n ₄ = an integer from 1 to 12; and/or,

L ₂ is selected from n ₅ = an integer of 7-24, L ₂ is connected to A in the general formula I; or

L ₂ is selected from L ₂ is connected to the O of -OH in the general formula I;

D is selected from the product group formed by the aforementioned camptothecin derivative losing one or more atoms or groups, that is, the product group formed by the compound of general formula I or its prodrug, pharmaceutically acceptable salt, ester, solvate, tautomer, stereoisomer, polymorph, nitrogen oxide or isotope label losing one or more atoms or groups.

In the general formula II, one or two of L1 and L2 are selected.

According to one embodiment of the present invention, M is selected from

According to one embodiment of the present invention, L ₁ is selected from

According to one embodiment of the present invention, _L2 is selected from

_L2 is connected to A in the general formula I.

According to one embodiment of the present invention,

L ₂ is selected from _L2 is connected to the O of -OH in the general formula (I).

According to one embodiment of the present invention, the compound of formula II is at least selected from one of the structures shown below, wherein _L2 is connected to A in formula I:

Alternatively, the compound of formula II is selected from the structure shown below, wherein _L2 is connected to the O of -OH in formula I:

According to one embodiment of the present invention, it also includes prodrugs, pharmaceutically acceptable salts, esters, solvates, tautomers, stereoisomers, polymorphs, nitrogen oxides or isotope labels of the compound of formula II.

Another aspect of the present invention also provides use of the aforementioned compound in preparing a drug for treating cancer.

Another aspect of the present invention provides an antibody-drug conjugate of formula III:

Wherein Ab is an antibody, an antibody fragment or a protein, wherein the antibody is selected from mouse antibodies, rabbit antibodies, phage display antibodies, yeast display antibodies, chimeric antibodies, humanized antibodies, fully human antibodies, antibody fragments, bispecific antibodies and multispecific antibodies; ML ₁ -L ₂ -D is selected from any compound of the aforementioned general formula II, or a prodrug, a pharmaceutically acceptable salt, an ester, a solvate, a tautomer, a stereoisomer thereof, a polymorph, a nitrogen oxide or an isotope label thereof; and n ₆ is selected from any value between 1 and 20.

In the general formula III, the carbon-carbon double bond of M in the ML ₁ -L ₂ -D is connected to the thiol group in the antibody Ab by Michael addition reaction to form a CS bond.

According to one embodiment of the present invention, the antibody is a monoclonal antibody, selected from the group consisting of, but not limited to, abciximab, alemtuzumab, anetumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatomumab, brentuximab, catumaxomab, cetuximab, cirmtuzumab, coltuximab, daclizumab, daratumumab, mumab), Denintuzumab, Denosumab, Depatuxizumab, Dinutuximab, Durvalumab, Elotuzumab, Enfortumab, Glembatumumab, Gemtuzumab, Ibritumomab, Indatuximab, Indusatumab, Inotuzumab, Ipilimumab, Labetuzumab, Latuzumab Ladiratuzumab, Laprituximab, Lifastuzumab, Lorvotuzumab, Milatuzumab, Mirvetuximab, Naratuximab, Necitumumab, Nimotuzumab, Nivolumab, Obinutuzumab, Ofatumumab, Olaratumab, Omalizumab, Palivizumab, Panitumumab, Patrutzumab ritumab), pembrolizumab, pertuzumab, pinatuzumab, polatuzumab, ramucirumab, rovalpituzumab, sacituzumab, siltuximab, sirtratumab, sofituzumab, vadastuximab, vorsetuzumab, trastuzumab, anti-CD4 antibody, anti-CD5 antibody, anti-CD13 antibody and anti-CD30 antibody or antigen-binding fragments or immunologically active portions thereof; ML ₁ -L ₂ -D is selected from the aforementioned compound, or a prodrug, a pharmaceutically acceptable salt, an ester, a solvate, a tautomer, a stereoisomer, a polymorph, a nitrogen oxide or an isotope label thereof; n ₆ is selected from any value between 4-10.

According to one embodiment of the present invention, the antibody is a monoclonal antibody, selected from, but not limited to, Patritumab, Sacituzumab, Mirvetuximab, Cetuximab, Trastuzumab, and Cirmtuzumab; ML ₁ -L ₂ -D is selected from the aforementioned compounds, or prodrugs, pharmaceutically acceptable salts, esters, solvates, tautomers, stereoisomers, polymorphs, nitrogen oxides, or isotope labels of the compounds; and n ₆ is selected from any value between 4 and 8.

According to one embodiment of the present invention, the conjugate is at least selected from one of the structures shown in the following formula:

Where n ₆ is any value between 4 and 8.

Another object of the present invention is to provide the camptothecin derivatives, their prodrugs, pharmaceutically acceptable salts, esters, solvates, tautomers, stereoisomers, polymorphs, nitrogen oxides and isotope labels described in the present invention for use in treating cancer, preferably treating sarcoma, lung cancer, ovarian cancer, gastric cancer, colorectal cancer and breast cancer.

Another object of the present invention is to provide the antibody-drug conjugate based on camptothecin derivatives of the present invention for use in treating cancer, preferably treating sarcoma, lung cancer, ovarian cancer, gastric cancer, colorectal cancer and breast cancer.

The camptothecin derivatives and antibody-drug conjugates of the present invention have good in vitro cell-killing activity and better anti-tumor effect.

Another object of the present invention is to provide a method for synthesizing the aforementioned camptothecin derivatives.

The compounds of the general formula I-1-1 and I-1-2 in the present invention can be synthesized by the following synthetic routes.

Wherein, R ₁ , R ₂ , R ₆ , R ₇ , R ₈ and R ₉ have the same meanings as described above;

Step 1

The compound of general formula I-intermediate 1 is obtained by a substitution reaction of the compound of general formula I-raw material 1.

In some embodiments, this step is carried out at a suitable temperature ranging from -15°C to 55°C, and the temperature may be -10°C, -5°C, 0°C, 5°C, 10°C, 25°C, 50°C, preferably -5°C to 5°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane and n-hexane, preferably N,N-dimethylformamide.

In some embodiments, this step is carried out under alkaline conditions, and the reagent providing alkaline conditions includes but is not limited to one or more of potassium carbonate, sodium carbonate, cesium carbonate, potassium tert-butoxide, sodium hydride, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide and potassium bistrimethylsilylamide, preferably cesium carbonate.

Step 2

The compound of the general formula I-intermediate 2 is obtained by hydrolyzing the compound of the general formula I-intermediate 1.

In some embodiments, this step is carried out at a suitable temperature ranging from -15°C to 55°C; the temperature may be -10°C, -5°C, 0°C, 5°C, 10°C, 25°C, 50°C; preferably 20°C to 40°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably methanol;

In some embodiments, this step is performed under acidic conditions, and the reagent providing acidic conditions includes but is not limited to one or more of hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably hydrochloric acid.

Step 3

The compounds of the general formula I-intermediate 3-1 and the general formula I-intermediate 3-2 are obtained by cyclization reaction of the compound of the general formula I-intermediate 2 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indolizine-3,6,10(4H)-trione.

In some embodiments, this step is carried out at a suitable temperature in the range of 15°C to 150°C; the temperature may be 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 130°C, 140°C; preferably, the temperature range is 110°C to 140°C;

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of toluene, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably toluene;

In some embodiments, this step is carried out under acidic conditions, and the reagent providing acidic conditions is selected from one or more of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably methanesulfonic acid.

Step 4

The compounds of the general formula I-1-1 and the general formula I-1-2 are obtained by hydrolyzing the compounds of the general formula I-intermediate 3-1 and the general formula I-intermediate 3-2.

In some embodiments, this step is carried out at a suitable temperature ranging from 15°C to 150°C, wherein the temperature is 20°C, 25°C, 40℃, 50℃, 60℃, 100℃, 130℃, 140℃; the preferred temperature range is 110℃-140℃;

In some embodiments, this step is carried out under acidic conditions, and the reagent providing acidic conditions is selected from one or more of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably methanesulfonic acid.

The compounds of the general formula I-2-1 and I-2-2 in the present invention can be synthesized by the following synthetic routes.

Wherein, R ₁ , R ₂ , R ₆ , R ₇ , R ₈ and R ₉ have the same meanings as described above;

Step 1

The compound of general formula I-intermediate 4 is obtained by a substitution reaction of the compound of general formula I-raw material 1.

In some embodiments, this step is carried out at a suitable temperature ranging from -15°C to 55°C; the temperature may be -10°C, -5°C, 0°C, 5°C, 10°C, 25°C, 50°C; preferably -5°C to 5°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane and n-hexane, preferably tetrahydrofuran.

In some embodiments, this step is carried out under alkaline conditions, and the reagent providing alkaline conditions is selected from one or more of potassium carbonate, sodium carbonate, cesium carbonate, potassium tert-butoxide, sodium hydride, n-butyl lithium, lithium diisopropylamide, lithium bistrimethylsilylamide, sodium bistrimethylsilylamide and potassium bistrimethylsilylamide, preferably sodium hydride.

Step 2

The compound of the general formula I-intermediate 4 undergoes a 1,2-elimination reaction to obtain the compound of the general formula I-intermediate 5.

In some embodiments, this step is carried out at a suitable temperature in the range of 15°C-150°C; the temperature can be 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 130°C, 140°C; preferably, the temperature range is 120°C-150°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of toluene, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably toluene.

Step 3

The compound of the general formula I-intermediate 6 is obtained by hydrolyzing the compound of the general formula I-intermediate 5.

In some embodiments, this step is carried out at a suitable temperature in the range of -5°C to 110°C, which may be 0°C, 10°C, 25°C, 50°C, 70°C, 80°C, 100°C, preferably in the range of 60°C to 90°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably methanol.

In some embodiments, this step is performed under acidic conditions, and the reagent providing acidic conditions is selected from one or more of hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably hydrochloric acid.

Step 4

The compounds of the general formula I-intermediate 7-1 and the general formula I-intermediate 7-2 are obtained by cyclization reaction of the compound of the general formula I-intermediate 6 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indolizine-3,6,10(4H)-trione.

In some embodiments, this step is carried out at a suitable temperature ranging from 15°C to 150°C, and the temperature can be 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 130°C, 140°C, preferably the temperature range is 110°C-150°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of toluene, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably toluene.

In some embodiments, this step is carried out under acidic conditions, and the reagent providing acidic conditions is selected from one or more of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably p-toluenesulfonic acid.

Step 5

The compounds of the general formula I-2-1 and the general formula I-2-2 are obtained by hydrolyzing the compounds of the general formula I-intermediate 7-1 and the general formula I-intermediate 7-2.

In some embodiments, this step is carried out at a suitable temperature in the range of 15°C-150°C, and the temperature can be 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 130°C, 140°C, preferably in the range of 110°C-150°C.

In some embodiments, this step is carried out under acidic conditions, and the reagent providing acidic conditions is selected from one or more of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably methanesulfonic acid.

Alternatively, the compound of formula I-3 in the present invention can be synthesized by the following synthetic route.

Wherein, R ₁ , R ₂ , R ₈ and R ₉ have the same meanings as described above;

Step 1

The compound of the general formula I-intermediate 8 is obtained by a deoxyfluorination reaction of the compound of the general formula I-raw material 2.

In some embodiments, this step is carried out at a suitable temperature ranging from -5°C to 120°C, wherein the temperature is 0°C, 10°C, 25°C, 50°C, 70°C, 80°C, 100°C; preferably, the temperature ranges from 60°C to 90°C.

In some embodiments, this step is carried out in a suitable fluorination agent, which can be selected from one or more of DAST, Deoxo-Fluor, TFEDMA, XtalFluor, FluoLead, PhenoFluor, BPSF, PyFluor and SulfoxFluor, preferably DAST.

Step 2

The compound of the general formula I-intermediate 9 is obtained by reducing the compound of the general formula I-intermediate 8.

In some embodiments, this step is carried out at a suitable temperature in the range of 15°C-120°C, such as 20°C, 25°C, 40°C, 50°C, 60°C, 70°C, 90°C, 100°C; preferably, the temperature range is 60°C-90°C.

In some embodiments, this step is carried out in the presence of a suitable reducing agent, which can be selected from one or more of palladium catalysts, platinum catalysts, rhodium catalysts, iron powder, zinc powder, stannous chloride, nickel catalysts, hydrosulfur powder and titanium trichloride, preferably iron powder.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of toluene, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably methanol.

Step 3

The compound of the general formula I-intermediate 10 is obtained by condensation reaction of the compound of the general formula I-intermediate 9.

In some embodiments, this step is carried out at a suitable temperature ranging from -5°C to 60°C, wherein the temperature is 0°C, 5°C, 10°C, 25°C, 50°C; preferably, the temperature ranges from 10°C to 30°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of tetrahydrofuran, 1,4-dioxane, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane and n-hexane, preferably dichloromethane.

In some embodiments, this step is carried out in a suitable base, including but not limited to one or more of potassium carbonate, sodium carbonate, cesium carbonate, sodium hydride, sodium hydroxide, DIPEA, triethylamine, potassium tert-butoxide and pyridine, preferably DIPEA.

Step 4

The compound of the general formula I-intermediate 11 is obtained by oxidation reaction of the compound of the general formula I-intermediate 10.

In some embodiments, this step is performed at a suitable temperature ranging from -5°C to 60°C, wherein the temperature is 0°C, 5°C, 10°C, 25°C, 50°C, preferably 25°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from tetrahydrofuran, 1,4-dioxane, dichloromethane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, acetone, n-hexane, preferably acetone.

In some embodiments, this step is carried out in a suitable oxidizing agent, including but not limited to potassium permanganate, chromium trioxide, hydrogen peroxide, tert-butyl hydroperoxide, pyridinium chlorochromate, sodium dichromate, DDQ, pyridinium dichromate, Jones reagent, manganese dioxide, cerium ammonium nitrate, preferably potassium permanganate.

Step 5

The compound of the general formula I-intermediate 12 is obtained by hydrolyzing the compound of the general formula I-intermediate 11.

In some embodiments, this step is carried out at a suitable temperature, such as 0°C, 10°C, 25°C, 50°C, 70°C, 80°C, 100°C; preferably, the temperature ranges from 10°C to 30°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably methanol.

In some embodiments, this step is performed under acidic conditions, and the reagent providing acidic conditions is selected from one or more of hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably hydrochloric acid.

Step 6

The compound of general formula I-3 is obtained by cyclization reaction of the compound of general formula I-intermediate 12 and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyran[3,4-f]indolizine-3,6,10(4H)-trione.

In some embodiments, this step is carried out at a suitable temperature in the range of 15°C-150°C, such as 20°C, 25°C, 40°C, 50°C, 60°C, 100°C, 130°C, 140°C; preferably, the temperature range is 110°C-150°C.

In some embodiments, this step is carried out in a suitable organic solvent, which can be selected from one or more of toluene, methanol, ethanol, tetrahydrofuran, 1,4-dioxane, N,N-dimethylformamide, N-methylpyrrolidone, dimethyl sulfoxide, n-heptane, n-hexane and ethyl acetate, preferably toluene.

In some embodiments, this step is carried out under acidic conditions, and the reagent providing acidic conditions is selected from one or more of p-toluenesulfonic acid, hydrochloric acid, trifluoroacetic acid, trifluoromethanesulfonic acid, methanesulfonic acid, formic acid and sulfuric acid, preferably p-toluenesulfonic acid.

DETAILED DESCRIPTION

Table 1: Examples of specific compounds of formula II

Synthesis of compounds

The following are examples of the synthesis and analysis of the compounds described in this patent (including intermediates, toxins, linkers and toxin linkers). It must be noted that the following examples are only some embodiments, and the present invention is not limited to the examples currently described.

1. Camptothecin derivatives

Example 1: Synthesis of P1 and P16

(9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione methanesulfonate.

Step 1: Synthesis of N,N'-(3-fluoro-7-(fluoromethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide.

To a solution of N,N'-(3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (4.38 g, 15.00 mmol) in DMF (45 mL, 100%) was added _Cs2CO3 (7.33 g, 22.50 mmol ₎ at 0°C. _ICH2F (6.0 g, 37.50 mmol) was added thereto after 30 minutes. After the addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was diluted with _H2O (50 mL) and extracted with ethyl acetate (50 mL x 3). The organic phase was separated, washed with saturated brine (30 mL), and anhydrous _{Na2SO 4} was dried, filtered and concentrated to give the product, which was purified by silica gel flash column chromatography (0-50% ethyl acetate in petroleum ether) to give N,N'-(3-fluoro-7-(fluoromethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (2.5 g, 51% yield) as a brown solid.

LC-MS(ESI)m/z:325.2[M+H] ⁺ .

Step 2: Synthesis of N-(8-amino-6-fluoro-2-(fluoromethyl)-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide.

To a solution of N,N'-(3-fluoro-7-(fluoromethyl)-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diacetamide (0.50 g, 1.54 mmol) in _H2O (5.00 mL) was added 2N HCl/MeOH (5.00 mL) at 0°C. After the addition was complete, the mixture was stirred at room temperature under _N2 protection for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give a crude product, which was purified by silica gel flash column chromatography (0-50% ethyl acetate in petroleum ether) to give N-(8-amino-6-fluoro-2-(fluoromethyl)-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalene-2-yl)acetamide (150 mg, 34% yield) as a yellow solid.

LC-MS(ESI)m/z:283.2[M+H] ⁺ .

Step 3: Synthesis of (9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12',15'-dihydro-2'-hydrospiro[aziridine-2'-benzopyran[3',4':6,7]indolizine[1,2-b]quinoline]-10',13'(3'H,9'H)-dione.

MsOH (10.18 mg, 0.10 mmol) and AcOH (34.98 mg, 0.58 mmol) were added to a solution of N-(8-amino-6-fluoro-2-(fluoromethyl)-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (150 mg, 0.53 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (167 mg, 0.63 mmol) in toluene (20.0 mL, 100%). The reaction mixture was stirred at 130° C. for 12 hours. LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature and concentrated to give a crude product, which was purified by silica gel flash column chromatography (0-10% methanol in dichloromethane) to give (9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12',15'-dihydro-2'-hydropyran[aziridine-2'-benzo[de]pyran[3',4':6,7]indolizino[1,2-b]quinoline]-10',13'(3'H,9'H)-dione (20 mg, 7% yield) as a brown solid.

LC-MS(ESI)m/z:490.2[M+H] ⁺ .

Step 4: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzopyran[3',4':6,7]indolizine[1,2-b]quinoline-10,13(1H,9H)-dione.

MsOH (1.00 mL) was added to a solution of (9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12',15'-dihydro-2'-pyrano[aziridine-2'-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10',13'(3'H,9'H)-dione (20.00 mg, 0.04 mmol) in H ₂ O (0.50 mL) at 0° C. After the addition was complete, the mixture was stirred at 130° C. for 12 hours. LCMS showed the reaction was complete. The mixture was concentrated to afford the product which was purified by reverse phase flash chromatography (0-70% acetonitrile in _H2O ) to afford (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H, 9H)-dione (1.68 mg, yield 7%) as a light yellow solid.

Characterization data of P1:

LC-MS(ESI)m/z:466.0[M+H] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.79-7.73 (m, 1H), 7.67 (d, J = 2.8Hz, 1H), 5.72-5.60 (m, 2H), 5.54-5.40 (m, 2H) ,4.15-3.93(m,2H),3.48-3.36(m,1H),3.31-3.20(m,1H),2.82-2.74(m,1H),2.49(s,3H),2.44-2.32(m, 1H),2.05-1.90(m,2H),1.08-1.00(m,3H).

Example 2: Synthesis of P2, P3, P17, and P18

(1R, 9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione and (1S, 9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione.

Step 1: Synthesis of (1’S, 9’S)-1-acetyl-9’-ethyl-5’-fluoro-9’-hydroxy-4’-methyl-12’, 15’-dihydro-2’-pyrano[aziridine-2,1’-benzopyrano[3’, 4’: 6,7]indolizine[1,2-b]quinoline]-10’, 13’(3’H, 9’H)-dione and (1’R, 9’S)-1-acetyl-9’-methyl-5’-fluoro-9’-hydroxy-4’-methyl-12’, 15’-dihydro-2’H-pyrano]aziridine-2,1’-benzopyrano[3’, 4’: 6,7]indolizine[1,2-b]quinoline]-10’, 13’(3’H, 9’H)-dione.

N-(8-amino-6-fluoro-2-(fluoromethyl)-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (1.10 g, 3.88 mmol), (S)-4-ethyl-4-hydroxy-7,8-dihydro-1h-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (1.23 g, 4.66 mmol) in toluene (80.00 mL), MsOH (74.5 mg, 0.77 mmol), and AcOH (256 mg, 4.3 mmol) were added. The reaction mixture was stirred at 130° C. for 12 hours, and LCMS showed that the reaction was complete. The reaction solution was cooled to room temperature and concentrated to give the product, which was purified by silica gel chromatography (0-4% MeOH in DCM) to give (1',9')-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12',15'-dihydro-2'-spiro[azapyridine-2,1'-benzo]pyrano[3',4':6,7]indolizine[1,2-b]quinoline]-10',13'(3'h,9'H)- Dione P17 (100 mg, 5%) was a yellow solid, and (1'r,9')-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12',15'-dihydro-2'-spiro[azapyridine-2,1'-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10',13'(3'h,9'H)-dione P18 (95 mg, 5% yield) was a yellow solid.

Characterization data of P17:

LC-MS(ESI)m/z:490.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.74 (d, J = 10.8 Hz, 1H), 7.29 (d, J = 8.8 Hz, 1H), 6.50 (d, J = 3.2 Hz, 1H), 5.40 (s,2H),5.25(d,J＝18.0Hz,1H),5.08(d,J＝18.0Hz,1H),4.59(d,J＝9.2Hz,1H),4.18(d,J＝8.8Hz ,1H),3.19-3.03(m,1H),2.37(s,3H),2.16(s,3H),2.14-2.06(m,2H),1.93-1.77(m,3H),0.88(t,J =7.2Hz,3H).

Characterization data of P18:

LC-MS(ESI)m/z:490.2[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ7.77 (d, J = 10.8 Hz, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 5.41 (s, 2H), 5.30-5.01 ( m,2H),4.63-4.54(m,1H),4.17-4.15(m,1H),3.13-3.05(m,1H),2.38(s,3H),2.16(s,3H),2.16-2.10( m,1H),1.99-1.98(m,1H),1.87-1.84(m,3H),0.90-0.83(m,3H).

Step 2: Synthesis of (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione.

MsOH (1.0 mL) was added to a solution of (1'S,9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12'-,15'-dihydro-2'-spiro[aziridine-2'-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10'-,13'(3'H,9'H)-dione (20 mg, 0.04 mmol) in H ₂ O (0.5 mL) at 0° C. After addition, the mixture was stirred at 130° C. for 12 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the product, which was purified with _C18 (0-70% ACN in _H2O ) to give (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (12 mg, 63% yield) as a light yellow solid.

Characterization data of P3:

LC-MS(ESI)m/z:466.4[M+H] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.81 (d, J = 10.4Hz, 1H), 7.69 (s, 1H), 5.68-5.60 (m, 2H), 5.47-5.41 (m, 2H), 4.04 -3.98(m,2H),3.42-3.36(m,1H),3.25-3.21(m,1H),2.80-2.73(m,1H),2.49(s,3H),2.41-2.32(m,1H) ,2.03-1.94(m,2H),1.05-1.01(m,3H).

Step 3: Synthesis of (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione.

MsOH (1.0 mL) was added to a solution of (1'R,9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12'-,15'-dihydro-2'-spiro[aziridine-2'-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10'-,13'(3'H,9'H)-dione (20 mg, 0.04 mmol) in H ₂ O (0.5 mL) at 0° C. After addition, the mixture was stirred at 130° C. for 12 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the product, which was purified with _C18 (0-70% ACN in _H2O ) to give (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-2,3,12,15-tetrahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13(1H,9H)-dione (7.0 mg, 37% yield) as a white solid.

Characterization data of P2:

LC-MS(ESI)m/z:466.4[M+H] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.65-7.62(m,2H),5.82-5.38(m,1H),5.60-5.53(m,2H),5.39-5.35(m,1H),3.76- 3.64(m,2H),3.42-3.31(m,1H),3.20-3.03(m,1H),2.53-2.51(m,1H),2.41(s,3H),2.09-2.07(m,1H), 1.96-1.94(m,1H),1.00-0.97(m,3H).

Example 3: Synthesis of P8

((1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetate methyl 2,2,2-trifluoroacetate.

Synthesis of P8

To a solution of (1'S,9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12'-,15'-dihydro-2'-spiro[aziridine-2'-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10'-,13'(3'H,9'H)-dione (10 mg, 0.02 mmol) in H ₂ O (0.5 mL) and acetonitrile (2 mL) was added TFA (0.5 mL) at 0° C. After addition, the mixture was stirred at 25° C. for 2 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the product, which was purified with C18 (0-70% acetonitrile in _H2O ) to give ((1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetate methyl 2,2,2-trifluoroacetate (5.95 mg, 47% yield) as a light yellow solid.

Characterization data of P8:

LC-MS(ESI)m/z:507.7[M+H] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.79 (d, J = 10Hz, 1H), 7.67 (s, 1H), 5.68-5.39 (m, 4H), 4.67-4.51 (m, 2H), 3.45- 3.35(m,1H),3.30-3.28(m,1H),2.74-2.71(m,1H),2.48(s,3H),2.38-2.19(m,1H),2.20(s,3H),1.99- 1.96(m,2H),1.03(t,J＝7.4Hz,3H).

Example 4: Synthesis of P9

((1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzopyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetate methyl 2,2,2-trifluoroacetate.

To a solution of (1'R,9'S)-1-acetyl-9'-ethyl-5'-fluoro-9'-hydroxy-4'-methyl-12'-,15'-dihydro-2'-spiro[aziridine-2'-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline]-10'-,13'(3'H,9'H)-dione (10 mg, 0.02 mmol) in H ₂ O (0.5 mL) and ACN (2 mL) was added TFA (0.5 mL) at 0° C. After addition, the mixture was stirred at 25° C. for 2 hours. LCMS showed the reaction was complete. The mixture was concentrated to give the product, which was purified with C18 (0-70% ACN in _H2O ) to give ((1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetate methyl 2,2,2-trifluoroacetate (4.10 mg, 33% yield) as a light yellow solid.

Characterization data of P9:

LC-MS(ESI)m/z:507.8[M+H] ⁺ .

¹ H NMR (400MHz, CD ₃ OD) δ7.81 (d, J = 10.4Hz, 1H), 7.69 (s, 1H), 5.61-5.78 (m, 2H), 5.45-5.41 (m, 2H), 4.65 -4.62(m,1H),4.53-4.50(m,1H),3.40-3.92(m,1H),3.28-3.23(m,1H),2.80-2.66(m,1H),2.49(s,3H) ,2.46-2.34(m,1H),2.33(s,1H),2.19(s,3H),1.99-1.96(m,2H),1.02-0.99(m,3H).

Example 5: Synthesis of P12, P13, P55, and P56

(1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione.

Step 1: Synthesis of N,N'-(3-fluoro-4-methyl-8-oxo-7-(2-(phenylsulfinyl)ethyl)-5,6,7-8-tetrahydronaphthalene-1,7-diyl) diacetamide

Under argon protection, NaH (1.6 g, 68.45 mmol) was added to a solution of N,N'-(3-fluoro-4-methyl-8-oxy-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diethylamide (1,4.0 g, 13.69 mmol) in THF (30 mL) at 0°C with stirring. Vinylphenyl sulfoxide (3.1 g, 20.54 mmol) was then added dropwise at the same temperature. The reaction mixture was stirred at room temperature for 6 hours. After the reaction was completed, the reactants were quenched with ice water and extracted with 5% MeOH in chloroform. The organic layer was dried over anhydrous Na ₂ SO ₄ and concentrated under reduced pressure to obtain a crude product, which was purified by silica gel flash column chromatography (ethyl acetate: heptane = 4:1-9:1 elution) to obtain the product (4.4 g, yield 65%).

LC-MS(ESI)m/z:445.28[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): δ11.2 (d, J = 15.2Hz, 1H), 8.20 (m, 2H), 7.55 (m, 5H), 3.19-3.13 (m, 1H), 2.88 (m,2H),2.71(m,1H),2.12(d,J＝2.0Hz,3H),2.07(s,3H),1.87(m,4H),1.74(d,J＝2.0Hz,3H) .

Step 2: Synthesis of N, N'-(3-fluoro-4-methyl-8-oxo-7-vinyl-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diacetamide

N,N'-(3-fluoro-4-methyl-8-oxy-7-(2-(phenylsulfinyl)ethyl)-5,6,7,8-tetrahydronaphthalene-1,7-diyl) diethylamide (4.4 g, 9.90 mmol) was dissolved in toluene (20 mL), stirred at 140 ° C for 16 hours under argon protection, and monitored by TLC. The obtained reactant was quenched with ice water, extracted with ethyl acetate, and the organic layer was concentrated under reduced pressure to obtain a crude residue, which was purified by silica gel column chromatography using 20-40% ethyl acetate/heptane as the eluent. (2.5 g, yield 79%).

LC-MS(ESI)m/z:319.1[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): δ11.57 (s, 1H), 8.34 (s, 1H), 8.28 (d, J = 12.8Hz, 1H), 6.05 (dd, J = 17.6Hz, 10.8Hz,1H),5.30(d,J＝10.4Hz,1H),5.15(d,J＝17.6Hz,1H),3.03-2.96(m,1H),2.81-2.73(m,1H),2.67- 2.59(m,1H),2.15(s,3H),2.14-2.11(m,1H),2.10(s,3H),1.82(s,3H).

Step 3: Synthesis of N-(8-amino-6-fluoro-5-methyl-1-oxo-2-vinyl-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

To N,N'-(3-fluoro-4-methyl-8-oxy-7-vinyl-5,6,7,8-tetrahydronaphthalene-1,7-diyl)diethylamide (7 g, 22.01 mmol) in MeOH (15 mL) was added 2N HCl (4 mL) under stirring at 0°C, and then the reaction was stirred at 70°C for 8 hours, and the reaction was monitored by TLC. After the reaction was completed, the reactants were azeotroped with methanol under reduced pressure until the water was evaporated and submitted to LCMS analysis. The crude material was triturated twice with diethyl ether and used as the next step without further purification. (3.8 g, yield 62%).

LC-MS(ESI)m/z:277.05[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): δ7.96 (s, 1H), 6.39 (d, J = 12.8Hz, 1H), 5.99 (dd, J = 17.6Hz, 10.8Hz, 2H), 5.23 (d,J＝10.8Hz,1H),5.02(d,J＝17.6Hz,1H),3.17(s,1H),2.86-2.81(m,1H),2.69-2.59(m,2H),2.21- 2.18(m,1H),1.95(s,3H),1.83(s,3H).

Step 4: Synthesis of N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)acetamide and N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)acetamide.

Under argon protection at room temperature, to a toluene (35 mL) solution of N-(8-amino-6-fluoro-5-methyl-1-oxy-2-vinyl-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide (3.8 g, 11.94 mmol) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-F]indolizine-3,6,10(4H)-one (3.1 g, 11.94 mmol) and Molecular sieves (2g), followed by the addition of PTSA˙H ₂ O (453mg, 2.3mmol), and stirred at 140℃ for 16 hours using a Dean Stark apparatus, and the reaction was monitored by TLC. After the reaction was completed, the reactant was filtered through diatomaceous earth, the filtrate was concentrated under reduced pressure, and the crude product was purified by silica gel flash column chromatography (methanol: dichloromethane = 0:1-1:11 elution) to obtain (1.8g crude product) including two diastereoisomers of P55 and P56. LC-MS (ESI) m/z: 504.33 [M+H] ⁺ .

Step 5: Synthesis of (1R, 9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione and (1S, 9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione

A mixture of N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide and N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide (1.8 g, 5.6 mmol) was dissolved in H ₂ O (5 mL), MsOH (10 mL) was added, and the mixture was stirred at 130°C for 24 h. LCMS showed that the reaction was complete. The mixture was concentrated to obtain a crude product, which was purified by preparative high performance liquid chromatography (0-50% CH ₃ CN/H ₂ O) purification gave (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (24 mg, 0.72% yield) as a white solid and (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1-vinyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizine[1,2-b]quinoline-10,13-dione (22 mg, 0.72% yield) as a white solid.

Characterization data of P12:

LC-MS(ESI)m/z:462.3[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): 8.98 (bs, 2H), 7.92 (d, J = 12.8Hz, 1H), 7.35 (s, 1H), 6.56 (s, 1H), 6.38 (dd, J＝ 17.2Hz,10.8Hz,1H),5.66(d,J＝10.4Hz,1H),5.50-5.29(m,4H),3.22-3.14(m,2H),2.53(s,2H),2.49-2.39( m, 4H), 1.90-1.84 (m, 2H), 0.87 (t, J = 7.2Hz, 3H).

Characterization data of P13:

LC-MS(ESI)m/z:462.4[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): 9.08 (bs, 2H), 7.93 (d, J = 10.8Hz, 1H), 7.36 (s, 1H), 6.54 (s, 1H), 6.36 (dd, J＝17.6Hz,6.4Hz,1H),5.66(d,J＝11.2Hz,1H),5.51-5.29(m,4H),3.25-3.14(m,2H),2.45-2.42(m,5H), 2.32-2.29(m,1H),1.91-1.83(m,2H),0.87(t,J=7.2Hz,3H).

Example 6: Synthesis of P21

Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4':6,7]indolyl[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

Steps 1 and 2: P2 (9 mg, 0.016 mmol, 1 eq) and triethylamine (3.24 mg, 0.032 mmol, 2 eq) were dissolved in anhydrous DMF (1 mL), and 2-((tert-butyldimethylsilyl)oxy)acetic acid (3.04 mg, 0.016 mmol, 1 eq) and HATU (12.2 mg, 0.032 mmol, 2 eq) were added. Stir at room temperature for 2.5 hours. LCMS showed that the reaction was complete. Triethylamine trihydrofluoride (0.5 mL) was added to remove the TBS protecting group. Purification by preparative HPLC gave (2.78 mg, yield 33.1%) as a white solid.

Characterization data of P21:

LC-MS (ESI) m/z: 524 [M+H] ⁺ .

Example 7: Synthesis of P25

Synthesis of (S)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizine[1,2-b]quinolin-1-yl)-2-hydroxypropanamide

P2 (15 mg, 0.027 mmol, 1 eq) and triethylamine (27 mg, 0.27 mmol, 10 eq) were added to anhydrous DMF (1 mL), followed by (S)-2-hydroxypropionic acid (12 mg, 0.134 mmol, 5 eq) and HATU (50.9 mg, 0.27 mmol, 2 eq). Stir at room temperature for 2.5 hours. LCMS showed that the reaction was complete. Purification by preparative HPLC gave (3 mg, yield 12.4%) of a white solid.

Characterization data of P25:

LC-MS (ESI) m/z: 538 [M+H] ⁺ .

Example 8: Synthesis of P39

Synthesis of N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

To a solution of P12 (3 mg, 0.06 mmol), 2-hydroxyacetic acid (1.2 mg, 0.016 mmol) and HATU (6 mg, 0.016 mmol) in anhydrous DMF (0.20 mL) was added DIEA (5.6 μL, 0.032 mmol). After the addition, the mixture was stirred at room temperature overnight. The target product (1.0 mg, yield 29.6%) was purified by preparative HPLC as a yellow solid.

Characterization data of P39:

LC-MS(ESI)m/z:560.22[M+H] ⁺ .

¹ H NMR(400MHz, DMSO-d ₆ )δ8.61(s,1H),7.82-7.80(m,1H),7.33(s,1H),7.22(s,1H),7.09(s,1H), 6.97(s,1H),6.52(m,1H),5.43-5.41(m,1H),5.26-5.23(s,1H),5.06-5.01(m,1H),3.98-3.95(m,1H), 2.81-2.62(m,2H),2.38(s,3H),2.00-1.76(m,3H),1.24(s,3H),0.91-0.87(m,3H).

Example 9: Synthesis of P43

(S)-9-Ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione.

Step 1: Synthesis of 1,1,7-trifluoro-8-methyl-5-nitro-1,2,3,4-tetrahydronaphthalene.

DAST (20 mL) was added to 7-fluoro-8-methyl-5-nitro-3,4-dihydronaphthalen-1(2H)-one (1) (2.0 g, 8.944 mmol) at 0°C, and the reaction solution was stirred at 70°C for 16 hours. The reaction was monitored by TLC. After the reaction was completed, the reactant was quenched with saturated aqueous NaHCO ₃ solution and extracted with ethyl acetate (2×15 mL). The organic phase was concentrated under reduced pressure and the crude product (2.4 g) was purified by flash silica gel column chromatography to obtain a sticky brown solid (1.7 g, yield: 73%).

¹ H-NMR (400MHz; DMSO-d ₆ ): δ8.07 (d, J = 9.6 Hz, 1H), 2.95-2.90 (m, 2H), 2.45 (d, J = 1.6 Hz, 3H), 2.39- 2.32(m,2H),1.84-1.79(m,2H).

Step 2: Synthesis of 3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthalen-1-amine.

At room temperature, H ₂ O (5 mL), Fe (858 mg, 15.37 mmol) and NH ₄ Cl (2.24 g, 41.94 mmol) were added to a solution of 1,1,7-trifluoro-8-methyl-5-nitro-1,2,3,4-tetrahydronaphthalene (1.7 g, 6.99 mmol) in MeOH (5 mL), and the reaction mixture was stirred at 70°C for 2 hours, and the progress of the reaction was monitored by TLC. After the reaction was completed, the reactant was diluted with ethyl acetate, filtered through celite and washed with H ₂ O. The organic phase was concentrated under reduced pressure to obtain a crude product (1.4 g, yield: 94%) as a brown viscous substance.

¹ H-NMR (400MHz; DMSO-d ₆ ): δ6.49 (d, J = 12.4Hz, 1H), 5.18 (s, 2H), 2.43-2.33 (m, 2H), 2.25-2.11 (m, 5H ), 1.99-1.82(m,2H).

Step 3: Synthesis of N-(3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

At 0°C, DIPEA (3.4 mL, 19.53 mmol) and Ac ₂ O (1.84 mL, 19.53 mmol) were added to a solution of 3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthalene-1-amine (1.4 g, 6.511 mmol) in dichloromethane (10 mL). The reaction mixture was stirred at room temperature for 2 hours and the reaction was monitored by TLC. After the reaction was completed, the reactant was diluted with saturated aqueous NaHCO ₃ solution and extracted with dichloromethane. The organic phase was concentrated under reduced pressure to obtain a crude product (1.2 g, yield: 71.8%) as a brown viscous oil.

LC-MS(ESI)m/z:258.03[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): δ9.40 (s, 1H), 7.45 (d, J = 11.2Hz, 1H), 2.64 (m, 2H), 2.34-2.22 (m 5H), 2.07 (s,3H),1.84-1.79(m,2H).

Step 4: Synthesis of N-(3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

To a solution of N-(3,5,5-trifluoro-4-methyl-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (4) (1.1 g, 4.28 mmol) in acetone (20 mL) was added MgSO ₄ (15% aqueous solution) (3.2 mL) and KMnO ₄ (993 mg, 17.12 mmol) at 0°C. The reactants were stirred at room temperature for 16 hours and the reaction was monitored by TLC. After the reaction was completed, the reaction mixture was concentrated under reduced pressure and the crude solid (1.8 g) was purified by flash silica gel column chromatography to obtain a white solid (800 mg, yield: 69%).

¹ H-NMR (400MHz; DMSO-d ₆ ): δ11.95 (s, 1H), 8.57 (d, J = 13.2Hz, 1H), 2.87-2.84 (m, 2H), 2.77-2.66 (m, 2H ), 2.37 (d, J = 2.4Hz, 3H), 2.19 (s, 3H).

Step 5: Synthesis of 8-amino-4,4,6-trifluoro-5-methyl-3,4-dihydronaphthalen-1(2H)-one

To a solution of N-(3,5,5-trifluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (200 mg, 0.73 mmol) in methanol (5.0 mL) was added dropwise 2(N)HCl (0.5 mL) in water at 0°C. The reactants were stirred at 80°C for 1.5 hours. The reaction was monitored by TLC. After the reaction was completed, the crude product was concentrated and slurried with ether to give a red solid (120 mg, yield: 71%).

LC-MS(ESI)m/z:230.01[M+H] ⁺ .

¹ H-NMR (400MHz; CDCl ₃ ): δ6.57 (bs, 2H), 6.42 (d, J = 11.6Hz, 1H), 2.79-2.76 (m, 2H), 2.59-2.50 (m, 2H), 2.33-2.31(m,3H).

Step 6: Synthesis of (S)-9-ethyl-3,3,5-trifluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione

To a stirred toluene solution of 8-amino-4,4,6-trifluoro-5-methyl-3,4-dihydronaphthalene-1(2H)-one (120 mg, 0.52 mmol) was added (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (152 mg, 0.63 mmol), followed by PTSA (45 mg, 0.26 mmol). The reaction mixture was heated at 130°C for 5 hours. The reaction was monitored by TLC. After the reaction was completed, the mixture was concentrated under reduced pressure and the crude product was purified by rapid silica gel column chromatography (eluted with ethyl acetate: hexane = 1:1) to give a light pink solid (44 mg, yield: 18%).

LC-MS(ESI)m/z:457.25[M+H] ⁺ .

¹ H-NMR (400MHz; DMSO-d ₆ ): δ8.11 (d, J = 10.8 Hz, 1H), 7.32 (s, 1H), 6.54 (s, 1H), 5.44 (s, 2H), 5.30 ( s,1H),3.33-3.32(m,2H),2.74-2.67(m,2H),2.63(s,3H),1.92-1.84(m,2H),0.87(t,J＝7.2Hz,3H) .

Example 10: Synthesis of P53

Synthesis of (1s, 3S)-N-((1R, 9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3-hydroxycyclobutane-1-carboxamide

P12 (3 mg, 0.006 mmol), (1S, 3S)-3-hydroxycyclobutane-1-carboxylic acid (1.2 mg, 0.01 mmol) and HATU (6 mg, 0.016 mmol) were dissolved in anhydrous DMF (200 μL), and DIEA (5.6 μL, 0.032 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. LCMS showed that the reaction was complete, and the target product (1.0 mg, yield 28%) was obtained as a yellow solid by purification by preparative HPLC.

Characterization data of P53:

LC-MS(ESI)m/z:560.22[M+H] ⁺ .

¹ H NMR(400MHz, DMSO-d ₆ )δ8.61(s,1H),7.82-7.80(m,1H),7.33(s,1H),7.22(s,1H),7.09(s,1H), 6.97(s,1H),6.52(m,1H),5.43-5.41(m,1H),5.26-5.23(s,1H),5.06-5.01(m,1H),3.98-3.95(m,1H), 2.81-2.62(m,2H),2.38(s,3H),2.00-1.76(m,3H),1.24(s,3H),0.91-0.87(m,3H).

Example 11: Synthesis of P54

Synthesis of (1R,3R)-n-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxy-1-vinyl-2,3,9,10,13,15-hexahydro-1h,12h-benzo[de]pyrano[3,4':6,7]indolyl[1,2-b]quinolin-1-yl)-3-hydroxycyclobutane-1-carboxamide

P12 (3 mg, 0.006 mmol), (1R, 3R)-3-hydroxycyclobutane-1-carboxylic acid (1.2 mg, 0.01 mmol) and HATU (6 mg, 0.016 mmol) were dissolved in anhydrous DMF (0.20 mL), and DIEA (5.6 μL, 0.032 mmol) was added. The reaction solution was stirred at room temperature overnight. LCMS showed that the reaction was complete, and the target product (0.86 mg, yield 24%) was purified by preparative HPLC as a yellow solid.

Characterization data of P54:

LC-MS (ESI) m/z: 560.22 [M+H] ⁺ .

¹ H NMR(400MHz, DMSO-d ₆ )δ8.55(s,1H),7.82-7.80(m,1H),7.33(s,1H),7.22(s,1H),7.09(s,1H), 6.96(s,1H),6.53-6.51(m,1H),6.16-6.09(m,1H),5.41(s,1H),5.26-5.23(m,1H),5.07-5.00(m,1H), 4.18-4.22(m,1H),2.94(s,33H),2.38(s,3H),2.00-1.76(m,3H),1.24(s,3H)1.08-1.05(s,3H),0.91-0.87 (m,3H).

Example 12: Synthesis of P57

Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3-trifluoro-2-hydroxypropionamide

P2 (20 mg, 0.036 mmol, 1 eq) and triethylamine (36 mg, 0.36 mmol, 10 eq) were dissolved in anhydrous DMF (1 mL), and (S)-3,3,3-trifluoro-2-hydroxypropionic acid (25.6 mg, 0.18 mmol, 5 eq) and HATU (67.6 mg, 0.18 mmol, 2 eq) were added. Stir at room temperature for 2.5 hours. LCMS showed that the reaction was complete. Purification by preparative HPLC gave (3 mg, yield 14%) as a white solid.

Characterization data of P57:

LC-MS (ESI) m/z: 592 [M+H] ⁺ .

2. Synthesis of intermediates

Example 13: Synthesis of K1

(S)-10-Benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaoxo Azatribenzoic acid.

Step 1: Synthesis of methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate.

To a solution of 2-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (20.0 g, 56.5 mmol) in THF (600 mL) and Tol (200 mL) was added Pb(OAc) ₄ (34.7 g, 78.3 mmol) and pyridine (5.4 mL) at room temperature. After addition, the mixture was stirred at 75 °C for 12 hours. LCMS showed that the reaction was complete. The mixture was filtered and concentrated to give a crude product, which was purified by silica gel flash column chromatography (0-100% ethyl acetate/petroleum ether) to give methyl (2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetate (13.6 g, 66% yield) as a white solid.

LC-MS(ESI)m/z:390.9[M+Na] ⁺ .

Step 2: Synthesis of 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-(4-nitrophenyl)benzyl carbonate

To a solution of methyl (2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (6.60 g, 17.9 mmol) in THF (150 mL) at 0°C were added benzyl 2-hydroxyacetate (5.95 g, 35.8 mmol) and PTSA (1.04 g, 6.05 mmol). After the addition, the mixture was stirred at room temperature for 4 hours. TLC showed that the reaction was complete. The mixture was added with H ₂ O (150 mL) and extracted with ethyl acetate (150 mL×3). The organic phase was separated, washed with saturated brine (80 mL), dried _over anhydrous _Na2SO4 , filtered and concentrated to give the crude product, which was purified by silica gel flash column chromatography (0-80% ethyl acetate in petroleum ether) to give 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oic acid benzyl ester (4.9 g, 58% yield) as a white solid.

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.79-8.73 (m, 1H), 7.89 (d, J = 8.0Hz, 2H), 7.71 (d, J = 8.0Hz, 2H), 7.60-7.50 (m,1H),7.33-7.42(m,9H),5.75(s,1H),4.63(d,J＝8.0Hz,2H),4.15-4.30(m,5H),3.63(d,J＝8.0 Hz, 2H), 3.18 (d, J = 4.0Hz, 1H).

Step 3: Synthesis of benzyl 2-((2-aminoacetamido)methoxy)acetate.

To a solution of benzyl 1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecane-11-oate (4.00 g, 8.40 mmol) in DMF (20 mL) was added piperidine (4 mL) at 0° C. The reaction mixture was stirred at room temperature for 2 hours and then concentrated in vacuo to give a crude product of benzyl 2-((2-aminoacetamido)methoxy)acetate (5.0 g, crude product) as a white solid, which was used directly in the next step without further purification.

Step 4: Synthesis of 4-nitrophenyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamide)acetate.

To a solution of 2-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetic acid (50.0 g, 141 mmol) in THF (800 ml) was added 4-nitrophenol (49.1 g, 353 mmol) and DCC (72.7 g, 353 mmol) at room temperature. After addition, the mixture was stirred at room temperature for 12 hours. LCMS showed that the reaction was complete. The mixture was filtered to give 2-(2-((((9H-fluorene-9-yl)methoxy)carbonyl)amino)acetamido)acetate (42.0 g, 63%) as a white solid which was used directly in the next step without further purification.

LC-MS(ESI)m/z:476.2[M+H] ⁺ .

Step 5: Synthesis of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxo-4,7,10-triazadodecane-12-oic acid

To a solution of 4-nitrophenyl 2-(2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)acetate (22.0 g, 46.3 mmol) in THF (300 mL) was added a solution of Na ₂ CO ₃ (7.94 g, 92.6 mmol) in H ₂ O (99 mL) at room temperature, followed by (S)-2-amino-3-phenylpropionic acid (6.11 g, 37.1 mmol). The resulting mixture was stirred at room temperature for 12 hours. LCMS showed the reaction was complete. H ₂ O (100 mL) was added to the mixture and extracted with ethyl acetate (200 mL). The organic phase was separated, dried _over anhydrous _Na2SO4 , filtered and concentrated to give the crude product, which was purified by silica gel flash column chromatography (0-15% methanol in dichloromethane) to give (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxo-2-oxo-4,7,10-triazadodecane-12-oic acid (11 g, 59% yield) as a white solid.

LC-MS(ESI)m/z:501.9[M+H] ⁺ .

Step 6: Synthesis of (S)-benzyl 11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazaeicosanoic acid ester.

At 0°C, (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9-trioxy-2-oxa-4,7,10-triazadodecane-12-oic acid (5.00 To a solution of 2-((2-aminoacetamido)methoxy)benzyl acetate (5.01 g, 19.8 mmol) and HATU (7.58 g, 19.9 mmol) in DMF (20 mL) was added benzyl 2-((2-aminoacetamido)methoxy)acetate (5.01 g, 19.8 mmol). After addition, the mixture was stirred at room temperature for 1 hour. LCMS showed that the reaction was complete. The mixture was concentrated to give a crude product, which was purified by silica gel flash column chromatography (0-10% methanol/dichloromethane) to give (S)-benzyl 11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,13,13,16-pentaazaeicosane-20-acid salt (1.00 g, 14% yield) as a yellow solid.

LC-MS(ESI)m/z:569.9[M+H] ⁺ .

Step 7: Synthesis of (S)-11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazaiconic acid.

Pd/C (10% water, 100 mg) was added to a solution of (S)-benzyl 11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazaeicosane-20-acid salt (1.00 g, 1.36 mmol) in methanol (20 mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under vacuum to give the crude product (S)-11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazaeicosane-20-acid (900 mg crude product) as a yellow solid, which was used directly in the next step without further purification.

LC-MS(ESI)m/z:569.8[M+H] ⁺ .

Step 8: Synthesis of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-1-oic acid

To (S)-benzyl (S)-11-benzyl-1-(9H-fluorene-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxo-4,7,10,13,16-pentaazaeicosane-20-acid (900mg, 1.39mmol) in DMF (10mL) solution was added TEA (2mL) at room temperature. The reaction mixture was stirred at room temperature for 12 hours. The reaction mixture was filtered and the filtrate was concentrated under vacuum to obtain a crude product (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecane-1-acid (1.50g, crude product) as a yellow solid, which was used directly in the next step without further purification.

LC-MS(ESI)m/z:424.2[M+H] ⁺ .

Step 9: Synthesis of (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaazatriazole-1-carboxylic acid.

To a solution of (S)-16-amino-10-benzyl-6,9,12,15-tetraoxo-3-oxo-5,8,11,14-tetraazahexadecane-1-oic acid (1.0 g, 2.36 mmol) in DMF (5 mL) at 0°C was added 2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl-6-(2,5-difluoro-2,5 dihydro-1H pyrrol-1-yl) hexanoate (1.08 g, 3.54 mmol) and N,N-diisopropylethylamine (1.20 g, 9.45 mmol). After the addition was complete, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product, which was purified by reverse phase flash column chromatography (0-70% acetonitrile/ _H2O ) to give (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaazatriazole-1-carboxylic acid (201 mg, 14% yield) as a white solid.

Characterization data of K1:

LC-MS(ESI)m/z:617.0[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ): δ8.60-8.50(m,1H),8.32-8.27(m,1H),8.15-7.97(m,3H),7.31-7.11(m,5H), 7.00(s,2H),4.61(d,J＝6.4Hz,1H),4.54-4.45(m,1H),3.98(s,2H),3.81-3.53(m,6H),3.10-3.00(m, 1H),2.84-2.74(m,2H),2.11(t,J=7.4Hz,2H),1.55-1.40(m,5H),1.25-1.00(m,3H).

3. Synthesis of drug linkers

Example 14: Synthesis of LP1

N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

Synthesis of LP1

At 0°C, to (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-10,13-dione (10.00 mg, 0.021 mmol) in DMF (10% HCl) was added. (S)-10-benzyl-23-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-6,9,12,15,18-pentaoxo-3-oxo-5,8,11,14,17-pentaazatrienoic acid (40.00 mg, 0.064 mmol), TCFH (18 mg, 0.064 mmol) and NMI (11.00 mmol) were added to the solution. After addition, the mixture was stirred at room temperature for 2 hours. LCMS showed that the reaction was complete. The mixture was concentrated to give the crude product which was purified by C18 (0-70% ACN in _H2O ) to give N-((S)-10-benzyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1-(hydroxymethyl)-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide (1.05 mg, 5% yield) as a white solid.

Characterization data of LP1:

LC-MS(ESI)m/z:1063.5[M+H] ⁺ .

¹ H NMR (400MHz, DMSO): δ8.58(t,J＝5.6Hz,1H),8.30(t,J＝7.2Hz,1H),8.13(d,J＝7.2Hz,1H),8.08(t ,J＝6.0Hz,1H),8.02(d,J＝5.6Hz,1H),7.89(d,J＝10.4Hz,1H),7.38(s,1H),7.29–7.15(m,6H),6.99 (s,2H),6.57-6.53(m,1H),5.57(s,1H),5.52(s,2H),4.60(d,J＝6.8H z,2H),4.50-4.49(m,2H),4.19-4.17(m,1H),3.79–3.61(m,5H),3.26–3.15(m,8H),3.07-3.04(m,1H), 2.85–2.76(m,1H),2.68(s,1H),2.39(s,3H),2.31–2.22(m,1H),2.12-2.08(m,2H),2.07-1.90(m,2H), 1.54-1.41(m,4H),1.2-1.08(m,3H),0.89(t,J＝7.2Hz,3H).

Example 15: Synthesis of LP3

N-((S)-10-benzyl-1-(((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1-vinyl-2,3,9,10,13,15-hexahydro-1H,12H-benzo[d]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecane-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide

Synthesis of LP3

P12 (5 mg, 0.0022 mmol), K1 (10 mg, 0.0033 mmol,), DMAP (0.65 mg, 0.0011 mmol) and HOAt (7.5 mg, 0.011 mmol) were dissolved in anhydrous DMF (0.50 mL). EDCI (10 mg, 0.011 mmol) was added to the reaction solution and stirred at room temperature for 2-3 hours. The reaction mixture was purified by preparative HPLC to obtain LP3 (4.3 mg, yield 37.5%) as a yellow solid.

Characterization data of LP3:

LC-MS(ESI)m/z:1060.33[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.68-8.65(m,1H),8.42(s,1H),8.14-8.12(m,1H),8.07-8.05(m,1H),8.00-8.96 (m,1H),7.82-7.79(m,1H),7.33(s,1H),7.26-7.17(m,6H),7.11(s,1H),6.99(s,1H),6.98(s,2H ),6.28-6.21(m,1H),5.40(s,2H),5.27-5.25(m,1H),5.04-5.01 (m,2H),4.65-4.63(m,2H),4.52-4.49(m,2H),3.97-3.91(m,4H),3.78-3,71(m,2H),3.68-3.60(m, 2H),3.36-3.34(m,2H),2.11(S,2H),2.09-2.07(s,2H),1.86-1.84(s,1H),1.49-1.44(s,4H),1.24(s, 1H),1.22-1.18(m,2H),0.98-0.86(m,3H).

Example 16: Synthesis of Compound LP6

Synthesis of compound LP6

P12 (3 mg, 0.0065 mmol), (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine glycine (7 mg, 0.013 mmol), DMAP (0.4 mg, 0.0033 mmol) and HOAt (4.5 mg, 0.033 mmol) were added to anhydrous DMF (0.50 mL), and EDCI (6 mg, 0.033 mmol) was added to the above reaction solution and stirred at room temperature for 2-3 hours. The reaction mixture was purified by preparative HPLC to give LP6 (1.6 mg, yield 25%) as a white solid.

Characterization data of LP6:

LC-MS(ESI)m/z:974.4[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.67(s,1H),8.14-8.05(m,1H),8.03-7.99(m,1H),7.99-7.91(m,1H),7.81-7.78 (m,1H),7.32(s,1H),7.19-7.11(m,6H),6.99(s,2H),7.26-7.17(m,6H),7.11(s,1H),6.51(s,1H ),6.19-6.12(m,1H),5.42-5.28(m,3H ),5.09(s,2H),4.62-4.57(m,1H),4.35-4.47(m,1H),3.85-3.54(m,4H),2.91-2.96(m,2H),2.71-2.68(m ,3H),2.38(s,3H),2.08-2.04(m,3H),1.85-1.82(m,2H),1.48-1.41(m,4H),1.24-1.14(m,3H),0.89-0.85 (m,3H).

Example 17: Synthesis of Compound LP7

Step 1: Synthesis of compound LP7-1

A mixture of P43 (10 mg, 0.22 mmol), DMAP (16 mg, 0.13 mmol) and triphosgene (5 mg, 0.017 mmol) was added to a 5 mL EP tube, and DCM (0.5 mL) was added to the mixture. The resulting mixture was stirred at room temperature for 10 minutes. Then tert-butyl (2-(methylamino)ethyl)carbamate (4 mg, 0.23 mmol) was added and stirred for 8 minutes. The mixture was purified by preparative HPLC to obtain LP7-1 (8.2 mg, yield 57%) as a white solid.

LC-MS(ESI)m/z:658.2[M+H] ⁺ .

Step 2: Synthesis of compound LP7-2

HCl/EA (2M, 1 mL) and LP7-1 (4 mg, 0.006 mmol) were added, and stirred at room temperature for 30 minutes. TLC showed no starting material. The mixture was concentrated to obtain LP7-2 as a crude product, which was directly used for the next step without purification.

Step 3: Synthesis of compound LP7

(6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)glycylglycyl-L-phenylalanine glycine (6 mg, 0.03 mmol), EDCI (6 mg, 0.03 mmol) and HOBT (4 mg, 0.03 mmol) were added to a solution of LP7-2 in DMF (0.5 mL). The mixture was stirred at room temperature for 16 hours. LCMS showed the target product, and the target product LP7 (2.3 mg, yield 30%) was purified by preparative HPLC as a white solid.

Characterization data of LP7:

LC-MS(ESI)m/z:1068.5[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.25-7.96(m,5H),7.24-7.17(m,6H),7.09-7.05(m,1H),6.98(s,2H),5.47(s ,1H),5.33(s,2H),3.66-3.58(m,8H),3,28-3.11(m,4H),22.79(s,3H),2.77-2.62(m,5H),2.34-2.08 (m,5H),1.46-1.48(m,4H),1.46(s,4H),11.24-1.18(m,2H),0.94-0.90(m,3H),0.86-0.84(m,1H).

Example 18: Synthesis of Compound LP8

Synthesis of compound LP8

P12 (5 mg, 0.011 mmol), (6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanoyl)-D-valine-D-alanine (8.2 mg, 0.022 mmol), DMAP (0.2 mg, 0.016 mmol) and HOAt (15 mg, 0.11 mmol) were dissolved in anhydrous DMF (200 μL), and EDCI (21 mg, 0.11 mmol) was added. Stirring was carried out at room temperature overnight. Purification by preparative HPLC gave LP8 (1.0 mg, yield 11%) as a white solid.

Characterization data of LP8:

LC-MS(ESI)m/z:825.28[M+H] ⁺ .

¹ H NMR (400MHz, DMSO-d ₆ ) δ8.25 (s, 1H), 8.21 (d, J = 8Hz, 1H), 7.92 (d, J = 8Hz, 1H), 7.80 (d, J = 12Hz, 1H),7.31(s,1H),7.08(s,2H),6.51(s,1H),6.25-6.20(m,1H),5.42(d,J＝8Hz,1H),5.28(d,J＝ 8Hz,1H),5.16-5.05(m,2H),4.32 -4.28(m,2H),4.02-3.98(m,2H),2.80(s,2H),2.37(s,3H),2.32(s,2H),2.11(s,1H),1.91-1.87(m ,2H),1.44-1.40(m,2H),1.24-1.16(m,3H),1.07-1.05(m,2H),0.90-0.86(m,3H),0.83-0.79(m,9H).

Preparation and characterization of antibody-drug conjugates

The following are examples of methods for preparing and analyzing antibody drug conjugates. It should be noted that the following examples are only partial embodiments and are not limited to the embodiments currently described. For example, the antibody is not limited to the antibodies currently displayed, but can also be any other antibody.

Conjugation process for preparing antibody drug conjugates

Place the monoclonal antibody (1 eq.) in a 0.5 mL centrifuge tube, add 30 mM His-HAc pH 5.5 buffer, dilute the antibody concentration to 5 mg/mL, and then add 100 mM EDTA aqueous solution at 5% of the total volume of the reaction solution. After shaking and mixing, add 2 mg/mL TCEP (1.5 eq. to 18 eq.) aqueous solution to the mixture to reduce the antibody. After shaking and mixing, place the reaction on a heating and cooling constant temperature mixer and reduce it at 20°C for 1.5 hours. Add a toxin linker solution (4.0 eq. to 20 eq.) dissolved in DMSO at a concentration of 10 mg/mL, add DMSO at 20% of the total volume of the final reaction solution, shake and mix, and place on a heating and cooling constant temperature mixer for reaction, and couple at 20°C for 0.5 hours.

Use 300 mg/mL dextran-coated activated carbon (manufacturer: Sigma) to remove the residual toxin linkers in the reaction solution that are not coupled to the antibody. Add 10% volume of activated carbon solution to the reaction solution, mix and shake, and shake at 4°C for 2 hours. Take the supernatant to detect the residual toxin linker content. If the free toxin linker content is greater than 1%, the above process should be repeated several times until the test result meets the standard (usually 3 treatments can meet the requirements). After the treatment is completed, the mixture is centrifuged, the supernatant is collected with a syringe and a hydrophilic membrane filter, and the activated carbon is filtered out. The buffer of the desired antibody-drug conjugate is replaced with a suitable storage buffer by 4 ultrafiltrations and stored at -80°C.

General Characterization Methods for Antibody Drug Conjugates

(a) RP-HPLC analysis of DAR values of ADC

High performance liquid chromatograph: e2695 high performance liquid chromatography system.

Chromatographic column: BioResolve RP mAb Polyphenyl (4.6×100mm, 2.7μm) (manufacturer: Waters).

Mobile phase: Mobile phase A (MPA): 0.1% TFA- _H2O ; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following elution program (35%-45%), wherein, from 0 to 3 min, the volume of mobile phase A was 90%-90%, and the volume of mobile phase B was 10%-10%; from 3 to 5 min, the volume of mobile phase A was 90%-65%, and the volume of mobile phase B was 10%-35%; from 5 to 20 min, the volume of mobile phase A was 5%-0%, and the volume of mobile phase B was 95%-100%; from 24 to 26 min, the volume of mobile phase A was 0%-100%, and the volume of mobile phase B was 100%-0%; from 26 to 30 min, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.

Detection conditions: Set the mobile phase flow rate to 1 ml/min, the detection wavelength to 280 nm, and the column temperature to 65 °C.

Experimental steps: Take 50μg of the coupled sample (converted into volume according to the concentration), add 5μL 1M DTT, add storage buffer to a final volume of 50μL, and vortex to mix. Incubate the treated sample at 37℃ for 30min, return to room temperature, centrifuge at 12000rpm for 5min, take 20μL of the supernatant and inject it into the HPLC, elute using the above elution procedure, and record the chromatogram.

DAR value calculation formula: DAR = (L0 peak area ratio × 0 + L1 peak area ratio × 1 + H0 peak area ratio × 0 + H1 peak area ratio × 1 + H2 peak area ratio × 2 + H3 peak area ratio × 3)/100 × 2.

(b) DAR value of ADC analyzed by HIC-HPLC

High performance liquid chromatograph: e2695 high performance liquid chromatography system.

Chromatographic column: MabPac ^™ HIC-Butyl 5 μm 4.6×100 mm (manufacturer: Thermo).

Mobile phase: Mobile phase A (MPA): 1.5M (NH4) ₂ SO ₄ +50mM potassium phosphate (pH 7.0); Mobile phase B (MPB): 50mM sodium phosphate (pH 7.0)/isopropanol (75:25V/V); Elution was performed according to the following elution program (5%-95%), wherein, from 0 to 2 min, the volume of mobile phase A was 100%-95%, and the volume of mobile phase B was 0%-5%; from 2 to 22 min, the volume of mobile phase A was 95%-5%, and the volume of mobile phase B was 5%-95%; from 22 to 24 min, the volume of mobile phase A was 5%-0%, and the volume of mobile phase B was 95%-100%; from 24 to 26 min, the volume of mobile phase A was 0%-100%, and the volume of mobile phase B was 100%-0%; from 26 to 30 min, the volume of mobile phase A was 100%-100%, and the volume of mobile phase B was 0%-0%.

Detection conditions: Set the mobile phase flow rate to 1 ml/min, the detection wavelength to 280 nm, and the column temperature to 30 °C.

Experimental steps: Take 50 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

DAR value calculation formula: DAR = Σ (relative peak area × number of loaded drugs) / 100

(c) DAR value of ADC analyzed by SEC-HPLC

High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm).

Mobile phase: 50mM PB + 200mM Arg (pH 6.80) + 10% IPA. Elution was performed according to the following elution procedure. The volume of mobile phase A in 0-30min was 100%-100%.

Detection conditions: Set the mobile phase flow rate to 0.5 ml/min, the detection wavelengths to 280 nm and 254 nm, and the column temperature to 26 °C.

Experimental steps: Take 20 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

Calculation formula: SEC-DAR = C _drug /C _mAb

The sum of the absorbance of small molecule drugs and antibodies at 280 nm

Total absorbance of small molecule drugs at maximum absorption λ(254)

(d) C18-HPLC analysis of Free Linker Payload (mol/mol%)

High performance liquid chromatograph: e2695 high performance liquid chromatography system.

Chromatographic column: C18 3.5μm 4.6×150mm (manufacturer: Waters).

Mobile phase: Mobile phase A (MPA): 0.1% TFA- _H2O ; Mobile phase B (MPB): 0.1% TFA-ACN; Elution was performed according to the following elution program (5%-95%), wherein, from 0 to 30 min, the volume of mobile phase A was 90%-20%, and the volume of mobile phase B was 10%-80%; from 30 to 31 min, the volume of mobile phase A was 20%-90%, and the volume of mobile phase B was 80%-10%; from 31 to 35 min, the volume of mobile phase A was 90%-90%, and the volume of mobile phase B was 10%-10%.

Detection conditions: Set the mobile phase flow rate to 0.5 ml/min, the detection wavelength to 254 nm, and the column temperature to 30 °C.

Preparation example of reagent I: Measure 30 mL of anhydrous methanol and 50 mL of acetonitrile, weigh 10 g of sodium chloride, mix in a container, stir at room temperature for more than 1 hour, and then let stand for 1 hour. Take the supernatant, filter with a 0.22 μm organic membrane, store at room temperature, and the validity period is 3 months.

Example of preparing reagent II: measure 100 mL of reagent I, 15 mL of DMSO, and 85 mL of ADC sample storage buffer respectively, mix them evenly in a container, and store at room temperature. The shelf life is 2 months.

Experimental steps:

Sample solution preparation:

1) Free toxin reference: Take the toxin corresponding to the test sample as the reference and prepare it with the above reagent II to a final concentration of 1.0 mg/ml.

2) Take 10 μl of the above-mentioned 1.0 mg/ml toxin reference substance and add it to 90 μl of Reagent II to prepare a concentration of 100 μg/ml; then dilute the samples in sequence with Reagent II according to the following table to prepare samples of the required concentration.

Note: Inject the samples prepared above to the required concentration into HPLC in ascending order.

Sample test: Take 85 μg of the coupled sample, mix the sample with 3 μl DMSO for 5 minutes, then take 60 μl of reagent I and add it to the sample system, mix for 5-10 minutes, centrifuge at 2000 rpm for 2 minutes, take 20 μL of the supernatant, inject it into the HPLC, elute using the above elution procedure, and record the chromatogram.

The content of free toxin in ADC was calculated based on the calculated standard binary linear regression curve equation.

Free Drug (mol/mol%) = residual small molecule molar concentration/antibody molar concentration × 100.

(e) SEC-HPLC analysis of ADC aggregates

High performance liquid chromatograph: Agilent 1260 liquid chromatograph.

Chromatographic column: Waters Xbridge BEH200 SEC (7.8×300mm, 3.5μm).

Mobile phase: 50mM PB + 200mM Arg (pH 6.80) + 10% IPA. Elution was performed according to the following elution procedure. The volume of mobile phase A in 0-30min was 100%-100%.

Detection conditions: Set the mobile phase flow rate to 0.5 ml/min, the detection wavelength to 280 nm, and the column temperature to 26 °C.

Experimental steps: Take 20 μg of the coupled sample (the volume depends on the sample concentration), inject it into the high performance liquid chromatograph, elute using the above elution procedure, and record the chromatogram.

Calculation formula: Monomer purity (%) = A monomer/A total × 100%; polymer purity (%) = A polymer/A total × 100%

Example 19: Preparation of Antibody Drug Conjugate ADC-1

_n6 ＝7.87

According to the general coupling process for preparing antibody drug conjugates, TCEP (2 mg/mL, 18.0 eq.) aqueous solution was added to 50 mM PBS pH 8.0 buffer of cisplatin (1.0 mg, 4.21 mg/mL, 1.0 eq.) and reduced at 20°C for 16 hours; then LP1 (10 mg/mL, 20.0 eq.) DMSO solution was added and coupled at 20°C for 0.5 hours. After purification by dextran-coated activated carbon treatment twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-1 (C _ADC (mg/mL): 2.52, V (mL): 0.084, yield 21.2%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR: 7.87, SEC purity: 92.98%, Free Linker Payload (mol/mol%): 0.98%.

Example 20: Preparation of Antibody Drug Conjugate ADC-2

_n6 ＝7.51

According to the general coupling process for preparing antibody drug conjugates, TCEP (2 mg/mL, 18.0 eq.) aqueous solution was added to trastuzumab (1.0 mg, 38.7 mg/mL, 1.0 eq.) in 50 mM PBS pH 8.0 buffer, and reduced at 20°C for 16 hours; then LP1 (10 mg/mL, 20.0 eq.) in DMSO was added, and the coupling reaction was carried out at 20°C for 0.5 hours. After purification by dextran-coated activated carbon treatment twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-2 (C _ADC (mg/mL): 3.79, V (mL): 0.084, yield 31.8%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR: 7.51, SEC purity: 96.7%, Free Linker Payload (mol/mol%): 0.61%.

Example 21: Preparation of Antibody Drug Conjugate ADC-3

_n6 ＝7.50

According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mg/mL, 15 eq.) aqueous solution was added to trastuzumab (1.5 mg, 21.07 mg/mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP3 (10 mg/mL, 15 eq.) in DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-3 (C _ADC (mg/mL): 6.92, V (mL): 0.12, yield 55.4%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR:7.50,SEC purity:95.71%, Free Linker Payload(mol/mol%):0.00805.

Example 22: Preparation of Antibody Drug Conjugate ADC-4

_n6 ＝8.0

According to the general coupling process for preparing antibody drug conjugates, TCEP (1mM, 15eq.) aqueous solution was added to trastuzumab (1.5mg, 10mg/mL, 1.0eq.) in 20mM PBS pH 7.4 buffer, and reduced at 37°C for 1.5 hours; placed on ice for 5 minutes, and then LP6 (1mM, 15.0eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment for 3 times, the buffer of the desired antibody drug conjugate was ultrafiltered 5 times to obtain ADC-4 (C _ADC (mg/mL): 10, V (mL): 0.1, yield 66%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR: 8.0, SEC purity: 98%, Free Linker Payload (mol/mol%): not detected.

Example 23: Preparation of Antibody Drug Conjugate ADC-5

_n6 ＝8.0

According to the general coupling process for preparing antibody drug conjugates, TCEP (1mM, 15eq.) aqueous solution was added to trastuzumab (1.5mg, 10mg/mL, 1.0eq.) in 20mM PBS pH 7.4 buffer, and reduced at 37°C for 1.5 hours; placed on ice for 5 minutes, and then LP7 (0.62mM, 9.4eq.) DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment three times, the buffer of the desired antibody drug conjugate was ultrafiltered five times to obtain ADC-5 (C _ADC (mg/mL): 4.5, V (mL): 0.2, yield 60%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR: 8.0, SEC purity: 98%, Free Linker Payload (mol/mol%): not detected.

Example 24: Preparation of Antibody Drug Conjugate ADC-6

_n6 ＝7.92

According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mg/mL, 15 eq.) aqueous solution was added to trastuzumab (1.5 mg, 21.07 mg/mL, 1.0 eq.) in 50 mM PBS pH 7.0 buffer, and reduced at 22°C for 2 hours; then LP8 (10 mg/mL, 15 eq.) in DMSO solution was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment three times, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain ADC-6 (C _ADC (mg/mL): 7.16, V (mL): 0.1, yield 47.7%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR: 7.92, SEC purity: 98.96%, Free Linker Payload (mol/mol%): not detected.

Example 25: Preparation of Antibody Drug Conjugate DS-8201a

_n6 ＝7.96

According to the general coupling process for preparing antibody drug conjugates, TCEP (10 mg/mL, 12 eq.) aqueous solution was added to 50 mM PBS pH 7.0 buffer of trastuzumab (5 mg, 21.07 mg/mL, 1.0 eq.), and reduced at 22°C for 2 hours; then, DMSO solution of Deruxtecan (10 mg/mL, 10 eq.) was added, and the coupling reaction was carried out at 22°C for 1 hour. After purification by dextran-coated activated carbon treatment twice, the buffer of the desired antibody drug conjugate was ultrafiltered three times to obtain DS-8201a (C _ADC (mg/mL): 6.74, V (mL): 0.6, yield 80.9%).

The following characterization results were obtained using the general characterization method for antibody drug conjugates:

HIC-DAR:7.96,SEC purity:97.7%, Free Linker Payload(mol/mol%):0.04221.

Biological evaluation

1) Experiment on tumor cell proliferation inhibition of camptothecin derivatives

Add a certain cell concentration of MDA-MB-231, OV90, NCI-N87 cells (1500 cells per well, in 80μl) to a 96-well plate. The cells were cultured overnight in a constant temperature incubator (37°C, 5% CO ₂ ). Prepare a camptothecin derivative solution with a maximum concentration of 2000nM and dilute it in a 1:4 gradient with PBS. Take the diluted solution (20μl per well) and incubate it with the cells (37°C, 5% CO 2) for five days, then add CellTitre-Glo reagent (40μl per well) and use i3X microplate reader to measure the fluorescence value.

In vitro cell killing activity of the camptothecin derivatives of the present invention (++++: <1nM; +++: 1-10nM; ++: 10-200nM; +: >200nM)

The in vitro cell killing activities of P12, P43 and P57 were greater than those of Dxd and exotecan, and the in vitro cell killing activity of P2 was stronger than or equivalent to those of Dxd and exotecan.

2) Tumor cell proliferation inhibition experiment based on antibody-drug conjugates based on camptothecin derivatives

PA-1, SK-BR-3 and NCI-N87 cells (1500 cells per well, in 80μl) at a certain cell concentration were added to a 96-well plate. The cells were cultured overnight in a constant temperature incubator (37°C, 5% CO2). The antibody-drug conjugate solution of camptothecin derivatives was prepared with a maximum concentration of 5000nM and diluted 1:4 with PBS. After the diluted solution (20μl per well) was incubated with the cells (37°C, 5% CO2) for five days, CellTitre-Glo reagent (40μl per well) was added and the fluorescence value was measured using an i3X microplate reader.

In vitro cell killing activity of ROR1-ADCs (++++: <1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM).

In vitro cell killing activity of HER2-ADCs (+++++: <0.1 nM; ++++: 0.1-1 nM; +++: 1-10 nM; ++: 10-200 nM; +: >200 nM).

All ADCs showed some antitumor activity, for example, ADC-4 showed in vitro cell proliferation in SK-BR-3 and NCI-N87 cells. The killing activity was comparable to that of DS-8201a in vitro.

The camptothecin derivatives and antibody-drug conjugates of the present invention have good in vitro cell-killing activity and better anti-tumor effect.

The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modification, equivalent substitution or improvement within the spirit of the present invention is included in the scope of the claims of the present invention.

Claims (24)

Camptothecin derivatives, characterized in that their structure is as shown in general formula I:
in: _R1 and _R2 are each independently selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy or cycloalkyl; A is selected from hydrogen, amino, W is selected from cycloalkyl, Y is selected from -C(=O)-, -S(=O) ₂ -, -CHCF ₃ -, -C(=CH ₂ )-, -P(=O)R ₁₀ -, -C(=NR ₁₁ )-, or -C(=O)NH-; n=an integer of 0-4; Z is selected from hydrogen or -OH; R ₃ in A is selected from hydrogen or alkyl; _R4 and _R5 in A are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl, cycloalkyl substituted alkyl, heterocyclyl, aryl or heteroaryl; B is selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkanoyloxy-substituted alkyl, glycosyl-substituted alkyl, mercapto-substituted alkyl, amino-substituted alkyl, carboxyl-substituted alkyl, ester-substituted alkyl, alkynyl, alkenyl, cyano or cyano-substituted alkyl; R ₆ and R ₇ are the same or different and are independently selected from hydrogen, fluorine or alkyl; R ₈ and R ₉ are the same or different and are independently selected from hydrogen, fluorine or alkyl; R ₁₀ and R ₁₁ are the same or different and are independently selected from saturated or unsaturated alkyl or aryl groups. The camptothecin derivative according to claim 1, characterized in that in the general formula I: _R1 and _R2 are each independently selected from hydrogen, halogen, hydroxy, cyano, alkyl, alkoxy or cycloalkyl; A is selected from hydrogen, amino, Y is selected from -C(=O)-, -S(=O) ₂ -, -CHCF ₃ -, -C(=CH ₂ )-, -P(=O)R ₁₀ -, -C(=NR ₁₁ )-, or -C(=O)NH-; n=an integer of 0 to 4; Z is selected from hydrogen or -OH; R ₃ in A is selected from hydrogen or alkyl; _R4 and _R5 in A are the same or different and are independently selected from hydrogen, trifluoromethyl, alkyl, cycloalkyl, cycloalkyl substituted alkyl, heterocyclyl, aryl or heteroaryl; B is selected from hydrogen, hydroxy, hydroxy-substituted alkyl, alkanoyloxy-substituted alkyl, glycosyl-substituted alkyl, mercapto-substituted alkyl, amino-substituted alkyl, carboxyl-substituted alkyl, ester-substituted alkyl, alkynyl, alkenyl, cyano or cyano-substituted alkyl; R ₆ and R ₇ are the same or different and are independently selected from hydrogen, fluorine or alkyl; R ₈ and R ₉ are the same or different and are independently selected from hydrogen, fluorine or alkyl; R ₁₀ and R ₁₁ are the same or different and are independently selected from saturated or unsaturated alkyl or aryl groups. The camptothecin derivative according to claim 1, characterized in that in the general formula I: _R1 is selected from fluorine; _R2 is selected from methyl; A is selected from hydrogen, amino, W is selected from cyclobutyl, Y is selected from -C(=O)-, -S(=O) ₂ - or - CHCF ₃ -; n=an integer of 0-3; Z is selected from hydrogen or -OH; R ₃ in A is selected from hydrogen or methyl; _R4 and _R5 in A are the same or different and are independently selected from hydrogen, methyl, trifluoromethyl, B is selected from hydrogen, _-CH2OH , _-CH2OAc , _-CH2SH , _-CH2NH2 , _-CH2C ( ₌ O)OH, _-CH2C (=O)OMe, ethynyl, vinyl, cyano or _-CH2CN ; R ₆ and R ₇ are the same or different and are independently selected from hydrogen or fluorine; _R8 and _R9 are the same or different and are independently selected from hydrogen or fluorine. The camptothecin derivative according to claim 1, 2 or 3, characterized in that R ₄ and R ₅ together with the carbon atom to which they are connected constitute a cyclopropyl group. The camptothecin derivative according to claim 1, 2 or 3, characterized in that _R4 and _R5 are not hydrogen at the same time. The camptothecin derivative according to claim 1, 2 or 3, characterized in that when B is hydrogen, A is also hydrogen; when A is amino, B is not hydrogen. The camptothecin derivative according to claim 1, characterized in that R ₃ , B and the carbon atom to which A and B are connected together and/or the nitrogen atom to which R ₃ is connected constitute an aziridine group or an azetidine group, and/or, _R1 and _R2 and their adjacent carbon atoms form a cycloalkyl or heterocyclic group, and/or, _R4 and _R5 together with the carbon atom to which they are attached constitute a cycloalkyl group or a heterocycloalkyl group. The camptothecin derivative according to claim 1, characterized in that the compound of general formula I is at least one of the structures shown in the following formulas:


The camptothecin derivative according to any one of claims 1 to 8, characterized in that it also comprises a prodrug of the compound of general formula I, or a pharmaceutically acceptable salt, ester, solvate, tautomer, stereoisomer, polymorph, nitrogen oxide or isotope label thereof. Use of the camptothecin derivative according to any one of claims 1 to 9 in the preparation of a drug for treating cancer. A compound of general formula II, characterized in that:
ML ₁ -L ₂ -D
Formula Ⅱ Among them, M is selected from and/or, L ₁ is selected from n ₁ = an integer from 1 to 10, n ₂ = an integer from 1 to 12, n ₃ = an integer from 1 to 10, n ₄ = an integer from 1 to 12; and/or L ₂ is selected from n ₅ = an integer of 7-24, L ₂ is connected to A in the general formula I; or L ₂ is selected from _L2 is connected to the O of OH in the general formula I. D is selected from the product group formed by the camptothecin derivative according to any one of claims 1 to 9 losing one or more atoms or groups; In the general formula II, one or two of _L1 and _L2 are selected. The compound according to claim 11, characterized in that M is selected from and/or L ₁ is selected from and/or L ₂ is selected from _L2 is connected to A in Formula I; or L ₂ is selected from _L2 is connected to the O of OH in the general formula I. The compound according to claim 11, characterized in that The compound of formula II is at least one of the following compounds, wherein _L2 is connected to A in formula I:
or, The compound of formula II is selected from the following compounds, wherein _L2 is connected to the O of OH in formula I:
The compound according to claim 11, 12 or 13, characterized in that it also includes a prodrug of the compound of general formula II, or a pharmaceutically acceptable salt, ester, solvate, tautomer, stereoisomer, polymorph, nitrogen oxide or isotope label thereof. Use of the compound of claim 11, 12, 13 or 14 in the preparation of a medicament for treating cancer. An antibody-drug conjugate of formula III, characterized in that:
Ab is an antibody, an antibody fragment or a protein, wherein the antibody is selected from a mouse antibody, a rabbit antibody, a phage display antibody, a yeast display antibody, a chimeric antibody, a humanized antibody, a fully human antibody, an antibody fragment, a bispecific antibody or a multispecific antibody; ML ₁ -L ₂ -D is selected from the compound of formula II according to claim 11, 12 or 13, or its pharmaceutically acceptable salt, ester, solvate, tautomer, stereoisomer, polymorph, nitrogen oxide and/or isotope label; n ₆ is selected from any value between 1-20. In the general formula III, the carbon-carbon double bond of M in the ML ₁ -L ₂ -D is connected to the thiol group in the antibody Ab by Michael addition reaction to form a CS bond. The antibody-drug conjugate according to claim 16, characterized in that the antibody is a monoclonal antibody. The antibody-drug conjugate according to claim 16, characterized in that: the antibody is selected from abciximab, alemtuzumab, anetuzumab, atezolizumab, avelumab, basiliximab, bevacizumab, blinatumomab, brentuximab, catumaxomab, cetuximab, cetuximab, cotuximab, daclizumab, daratumumab, deninutuzumab, denosumab, depatuxizumab, denutumab, durvalumab, elotuzumab, enroku, gebatumumab, gemtuzumab, ibritumomab tiuxetan, indalatuzumab, indoximab, inotuzumab, ipilimumab, labetuzumab, latuzumab, latuximab, rifatuzumab, lovotuzumab, miratumumab, metuximab, natalizumab, natuximab, The invention relates to the invention to the invention to at least one of the following: anti-CD4 antibodies, anti-CD5 antibodies, anti-CD13 antibodies, and anti-CD30 antibodies, or antigen-binding fragments or immunologically active portions thereof. The antibody-drug conjugate according to claim 16, characterized in that the antibody is selected from a monoclonal antibody. The antibody-drug conjugate according to any one of claims 16 to 19, characterized in that _n6 is selected from any value between 4 and 10. The antibody-drug conjugate according to claim 20, characterized in that: _n6 is selected from any value between 4 and 8. The antibody-drug conjugate according to claim 16, characterized in that the conjugate is at least one of the structures shown in the following formula:
; where n ₆ is any value between 4 and 8. Use of the antibody-drug conjugate of general formula III as described in claims 16 to 22 in the preparation of a drug for treating cancer. The method for preparing a camptothecin derivative according to any one of claims 1 to 9, characterized in that the synthesis route is: