NOVEL CAMPTOTHECIN DERIVATIVE, COMPOSITION COMPRISING SAME AND USE THEREOF

Abstract

The present invention provides a camptothecin derivative, a composition comprising the same and use thereof. Specifically, the present invention provides a compound of formula (1) and a preparation method therefor, and use of the compound of general formula (1) and optical isomers, crystalline forms and pharmaceutically acceptable salts thereof in the preparation of a medicament for treating cancer.

##STR00001##

Claims

1. A camptothecin derivative compound of general formula (1) or optical isomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates thereof: ##STR00228## wherein, in general formula (1): m is an integer of 0, 1 or 2; X is selected from O, S, S(O), S(O.sub.2), and N(R.sup.4); R.sup.1 and R.sup.2 are independently selected from H, halogen, OH, C.sub.1-6 alkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, C.sub.3-6 cycloalkyl, NH.sub.2, NO.sub.2, and CN, or R.sup.1 and R.sup.2, together with the phenyl ring attached thereto, form ##STR00229## by cyclization; R.sup.3 is selected from C.sub.1-6 alkyl, C.sub.2-6 alkenyl, C.sub.1-3 alkoxy-substituted C.sub.1-3 alkyl, and C.sub.1-6 haloalkyl; R.sup.4 is selected from H, C.sub.1-6 alkyl and C.sub.1-6 haloalkyl; R.sup.5 is selected from H, C.sub.1-6 alkyl and C.sub.3-6 cycloalkyl; R.sup.6 and R.sup.7 are independently selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, and C.sub.3-6 cycloalkyl; or R.sup.6 and R.sup.7, together with the carbon atom attached thereto, form C.sub.3-6 cycloalkyl or 4-7 membered heterocycloalkyl by cyclization; or R.sup.6 and R.sup.5 are linked to form a 5-7 membered lactam ring, and R.sup.7 is selected from H, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, and C.sub.3-6 cycloalkyl; R.sup.8 is selected from OH and NR.sup.9R.sup.10, and R.sup.9 and R.sup.10 are independently selected from H, C.sub.1-6 alkyl and C.sub.3-6 cycloalkyl; or R.sup.9 and R.sup.10, together with the N atom attached thereto, form 4-7 membered heterocycloalkyl by cyclization, and the 4-7 membered heterocycloalkyl is unsubstituted or substituted with 1-3 groups selected from the following: C.sub.1-6 alkyl, halogen, OH, CN, and NH.sub.2.

2. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), R.sup.8 is OH.

3. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), R.sup.1 and R.sup.2 are independently selected from H, halogen, OH, Me, Et, OMe, OEt, CF.sub.3, NH.sub.2, NO.sub.2, and CN; or R.sup.1 and R.sup.2, together with the phenyl ring attached thereto, form ##STR00230## by cyclization.

4. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), R.sup.3 is selected from Me, Et, ##STR00231##

5. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), X is selected from O, S, S(O), S(O.sub.2), N(H), and N(Me)-.

6. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), R.sup.5 is selected from H, Me, Et, and ##STR00232##

7. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), R.sup.6 and R.sup.7 are independently selected from H, Me, Et, CHF.sub.2, CF.sub.3, CH.sub.2CF.sub.3, ##STR00233##

8. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein in the formula (1), ##STR00234## is selected from ##STR00235## ##STR00236##

9. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 2, wherein in the formula (1), R.sup.8 is OH, and ##STR00237## is selected from ##STR00238## ##STR00239##

10. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein the compound has one of the following structures: ##STR00240## ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248##

11. The camptothecin derivative compound of formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, wherein the compound has one of the following structures: ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256## ##STR00257##

12. A camptothecin derivative compound or optical isomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates thereof, wherein the compound has one of the following structures: ##STR00258## ##STR00259## ##STR00260## ##STR00261## ##STR00262## ##STR00263## ##STR00264##

13. A camptothecin derivative compound or optical isomers, crystalline forms, pharmaceutically acceptable salts, hydrates or solvates thereof, wherein the compound has one of the following structures: ##STR00265## ##STR00266## ##STR00267##

14. Use of the camptothecin derivative compound of general formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1 as a small molecule toxin in the preparation of an antibody-drug conjugate.

15. A pharmaceutical composition, comprising a pharmaceutically acceptable excipient or carrier, and the camptothecin derivative compound of general formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1 as an active ingredient.

16. Use of the camptothecin derivative compound of general formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1 in the preparation of a medicament for treating cancer diseases.

17. An antibody-drug conjugate, comprising an antibody, a small molecule toxin, and a linker, wherein the small molecule toxin is the camptothecin derivative compound of general formula (1) or the optical isomers, the crystalline forms, the pharmaceutically acceptable salts, the hydrates or the solvates thereof according to claim 1, and the linker links the antibody and the small molecule toxin via a covalent bond.

18. The antibody-drug conjugate according to claim 17, wherein the antibody-drug conjugate has one of the following structures: ##STR00268## ##STR00269## ##STR00270## wherein Ab represents a monoclonal antibody, preferably an anti-her2 antibody, and more preferably trastuzumab; n is a number from 2 to 8, preferably from 4 to 8, and more preferably from 7 to 8.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0084] FIG. 1 shows the results of the anti-tumor activity in mice of Example 11 according to the present invention.

DETAILED DESCRIPTION

[0085] Various specific aspects, features and advantages of the compounds, methods and pharmaceutical compositions described above will be set forth in detail in the following description, which will make the content of the present invention very clear. It should be understood that the detailed description and examples below describe specific embodiments for reference only. After reading the description of the present invention, those skilled in the art can make various changes or modifications to the present invention, and such equivalents also fall within the scope of the present invention defined herein.

[0086] In all examples, .sup.1H-NMR spectra were recorded with a Varian Mercury 400 nuclear magnetic resonance spectrometer, and chemical shifts are expressed in (ppm); silica gel for separation was 200-300 mesh silica gel if not specified, and the ratio of the eluents was volume ratio. In the present invention, the following abbreviations are used: room temperature (RT, rt); aqueous solution (aq.); petroleum ether (PE); ethyl acetate (EA); dichloromethane (DCM); 1,4-dioxane (dioxane); methanol (MeOH); methyl tert-butyl ether (MTBE); ethanol (EtOH); tetrahydrofuran (THF); dimethylformamide (DMF); N-methylpyrrolidone (NMP); dimethyl sulfoxide (DMSO); triethylamine (TEA); diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); carbon tetrachloride (CCl.sub.4); palladium on carbon (Pd/C); Eaton's reagent (Eaton reagent, 7.7 wt % of phosphorus pentoxide in methanesulfonic acid); iron powder (Fe); zinc powder (Zn); Raney nickel (Ranyi Ni); acetyl chloride (AcCl); acetic acid (AcOH); acetic anhydride (Ac.sub.2O); m-chloroperoxybenzoic acid (m-CPBA); butyl nitrite (n-BuNO); sodium nitrite (NaNO.sub.2); sodium hydride (NaH); magnesium sulfate (MgSO.sub.4); N-bromosuccinimide (NBS); -toluenesulfonic acid monohydrate (TsOH.Math.H.sub.2O); sodium carbonate (Na.sub.2CO.sub.3); potassium carbonate (K.sub.2CO.sub.3); equivalent (eq); gram/milligram (g/mg); mole/millimole (mol/mmol); liter/milliliter (L/mL); minutes (min (s)); hours (h, hr, hrs); nitrogen (N.sub.2); nuclear magnetic resonance (NMR); liquid-mass spectrometry (LC-MS); thin-layer chromatography (TLC); preparative liquid chromatography (pre-HPLC).

Preparation Example 1: Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-Yl)acetamide (A7-a)

[0087] ##STR00057##

Step 1: Synthesis of methyl 3-((3-fluoro-2-methyl-5-nitrophenyl)thio)propanoate

[0088] ##STR00058##

[0089] To a 50-mL three-necked flask were added A1-a (1.73 g, 10 mmol, 1 eq), methyl 3-mercaptopropanoate (1.8 g, 15 mmol, 1.5 eq) and NMP (10 mL), and after the system was dissolved, potassium carbonate (2 g, 15 mmol, 1.5 eq) was added and stirred at 60 C. for 8 hours under the atmosphere of argon. After the system was cooled to room temperature, water (30 mL) was added to dilute the system, and the precipitated solid was filtered and washed with water. The filter cake was separated by column chromatography (PE/EA= 1/12- 1/7) to give a yellow solid A2-a (1.55 g, 56.8% yield), LC-MS: 274.2 [M+H].sup.+.

Step 2: Synthesis of 3-((3-fluoro-2-methyl-5-aminophenyl)thio)propionic acid

[0090] ##STR00059##

[0091] To a 250-mL three-necked flask were added A2-a (1.55 g, 5.67 mmol, 1 eq), iron powder (1.27 g, 22.69 mmol, 4 eq), ethanol (80 mL) and aqueous ammonium chloride (2.8 M, 28 mL, 5 eq), and the system was stirred at 90 C. for 16 hours under the atmosphere of argon. After the system was cooled to room temperature, the system was filtered through celite and washed with ethanol. The filtrate was concentrated, and the crude product was diluted with water (30 mL) and extracted with EA (30 mL2). The organic phase was washed with saturated brine, dried over sodium sulfate and concentrated to give the crude product (1.5 g, equivalent yield). LC-MS: 244.3 [M+H].sup.+.

[0092] To a 50-mL three-necked flask were added the above crude product (1.5 g, 5.67 mmol, 1 eq) and 1,4-dioxane (15 mL), and after the system was dissolved, concentrated hydrochloric acid (37%, 10 mL) was added and stirred at 65 C. for 4 hours under the atmosphere of argon. After the system was cooled to room temperature, 3 N sodium carbonate solution was added to adjust the pH to 5. The system was extracted with EA (30 mL2), and the organic phase was washed with saturated brine, dried over sodium sulfate and concentrated. The crude product was slurried (EA/PE=) to give a white solid A3-a (985 mg, 75.8% yield over two steps), LC-MS: 228.2 [MH].sup.+.

Step 3: Synthesis of 5-amino-7-fluoro-8-methylthiochroman-4-one

[0093] ##STR00060##

[0094] To a 50-mL three-necked flask were added A3-a (985 mg, 4.3 mmol, 1 eq) and Eaton's Reagent (15 mL), and the system was stirred at 60 C. for 1 hour under the atmosphere of argon. After the system was cooled to room temperature, the reaction solution was poured into ice water, and 3 N sodium carbonate solution was added to adjust the pH to 8. The system was extracted with EA (30 mL2), and the organic phase was washed with saturated brine, dried over sodium sulfate and concentrated to give the crude product A4-a (1.05 g, equivalent yield), LC-MS: 212.3 [M+H].sup.+.

Step 4: Synthesis of N-(7-fluoro-8-methyl-4-oxothiochroman-5-yl)acetamide

[0095] ##STR00061##

[0096] To a 50-mL three-necked flask were added A4-a (1.05 g, 4.3 mmol, 1 eq), DMAP (52.5 mg, 0.43 mmol, 0.1 eq) and DCM (15 mL), acetyl chloride (674 mg, 8.6 mmol, 2 eq) and triethylamine (869 mg, 8.6 mmol, 2 eq) were added in sequence under an ice bath. The system naturally returned to room temperature and was then stirred for 1 hour until the starting materials were completely consumed. The system was quenched with water (20 mL), followed by separation.

[0097] The aqueous phase was extracted with DCM (20 mL2), and the organic phases were combined, washed with saturated brine, dried and concentrated. The residue was subjected to column chromatography (EA/PE=1/1) to give a yellow solid A5-a (910 mg, 89% yield over two steps), LC-MS: 254.3 [M+H].sup.+.

Step 5: Synthesis of N,N-(7-fluoro-8-methyl-4-oxothiochromane-3,5-diyl)diacetamide

[0098] ##STR00062##

[0099] To a 50-mL three-necked flask were added potassium tert-butoxide (191 mg, 1.7 mmol, 1.1 eq) and anhydrous THF (5 mL), and A5-a (375 mg, 1.48 mmol, 1 eq) and butyl nitrite (190 mg, 1.85 mmol, 1.25 eq) were added in sequence at 20 C. The system was heated to 5 C. and then stirred for 2 hours until the starting materials were completely consumed. The system was added with MTBE (15 mL) for dilution and filtered, and the solid was dissolved in acetic acid (5 mL), added with zinc powder (200 mg, 3.1 mmol, 2.1 eq), stirred at room temperature for 5 minutes, added with acetic anhydride (1 mL), and stirred for 2 hours. The system was washed with MeOH/DCM ( 3/30 mL) and concentrated, and the crude product was separated by column chromatography (EA/DCM= 1/10-) to give a light brown solid A6-a (175 mg, 41%), LC-MS: 311.1 [M+H].sup.+.

Step 6: Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-yl)acetamide

[0100] ##STR00063##

[0101] To a 50-mL three-necked flask were added A6-a (175 mg, 0.56 mmol, 1 eq) and methanol/1,4-dioxane ( 4/8 mL), and after the system was dissolved, concentrated hydrochloric acid (37%, 4 mL) was added and stirred at 40 C. for 2 hours under the atmosphere of argon. After the system was cooled to room temperature, 3 N sodium carbonate solution was added to adjust the pH to 8. The system was filtered, and the solid was dried to give A7-a (137 mg, 91% yield). LC-MS: 269.2 [M+H].sup.+.

[0102] Similar to the synthesis of A7-a, the intermediates listed in the following table can be obtained:

TABLE-US-00001 TABLE 1 Intermediates A7-b to A7-y MS No. Structure [M + H].sup.+ A7-b [00064] 255.2 A7-c [00065] 283.1 A7-d [00066] 285.1 A7-e [00067] 281.1 A7-f [00068] 295.1 A7-g [00069] 253.2 A7-h [00070] 285.2 A7-i [00071] 301.2 A7-j [00072] 269.1 A7-k [00073] 226.2 A7-l [00074] 252.1 A7-m [00075] 230.1 A7-n [00076] 242.2 A7-o [00077] 237.1 A7-p [00078] 228.3 A7-q [00079] 267.2 A7-r [00080] 281.2 A7-s [00081] 295.2 A7-t [00082] 283.2 A7-u [00083] 297.2 A7-v [00084] 297.2 A7-w [00085] 311.2 A7-x [00086] 311.1 A7-y [00087] 325.1

Preparation Example 2: Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (B7-a)

[0103] ##STR00088## ##STR00089##

Step 1: Synthesis of ethyl 2-bromo-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2-[1,3]dioxolan]-7-yl)acetate

[0104] ##STR00090##

[0105] B1-a (3 g, 9.9 mmol, 1 eq) was dissolved in DMF (75 mL), NBS (1.5 g, 12 mmol, 1.2 eq) and m-CPBA (170 mg, 1 mmol, 0.1 eq) were added, and the system was stirred at room temperature overnight, then poured into 500 mL of ice water, and filtered. The solid was washed with water and dried to give a gray solid B2-a (3.8 g, equivalent yield), LC-MS: 383.2 [M+H].sup.+.

Step 2: Synthesis of 1-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2-[1,3]dioxolan]-7-yl)-2-ethoxy-2-oxoethyl tosyl-D-prolinate

[0106] ##STR00091##

[0107] B2-a (1 g, 2.6 mmol, 1 eq), sodium tosyl-D-prolinate (1 g, 3.9 mmol, 1.5 eq) and K.sub.2CO.sub.3 (362 mg, 2.6 mmol, 1 eq) are dissolved in DMF (20 mL), and the system was stirred at 65 C. for 2 hours under the atmosphere of argon until the starting materials were completely consumed. The system was added with water (100 mL) for dilution and extracted with EA (100 mL3), and the organic phases were combined and washed twice with water, washed with saturated brine, dried and concentrated. The residue was separated by column chromatography (EA/DCM=) to give a white solid B3-a (1.1 g, 74% yield), LC-MS: 572.2 [M+H].sup.+.

Step 3: Synthesis of (S)-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2-[1,3]dioxolan-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate

[0108] ##STR00092##

[0109] B3-a (1 g, 1.7 mmol, 1 eq) was dissolved in DMF (20 mL), and NaH (101 mg, 60%, 2.5 mmol, 1.5 eq) was added under an ice bath. The system returned to room temperature, stirred for 1 hour, then added with 2-fluoroiodoethane (1.5 g, 8.6 mmol, 5 eq) under an ice bath, naturally returned to room temperature, and stirred overnight. After the reaction was completed, the system was poured into ice water (100 mL) and extracted with EA (100 mL3), and the organic phases were combined, washed twice with water, washed with saturated saline, dried and concentrated. The residue was separated by column chromatography (EA/DCM=) to give 743 mg of crude product, which was subjected to Pre-HPLC to give a white solid B4-a (150 mg, 70% de, 15% yield), LC-MS: 618.2 [M+H].sup.+.

Step 4: Synthesis of (S)-2-(6-(acetamidomethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2-[1,3]dioxolan]-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate

[0110] ##STR00093##

[0111] To a 50-mL three-necked flask was added Raney Ni (600 mg, water content of 50%), and the system was washed three times with HOAc, added with a solution of B4-a (150 mg, 0.24 mmol, 1 eq) in Ac.sub.2O/HOAc (4/1 mL) under the atmosphere of argon, purged three times with hydrogen, reacted at 65 C. for 3 hours and filtered. The solid was washed with AcOH, and the filtrate was concentrated and separated by column chromatography (MeOH/DCM= 1/20) to give a colorless oily solution B5-a (130 mg, 83% yield), LC-MS: 664.2 [M+H].sup.+.

Step 5: Synthesis of (S)-2-(6-(acetoxymethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2-[1,3]dioxolan]-7-yl)-1-ethoxy-4-fluoro-1-oxobutan-2-yl tosyl-D-prolinate

[0112] ##STR00094##

[0113] B5-a (130 mg, 0.2 mmol, 1 eq) was dissolved in Ac.sub.2O/HOAc (3/1 mL), and NaNO.sub.2 (68 mg, 1 mmol, 5 eq) was added under an ice bath. The system returned to room temperature and stirred for 1 hour. After the reaction was completed, the system was filtered, the solid was washed with AcOH, and the filtrate was concentrated, added with CCl.sub.4 (15 mL), and stirred under reflux overnight. The system was washed with water, washed with saturated brine, dried and concentrated. The residue was separated by column chromatography (MeOH/DCM= 1/20) to give 90 mg of colorless oily solution, which was subjected to Pre-HPLC to give a white solid B6-a (90 mg, 69% yield), LC-MS: 665.2 [M+H].sup.+.

Step 6: Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-J]indolizine-3,6,10(4H)-trione

[0114] ##STR00095##

[0115] B6-a (90 mg, 0.14 mmol, 1 eq) was dissolved in EtOH (3 mL), and 1 N aqueous sodium carbonate (1 mL) was added. The system was stirred at room temperature for 1 hour until the starting materials were completely consumed, then concentrated at room temperature and lyophilized. The crude product was dissolved in 85% aqueous TFA (5 mL) and stirred at 85 C. for 1 hour. After the reaction was completed, the system was concentrated, and the crude product was separated by Pre-HPLC to give a white solid B7-a (7 mg, 17% yield, 68% de), LC-MS: 282.2 [M+H].sup.+.

[0116] Similar to the synthesis of B7-a, the intermediates listed in the following table can be obtained:

TABLE-US-00002 TABLE 2 Intermediates B7-b to B7-d No. Structure MS [M + H].sup.+ B7-b [00096] 264.1 B7-c [00097] 276.1 B7-d [00098] 294.1

Example 1: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3, 4:6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione (Compound 1)

[0117] ##STR00099##

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

[0118] ##STR00100##

[0119] To a 50-mL three-necked flask were added A7-a (108 mg, 0.4 mmol, 1 eq), B7-b (156 mg, 0.6 mmol, 1.5 eq), -toluenesulfonic acid monohydrate (45 mg, 0.24 mmol, 0.6 eq), anhydrous magnesium sulfate (1 g) and acetic acid (10 mL), and the system were stirred at 105 C. for 24 hours under the atmosphere of argon until the starting materials were completely consumed. The system was filtered, and the filter cake was washed with EA and concentrated. The crude product was separated by column chromatography (MeOH/DCM= 1/40- 1/20) to give a light brown solid B8-a (131 mg, 67%), LC-MS: 496.2 [M+H].sup.+.

[0120] Step 2: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3,4: 6,7]indolizino[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13 (2H)-dione

##STR00101##

[0121] To a 50-mL three-necked flask were added B8-a (131 mg, 0.26 mmol, 1 eq) and 1,4-dioxane (5 mL), and after the system was dissolved, concentrated hydrochloric acid (37%, 5 mL) was added and stirred at 80 C. for 24 hours under the atmosphere of argon. The system was cooled to room temperature and then concentrated. The crude product was slurried with ACN/EA (1/1) to give a brown solid 1 (hydrochloride) (107 mg, 84.6% yield), LC-MS: 454.2 [M+H].sup.+.

[0122] Similar to the synthesis of compound 1, the compounds listed in the following table can be obtained:

TABLE-US-00003 TABLE 3 List of compounds 2-31 MS No. Structure [M + H].sup.+ 2 [00102] 440.1 3 [00103] 468.2 4 [00104] 470.2 5 [00105] 466.1 6 [00106] 480.2 7 [00107] 438.2 8 [00108] 470.2 9 [00109] 486.1 10 [00110] 451.2 11 [00111] 466.1 12 [00112] 472.1 13 [00113] 484.2 14 [00114] 468.2 15 [00115] 453.2 16 [00116] 479.2 17 [00117] 457.1 18 [00118] 469.2 19 [00119] 464.1 20 [00120] 455.1 21 [00121] 494.2 22 [00122] 508.2 23 [00123] 522.2 24 [00124] 510.1 25 [00125] 524.2 26 [00126] 524.2 27 [00127] 538.2 28 [00128] 538.2 29 [00129] 552.2 30 [00130] 538.2 31 [00131] 552.2

Example 2: Chiral Separation of Compound 1

[0123] Compound 1 is a pair of diastereomeric mixture, and two diastereoisomers 1-1 and 1-2 of compound 1 can be obtained by adopting a method of salification and recrystallization or the separation and purification with pre-HPLC.

##STR00132##

[0124] Conditions for chromatography: the preparative liquid chromatograph equipped with Shimadzu LC-20AP; chromatographic column: Waters SunFire Prep C18 OBD (50150 mm, 5 m); mobile phase: acetonitrile-0.5 aqueous trifluoroacetic acid solution=66:34; flow rate: 48.0 mL/min; detection wavelength: 254 nm; injection volume: 3000 L.

[0125] The experimental procedures were as follows: a proper amount of 1 was taken and brought to a certain volume by using a 50% aqueous acetonitrile solution. A test sample solution with a concentration of 25 mg/mL was prepared. The test sample solution was taken and placed into the preparative liquid chromatograph for detection according to the conditions for chromatography of the present invention and then data were recorded.

[0126] As a result, the above solution was separated by pre-HPLC to give 1-1 (33 mg) and 1-2 (31 mg). The retention times of the two components were 5.419 minutes and 7.614 minutes, respectively, and the purities thereof were 99.38% and 99.21%, respectively.

[0127] Similar to the chiral separation method of compound 1, the compounds listed in the following table can be obtained:

TABLE-US-00004 TABLE 4 Separation of compounds by Pre-HPLC No. Structure 2-1 [00133] 2-2 [00134] 3-1 [00135] 3-2 [00136] 4-1 [00137] 4-2 [00138] 5-1 [00139] 5-2 [00140] 6-1 [00141] 6-2 [00142] 7-1 [00143] 7-2 [00144] 9-1 [00145] 9-2 [00146] 11-1 [00147] 11-2 [00148] 12-1 [00149] 12-2 [00150] 13-1 [00151] 13-2 [00152] 14-1 [00153] 14-2 [00154]

Example 3: Synthesis of Compound 32-1 and Compound 32-2

[0128] ##STR00155##

[0129] To a 50-mL three-necked flask were added component 1-2 (98 mg, 0.2 mmol, 1 eq) with a long retention time, 2-hydroxyacetic acid (18.4 mg, 0.24 mmol, 1.2 eq) and anhydrous DCM (5 mL), and HATU (84 mg, 0.3 mmol, 1.5 eq) and DIPEA (90.3 mg, 0.7 mmol, 3.5 eq) were added in sequence under an ice bath. After the temperature was maintained for 0.5 hours, the system was added with water (5 mL) for dilution, followed by liquid separation and extraction with DCM (5 mL2). The organic phase was washed with saturated saline, dried and concentrated. The residue was subjected to column chromatography (MeOH/DCM= 1/40) to give compound 32-2 (75 mg, 73% yield).

[0130] .sup.1H NMR (400 MHz, DMSO-d 6) : 8.21 (dd, J=13.3, 8.5 Hz, 1H), 7.78 (d, J=10.6 Hz, 1H), 7.31 (d, J=1.2 Hz, 1H), 6.53 (s, 1H), 5.89-5.79 (m, 1H), 5.56-5.49 (m, 1H), 5.43 (s, 2H), 5.38 (s, 1H), 5.31 (d, J=4.8 Hz, 1H), 3.90 (d, J=4.7 Hz, 2H), 3.50-3.35 (m, 3H), 2.44 (s, 3H), 1.91-1.80 (m, 2H), 0.87 (t, J=6.5 Hz, 3H), LC-MS: 512.2 [M+H].sup.+.

[0131] Similar to the synthesis of compound 32-2, a diastereoisomer 32-1 of compound 32-2 can be obtained by using another component 1-1 with a short retention time.

[0132] Similar to the synthesis of compound 32-1 and compound 32-2, the compounds listed in the following table can be obtained by using different intermediates:

TABLE-US-00005 TABLE 5 List of compounds 33-96 MS No. Structure [M + H].sup.+ 33 [00156] 498.1 34 [00157] 526.2 35 [00158] 528.1 36 [00159] 524.1 37 [00160] 538.1 38 [00161] 524.1 39 [00162] 530.1 40 [00163] 542.1 41 [00164] 496.2 42 [00165] 544.1 43 [00166] 526.2 44 [00167] 526.2 45 [00168] 526.2 46 [00169] 580.1 47 [00170] 580.1 48 [00171] 552.2 49 [00172] 552.2 50 [00173] 538.2 51 [00174] 552.2 52 [00175] 526.2 53 [00176] 552.2 54 [00177] 566.2 55 [00178] 528.1 56 [00179] 511.1 57 [00180] 525.2 58 [00181] 553.2 59 [00182] 525.2 60 [00183] 551.2 61 [00184] 525.2 62 [00185] 539.2 63 [00186] 539.2 64 [00187] 553.2 65 [00188] 498.1 66 [00189] 526.2 67 [00190] 528.1 68 [00191] 524.1 69 [00192] 538.1 70 [00193] 524.1 71 [00194] 530.1 72 [00195] 542.1 73 [00196] 496.2 74 [00197] 544.1 75 [00198] 526.2 76 [00199] 526.2 77 [00200] 526.2 78 [00201] 580.1 79 [00202] 580.1 80 [00203] 552.2 81 [00204] 552.2 82 [00205] 538.2 83 [00206] 552.2 84 [00207] 526.2 85 [00208] 552.2 86 [00209] 566.2 87 [00210] 512.2 88 [00211] 511.1 89 [00212] 525.2 90 [00213] 553.2 91 [00214] 525.2 92 [00215] 551.2 93 [00216] 525.2 94 [00217] 539.2 95 [00218] 539.2 96 [00219] 553.2

Example 4: Preparation of Antibody-Drug Conjugate (ADC-1)

[0133] ##STR00220##

[0134] Step 1: Preparation of compound L-D-1

##STR00221##

[0135] To a 50-mL single-necked flask were added L-1 (76 mg, 0.12 mmol, 1.0 eq), component compound 1-2 (55 mg, 0.12 mmol, 1.0 eq) with a long retention time, NMI (50.6 mg, 0.62 mmol, eq) and DMF (2 mL), and the system was stirred well and cooled to 0 C. The reaction solution was added with TCFH (41.5 mg, 0.15 mmol, 1.2 eq) and stirred for 30 minutes, followed by detection by LC-MS. After the reaction was completed, the reaction solution was purified by reversed-phase C18 column chromatography (MeCN/water=0-60%), and fractions of the target substance were lyophilized to give a yellow solid L-D-1 (80 mg, 61.5% yield).

[0136] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 8.56 (t, J=6.4 Hz, 1H), 8.30 (dd, J=14.0, 8.3 Hz, 2H), 8.11 (d, J=7.9 Hz, 1H), 8.06 (t, J=5.7 Hz, 1H), 7.99 (t, J=5.7 Hz, 1H), 7.76 (d, J=10.7 Hz, 1H), 7.31 (d, J=2.8 Hz, 1H), 7.27-7.12 (m, 5H), 6.98 (s, 2H), 6.54 (d, J=1.8 Hz, 1H), 5.84-5.83 (m, 1H), 5.49-5.31 (m, 3H), 5.27-5.21 (m, 1H), 4.66-4.53 (m, 2H), 4.48-4.41 (m, 1H), 3.98 (s, 2H), 3.75-3.54 (m, 6H), 3.42 (s, 2H), 3.36-3.35 (m, 2H), 3.04-2.98 (m, 1H), 2.80-2.74 (m, 1H), 2.42 (s, 3H), 2.08 (t, J=7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J=13.2, 5.9 Hz, 4H), 1.25-1.12 (m, 2H), 0.88-0.84 (m, 3H), LC-MS: 1052.1 [M+H].sup.+, 1050.1 [MH].sup..

[0137] Step 2: Preparation of ADC-1

[0138] To an aqueous PBS buffer (0.05 PBS buffer at pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 nmol) of antibody trastuzumab was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL) at 37 C., and the system was placed in a water bath shaker, and shaken and reacted at 37 C. for 3 hours before the reaction was stopped; the reaction solution was cooled to C. under a water bath and diluted to 5.0 mg/mL.

[0139] Compound L-D-1 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution, and the system was placed in a water bath shaker, and shaken and reacted at C. for 3 hours before the reaction was stopped. The reaction solution was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give a PBS buffer (5.0 mg/mL, 1.1 mL) of ADC, which was frozen and stored at 4 C. Mean calculated by UV-HPLC: n=7.2.

Example 5: Preparation of Antibody-Drug Conjugate (ADC-2)

[0140] ##STR00222##

[0141] Step 1: Preparation of Compound L-D-2

##STR00223##

[0142] To a 50-mL single-necked flask were added L-1 (76 mg, 0.12 mmol, 1.0 eq), component compound 1-1 (55 mg, 0.12 mmol, 1.0 eq) with a short retention time, NMI (50.6 mg, 0.62 mmol, eq) and DMF (2 mL), and the system was stirred well and cooled to 0 C. The reaction solution was added with TCFH (41.5 mg, 0.15 mmol, 1.2 eq) and stirred for 30 minutes, followed by detection by LC-MS. After the reaction was completed, the reaction solution was purified by reversed-phase C18 column chromatography (MeCN/water=0-60%), and fractions of the target substance were lyophilized to give a yellow solid L-D-2 (80 mg, 61.5% yield).

[0143] .sup.1H-NMR (400 MHz, DMSO-d.sub.6) .sup.1H-NMR (400 MHz, DMSO-d.sub.6) : 8.56 (t, J=6.4 Hz, 1H), 8.30 (dd, J=14.0, 8.3 Hz, 2H), 8.11 (d, J=7.9 Hz, 1H), 8.06 (t, J=5.7 Hz, 1H), 7.99 (t, J=5.7 Hz, 1H), 7.76 (d, J=10.7 Hz, 1H), 7.31 (d, J=2.8 Hz, 1H), 7.30-7.16 (m, 5H), 6.98 (s, 2H), 6.54 (d, J=1.8 Hz, 1H), 5.90-5.86 (m, 1H), 5.46-5.30 (m, 3H), 5.26-5.21 (m, 1H), 4.68-4.53 (m, 2H), 4.46-4.41 (m, 1H), 3.98 (s, 2H), 3.75-3.54 (m, 6H), 3.42 (s, 2H), 3.36-3.35 (m, 2H), 3.04-2.98 (m, 1H), 2.80-2.74 (m, 1H), 2.42 (s, 3H), 2.08 (t, J=7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J=13.2, 5.9 Hz, 4H), 1.25-1.12 (m, 2H), 0.88-0.84 (m, 3H), LC-MS: 1052.1 [M+H].sup.+, 1050.1 [MH].sup..

[0144] Step 2: Preparation of ADC-2

[0145] To an aqueous PBS buffer (0.05 PBS buffer at pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 nmol) of antibody trastuzumab was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL) at 37 C., and the system was placed in a water bath shaker, and shaken and reacted at 37 C. for 3 hours before the reaction was stopped; the reaction solution was cooled to C. under a water bath and diluted to 5.0 mg/mL.

[0146] Compound L-D-2 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution, and the system was placed in a water bath shaker, and shaken and reacted at C. for 3 hours before the reaction was stopped. The reaction solution was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give a PBS buffer (5.0 mg/mL, 1.1 mL) of ADC, which was frozen and stored at 4 C. Mean calculated by UV-HPLC: n=7.2.

Example 6: Preparation of Antibody-Drug Conjugates (ADC-3 and ADC-4)

[0147] ##STR00224##

[0148] Step 1: Preparation of Compounds L-D-3 and L-D-4

##STR00225##

[0149] To a 50-mL single-necked flask were added L-2 (80 mg, 0.12 mmol, 1.0 eq), component compound 1-1 (55 mg, 0.12 mmol, 1.0 eq) with a short retention time, NMI (50.6 mg, 0.62 mmol, eq) and DMF (2 mL), and the system was stirred well and cooled to 0 C. The reaction solution was added with TCFH (41.5 mg, 0.15 mmol, 1.2 eq) and stirred for 30 minutes, followed by detection by LC-MS. After the reaction was completed, the reaction solution was purified by reversed-phase C18 column chromatography to give two fractions, with the fraction with a short retention time being L-D-3 and the fraction with a long retention time being L-D-4. The fractions of the target substance were lyophilized to give yellow solids L-D-3 (20 mg) and L-D-4 (25 mg), LC-MS: 1092.4 [M+H].sup.+.

[0150] Conditions for chromatography: the semi-preparative liquid chromatograph U3000 from Thermo Fisher; chromatographic column: Welch Ultimate XB-Phenyl; mobile phase: acetonitrile containing 0.1% formic acid-0.1% aqueous formic acid=50:50; flow rate: 30.0 mL/min; detection wavelength: 370 nm; injection amount: 100 L.

[0151] The experimental procedures were as follows: a proper amount of a mixture of L-D-3 and L-D-4 was taken and dissolved in DMF. A test sample solution with a concentration of 10 mg/mL was prepared. The test sample solution was taken and placed into the preparative liquid chromatograph for detection according to the conditions for chromatography of the present invention and then data were recorded. The injection was carried out for multiple times. As a result, L-D-3 and L-D-4 were separated by pre-HPLC, where the retention times of the two components were 5.29 minutes and 5.87 minutes, respectively, and the purities thereof were 99.38% and 99.21%, respectively.

[0152] Step 2: Preparation of ADC-3 and ADC-4

[0153] To an aqueous PBS buffer (0.05 PBS buffer at pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 nmol) of antibody trastuzumab was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL) at 37 C., and the system was placed in a water bath shaker, and shaken and reacted at 37 C. for 3 hours before the reaction was stopped; the reaction solution was cooled to 25 C. under a water bath and diluted to 5.0 mg/mL. Two aliquots were prepared in parallel. Compound L-D-3 (2.0 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution, and the system was placed in a water bath shaker, and shaken and reacted at 25 C. for 3 hours before the reaction was stopped. The reaction solution was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give a PBS buffer (5.0 mg/mL, 1 mL) of ADC, which was frozen and stored at 4 C. Mean calculated by UV-HPLC: n=7.3.

[0154] ADC-4 was prepared by using compound L-D-4 in the same manner, where n=7.3.

Example 7: Preparation of Antibody-Drug Conjugates (ADC-5 and ADC-6)

[0155] ##STR00226##

Step 1: Preparation of Compounds L-D-5 and L-D-6

[0156] ##STR00227##

[0157] To a 50-mL single-necked flask were added L-2 (80 mg, 0.12 mmol, 1.0 eq), component compound 1-2 (55 mg, 0.12 mmol, 1.0 eq) with a long retention time, NMI (50.6 mg, 0.62 mmol, eq) and DMF (2 mL), and the system was stirred well and cooled to 0 C. The reaction solution was added with TCFH (41.5 mg, 0.15 mmol, 1.2 eq) and stirred for 30 minutes, followed by detection by LC-MS. After the reaction was completed, the reaction solution was purified by reversed-phase C18 column chromatography to give two fractions, with the fraction with a short retention time being L-D-5 and the fraction with a long retention time being L-D-6. The fractions of the target substance were lyophilized to give yellow solids L-D-5 (20 mg) and L-D-6 (23 mg).

[0158] Conditions for chromatography: the semi-preparative liquid chromatograph U3000 from Thermo Fisher; chromatographic column: Welch Ultimate XB-Phenyl; mobile phase: acetonitrile containing 0.1% formic acid-0.1% aqueous formic acid=50:50; flow rate: 30.0 mL/min; detection wavelength: 370 nm; injection amount: 100 L.

[0159] The experimental procedures were as follows: a proper amount of a mixture of L-D-5 and L-D-6 was taken and dissolved in DMF. A test sample solution with a concentration of 10 mg/mL was prepared. The test sample solution was taken and placed into the preparative liquid chromatograph for detection according to the conditions for chromatography of the present invention and then data were recorded. The injection was carried out for multiple times.

[0160] As a result, L-D-5 and L-D-6 were separated by pre-HPLC, where the retention times of the two components were 6.04 minutes and 6.48 minutes, respectively, and the purities thereof were 98.58% and 99.13%, respectively.

[0161] Step 2: Preparation of ADC-5 and ADC-6

[0162] To an aqueous PBS buffer (0.05 PBS buffer at pH=6.5; 2.5 mL, 9.96 mg/mL, 0.168 nmol) of antibody trastuzumab was added a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL) at 37 C., and the system was placed in a water bath shaker, and shaken and reacted at 37 C. for 3 hours before the reaction was stopped; the reaction solution was cooled to 25 C. under a water bath and diluted to 5.0 mg/mL. Two aliquots were prepared in parallel. Compound L-D-5 (2.0 nmol) was dissolved in DMSO (0.10 mL) and added to 2.0 mL of the above solution, and the system was placed in a water bath shaker, and shaken and reacted at 25 C. for 3 hours before the reaction was stopped. The reaction solution was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer at pH 6.5, containing 0.001 M EDTA) to give a PBS buffer (5.0 mg/mL, 1.1 mL) of ADC, which was frozen and stored at 4 C. Mean calculated by UV-HPLC: n=7.3.

[0163] ADC-6 was prepared by using compound L-D-6 in the same manner, where n=7.3.

Example 8: Other ADCs

[0164] Other compounds similar to L-D-1, L-D-2, L-D-3, L-D-4, L-D-5 or L-D-6 (the camptothecin derivative of the present application is a small molecule toxin) can be prepared in the same manner. L-D-1, L-D-2, L-D-3, L-D-4, L-D-5 or L-D-6 and similar compounds can be further combined with antibody trastuzumab or other similar antibodies to prepare an antibody-drug conjugate comprising the camptothecin derivative of the present application as a small molecule toxin.

Example 9: Assay for Antiproliferative Activity Against SK-BR-3 Cells

[0165] The activity of the antibody-drug conjugate of the present invention can be determined by the assay for the in-vitro antiproliferative activity against SK-BR-3 cells of a camptothecin derivative as a small molecule toxin.

[0166] SK-BR-3 cells were seeded into a 384-well plate (Fisher 142762) at 3000 cells per well. The next day, serially diluted compounds were added, and 72 hours after the addition, CellTiter-Lumi (Beyotime C0068XL) was added to measure the ATP content in the cells. The growth of the cells was evaluated, and relative IC.sub.50 values of the compounds against cell growth was calculated. The screening results are shown in Table 6.

TABLE-US-00006 TABLE 6 Antiproliferative activity of the compounds of the present invention against SK-BR-3 cells No. IC.sub.50 (nM) No. IC.sub.50 (nM) 1 2.98 1-1 5.81 1-2 1.73 3-2 2.85 4-2 2.74 7-2 2.68 14-2 4.13 20 5.83 32 4.30 34 5.84 35 6.53 39 4.78 41 6.47 44 6.29 48 6.73 49 7.32 Exatecan 5.35 Deruxtecan 13.61 Topotecan 58.30

[0167] Compared with exatecan, deruxtecan and topotecan which is a camptothecin medicament on the market, the compounds of the present invention have strong in-vitro antiproliferative activity against SK-BR-3 cells, and particularly when X in general formula (1) is S or O, have strong antiproliferative activity against cells. For example, compound 1-2 is 2-fold more active than exatecan, and compound 32 is 3-fold more active than deruxtecan. In particular, the compound of general formula (1), containing easily attached groups such as OH or NH.sub.2 in the side chains and having strong cell activity, is suitable to be used as a small molecule toxin of ADCs.

Example 10: In-Vitro Anti-Tumor Activity of Antibody-Drug Conjugates of the Present Invention

[0168] SK-BR-3 cells with high expression of HER2 were selected as cell strains for in-vitro activity detection in the experiment and were used for evaluating the dose-effect relationship of the antibody-drug conjugates (ADCs) of the present invention on cell killing. The plating density for each type of cells was initially selected to be 1500-2000 cells/well, and the assay for cell cytotoxicity was performed after 12 hours; the ADCs were added at 10 nM as the starting concentration for a 3-10 fold serial dilution to give a final concentration, the killing effect was then observed for 144 hours, CellTiter-Glo Luminescent Cell Viability Assay was used for chemiluminescent staining, and IC.sub.50 was calculated after the fluorescence data were read. From the results of the activity test, all of the ADCs showed certain anti-tumor activity, and the activity of part of the ADCs was greater than that of DS-8201a.

TABLE-US-00007 TABLE 7 Antiproliferative activity of the antibody-drug conjugates of the present invention against SK-BR-3 cells Sample IC.sub.50 (nM) ADC-1 0.26 ADC-2 0.13 ADC-3 0.08 ADC-4 0.06 ADC-5 0.02 ADC-6 0.10 Exatecan 0.73 DS-8201a 0.09

[0169] From the results of the activity test, all of the ADCs showed certain anti-tumor activity, and the activity of part of the ADCs was greater than that of DS-8201a.

Example 11: In-Vivo Anti-Tumor Activity of Antibody-Drug Conjugates of the Present Invention

[0170] Human gastric carcinoma cells (NCI-N87) dissolved in 100 L of PBS solution were injected subcutaneously into the right sides of the necks or dorsa of female Balb/c nude mice aged 6-8 weeks. When the average tumor volume was about 150-200 mm.sup.3, the 32 nude mice were randomly divided into 4 groups according to the tumor size, with 8 animals in each group, and injection administration was performed via tail veins, where 01 was blank control group, 02 was DS-8201a (4.5 mg/kg) group, 03 was ADC-1 (4.5 mg/kg) group, and 04 was ADC-2 (4.5 mg/kg) group. The weight and the tumor volume of the experimental animals were measured twice a week and the survival state of the animals in the experimental process was observed, wherein the specific results of the change in the tumor volume of each group are shown in FIG. 1. As can be seen from FIG. 1, both of the ADC samples of the present invention showed in-vivo anti-tumor activity comparable to DS-8201a.

[0171] Although specific embodiments of the present invention have been described above, it will be appreciated by those skilled in the art that these embodiments are merely illustrative and that many changes or modifications can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of protection of the present invention is therefore defined by the appended claims.