RU2793316C2 - Ligand-drug conjugate of exatecan analogue, its production method and its use - Google Patents

Abstract

FIELD: organic chemistry.

SUBSTANCE: invention relates to the field of organic chemistry, namely to a ligand-drug conjugate or its pharmaceutically acceptable salt of the formula (Pc-L-Y-Dr), where Y is -O-(CR^aR^b)_m-CR¹R²-C(O)-; R^a and R^b are a hydrogen atom; R¹ is C_3-6 cycloalkyl or C_3-6 cycloalkyl-C_1-6 alkyl; R² is a hydrogen atom; or R¹ and R², together with a carbon atom, to which they are attached, form C_3-6 cycloalkyl; m is 0 or 1; n is from 1 to 10, which can be an integer or a decimal number; Pc is an antibody; and L is a linker. The invention also relates to a compound of the formula (D₁) or to its pharmaceutically acceptable salt, a compound of the formula (L_a-Y-Dr) or to its pharmaceutically acceptable salt, a pharmaceutical composition for the treatment of cancer associated with expression of HER2 or B7H3, containing a ligand-drug conjugate of the formula (Pc-L-Y-Dr) or its pharmaceutically acceptable salt, or a compound of the formula (D₁), or its pharmaceutically acceptable salt, the use of a ligand-drug conjugate of the formula (Pc-L-Y-Dr) or its pharmaceutically acceptable salt, or a compound of the formula (D₁).

EFFECT: treatment of cancer associated with expression of HER2 or B7H3 with a conjugate of the formula (Pc-L-Y-Dr) or a compound of the formula (D₁).

,

,

28 cl, 9 dwg, 4 tbl, 59 ex

Description

FIELD OF TECHNOLOGY

The present invention relates to a ligand-drug conjugate of an exatecan analogue with a novel structure. In particular, the present invention relates to a ligand-drug conjugate of an exatecan analogue with a Y structural unit, a method for its preparation, a pharmaceutical composition containing said conjugate, and the use of the conjugate or pharmaceutical composition.

BACKGROUND OF THE INVENTION

Chemotherapy remains one of the most important anti-cancer therapies, along with surgery, radiation therapy, and targeted therapy. Although there are many types of highly effective cytotoxins, there is very little difference between tumor and normal cells, which limits the wide clinical use of these anticancer compounds due to toxic side effects. Antibody-based drugs have become first-line drugs for antitumor therapy due to the specificity of antitumor monoclonal antibodies to the surface antigen of tumor cells. However, when the antibody is used alone as an anticancer drug, the efficacy is often unsatisfactory.

Antibody-drug conjugates (ADCs) allow a monoclonal antibody or antibody fragment to be combined with a biologically active cytotoxin via a chemically stable linker, taking full advantage of the specificity of antibody binding to surface antigens of normal cells or tumor cells and the high efficiency of the cytotoxin, while avoiding the low efficiency of antibodies and toxic side effect of cytotoxin. This means that, compared to conventional chemotherapy drugs, antibody-drug conjugates can accurately bind to tumor cells and reduce the effects on normal cells (Milliard A, (2013) Nature Reviews Drug Discovery, 12:329-332; DiJoseph JF, Armellino DC, (2004) Blood, 103:1807-1814).

In 2000, the first Mylotarg antibody-drug conjugate (gemtuzumab ozogamicin, Wyeth Pharmaceuticals) was approved by the US Food and Drug Administration (FDA) for the treatment of acute myeloid leukemia (Drugs of the Future (2000) 25(7):686 ; US 4970198; US 5079233; US 5585089; US 5606040; US 5693762; US 5739116; US 5767285; US 5773001).

In August 2011, Adcetris (brentuximab vedotin, Seattle Genetics Inc.) was approved under the US FDA's accelerated review process for the treatment of Hodgkin's lymphoma and recurrent anaplastic large cell lymphoma (Wet Biotechnol (2003) 21(7):778-784; WO 2004010957 ; WO 2005001038; US 7090843A; US 7659241; WO 2008025020). Adcetris® is a new targeted ADC drug that allows the drug to directly target CD30 in lymphoma cells, trigger endocytosis and therefore induce tumor cell apoptosis.

Both Mylotarg and Adcetris are targeted drugs for the treatment of hematological tumors, which have a relatively simple organizational structure compared to solid tumors. In February 2013, Kadcyla (ado-trastuzumab emtansine, T-DM1) was approved by the FDA for the treatment of patients with advanced or metastatic breast cancer who are HER ² positive, trastuzumab (trade name: Herceptin) resistant, and paclitaxel resistant. (WO 2005037992; US 8088387). Kadcyla is the first ADC drug approved by the FDA for the treatment of solid tumors.

There are several types of cytotoxic small molecules used in the antibody-drug conjugate, one of which is camptothecin derivatives, which exhibit an antitumor effect by inhibiting topoisomerase I. Documents reporting the use of the camptothecin derivative, exatecan (chemical name: (1S,9S) -1-amino-9-ethyl-5-fluoro-2,3-dihydro-9-hydroxy-4-methyl-1Н,12Н-benzo[de]pyrano[3',4':6,7]imidazo[1 ,2-b]quinoline-10,13(9H,15H)-dione) in an antibody drug conjugate (ADC) include WO 2014057687, Clinical Cancer Research (2016) 22 (20): 5097-5108, and Cancer Sci (2016) 107: 1039-1046. However, further development of ADC drugs with greater efficacy is still needed.

DISCLOSURE OF THE INVENTION

To improve the ligand, especially the binding effect between the antibody and the drug, provided herein is a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises the structure of formula (-D):

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-, -O- CR ¹ R ² -( CR ^a R ^b )m-, -O-CR ¹ R ² -, -NH-(CR ^a R ^b )m-CR ¹ R ² -C(O)- and -S-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

R ^a and R ^b are the same or different and are each independently selected from the group consisting of hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclyl;

or R ^a and R ^b together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

R ¹ is selected from the group consisting of halogen, deuterated alkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl, aryl and heteroaryl;

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

or R ¹ and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

or R ^a and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

where the wavy line in -D represents a hydrogen atom or a covalent bond with a linker unit or antibody that binds to the antigen expressed by the target cell;

m is an integer from 0 to 4.

In some embodiments of the present invention, the proposed ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, is a compound of formula (-D ₁ or a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof:

Where:

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

the wavy line in formula -D ₁ represents a hydrogen atom or a covalent bond to a linker or antibody that binds to an antigen expressed by the target cell;

m is 0 or 1.

In some embodiments of the present invention, the proposed ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, is a ligand-drug conjugate of formula (Pc-L-Y-Dr), or a pharmaceutically acceptable salt or solvate thereof:

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-, -O- CR ¹ R ² -( CR ^a R ^b ) _m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)- and -S-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-;

R ^a and R ^b are the same or different and are each independently selected from the group consisting of hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclyl;

or R ^a and R ^b together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

R ¹ is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

or R ¹ and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

or R ^a and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

m is an integer from 0 to 4;

n is from 1 to 10, which may be an integer or a decimal number;

PC is a ligand; and L is a linker unit.

In some embodiments of the present invention, in the proposed ligand-drug conjugate or its pharmaceutically acceptable salt or solvate

-Y- is -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

R ^a and R ^b are the same or different and are each independently selected from the group consisting of hydrogen atom, deuterium atom, halogen and alkyl;

R ¹ is C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

m is 0 or 1.

In some embodiments of the present invention, in the proposed ligand-drug conjugate, or a pharmaceutically acceptable 30 salt or solvate thereof, the -Y- structural unit is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

R ¹ is C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of hydrogen, haloalkyl and C _3-6 cycloalkyl;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

m is 0 or 1.

In some embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the -Y- structural unit is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

R ¹ is C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² represents a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1.

In some embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the -Y- structural unit is -O-(CH ₂ )m-CR ¹ R ² -C(O)-;

R ¹ is C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² represents a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, the -Y- structural unit is selected from the group consisting of:

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the terminal O in -Y- is attached to the clinker unit L.

In some other embodiments of the present invention, the proposed ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, is a ligand-drug conjugate of the formula (Pc-L-D1), or a pharmaceutically acceptable salt or solvate thereof:

Where:

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

m is 0 or 1;

n is from 1 to 10, which may be an integer or a decimal number;

PC is a ligand; and L is a linker unit.

In another preferred embodiment of the present invention, in the ligand-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to the present invention, n is from 2 to 8, which may be an integer or a decimal number; and preferably n is from 3 to 8, which may be an integer or a decimal number.

In another preferred embodiment of the present invention, in the ligand-drug conjugate or pharmaceutically acceptable salt or solvate thereof according to the present invention, the linker unit L is -L ¹ -L ² -L ³ -L ⁴ -,

L ¹ is selected from the group consisting of -(succinimide-3-yl-N)-W-C(O)-, -CH ₂ -C(O)-NR ₃ -WC(O)- and -C(O) -WC(O)-, where W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkylcycloalkyl and linear heteroalkyl containing from 1 to 8 atoms, heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of of N, O and S, where C _1-8 alkyl, cycloalkyl and linear heteroalkyl are each independently optionally further substituted with one or more substituents selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl , alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)- and a chemical bond, where p ¹ is an integer from 1 to 20; and L ² preferably represents a chemical bond;

L ³ is a peptide residue of 2-7 amino acids, the amino acids optionally further substituted with one or more substituents selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl;

L ⁴ is selected from the group consisting of -NR ⁵ (CR ⁶ R ⁷ )t-, -C(O)NR ⁵ , -C(O)NR ⁵ (CH ₂ ) _t - and a chemical bond, where t is an integer a number from 1 to 6; and L ⁴ is preferably -NR ⁵ (CR ⁶ R ⁷ )t-;

R ³ , R ⁴ and R ⁵ are the same or different and each is independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl, and hydroxyalkyl.

In some other embodiments of the present invention, in the ligand-drug conjugate or pharmaceutically acceptable salt or solvate thereof, the linker unit L ¹ is selected from the group consisting of -(succinimide-3-yl-N)-(CH ₂ )s ¹ -C( O)-, -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C(O)-, -(succinimide-3-yl-N)-(CH ₂ CH ₂ O)s ² -CH ₂ CH ₂ -C(O)-, -CH ₂ -C(O)-NR ³ -(CH ₂ )s ³ -C(O)- and -C(O)-(CH ₂ )s ⁴ C(O)- where s ¹ is an integer from 2 to 8, s ² is an integer from 1 to 3, s ³ is an integer from 1 to 8, and s ⁴ is an integer from 1 to 8; and s ¹ is preferably 5.

In some other embodiments of the present invention, in the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the linker unit L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- and chemical bond, where p ¹ is an integer from 6 to 12.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, L ⁴ is selected from -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is selected from the group consisting of hydrogen and alkyl, R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen and alkyl, t is 1 or 2, and preferably 2; L ⁴ is preferably NR ⁵ CR ⁶ R ⁷ -; and L ⁴ is more preferably -NHCH ₂ -.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -,

и s¹ представляет собой целое число от 2 До 8;L ¹ is

and s ¹ is an integer from 2 to 8;

L ² is a chemical bond;

L ³ is a tetrapeptide residue;

L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is selected from the group consisting of hydrogen and alkyl, R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl, and t is 1 or 2.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -,

L ¹ represents -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C(O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

L ³ is a tetrapeptide residue;

L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ is selected from the group consisting of hydrogen and alkyl, R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl, and t is 1 or 2.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the peptide residue L ³ is an amino acid residue consisting of one, two or more amino acids selected from the group consisting of phenylalanine (E), glycine (G), valine (V), lysine (K), citrulline, serine (S), glutamic acid (E) and aspartic acid (N), preferably an amino acid residue consisting of one, two or more amino acids selected from the group , consisting of phenylalanine and glycine, more preferably a tetrapeptide residue, and most preferably a GGFG (glycine-glycine-phenylalanine-glycine) tetrapeptide residue.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the L ¹ end of the -L- linker unit is connected to the ligand, and the L ⁴ end of the -L- linker unit is connected to Y.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or its pharmaceutically acceptable salt or solvate -L-Y- is:

L ¹ is selected from the group consisting of -(succinimide-3-yl-N)-(CH ₂ )s ¹ -C(O)- and -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C( O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, and p ¹ is an integer from 6 to 12;

L ³ is a tetrapeptide residue of GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl;

s ¹ is an integer from 2 to 8; and preferably 5;

m is an integer from 0 to 4.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate or its pharmaceutically acceptable salt or solvate -L-Y- is:

and preferably is:

L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-;

L ³ is a tetrapeptide residue of GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl;

m is an integer from 0 to 4.

In some other embodiments of the present invention, in the proposed ligand-drug conjugate of the formula (Pc-L-Y-Dr) or its pharmaceutically acceptable salt or solvate -L-Y- is:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl;

s ¹ is an integer from 2 to 8; and preferably 5;

m is an integer from 0 to 4.

In another aspect of the present invention, there is provided a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises the structure of formula (-L-Y-):

which can be used to form a ligand-drug conjugate by connecting the drug and the ligand through a linker moiety;

Where:

L ¹ is selected from the group consisting of -(succinimide-3-yl-N)-(CH ₂ )s ¹ -C(O)- and -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C( O)-;

L ² is -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- or a chemical bond, and p ¹ is an integer from 1 to 20;

L ³ is a tetrapeptide residue of GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ⁵ , R ⁶ and R ⁷ are the same or different and each is independently selected from the group consisting of hydrogen, alkyl;

s ¹ is an integer from 2 to 8;

m is an integer from 0 to 4.

In another aspect of the present invention, there is provided a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, wherein the ligand-drug conjugate comprises the structure of formula (-L-Y-):

Where:

L ² is a chemical bond;

L ³ is a tetrapeptide residue of GGFG;

R ¹ is cycloalkylalkyl or cycloalkyl, and preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of a hydrogen atom, haloalkyl and C _3-6 cycloalkyl, and preferably a hydrogen atom;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl;

R ⁵ is selected from the group consisting of hydrogen and alkyl, and R ⁶ and R ⁷ are the same or different, and each is independently selected from the group consisting of hydrogen and alkyl;

s1 is an integer from 2 to 8;

m is an integer from 0 to 4.

In some other embodiments of the present invention, the provided ligand-drug conjugate of formula (Pc-L-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof is a ligand-drug conjugate of formula (Pc-C-Y-Dr) or a pharmaceutically acceptable salt thereof or solvate:

Where:

W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkylcycloalkyl and linear heteroalkyl containing from 1 to 8 atoms, heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of N, O and S, where C _1-8 alkyl, cycloalkyl and linear heteroalkyl, each independently optionally further substituted with one or more substituents selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)- and a chemical bond, where p ¹ is an integer from 1 to 20;

L ³ is a peptide residue consisting of 2-7 amino acids, the amino acid may be substituted or unsubstituted, upon substitution, the substituent group(s) may be substituted at any available point on the junction, the substituent group(s) may be one or more groups (group) independently selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R ¹ is selected from the group consisting of halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, preferably cycloalkylalkyl or cycloalkyl, and more preferably C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, heterocyclyl, aryl and heteroaryl, and preferably hydrogen; or R ¹ and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

m is an integer from 0 to 4;

n is a non-zero integer or decimal number from 0 to 10, preferably an integer or decimal number from 1 to 10;

PC is a ligand.

In some other embodiments of the present invention, the provided ligand-drug conjugate of formula (Pc-La-Y-Dr) or a pharmaceutically acceptable salt or solvate thereof is a ligand-drug conjugate of formula (Pc-L _b -Y-Dr) or its pharmaceutically acceptable salt or solvate:

Where:

s ¹ is an integer from 2 to 8; and preferably 5;

PC, R ¹ , R ² , R ⁵ ~R ⁷ type are as defined in the formula (Pc-La-Y-Dr).

The -L-Y- linker unit of the ligand-drug conjugate of the present invention includes, but is not limited to:

Ligand-drug conjugates of formula (Pc-L-Y-Dr) according to the present invention include, but are not limited to:

where Pc and n are as defined in the formula (Pc-La-Y-Dr).

In some other embodiments of the invention, in the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the Pc is an antibody or an antigen-binding fragment thereof, wherein the antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, and a fully humanized antibody, preferably a monoclonal antibody. antibodies.

In some other embodiments of the present invention, in the ligand-drug conjugate or pharmaceutically acceptable salt or solvate thereof, the antibody or antigen-binding fragment thereof is selected from the group consisting of aHTH-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody , anti-c-Met antibodies, aHTH-HER3 (ErbB3) antibodies, aHTH-HER4 (ErbB4) antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD44 antibodies, anti -CD56 antibodies, anti-CD70 antibodies, anti-CD73 antibodies, anti-CD105 antibodies, anti-CEA antibodies, anti-AZZ antibodies, anti-Cripto antibodies, anti-EphA2 antibodies, anti-G250 antibodies, anti-MUCI antibodies, anti -Lewis Y antibodies, anti-VEGFR antibodies, anti-GPNMB antibodies, anti-Integrin antibodies, anti-PSMA antibodies, anti-Tenascin C antibodies, aHTH-SLC44A4 antibodies, and anti-Mesothelin antibodies and antigen-binding fragments thereof.

In some other embodiments, the ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, the antibody or antigen-binding fragment thereof is selected from the group consisting of Trastuzumab, Pertuzumab, Nimotuzumab, Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab , Lorvotuzumab, cBR96, and Glematumamab and their antigen-binding fragments.

Ligand-drug conjugates of formula (Pc-L-Y-Dr) according to the present invention include, but are not limited to, the following formulas:

where n is a non-zero integer or decimal number from 0 to 10, preferably n is an integer or decimal number from 1 to 10; more preferably, n is from 2 to 8, which may be an integer or a decimal number; and most preferably, n is from 3 to 8, which may be an integer or a decimal number.

In another aspect, the present invention provides a compound of formula (D), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof,

Where:

Y is selected from the group consisting of -O-(CR ^a R ^b )m-CR ¹ R ² -C(O)-, -O-CR ¹ R ² -(CRaRb)m -, -O-CR ¹ R ² -, -NH-(CR ^a R ^b )m-CR ¹ R ² -C(O)- and -S-(CR ^a R ^b )m-CR ¹ R ² -C(O)-;

R ^a and R ^b are the same or different and are each independently selected from the group consisting of hydrogen atom, deuterium atom, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl, and heterocyclyl;

or R ^a and R ^b together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

R ¹ is selected from the group consisting of halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl;

or R ¹ and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

or R ^a and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

m is an integer from 0 to 4.

In a preferred embodiment of another aspect of the present invention, the compound of formula (D) or its tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof is a compound of formula (D^ or its tautomer, mesomer, racemate, enantiomer , a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof,

where: R ¹ is C _3-6 cycloalkylalkyl or C _3-6 cycloalkyl;

R ² is selected from the group consisting of hydrogen, haloalkyl and C _3-6 cycloalkyl;

or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1.

The compounds of formula (D) according to the present invention include, but are not limited to:

or their tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

In a preferred embodiment of another aspect of the present invention, there is provided a compound of formula (L _a -Y-Dr), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof:

Where

W is selected from the group consisting of C _1-8 alkyl, C _1-8 alkylcycloalkyl and linear heteroalkyl containing from 1 to 8 atoms, heteroalkyl contains from 1 to 3 heteroatoms selected from the group consisting of N, O and S, where C _1-8 alkyl, cycloalkyl and linear heteroalkyl, each independently optionally further substituted with one or more substituents selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ CH ₂ C(O)-, -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)-, -S(CH ₂ )p ¹ C(O)- and a chemical bond, where p1 is an integer from 1 to 20;

L ³ is a peptide residue of 2-7 amino acids, the amino acids optionally further substituted with one or more substituents selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy, and cycloalkyl, with substitution, the substituent group(s) may be substituted at any accessible point on the compound, the substituent group(s) is one or more groups independently selected from the group consisting of halo, hydroxy, cyano, amino, alkyl, chloroalkyl, deuterated alkyl, alkoxy and cycloalkyl;

R ¹ is selected from the group consisting of halogen, cycloalkylalkyl, deuterated alkyl, cycloalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl;

R ² is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl, and heteroaryl; or R ¹ and R ² together with the carbon atom to which they are attached form cycloalkyl or heterocyclyl;

R ⁴ and R ⁵ are the same or different and are each independently selected from the group consisting of hydrogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

R ⁶ and R ⁷ are the same or different and are each independently selected from the group consisting of hydrogen, halogen, alkyl, haloalkyl, deuterated alkyl and hydroxyalkyl;

m is an integer from 0 to 4.

In a preferred embodiment of another aspect of the present invention, the subject compound of formula (L _a -Y-Dr) or its tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof is a compound of formula (L _b -Y-Dr ) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof, or a pharmaceutically acceptable salt thereof,

where R ¹ , R ² , R ⁵ ~R ⁷ , s ¹ and m are as defined in the formula (L _a -Y-Dr). Compounds of formula (L _a -Y-Dr) according to the present invention include, but are not limited to:

or their tautomer, mesomer, racemate, enantiomer, diastereomer, or a mixture thereof, or a pharmaceutically acceptable salt thereof.

In another aspect of the present invention, there is provided a process for the preparation of a compound of formula (D ₁ ), or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the following steps:

condensation of a compound of formula (Y ₁ ) and a compound of formula (Dr) to obtain a compound of formula (D ₁ ),

where: R ¹ , R ² and m are as defined in formula (D ₁ ).

In another aspect of the present invention, there is provided a process for the preparation of a compound of formula (L _b -Y-Dr) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

condensation of a compound of formula (IA) and a compound of formula (IB) to obtain a compound of formula (L _b -Y-Dr),

where: R ¹ , R ² , R ⁵ ~R ⁷ , s ¹ and m are as defined in the formula (L _b -Y-Dr).

In another aspect of the present invention, there is provided a process for the preparation of a compound of formula (L _b -Y-Dr) or a tautomer, mesomer, racemate, enantiomer, diastereomer, or mixture thereof, or a pharmaceutically acceptable salt thereof, comprising the steps of:

condensation of a compound of formula (IA) and a compound of formula (IB) to obtain a compound of formula (L _b -Y-Dr),

where: R ¹ , R ² , R ⁵ ~R ⁷ , s ¹ and m are as defined in the formula (L _b -Y-Dr).

In another aspect of the present invention, there is provided a method for preparing a ligand-drug conjugate of the formula (Pc-L _a -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof, comprising the step of:

PC is coupled to a compound of formula (L _a -Y-Dr) after reduction to give a compound of formula (Pc-L _a -Y-Dr); the reducing agent is preferably TCEP;

Where:

PC is a ligand;

W, L ² , L ³ , R ¹ , R ² , R ⁵ ~R ⁷ , m and n are as defined in the formula (Pc-L _a -Y-Dr).

Another aspect of the present invention also relates to a pharmaceutical composition comprising a therapeutically effective amount of a ligand-drug conjugate or compound or a pharmaceutically acceptable salt or solvate thereof according to the present invention, and one or more pharmaceutically acceptable carriers, diluents or excipients.

Another aspect of the present invention also relates to a pharmaceutical composition comprising a compound of formula (D) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof or a pharmaceutically acceptable salt thereof according to the present invention, and one or more pharmaceutically acceptable carriers, diluents or excipients. substances.

Another aspect of the present invention also relates to a ligand-drug conjugate, or a pharmaceutically acceptable salt or solvate thereof, comprising a ligand and a ligand-linked drug, the drug being selected from the group consisting of a compound of formula (D), a compound of formula (L _a -Y-Dr) or a tautomer, mesomer, racemate, enantiomer, diastereomer or mixture thereof or a pharmaceutically acceptable salt thereof according to the present invention, wherein the drug is preferably linked to a ligand via a linker and the ligand is preferably a monoclonal antibody.

Another aspect of the present invention also relates to a method for preparing a ligand-drug conjugate or a pharmaceutically acceptable salt or solvate thereof, comprising the step of coupling a compound of formula (D), a compound of formula (L _a -Y-Dr) or its tautomer, mesomer, racemate, enantiomer , a diastereomer or a mixture thereof, or a pharmaceutically acceptable salt thereof according to the present invention, with a ligand, preferably via a linker, and the ligand is preferably a monoclonal antibody.

Another aspect of the present invention also relates to a ligand-drug conjugate or compound or a pharmaceutically acceptable salt or solvate thereof according to the present invention, for use as a medicine.

Another aspect of the present invention also relates to the use of a ligand-drug conjugate or compound, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition containing the same according to the present invention, for the preparation of a medicament for the treatment or prevention of a tumor, and preferably the tumor is is a cancer associated with the expression of HER2, HER3 or EGFR.

Another aspect of the present invention also relates to the use of a ligand-drug conjugate or compound, or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition containing the same according to the present invention, for the preparation of a medicament for the treatment or prevention of cancer, wherein the cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethra cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland cancer, esophageal cancer, melanoma , gliomas, neuroblastomas, sarcomas, lung cancer (eg, small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, colorectal cancer, leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia, acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, prostate cancer, and lymphoma (eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma).

Another aspect of the present invention also relates to a method of treating and/or preventing a tumor comprising administering to a patient in need thereof a therapeutically effective amount of a ligand-drug conjugate or compound or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition containing the same, according to the present invention, and preferably the tumor is a cancer associated with the expression of HER2, HER3 or EGFR.

Another aspect of the present invention also relates to a method for treating or preventing cancer comprising administering to a patient in need thereof a therapeutically effective amount of a ligand-drug conjugate or compound or a pharmaceutically acceptable salt or solvate thereof, or a pharmaceutical composition comprising the same according to the present invention. of the invention, wherein the cancer is preferably selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethra cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary cancer cancer of the esophagus, melanoma, glioma, neuroblastoma, sarcoma, lung cancer (eg, small cell lung cancer and non-small cell lung cancer), colon cancer, rectal cancer, co-rectal cancer, leukemia (eg, acute lymphocytic leukemia, acute myeloid leukemia , acute promyelocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia), bone cancer, skin cancer, thyroid cancer, pancreatic cancer, and lymphoma (eg, Hodgkin's lymphoma, non-Hodgkin's lymphoma, or recurrent anaplastic large cell lymphoma).

The active compound may be in a form suitable for administration by any suitable route, and the active compound is preferably in unit dose form or in a form in which the patient may self-administer a single dose. The unit dose form of a compound or composition of the present invention may be a tablet, capsule, sachet, bottled mixture, powder, granule, lozenge, suppository, reconstitution powder, or liquid preparation.

The dosage of a compound or composition used in a method of treatment according to the present invention will generally vary depending on the severity of the disease, the body weight of the patient, and the relative potency of the compound. However, in general, a suitable unit dose may be from 0.1 to 1000 mg.

In addition to the active compound, the pharmaceutical composition of the present invention may also contain one or more excipients, including a filler (diluent), a binder, a wetting agent, a disintegrant, an excipient, and the like. Depending on the route of administration, the composition may contain from 0.1 to 99% by weight of the active compound.

The pharmaceutical composition containing the active ingredient may be in a form suitable for oral administration, for example, in the form of a tablet, troche, lozenge, aqueous or oily suspension, dispersible powder or granule, emulsion, hard or soft capsule, syrup or elixir. The composition for oral administration may be prepared by any method known in the art for the preparation of a pharmaceutical composition. Such a composition may contain binders, fillers, lubricants, disintegrants or pharmaceutically acceptable wetting agents, and the like. Such a composition may also contain one or more components selected from the group consisting of sweeteners, flavoring agents, coloring agents and preservatives to provide a pleasing and palatable pharmaceutical composition.

The aqueous suspension contains the active ingredient in admixture with excipients suitable for the preparation of an aqueous suspension. The aqueous suspension may also contain one or more preservatives, one or more colorants, one or more flavoring agents, and one or more sweeteners.

An oil suspension can be prepared by suspending the active ingredient in vegetable oil. The oil suspension may contain a thickener. The above sweeteners and flavoring agents may be added to obtain a palatable composition.

The pharmaceutical composition may also be a dispersible powder and granules for the preparation of an aqueous suspension, it is proposed to add water to the active ingredient for mixing with one or more dispersing agents, wetting agents, suspending agents or preservatives. Other adjuvants such as sweeteners, flavoring agents and colorants may also be added. These compositions can be preserved by the addition of antioxidants such as ascorbic acid.

The pharmaceutical composition of the present invention may also be in the form of an oil-in-water emulsion.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable vehicles or solvents that can be used are water, Ringer's solution or isotonic sodium chloride solution. The sterile injectable formulation may be a sterile oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injection solution or microemulsion can be administered into the patient's bloodstream by local bolus injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS IV pump. TM. 5400.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. Such a suspension may be formulated with suitable dispersants or wetting agents and suspending agents as described above, in accordance with known methods. The sterile injectable formulation may also be a sterile injectable solution or suspension formulated in a non-toxic parenterally acceptable diluent or vehicle. Moreover, sterile fixed oils can easily be used as a solvent or suspending medium.

The compound of the present invention may be administered in the form of a rectal suppository. These pharmaceutical compositions can be prepared by mixing the drug with a suitable non-irritating excipient which is solid at ordinary temperatures but liquid in the rectum, thereby melting in the rectum to release the drug. Such materials include cocoa butter, glycerin gelatin, hydrogenated vegetable oil, a mixture of polyethylene glycols of various molecular weights and their fatty acid esters.

It is well known to those skilled in the art that the dosage of a drug depends on a variety of factors, including, but not limited to, the following factors: potency of the particular compound, age of the patient, body weight of the patient, general health of the patient, behavior of the patient, diet of the patient, time of administration, route administration, excretion rate, drug combination, and the like. In addition, the optimal treatment, such as the treatment regimen, the daily dose of the compound of formula (I) or the type of pharmaceutically acceptable salt thereof, can be checked in accordance with conventional therapeutic regimens.

BRIEF DESCRIPTION OF GRAPHICS

Figure 1A: Results of stability tests of ADC-19 according to the present invention in plasma.

Figure 1B: Results of stability tests of ADC-18 according to the present invention in plasma.

Figure 1C: Results of stability tests of ADC-20 according to the present invention in plasma.

Figure 2: Evaluation of the effectiveness of ADC-21 and ADC-24 according to the present invention in mice with JIMT-1 tumor.

Figure 3: Evaluation of the efficacy of the ADCs of the present invention in nude mice with tumor xenograft of human breast cancer cells SK-BR-3.

Figure 4: Results of stability tests of ADC-25 according to the present invention in plasma.

Figure 5: Efficacy of the ADCs of the present invention in nude mice with U87MG human brain astroblastoma tumor xenograft.

Figure 6: Efficacy of the ADCs of the present invention in nude mice with tumor xenograft of metastatic pleural fluid cells of human esophageal carcinoma Detroit 562.

Figure 7: Efficacy of the ADC of the present invention in nude mice with U87MG human glioma tumor xenograft.

DETAILED DESCRIPTION OF THE INVENTION

Definitions

Unless otherwise indicated, all technical and scientific terms used in this application are consistent with the general understanding of experts in the art to which the present invention belongs. While any methods and materials similar or equivalent to those described herein may be used in the practice or testing of the present invention, the present application describes the preferred methods and materials. In describing and previewing the present invention, the following terms are used in accordance with the following definitions.

When a trade name is used in the present invention, the applicant means to include drugs, generic drug and active ingredients of the product under the trade name.

Unless otherwise indicated, terms used in the description and claims have the meanings described below.

A "ligand" is a macromolecular compound capable of recognizing and binding to antigens or receptors associated with a target cell. The role of the ligand is to deliver the drug bound to the ligand to the target cell population. Such ligands include, but are not limited to, proteinaceous hormones, lectins, growth factors, antibodies, or other molecules capable of binding to cells. In an embodiment of the present invention, the ligand is represented by Pc. The ligand may form a bond with the link through a heteroatom on the ligand. The ligand is preferably an antibody or antigen-binding fragment thereof. The antibody is selected from the group consisting of a chimeric antibody, a humanized antibody, a fully humanized antibody, or a mouse antibody, and preferably a monoclonal antibody.

The term "drug" refers to a cytotoxic drug, which is represented as Dr, is a chemical molecule that can greatly interfere with the normal growth of tumor cells. In principle, all cytotoxic drugs can kill tumor cells at sufficiently high concentrations. However, it can cause apoptosis of normal cells and serious side effects by killing tumor cells due to lack of specificity. This term includes toxins such as low molecular weight toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, radioisotopes (for example, radioisotopes At ²¹¹ , I ¹³¹ , I ¹²⁵ , Y ⁹⁰ , Re ¹⁸⁶ , Re ¹⁸⁸ , Sm ¹⁵³ , Bi ²¹² , P ³² and Lu), toxic drugs, chemotherapeutic drugs, antibiotics and nucleolytic enzymes, and preferably toxic drugs.

The term "linker unit", "linking moiety", or "connector" refers to a chemical structural moiety or bond that is linked to a ligand at one end and linked to a drug at the other end, or linked to other linkers and then linked to a drug . Preferred embodiments of the present invention are represented by L and L ¹ -L ⁴ where the L ¹ end is linked to a ligand and the L ⁴ end is linked to a drug (Dr) via a structural unit Y.

The linker, including the extension unit, the spacer unit and the amino acid unit, can be synthesized by methods known in the art, such as those described in US 2005-0238649 A1. The linker may be a "cleavable linker" that promotes drug release in the cell. For example, an acid-labile linker (eg, hydrazone), a protease-sensitive (eg, peptidase-sensitive) linker, a light-sensitive linker, a dimethyl linker, or a disulfide-containing linker can be used (Chari et al, Cancer Research 52: 127-131 (1992 ); U.S. Pat. No. 5,208,020).

The term "ligand-drug conjugate" means that the ligand is linked to the biologically active drug through a stable linker. As used herein, a "ligand-drug conjugate" is preferably an antibody-drug conjugate (ADC), meaning that the monoclonal antibody or antibody fragment is linked to a toxic drug with biological activity through a stable linker.

Three-letter codes and single-letter codes for amino acids used in the present description are described in J. biol. chem, 243, p 3558 (1968).

The term "antibody" refers to an immunoglobulin, a structure of four peptide chains linked by an interchain disulfide bond between two identical heavy chains and two identical light chains. Different constant regions of the immunoglobulin heavy chain have different amino acid compositions and sequences, and therefore have different antigenicity. Accordingly, immunoglobulins can be divided into five types or so-called immunoglobulin isotypes, namely IgM, IgD, IgG, IgAn IgE, with corresponding heavy chains μ, δ, γ, α and ε, respectively. According to the amino acid composition of the hinge region and the number and location of heavy chain disulfide bonds, the same Ig type can be further divided into different subtypes, for example, IgG can be divided into IgG1, IgG2, IgG3 and IgG4. The light chain can be divided into k- or A-chains based on different constant regions. Every five lg types can have a chain k or λ. The antibodies described in the present invention are preferably specific antibodies against cell surface antigens on target cells, non-limiting examples are one or more of the following antibodies: anti-HER2 (ErbB2) antibody, anti-EGFR antibody, anti-B7-H3 antibody , anti-c-Met antibody, anti-HER3 (ErbB3) antibody, anti-HER4 (ErbB4) antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD44 antibody, anti -CD56 antibody, anti-CD70 antibody, anti-CD73 antibody, anti-CD105 antibody, anti-CEA antibody, anti-A33 antibody, anti-Cripto antibody, anti-EphA2 antibody, anti-G250 antibody, anti-MUCI antibody, anti -Lewis Y antibody, anti-VEGFR antibody, anti-GPNMB antibody, anti-integrin antibody, anti-PSMA antibody, anti-Tenascin C antibody, anti-SLC44A4 antibody, or anti-Mesothelin antibody, and preferably Trastuzumab (trade name Herceptin), Pertuzumab (also known as 2C4, trade name Perjeta), Nimotuzumab (trade name Taixinsheng), Enoblituzumab, Emibetuzumab, Inotuzumab, Pinatuzumab, Brentuximab, Gemtuzumab, Bivatuzumab, Lorvotuzumab, cBR96, and Glematumamab.

The sequence of about 110 amino acids adjacent to the N-terminus of heavy chains or light chains of an antibody is highly variable, known as the variable region (Fv region); the remainder of the amino acid sequence adjacent to the C-terminus is relatively stable and is known as the constant region. The variable region includes three hypervariable regions (HVR) and four relatively conserved framework regions (FR). The three hypervariable regions that determine the specificity of an antibody are also known as complementarity determining regions (CDRs). Each light chain variable region (LCVR) or each heavy chain variable region (HCVR) consists of three CDRs and four FR regions, which are sequentially arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3 , FR4. The three light chain CDR regions are referred to as LCDR1, LCDR2 and LCDR3; and the three CDR regions of the heavy chain are referred to as HCDR1, HCDR2 and HCDR3.

The antibodies of the present invention include mouse antibodies, chimeric antibodies, humanized antibodies and fully humanized antibodies, and preferably humanized antibodies and fully humanized antibodies.

The term "mouse antibody" in the present invention refers to an antibody obtained from a mouse in accordance with the knowledge and skill in the art. During preparation, a specific antigen is administered to the test subject, and then a hybridoma is isolated expressing an antibody that has the desired sequence or functional characteristics.

The term "chimeric antibody" means an antibody obtained by fusing the variable region of a mouse antibody with the constant region of a human antibody, and the chimeric antibody can attenuate the immune response induced by the mouse antibody. To create a chimeric antibody, a hybridoma secreting a mouse specific monoclonal antibody is created, and the variable region gene is cloned from a mouse hybridoma cell; then, if necessary, the human antibody constant region gene is cloned; and a human constant region gene is fused with a mouse variable region gene to form a chimeric gene, which is subsequently inserted into an expression vector; finally, the chimeric antibody molecule is expressed in a eukaryotic or prokaryotic system.

The term "humanized antibody", which is also known as a CDR-grafted antibody, refers to an antibody obtained by grafting murine CDR sequences into the framework region of a human antibody variable region, i.e. to an antibody produced with various types of human germline antibody framework region sequences. The humanized antibody can overcome the heterologous responses caused by the high amount of mouse protein components carried by the chimeric antibody. Such framework sequences may be obtained from a public DNA database covering germline antibody gene sequences or from published references. For example, the germline DNA sequences of the human heavy and light chain variable region genes can also be found in the VBase human germline sequence database (available online at www.mrccpe.com.ac.uk/vbase), as well as in Kabat, E.A., et al. 1991, Sequences of Proteins of Immunological Interest, ^5th Ed. To avoid a decrease in activity caused by a decrease in immunogenicity, framework sequences in the variable region of a human antibody can be subjected to minimal reversible mutations or back mutations to maintain activity. The humanized antibody of the present invention also includes a humanized antibody in which CDR affinity maturation is performed by phage display. Documents that further describe methods for using murine antibodies involved in humanization include, for example, Queen et al., Proa, Natl.Acad.Sci.USA, 88, 2869, 1991, and Winter and colleagues' method [Jones et al. , Nature, 321, 522 (1986), Riechmann et al., Nature, 332, 323-327 (1988), Verhoeyen et al., Science, 239, 1534 (1988)].

The term "fully humanized antibody" is also known as "fully humanized monoclonal antibody", where the variable region and constant region of the antibody are of human origin, which excludes immunogenicity and side effects. The development of monoclonal antibodies goes through four stages, namely: mouse monoclonal antibody, chimeric monoclonal antibody, humanized monoclonal antibody and fully humanized monoclonal antibody. The antibody of the present invention is a fully humanized monoclonal antibody. Related technologies for producing fully humanized antibodies mainly include human hybridoma technology, EBV-transformed B lymphocyte technology, phage display technology, transgenic mouse antibody technology, single B cell technology, and the like.

The term "antigen-binding fragment" refers to one or more antibody fragments that retain the ability to specifically bind to an antigen. It has been shown that full length antibody fragments can be used to achieve antigen binding function. Examples of binding fragments in the term "antigen binding fragment" include (i) a Fab fragment, a monovalent fragment consisting of a VL domain, VH, CL and CH1; (ii) an F(ab') ₂ fragment, a bivalent fragment containing two Fab fragments linked by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VH and VL domains of a single antibody; (v) single domain or fragment dAb (Ward et al. (1989) Nature 341:544-546) consisting of a VH domain; and (vi) a dedicated complementarity determining region (CDR) or (vii) a combination of two or more dedicated CDRs, optionally joined by a synthetic linker. In addition, although the VL domain and the VH domain of the Fv fragment are encoded by two separate genes, they can be joined by a synthetic linker using recombinant methods, thereby generating a single protein chain of a monovalent molecule formed by pairing the VL and VH domain (referred to as single-chain Fv (scFv); see, for example, Bird et al (1988) Science: 242:423-426, and Huston et al (1988) Proc Natl Acad Sci USA 85:5879-5883). This single chain antibody is also intended to be included in the term "antigen binding fragment" of an antibody. Such antibody fragments are prepared using conventional methods known to those skilled in the art and tested for functional fragments using the same method as for an intact antibody. Antigen-binding sites can be obtained using recombinant DNA technology or enzymatic or chemical destruction of intact immunoglobulin. Antibodies can be antibodies of different isotypes, for example, IgG (eg, IgG1, IgG2, IgG3 or IgG4 subtype), IgA1, IgA2, IgD, IgE or IgM antibodies.

Fab is an antibody fragment obtained by treating an IgG antibody molecule with papain (which cleaves the amino acid residue at position 224 of the H chain). The Fab fragment has a molecular weight of about 50,000 and has antigen-binding activity in which about half of the N-terminal side of the H chain and the entire L chain are linked to each other via a disulfide bond.

F(ab')2 is an antibody fragment prepared by downstream hydrolysis of two disulfide bonds in the hinge region of IgG with pepsin, which has a molecular weight of about 100,000, has antigen-binding activity, and includes two Fab regions that are linked at the hinge position.

Fab' is an antibody fragment obtained by cleavage of the disulfide bond in the hinge region of the above F(ab')2, which has a molecular weight of about 50,000 and has antigen-binding activity.

Moreover, Fab' can be obtained by inserting DNA encoding the Fab' antibody fragment into a prokaryotic expression vector or eukaryotic expression vector, which is then introduced into prokaryotes or eukaryotes to express Fab'.

The term "single chain antibody", "single chain Fv", or "scFv" refers to a molecule containing an antibody heavy chain variable domain (or region; VH) and an antibody light chain variable domain (or region; VL) connected by a linker. Such scFv molecules may have the general structure NH ₂ -VL-linker-VH-COOH or NH ₂ -VH-linker-VL-COOH. A suitable linker in the prior art consists of the repeat amino acid sequence GGGGS or a variant thereof, e.g. using a 1-4 repeat variant (Holliger et al. (1993), Proc. Natl. Acad. Sci. USA 90:6444-6448) . Other linkers that can be used in the present invention are described in Alfthan et al. (1995), Protein Eng. 8:725-731, Choi et al. (2001), Euro. J. Immunol. 31:94-106, Hu et al. (1996), Cancer Res. 56:3055-3061, Kipriyanov et al. (1999), J. Mol. Biol. 293:41-56 and Roovers et al. (2001), Cancer Immunol.

The term "CDR" refers to one of the six hypervariable regions in the variable domain of an antibody that primarily promotes antigen binding. One of the most commonly used definitions of the six CDRs is provided by Kabat E. A. et al. (1991) Sequences of proteins of immunological interest, NIH Publication 91-3242. In this context, the definition of CDR by Cabot applies only to CDR1, CDR2 and CDR3 of the light chain variable domain (CDR L1, CDR L2, CDR L3 or L1, L2, L3), as well as CDR ² and CDR3 of the heavy chain variable domain (CDR H2 , CDR H3 or H2, H3).

The term "antibody scaffold" refers to the portion of the VL or VH variable domain that serves as the scaffold for the antigen-binding loop (CDR) of the variable domain. In fact, it is a variable domain without a CDR.

The term "epitope" or "antigenic determinant" refers to the site of an antigen to which an immunoglobulin or antibody specifically binds. Epitopes typically include at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 contiguous or non-contiguous amino acids in a unique spatial conformation. See, for example, Epitope Mapping Protocols in Methods in Molecular Biology, Vol. 66, G. E. Morris, Ed. (1996).

The terms "specific binding", "selective binding", "selectively binds" and "specific binding" refer to the binding of an antibody to an epitope on a predetermined antigen. Typically, the antibody binds with an affinity (KD) of less than about 10 ^-7 M, such as less than about 10 ^-8 M, 10 ^-9 M, or 10 ^-10 M or less.

The term "nucleic acid molecule" refers to a DNA molecule and an RNA molecule. The nucleic acid molecule may be single or double stranded, but is preferably double stranded DNA. A nucleic acid is "effectively linked" when it is operably linked to another nucleic acid sequence. For example, if a promoter or enhancer affects the transcription of a coding sequence, the promoter or enhancer effectively binds to the coding sequence.

The term "vector" refers to a nucleic acid molecule capable of carrying another nucleic acid to which it is linked. In one embodiment, the vector is a "plasmid", which refers to a circular double-stranded loop of DNA into which an additional segment of DNA can be ligated. In another embodiment, the vector is a viral vector in which an additional segment of DNA can be ligated into the viral genome. The vectors disclosed herein are capable of self-replication in the host cell into which they have been introduced (e.g., a bacterial vector having a bacterial origin of replication and a mammalian episomal vector), or can be integrated into the genome of a host cell when introduced into the host cell. , thereby replicating along with the host genome (eg, a non-episomal mammalian vector).

Methods for making and purifying antibodies and antigen-binding fragments are well known in the art, for example, in the Cold Spring Harbor Antibody Technical Guide, Chapters 5-8 and 15. The antigen-binding fragment can also be prepared by conventional methods. The antibodies or antigen-binding fragments of the present invention are genetically engineered to add one or more human FR regions to non-human CDR regions. The human germline FR sequence(s) can be obtained by aligning the IMGT germline variable region gene databases and using the MOE software from the ImMunoGeneTics (IMGT) website available at http://imgt.cines. fr or in the Journal of Immunoglobulins 20011SBN012441351.

The term "host cell" refers to a cell into which an expression vector has been introduced. Host cells may include bacterial, microbial, plant or animal cells. Bacteria subject to transformation include members of the Enterobacteriaceae such as strains of Escherichia coli or Salmonella; Bacillaceae such as Bacillus subtilis; pneumococcus; Streptococcus and Haemophilus influenzae. Suitable microorganisms include Saccharomyces cerevisiae and Pichia pastoris. Suitable animal host cell lines include CHO cells (Chinese Hamster Ovary cell line) and NSO cells.

The engineered antibody or antigen-binding fragment of the present invention can be prepared and purified by conventional methods. For example, the cDNA sequence(s) encoding the heavy chain and light chain can be cloned and recombined into a GS expression vector. The recombinant immunoglobulin expression vector can be stably transfected into CHO cells. In the case of the recommended existing technology, mammalian expression systems can lead to antibody glycosylation, especially at the highly conserved N-terminal site of the Fc region. Positive clones accumulate in serum-free medium in a bioreactor to obtain antibodies. The culture medium containing the secreted antibody can be purified in the usual way. For example, purification is carried out using a Sepharose FF A or G column that contains a matched buffer. Non-specifically bound components are removed by elution. The bound antibody is eluted by the pH gradient method, and the antibody fragments are detected by SDS-PAGE and collected. The antibody can be filtered and concentrated in the usual way. Soluble aggregates and multimers can also be removed by conventional methods such as gel filtration or ion exchange. The resulting product should be frozen immediately, for example at -70°C, or lyophilized.

The term "peptide" refers to a fragment of the connection between an amino acid and a protein, consisting of two or more amino acid molecules linked to each other by peptide bonds. Peptides are structural and functional fragments of proteins. Hormones, enzymes and the like are essentially peptides.

The term "saccharide" refers to a biological macromolecule consisting of three elements: C, H and O, which can be divided into monosaccharides, disaccharides and polysaccharides.

The term "fluorescent probe" refers to a variety of fluorescent molecules with characteristic fluorescence in the ultraviolet, visible and near infrared regions. The fluorescence properties of a fluorescent probe (excitation and emission wavelengths, intensity, lifetime and polarization, etc.) can vary sensitively depending on environmental properties such as polarity, refractive index, viscosity, etc. Non-covalent interaction between a fluorescent probe and a nucleic acid (DNA or RNA), protein, or other macromolecular structure allows one or more fluorescent properties to be altered, which can be used to study the property and behavior of the macromolecular substance.

The term "toxic drug" refers to a substance that inhibits or stops the functioning of cells and/or causes cell death or destruction. Toxic drugs include toxins and other compounds that can be used to treat tumors.

The term "toxin" refers to any substance that can have an adverse effect on cell growth or proliferation. The toxins may be small molecule toxins and their derivatives from bacteria, fungi, plants or animals, including camptothecin derivatives such as exatecan, maytansinoid and its derivatives (CN 101573384) such as DM1, DM3, DM4, auristatin F (AF) and its derivatives such as MMAF, MMAE, 3024 (WO 2016/127790 A1, compound 7), diphtheria toxin, exotoxin, ricin A chain, abrin A chain, modecine, a-sarcin, Aleutites fordii toxic protein, diantine toxic protein, toxic protein Phytolaca americana (PAPI, PAPII and PAP-S), Momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogellin, restrictocin, fenomycin, enomycin and trichothecenes.

The term "chemotherapeutic drug" refers to a chemical compound that can be used to treat tumors. This definition also includes antihormonal agents that act by modulating, reducing, blocking, or suppressing the effects of cancer-promoting hormones, which are often used in the form of systemic or holistic therapy. They may be hormones. Examples of chemotherapeutic drugs include alkylating agents such as thiotepa; cyclophosphamide (CYTOXAN™); alkylsulfonate such as busulfan, improsulfan and piposulfan; aziridine such as benzodopa, carbokwon, meturedopa and uredopa; aziridine and methylamelamine, including altretamine, triethylenemelamine, triethylenephosphoramide,

triethylenethiophosphoramide and trimethylolmelamine; nitrogen mustards such as chlorambucil, chlornaphasine, holofosfamide, estramustine, ifosfamide, mechlorethamine, nitrobine hydrochloride; melphalan, novembihin, phenesterin, prednimustine, trofosfamide, uramustine; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, ranimustine; antibiotics such as aclacinomycin, actinomycin, autramycin, azaserin, bleomycin, cactinomycin C, calicheamicin, carabicin, chromomycin, carsinophilin, chromomycin, actinomycin D, daunorubicin, detorubicin, 6-diazo-5-hydroxy-1_-norleucine, doxorubicin, epirubicin, esorubicin, idarubicin, marcellomycin, mitomycin, mycophenolic acid, nogalamycin, olivomycin, peplomycin, potfiromycin, puromycin, quelamycin, rhodorubicin, streptonigrin; streptozocin, tuberculocidin, ubenimex, zinostatin, zorubicin; antimetabolites such as methotrexate, 5-fluorouracil (5-FU); folic acid analogs such as denopterin, methotrexate, pteropterin, trimetrexate; pterin analogs such as fludarabine, 6-mercaptopterin, thiomethopterin, thioguanopterin; pyrimidine analogs such as ancitabine, azacitidine, 6-azuridine, carmofur, cytarabine, dideoxyuridine, doxyuridine, enocytabine, floxuridine, 5-FU; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; antiadrenalines such as aminoglutethimide, mitotane, trilostane; folic acid supplements such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; amsacrine; bestrabucil; bisantrene; edatraksat; defofamine; demecolcin; diaziquon; elfomitin; elliptinium acetate; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidamine; mitoguazone; mitoxantrone; mopidamol; nitracrine; pintostatin; phenamet; pyrarubicin; podophyllic acid; 2-ethylhydrazide; procarbazine; PSK®; razoxane; sisofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2"-trichlorotriethylamine; urethane; vindesine; dacarbazine; mannomustine; mitobronitol; dibromodulcite; pipobroman; hacytosine; arabinoside ("Ara-C"); cyclophosphamide; thiotepa; taxanes such as paclitaxel (TAXOL®, Bristol- Myers Squibb Oncology, Princeton, NJ) and docetaxel (TAXOTERE®, Rhone-Poulenc Rorer, Antony, France); chlorambucil; gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogues such as cisplatin and carboplatin; vinblastine; platinum ; etoposide (VP-16); ifosfamide; mitomycin C; mitoxantrone; vincristine; vinorelbine; navelbin; novantron; teniposide; daunorubicin; aminopterin; xeloda; ibandronate; CPT-11; topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoic acid, esperamycins capecitabine, and a pharmaceutically acceptable salt, acid, or derivative of any of the foregoing This definition also includes antihormonal agents that can modulate or inhibit the action of hormones on tumors, such as antiestrogens, including tamoxifen, raloxifene, the aromatase inhibitor 4(5)-imidazole , 4-hydroxy tamoxifen, trioxifene, keoxifene, LY117018, onapristone and fareston; and antiandrogens such as flutamide, nilutamide, bicalutamide, leuprolide and goserelin; and a pharmaceutically acceptable salt, acid or derivative of any of the above.

The term "alkyl" refers to a saturated aliphatic hydrocarbon group which is a straight or branched chain group containing 1 to 20 carbon atoms, preferably alkyl containing 1 to 12 carbon atoms, more preferably alkyl containing 1 to 10 carbon atoms. carbon, and most preferably alkyl containing from 1 to 6 carbon atoms. Non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1 -ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1 ,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 2.3 -dimethylpentyl, 2,4-dimethylpentyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2-ethylpentyl, 3-ethylpentyl, n-octyl, 2,3-dimethylhexyl, 2,4-dimethylhexyl, 2,5-dimethylhexyl , 2,2-dimethylhexyl, 3,3-dimethylhexyl, 4,4-dimethylhexyl, 2-ethylhexyl, 3-ethylhexyl, 4-ethylhexyl, 2-methyl-2-ethylpentyl, 2-methyl-3-ethylpentyl, n-nonyl, 2 -methyl-2-ethylhexyl, 2-methyl-3-ethylhexyl, 2,2-diethylpentyl, n-decyl, 3,3-diethylhexyl, 2,2-diethylhexyl and their various branched isomers. More preferably, the alkyl group is a lower alkyl having 1 to 6 carbon atoms, and non-limiting examples include methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1,1,2-trimethylpropyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 2,2-dimethylbutyl, 1,3-dimethylbutyl, 2-ethylbutyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 2,3-dimethylbutyl and the like. The alkyl may be substituted or unsubstituted. In substitution, the substituent group(s) may be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo.

The term "heteroalkyl" refers to alkyl containing one or more heteroatoms selected from the group consisting of N, O and S, where alkyl is as defined above.

The term "alkylene" refers to a saturated linear or branched aliphatic hydrocarbon group having two residues formed by the removal of two hydrogen atoms from the same carbon atom or two different carbon atoms of the parent alkane. Alkylene is a linear or branched group containing 1 to 20 carbon atoms, preferably 1 to 12 carbon atoms, and more preferably 1 to 6 carbon atoms. Non-limiting examples of alkylene include, but are not limited to, methylene (-CH ₂ -), 1,1-ethylene (-CH(CH ₃ )-), 1,2-ethylene (-CH ₂ CH ₂ )-, 1,1 -propylene (-CH (CH ₂ CH ₃ )-), 1,2-propylene (-CH ₂ CH (CH ₃ )-), 1,3-propylene (-CH ₂ CH ₂ CH ₂ -), 1.4 -butylene (-CH ₂ CH ₂ CH ₂ CH ₂ -), 1,5-pentylene (-CH ₂ CH ₂ CH ₂ CH ₂ CH ₂ -) and the like. The alkylene may be substituted or unsubstituted. In substitution, the substituent group(s) may be substituted at any available connection point. The substituent group(s) is preferably one or more groups independently optionally selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl , cycloalkoxy, heteroalkoxy, cycloalkylthio, heterocyclylthio and oxo.

The term "alkoxy" refers to the group -O-(alkyl) or -O-(unsubstituted cycloalkyl), where alkyl and cycloalkyl are as defined above. Non-limiting examples of alkoxy include methoxy, ethoxy, propoxy, butoxy, cyclopropyloxy, cyclobutyloxy, cyclopentyloxy, cyclohexyloxy. Alkoxy may be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocyclylthio.

The term "cycloalkyl" refers to a saturated or partially unsaturated monocyclic or polycyclic hydrocarbon substituent group having 3 to 20 carbon atoms, preferably 3 to 12 carbon atoms, more preferably 3 to 10 carbon atoms, and most preferably 3 to 8 carbon atoms. . Non-limiting examples of monocyclic cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. Polycyclic cycloalkyl includes cycloalkyl having a spiro ring, a fused ring, or an internally bridged ring.

The term "heterocyclyl" refers to a 3-20 membered saturated or partially unsaturated monocyclic or polycyclic hydrocarbon group in which one or more atoms in the ring are heteroatoms selected from the group consisting of N, O and S(O) _m (wherein m is an integer from 0 to 2), but with the exception of -O-O-, -OS- or -SS- in the ring, and the remaining atoms in the ring are carbon atoms. Preferably, the heterocyclyl has 3 to 12 ring atoms, where 1 to 4 atoms are heteroatoms; and more preferably 3 to 10 ring atoms. Non-limiting examples of monocyclic heterocyclyl include pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Polycyclic heterocyclyl includes a heterocyclyl having a spiro ring, a fused ring, or an internally bridged ring.

- электронной системы. Спирогетероциклил предпочтительно представляет собой 6-14-членный спирогетероциклил, и более предпочтительно 7-10-членный спирогетероциклил. По количеству спироатомов, общих между кольцами, спирогетероциклил можно разделить на моноспиро-гетероциклил, диспиро-гетероциклил или полиспиро-гетероциклил, и спирогетероциклил предпочтительно представляет собой моноспиро-гетероциклил или диспиро-гетероциклил, и более предпочтительно 4-членный/4-членный, 4-членный/5-членный, 4-членный/6-членный, 5-членный/5-членный или 5-членный/6-членный моноспиро-гетероциклил. Неограничивающие примеры спирогетероциклила включают:The term "spiroheterocyclyl" refers to a 5-20 membered polycyclic heterocyclyl group with individual rings connected via a single common atom (called a spiro atom), where one or more ring atoms are heteroatoms selected from the group consisting of N, O, and S (O) _m (where m is an integer from 0 to 2), with the remaining atoms in the ring being carbon atoms, where the rings may contain one or more double bonds, but none of the rings is fully conjugated

- electronic system. Spiroheterocyclyl is preferably 6-14 membered spiroheterocyclyl, and more preferably 7-10 membered spiroheterocyclyl. According to the number of spiroatoms shared between the rings, spiroheterocyclyl can be divided into monospiroheterocyclyl, dispiroheterocyclyl or polyspiroheterocyclyl, and spiroheterocyclyl is preferably monospiroheterocyclyl or dispiroheterocyclyl, and more preferably 4-membered/4-membered, 4- membered/5-membered, 4-membered/6-membered, 5-membered/5-membered or 5-membered/6-membered monospiro-heterocyclyl. Non-limiting examples of spiroheterocyclyl include:

The term "fused heterocyclyl" refers to a 5-20 membered polycyclic heterocyclyl group where each ring in the system shares an adjacent pair of atoms with another ring, where one or more rings may contain one or more double bonds, but none of the rings does not have a fully conjugated tt-electron system, and wherein one or more atoms in the ring are heteroatoms selected from the group consisting of N, O and S(O) _m (where m is an integer from 0 to 2), and the remaining atoms in the ring are carbon atoms. The fused heterocyclyl is preferably a 6-14 membered fused heterocyclyl, and more preferably a 7-10 membered fused heterocyclyl. According to the number of member rings, the fused heterocyclyl can be divided into bicyclic, tricyclic, tetracyclic or polycyclic fused heterocyclyl, and the fused heterocyclyl is preferably bicyclic or tricyclic fused heterocyclyl, and more preferably 5-membered/5-membered or 5-membered/6-membered bicyclic fused heterocyclyl. Non-limiting examples of fused heterocyclyl include:

The term "bridged heterocyclyl" refers to a 5-14 membered polycyclic heterocyclyl group in which every two rings in the system have two atoms in common that are not bonded to each other, and the rings may have one or more double bonds, but none of the rings has a fully conjugated tt-electron system, and in which one or more atoms in the ring are heteroatoms selected from the group consisting of N, O, and S(O) _m (where m is an integer from 0 to 2), and the remaining atoms in the ring are carbon atoms. The bridged heterocyclyl is preferably a 6-14 membered bridged heterocyclyl, and more preferably a 7-10 membered bridged heterocyclyl. According to the number of ring members, bridged heterocyclyl can be divided into bicyclic, tricyclic, tetracyclic or polycyclic bridged heterocyclyl, and bridged heterocyclyl is preferably bicyclic, tricyclic or tetracyclic bridged heterocyclyl or more preferably bicyclic or tricyclic bridged heterocyclyl. Non-limiting examples of internally bridged heterocyclyl include:

The heterocyclyl ring may be fused to an aryl, heteroaryl, or cycloalkyl ring, where the ring linked to the parent structure is heterocyclyl. Their non-limiting examples include:

и подобные.

and the like.

The heterocyclyl may be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio, heterocyclylthio and oxo.

-электронную систему, предпочтительно 6-10-членному арилу, например фенилу и нафтилу, и предпочтительно фенилу. Арильное кольцо может быть конденсировано с кольцом гетероарила, гетероциклила или циклоалкила, где кольцо, связанное с исходной структурой, представляет собой арильное кольцо. Их неограничивающие примеры включают:The term "aryl" refers to a 6- to 14-membered all-carbon monocyclic ring or polycyclic fused ring (i.e., each ring in the system shares an adjacent pair of carbon atoms with other rings in the system) having a conjugated

-electron system, preferably 6-10 membered aryl, such as phenyl and naphthyl, and preferably phenyl. The aryl ring may be fused to a heteroaryl, heterocyclyl, or cycloalkyl ring, where the ring associated with the parent structure is an aryl ring. Their non-limiting examples include:

Aryl may be substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocyclylthio.

The term "heteroaryl" refers to a 5-14 membered heteroaromatic system containing 1 to 4 heteroatoms selected from the group consisting of O, S and N. Heteroaryl is preferably a 5-10 membered heteroaryl, more preferably 5 or 6- membered heteroaryl, for example, furyl, thienyl, pyridyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring may be fused to an aryl, heterocyclyl, or cycloalkyl ring, where the ring linked to the parent structure is a heteroaryl ring. Their non-limiting examples include:

Heteroaryl may be optionally substituted or unsubstituted. When substituted, the substituent group(s) is preferably one or more groups independently selected from the group consisting of alkyl, alkenyl, alkynyl, alkoxy, alkylthio, alkylamino, halogen, thiol, hydroxy, nitro, cyano, cycloalkyl, heterocyclyl, aryl, heteroaryl, cycloalkoxy, heterocycloalkoxy, cycloalkylthio and heterocyclylthio.

The term "amino protecting group" refers to a group that prevents an amino group from reacting when other parts of the molecule are reacted and that can be easily removed. Non-limiting examples include 9-fluorenylmethyloxycarbonyl, t-butoxy carbonyl, acetyl, benzyl, allyl, p-methoxybenzyl, and the like. These groups may optionally be substituted with one to three substituents selected from the group consisting of halo, alkoxy and nitro. The amino protecting group is preferably 9-fluorenylmethyloxycarbonyl.

The term "cycloalkylalkyl" refers to an alkyl group substituted with one or more, preferably one, cycloalkyl(s), where alkyl and cycloalkyl are as defined above.

The term "haloalkyl" refers to an alkyl group substituted with one or more halogens, where alkyl is as defined above.

The term "deuterated alkyl" refers to an alkyl group substituted with one or more deuterium atoms, where alkyl is as defined above.

The term "hydroxy" refers to the -OH group.

The term "halogen" refers to fluorine, chlorine, bromine or iodine.

The term "amino" refers to the -NH ₂ group.

The term "nitro" refers to the -NO ₂ group.

The term "amide" refers to a -C(O)N(alkyl) or -C(O)N(cycloalkyl) group, where alkyl and cycloalkyl are as defined above.

The term "alkoxycarbonyl" refers to the group -C(O)O(alkyl) or -C(O)O(cycloalkyl), where alkyl and cycloalkyl are as defined above.

The present invention also includes compounds of formula (I) in various deuterated forms. Each of the available hydrogen atoms attached to a carbon atom can be independently replaced by a deuterium atom. Those skilled in the art can synthesize the compound of formula (I) in deuterated form with reference to the relevant literature. The compound of formula (I) in deuterated form can be prepared using commercially available deuterated starting materials, or they can be synthesized by conventional methods using deuterated reagents, including, but not limited to, deuterated borane, trideuterated borane in tetrahydrofuran, deuterated lithium aluminum hydride, deuterated iodoethane, deuterated iodomethane and the like.

"Optional" or "optional" means that the event or circumstance described subsequently may, but need not, occur, and such description includes the situation in which the event or circumstance occurs or does not occur. For example, "heterocyclyl optionally substituted with alkyl" means that an alkyl group may be present, but need not be, and such description includes the situation where the heterocyclyl is substituted with alkyl and the heterocyclyl is not substituted with alkyl.

"Substituted" refers to one or more hydrogen atoms in a group, preferably up to 5, and more preferably 1 to 3 hydrogen atoms, independently substituted with the appropriate number of substituents. Of course, substituents exist only in the chemical position possible for them. A person skilled in the art can, without undue effort, determine whether substitution is possible or not possible, experimentally or theoretically. For example, an amino or hydroxy combination having a free hydrogen and carbon atoms having unsaturated bonds (eg, olefins) may be unstable.

The term "pharmaceutical composition" refers to a mixture of one or more compounds described in this application or their physiologically/pharmaceutically acceptable salts or prodrugs with other chemical components and other components, such as physiologically/pharmaceutically acceptable carriers and excipients. The purpose of the pharmaceutical composition is to facilitate the administration of the compound to the body, thereby facilitating the absorption of the active ingredient in order to exhibit biological activity.

The term "pharmaceutically acceptable salt" or "pharmaceutical salt" refers to a salt of a ligand-drug conjugate of the present invention or a salt of a compound of the present invention that is safe and effective in mammals and has the desired biological activity. The ligand-drug conjugate of the present invention contains at least one amino group, so it can form a salt with an acid. Non-limiting examples of pharmaceutically acceptable salts include hydrochloride, hydrobromide, hydroiodide, sulfate, bisulfate, citrate, acetate, succinate, ascorbate, oxalate, nitrate, sorbate, hydrogen phosphate, dihydrogen phosphate, salicylate, hydrogen citrate, tartrate, maleate, fumarate, formate, benzoate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-tol wol sulfonate.

The term "solvate" refers to a pharmaceutically acceptable solvate formed by a ligand-drug conjugate of the present invention with one or more solvent molecules. Non-limiting examples of solvent molecules include water, ethanol, acetonitrile, isopropanol, DMSO, ethyl acetate.

The term "drug loading" refers to the average amount of cytotoxic drugs loaded per ligand in a compound of formula (I) and can also be expressed as the ratio of drug to antibody. Drug loading may range from 0 to 12, preferably 1 to 10 cytotoxic drugs (D) per ligand (Pc). In one embodiment of the present invention, drug loading is expressed as n, and exemplary values may average 1, 2, 3, 4, 5, 6, 7, 8, 9, 10. Average number of drugs per ADC molecule after reaction the combinations can be determined by conventional methods such as UV/visible spectroscopy, mass spectrometry, ELISA test and HPLC characterization.

In one embodiment of the present invention, the cytotoxic drug is conjugated to the N-terminal amino group and/or e-amino group of lysine residues in the ligand via a linker. Typically, the number of drug molecules conjugated to the antibody in a coupling reaction will be less than the theoretical maximum.

The following non-limiting methods can be used to control the loading of ligand-cytotoxic drug conjugates:

(1) adjusting the molar ratio of binding reagent to monoclonal antibody,

(2) reaction time and temperature control,

(3) selection of different reaction reagents.

The preparation of conventional pharmaceutical compositions can be found in the Chinese Pharmacopoeia.

The term "carrier" as used in the composition of the present invention refers to a system that can change the way the drug enters the human body and distributes it, controls the release rate of the drug, and delivers the drug to the target organ. Carrier systems for drug release and targeting can reduce drug degradation and loss, reduce side effects, and improve bioavailability. For example, polymeric surfactants that can be used as carriers can self-assemble into various forms of aggregates due to their unique amphiphilic structure. Preferred examples include micelles, microemulsions, gels, liquid crystals, bubbles, and the like. These aggregates have the ability to encapsulate drug molecules while having good membrane permeability and can be used as an excellent drug carrier.

The term "adjuvant" is an additive in a pharmaceutical formulation other than the main drug, which may also be referred to as an adjuvant, such as adhesives, fillers, disintegrants, lubricants in tablets; matrix parts in semi-solid ointment and cream preparations; preservatives, antioxidants, flavoring agents, flavoring agents, co-solvents, emulsifiers, solubilizers, osmotic pressure regulators, colorants in liquid formulations, and the like.

The term "diluent", also known as filler, is primarily intended to increase the weight and volume of a tablet. The addition of a diluent provides a defined volume, reduces dose variation of the main components, and improves the compression profile of the drug. When the tablet contains an oil component, an absorbent is added to absorb the oily substance, thereby maintaining a "dry" state to facilitate tablet formation. For example, the diluent includes starch, lactose, inorganic calcium salts, microcrystalline cellulose, and the like.

The pharmaceutical composition may be in the form of a sterile injectable aqueous solution. Acceptable vehicles or solvents that can be used are water, Ringer's solution or isotonic sodium chloride solution. The sterile injectable formulation may be a sterile oil-in-water microemulsion in which the active ingredient is dissolved in the oil phase. For example, the active ingredient is dissolved in a mixture of soybean oil and lecithin. The oil solution is then added to a mixture of water and glycerin and processed to form a microemulsion. The injection solution or microemulsion can be administered into the patient's bloodstream by local bolus injection. Alternatively, the solution and microemulsion are preferably administered in a manner that maintains a constant circulating concentration of the compound of the present invention. To maintain this constant concentration, a continuous intravenous delivery device may be used. An example of such a device is the Deltec CADD-PLUS IV pump. TM. 5400.

The pharmaceutical composition may be in the form of a sterile injectable aqueous or oily suspension for intramuscular and subcutaneous administration. Such a suspension may be formulated with suitable dispersants or wetting agents and suspending agents as described above, in accordance with known methods. The sterile injectable formulation may also be a sterile injectable solution or suspension formulated in a non-toxic parenterally acceptable diluent or solvent, for example, the solution is prepared in 1,3-butanediol. Moreover, sterile fixed oils can easily be used as a solvent or suspending medium. Any mixed fixed oils can be used for this purpose, including synthetic mono- or diglycerides. Moreover, fatty acids such as oleic acid can also be used to obtain an injection.

The present invention relates to a cleavable linker arm with a defined structure and an active substance with a defined structure, as well as to an antibody-drug conjugate (ADC) consisting of a linker arm, an active substance and an antibody. This ADC is a complex formed by binding a toxic substance to an antibody through a spacer. The antibody-drug conjugate (ADC) is degraded in the body to release active molecules, thereby exhibiting an antitumor effect.

Synthesis method according to the present invention

To implement the task of the present invention, the following technical solutions are used in the present invention:

Scheme I:

The process for preparing a compound of formula (D1) or a pharmaceutically acceptable salt or solvate thereof according to the present invention includes the following step:

reacting a compound of formula (Y1) and a compound of formula (Dr) in the presence of a condensing agent and optionally under basic conditions to obtain a compound of formula (D1),

where: R ¹ , R ² and m are as defined in formula (D1).

The alkaline agent includes organic and inorganic bases. Organic bases include, but are not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium t-butoxide, and potassium t-butoxide. Inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

The condensing agent may be selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3 -ethylcarbodiimide hydrochloride, N,N-dicyclohexylcarbodiimide, N,N-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N ,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and benzotriazol-1-yl- hydroxytripyrrolidinyl phosphorus hexafluorophosphate, and preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Scheme II:

The process for preparing a compound of formula (L _b -Y-Dr) or a pharmaceutically acceptable salt or solvate thereof according to the present invention includes the following steps:

Step 1: a compound of formula (IB-1) and exatecan methanesulfonate (1b) are reacted in the presence of a condensing agent and optionally under alkaline conditions to obtain a compound of formula (IB-2);

Step 2: The compound of formula (IB-2) is deprotected to obtain a compound of formula (IB);

Step 3: A compound of formula (IA) and a compound of formula (IB) are reacted in the presence of a condensing agent and optionally under alkaline conditions to give a compound of formula (L _b -Y-Dr),

Where:

R ⁺ is an amino protecting group, and is preferably 9-fluorenylmethyloxycarbonyl (Fmoc);

R ¹ , R ² , R ⁵ ~R ⁷ , s ¹ and m are as defined in the formula (L _b -Y-Dr). The alkaline agent includes organic and inorganic bases. Organic bases include, but are not limited to, triethylamine, diethylamine, N-methylmorpholine, pyridine, hexahydropyridine, N,N-diisopropylethylamine, n-butyllithium, lithium diisopropylamide, potassium acetate, sodium t-butoxide, and potassium t-butoxide. Inorganic bases include, but are not limited to, sodium hydride, potassium phosphate, sodium carbonate, potassium carbonate, cesium carbonate, sodium hydroxide, and lithium hydroxide.

The condensing agent is selected from the group consisting of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, N,N'-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide, O-benzotriazole-N,N,N',N'-tetramethylurea tetrafluoroborate, 1-hydroxybenzotriazole, 1-hydroxy-7-azobenzotriazole, O-benzotriazole-N ,N,N',N'-tetramethylurea hexafluorophosphate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate, and benzotriazol-1-yl- hydroxytripyrrolidinyl phosphorus hexafluorophosphate, and preferably 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride, 1-hydroxybenzotriazole and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride.

Scheme III:

The process for preparing a compound of formula (Pc-L _a -Y-Dr) of the present invention includes the following step:

The PC is reacted with a compound of formula (L _a -Y-Dr) after reduction to give a compound of formula (Pc-L _a -Y-Dr); the reducing agent is preferably TCEP, in particular preferably reduces the disulfide bond on the antibody; Where:

PC is a ligand;

W, L ² , L ³ , R ¹ , R ² , R ⁵ ~R ⁷ , m and n are as defined in the formula (Pc-L _a -Y-Dr).

The present invention will be further described with reference to the following examples, but the examples should not be construed as limiting the scope of the present invention.

The test methods in the examples of the present invention, for which no specific conditions are indicated, were carried out in accordance with the usual conditions or conditions recommended by the manufacturers of materials or products. Reagents for which specific sources are not indicated are common commercially available reagents.

EXAMPLES

Compound structures are identified by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR is determined on a Bruker AVANCE-400 instrument. The detection solvents are deuterated dimethyl sulfoxide (DMSO-d ₆ ), deuterated chloroform (CDCl ₃ ) and deuterated methanol (CD ₃ OD) and the internal standard is tetramethylsilane (TMS). Chemical shifts are given in 10 ^-6 (ppm).

MS is determined using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, type: Finnigan LCQ advantage MAX).

UHPLC is carried out using a Waters Acquity UPLC SQD liquid chromatograph/mass spectrometer.

High performance liquid chromatography (HPLC) was performed on an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 chromatographic column 150 x 4.6 mm) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 chromatographic column 150 x 4.6 mm).

UV-HPLC is determined on a Thermo nanodrop2000 UV spectrophotometer. The extent of proliferation inhibition and IC ₅₀ values were determined using a PHERAstarFS microplate reader (BMG Co., Germany). Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates were used as a thin layer silica gel chromatography (TLC) plate. The size of the silica gel plate used in TLC is 0.15 mm to 0.2 mm, and the size of the silica gel plate used in product purification is 0.4 mm to 0.5 mm.

200 to 300 mesh Yantai Huanghai silica gel is generally used as a support for column chromatography.

Known starting materials according to the present invention can be obtained by methods known in the art or can be purchased from ABCR GmbH & Co. KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc., Dari chemical Company, etc.

Unless otherwise indicated, the reactions are carried out under an argon or nitrogen atmosphere.

"Argon atmosphere" or "nitrogen atmosphere" means that the reaction flask is equipped with a cylinder of argon or nitrogen (approximately 1 L).

"Atmosphere of hydrogen" means that the reaction flask is equipped with a cylinder of hydrogen (about 1 liter).

The pressure hydrogenation reaction was carried out using a Parr 3916EKX hydrogenation apparatus and a Qinglan QL-500 hydrogen generator, or using an HC2-SS hydrogenation apparatus.

In hydrogenation reactions, the reaction system is usually evacuated and filled with hydrogen, and the above operation is repeated three times.

A microwave reactor type CEM Discover-S 908860 was used in reactions using microwaves.

Unless otherwise indicated, the reaction solution refers to an aqueous solution.

Unless otherwise indicated, the reaction temperature is room temperature.

Room temperature from 20°C to 30°C is the most suitable reaction temperature.

Preparation of PBS buffer (pH=6.5) in the examples: 8.5 g KH ₂ PO ₄ , 8.56 g K ₂ HPO ₄ 3H ₂ O, 5.85 g NaCl and 1.5 g EDTA were placed in a flask with a volume 2 L, the mixture was sonicated to dissolve completely and shaken well to obtain a buffer.

The eluent system in column chromatography and the developing solvent system in thin layer chromatography to purify compounds include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, C: petroleum ether and ethyl acetate system. The solvent volume ratio is adjusted according to the polarity of the compounds, and a small amount of an acidic reagent or an alkaline reagent such as triethylamine can also be added to adjust.

Some of the compounds of the present invention have been characterized by Q-TOF LC/MS. For Q-TOF LC/MS, an Agilent 6530 Accurate-Mass Quadrupole-Time of Flight mass spectrometer and an Agilent 1290-lnfinity UHPLC (Agilent Poroshell 300SB-C8 5 µm, 2.1 × 75 mm column) were used.

Example 1

]пирано[3',4':6,7]индолизино[1,2-b]хинолин-1-ил)-1-гидроксициклопропан-1-карбоксамид 1N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo [

]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopropan-1-carboxamide 1

1 ml of N,N-dimethylformamide was added to exatecan methanesulfonate 1b (2.0 mg, 3.76 μmol, prepared according to the method described in patent application "EP 0737686 A1"), and the solution was cooled to 0-5°C in a bath with ice water. One drop of triethylamine was added dropwise, and the reaction solution was stirred until clear. 1-Hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 µmol, prepared according to the known method described in "Tetrahedron Letters, 25(12), 1269-72; 1984") and 4-(4,6-dimethoxy -1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (3.8 mg, 13.7 μmol) was sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5° C. for 2 hours. 5 ml of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 ml×3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using the developing solvent system B to obtain the title product 1 (1.6 mg, yield: 82.1%).

MS m/z (ESI): 520.2 [M+1]

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.90-7.84 (m, 1H), 7.80-7.68(m, 1H), 5.80-5.70 (m, 1H) , 5.62-5.54(m, 2H), 5.44-5.32(m, 2H), 5.28-5.10(m, 2H), 3.40-3.15(m, 3H), 2.44 (s, 3H), 2.23(t, 1H), 2.06-1.75 (m, 2H), 1.68-1.56 (m, 1H), 1.22 -1.18 (m, 2H), 1.04-0.98 (m, 2H), 0.89 (t, 3H).

Example 2

(S)-2-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indole isino[1,2-b]quinol in-1-yl)-2-hydroxyacetamide 2-A

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

2 ml of ethanol and 0.4 ml of NN-dimethylformamide were added to 1b (4 mg, 7.53 µmol). The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 0.3 ml of N-methylmorpholine was added dropwise, and the reaction solution was stirred until clear. 2-Cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 µmol, obtained according to the method described in patent application "WO 2013106717"), 1-hydroxybenzotriazole (3 mg, 22.4 µmol) and 1-( 3-Dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol) was added sequentially to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5° C. for one hour. The ice water bath was removed, and the reaction solution was heated to 30° C. and stirred for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 2 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B -acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product (2-A: 1.5 mg, 2-B: 1.5 mg).

MS m/z (ESI): 534.0 [M+1].

2-B compound with single configuration (shorter retention time)

UHPLC analysis: retention time: 1.06 minutes, purity: 88% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.37 (d, 1H), 7.76 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.58-5.56 (m, 1H), 5.48 (d, 1H), 5.41 (s, 2H), 5.32-5.29 (m, 2H), 3.60 (t, 1H), 3.19-3.13 (m, 1H), 2.38 (s, 3H), 2.20-2.14 (m, 1H), 1.98 (q, 2H), 1.87 -1.83 (m, 1H), 1.50-1.40 (m, 1H), 1.34-1.28 (m, 1H), 0.86 (t, 3H), 0.50- 0.39 (m, 4H).

Compound 2-A with single configuration (longer retention time)

UHPLC analysis: retention time: 1.10 minutes, purity: 86% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.35 (d, 1H), 7.78 (d, 1H), 7.31 (s, 1H), 6.52 (s, 1H), 5.58-5.53 (m, 1H), 5.42 (s, 2H), 5.37 (d, 1H), 5.32 (t, 1H), 3.62 (t, 1H), 3 .20-3.15 (m, 2H), 2.40 (s, 3H), 2.25-2.16 (m, 1H), 1.98 (q, 2H), 1.87-1.82 (m, 1H), 1.50-1.40 (m, 1H), 1.21-1.14 (m, 1H), 0.87 (t, 3H), 0.47-0.35 (m , 4H).

Example 3

(S)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-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)-3,3,3-trifluoro-2-hydroxypropanamide 3-A

(R)-N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-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)-3,3,3-trifluoro-2-hydroxypropanamide 3-B

2 ml of ethanol and 0.4 ml of N,N-dimethylformamide were added to 1b (5.0 mg, 9.41 μmol) and the solution was cooled to 0-5° C. in an ice water bath. 0.3 ml of N-methylmorpholine was added dropwise, and the reaction solution was stirred until clear. 3,3,3-trifluoro-2-hydroxypropionic acid 3а (4.1 mg, 28.4 µmol, supplier: Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 µmol) and 1-(3-dimethylaminopropyl )-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol) was sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5° C. for 10 minutes. The ice water bath was removed, and the reaction solution was heated to 30° C. and stirred for 8 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 3 was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B -acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product (1.5 mg, 1.5 mg).

MS m/z (ESI): 561.9 [M+1].

Single configuration compound (shorter retention time)

UHPLC analysis: retention time: 1.11 minutes, purity: 88% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.94 (d, 1H), 7.80 (d, 1H), 7.32 (s, 1H), 7.20 (d, 1H), 6.53 (s, 1H), 5.61-5.55 (m, 1H), 5.45-5.23 (m, 3H), 5.15-5.06 (m, 1H), 4. 66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.26-2.20 (m, 1H), 2.16- 2.08 (m, 1H), 2.02-1.94 (m, 1H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Connection with a single configuration (longer retention time)

UHPLC analysis: retention time: 1.19 minutes, purity: 90% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.97 (d, 1H), 7.80 (d, 1H), 7.31 (s, 1H), 7.16 (d, 1H), 6.53 (s, 1H), 5.63-5.55 (m, 1H), 5.45-5.20 (m, 3H), 5.16-5.07 (m, 1H), 4. 66-4.57 (m, 1H), 3.18-3.12 (m, 1H), 2.40 (s, 3H), 2.22-2.14 (m, 1H), 2.04- 1.95 (m, 2H), 1.89-1.82 (m, 1H), 1.50-1.40 (m, 1H), 0.87 (t, 3H).

Example 4

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12H-benzo [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-hydroxycyclopentan-1-carboxamide 4

1 ml of N.N-dimethylformamide was added to 1b (3.0 mg, 5.64 μmol) and the solution was cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added dropwise, and the reaction solution was stirred until clear. 1-Hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 µmol, prepared according to the method described in patent application "WO 2013106717") and 4-(4,6-dimethoxy-1,3,5-triazine-2 -yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) was sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5°C for 1 hour, 5 ml of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 4 (2.5 mg, yield: 80.9%).

MS m/z (ESI): 548.0 [M+1].

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.73-7.62 (m, 2H), 5.75-5.62 (m, 1H), 5.46-5.32 (m, 2H) , 5.26-5.10 (m, 1H), 3.30-3.10 (m, 1H), 2.43 (s, 3H), 2.28-2.20 (m, 2H), 2 .08-1.84 (m, 8H), 1.69-1.58 (m, 2H), 1.04-1.00 (m, 2H), 0.89 (t, 3H).

Example 5

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12H-benzo [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclopropane-1-carboxamide 5

1 ml of N,N-dimethylformamide was added to 1b (2.0 mg, 3.76 μmol) and the solution was cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added dropwise, and the reaction solution was stirred until clear. 1-(Hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 µmol, prepared according to the method described in patent application "WO 201396771") and 4-(4,6-dimethoxy-1,3,5-triazine -2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol) was sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5° C. for 2 hours, 5 ml of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (8 ml×3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using the developing solvent system B to obtain the title product 5 (1.0 mg, yield: 50%).

MS m/z (ESI): 533.9 [M+1].

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.07 (s, 1H), 7.23-7.18 (m, 2H), 6.71-6.64 (m, 1H), 6.55 -6.51(m, 1H), 5.36-5.27(m, 2H), 4.67-4.61(m, 2H), 3.53-3.48(m, 1H), 3 .30-3.22 (m, 2H), 3.18-3.13 (m, 1H), 2.71-2.61 (m, 2H), 2.35-2.28 (m, 1H) , 2.04-1.91(m, 4H), 1.53-1.40(m, 3H), 0.91-0.75(m, 4H).

Example 6

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12H-benzo [de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-1-(hydroxymethyl)cyclobutane-1-carboxamide 6

1 ml of N,N-dimethylformamide was added to 1b (3.0 mg, 5.64 µmol) and the solution was cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added dropwise, and the reaction solution was stirred until clear. 1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 µmol, obtained according to the known method described in the patent application "Journal of the American Chemical Society, 2014, vol. 136, #22, p 8138-8142") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol) were successively added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5°C for 1 hour, 5 ml of water was added to the reaction solution to quench the reaction, and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phases were combined, washed with saturated sodium chloride solution (5 ml × 2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 6 (2.1 mg, yield: 67.9%).

MS m/z (ESI): 548.0 [M+1].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 7.85-7.62 (m, 1H), 6.88 (br, 1H), 5.87-5.48 (m, 2H), 5 .47-5.33(m, 1H), 5.31-5.06(m, 1H), 4.25-3.91(m, 2H), 3.25(br, 1H), 2, 60-2.32 (m, 3H), 2.23 (t, 1H), 2.15-1.95 (m, 3H), 1.70-1.56 (m, 2H), 1.41- 1.17 (m, 9H), 1.03 (s, 1H), 0.95-0.80 (m, 2H).

Example 7

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-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)-1-hydroxycyclobutan-1-carboxamide 7

2 ml of ethanol and 0.4 ml of N.N-dimethylformamide were added to 1b (3.0 mg, 5.64 μmol) and the solution was cooled to 0-5° C. in an ice water bath. 0.3 ml of N-methylmorpholine was added dropwise, and the reaction solution was stirred until clear. 1-Hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 µmol, supplier: PharmaBlock Sciences), 1-hydroxybenzotriazole (2.3 mg, 17.0 µmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ( 3.2 mg, 16.7 μmol) were sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at 0-5° C. for 10 minutes. The ice water bath was removed, and the reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using the developing solvent system B to obtain the title product 7 (2.5 mg, yield: 83.1%).

MS m/z (ESI): 534.0 [M+1].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.28 (d, 1H), 7.75 (d, 1H), 7.29 (s, 1H), 6.51 (s, 1H), 6.12 (s, 1H), 5.59-5.51 (m, 1H), 5.41 (s, 2H), 5.20-5.01 (m, 2H), 3.27-3, 17 (m, 1H), 3.15-3.05 (m, 1H), 2.71-2.63 (m, 1H), 2.37 (s, 3H), 2.12-2.05 ( m, 1H), 2.03-1.94 (m, 2H), 1.92-1.78 (m, 4H), 1.50-1.42 (m, 1H), 0.90-0 .83 (m, 4H).

Example 8

1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2, 5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-hexahydro-1Н,12Н-benzo[be]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropan-1-carboxamide 8

Stage 1

Benzyl

1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)i,iclopropane-1-carboxylate 8с

Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol, prepared according to the method described in US 2005/20645) and (2-((((9H-fluoren-9-yl)methoxy )carbonyl)amino)acetamido)methyl acetate 8b (100 mg, 0.27 mmol, prepared according to the method described in patent application "CN 105829346") was added to the reaction flask, after which 5 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which potassium tert-butoxide (61 mg, 0.54 mmol) was added. The ice water bath was removed, and the reaction solution was warmed to room temperature and stirred for 10 minutes. 20 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (5 ml×2) and chloroform (5 ml×5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 ml of 1,4-dioxane. 0.6 ml of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (70 mg, 0.27 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. 20 ml of water was added, and the reaction solution was extracted with ethyl acetate (8 ml×3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system B to obtain the title product 8 sec (100 mg, yield: 73.6%).

MS m/z (ESI): 501.0 [M+1]

Stage 2

1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylic acid 8d

8c (50 mg, 0.10 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), after which a carbon-supported palladium catalyst (25 mg, content: 10%) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through Celite, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 8d (41 mg, yield: 100%).

MS m/z (ESI): 411.0 [M+1].

Stage 3

(9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)cyclopropoxy )methyl)amino)-2-oxoethyl)carbamate 8e

1b (7 mg, 0.013 mmol) was added to the reaction flask, after which 1 ml of N,N-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, 8d (7 mg, 0.017 mmol) in 0.5 ml of N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium was added chloride (7 mg, 0.026 mmol) and the reaction solution was stirred in an ice bath for 35 minutes. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (5 ml×3). The organic phase was washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using a developing solvent system B to obtain the title product 8e (8.5 mg, yield: 78.0%).

MS m/z (ESI): 828.0 [M+1].

Stage 4

1-((2-Aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)cyclopropan-1-carboxamide 8f

8e (4 mg, 4.84 µmol) was dissolved in 0.2 ml of dichloromethane, after which 0.1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give crude product 8f (2.9 mg) which was used directly in the next step without purification.

MS m/z (ESI): 606.0 [M+1].

Stage 5

1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2, 5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-hexahydro-1Н,12Н-benzo[ae]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropan-1-carboxamide 8

The crude compound 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 ml of NN-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetamido)acetamido)-3-phenylpropionic acid 8g (2.7 mg, 5.80 µmol obtained according to the method described in patent application "EP 2907824") in 0.3 ml of N,N-dimethylformamide, and added 4-(4,6-dimethoxy-1,3,5-triazine-2- yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol) and the reaction solution was stirred in an ice bath for 30 minutes. The ice bath was removed and the reaction solution was warmed to room temperature and stirred for 15 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product 8 (2 mg, yield: 39.0%).

MS m/z (ESI): 1060.0 [M+1].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.01 (d, 1H), 8.77 (t, 1H), 8.21 (t, 1H), 8.08-7.92 (m , 2H), 7.73 (d, 1H), 7.28 (s, 1H), 7.24-7.07 (m, 4H), 6.98 (s, 1H), 6.50 (s, 1H), 5.61 (q, 1H), 5.40 (s, 2H), 5.32 (t, 1H), 5.12 (q, 2H), 4.62 (t, 1H), 4, 52 (t, 1H), 4.40-4.32 (m, 1H), 3.73-3.47 (m, 8H), 3.16-3.04 (m, 2H), 2.89 ( dd, 1H), 2.69-2.55 (m, 2H), 2.37-2.23 (m, 4H), 2.12-1.93 (m, 4H), 1.90-1, 74 (m, 2H), 1.52-1.38 (m, 4H), 1.33-1.11 (m, 5H), 0.91-0.81 (m, 4H).

Example 9

N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl)hexanamide 9-А N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl -10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline -1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

Stage 1

Benzyl 2-cyclopropyl-2-hydroxyacetate 9а

2a (1.3 g, 11.2 mmol, obtained according to the method described in patent application "WO 2013/106717") was dissolved in 50 ml of acetonitrile, and then potassium carbonate (6.18 g, 44.8 mmol) was added successively , benzyl bromide (1.33 ml, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol). The reaction solution was stirred at room temperature for 48 hours, and filtered through Celite. The filter cake was washed with ethyl acetate (10 ml) and the filtrates were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 9a (2 g, yield: 86.9%).

Stage 2

Benzyl

10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat 9b

9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to the reaction flask, after which 4 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. Potassium tert-butoxide (109 mg, 0.98 mmol) was added and the ice water bath removed. The reaction solution was warmed to room temperature and stirred for 40 minutes. 10 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (20 ml×2) and chloroform (10 ml×5). The organic phases were combined and concentrated. The resulting residue was dissolved in 4 ml of dioxane. 2 ml of water, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorene methyl chloroformate (126 mg, 0.49 mmol) were added, and the reaction solution was stirred at room temperature for 2 hours. 20 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 9b (48 mg, yield: 19%).

MS m/z (ESI): 515.0 [M+1].

Stage 3

10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 9с

9b (20 mg, 0.038 mmol) was dissolved in 4.5 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), after which a carbon-supported palladium catalyst (12 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through Celite, and the filter residue was washed with ethyl acetate. The filtrate was concentrated to give the crude product 9 s (13 mg) which was used directly in the next step without purification.

MS m/z (ESI): 424.9 [M+1].

Stage 4

(9H-Fluoren-9-yl)methyl (2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)-2-oxoethoxy)methyl)amino)-2-oxoethyl)carbamate 9d

1b (10 mg, 18.8 µmol) was added to the reaction flask, after which 1 ml of 1\1,1M-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, crude compound 9c (13 mg, 30.6 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (16.9 mg, 61.2 μmol), and the reaction solution was stirred in an ice bath for 40 minutes. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 9d (19 mg, yield: 73.6%).

MS m/z (ESI): 842.1 [M+1].

Stage 5

2-((2-Aminoacetamido)methoxy)-2-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3, 9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)acetamide 9е

9d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane, after which 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. 1 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the residue to form a slurry, and the supernatant was discarded after settling to retain a solid. The solid residue was concentrated under reduced pressure with an oil pump to dryness to give crude product 9e (17 mg) which was used directly in the next step without purification.

MS m/z (ESI): 638DM+18].

Stage 6

N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl)hexanamide 9-A

N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2 ,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl)hexanamide 9-B

The crude compound 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 ml of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 8d (21.2 mg, 44.8 µmol) in 0.3 ml N,N-dimethylformamide and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium were added chloride (18.5 mg, 67.3 μmol) and the reaction solution was stirred in an ice bath for 10 minutes. The ice bath was removed and the reaction solution was warmed to room temperature and stirred for 1 hour to obtain compound 9. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 µm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product (9-A: 2.4 mg, 9-B: 1.7 mg).

MS m/z (ESI): 1074.4 [M+1].

Compound 9-A with a single configuration (shorter retention time):

UHPLC analysis: retention time: 1.14 minutes, purity: 85% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.60 (t, 1H), 8.51-8.49 (d, 1H), 8.32-8.24 (m, 1H), 8 .13-8.02(m, 2H), 8.02-7.96(m, 1H), 7.82-7.75(m, 1H), 7.31(s, 1H), 7.26 -7.15(m, 4H), 6.99(s, 1H), 6.55-6.48(m, 1H), 5.65-5.54(m, 1H), 5.41(s , 2H), 5.35-5.15 (m, 3H), 4.74-4.62 (m, 1H), 4.54-4.40 (m, 2H), 3.76-3.64 (m, 4H), 3.62-3.48 (m, 2H), 3.20-3.07 (m, 2H), 3.04-2.94 (m, 1H), 2.80-2 .62 (m, 1H), 2.45-2.30 (m, 3H), 2.25-2.15 (m, 2H), 2.15-2.04 (m, 2H), 1.93 -1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 5H), 0.87 (t, 3H), 0.64-0 .38 (m, 4H).

Compound 9-B with single configuration (longer retention time):

UHPLC analysis: retention time: 1.16 minutes, purity: 89% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.68-8.60 (m, 1H), 8.58-8.50 (m, 1H), 8.32-8.24 (m, 1H), 8.13-8.02 (m, 2H), 8.02-7.94 (m, 1H), 7.82-7.75 (m, 1H), 7.31 (s, 1H) , 7.26-7.13(m, 3H), 6.99(s, 1H), 6.55-6.48(m, 1H), 5.60-5.50(m, 1H), 5 .41 (s, 2H), 5.35-5.15 (m, 2H), 4.78-4.68 (m, 1H), 4.60-4.40 (m, 2H), 3.76 -3.58 (m, 4H), 3.58-3.48 (m, 1H), 3.20-3.10 (m, 2H), 3.08-2.97 (m, 2H), 2 .80-2.72 (m, 2H), 2.45-2.30 (m, 3H), 2.25-2.13 (m, 2H), 2.13-2.04 (m, 2H) , 2.03-1.94 (m, 2H), 1.91-1.78 (m, 2H), 1.52-1.39 (m, 3H), 1.34-1.12 (m, 4H), 0.91-0.79 (m, 3H), 0.53-0.34 (m, 4H).

Example 10

N-((25.105)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10 ,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-1,1,1- trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrole- 1-yl)hexanamide 10-A

N-((2R,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)-1,1, 1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 10-B

Benzyl 3,3,3-trifluoro-2-hydroxypropanoate 10a

3a (1.80 g, 12.5 mmol) was dissolved in 100 ml of acetonitrile, and then potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 ml, 37.5 mmol) and tetrabutylammonium iodide (231 mg, 0.63 mmol). The reaction solution was heated to 60° C. and stirred for 5 hours. The reaction solution was cooled to room temperature and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 10a (980 mg, yield: 33.5%).

¹ H NMR (400 MHz, CDCl ₃ ): δ 7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H ).

Stage 2

Benzyl

1-(9Н-fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecan-11-oat 10b

8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to the reaction flask, after which 3 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. Potassium tert-butoxide (38 mg, 0.34 mmol) was added and the ice water bath was removed. The reaction solution was warmed to room temperature and stirred for 20 minutes. 10 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (20 ml×2) and chloroform (10 ml×5). The organic phases were combined and concentrated, and the resulting residue was dissolved in 2 ml of dioxane. 0.4 ml of water, sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorene methyl chloroformate (49 mg, 0.19 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. 20 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 10b (51 mg, yield: 55.3%).

MS m/z (ESI): 559.9 [M+18].

Stage 3

1-(9H-Fluoren-9-yl)-3,6-dioxo-10-(trifluoromethyl)-2,9-dioxa-4,7-diazaundecan-11-oic acid 10 s

10b (15 mg, 0.28 mmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), after which a carbon-supported palladium catalyst (15 mg, content: 10%) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through Celite, and the filter residue was washed with tetrahydrofuran. The filtrate was concentrated to give the crude product 10 s (13 mg).

MS m/z (ESI): 452.9 [M+1].

Stage 4 (9H-Fluoren-9-yl)methyl (2-((((3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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 )-1,1,1-trifluoro-3-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

1b (10 mg, 18.8 µmol) was added to the reaction flask, followed by the addition of 1 ml of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, 10 s (13 mg, 28.7 µmol) in 0.5 ml of N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)- 4-methylmorpholinium chloride (11 mg, 39.7 μmol) and the reaction solution was stirred in an ice bath for 30 minutes. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phase was combined, washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 10d (16 mg, yield: 97.8%).

MS m/z (ESI): 870 DM+1].

Stage 5

2-((2-Aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15- hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)-3,3,3-trifluoropropanamide 10e

10d (16 mg, 18.4 μmol) was dissolved in 0.6 ml of dichloromethane, after which 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the residue to form a slurry, and the supernatant was discarded after settling for some time to retain a solid, which was repeated three times. The solid residue was concentrated under reduced pressure with an oil pump to dryness to give crude product 10e (12 mg) which was used directly in the next step without purification.

MS m/z (ESI): 647.9 [M+1].

Stage 6

N-((2S,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)carbamoyl)-1,1, 1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 10-A

N-((2R,10S)-10-Benzyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)-1,1, 1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 10-B

The crude compound 10e (12 mg, 18.5 μmol) was dissolved in 1.0 ml of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 8g (14 mg, 29.6 µmol) in 0.3 ml N,N-dimethylformamide was added, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride ( 15 mg, 54.2 μmol) and the reaction solution was stirred in an ice bath for 30 minutes. The ice bath was removed and the reaction solution was warmed to room temperature and stirred for 1 hour to give compound 10. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 µm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title products (2.7 mg, 2.6 mg).

MS m/z (ESI): 1102.0 [M+1].

Connection with a single configuration (with a shorter retention time):

UHPLC analysis: retention time: 1.18 minutes, purity: 91% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.97 (d, 1H), 8.85-8.76 (m, 1H), 8.37-8.27 (m, 1H), 8 .12-8.02(m, 1H), 8.02-7.95(m, 1H), 7.80(d, 1H), 7.31(s, 1H), 7.26-7.10 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.52 (s, 1H), 5.65-5.54 (m, 1H), 5.41 ( s, 1H), 5.37-5.25 (m, 3H), 5.23-5.13 (m, 1H), 4.81-4.68 (m, 2H), 4.51-4, 41 (m, 1H), 3.78-3.45 (m, 6H), 3.21-3.13 (m, 1H), 3.02-2.93 (m, 1H), 2.77- 2.63 (m, 2H), 2.45-2.29 (m, 3H), 2.24-2.05 (m, 3H), 2.04-1.93 (m, 5H), 1.90- 1.75 (m, 2H), 1.52-1.38 (m, 4H), 0.90-0.78 (m, 5H).

Connection with a single configuration (longer retention time):

UHPLC analysis: retention time: 1.23 minutes, purity: 90% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 9.05 (d, 1H), 8.97-8.88 (m, 1H), 8.35-8.27 (m, 1H), 8 .11-8.03(m, 1H), 8.02-7.95(m, 1H), 7.80(d, 1H), 7.34(s, 1H), 7.29-7.13 (m, 4H), 6.99 (s, 1H), 6.66 (br, 1H), 6.54 (s, 1H), 5.64-5.55 (m, 1H), 5.43 ( s, 1H), 5.36-5.20 (m, 3H), 4.92-4.85 (m, 1H), 4.82-4.72 (m, 2H), 4.52-4, 42 (m, 1H), 3.77-3.48 (m, 6H), 3.21-3.14 (m, 1H), 3.03-2.95 (m, 1H), 2.79- 2.65 (m, 2H), 2.47-2.28 (m, 3H), 2.25-2.05 (m, 3H), 2.05-1.94 (m, 5H), 1.91- 1.76 (m, 2H), 1.52-1.37 (m, 4H), 0.92-0.77 (m, 5H).

Example 11

1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2, 5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-hexahydro-1Н,12Н-benzo[ae]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutan-1-carboxamide 11

Benzyl

1-((2-((((9Н-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclobutane-1-carboxylate 11b

Benzyl 1-hydroxycyclobutane carboxylate 11a (167 mg, 0.81 mmol, prepared according to the known method described in the patent application "Journal of Medicinal Chemistry, 2013, vol. 56, # 13, p. 5541-5552") and 8b ( 150 mg, 0.41 mmol) was added to the reaction flask, after which 5 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which potassium tert-butoxide (92 mg, 0.82 mmol) was added. The ice water bath was removed, and the reaction solution was warmed to room temperature and stirred for 10 minutes. 20 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (5 ml×2) and chloroform (5 ml×5). The organic phases were combined and concentrated, and the resulting residue was dissolved in 3 ml of dioxane. 0.6 ml of water, sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorene methyl chloroformate (105 mg, 0.41 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. 20 ml of water was added, and the reaction solution was extracted with ethyl acetate (8 ml×3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 11 b (37 mg, yield: 17.6%).

MS m/z (ESI): 514.6 [M+1].

Stage 2

1-((2-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclobutane-1-carboxylic acid 11c

11b (37 mg, 71.9 μmol) was dissolved in 3 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), after which a carbon-supported palladium catalyst (15 mg, content: 10%) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 2 hours. The reaction solution was filtered through Celite, and the filter residue was washed with tetrahydrofuran. The filtrate was concentrated to give the title product 11 s (35 mg, yield: 82%), which was used directly in the next step.

Stage 3

(9H-Fluoren-9-yl)methyl (2-(((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)cyclobutoxy) methyl)amino)-2-oxoethyl)carbamate 11d

1b (10 mg, 0.018 mmol) was added to the reaction flask, after which 1 ml of N,N-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, 11 s (13 mg, 0.031 mmol) in 0.5 ml N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride (25 mg, 0.091 mmol) and the reaction solution was stirred in an ice bath for 40 minutes. 8 ml of water was added and the reaction solution was extracted with ethyl acetate (5 ml×3). The organic phase was washed with saturated sodium chloride solution (8 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using an Ac developing solvent system to give the title product 11d (19 mg, yield: 73.9%).

MS m/z (ESI): 842.3 [M+1].

Stage 4

1-((2-Aminoacetamido)methoxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)cyclobutan-1-carboxamide 11e

11d (19 mg, 22.6 μmol) was dissolved in 2 ml of dichloromethane, after which 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure. 1 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 4 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give the crude product 11e (15 mg) which was used directly in the next step without purification.

Stage 5

1-(((S)-7-Benzyl-20-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-3,6,9,12,15-pentaoxo-2, 5,8,11,14-pentaazaicosyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9, 10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutan-1-carboxamide 11

The crude compound 11e (2 mg, 3.22 μmol) was dissolved in 0.5 ml of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 8g (1.5 mg, 3.17 µmol) in 0.3 ml N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium were added chloride (2.7 mg, 9.67 μmol) and the reaction solution was stirred at room temperature for 30 minutes. The reaction solution was concentrated to dryness by rotary evaporation with an oil pump to remove DMF. The residues were dissolved in DCM and purified by thin layer chromatography twice (developing solution polarity: DCM/MeOH=10/1) to give the title product 11 (1 mg, yield: 28.8%).

MS m/z (ESI): 1073.6 [M+1].

¹ H NMR (400 MHz, CDCl ₃ ): δ 8.70-8.60 (m, 1H), 8.28-8.19 (m, 1H), 8.13-7.91 (m, 3H) , 7.79-7.71 (d, 1H), 7.29 (s, 1H), 7.25-7.09 (m, 4H), 6.98 (s, 1H), 6.71-6 .62 (m, 1H), 6.55-6.47 (m, 1H), 5.64-5.54 (m, 2H), 5.40 (s, 1H), 5.35-5.27 (t, 2H), 5.17-5.10 (m, 2H), 4.60-4.51 (m, 1H), 4.51-4.35 (m, 2H), 3.93-3 .78 (m, 3H), 3.71-3.59 (m, 3H), 3.01-2.88 (m, 3H), 2.70-2.64 (m, 2H), 2.44 -2.30 (m, 3H), 2.28-2.14 (m, 3H), 2.11-1.92 (m, 6H), 1.90-1.76 (m, 3H), 1 .51-1.39 (m, 4H), 0.92-0.75 (m, 6H).

Example 12

(S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxypropanamide 12-A

(R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxypropanamide 12-B

Stage 1

3-Cyclopropyl-2-hydroxypropanoic acid 12b

12a (0.5 g, 3.87 mmol, supplier: Adamas) was dissolved in 35 ml of a solvent mixture of water and acetic acid (V:V=4:1) and the solution was cooled to 0-5° C. in an ice water bath . A 2M sodium nitrite aqueous solution (0.53 g, 7.74 mmol) was added dropwise, and the reaction solution was warmed to room temperature and stirred for 3 hours. Solid sodium chloride was added to the reaction solution to saturate the aqueous phase. The solution was extracted with ethyl acetate (8 ml×8), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give the title product 12b (0.45 g, yield: 89.3%).

Stage 2

(S)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15 -hexahydro-1H,12H-benzo[de]pyrano[3',4':6,7]indolizino[1,2-i]quinolin-1-yl)-2-hydroxypropanamide 12-A

(R)-3-Cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)-2-hydroxypropanamide 12-B

1.5 ml of ethanol and 1.5 ml of N,N-dimethylformamide were added to 1b (45 mg, 0.085 mmol). The solution was purged with argon three times. 0.1 ml of N-methylmorpholine was added dropwise, and the reaction solution was stirred until clear. 12b (90 mg, 0.691 mmol), 1-hydroxybenzotriazole (34 mg, 0.251 mmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (49 mg, 0.256 mmol) were sequentially added to the reaction solution. After completion of the addition, the reaction solution was stirred at room temperature for 3 hours. The reaction solution was concentrated under reduced pressure, and the resulting crude compound 12 was purified by high performance liquid chromatography (separation conditions: column: Sharpsil-T C18 5 μm 21.2*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc) , B-acetonitrile, gradient elution, flow rate: 18 ml/min) to give the title product (7 mg, 15 mg).

MS m/z (ESI): 547.9 [M+1].

Single configuration compound (shorter retention time)

UHPLC analysis: retention time: 1.345 minutes, purity: 72% (column: ZORBAX Ecliphase Plus C18 1.8 µm 2.1*50mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.42 (d, 1H), 7.78 (d, 1H), 7.30 (s, 1H), 6.51 (s, 1H), 5.60-5.50 (m, 2H), 5.42 (s, 1H), 5.19 (q, 2H), 4.02-4.00 (m, 1H), 3.21-3, 11 (m, 2H), 2.39 (s, 3H), 2.21-2.07 (m, 2H), 2.05-1.95 (m, 1H), 1.92-1.68 ( m, 4H), 1.53-1.41 (m, 1H), 0.87 (t, 3H), 0.48-0.34 (m, 2H), 0.14-0.01 (m, 2H).

Connection with a single configuration (longer retention time)

UHPLC analysis: retention time: 1.399 minutes, purity: 88% (column: ZORBAX Ecliphase Plus C18 1.8 µm 2.1*50mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.36 (d, 1H), 7.77 (d, 1H), 7.31 (s, 1H), 6.51 (s, 1H), 5.58-5.51(m, 1H), 5.48(d, 1H), 5.42(s, 1H), 5.20(q, 2H), 4.09-4.02( m, 1H), 3.22-3.11(m, 2H), 2.39(s, 3H), 2.27-2.06(m, 2H), 2.05-1.95(m, 1H), 1.93-1.81(m, 2H), 1.65-1.43(m, 2H), 1.32-1.21(m, 1H), 0.87(t, 3H ), 0.48-0.33 (m, 2H), 0.14-0.01 (m, 2H).

Example 13 (reference example)

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-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)-2-hydroxyacetamide

The title compound 13 was obtained according to the method described in Example 76 on page 147 of the specification of patent application EP 2907824 A1.

Example 14

N-((2R,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 14-A

N-((2S,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 14-B

Stage 1

Benzyl 3-cyclopropyl-2-hydroxypropanoate 14a

12b (200 mg, 1.54 mmol) was dissolved in 20 ml of acetonitrile and then potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 ml, 1.34 mmol) and tetrabutylammonium iodide were added successively (28 mg, 0.07 mmol). The reaction solution was stirred at room temperature for 48 hours, and filtered through Celite. The filter cake was washed with ethyl acetate (10 ml) and the filtrates were combined and concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 14a (140 mg, yield: 41.3%).

Stage 2

Benzyl

10-(cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat 14b

14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to the reaction flask, after which 10 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which potassium tert-butoxide (79 mg, 0.704 mmol) was added. The ice water bath was removed, and the reaction solution was warmed to room temperature and stirred for 10 minutes. 20 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (10 ml×4). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 14b (50 mg, yield: 26.8%).

MS m/z (ESI): 529.2 [M+1].

Stage 3

10-(Cyclopropylmethyl)-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 14с

14b (27 mg, 0.051 mmol) was dissolved in 3 ml of ethyl acetate, after which a carbon-supported palladium catalyst (7 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 1 hour. The reaction solution was filtered through Celite, and the filter residue was washed with ethyl acetate. The filtrate was concentrated to give the crude product 14 s (23 mg) which was used directly in the next step without purification.

MS m/z (ESI): 439.1 [M+1].

Stage 4

(9H-Fluoren-9-yl)methyl (2-((((3-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10.13 -dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl )amino)-1-oxopropan-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 14d

1b (22 mg, 42.38 µmol) was added to the reaction flask, after which 3 ml of N,N-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. Triethylamine (4.3 mg, 42.49 µmol) was added dropwise and then crude compound 14c (23 mg, 51.1 µmol) and 4-(4,6-dimethoxy-1,3,5-triazine- 2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 µmol). The reaction solution was stirred in an ice bath for 40 minutes. 15 ml of water was added and the reaction solution was extracted with ethyl acetate (8 ml×3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 14d (29 mg, yield: 79.9%).

MS m/z (ESI): 856.1 [M+1].

Stage 5

2-((2-Aminoacetamido)methoxy)-3-i, iklopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)propanamide 14е

14d (29 mg, 33.9 μmol) was dissolved in 0.8 ml dichloromethane, after which 0.4 ml diethylamine was added. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure. 1 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the residue to form a slurry, and the supernatant was discarded after settling for some time, which was repeated three times. The residues were concentrated under reduced pressure with an oil pump to dryness to give crude product 14e (22 mg) which was used directly in the next step without purification.

MS m/z (ESI): 634.1 [M+1].

Stage 6

N-((2R,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 14-A

N-((2S,10S)-10-Benzyl-2-(cyclopropylmethyl)-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo -2,3,9,10,13,15-hexahydro-1Н,12Н-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-tetraazahexadecan-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H- pyrrol-1-yl)hexanamide 14-B

The crude compound 14e (22 mg, 33.9 μmol) was dissolved in 2.5 ml of N,N-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 8d (24 mg, 50.8 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (14 mg, 50.6 µmol) were added successively. The ice water bath was removed, and the reaction solution was warmed to room temperature and stirred for 1 hour to give compound 14. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 µm 19*250 mm; movable phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min) to give the title products (2 mg, 2 mg).

MS m/z (ESI): 1088.4 [M+1].

Connection with a single configuration (with a shorter retention time):

UHPLC analysis: retention time: 1.18 minutes, purity: 88% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

Connection with a single configuration (longer retention time):

UHPLC analysis: retention time: 1.23 minutes, purity: 96% (column: ACQUITY UPLC BEC18 1.7 µm 2.1*50 mm, mobile phase: A-water (5 mmol NH ₄ OAc), B-acetonitrile) .

Example 15

1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa -4,7,10,13,16-pentaazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3,4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropan-1-carboxamide 15

Stage 1

Benzyl

1-(10-(9Н-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylate 15b

8b (500 mg, 1.35 mmol) was added to the reaction flask, after which 6 ml of tetrahydrofuran was added. Benzyl 1-hydroxymethylcyclopropane-1-carboxylate 15a (233 mg, 1.13 mmol; obtained according to the method described in Example 22-2 on page 262 of the specification of patent application "EP 2862856 A1") was added to the reaction flask. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which sodium hydride (54 mg, 1.35 mmol) was added. The ice water bath was removed, and the reaction solution was warmed to room temperature and stirred for 40 minutes. The reaction solution was cooled to 0°C, to which was added 20 ml of ice water. The solution was extracted with ethyl acetate (5 ml x 2) and chloroform (5 ml x 5). The organic phases were combined, washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography using developing solvent system B to obtain the title product 15b (15 mg, yield: 2.5%).

MS m/z (ESI): 515.2 [M+1].

Stage 2

1-(10-(9H-Fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclopropane-1-carboxylic acid 15с

15b (15 mg, 0.029 mmol) was dissolved in 2 ml of ethyl acetate, after which a carbon-supported palladium catalyst (3 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 4.5 hours. The reaction solution was filtered through Celite, and the filter residue was washed with ethyl acetate. The filtrate was concentrated to give the title product 15 sec (11 mg, yield: 89%).

MS m/z (ESI): 425.2 [M+1].

Stage 3

(9H-Fluoren-9-yl)methyl (2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)cyclopropyl )methoxy)methyl)amino)-2-oxoethyl)carbamate 15d

1b (10 mg, 0.021 mmol) was added to the reaction flask, after which 1 ml of N,N-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, 15c (11 mg, 0.026 mmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (10.7 mg, 0.039 mmol) were added. After completion of the addition, the reaction solution was stirred at room temperature for 60 minutes. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (5 ml×3). The organic phase was washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 15d (19 mg, yield: 87.0%).

MS m/z (ESI): 842.2 [M+1].

Stage 4

1-(((2-Aminoacetamido)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)cyclopropan-1-carboxamide 15e

15d (19 mg, 22.56 µmol) was dissolved in 2 ml of dichloromethane, after which 1 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure at 0°C. 1 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give the crude product 15e (13.9 mg) which was used directly in the next step without purification.

MS m/z (ESI): 620.1 [M+1].

Stage 5

1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa -4,7,10,13,16-pentaazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropan-1-carboxamide 15

The crude compound 15e (13.9 mg, 22.4 µmol) was dissolved in 1 ml of g\1,G\1-dimethylformamide. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. 8g (15.8 mg, 33.4 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (9.3 mg, 33.6 µmol) were added ). The reaction solution was warmed to room temperature and stirred for 60 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product 15 (2.5 mg, yield: 10.3%).

MS m/z (ESI): 1074.2 [M+1].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.51-8.37 (m, 1H), 8.22 (t, 1H), 8.14-8.02 (m, 2H), 8.011 -7.94(m, 1H), 7.82-7.73(m, 1H), 7.29(s, 1H), 7.26-7.10(m, 3H), 6.98(s , 1H), 6.53-6.47 (m, 1H), 5.62-5.50 (m, 1H), 5.45-5.36 (m, 1H), 5.35-5.23 (m, 2H), 5.13-5.02 (m, 2H), 4.61-4.50 (m, 2H), 4.42-4.28 (m, 2H), 3.76-3 .61 (m, 3H), 3.60-3.45 (m, 3H), 3.27-3.23 (m, 1H), 3.20-2.81 (m.7H), 2.75 -2.61 (m, 3H), 241-2.28 (m, 3H), 2.23-2.13 (m, 2H), 2.11-2.01 (m, 1H), 2.03 -1.94 (m, 1H), 1.90 (s, 1H), 1.87-1.74 (m, 2H), 1.53-1.36 (m, 3H), 1.29-1 .08 (m, 4H), 0.90-0.68 (m, 4H).

Example 16

1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa -4,7,10,13,16-pentaazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutan-1-carboxamide 16

Stage 1

1-(Hydroxymethyl)cyclobutane-1-carboxylic acid 16b

Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplier: Alfa) was dissolved in methanol (2 ml) and water (1 ml), after which sodium hydroxide (126 mg, 3.15 mmol) was added. The reaction solution was heated to 40° C. and stirred for 3 hours. The reaction solution was cooled to room temperature and concentrated under reduced pressure to remove the organic solvent. The solution was extracted with ether (10 ml) and the aqueous phase was collected. The aqueous phase was adjusted to pH 3-4 with 6N. an aqueous solution of hydrochloric acid and concentrated under reduced pressure to obtain a solid. 3 ml of toluene was added and the solution was concentrated under reduced pressure to dryness, which was repeated three times. The residue was dried with an oil pump to give crude product 16b (206 mg) which was used directly in the next step without purification.

MSm/z (ESI, NEG): 129.2 [M-1].

Stage 2

Benzyl 1-(hydroxymethyl)cyclobutane-1-carboxylate 16c

The crude compound 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 ml), after which anhydrous potassium carbonate (1.09 g, 7.90 mmol), tetrabutylammonium iodide (29 mg, 78.51 µmol) and benzyl bromide (216 mg, 1.26 mmol). The reaction solution was stirred at room temperature overnight. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 16c (112 mg, yield: 32.1%).

MS m/z (ESI): 221.1 [M+1].

Stage 3

Benzyl

1-(10-(9H-fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylate 16d

16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to the reaction flask, after which 3 ml of tetrahydrofuran was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which potassium tert-butoxide (61 mg, 0.54 mmol) was added. The reaction solution was stirred in an ice water bath for 10 minutes. 20 ml of ice water was added to the reaction solution, which was then extracted with ethyl acetate (5 ml) and chloroform (5 ml×5). The organic phases were combined and concentrated. The resulting residue was dissolved in 3 ml of 1,4-dioxane. 0.5 ml of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorene methyl chloroformate (70 mg, 0.27 mmol) were added, and the reaction solution was stirred at room temperature for 1 hour. 20 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 16d (24 mg, yield: 16.7%).

MS m/z (ESI): 551.3 [M+23].

Stage 4

1-(10-(9Н-Fluoren-9-yl)-5,8-dioxo-2,9-dioxa-4,7-diazadecyl)cyclobutane-1-carboxylic acid 16е

16d (12 mg, 22.7 μmol) was dissolved in 1.5 ml of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), after which a carbon-supported palladium catalyst (5 mg, content: 10%) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 2 hours. The reaction solution was filtered through Celite, and the filter residue was washed with ethyl acetate. The filtrate was concentrated under reduced pressure to give crude product 16e (10 mg) which was used directly in the next step without purification.

Stage 5

(9H-Fluoren-9-yl)methyl (2-((((1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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)carbamoyl)cy class butyl)methoxy)methyl)amino)-2-oxoethyl)carbamate 16f

1b (7.5 mg, 0.014 mmol) was added to the reaction flask, after which 1 ml of N,N-dimethylformamide was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine, crude compound 16e (10 mg) in 0.5 ml of N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride were added (6 mg, 0.026 mmol) and the reaction solution was stirred in an ice bath for 30 minutes. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phase was washed with saturated sodium chloride solution (10 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 16f (10.6 mg, yield: 87.8%).

MS m/z (ESI): 856.2 [M+1].

Stage 6

1-(((2-Aminoacetamido)methoxy)methyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutan-1-carboxamide 16g

16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 ml dichloromethane, after which 0.3 ml diethylamine was added. The reaction solution was stirred at room temperature for 2 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give crude product 16e (8 mg) which was used directly in the next step without purification.

MS m/z (ESI): 634.1 [M+1].

Stage 7

1-((S)-9-Benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo-2-oxa -4,7,10,13,16-pentaazadocosyl)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-2,3,9 ,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutan-1-carboxamide 16

The crude compound 16g (8 mg) was dissolved in 1 ml of N,N-dimethylformamide. 8d (8.8 mg, 18.6 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (5.2 mg, 18.8 µmol) were added ). The reaction solution was stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min) to give the title product 16 (1.0 mg, yield: 7.2%).

MS m/z (ESI): 1088.0 [M+1].

Example 17

(1r,4f)-N-((S)-7-Benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) carbamoyl)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatritetetracontan-43 -yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

Stage 1

tert-Butyl 1-phenyl-2,5,8,11,14,17,20,23,26,29-dekaoxagentriacontan-31-oat 17b

1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosan-28-ol 17a (0.34 g, 0.67 mmol, supplier: Bide Pharmatech Ltd.) was dissolved in 10 ml dichloromethane, and then silver oxide (0.24 g, 1.01 mmol), tert-butyl bromoacetate (0.16 g, 0.81 mmol) and potassium iodide (0.07 g, 0.40 mmol) were added successively. The reaction solution was stirred at room temperature for 3 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to obtain the title product 17b (0.42 g, yield: 100%).

MS m/z (ESI): 636.3 [M+18].

Stage 2

tert-Butyl 29-hydroxy-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1-oate 17с

17b (417 mg, 0.67 mmol) was dissolved in 15 ml of tetrahydrofuran, after which a carbon-supported palladium catalyst (110 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times, heated to 60° C., and stirred for 3 hours. The reaction solution was filtered through Celite, and the filter residue was washed with tetrahydrofuran. The filtrate was concentrated to give the crude product 17 s (357 mg) which was used directly in the next step without purification.

MS m/z (ESI): 546.2 [M+18].

Stage 3

tert-butyl 29-azido-3,6,9,12,15,18,21,24,27-nonaoxanonocosan-1-oate 17d

17c (357 mg, 0.675 mmol) was dissolved in 10 ml of toluene, after which diphenyl azide phosphate (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added. The reaction solution was purged with argon three times and stirred at room temperature for 2 hours, followed by stirring at 105° C. for 19 hours. The reaction solution was cooled to room temperature and concentrated. 20 ml of water was added and the solution was extracted with ethyl acetate (10 ml x 4). The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to give crude product 17d (412 mg).

MS m/z (ESI): 571.3 [M+18].

Stage 4

tert-Butyl 29-amino-3,6,9,12,15,18,21,24,27-nonaoxanonacosan-1-oate 17e

17d (230 mg, 0.415 mmol) was dissolved in 8 ml of tetrahydrofuran, after which a carbon-supported palladium catalyst (58 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times, and stirred at room temperature for 2 hours. The reaction solution was filtered through Celite, and the filter cake was washed with tetrahydrofuran. The filtrate was concentrated to give crude product 17e (220 mg) which was used directly in the next step without purification.

MS m/z (ESI): 528.2 [M+1].

Stage 5

tert-butyl

1-((1r,4r)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14, 17,20,23,26,29-nonaoxa-2-azagentriacontane-31-oat 17f

(1r,4r)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxylic acid (98.5 mg, 0.415 mmol) was dissolved in 10 ml of dichloromethane followed by the addition of 2-(7-oxabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol). The reaction solution was purged with argon three times, and then the crude compound 17e (220 mg, 0.417 mmol) was added. The reaction solution was stirred at room temperature for 1 hour. 15 ml of water was added and the reaction solution was extracted with dichloromethane (8 mlx3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (15 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system B to obtain the title product 17f (122 mg, yield: 39.2%).

MS m/z (ESI): 747.2 [M+1].

Stage 6

1-((1r,4r)-4-((2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexyl)-1-oxo-5,8,11,14, 17,20,23,26,29-nonaoxa-2-azagentriacontan-31-oic acid 17d

17f (122 mg, 0.163 mmol) was dissolved in 0.8 ml dichloromethane, after which 0.4 ml trifluoroacetic acid was added. The reaction solution was stirred at room temperature for 1 hour. 15 ml of dichloromethane was added to the diluted reaction solution, and then concentrated under reduced pressure. 10 ml of n-hexane was added and the solution was concentrated under reduced pressure, which was repeated twice. 10 ml of toluene was added and the solution was concentrated under reduced pressure. 10 ml of a mixture of solvents l-hexane:ether=5:1 was added to the slurry, which was repeated three times until a pH close to 7. The solution was concentrated with an oil pump to dryness to give the title product 17g (98 mg, yield: 86.8 %).

MS m/z (ESI): 691.2 [M+1].

Stage 7

2,4-dimethoxybenzyl 1-((2-((((9Н-fluoren-9-yl)methoxy)carbonyl)amino)acetamido)methoxy)cyclopropane-1-carboxylate 17h

8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 ml) and then 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol) was added,

1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (115 mg, 0.60 mmol) and 4-dimethylaminopyridine (5 mg, 0.041 mmol). After completion of the addition, the reaction solution was stirred at room temperature for 1 hour. 20 ml of water was added and the solution was separated after shaking. The aqueous phase was extracted with dichloromethane (8 ml x 3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (20 ml), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 17h (124 mg, yield: 55.4%).

MS m/z (ESI): 583.1 [M+23].

Stage 8

2,4-dimethoxybenzyl(S)-1-((11-benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10 ,13,16-pentaazaheptadecan-17-yl)oxy)cyclopropan-1-carboxylate 17j

17h (39 mg, 69.6 μmol) was dissolved in 0.6 ml dichloromethane, after which 0.3 ml diethylamine was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure. The obtained crude product was dissolved in 2 ml of N,N-dimethylformamide, after which (((9H-fluoren-9-yl)methoxy)carbonyl)glycylglycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, obtained according to the method described in Example 7-12 on page 13 of the patent application "CN 108853514 A") and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83 .1 µmol). The reaction solution was stirred at room temperature for 1 hour. 10 ml water was added and the solution was extracted with ethyl acetate (10 ml x 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 17j (48 mg, yield: 83.9%).

MS m/z (ESI): 822DM+1].

Stage 9

(S)-1-((11-Benzyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2-oxa-4,7,10,13,16- pentaazaheptadecan-17-yl)oxy)cyclopropane-1-carboxylic acid 17k

17j (48 mg, 58.4 μmol) was dissolved in 1.4 ml of 3% (v/v) dichloroacetic acid in dichloromethane and the solution was cooled to 0-5° C. in an ice water bath. Triethylsilane (21 mg, 180.6 µmol) was added and the reaction solution was stirred in an ice bath for 3 hours. The reaction solution was concentrated under reduced pressure in an ice bath to remove half of the organic solvent. Added 5 ml of ether, and the solution naturally warmed to room temperature and suspended. A white solid was precipitated and filtered. The filter cake was collected and dried with an oil pump to give the title product 17k (33 mg, yield: 84.1%).

Stage 10

(9H-Fluoren-9-yl)methyl ((S)-7-benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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]quinoline-1- yl)carbamoyl)cyclopropoxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)carbamate 17l

1b (20 mg, 42.4 μmol) was added to the reaction flask, after which 1 ml of 10% (v/v) methanol in dichloromethane was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added and the reaction solution was stirred until 1b dissolved. 17k (33 mg, 49.1 μmol) was dissolved in 1 ml of 10% (v/v) methanol in dichloromethane. The resulting solution was added dropwise to the above reaction solution, followed by the addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 µmol) . The reaction solution was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added and the solution was stirred for 5 minutes and left to separate. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 ml x 3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to give the title product 171 (37 mg, yield: 80.2%).

MS m/z (ESI): 1090.1 [M+1].

Stage 11

(1r,4r)-N-((S)-7-Benzyl-1-(1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13- dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinolin-1-yl) carbamoyl)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatritetetracontan-43 -yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

17l (15.5 mg, 14.23 μmol) was dissolved in 0.6 ml of dichloromethane, after which 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure and dried with an oil pump. The obtained crude product was dissolved in 1 ml of N,N-dimethylformamide. 17g (11 mg, 15.92 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (6.0 mg, 21.68 µmol) were added. The reaction solution was purged with argon three times, and stirred at room temperature for 30 minutes. The reaction solution was purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product 17 (16 mg, yield: 27.4%).

MS m/z (ESI): 1556.4 [M+18].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.98 (d, 1H), 8.76 (s, 1H), 8.20 (br, 1H), 8.12-7.95 (m , 3H), 7.93-7.76 (m, 2H), 7.75-7.66 (m, 2H), 7.24 (s, 1H), 7.20-7.05 (m, 6H ), 6.97 (s, 1H), 6.64 (br, 1H), 6.55 (d, 1H), 6.47 (s, 1H), 5.61-5.52 (m, 2H) , 5.37 (s, 1H), 5.33-5.23 (m, 2H), 5.18 (s, 1H), 5.13 (s, 1H), 5.05 (s, 1H), 5.00 (s, 1H), 4.65-4.55 (m, 2H), 4.53-4.45 (m, 1H), 4.38-4.28 (m, 2H), 3, 84 (s, 2H), 3.67 (d, 3H), 3.60-3.40 (m, 33H), 3.18 (d, 1H), 3.15-3.08 (m, 3H) , 2.28 (s, 3H), 2.00-1.92 (m, 3H), 1.85 (s, 2H), 1.82-1.73 (m, 2H), 1.68-1 .52 (m, 4H), 1.29-1.15 (m, 3H), 0.86-0.76 (m, 5H).

Example 18

(1r,4r)-N-((2R, 10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14, 17-pentaazahexatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

Stage 1

Benzyl (R)-2-cyclopropyl-2-hydroxyacetate 18a

Benzyl (S)-2-cyclopropyl-2-hydroxyacetate 18b

2a (7.4 g, 63.7 mmol) was dissolved in 200 ml of acetonitrile, and then potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide were added successively (500 mg, 1.36 mmol). The reaction solution was stirred at room temperature for 16 hours, and filtered through Celite. The filter cake was washed with ethyl acetate (10 ml) and the filtrates were combined and concentrated under reduced pressure. 4.1 g of the resulting residue was purified by silica gel column chromatography using developing solvent system C. Chiral separation was further performed to give the title products 18a (1.1 g) and 18b (1.2 g).

Stage 2

Benzyl

(R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat18 c

8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 ml), after which 18a (2.0 g, 9.70 mmol) was added. The reaction solution was cooled to 0-5° C. in an ice water bath, after which potassium tert-butoxide (1.89 g, 16.84 mmol) was added. The reaction solution was stirred in an ice water bath for 10 minutes. Ethyl acetate (30 ml) and water (20 ml) were added and the solution was left to separate. The aqueous phase was extracted with chloroform (30 ml x 5) and the organic phases were combined. The organic phase was concentrated under reduced pressure and the resulting residue was dissolved in 1,4-dioxane (32 ml) and water (8 ml). Sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorene methyl chloroformate (2.18 g, 8.42 mmol) were added, and the reaction solution was stirred at room temperature for 2 hours. Water (30 ml) was added to the reaction solution, which was extracted with ethyl acetate (50 ml×3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography using developing solvent system C to obtain the title product 18 sec (1.3 g, yield: 30.0%).

MS m/z (ESI): 515.2 [M+1].

Stage 3

(R)-10-Cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 18d

18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 ml), after which carbon-supported palladium catalyst (260 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 5 hours. The reaction solution was filtered through Celite, and the filter cake was washed with ethyl acetate (20 ml) and methanol (20 ml). The filtrate was concentrated to give crude product 18d (980 mg) which was used directly in the next step without purification.

MS m/z (ESI): 425.1 [M+1].

Stage 4

2,4-dimethoxybenzyl

(R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat 18e

The crude compound 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 ml) and then 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride was added (664 mg, 3.46 mmol) and 4-dimethylaminopyridine (28 mg, 0.23 mmol). The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure to remove the organic solvent. 20 ml of water was added and the solution was extracted with ethyl acetate (50 ml x 3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (30 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by column chromatography using developing solvent system C to obtain the title product 18e (810 mg, yield: 61.1%).

MS m/z (ESI): 575.0 [M+1].

Stage 5

2,4-Dimethoxybenzyl

(R)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 18f

18e (33 mg, 57.4 μmol) was dissolved in 0.6 ml of dichloromethane, after which 0.3 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give crude product 18f (21 mg) which was used directly in the next step without purification.

Stage 6

2,4-dimethoxybenzyl

(11S,19R)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10, 13,16-pentaazaicosan-20-oat 18d

The crude compound 18f (21 mg, 57.4 μmol) was dissolved in 3 ml of N,N-dimethylformamide. 17i (29 mg, 57.8 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (19 mg, 68.7 µmol) were added. The reaction solution was stirred at room temperature for 1 hour. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 18e (37 mg, yield: 77.1%).

MS m/z (ESI): 853.0 [M+18].

Stage 7

(11S,19R)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10, 13,16-pentaazaicosan-20-oic acid 18h

18g (37 mg, 44.3 µmol) was dissolved in 1.4 ml of 3% (v/v) dichloroacetic acid, dichloromethane, and the solution was cooled to 0-5° C. in an ice water bath. Triethylsilane (15.4 mg, 132.4 µmol) was added and the reaction solution was stirred in an ice bath for 3 hours. The reaction solution was concentrated under reduced pressure in an ice bath to remove half of the organic solvent. Added 5 ml of ether, and the solution naturally warmed to room temperature and suspended. A white solid was precipitated and filtered. The filter cake was collected and dried with an oil pump to give the title product 18h (24 mg, yield: 79.1%).

MS m/z (ESI): 708.2 [M+23].

Stage 8

(9H-Fluoren-9-yl)methyl

((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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-tetraazahexadecan-16-yl)carbamate 18i

1b (30 mg, 63.6 μmol) was added to the reaction flask, after which 1 ml of 10% (v/v) methanol in dichloromethane was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added and the reaction solution was stirred until 1b dissolved. 18h (65 mg, 94.8 µmol) was dissolved in 1 ml of 10% (v/v) methanol in dichloromethane, and the resulting solution was added dropwise to the above reaction solution, after which 4-(4,6-dimethoxy- 1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 µmol). The reaction solution was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added and the solution was stirred for 5 minutes and left to separate. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 ml x 3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 1 Si (25 mg, yield: 35.6%).

MS m/z (ESI): 1104.4 [M+1].

Stage 9

(S)-2-(2-(2-Aminoacetamido)acetamido)-N-(2-((((R)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 18j

18i (12 mg, 10.9 μmol) was dissolved in 0.6 ml dichloromethane, after which 0.3 ml diethylamine was added. The reaction solution was stirred at room temperature for 1.5 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give crude product 18j (10 mg) which was used directly in the next step without purification.

MS m/z (ESI): 881.0 [M+1].

Stage 10

(1r,4r)-N-((2R,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14, 17-pentaazahexatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

The crude compound 18j (10 mg) was dissolved in 1 ml of N,N-dimethylformamide. 17g (8.5 mg, 12.3 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.6 mg, 16.6 µmol) were added ). The reaction solution was stirred at room temperature for 30 minutes. The reaction solution was filtered, and purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 µm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, flow rate: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product 18 (9.5 mg, yield: 56.2%).

MS m/z (ESI): 1570.2 [M+18].

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.77 (d, 1H), 8.59-8.55 (m, 1H), 8.42 (d, 1H), 8.37-8 .28 (m, 1H), 8.25-8.06 (m, 2H), 7.96-7.86 (m, 1H), 7.86-7.70 (m, 2H), 7.32 -7.28 (m, 1H), 7.25-7.14 (m, 3H), 6.67 (m, 1H), 5.96 (s, 1H), 5.80-5.72 (m , 1H), 5.62-5.52 (m, 2H), 5.43-5.30 (m, 3H), 5.28-5.17 (m, 2H), 5.12-5.08 (m, 1H), 4.72-4.35 (m, 8H), 3.95-3.70 (m, 13H), 3.35-3.22 (m, 14H), 2.42-2 .32 (m, 3H), 2.05-1.98 (m, 4H), 1.88-1.82 (m, 12H), 1.47-1.39 (m, 3H), 1.32 -1.18(m, 11H), 0.90-0.80(m, 4H), 0.52-0.37(m, 3H), 0.32-0.18(m, 2H).

Example 19

(1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14, 17-pentaazahexatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

Stage 1

Benzyl

(S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat 19a

18b (252 mg, 1.22 mmol) was added to the reaction flask, after which 4 ml of dichloromethane was added. The reaction solution was purged with argon three times and cooled to 0-5° C. in an ice water bath, after which lithium tert-butoxide (98 mg, 1.22 mmol) was added. The reaction solution was stirred in an ice water bath for 15 minutes until clear. 8b (300 mg, 814.3 µmol) was added and the reaction solution was stirred in an ice water bath for 2.5 hours. Water (10 ml) was added and the solution was separated. The aqueous phase was extracted with dichloromethane (8 ml x 2). The organic phases were combined, washed with water (10 ml×1) and brine (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated to give the crude product. The resulting residue was purified by silica gel column chromatography using developing solvent system C to obtain the title product 19a (282 mg, yield: 67.2%).

Stage 2

(S)-10-Cyclopropyl-1-(9Н-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oic acid 19b

19a (280 mg, 0.554 mmol) was dissolved in 8 ml of ethyl acetate, after which a carbon-supported palladium catalyst (84 mg, content: 10%, dry) was added. The reaction solution was purged with hydrogen three times and stirred at room temperature for 3 hours. The reaction solution was filtered through Celite, and the filter residue was washed with ethyl acetate. The filtrate was concentrated to give the crude product 19b (230 mg) which was used directly in the next step without purification.

Stage 3 2,4-dimethoxybenzyl

(S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-dioxo-2,9-dioxa-4,7-diazaundecan-11-oat 19c

The crude compound 19b (230 mg, 541.8 µmol) was dissolved in 7 ml of dichloromethane, and then 2,4-dimethoxybenzyl alcohol (136.7 mg, 812.7 µmol), 1-ethyl-(3-dimethylaminopropyl)carbodiimide was added successively hydrochloride (155 mg, 808.5 µmol) and 4-dimethylaminopyridine (6.6 mg, 53.5 µmol). The reaction solution was stirred at room temperature for 16 hours. The reaction solution was diluted with 10 ml dichloromethane, washed with water (10 ml×1) and brine (10 ml×2), dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to give the crude product. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 19 s (159 mg, yield: 51.0%).

Stage 4

2,4-dimethoxybenzyl (S)-2-((2-aminoacetamido)methoxy)-2-cyclopropyl acetate 19d

19c (60 mg, 104.4 µmol) was dissolved in 1 ml of dichloromethane, after which 0.5 ml of diethylamine was added. The reaction solution was stirred at room temperature for 1 hour. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give the crude product 19d (21 mg) which was used directly in the next step without purification.

Stage 5

2,4-dimethoxybenzyl

(11S,19S)-11-benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10, 13,16-pentaazaicosan-20-oate 19e

The crude compound 19d (36 mg, 102.2 μmol) was dissolved in 4 ml of N,N-dimethylformamide. 17i (52 mg, 103.6 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (34.6 mg, 125.0 µmol) were added. The reaction solution was stirred at room temperature for 1 hour. 10 ml of water was added and the reaction solution was extracted with ethyl acetate (10 ml×3). The organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 19e (70 mg, yield: 80.2%).

Stage 6

(11S,19S)-11-Benzyl-19-cyclopropyl-1-(9H-fluoren-9-yl)-3,6,9,12,15-pentaoxo-2,18-dioxa-4,7,10, 13,16-pentaazaicosan-20-oic acid 19f

19e (70 mg, 83.7 μmol) was dissolved in 2.5 ml of 3% (v/v) dichloroacetic acid in dichloromethane and the solution was cooled to 0-5° C. in an ice water bath. Triethylsilane (29 mg, 249.4 µmol) was added and the reaction solution was stirred in an ice bath for 3 hours. The reaction solution was concentrated under reduced pressure in an ice bath to remove half of the organic solvent. Added 5 ml of ether, and the solution naturally warmed to room temperature and suspended. A white solid was precipitated and filtered. The filter cake was collected and dried with an oil pump to give the title product 19f (57 mg, yield: 99.2%).

Stage 7

(9H-Fluoren-9-yl)methyl

((2S,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-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-tetraazahexadecan-16-yl)carbamate 19g

1b (30 mg, 63.6 μmol) was added to the reaction flask, after which 1 ml of 10% (v/v) methanol in dichloromethane was added. The solution was purged with argon three times and cooled to 0-5° C. in an ice water bath. One drop of triethylamine was added and the reaction solution was stirred until 1b dissolved. 19f (57 mg, 83.1 µmol) was dissolved in 1 ml of 10% (v/v) methanol in dichloromethane, and the resulting solution was added dropwise to the above reaction solution, after which 4-(4,6-dimethoxy- 1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 µmol). The reaction solution was warmed to room temperature and stirred for 1 hour. 10 ml of dichloromethane and 5 ml of water were added and the solution was stirred for 5 minutes and left to separate. The organic phase was collected. The aqueous phase was extracted with dichloromethane (10 ml x 3) and the organic phases were combined. The organic phase was washed with saturated sodium chloride solution (10 ml×2), dried over anhydrous sodium sulfate and filtered. The filtrate was concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography using developing solvent system B to obtain the title product 19e (56 mg, yield: 79.8%).

MS m/z (ESI): 1103.1 [M+1]

Stage 8

(S)-2-(2-(2-Aminoacetamido)acetamido)-N-(2-((((5)-1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro -9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino [1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethyl)-3-phenylpropanamide 19h

19g (4.6 mg, 4.16 µmol) was dissolved in 1.5 ml dichloromethane, after which 0.75 ml diethylamine was added. The reaction solution was stirred at room temperature for 1.6 hours. The reaction solution was concentrated under reduced pressure. 2 ml of toluene was added and the solution was concentrated under reduced pressure, which was repeated twice. 3 ml of n-hexane was added to the slurry and the top layer of hexane was decanted, which was repeated three times. The solution was concentrated under reduced pressure to give crude product 19h (4.0 mg) which was used directly in the next step without purification.

Stage 9

(1r,4r)-N-((2S,10S)-10-Benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10 ,13-dioxo-2,3,9,10,13,15-hexahydro-1Н,12Н-benzo[de]pyrano[3',4':6,7]indolizino[1,2-b]quinoline-1 -yl)amino)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14, 17-pentaazahexatetracontan-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexan-1-carboxamide 19

The crude compound 19h (4.0 mg) was dissolved in 1 ml of N,N-dimethylformamide. 17d (2.9 mg, 4.2 µmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (1.5 mg, 5.4 µmol) were added ). The reaction solution was stirred at room temperature for 40 minutes. The reaction solution was filtered and purified by high performance liquid chromatography (separation conditions: column: XBridge Prep C18 OBD 5 μm 19*250 mm; mobile phase: A-water (10 mmol NH ₄ OAc), B-acetonitrile, gradient elution, speed flow: 18 ml/min). Appropriate fractions were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, yield: 32.4%).

¹ H NMR (400 MHz, DMSO-d ₆ ): δ 8.71-8.62 (m, 1H), 8.59-8.51 (m, 1H), 8.34-8.26 (m, 1H), 8.14-8.02 (m, 2H), 7.95-7.86 (m, 1H), 7.83-7.69 (m, 2H), 7.35-7.31 ( m, 1H), 7.29-7.11(m, 3H), 7.01(s, 1H), 6.72-6.50(m, 3H), 5.59-5.50(m, 2H), 5.42 (s, 2H), 5.38-5.18 (m, 3H), 4.79-4.69 (m, 2H), 4.61-4.42 (m, 3H) , 3.91 (s, 2H), 3.79-3.65 (m, 4H), 3.63-3.44 (m, 13H), 3.41-3.30 (m, 2H), 3 .26-3.09 (m, 5H), 3.08-2.84 (m, 4H), 2.81-2.64 (m, 3H), 2.42-2.28 (m, 3H) , 2.24-2.12(m, 2H), 2.05-1.93(m, 4H), 1.89-1.77(m, 2H), 1.72-1.56(m, 3H), 1.53-1.38 (m, 3H), 1.34-1.10 (m, 11H), 0.94-0.78 (m, 5H), 0.52-0.35 ( m, 3H).

Example 20 (reference example)

The title compound 20 was obtained based on the method described in Example 58 on page 163 of the specification of patent application CN 104755494 A.

The following antibodies were generated according to conventional methods, eg, vector construction, transfection of eukaryotic cells, eg, HEK293 transfection (Life Technologies Cat. No. 11625019), cell purification and expression.

Following is the sequence of trastuzumab:

light chain

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 ml, 0.82 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 2.5 ml , 9.96 mg/ml, 0.168 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 2.0 ml of the solution was taken for the next reaction.

Compound 10, which has a shorter retention time (2.1 mg, 2.02 μmol), was dissolved in 0.10 ml of DMSO, and then added to 2.0 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-1 product of formula FADC-1 ( 5.0 mg/ml, 1.1 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=5.09.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 ml, 0.82 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 2.5 ml , 9.96 mg/ml, 0.168 μmol) at 37°C. The reaction solution was placed in a shaker with a water bath, and shaken at 37°C for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 2.0 ml of the solution was taken for the next reaction.

Compound 10, which has a longer retention time (2.1 mgt 2.02 μmol), was dissolved in 0.10 ml of DMSO, and then added to 2.0 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-2 product of formula FADC-1 ( 4.95 mg/ml, 1.1 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=7.39.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 ml, 0.82 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 2.5 ml , 9.96 mg/ml, 0.168 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 2.0 ml of the solution was taken for the next reaction.

Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 ml of DMSO, and then added to 2.0 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-3 product of formula FADC-3 ( 5.24 mg/ml, 1.1 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=7.36.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 ml, 1.73 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 3.74 ml , 13.38 mg/ml, 0.338 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg/ml. 1.3 ml of the solution was taken for the next reaction.

Compound 9, having a shorter retention time, compound 9-A (1.0 mg, 0.93 μmol) was dissolved in 0.10 ml of DMSO, and then added to 1.3 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction.

The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to obtain a PBS buffered solution of exemplary ADC-4 product of formula FADC-4A( 1.72 mg/ml, 2.36 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=7.39.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 ml, 0.67 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 3.0 ml , 6.70 mg/ml, 0.136 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath, and 0.614 ml of the solution was taken for the next reaction.

Compound 9, having a shorter retention time, compound 9-A (0.5 mg, 0.42 μmol) was dissolved in 0.031 ml of DMSO, and then added to 0.614 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-5 product of formula FADC-4A ( 3.08 mg/ml, 0.82 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=3.16.

Example 26 ADC-6

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 ml, 1.73 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 3.74 ml , 13.38 mg/ml, 0.338 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 6.7 mg/ml. 0.75 ml of the solution was taken for the next reaction.

Compound 9, having a longer retention time, Compound 9-B (0.68 mg, 0.63 μmol) was dissolved in 0.10 ml of DMSO, and then added to 0.75 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-6 product of formula FADC-4B ( 1.78 mg/ml, 1.78 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=3.94.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 ml, 1.73 µmol) was added to PBS buffered aqueous solution of Pertuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 5.0 ml , 10 mg/ml, 0.338 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 1.0 ml of the solution was taken for the next reaction.

Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 ml of DMSO, and then added to 1.0 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-7 product of formula FADC-7 ( 1.42 mg/ml, 2.15 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=6.91.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 ml, 1.73 µmol) was added to PBS buffered aqueous solution of Pertuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 5.0 ml , 10 mg/ml, 0.338 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 1.6 ml of the solution was taken for the next reaction.

Compound 10, which has a shorter retention time (1.04 mg, 1.0 μmol) was dissolved in 0.1 ml of DMSO, and then added to 1.6 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-8 product of formula FADC-8 ( 2.14 mg/ml, 2.31 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=6.58.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 ml, 1.73 µmol) was added to PBS buffered aqueous solution of Pertuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 5.0 ml , 10 mg/ml, 0.338 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg/ml. 0.8 ml of the solution was taken for the next reaction.

Compound 9, having a shorter retention time, Compound 9-A (0.55 mg, 0.5 µmol) was dissolved in 0.1 ml of DMSO, and then added to 0.8 ml of the above solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-9 product of formula FADC-9A ( 2.27 mg/ml, 1.11 ml) which was stored at 4°C.

Average calculated by UV-HPLC: n=3.16.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 ml, 197.6 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.574 mg/ml, 38.78 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 14, which has a shorter retention time (0.64 mg, 588 nmol) was dissolved in 40 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-10 of formula FADC-10 ( 5.48 mg/ml, 1.03 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.25.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 ml, 222.4 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.646 mg/ml, 43.64 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 14, a compound having a longer retention time (0.72 mg, 662 nmol), was dissolved in 40 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-11 of formula FADC-10 ( 2.13 mg/ml, 1.87 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.03.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μl, 250.0 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.726 ml, 49.05 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-12 of formula FADC-12 ( 3.34 mg/ml, 1.45 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.93.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μl, 98.8 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.287 ml, 19.39 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-13 of formula FADC-13 ( 2.37 mg/ml, 0.88 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.53.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μl, 203.8 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.592 ml, 40.0 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-14 of formula FADC-14 ( 0.30 mg/ml, 12.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.61.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μl, 203.8 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.592 ml, 40.0 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-15 of formula FADC-15 ( 0.32 mg/ml, 11.8 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.89.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 µl, 182.5 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.53 ml, 35.8 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-16 of formula FADC-16 ( 0.32 mg/ml, 12.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.43.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μl, 432 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 2.0 ml, 135.12 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (2.22 mg, 2067 nmol) was dissolved in 175 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-17 product of formula FADC-4A ( 1.32 mg/ml, 12.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=5.42.

Example 38ADC-18 (reference example)

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μl, 517 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 1.5 ml, 101.3 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-18 of formula FADC-18 ( 0.79 mg/ml, 13.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.23.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μl, 469 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 1.36 ml, 91.9 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-19 of formula FADC-4A ( 0.73 mg/ml, 13.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.26.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μl, 517 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 1.5 ml, 101.3 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 10, a compound having a longer retention time (2.0 mg, 1815 nmol) was dissolved in 100 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-20 of formula FADC-1 ( 0.73 mg/ml, 13.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.43.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μl, 639 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 1.86 ml, 125.4 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-21 of formula FADC-18 ( 2.91 mg/ml, 4.44 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.23.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μl, 649 nmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5; 10 .0 mg/ml, 1.88 ml, 127.2 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-22 product of formula FADC-4A ( 3.56 mg/ml, 3.98 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.79.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 ml, 118.9 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 345 ml, 23.31 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3.5 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 ml of MeCN and 3.56 ml of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified on an ultrafiltration unit with a PBS buffered aqueous solution containing 2% (v/v) MeCN and 1% (v/v) DMSO (0.05 M PBS buffered aqueous solution pH=6, 5) and an aqueous solution buffered with succinic acid (0.01 M aqueous solution buffered with succinic acid, pH=5.3) Sucrose was added to 60 mg/ml, and Tween 20 was added to 0.2 mg/ml. The solution was bottled and lyophilized to provide a lyophilized powder sample of exemplary product ADC-23 of formula FADC-18, which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.05.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 ml, 114.4 µmol) was added to PBS buffered aqueous solution of Trastuzumab antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 332 ml, 22.43 µmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3.5 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (241 mg, 224 μmol) was dissolved in 13.76 ml of MeCN and 6.88 ml of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified on an ultrafiltration unit with a PBS buffered aqueous solution containing 4% (v/v) MeCN and 2% (v/v) DMSO (0.05 M PBS buffered aqueous solution pH=6, 5) and an aqueous solution buffered with succinic acid (0.01 M aqueous solution buffered with succinic acid, pH=5.3) Sucrose was added to 60 mg/ml, and Tween 20 was added to 0.2 mg/ml. The solution was bottled and lyophilized to provide a lyophilized powder sample of exemplary product ADC-24 of formula FADC-4A, which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.07.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μl, 740 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 2.14 ml, 144.60 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (3.0 mg, 2793 nmol) was dissolved in 150 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-25 of formula FADC-25 ( 1.28 mg/ml, 13.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.87.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μl, 300 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.89 ml, 60.14 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-26 of formula FADC-26 ( 1.61 mg/ml, 4.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.15.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μl, 300 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.89 ml, 60.14 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary ADC-27 product of formula FADC-25 ( 1.94 mg/ml, 3.5 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.11.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μl, 810 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 2.36 ml, 159.47 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 µl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-28 of formula FADC-26 ( 1.29 mg/ml, 13.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.46.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μl, 290 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.80 ml, 50.06 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 9 having a shorter retention time, compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-29 of formula FADC-25 ( 2.63 mg/ml, 2.4 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=7.24.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μl, 290 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.86 ml, 58.4 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-30 of formula FADC-26 ( 1.61 mg/ml, 4.0 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.15.

The prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μl, 300 nmol) was added to PBS buffered aqueous solution of B7H3 antibody 1F9DS antibody (0.05 M PBS buffered aqueous solution pH=6.5 ; 10.0 mg/ml, 0.89 ml, 60.14 nmol) at 37°C. The reaction solution was placed in a water bath shaker and shaken at 37° C. for 3 hours before stopping the reaction. The reaction solution was cooled to 25° C. in a water bath.

Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μl of DMSO, and then added to the above reaction solution. The reaction solution was placed in a water bath shaker and shaken at 25° C. for 3 hours before stopping the reaction. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffered aqueous pH 6.5 containing 0.001 M EDTA) to give a PBS buffered solution of exemplary product ADC-31 of formula FADC-31 ( 1.47 mg/ml, 4.5 ml) which was stored at 4°C.

Mean value calculated with UV-Vis: n=6.33.

Drug loading analysis in ADC stock solution Experimental purpose and principle

The ADC stock solution is a kind of drug conjugated to antibodies. Its mechanism for treating disease is to deliver toxin molecules to cells by targeting antibodies, thereby killing the cells. Drug loading plays a critical role in drug efficacy. The drug loading in the stock ADC solution was determined by the ultraviolet method.

Experiment method

The cuvette containing the sodium succinate buffer was placed in the absorbance control well and in the absorbance well for sample measurement, respectively. After the blank was subtracted, the cuvette containing the test solution was placed in the sample absorbance cell, and the optical density was measured at 280 nm and 370 nm.

Calculation of results: Drug loading in the stock ADC solution was determined by ultraviolet spectrophotometry (instrument: Thermo nanodrop2000 ultraviolet spectrophotometer). Its principle is that the total absorbance of an ADC stock solution at a certain wavelength is equal to the sum of the absorbance of the cytotoxic drug and the absorbance of the monoclonal antibody at that wavelength, i.e.:

ε _Drug-280 : the average value of the molar absorption coefficient of the drug at 280 nm is 5100;

C _drugs : drug concentration;

ε _mab-280 : mean molar absorbance of trastuzumab stock solution or pertuzumab stock solution at 280 nm is 214600;

C _mab : concentration of trastuzumab stock solution or pertuzumab stock solution;

b: The optical path length is 1 cm.

In the same way, one can obtain the equation for the total absorption of the sample at 370 nm:

ε _Drug-370 : the average value of the molar absorption coefficient of the drug at 370 nm is 19000;

C _drugs : drug concentration;

ε _mab-370 : mean molar absorbance of trastuzumab stock solution or pertuzumab stock solution at 370 nm is 0;

C _mab : concentration of stock solution of trastuzumab;

b: The optical path length is 1 cm.

Drug loading can be calculated using two equations (1) and (2) in combination with extinction coefficient and monoclonal antibody and drug concentration data at two detection wavelengths.

Drug load = C _drug / C _mab .

Biological analysis

Test Example 1: In Vitro Test for Inhibition of Tumor Cell Proliferation by a Compound of Formula (D)

I. Challenge of the test

The purpose of this test is to test in vitro the inhibitory effect of the drug compound of formula (D) according to the present invention on the proliferation of U87MG cells (Cell Bank of the Chinese Academy of Sciences, catalog No. TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Art. No. NTV-30). Cells were treated with various concentrations of the compound in vitro. After 6 days of culture, cell proliferation was determined with the CTG reagent (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, art. no.: G7573) and the in vitro activity of the compound was evaluated according to the IC ₅₀ value.

II. Test method

The test method for testing in vitro proliferation inhibition of the present compounds on tumor cells is described below using the method for testing in vitro proliferation inhibition on U87MG cells as an example. The method is also applicable to, but not limited to, in vitro proliferation inhibition assays on other tumor cells.

1. Cell culture: U87MG cells and SK-BR-3 cells were cultured in EMEM medium containing 10% FBS (GE, art. no. SH30024.01) and McCoy 5A medium containing 10% FBS (Gibco, art. no. 16600 -108), respectively.

2. Cell preparation: U87MG and SK-BR-3 cells in the logarithmic growth phase were washed once with PBS (phosphate buffer, Shanghai Basal Media Technologies Co., Ltd.), and then 2-3 ml of trypsin (0.25% Trypsin-EDTA (1×), Gibico, Life Technologies) for hydrolysis for 2-3 min. After complete hydrolysis of cells, 10-15 ml of cell culture medium was added. The digested cells were eluted and centrifuged at 1000 rpm for 5 minutes. The supernatant was discarded and 10-20 ml of cell culture medium was added to resuspend the cells to obtain a single cell suspension.

3. Cell seeding: Monosuspensions of U87MG and SK-BR-3 cells were mixed well and the cell density was adjusted to 2.75 x 10 ³ cells/ml and 8.25 x 10 ³ cells/ml with cell culture medium, respectively. The density-adjusted cell suspension was mixed well and added to a 96-well cell culture plate at 180 μl/well. 200 μl of culture medium was added to the peripheral wells of a 96-well plate. The tablet was incubated in an incubator for 24 hours (37°C, 5% CO ₂ ).

4. Compound composition: The compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Technology Co., Ltd.) to obtain a stock solution with an initial concentration of 10 mM.

The initial concentration of the low molecular weight compound was 500 nM, and the preparation method was as follows.

30 μl of various test samples were respectively added to the first column of a 96-well U-bottom plate to prepare a preparation with a sample concentration of 100 μM. 20 μl of DMSO was added to each well from the second to the 11th column. 10 µl of the sample from the first column was added to 20 µl of DMSO in the second column and mixed well, from which 10 µl was taken and added to the third column, and so on up to the 10th column. 5 µl of drug from each well of the formulation plate was added to 95 µl of EMEM culture medium and mixed well for subsequent use.

The starting concentration of ADC was 10 nM or 500 nM, and the preparation method was as follows.

In the first column of a 96-well plate, 100 μl of various test samples were respectively added at a sample concentration of 100 nM or 5 μM. 100 μl of PBS was added to each well from the second to the 11th column. 50 µl of the sample from the first column was added to 100 µl of PBS in the second column and mixed well, from which 50 µl was taken and added to the third column, and so on up to the 10th column with a 3-fold dilution.

5. Sample content: 20 µl of the prepared test samples at various concentrations were added to the culture plate, each sample was tested in duplicate. The tablet was incubated in an incubator for 6 days (37°C, 5% CO ₂ ).

6. Staining procedure: Take out a 96-well cell culture plate, add 90 µl of CTG solution to each well, and incubate at room temperature for 10 minutes.

7. Plate Reading: A 96-well cell culture plate was taken out and placed in a microplate reader (BMG labtech, PHERAstar FS) to measure chemiluminescence.

III. Data analysis

The data was analyzed using Microsoft Excel and Graphpad Prism 5. The results are shown in the following table.

Table 1. IC ₅₀ value of in vitro inhibition of small molecular weight fragments according to the present invention in relation to the proliferation of SK-BR-3 cells and U87 cells

Conclusion: The low molecular weight fragment according to the present invention has an obvious inhibitory activity against the proliferation of SK-BR-3 cells and U87 cells, and the chiral center has a certain effect on the inhibitory activity of the compound.

Test Example 2: In Vitro Test for Inhibition of Tumor Cell Proliferation with the Antibody Drug Conjugate of the Invention Targeting HER ²

The purpose of this assay is to test the in vitro inhibitory effect of the antibody-drug conjugate of the present invention targeting HER2 on the proliferation of SK-BR-3 cells (human breast cancer cells, ATCC, art. no. HTB-30) and MDA- MB-468 (human breast cancer cells, ATCC, art. no. NTV-132). Cells were treated with various concentrations of compounds in vitro. After 6 days of culture, cell proliferation was determined using the CTG reagent and the in vitro activity of the compounds was evaluated according to the IC ₅₀ value.

According to the test method in Test Example 1, the test cells were SK-BR-3 cells and MDA-MB-468 cells, and McCoy 5A medium containing 10% FBS (Gibco, ref no. 16600-108), EMEM medium containing 10% FBS (GE, part number SH30024.01) and L-15 medium containing 10% FBS (ThermoFisher, part number 11415-114). The viable cell densities of the three cell lines were adjusted to 8.33×10 ³ cells/ml, 8.33×10 ³ cells/ml, and 1.39×10 ⁴ cells/ml using cell culture medium, respectively. The density-adjusted cell suspension was mixed well and added to a 96-well cell culture plate at 180 μl/well. Related compounds were tested and the results are shown in the table below.

Table 2. In vitro IC ₅₀ value of inhibition of tumor cell proliferation by an antibody drug conjugate of the present invention targeting HER2

Conclusion: The HER2-targeted antibody-drug conjugate of the present invention has a clear inhibitory activity on the proliferation of HER2-positive SK-BR-3 cells. Meanwhile, it has a weak inhibitory activity against the proliferation of HER ² -negative MDA-MB-468 cells. Therefore, it has good selectivity.

Test Example 3: Her2-ADC Plasma Stability Test

ADC-19 sample, ADC-18 sample, ADC-20 sample, human plasma, monkey plasma (Shanghai Medicilon Inc.) and 1% BSA solution (Sigma) PBS (Sangon Biotech (Shanghai) Co., Ltd.) were respectively filtered through a 0.22 µm filter for sterilization. ADC-19, ADC-18, and ADC-20 were added to the above sterile plasma or 1% BSA PBS solution to a final concentration of 200 μg/ml, respectively, which was then incubated in a cell incubator at 37°C, and the day the incubation started was recorded as day 0. Samples were collected on day 7, day 14 and day 21 for the detection of free toxin.

25 µl of sample was added to a 96 well plate. 50 µl of an internal standard working solution (100 ng/ml camptothecin in acetonitrile) and 150 µl of acetonitrile were added. The solution was shaken for 5 minutes and centrifuged for 10 minutes (4000 rpm). 5 μl of solution for LC/MS/MS analysis was taken (Applied Biosystems, Inc., USA).

The results show that ADC-19 is quite stable in human plasma, monkey plasma and 1% BSA PBS. The release rate of free toxin did not exceed 2.1% and became stable on the 14th day. The results are shown in Figure 1A.

ADC-18 has low stability in human plasma and monkey plasma, and the maximum free toxin release rate was 14.5% and 8.10%, respectively. It is stable in 1% BSA PBS solution. The results are shown in Figure 1B.

ADC-20 has low stability in human plasma, monkey plasma and 1% BSA PBS solution, and the highest free toxin release rate was 21.7%, 29.7% and 21.7% respectively. It was always in a state of decomposition in 1% BSA PBS solution. The results are shown in Figure 1C. The results are shown in Figure 1C.

Test Example 4: Efficacy Evaluation in JIMT-1 Tumor Mice I. Test Objective

Nunu nude mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies T-DM1, ADC-21 and ADC-24 against tumor nude mice transplanted with trastuzumab (Herceptin)-resistant JIMT-1 human breast cancer cells, after intraperitoneal injection.

II. Tested medicinal products and materials

1. Tested medicinal products

T-DM1 (obtained according to patent application US 20050169933)

ADC-21:3 mg/kg

ADC-21:10 mg/kg

ADC-24:3 mg/kg

ADC-24:10 mg/kg

Idle: PBS

2. Method of preparation: all drugs were diluted and prepared with PBS.

3. Test animals

Nunu nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

III. Test method

JIMT-1 cells (Nanjing Cobioer Biosciences Co., Ltd.) (5×10 ⁶ cells/mouse, with 50% Matrigel content) were inoculated subcutaneously into the right rib of the mouse. After tumor growth for 8 days and reaching a size of 203.09±11.94 mm ³ , the animals were randomly divided into 6 groups of 8 animals per group (d1).

Drugs were administered intraperitoneally only 2 times. Tumor volume and body weight were measured twice a week and the data was recorded.

Statistical software Excel 2003 was used for data statistics: mean value was calculated as avg; the SD value was calculated as STDEV; SEM value was calculated as STDEV/SQRT; P value between different groups was calculated as TTEST.

Tumor volume (V) was calculated using the formula: V=1/2×L _length ×L _short ²

Relative Volume (RTV)=V _T /V ₀

Degree of tumor inhibition (%)=(C _RTV -T _RTV )/C _RTV (%),

where V ₀ and V _T represent the volume of the tumor at the beginning of the test and at the end of the test, respectively. C _RTV and T _RTV are the relative tumor volume of the blank control group (vehicle, PBS) and test group at the end of the trial, respectively.

IV. Test results

The results of the test are shown in Figure 2. Drugs were administered intraperitoneally 2 times, and the test was completed on the 34th day of observation. T-DM1 (10 mg/kg) has no inhibitory effect on the tumor; ADC-21 (3 mg/kg) showed a tumor inhibition rate of 46.22% (P<0.01); ADC-21 (10 mg/kg) showed a tumor inhibition rate of 56.77% (P<0.001); ADC-24 (3 mg/kg) showed a tumor inhibition rate of 62.77% (P<0.001); and ADC-24 (10 mg/kg) showed a tumor inhibition rate of 76.32% (P<0.001). At the same dose, the tumor inhibition effect of ADC-24 is significantly better than that of ADC-21.

Test Example 5 Efficacy Evaluation in SK-BR-3 Tumor Mice

I. Challenge of the test

Nunu nude mice were used as test animals to evaluate the efficacy of Her2-ADC antibodies ADC-21 and ADC-24 against human breast cancer tumor transplanted nude mice SK-BR-3 after intraperitoneal injection.

II. Tested medicinal products and materials

1. Tested medicinal products

ADC-21:1 mg/kg

ADC-21: 6 mg/kg

ADC-22:1 mg/kg

ADC-22: 6 mg/kg

Idle: PBS

2. Method of preparation: all drugs were diluted and prepared with PBS.

3. Test animals

Nunu nude mice purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

III. Test method

SK-BR-3 cells (ATCC) (5×10 ⁶ cells/mouse, with 50% Matrigel content) were inoculated subcutaneously into the right rib of the mouse. After tumor growth for 20 days and reaching a size of 153.34±11.73 mm ³ , the animals were randomly divided into 5 groups of 8 animals per group (d0).

The drugs were administered by intraperitoneal injection once. Tumor volume and body weight were measured twice a week and the data were recorded.

Statistical software Excel 2003 was used for data statistics: mean value was calculated as avg; the SD value was calculated as STDEV; SEM value was calculated as STDEV/SQRT; P value between different groups was calculated as TTEST.

Tumor volume (V) was calculated using the formula: V=1/2×L _length ×L _short ²

Relative Volume (RTV)=V _T /V ₀

Degree of tumor inhibition (%)=(C _RTV -T _RTV )/C _RTV (%),

where V ₀ and V _T represent the volume of the tumor at the beginning of the test and at the end of the test, respectively. C _RTV and T _RTV are the relative tumor volume of the blank control group and test group at the end of the trial, respectively.

IV. Test results

The results of the test are shown in Figure 3. The drugs were administered by intraperitoneal injection once, and the test was completed on day 28 of observation. ADC-21 (1 mg/kg) showed a tumor inhibition rate of 15.01%; and ADC-21 (6 mg/kg) showed a tumor inhibition rate of 77.4%, which was significantly different from that of the blank control (P<0.001). ADC-22 (1 mg/kg) showed a tumor inhibition rate of 19.82%; and ADC-22 (6 mg/kg) showed a tumor inhibition rate of 98.38% (P<0.001). At the same dose of 6 mg/kg, the tumor inhibiting effect of ADC-22 is significantly better than that of ADC-21.

Test Example 6: Plasma Stability Test

A sample of ADC-25 was mixed well with human plasma, monkey plasma and 1% BSA PBS, respectively, to a final concentration of 100 μg/ml and filtered to sterilize. The mixture was incubated in a water bath at 37° C. and the day the incubation started was recorded as day 0. Samples were collected on day 7, day 14 and day 21 for detection of free toxin.

Samples collected at different time points were cooled to room temperature and mixed well on a vortex. 25 µl of sample was added to a 96 well plate. 50 µl of an internal standard working solution (100 ng/ml camptothecin in acetonitrile) and 150 µl of acetonitrile were added. The solution was shaken for 5 minutes and centrifuged for 10 minutes (4000 rpm). 5 µl of solution was taken for LC/MS/MS analysis.

The results are shown in Figure 4. ADC-25 is fairly stable in human plasma, monkey plasma and 1% BSA PBS. The release rate of free toxin did not exceed 2% and became stable on the 14th day.

Test Example 7: Evaluation of ADC Efficacy in Nude Mice with U87MG Human Brain Astroblastoma Tumor Xenograft

I. Challenge of the test

BALB/cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compound of the present invention against U87MG human astroblastoma brain tumor xenograft in nude mice.

II. Tested medicinal products and materials

1. Tested medicinal products

ADC-27 (3 mg/kg)

ADC-26 (3 mg/kg)

Blank: PBS buffer solution (pH 7.4)

2. Method of preparation: PBS buffer solution (pH 7.4).

3. Test animals

BALB/cA-nude nude mice purchased from Shanghai JieSiJie Laboratory Animal Co., Ltd.

III. Test method

BALB/cA-nude nude mice (female, 6 to 7 weeks of age) were used for the test. Human brain astroblastoma U87MG cells (Human Brain Astroblastoma, Chinese Academy of Sciences Cell Bank, Cat No. TCHi138) were inoculated subcutaneously. On day 10 after inoculation, animals were randomly grouped at 8 animals per group (DO) and drugs were administered by intraperitoneal injection once a week 3 times. Tumor volume and body weight were measured 2 to 3 times a week and the data were recorded. The formula for calculating tumor volume (V) is as follows:

V=1/2×a×b ²

where a and b are the length and width, respectively.

Relative Volume (RTV)=V _T /V ₀

Degree of tumor inhibition (%)=(C _RTV -T _RTV )/C _RTV (%)

where V ₀ and V _T represent the volume of the tumor at the beginning of the test and at the end of the test, respectively. C _RTV and T _RTV are the relative tumor volumes of the control group (blank) and test group at the end of the trial, respectively.

IV. Test results

Intraperitoneal administration (i.p.) was performed 1 time per week 3 times. On day 22 of follow-up, the degree of tumor inhibition for ADC-27 (3 mg/kg) reached 63.3% (P<0.0001); and the degree of tumor inhibition for ADC-26 (3 mg/kg) reached 49.1%. ADC-27 shows stronger antitumor efficacy than ADC-26.

At the time of administration, animals in each group show normal body weight, suggesting that ADC has no obvious side effects. The test results are shown in Table 3 and Figure 5. The antibodies tested can effectively inhibit the growth of U87MG tumor xenograft in tumor-bearing nude mice and exhibit a dose-dependent pattern.

Test Example 8: Performance of ADC Evaluation in Nude Mice with Detroit 562 Human Esophageal Carcinoma Metastatic Pleural Fluid Tumor Xenograft.

I. Challenge of the test

BALB/cA-nude nude mice were used as test animals to evaluate the efficacy of the ADC compound of the present invention against Detroit 562 human esophageal carcinoma metastatic pleural fluid tumor xenograft in nude mice.

II. Tested medicinal products and materials 1.

Tested medicinal products

ADC-29 (3 mg/kg)

ADC-28 (3 mg/kg)

Negative control ADC (3 mg/kg): ligand-toxin conjugate formed by binding a non-B7H3 target to compound 20.

2. Method of preparation: all drugs were diluted and prepared with PBS.

3. Test animals

BALB/cA-nude nude mice purchased from Changzhou Cavens Laboratory Animal Co.,Ltd.

III. Test method

BALB/cA-nude nude mice (female, 6 to 7 weeks of age) were used for the test. Human esophageal carcinoma Detroit 562 human esophageal carcinoma pleural fluid metastatic cells (ATCC, catalog number #ATCC® CCL-138™) were inoculated subcutaneously. On day 10 after inoculation, animals were randomly grouped at 8 animals per group (DO) and drugs were administered by intraperitoneal injection once a week 3 times. Tumor volume and body weight were measured 2 to 3 times a week and the data were recorded. The formula for calculating tumor volume (V) is as follows:

V=1/2×a×b ²

where a and b are the length and width, respectively.

Relative Volume (RTV)=V _T /V ₀

Degree of tumor inhibition (%)=(C _RTV -T _RTV )/C _RTV (%)

where V ₀ and V _T represent the volume of the tumor at the beginning of the test and at the end of the test, respectively. C _RTV and T _RTV are the relative tumor volume of the control group (negative control) and test group at the end of the trial, respectively.

IV. Test results

Intraperitoneal administration was carried out 1 time per week 3 times. On the 28th day of observation, the degree of tumor inhibition for ADC-29 (3 mg/kg, 3mrk) reached 72.27% (P<0.001); and the degree of tumor inhibition for ADC-28 (3 mg/kg, 3mrk) reached 56.2% (P<0.001). ADC-29 shows stronger antitumor efficacy than ADC-28.

At the time of administration, animals in each group show normal body weight, suggesting that ADC has no obvious side effects. The test results are shown in Table 4 and Figure 6. The antibodies tested can effectively inhibit the growth of Detroit 562 tumor xenograft in tumor nude mice and exhibit a dose-dependent pattern.

Test Example 9 Efficacy Evaluation in U87-MG Tumor Mice

I. Challenge of the test

BALB/c nude mice were used as test animals to evaluate the efficacy of B7H3-antibody-drug conjugate administered by intraperitoneal injection in the U87MG glioma cell tumor xenograft model.

II. Tested medicinal products and materials

1. Tested medicinal products

ADC-30 1 mg/kg

ADC-30 3 mg/kg

ADC-31 1 mg/kg

ADC-31 3 mg/kg

Idle: PBS

2. Method of preparation: all drugs were diluted and prepared with PBS.

3. Test animals

BALB/cA-nude nude mice purchased from Shanghai Slac Laboratory Animal Co. Ltd.

III. Test method

U87MG cells (human brain astroblastoma, Chinese Academy of Sciences Cell Bank, Cat No. TCHu138) (2.5×10 ⁶ cells/mouse) were inoculated subcutaneously into the right rib of the mouse. After tumor growth for 14 days and reaching a size of 167.49 mm ³ , the animals were randomly divided into 5 groups of 8 animals per group (d1).

The drugs were administered by intraperitoneal injection once a week for a total of 3 times. Tumor volume and body weight were measured twice a week and the data were recorded.

Statistical software Excel 2003 was used for data statistics: mean value was calculated as avg; the SD value was calculated as STDEV; SEM value was calculated as STDEV/SQRT; P value between different groups was calculated as TTEST.

Tumor volume (V) was calculated using the formula: V=1/2×L _length ×L _short ²

Relative Volume (RTV)=V _T /V ₀

Degree of tumor inhibition (%)=(C _RTV -T _RTV )/C _RTV (%)

where V ₀ and V _T represent the volume of the tumor at the beginning of the test and at the end of the test, respectively. C _RTV and T _RTV are the relative tumor volume of the blank control group (vehicle) and test group at the end of the trial, respectively.

IV. Test results

The test results are shown in Figure 7. Intraperitoneal administration was carried out once a week for a total of 3 times. On the 18th day of observation, the degree of tumor inhibition for ADC-30 (1 mg/kg) reached 0.31%; the degree of tumor inhibition for ADC-30 (3 mg/kg) reached 45.23% (P<0.0001); the degree of tumor inhibition for ADC-31 (1 mg/kg) reached 39.22% (P<0.01); and the degree of tumor inhibition for ADC-31 (3 mg/kg) reached 80.24% (P<0.0001). At the same dose, the tumor inhibiting effect of ADC-31 is significantly better than that of ADC-30.

--->

SEQUENCE LIST

<110> JIANGSU HENGRUI MEDICINE CO., LTD.

SHANGHAI HENGRUI PHARMACEUTICAL CO., LTD.

<120> EXATECAN ANALOGUE LIGAND-DRUG CONJUGATE,

METHOD OF ITS OBTAINING AND ITS APPLICATION

<160> 6

<170> SIPOSequenceListing 1.0

<210> 1

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> Trastuzumab light chain

<400> 1

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr Pro Pro

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 2

<211> 450

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> Trastuzumab heavy chain

<400> 2

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp Thr

20 25 30

Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln

100 105 110

Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val

115 120 125

Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala

130 135 140

Leu Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser

145 150 155 160

Trp Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val

165 170 175

Leu Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro

180 185 190

Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys

195 200 205

Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp

210 215 220

Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly

225 230 235 240

Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

245 250 255

Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu

260 265 270

Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

275 280 285

Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg

290 295 300

Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys

305 310 315 320

Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

325 330 335

Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr

340 345 350

Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu

355 360 365

Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

370 375 380

Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val

385 390 395 400

Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp

405 410 415

Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His

420 425 430

Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro

435 440 445

Gly Lys

450

<210> 3

<211> 214

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> Pertuzumab light chain

<400> 3

Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly

1 5 10 15

Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gln Asp Val Ser Ile Gly

20 25 30

Val Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile

35 40 45

Tyr Ser Ala Ser Tyr Arg Tyr Thr Gly Val Pro Ser Arg Phe Ser Gly

50 55 60

Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro

65 70 75 80

Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Tyr Ile Tyr Pro Tyr

85 90 95

Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg Thr Val Ala Ala

100 105 110

Pro Ser Val Phe Ile Phe Pro Ser Asp Glu Gln Leu Lys Ser Gly

115 120 125

Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe Tyr Pro Arg Glu Ala

130 135 140

Lys Val Gln Trp Lys Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln

145 150 155 160

Glu Ser Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser

165 170 175

Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr

180 185 190

Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser

195 200 205

Phe Asn Arg Gly Glu Cys

210

<210> 4

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> Pertuzumab heavy chain

<400> 4

Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Thr Asp Tyr

20 25 30

Thr Met Asp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val

35 40 45

Ala Asp Val Asn Pro Asn Ser Gly Gly Ser Ile Tyr Asn Gln Arg Phe

50 55 60

Lys Gly Arg Phe Thr Leu Ser Val Asp Arg Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Asn Leu Gly Pro Ser Phe Tyr Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<210> 5

<211> 215

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> B7H3 light chain of 1F9DS antibody

<400> 5

Asp Thr Val Val Thr Gln Glu Pro Ser Phe Ser Val Ser Pro Gly Gly

1 5 10 15

Thr Val Thr Leu Thr Cys Gly Leu Ser Ser Gly Ser Val Ser Thr Ser

20 25 30

His Tyr Pro Ser Trp Tyr Gln Gln Thr Pro Gly Gln Ala Pro Arg Met

35 40 45

Leu Ile Tyr Asn Thr Asn Thr Arg Ser Ser Gly Val Pro Asp Arg Phe

50 55 60

Ser Gly Ser Ile Leu Gly Asn Lys Ala Ala Leu Thr Ile Thr Gly Ala

65 70 75 80

Gln Ala Asp Asp Glu Ser Asp Tyr Tyr Cys Ala Ile His Val Asp Arg

85 90 95

Asp Ile Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu Gly Gln

100 105 110

Pro Lys Ala Asn Pro Thr Val Thr Leu Phe Pro Pro Ser Ser Glu Glu

115 120 125

Leu Gln Ala Asn Lys Ala Thr Leu Val Cys Leu Ile Ser Asp Phe Tyr

130 135 140

Pro Gly Ala Val Thr Val Ala Trp Lys Ala Asp Gly Ser Pro Val Lys

145 150 155 160

Ala Gly Val Glu Thr Thr Lys Pro Ser Lys Gln Ser Asn Asn Lys Tyr

165 170 175

Ala Ala Ser Ser Tyr Leu Ser Leu Thr Pro Glu Gln Trp Lys Ser His

180 185 190

Arg Ser Tyr Ser Cys Gln Val Thr His Glu Gly Ser Thr Val Glu Lys

195 200 205

Thr Val Ala Pro Thr Glu Cys

210 215

<210> 6

<211> 449

<212> PRT

<213> Artificial sequence

<220>

<221> Chain

<223> B7H3 heavy chain of 1F9DS antibody

<400> 6

Gln Val Gln Leu Val Gln Ser Gly Gly Gly Val Val Gln Pro Gly Thr

1 5 10 15

Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Ile Phe Ser Ser Ser

20 25 30

Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Glu Trp Val

35 40 45

Ala Val Ile Ser Tyr Asp Gly Ser Asn Lys Tyr Tyr Val Asp Ser Val

50 55 60

Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr

65 70 75 80

Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys

85 90 95

Ala Arg Ser Ala Arg Leu Tyr Ala Ser Phe Asp Tyr Trp Gly Gln Gly

100 105 110

Ala Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val Phe

115 120 125

Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly Gly Thr Ala Ala Leu

130 135 140

Gly Cys Leu Val Lys Asp Tyr Phe Pro Glu Pro Val Thr Val Ser Trp

145 150 155 160

Asn Ser Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val Leu

165 170 175

Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro Ser

180 185 190

Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys Pro

195 200 205

Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser Cys Asp Lys

210 215 220

Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly Pro

225 230 235 240

Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser

245 250 255

Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp

260 265 270

Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn

275 280 285

Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val

290 295 300

Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu

305 310 315 320

Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys

325 330 335

Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr

340 345 350

Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr

355 360 365

Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu

370 375 380

Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu

385 390 395 400

Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys

405 410 415

Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu

420 425 430

Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly

435 440 445

Lys

<---

Claims (111)

1. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof, wherein said ligand-drug conjugate contains the structure of formula (Pc-L-Y-Dr):

, Where: Y is -O-(CR ^a R ^b ) _m -CR ¹ R ² -C(O)-; R ^a and R ^b represent a hydrogen atom; R ¹ is C _3-6 cycloalkyl or C _3-6 cycloalkyl-C _1-6 alkyl; R ² represents a hydrogen atom; or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1; n is from 1 to 10, which may be an integer or a decimal number; Pc is an antibody; And L is a linker unit. 2. The ligand-drug conjugate or pharmaceutically acceptable salt thereof, according to claim 1, wherein Y is selected from the group consisting of

3. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-2, in which the terminal O in Y is attached to the linker unit L. 4. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 1, which is a ligand-drug conjugate of the formula (Pc-LD ₁ ) or a pharmaceutically acceptable salt thereof:

, where R ¹ represents C _3-6 cycloalkyl-C _1-6 alkyl or C _3-6 cycloalkyl; R ² represents a hydrogen atom; or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1; n is from 1 to 10, which may be an integer or a decimal number; PC is an antibody; And L is a linker unit. 5. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-4, in which n is from 2 to 8, which may be an integer or a decimal number. 6. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 5, wherein n is 3 to 8, which may be an integer or a decimal number. 7. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-6, in which the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ represents -(succinimide-3-yl-N)-W-C(O)-, where W is selected from the group consisting of C _1-8 alkyl and C _1-8 alkyl-C _3-6 cycloalkyl; L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- and a chemical bond, where p ¹ is an integer from 6 to 12; L ³ is a peptide residue consisting of 2-7 amino acids selected from phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁴ and R ⁵ represent a hydrogen atom; R ⁶ and R ⁷ represent a hydrogen atom; t is 1 or 2. 8. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 7, wherein L ¹ is selected from the group consisting of -(succinimide-3-yl-N)-(CH ₂ )s ¹ -C(O)- and -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C(O)-, where s ¹ is an integer from 2 to 8. 9. The ligand-drug conjugate or pharmaceutically acceptable salt thereof according to claim 7 or 8, wherein L ² is a chemical bond. 10. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 7-9, in which L ³ represents a tetrapeptide residue. 11. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 10, wherein L ³ is a glycine-glycine-phenylalanine-glycine tetrapeptide residue. 12. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-7, in which the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ is

and s ¹ is an integer from 2 to 8; L ² is a chemical bond; L ³ is a tetrapeptide residue; L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, R ⁵ , R ⁶ and R ⁷ is a hydrogen atom, and t is 1 or 2. 13. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-7, in which the linker unit -L- is -L ¹ -L ² -L ³ -L ⁴ -, where L ¹ represents -(succinimide-3-yl-N)-CH ₂ -cyclohexyl-C(O)-; L ² is -NR ⁴ (CH ₂ CH ₂ O) ₉ CH ₂ C(O)-; L ³ is a tetrapeptide residue; L ⁴ is -NR ⁵ (CR ⁶ R ⁷ )t-, wherein R ⁵ , R ⁶ and R ⁷ are hydrogen and t is 1 or 2. 14. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 7-13, in which the L ¹ end of the -L- linker unit is connected to the antibody, and the L ⁴ end of the -L- linker unit is connected to Y. 15. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to claim 14, which is a ligand-drug conjugate of the formula (Pc-L _b -Y-Dr) or a pharmaceutically acceptable salt thereof:

Where: s ¹ is an integer from 2 to 8; R ¹ is C _3-6 cycloalkyl-C _1-6 alkyl or C _3-6 cycloalkyl; R ² represents a hydrogen atom; or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1; n is from 1 to 10, which may be an integer or a decimal number; R ⁵ , R ⁶ and R ⁷ represent a hydrogen atom, and t is 1 or 2; Pc is an antibody. 16. The ligand-drug conjugate, or pharmaceutically acceptable salt thereof, of claim 15, wherein s ¹ is 5. 17. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-16 selected from the group consisting of:

Where: n is from 1 to 10, which may be an integer or a decimal number; Pc is an antibody. 18. The ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-17, wherein Pc is an antibody selected from the group consisting of a chimeric antibody, a humanized antibody, and a fully humanized antibody. 19. The ligand-drug conjugate, or pharmaceutically acceptable salt thereof, of claim 18, wherein the antibody is an anti-HER2 (ErbB2) antibody or an anti-B7-H3 antibody. 20. A ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-19 selected from the group consisting of:

where n is as specified in paragraph 1. 21. Compound of formula (D ₁ ):

or a pharmaceutically acceptable salt thereof, Where: R ¹ is C _3-6 cycloalkyl-C _1-6 alkyl or C _3-6 cycloalkyl; R ² represents a hydrogen atom; or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; m is 0 or 1. 22. A compound of formula (D ₁ ) according to claim 21, selected from the group consisting of:

23. Compound of formula (L _a -Y-Dr):

or a pharmaceutically acceptable salt thereof, Where: W is selected from the group consisting of C _1-8 alkyl and C _1-8 alkyl-C _3-6 cycloalkyl; L ² is selected from the group consisting of -NR ⁴ (CH ₂ CH ₂ O)p ¹ CH ₂ C(O)- and a chemical bond, where p ¹ is an integer from 1 to 20; L ³ is a peptide residue consisting of 2-7 amino acids selected from the group consisting of phenylalanine, glycine, valine, lysine, citrulline, serine, glutamic acid and aspartic acid; R ¹ is C _3-6 cycloalkyl or C _3-6 cycloalkyl-C _1-6 alkyl; R ² represents a hydrogen atom; or R ¹ and R ² together with the carbon atom to which they are attached form C _3-6 cycloalkyl; R ⁴ and R ⁵ represent a hydrogen atom; R ⁶ and R ⁷ represent a hydrogen atom; m is 0 or 1. 24. The compound of formula (L _a -Y-Dr) according to claim 23, which is a compound of formula (L _b -Y-Dr):

or a pharmaceutically acceptable salt thereof, where R ¹ , R ² , R ⁵ ~R ⁷ and m are as defined in paragraph 23, s ¹ is an integer from 2 to 8. 25. A compound of formula (L _a -Y-Dr) according to claim 23, selected from the group consisting of:

26. Pharmaceutical composition for the treatment of cancer associated with the expression of HER2 or B7H3, containing a therapeutically effective amount of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-20, or a compound of formula (D ₁ ), or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 21-22 and a pharmaceutically acceptable carrier(s), diluent(s) or excipient(s). 27. The use of a ligand-drug conjugate or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 1-20, a compound of formula (D1) or a pharmaceutically acceptable salt thereof according to any one of paragraphs. 21-22 or a pharmaceutical composition according to claim 26 for the preparation of a medicament for the treatment of cancer associated with the expression of HER2 or B7H3. 28. Use according to claim 27, wherein the cancer is selected from the group consisting of breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urethra cancer, bladder cancer, liver cancer, gastric cancer, cancer endometrial cancer, salivary gland cancer, esophageal cancer, melanoma, glioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

Images