PHARMACEUTICAL COMPOSITION COMPRISING ANTIBODY DRUG CONJUGATE AND USE THEREOF

Abstract

A pharmaceutical composition, comprising an antibody drug conjugate in a buffer solution. The antibody drug conjugate has a structure represented by the general formula (Pc-L-Y-D). The pharmaceutical composition further comprises sugar and a surfactant.

##STR00001##

Claims

1. A pharmaceutical composition, comprising an antibody drug conjugate and a buffer, wherein the antibody drug conjugate has a structure of general formula (Pc-L-Y-D): ##STR00135## wherein: Y is selected from the group consisting of —O—(CR.sup.aR.sup.b).sub.m—CR.sup.1R.sup.2—C(O)—, —O—CR.sup.1R.sup.2—(CR.sup.aR.sup.b).sub.m—, —O—CR.sup.1R.sup.2—, —NH—(CR.sup.aR.sup.b).sub.m—CR.sup.1R.sup.2—C(O)— and —S—(CR.sup.aR.sup.b).sub.m—CR.sup.1R.sup.2—C(O)—; R.sup.a and R.sup.b are identical or different and are each independently selected from the group consisting of hydrogen, deuterium, halogen, alkyl, haloalkyl, deuterated alkyl, alkoxy, hydroxy, amino, cyano, nitro, hydroxyalkyl, cycloalkyl and heterocyclyl; or, R.sup.a and R.sup.b, together with carbon atoms linked thereto, form cycloalkyl or heterocyclyl; R.sup.1 is selected from the group consisting of halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; R.sup.2 is selected from the group consisting of hydrogen, halogen, haloalkyl, deuterated alkyl, cycloalkyl, cycloalkylalkyl, alkoxyalkyl, heterocyclyl, aryl and heteroaryl; or, R.sup.1 and R.sup.2, together with carbon atoms linked thereto, form cycloalkyl or heterocyclyl; or, R.sup.a and R.sup.2, together with carbon atoms connected thereto, form cycloalkyl or heterocyclyl; m is an integer from 0 to 4; n is a decimal or an integer from 1 to 10; L is a linker unit; Pc is an antibody or an antigen-binding fragment thereof; the pharmaceutical composition is at a pH of 4.5 to 5.2.

2. The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition further comprises a surfactant selected from polysorbate.

3. The pharmaceutical composition according to claim 2, wherein the surfactant is at a concentration of about 0.01 mg/mL to about 1.0 mg/mL.

4. The pharmaceutical composition according to claim 1, wherein the composition further comprises a saccharide selected from the group consisting of sucrose, mannitol, sorbitol and trehalose.

5. The pharmaceutical composition according to claim 4, wherein the saccharide is at a concentration of about 60 mg/mL to about 90 mg/mL.

6. The pharmaceutical composition according to claim 1 to 5, wherein the antibody drug conjugate is at a concentration of about 1 mg/mL to about 100 mg/mL.

7. The pharmaceutical composition according to claim 1, wherein the buffer is selected from the group consisting of a histidine salt buffer, a succinate buffer and a citrate buffer, preferably a succinate buffer.

8. The pharmaceutical composition according to claim 1, wherein the buffer is at a concentration of about 5 mM to about 50 mM.

9. The pharmaceutical composition according to claim 1, comprising the following components: (a) the antibody drug conjugate at about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at about 60 mg/mL to about 90 mg/mL, and (d) a buffer at about 5 mM to about 20 mM; the pharmaceutical composition being at a pH of 4.8-5.2.

10. The pharmaceutical composition according to claim 1, wherein the antibody drug conjugate has the following structure: ##STR00136## wherein: n is a decimal or an integer from 3 to 8; Pc is an antibody or an antigen-binding fragment thereof.

11. The pharmaceutical composition according to claim 1, wherein the antibody or the antigen-binding fragment thereof is selected from the group consisting of an anti-HER2 (ErbB2) antibody, an anti-EGFR antibody, an anti-B7-H3 antibody, an anti-c-Met antibody, an anti-HER3 (ErbB3) antibody, an anti-HER4 (ErbB4) antibody, an anti-CD20 antibody, an anti-CD22 antibody, an anti-CD30 antibody, an anti-CD33 antibody, an anti-CD44 antibody, an anti-CD56 antibody, an anti-CD70 antibody, an anti-CD73 antibody, an anti-CD105 antibody, an anti-CEA antibody, an anti-A33 antibody, an anti-Cripto antibody, an anti-EphA2 antibody, an anti-G250 antibody, an anti-MUC1 antibody, an anti-Lewis Y antibody, an anti-VEGFR antibody, an anti-GPNMB antibody, an anti-integrin antibody, an anti-PSMA antibody, an anti-tenascin-C antibody, an anti-SLC44A4 antibody and an anti-mesothelin antibody, or antigen-binding fragments thereof.

12. The pharmaceutical composition according to claim 1, wherein the antibody conjugate has the following structure: ##STR00137## wherein n is a decimal or an integer from 3 to 8.

13. A pharmaceutical composition, comprising an antibody drug conjugate having the following structure: ##STR00138## wherein n is a decimal or an integer from 3 to 8; and the pharmaceutical composition comprises: (a) the antibody drug conjugate at about 10 mg/mL to about 30 mg/mL, (b) a polysorbate at about 0.05 mg/mL to about 0.5 mg/mL, (c) a saccharide at about 60 mg/mL to about 90 mg/mL, and (d) a buffer at about 5 mM to about 20 mM; the pharmaceutical composition being at a pH of 4.8 to 5.2.

14. A lyophilized formulation comprising an antibody drug conjugate, wherein the formulation can be reconstituted to form the pharmaceutical composition according to claim 1.

15. A method for preparing a lyophilized formulation comprising an antibody drug conjugate, comprising a step of lyophilizing the pharmaceutical composition according to claim 1.

16. A lyophilized formulation comprising an antibody drug conjugate, obtained by lyophilizing the pharmaceutical composition according to claim 1.

17. A reconstituted solution comprising an antibody drug conjugate, obtained by reconstituting the lyophilized formulation according to claim 14.

18. An article of manufacture, comprising a container containing the pharmaceutical composition according to claim 1.

19. A method for treating a cancer in a patient in need thereof, comprising administering to the patient an effective amount of the pharmaceutical composition according to claim 1, wherein, the cancer is associated with HER2, HER3, B7H3 or EGFR expression.

20. The method of claim 19, wherein the cancer is selected from the group consisting of: breast cancer, ovarian cancer, cervical cancer, uterine cancer, prostate cancer, kidney cancer, urinary tract cancer, bladder cancer, liver cancer, stomach cancer, endometrial cancer, salivary gland carcinoma, esophageal cancer, melanoma, neuroglioma, neuroblastoma, sarcoma, lung cancer, colon cancer, rectal cancer, colorectal cancer, leukemia, bone cancer, skin cancer, thyroid cancer, pancreatic cancer and lymphoma.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0279] FIG. 1A shows the results of plasma stability test for ADC-19 of the present disclosure.

[0280] FIG. 1B shows the results of plasma stability test for ADC-18 of the present disclosure.

[0281] FIG. 1C shows the results of plasma stability test for ADC-20 of the present disclosure.

[0282] FIG. 2 shows the evaluation of the efficacy of ADC-21 and ADC-24 of the present disclosure in JIMT-1 tumor-bearing mice.

[0283] FIG. 3 shows the evaluation of the therapeutic effect of ADCs on human breast cancer cell SK-BR-3 xenograft tumor nude mice.

[0284] FIG. 4 shows the results of plasma stability test for ADC-25 of the present disclosure.

[0285] FIG. 5 shows the efficacy of the ADCs of the present disclosure against human brain astrocytoma U87MG nude mouse xenograft tumors.

[0286] FIG. 6 shows the efficacy of the ADCs of the present disclosure against pleural effusion metastatic human pharyngeal cancer cell Detroit 562 nude mouse xenograft tumors.

[0287] FIG. 7 shows the efficacy of the ADCs of the present disclosure against human glioblastoma U87MG nude mouse xenograft tumors.

[0288] FIG. 8 shows fitted tendency graphs for a formula screening experiment, wherein the PS80 is in a unit of 10.sup.−4 g/mL, and the ADC-32 protein concentration (based on naked antibody) is in a unit of mg/mL.

DETAILED DESCRIPTION

[0289] The present disclosure is further described below with reference to examples, which, however, are not intended to limit the scope of the present disclosure. The experimental methods in the examples of the present disclosure in which specific conditions are not specified are generally performed under conventional conditions, for example, by referring to Antibodies: A Laboratory Manual and Molecular Cloning: A Laboratory Manual by Cold Spring Harbor Laboratory, or under conditions recommended by the manufacturer of the raw material or the goods. Reagents without specific origins indicated are commercially available conventional reagents.

[0290] I. Antibody Drug Conjugates

[0291] The structures of the compounds were determined by nuclear magnetic resonance (NMR) or mass spectrometry (MS). NMR spectra were measured using a Bruker AVANCE-400 nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d6), deuterated chloroform (CDCl.sub.3) and deuterated methanol (CD.sub.3OD) as determination solvents and tetramethylsilane (TMS) as internal standard. Chemical shifts were given in unit of 10.sup.−6 (ppm).

[0292] MS analysis was performed using a FINNIGAN LCQAd (ESI) mass spectrometer (manufacturer: Thermo, model: Finnigan LCQ advantage MAX).

[0293] UPLC analysis was performed using a Waters Acquity UPLC SQD liquid chromatography-mass spectrometry system.

[0294] HPLC analysis was performed using an Agilent 1200DAD high pressure liquid chromatograph (Sunfire C18 150×4.6 mm chromatography column) and a Waters 2695-2996 high pressure liquid chromatograph (Gimini C18 150×4.6 mm chromatography column).

[0295] UV-HPLC analysis was performed using a Thermo nanodrop2000 ultraviolet spectrophotometer.

[0296] Proliferation inhibition rates and IC.sub.50 values were determined using a PHERA starFS microplate reader (BMG, Germany).

[0297] Yantai Huanghai HSGF254 or Qingdao GF254 silica gel plates of specifications 0.15 mm to 0.2 mm were adopted for thin layer chromatography (TLC) analysis and 0.4 mm to 0.5 mm for TLC separation and purification.

[0298] Yantai Huanghai silica gel of 200-300 mesh is generally used as a carrier in column chromatography.

[0299] Known starting materials of the present disclosure may be synthesized using or according to methods known in the art, or may be purchased from ABCR GmbH & Co.KG, Acros Organnics, Aldrich Chemical Company, Accela ChemBio Inc, Chembee Chemicals, etc. In the Examples, the reactions were performed in an argon atmosphere or a nitrogen atmosphere unless otherwise stated.

[0300] The argon atmosphere or nitrogen atmosphere means that the reaction flask is linked to a balloon containing about 1 L of argon or nitrogen.

[0301] The hydrogen atmosphere means that the reaction flask is linked to a balloon containing about 1 L of hydrogen.

[0302] Parr 3916EKX hydrogenator, Qinglan QL-500 hydrogenator or HC2-SS hydrogenator was used in the pressurized hydrogenation reactions.

[0303] The hydrogenation reactions usually involve 3 cycles of vacuumization and hydrogen purge.

[0304] A CEM Discover-S 908860 microwave reactor was used in the microwave reactions. In the Examples, the solution in the reaction refers to an aqueous solution unless otherwise stated.

[0305] In the Examples, the reaction temperature is room temperature unless otherwise stated. The room temperature is the optimum reaction temperature, ranging from 20° C. to 30° C. Preparation of PBS buffer at pH 6.5 in Examples: 8.5 g of KH.sub.2PO.sub.4, 8.56 g of K.sub.2HPO.sub.4.3H.sub.2O, 5.85 g of NaCl and 1.5 g of EDTA were added to a flask, and the volume was brought to 2 L. The additions were all ultrasonically dissolved, and the solution was well mixed by shaking to give the desired buffer.

[0306] The eluent system for column chromatography and the developing solvent system for thin layer chromatography used for compound purification include: A: dichloromethane and isopropanol system, B: dichloromethane and methanol system, and C: petroleum ether and ethyl acetate system. The volume ratio of solvents was adjusted according to the polarity of the compound, or by adding a small amount of triethylamine and acidic or basic reagent.

[0307] Some of the compounds of the present disclosure are characterized by Q-TOF LC/MS. Q-TOF LC/MS analysis was performed using an Agilent 6530 accurate-mass quadrupole time-of-flight mass spectrometer and an Agilent 1290-Infinity ultra-high performance liquid chromatograph (Agilent Poroshell 300SB-C8 5 μm, 2.1×75 mm chromatography column).

Example 1-1

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]indolizinol[1,2-b]quinolin-1-yl)-1-hydroxycyclopropane-1-carboxamide 1

[0308] ##STR00069##

[0309] To exatecan mesylate 1b (2.0 mg, 3.76 μmol, prepared as disclosed in Patent Application “EP0737686A1”) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5° C. in an ice-water bath, and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxycyclopropylcarboxylic acid 1a (1.4 mg, 3.7 μmol, prepared using known method “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). After addition, the reaction mixture was stirred at 0-5° C. for 2 h, quenched with 5 mL of water, and 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.

[0310] The filtrate was concentrated under reduced pressure, and the resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 1 (1.6 mg, 82.1% yield).

[0311] MS m/z (ESI): 520.2 [M+1].

[0312] .sup.1H NMR (400 MHz, CDCl.sub.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 1-2

(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]indolizino[1,2-b]quinolin-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,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide 2-B

[0313] ##STR00070##

[0314] To compound 1b (4 mg, 7.53 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 2-cyclopropyl-2-hydroxyacetic acid 2a (2.3 mg, 19.8 μmol, prepared as disclosed in Patent Application “WO2013106717”), 1-hydroxybenzotriazole (3 mg, 22.4 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (4.3 mg, 22.4 μmol). After addition, the reaction mixture was stirred at 0-5° C. for 1 h. The ice-water bath was removed, and the reaction mixture was heated to 30° C., stirred for 2 h, and concentrated under reduced pressure. The resulting crude compound 2 was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title products (2-A: 1.5 mg, 2-B: 1.5 mg).

[0315] MS m/z (ESI): 534.0 [M+1].

[0316] Single-configuration compound 2-B (shorter retention time):

[0317] UPLC analysis: retention time: 1.06 min; purity: 88% (chromatography column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0318] .sup.1H NMR (400 MHz, DMSO-d.sub.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).

[0319] Single-configuration compound 2-A (longer retention time):

[0320] UPLC analysis: retention time: 1.10 min; purity: 86% (chromatography column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0321] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-3

(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]indolizinol[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 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]indolizinol[1,2-b]quinolin-1-yl)-3,3,3-trifluoro-2-hydroxypropionamide 3-B

[0322] ##STR00071##

[0323] To compound 1b (5.0 mg, 9.41 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 3,3,3-trifluoro-2-hydroxypropionic acid 3a (4.1 mg, 28.4 μmol, supplied by Alfa), 1-hydroxybenzotriazole (3.8 mg, 28.1 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (5.4 mg, 28.2 μmol). After addition, the reaction mixture was stirred at 0-5° C. for 10 min. The ice-water bath was removed, and the reaction mixture was heated to 30° C., stirred for 8 h, and concentrated under reduced pressure. The resulting crude compound 3 was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min), and the corresponding fractions were collected and concentrated under reduced pressure to give the title products (1.5 mg, 1.5 mg).

[0324] MS m/z (ESI): 561.9 [M+1].

[0325] Single-configuration compound (shorter retention time):

[0326] UPLC analysis: retention time: 1.11 min; purity: 88% (chromatography column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0327] .sup.1H NMR (400 MHz, DMSO-d.sub.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).

[0328] Single-configuration compound (longer retention time):

[0329] UPLC analysis: retention time: 1.19 min; purity: 90% (chromatography column: ACQUITY UPLC BEHC18 1.7 pin 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0330] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-4

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-hydroxycyclopentane-1-carboxamide 4

[0331] ##STR00072##

[0332] To compound 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5° C. in an ice-water bath, and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxy-cyclopentanecarboxylic acid 4a (2.2 mg, 16.9 μmol, prepared as disclosed in Patent Application “WO2013106717”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (4.7 mg, 16.9 μmol). After addition, the reaction mixture was stirred at 0-5° C. for 1 h, quenched with 5 mL of water, and 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 with developing solvent system B to give the title product 4 (2.5 mg, 80.9% yield).

[0333] MS m/z (ESI): 548.0 [M+1].

[0334] .sup.1H NMR (400 MHz, CDCl.sub.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 1-5

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-(hydroxymethyl)cyclopropane-1-carboxamide 5

[0335] ##STR00073##

[0336] To compound 1 b (2.0 mg, 3.76 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5° C. in an ice-water bath, and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-(hydroxymethyl)-cyclopentanecarboxylic acid 5a (0.87 mg, 7.5 μmol, prepared as disclosed in Patent Application “WO201396771”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2 mg, 7.24 μmol). After addition, the reaction mixture was stirred at 0-5° C. for 2 h, quenched with 5 mL of water, and 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 with developing solvent system B to give the title product 5 (1.0 mg, 50% yield).

[0337] MS m/z (ESI): 533.9 [M+1].

[0338] .sup.1H NMR (400 MHz, CDCl.sub.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 1-6

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-(hydroxymethyl)cyclobutane-1-carboxamide 6

[0339] ##STR00074##

[0340] To compound 1b (3.0 mg, 5.64 μmol) was added 1 mL of N,N-dimethylformamide. The mixture was cooled to 0-5° C. in an ice-water bath, and a drop of triethylamine was added. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-(hydroxymethyl)cyclobutane-1-carboxylic acid 6a (2.2 mg, 16.9 μmol, prepared as disclosed in “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). After addition, the reaction mixture was stirred at 0-5° C. for 1 h, quenched with 5 mL of water, and 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 with developing solvent system B to give the title product 6 (2.1 mg, 67.9% yield).

[0341] MS m/z (ESI): 548.0 [M+1].

[0342] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-7

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-hydroxycyclobutane-1-carboxamide 7

[0343] ##STR00075##

[0344] To compound 1b (3.0 mg, 5.64 μmol) were added 2 mL of ethanol and 0.4 mL of N,N-dimethylformamide, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by dropwise addition of 0.3 mL of N-methylmorpholine. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added 1-hydroxycyclobutanecarboxylic acid 7a (2.0 mg, 17.22 μmol, supplied by PharmaBlock), 1-hydroxybenzotriazole (2.3 mg, 17.0 μmol) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (3.2 mg, 16.7 μmol). After addition, the reaction mixture was stirred at 0-5° C. for 10 min. The ice-water bath was removed, and the reaction mixture was stirred at room temperature for 2 h, and concentrated under reduced pressure. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 7 (2.5 mg, 83.1% yield).

[0345] MS m/z (ESI): 534.0 [M+1].

[0346] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-8

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-pentaazaeicosyl)oxy)-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)cyclopropane-1-carboxamide 8

[0347] ##STR00076##

Step 1

Benzyl 1-((2-((((9H-fluoren yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclopropane-1-carboxylate 8c

[0348] Benzyl 1-hydroxycyclopropane-1-carboxylate 8a (104 mg, 0.54 mmol; prepared as disclosed in Patent Application “US2005/20645”) and 2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methyl acetate 8b (100 mg, 0.27 mmol; prepared as disclosed in Patent Application “CN105829346A”) were added to a reaction flask, and 5 mL of tetrahydrofuran was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (61 mg, 0.54 mmol). The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by addition of 20 mL of ice water and by extraction 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, and 0.6 mL of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (70 mg, 0.27 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. 20 mL of water was added, followed by extraction 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 with developing solvent system B to give the title product 8c (100 mg, 73.6% yield).

[0349] MS m/z (ESI): 501.0 [M+1].

Step 2

1-((2-((((9H-fluoren-9-yl)methoxy)carbonylamino)acetylamino)methoxy)cyclopropane-1-carboxylic acid 8d

[0350] Compound 8c (50 mg, 0.10 mmol) was dissolved in 3 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), and palladium on carbon (25 mg, 10% loading) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with tetrahydrofuran. The filtrate was concentrated to give the title product 8d (41 mg, 100% yield).

[0351] MS m/z (ESI): 411.0 [M+1].

Step 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)aminocarbonyl)cyclopropoxy)methyl)amino)-2-oxoethyl)carbamate 8e

[0352] Compound 1b (7 mg, 0.013 mmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, a solution of compound 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 chloride (7 mg, 0.026 mmol). The reaction mixture was stirred in an ice bath for 35 min. 10 mL of water was added, followed by extraction 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 with developing solvent system B to give the title product 8e (8.5 mg, 78.0% yield). MS m/z (ESI): 828.0 [M+1].

Step 4

1-((2-Aminoacetylamino)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)cyclopropane-1-carboxamide 8f

[0353] Compound 8e (4 mg, 4.84 μmol) was dissolved in 0.2 mL of dichloromethane, and 0.1 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 8f (2.9 mg), which was directly used in the next step without purification.

[0354] MS m/z (ESI): 606.0 [M+1].

Step 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-pentaazaeicosyl)oxy)-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)cyclopropane- 1-carboxamide 8

[0355] Crude 8f (2.9 mg, 4.84 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath. A solution of (S)-2(-2-(-2-(6-(2,5-dioxo-1H-pyrrol-1-yl)hexanamido)acetylaminolacetylamino)-3-phenylpropionic acid 8g (2.7 mg, 5.80 μmol, prepared as disclosed in Patent Application “EP2907824”) in 0.3 mL of N,N-dimethylformamide was added, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction mixture was stirred in an ice bath for 30 min. Then, the ice bath was removed, and the reaction mixture was warmed to room temperature, stirred for 15 min, and purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 8 (2 mg, 39.0% yield).

[0356] MS m/z (ESI): 1060.0 [M+1].

[0357] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-9

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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin- 1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 9-B

[0358] ##STR00077## ##STR00078## ##STR00079##

Step 1

Benzyl 2-cyclopropyl-2-hydroxyacetate 9a

[0359] Compound 2a (1.3 g, 11.2 mmol; prepared as disclosed in Patent Application “WO2013/106717”) was dissolved in 50 mL of acetonitrile, and potassium carbonate (6.18 g, 44.8 mmol), benzyl bromide (1.33 mL, 11.2 mmol) and tetrabutylammonium iodide (413 mg, 1.1 mmol) were successively added. The reaction mixture was stirred at room temperature for 48 h and filtered through celite, and the filter cake was rinsed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 9a (2 g, 86.9% yield).

Step 2

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

[0360] Compounds 9a (120.9 mg, 0.586 mmol) and 8b (180 mg, 0.489 mmol) were added to a reaction flask, and 4 mL of tetrahydrofuran was added. The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (109 mg, 0.98 mmol). The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 40 min, followed by addition of 10 mL of ice water and by extraction 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, and 2 mL of water, sodium bicarbonate (49.2 mg, 0.586 mmol) and 9-fluorenylmethyl chloroformate (126 mg, 0.49 mmol) were added. The reaction mixture was stirred at room temperature for 2 h. 20 mL of water was added, followed by extraction 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 with developing solvent system C to give the title product 9b (48 mg, 19% yield).

[0361] MS m/z (ESI): 515.0 [M+1].

Step 3

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

[0362] Compound 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), and palladium on carbon (12 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the crude title product 9c (13 mg), which was directly used in the next step without purification.

[0363] MS m/z (ESI): 424.9 [M+1].

Step 4

(9H-fluoren-9-yl)methyl(2-(((1-cyclopropyl-2-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy 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

[0364] Compound 1b (10 mg, 18.8 μmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, crude 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). The reaction mixture was stirred in an ice bath for 40 min. 10 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 9d (19 mg, 73.6% yield).

[0365] MS m/z (ESI): 842.1 [M+1].

Step 5

2-((2-Aminoacetylamino)methoxy)-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]indolizino[1,2-b]quinolin-1-yl)acetamide 9e

[0366] Compound 9d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 1 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane and let stand. Then, the supernatant was removed, and the solid was kept. The solid residue was concentrated under reduced pressure and dried using an oil pump to give the crude title product 9e (17 mg), which was directly used in the next step without purification.

[0367] MS m/z (ESI): 638.0 [M+18].

Step 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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)hexanamide 9-B

[0368] Crude 9e (13.9 mg, 22.4 μmol) was dissolved in 0.6 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath. A solution of 8g (21.2 mg, 44.8 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (18.5 mg, 67.3 μmol). The reaction mixture was stirred in an ice bath for 10 min. Then, the ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 1 h to produce compound 9. The reaction mixture was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title products (9-A: 2.4 mg, 9-B: 1.7 mg).

[0369] MS m/z (ESI): 1074.4 [M+1].

[0370] Single-configuration compound 9-A (shorter retention time):

[0371] UPLC analysis: retention time: 1.14 min; purity: 85% (chromatography column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0372] .sup.1H NMR (400 MHz, DMSO-d.sub.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).

[0373] Single-configuration compound 9-B (longer retention time):

[0374] UPLC analysis: retention time: 1.16 min; purity: 89% (chromatography column: ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0375] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-10

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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-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)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0376] ##STR00080## ##STR00081## ##STR00082##

Step 1

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

[0377] Compound 3a (1.80 g, 12.5 mmol) was dissolved in 100 mL of acetonitrile, and potassium carbonate (5.17 g, 37.5 mmol), benzyl bromide (4.48 mL, 37.5 mmol) and tetrabutylammonium iodide (231 mg, 0.63 mmol) were added successively. The reaction mixture was heated to 60° C., stirred for 5 h, cooled to room temperature, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 10a (980 mg, 33.5% yield).

[0378] .sup.1H NMR (400 MHz, CDCl.sub.3): δ 7.43-7.36 (m, 5H), 5.34 (s, 2H), 4.53 (s, 1H), 3.44 (s, 1H).

Step 2

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

[0379] Compounds 8b (63 mg, 0.17 mmol) and 10a (80 mg, 0.34 mmol) were added to a reaction flask, and 3 mL of tetrahydrofuran was added. The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (38 mg, 0.34 mmol). The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 20 min, followed by addition of 10 mL of ice water and by extraction with ethyl acetate (20 mL×2) and chloroform (10 mL×5). The organic phases were combined and concentrated. The resulting residue was dissolved in 2 mL of dioxane, and 0.4 mL of water, sodium bicarbonate (19 mg, 0.23 mmol) and 9-fluorenylmethyl chloroformate (49 mg, 0.19 mmol) were added. The mixture was stirred at room temperature for 1 h. 20 mL of water was added, followed by extraction 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, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 10b (51 mg, 55.3% yield).

[0380] MS m/z (ESI): 559.9 [M+18].

Step 3

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

[0381] Compound 10b (15 mg, 0.28 mmol) was dissolved in 3 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), and palladium on carbon (15 mg, 10% loading) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with tetrahydrofuran. The filtrate was concentrated to give the crude title product 10c (13 mg).

[0382] MS m/z (ESI): 452.9 [M+1].

Step 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-oxoprop-2-yl)oxy)methyl)amino)-2-oxoethyl)carbamate 10d

[0383] Compound 1b (10 mg, 18.8 μmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, a solution of 10c (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). The reaction mixture was stirred in an ice bath for 30 min. 10 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phases were 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 with developing solvent system B to give the title product 10d (16 mg, 97.8% yield).

[0384] MS m/z (ESI): 870.0 [M+1].

Step 5

2-((2-Aminoacetylamino)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)-3,3,3-trifluoropropionamide 10e

[0385] Compound 10d (16 mg, 18.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane and let stand. Then, the supernatant was removed, and the solid was kept; the procedures were repeated three times. The solid residue was concentrated under reduced pressure and dried using an oil pump to give the crude title product 10e (12 mg), which was directly used in the next step without purification.

[0386] MS m/z (ESI): 647.9 [M+1].

Step 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-1H,12H-benzo 1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-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 1-yl)aminocarbonyl)-1,1,1-trifluoro-6,9,12,15-tetraoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 10-B

[0387] Crude 10e (12 mg, 18.5 μmol) was dissolved in 1.0 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath. A solution of 8g (14 mg, 29.6 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (15 mg, 54.2 μmol). The reaction mixture was stirred in an ice bath for 30 min. Then, the ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 1 h to produce compound 10. The reaction mixture was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title products (2.7 mg, 2.6 mg).

[0388] MS m/z (ESI): 1102.0 [M+1].

[0389] Single-configuration compound (shorter retention time): UPLC analysis: retention time: 1.18 min; purity: 91% (chromatography column:

[0390] ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0391] .sup.1H NMR (400 MHz, DMSO-d.sub.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).

[0392] Single-configuration compound (longer retention time):

[0393] UPLC analysis: retention time: 1.23 min; purity: 90% (chromatography column:

[0394] ACQUITY UPLC BEHC18 1.7 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0395] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-11

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-pentaazaeicosyl)oxy)-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)cyclobutane-1-carboxamide 11

[0396] ##STR00083## ##STR00084##

Step 1

Benzyl 1-((2-4((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylate 11b

[0397] Benzyl 1-hydroxycyclobutane-carboxylate 11a (167 mg, 0.81 mmol, prepared as disclosed in “Journal of Medicinal Chemistry, 2013, vol. 56, #13, p.5541-5552”) and 8b (150 mg, 0.41 mmol) were added to a reaction flask, and 5 mL of tetrahydrofuran was added. The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (92 mg, 0.82 mmol). The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by addition of 20 mL of ice water and by extraction 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 dioxane, and 0.6 mL of water, sodium bicarbonate (41 mg, 0.48 mmol) and 9-fluorenylmethyl chloroformate (105 mg, 0.41 mmol) were added. The mixture was stirred at room temperature for 1 h. 20 mL of water was added, followed by extraction 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 with developing solvent system C to give the title product 11b (37 mg, 17.6% yield).

[0398] MS m/z (ESI): 514.6 [M+1].

Step 2

1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)acetylamino)methoxy)cyclobutane-1-carboxylic acid 11e

[0399] Compound 11b (37 mg, 71.9 μmol) was dissolved in 3 mL of a solvent mixture of tetrahydrofuran and ethyl acetate (V:V=2:1), and palladium on carbon (15 mg, 10% loading) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with tetrahydrofuran. The filtrate was concentrated to give the title product 11e (35 mg, 82% yield), which was directly used in the next step.

Step 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)aminocarbonyl)cyclobutoxy)methyl)amino)-2-oxoethyl)carbamate 11d

[0400] Compound 1b (10 mg, 0.018 mmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, a solution of compound 11c (13 mg, 0.031 mmol) in 0.5 mL of N,N-dimethylformamide, and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (25 mg, 0.091 mmol). The reaction mixture was stirred in an ice bath for 40 min. 8 mL of water was added, followed by extraction 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 with developing solvent system A to give the title product 11d (19 mg, 73.9% yield).

[0401] MS m/z (ESI): 842.3 [M+1].

Step 4

1-((2-Aminoacetylamino)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)cyclobutane-1-carboxamide 11e

[0402] Compound 11d (19 mg, 22.6 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 1 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 4 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 11e (15 mg), which was directly used in the next step without purification.

Step 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-pentaazaeicosyl)oxy)-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)cyclobutane-1-carboxamide 11

[0403] Crude 11e (2 mg, 3.22 μmol) was dissolved in 0.5 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath. A solution of 8g (1.5 mg, 3.17 μmol) in 0.3 mL of N,N-dimethylformamide was added, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (2.7 mg, 9.67 μmol). The reaction mixture was stirred at room temperature for 30 min and concentrated to dryness with an oil pump to remove DMF, and the residue was dissolved in DCM and purified directly by thin layer chromatography twice (polarity of developing solvent: DCM/MeOH=10/1) to give the title product 11 (1 mg, 28.8% yield).

[0404] MS m/z (ESI): 1073.6 [M+1].

[0405] .sup.1H NMR (400 MHz, CDCl.sub.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 1-12

(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-hydroxypropionamide 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-hydroxypropionamide 12-B

[0406] ##STR00085## ##STR00086##

Step 1

3-Cyclopropyl-2-hydroxypropionic acid 12b

[0407] Compound 12a (0.5 g, 3.87 mmol, supplied by 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, followed by dropwise addition of a 2 M aqueous solution of sodium nitrite (0.53 g, 7.74 mmol). The reaction mixture was warmed to room temperature and stirred for 3 h. Solid sodium chloride was added to the reaction mixture to saturate the aqueous phase. The reaction mixture 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, 89.3% yield).

Step 2

(S)-3-cyclopropyl-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-di oxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide 12-A

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

[0408] To compound 1b (45 mg, 0.085 mmol) were added 1.5 mL of ethanol and 1.5 mL of N,N-dimethylformamide. The system was purged with argon three times, and 0.1 mL of N-methylmorpholine was added dropwise. The reaction mixture was stirred until it became clear. To the reaction mixture were successively added compound 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). After addition, the reaction mixture was stirred at room temperature for 3 h and concentrated under reduced pressure, and the resulting crude compound 12 was purified by high performance liquid chromatography (separation conditions: chromatography column: Sharpsil-T C18 5 μm 21.2×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to give the title products (7 mg, 15 mg).

[0409] MS m/z (ESI): 547.9 [M+1].

[0410] Single-configuration compound (shorter retention time):

[0411] UPLC analysis: retention time: 1.345 min; purity: 72% (chromatography column:

[0412] ZORBAX Ecliphase Plus C18 1.8 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0413] .sup.1H NMR (400 MHz, DMSO-d.sub.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).

[0414] Single-configuration compound (longer retention time):

[0415] UPLC analysis: retention time: 1.399 min; purity: 88% (chromatography column: ZORBAX Ecliphase Plus C18 1.8 μm 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0416] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-13 (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

[0417] ##STR00087##

[0418] The title compound 13 was prepared as disclosed in “Example 76 on page 147 of the specification of Patent EP2907824A1”.

Example 1-14

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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol 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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol yl)hexanamide 14-B

[0419] ##STR00088## ##STR00089## ##STR00090##

Step 1

Benzyl 3-cyclopropyl-2-hydroxypropionate 14a

[0420] Compound 12b (200 mg, 1.54 mmol) was dissolved in 20 mL of acetonitrile, and potassium carbonate (1.06 g, 7.68 mmol), benzyl bromide (0.16 mL, 1.34 mmol) and tetrabutylammonium iodide (28 mg, 0.07 mmol) were added successively. The reaction mixture was stirred at room temperature for 48 h and filtered through celite, and the filter cake was rinsed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 14a (140 mg, 41.3% yield).

Step 2

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

[0421] Compounds 14a (94 mg, 0.427 mmol) and 8b (130 mg, 0.353 mmol) were added to a reaction flask, and 10 mL of tetrahydrofuran was added. The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (79 mg, 0.704 mmol). The ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 10 min, followed by addition of 20 mL of ice water and by extraction 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, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 14b (50 mg, 26.8% yield).

[0422] MS m/z (ESI): 529.2 [M+1].

Step 3

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

[0423] Compound 14b (27 mg, 0.051 mmol) was dissolved in 3 mL of ethyl acetate, and palladium on carbon (7 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times. The reaction mixture was stirred at room temperature for 1 h and filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the crude title product 14c (23 mg), which was directly used in the next step without purification.

[0424] MS m/z (ESI): 439.1 [M+1].

Step 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-1H,12H-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

[0425] Compound 1b (22 mg, 42.38 μmol) was added to a reaction flask, and 3 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by dropwise addition of triethylamine (4.3 mg, 42.49 μmol) and addition of crude 14c (23 mg, 51.1 μmol) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (17.6 mg, 63.6 μmol). The reaction mixture was stirred in an ice bath for 40 min. 15 mL of water was added, followed by extraction with ethyl acetate (8 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 14d (29 mg, 79.9% yield).

[0426] MS m/z (ESI): 856.1 [M+1].

Step 5

2-((2-Aminoacetylamino)methoxy)-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)propanamide 14e

[0427] Compound 14d (29 mg, 33.9 μmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 1 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane and let stand. Then, the supernatant was removed; and the procedures were repeated three times. The residue was concentrated under reduced pressure and dried using an oil pump to give the crude title product 14e (22 mg), which was directly used in the next step without purification.

[0428] MS m/z (ESI): 634.1 [M+1].

Step 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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol 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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadec-16-yl)-6-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)hexanamide 14-B

[0429] Crude 14e (22 mg, 33.9 μmol) was dissolved in 2.5 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath, and 8g (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. Then, the ice bath was removed, and the reaction mixture was warmed to room temperature and stirred for 1 h to produce compound 14. The reaction mixture was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 pin 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to give the title products (2 mg, 2 mg).

[0430] MS m/z (ESI): 1088.4 [M+1].

[0431] Single-configuration compound (shorter retention time):

[0432] UPLC analysis: retention time: 1.18 min; purity: 88% (chromatography column: ACQUITY UPLC BEHC18 1.7 pin 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

[0433] Single-configuration compound (longer retention time):

[0434] UPLC analysis: retention time: 1.23 min; purity: 96% (chromatography column: ACQUITY UPLC BEHC18 1.7 pin 2.1×50 mm; mobile phase: A-water (5 mmol of NH.sub.4OAc), B-acetonitrile).

Example 1-15

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-1H,12H-benzo[de]pyrano[3,4′:6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0435] ##STR00091## ##STR00092##

Step 1

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

[0436] Compound 8b (500 mg, 1.35 mmol) was added to a reaction flask, followed by addition of 6 mL of tetrahydrofuran, benzyl 1-hydroxymethylcyclopropane-1-carboxylate 15a (233 mg, 1.13 mmol; prepared as disclosed in “Example 22-2 on page 262 of the specification of Patent Application EP2862856A1”). The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of sodium hydride (54 mg, 1.35 mmol). Then, the ice-bath was removed, and the reaction mixture was warmed to room temperature, stirred for 40 min, and cooled to 0° C., followed by addition of 20 mL of ice water and by extraction with ethyl acetate (5 mL×2) and chloroform (5 mL×5). The organic phases were combined, washed with saturated sodium chloride solution (20 mL), dried over anhydrous sodium sulfate, and filtered, and the filtrate was concentrated under reduced pressure. The resulting residue was purified by silica gel column chromatography with developing solvent system B to give the title product 15b (15 mg, 2.5% yield).

[0437] MS m/z (ESI): 515.2 [M+1].

Step 2

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

[0438] Compound 15b (15 mg, 0.029 mmol) was dissolved in 2 mL of ethyl acetate, and palladium on carbon (3 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 4.5 h and filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the title product 15c (11 mg, 89% yield).

[0439] MS m/z (ESI): 425.2 [M+1].

Step 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)aminocarbonyl)cyclopropyl)methoxy)methyl)amino)2-oxoethyl)carbamate 15d

[0440] Compound 1b (10 mg, 0.021 mmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, compound 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). After addition, the reaction mixture was stirred at room temperature for 60 min. 10 mL of water was added, followed by extraction 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 with developing solvent system B to give the title product 15d (19 mg, 87.0% yield).

[0441] MS m/z (ESI): 842.2 [M+1].

Step 4

1-(((2-Amino acetylamino)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)cyclopropane-1-carboxamide 15e

[0442] Compound 15d (19 mg, 22.56 μmol) was dissolved in 2 mL of dichloromethane, and 1 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated at 0° C. under reduced pressure. 1 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 15e (13.9 mg), which was directly used in the next step without purification.

[0443] MS m/z (ESI): 620.1 [M+1].

Step 5

1-((S)-9-benzyl-22-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-5,8,11,14,17-pentaoxo 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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)cyclopropane-1-carboxamide 15

[0444] Crude 15e (13.9 mg, 22.4 μmol) was dissolved in 1 mL of N,N-dimethylformamide. The system was purged with argon three times, and the solution was cooled to 0-5° C. in an ice-water bath, followed by addition of 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). The reaction mixture was warmed to room temperature, stirred for 60 min, and purified by high performance liquid chromatography (separation conditions: chromatography column XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 15 (2.5 mg, 10.3% yield).

[0445] MS m/z (ESI): 1074.2 [M+1].

[0446] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-16

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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)cyclobutane-1-carboxamide 16

[0447] ##STR00093## ##STR00094##

Step 1

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

[0448] Ethyl 1-(hydroxymethyl)cyclobutanecarboxylate 16a (250 mg, 1.58 mmol, supplied by Alfa) was dissolved in methanol (2 mL) and water (1 mL), and sodium hydroxide (126 mg, 3.15 mmol) was added. The reaction mixture was warmed to 40° C., stirred for 3 h, cooled to room temperature, and concentrated under reduced pressure to remove the organic solvent. The reaction mixture was reversely extracted with diethyl ether (10 mL) to collect the aqueous phase. The aqueous phase was adjusted to pH 3-4 with 6 N aqueous hydrochloric acid and concentrated under reduced pressure to give a solid. 3 mL of toluene was added, followed by concentration under reduced pressure to dryness; the procedures were repeated three times. The residue was dried using an oil pump to give the crude title product 16b (206 mg), which was directly used in the next step without purification.

[0449] MS mlz (ESI, NEG):129.2 [M−1].

Step 2

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

[0450] Crude 16b (206 mg, 1.58 mmol) was dissolved in acetonitrile (15 mL), and anhydrous potassium carbonate (1.09 g, 7.90 mmol), tetrabutylammonium iodide (29 mg, 78.51 μmol) and benzyl bromide (216 mg, 1.26 mmol) were added. The reaction mixture was stirred at room temperature overnight and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 16c (112 mg, 32.1% yield).

[0451] MS m/z (ESI): 221.1 [M+1].

Step 3

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

[0452] Compounds 16c (77 mg, 0.35 mmol) and 8b (100 mg, 0.27 mmol) were added to a reaction flask, and 3 mL of tetrahydrofuran was added. The system was purged with argon three times, and the reaction mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of potassium tert-butoxide (61 mg, 0.54 mmol). The reaction mixture was stirred in the ice bath for 10 min 20 mL of ice water was added, followed by extraction 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, and 0.5 mL of water, sodium bicarbonate (27 mg, 0.32 mmol) and 9-fluorenylmethyl chloroformate (71 mg, 0.27 mmol) were added. The reaction mixture was stirred at room temperature for 1 h. 20 mL of water was added, followed by extraction 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, and the resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 16d (24 mg, 16.7% yield).

[0453] MS m/z (ESI): 551.3 [M+23].

Step 4

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

[0454] Compound 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), and palladium on carbon (5 mg, 10% loading) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 2 h. The reaction mixture was filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated under reduced pressure to give the crude title product 16e (10 mg), which was directly used in the next step without purification.

Step 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)aminocarbonyl)cyclobutyl)methoxy)methyl)amino)-2-oxoethyl)carbamate 16f

[0455] Compound 1b (7.5 mg, 0.014 mmol) was added to a reaction flask, and 1 mL of N,N-dimethylformamide was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine, a solution of crude 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 (6 mg, 0.026 mmol). The reaction mixture was stirred in an ice bath for 30 min 10 mL of water was added, followed by extraction 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 with developing solvent system B to give the title product 16f (10.6 mg, 87.8% yield).

[0456] MS m/z (ESI): 856.2 [M+1].

Step 6

1-(((2-Aminoacetylamino)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)cyclobutane-1-carboxamide 16g

[0457] Compound 16f (10.6 mg, 12.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 2 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The residue was concentrated under reduced pressure to give the crude title product 16g (8 mg), which was directly used in the next step without purification.

[0458] MS m/z (ESI): 634.1 [M+1].

Step 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-41 S,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)cyclobutane-1-carboxamide 16

[0459] Crude 16g (8 mg) was dissolved in 1 mL of N,N-dimethylformamide, and 8g (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 mixture was stirred at room temperature for 30 min and purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min) to give the title product 16 (1.0 mg, 7.2% yield).

[0460] MS m/z (ESI): 1088.0 [M+1].

Example 1-17

(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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatetratridec-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0461] ##STR00095## ##STR00096##

Step 1

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

[0462] 1-Phenyl-2,5,8,11,14,17,20,23,26-nonaoxaoctacosane-28-ol 17a (0.34 g, 0.67 mmol, supplied by Bide) was dissolved in 10 mL of dichloromethane, and 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 successively added. The reaction mixture was stirred at room temperature for 3 h, and filtered. The filtrate was concentrated under reduced pressure, and the resulting residue was purified by silica gel column chromatography with developing solvent system B to give the title product 17b (0.42 g, 100% yield).

[0463] MS m/z (ESI): 636.3 [M+18].

Step 2

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

[0464] Compound 17b (417 mg, 0.67 mmol) was dissolved in 15 mL of tetrahydrofuran, and palladium on carbon (110 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was warmed to 60° C., stirred for 3 h, filtered through celite, and the filter cake was rinsed with tetrahydrofuran. The filtrate was concentrated to give the crude title product 17c (357 mg), which was directly used in the next step without purification.

[0465] MS m/z (ESI): 546.2 [M+18].

Step 3

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

[0466] Compound 17c (357 mg, 0.675 mmol) was dissolved in 10 mL of toluene, and diphenyl phosphoryl azide (279 mg, 1.014 mmol) and 1,8-diazabicycloundec-7-ene (206 mg, 1.353 mmol) were added. The system was purged with argon three times, and the reaction mixture was stirred at room temperature for 2 h, then warmed to 105° C., reacted for 19 h, cooled to room temperature and concentrated. 20 mL of water was added, followed by extraction 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 with developing solvent system B to give the crude title product 17d (412 mg).

[0467] MS m/z (ESI): 571.3 [M+18].

Step 4

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

[0468] Compound 17d (230 mg, 0.415 mmol) was dissolved in 8 mL of tetrahydrofuran, and palladium on carbon (58 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times. The reaction mixture was stirred at room temperature for 2 h and filtered through celite, and the filter cake was rinsed with tetrahydrofuran. The filtrate was concentrated to give the crude title product 17e (220 mg), which was directly used in the next step without purification.

[0469] MS m/z (ESI): 528.2 [M+1].

Step 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-azatriundec-31-oate 17f

[0470] (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, and 2-(7-benzotriazol oxide)-N,N,N′,N′-tetramethyluronium hexafluorophosphate (190 mg, 0.500 mmol) and N,N-diisopropylethylamine (162 mg, 1.253 mmol) were added. The system was purged with argon three times, and crude 17e (220 mg, 0.417 mmol) was added. The reaction mixture was stirred at room temperature for 1 h. 15 mL of water was added, followed by extraction with dichloromethane (8 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 17f (122 mg, 39.2% yield).

[0471] MS m/z (ESI): 747.2 [M+1].

Step 6

1-((1 r,4 r)-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-azatriundec-31-oic acid 17g

[0472] Compound 17f (122 mg, 0.163 mmol) was dissolved in 0.8 mL of dichloromethane, and 0.4 mL of trifluoroacetic acid was added. The reaction mixture was stirred at room temperature for 1 h, diluted with 15 mL of dichloromethane, and concentrated under reduced pressure; 10 mL of n-hexane was added, followed by concentration under reduced pressure; the procedures were repeated twice. Then 10 mL of toluene was added, and the resulting mixture was concentrated under reduced pressure, slurried three times with 10 mL of a solvent mixture of n-hexane:diethyl ether=5:1 until the pH reached 7, concentrated, and dried with an oil pump to give the title product 17g (98 mg, 86.8% yield).

[0473] MS m/z (ESI): 691.2 [M+1].

Step 7

2,4-Dimethoxybenzyl 1-((2-((((9H-fluoren-9-yl)methoxy)carbonyl)amino) acetylamino)methoxy)cyclopropyl-1-carboxylate 17h

[0474] Compound 8d (164 mg, 0.40 mmol) was dissolved in dichloromethane (5 mL), and 2,4-dimethoxybenzyl alcohol (81 mg, 0.48 mmol), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimine hydrochloride (115 mg, 0.60 mmol) and 4-dimethylaminopyridine (5 mg, 0.041 mmol) were added successively. After addition, the reaction mixture was stirred at room temperature for 1 h. 20 mL of water was added, and layers formed after shaking. The aqueous phase was extracted with dichloromethane (8 mL×3). 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. The resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 17h (124 mg, 55.4% yield).

[0475] MS m/z (ESI): 583.1 [M+23].

Step 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-pentaazaheptadecane-17-yl)oxylcyclopropyl-1-carboxylate 17j

[0476] Compound 17h (39 mg, 69.6 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times, followed by concentration under reduced pressure. The resulting crude product was dissolved in 2 mL of N,N-dimethylformamide, and (((9H-fluoren-9-yl)methoxy)carbonyl)glycyl-L-phenylalanine 17i (35 mg, 69.8 μmol, prepared as disclosed in “Example 7-12 on page 13 of the specification of Patent Application CN108853514A”) and 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (23 mg, 83.1 μmol) were added. The reaction mixture was stirred at room temperature for 1 h. 10 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 17j (48 mg, 83.9% yield).

[0477] MS m/z (ESI): 822.0 [M+1].

Step 9

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

[0478] Compound 17j (48 mg, 58.4 μmol) was dissolved in 1.4 mL of a 3% (v/v) solution of dichloroacetic acid in dichloromethane, and the solution was cooled to 0-5° C. in an ice-water bath, followed by addition of triethylsilane (21 mg, 180.6 μmol). The reaction mixture was stirred in the ice bath for 3 h. The reaction mixture was concentrated under reduced pressure in the ice bath to remove half of the organic solvent. 5 mL of diethyl ether was added, and the resulting mixture was warmed to room temperature naturally and slurried to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 17k (33 mg, 84.1% yield).

Step 10

(9H-fluoren-9-yl)methyl((S)-7-benzyl-1-(1-(((1S,9 S)-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)aminocarbonyl)cyclopropoxy)-3,6,9,12-tetraoxo-2,5,8,11-tetraazatridecan-13-yl)carbamate 17l

[0479] Compound 1b (20 mg, 42.4 μmol) was added to a reaction flask, and 1 mL of a 10% (v/v) solution of methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine. The mixture was stirred until compound 1b was dissolved. Compound 17k (33 mg, 49.1 μmol) was dissolved in 1 mL of a 10% (v/v) solution of methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl) methylmorpholinium chloride (17.6 mg, 63.6 μmol). The reaction mixture was warmed to room temperature and stirred for 1 h. 10 mL of dichloromethane and 5 mL of water were added, and the resulting mixture was stirred for 5 min, and let stand to form layers. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 17l (37 mg, 80.2% yield).

[0480] MS m/z (ESI): 1090.1 [M+1].

Step 11

(1 r,4 r)-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-1H,12H-benzo[de]pyrano[3′,4′: 6,7]indolizino[1,2-b]quinolin-1-yl)aminocarbonyl)cyclopropoxy)-3,6,9,12,15-pentaoxo-17,20,23,26,29,32,35,38,41-nonaoxa-2,5,8,11,14-pentaazatetratridec-43-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 17

[0481] Compound 17l (15.5 mg, 14.23 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The reaction mixture was concentrated under reduced pressure and then dried with an oil pump. The resulting crude product was dissolved in 1 mL of N,N-dimethylformamide, and compound 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 system was purged with argon three times, and the reaction mixture was stirred at room temperature for 30 min and purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 17 (6 mg, 27.4% yield).

[0482] MS m/z (ESI): 1556.4 [M+18].

[0483] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-18

(1r,4r)-N-((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy 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)-1,6,9,12,15,18-hexaoxo-3,20,23,26,29,32,35,38,41,44-decaoxa-5,8,11,14,17-pentaaza-tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0484] ##STR00097## ##STR00098##

Step 1

Benzyl (R)-2-cyclopropyl-2-hydroxy acetate 18a

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

[0485] Compound 2a (7.4 g, 63.7 mmol) was dissolved in 200 mL of acetonitrile, and potassium carbonate (35 g, 253.6 mmol), benzyl bromide (9.3 g, 54.4 mmol) and tetrabutylammonium iodide (500 mg, 1.36 mmol) were added successively. The reaction mixture was stirred at room temperature for 16 h and filtered through celite, and the filter cake was rinsed with ethyl acetate (10 mL). The filtrates were combined and concentrated under reduced pressure, and the resulting residue (4.1 g) was purified by silica gel column chromatography with developing solvent system C and further by chiral resolution to give the title products 18a (1.1 g) and 18b (1.2 g).

Step 2

Benzyl (R)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-di oxo-2,9-di oxa-4,7-diazaundecan-11-oate 18c

[0486] Compound 8b (3.1 g, 8.41 mmol) was dissolved in tetrahydrofuran (55 mL), and compound 18a (2.0 g, 9.70 mmol) was added. The mixture was cooled to 0-5° C. in an ice-water bath, and potassium tert-butoxide (1.89 g, 16.84 mmol) was added. The mixture was stirred in the ice-water bath for 10 min. Ethyl acetate (30 mL) and water (20 mL) were added, and the resulting mixture was let stand to form layers. The aqueous phase was extracted with chloroform (30 mL×5), and the organic phases were combined, and concentrated under reduced pressure. The resulting residue was dissolved in 1,4-dioxane (32 mL) and water (8 mL), and sodium carbonate (1.78 g, 16.79 mmol) and 9-fluorenylmethyl chloroformate (2.18 g, 8.42 mmol) were added. The resulting mixture was stirred at room temperature for 2 h. Water (30 mL) was added, followed by extraction with ethyl acetate (50 mL×3). The organic phases were combined, 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 with developing solvent system C to give the title product 18c (1.3 g, 30.0% yield).

[0487] MS m/z (ESI): 515.2 [M+1].

Step 3

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

[0488] Compound 18c (1.29 g, 2.51 mmol) was dissolved in ethyl acetate (15 mL), and palladium on carbon (260 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 5 h and filtered through celite, and the filter cake was rinsed with ethyl acetate (20 mL) and methanol (20 mL). The filtrate was concentrated to give the crude title product 18d (980 mg), which was directly used in the next step without purification.

[0489] MS m/z (ESI): 425.1 [M+1].

Step 4

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

[0490] Crude 18d (980 mg, 2.31 mmol) was dissolved in dichloromethane (15 mL), and 2,4-dimethoxybenzyl alcohol (777 mg, 4.62 mmol), 1-ethyl-(3-dimethylaminopropyl) carbonyldiimine hydrochloride (664 mg, 3.46 mmol) and 4-dimethylaminopyridine (28 mg, 0.23 mmol) were added. The reaction mixture was stirred at room temperature for 1 h, and concentrated under reduced pressure to remove the organic solvent. 20 mL of water was added, followed by extraction with ethyl acetate (50 mL×3). The organic phases were combined, 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 with developing solvent system C to give the title product 18e (810 mg, 61.1% yield).

[0491] MS m/z (ESI): 575.0 [M+1].

Step 5

2,4-Dimethoxybenzyl(R)-2-(((2-aminoacetylamino)methoxy)-2-cyclopropylacetate 18f

[0492] Compound 18e (33 mg, 57.4 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 18f (21 mg), which was directly used in the next step without purification.

Step 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-oate 18g

[0493] Crude 18f (21 mg, 57.4 μmol) was dissolved in 3 mL of N,N-dimethylformamide, and compound 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 mixture was stirred at room temperature for 1 h. 10 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 18g (37 mg, 77.1% yield).

[0494] MS m/z (ESI): 853.0 [M+18].

Step 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

[0495] Compound 18g (37 mg, 44.3 μmol) was dissolved in 1.4 mL of a 3% (v/v) solution of dichloroacetic acid in dichloromethane, and the solution was cooled to 0-5° C. in an ice-water bath, followed by addition of triethylsilane (15.4 mg, 132.4 μmol). The reaction mixture was stirred in the ice bath for 3 h. The reaction mixture was concentrated under reduced pressure in the ice bath to remove half of the organic solvent, and 5 mL of diethyl ether was added. The resulting mixture was warmed to room temperature naturally and slurried to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 18h (24 mg, 79.1% yield).

[0496] MS m/z (ESI): 708.2 M+23].

Step 8

(9H-fluoren-9-yl)methyl((2R,10S)-10-benzyl-2-cyclopropyl-1-(((1S,9S)-9-ethyl 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-tetraazahexadec-16-ylicarbamate 18i

[0497] Compound 1b (30 mg, 63.6 μmol) was added to a reaction flask, and 1 mL of a 10% (v/v) solution of methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine. The mixture was stirred until compound 1b was dissolved. Compound 18h (65 mg, 94.8 μmol) was dissolved in 1 mL of a 10% (v/v) solution of methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (27 mg, 97.6 μmol). The reaction mixture was warmed to room temperature and stirred for 1 h. 10 mL of dichloromethane and 5 mL of water were added, and the resulting mixture was stirred for 5 min, and let stand to form layers. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 18i (25 mg, 35.6% yield).

[0498] MS m/z (ESI): 1104.4 [M+1].

Step 9

(S)-2-(2-(2-aminoacetyl amino)acetylamino)-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-1H,12H-benzo[de]pyrano[3 ‘,4’: 6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-oxoethoxy)methyl)amino)-2-oxoethoxy)-3-phenylpropanamide 18j

[0499] Compound 18i (12 mg, 10.9 μmol) was dissolved in 0.6 mL of dichloromethane, and 0.3 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.5 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 18j (10 mg), which was directly used in the next step without purification.

[0500] MS m/z (ESI): 881.0 [M+1].

Step 10

(1 r,4 r)-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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-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-pentaaza-tetrahexadec yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 18

[0501] Crude 18j (10 mg) was dissolved in 1 mL of N,N-dimethylformamide, and compound 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 mixture was stirred at room temperature for 30 min and filtered. The filtrate was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 18 (9.5 mg, 56.2% yield).

[0502] MS m/z (ESI): 1570.2 [M+18].

[0503] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-19

(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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-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-pentaaza-tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0504] ##STR00099## ##STR00100##

Step 1

Benzyl (S)-10-cyclopropyl-1-(9H-fluoren-9-yl)-3,6-di oxo-2,9-di oxa-4,7-diazaundecan-11-oate 19a

[0505] Compound 18b (252 mg, 1.22 mmol) was added to a reaction flask, and 4 mL of dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice water bath, followed by addition of lithium tert-butoxide (98 mg, 1.22 mmol). The reaction mixture was stirred in the ice water bath for 15 min and became clear, and 8b (300 mg, 814.3 μmol) was added. The reaction mixture was stirred in the ice-water bath for 2.5 h. Water (10 mL) was added for liquid separation. The aqueous phase was extracted with dichloromethane (8 mL×2), and the organic phases were combined and washed with water (10 mL×1) and saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and filtered and concentrated to give the crude product. The resulting residue was purified by silica gel column chromatography with developing solvent system C to give the title product 19a (282 mg, 67.2% yield).

Step 2

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

[0506] Compound 19a (280 mg, 0.554 mmol) was dissolved in 8 mL of ethyl acetate, and palladium on carbon (84 mg, 10% loading, dry basis) was added. The system was purged with hydrogen three times, and the reaction mixture was stirred at room temperature for 3 h and filtered through celite, and the filter cake was rinsed with ethyl acetate. The filtrate was concentrated to give the crude title product 19b (230 mg), which was directly used in the next step without purification.

Step 3

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

[0507] Crude 19b (230 mg, 541.8 μmol) was dissolved in 7 mL of dichloromethane, and 2,4-dimethoxybenzyl alcohol (136.7 mg, 812.7 μmol), 1-ethyl-(3-dimethylaminopropyl)carbonyldiimine hydrochloride (155 mg, 808.5 μmol) and 4-dimethylaminopyridine (6.6 mg, 53.5 μmol) were added successively. The reaction mixture was stirred at room temperature for 16 h, diluted with 10 mL of dichloromethane, washed with water (10 mL×1) and saturated brine (10 mL×2), dried over anhydrous sodium sulfate, and filtered and concentrated to give the crude product. The resulting residue was purified by thin layer chromatography with developing solvent system B to give the title product 19c (159 mg, 51.0% yield).

Step 4

2,4-Dimethoxybenzyl(S)-2-((2-aminoacetylamino)methoxy)-2-cyclopropyl acetate 19d

[0508] Compound 19c (60 mg, 104.4 μmol) was dissolved in 1 mL of dichloromethane, and 0.5 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 19d (21 mg), which was directly used in the next step without purification.

Step 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-pentaazaeicosa-20-oate 19e

[0509] Crude 19d (36 mg, 102.2 μmol) was dissolved in 4 mL of N,N-dimethylformamide, and compound 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 mixture was stirred at room temperature for 1 h. 10 mL of water was added, followed by extraction with ethyl acetate (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 19e (70 mg, 80.2% yield).

Step 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-pentaazaeicosa-20-oic acid 19f

[0510] Compound 19e (70 mg, 83.7 μmol) was dissolved in 2.5 mL of a 3% (v/v) solution of dichloroacetic acid in dichloromethane, and the solution was cooled to 0-5° C. in an ice-water bath, followed by addition of triethylsilane (29 mg, 249.4 μmol). The reaction mixture was stirred in the ice bath for 3 h. The reaction mixture was concentrated under reduced pressure in an ice bath to remove half of the organic solvent, and 5 mL of diethyl ether was added. The resulting mixture was warmed to room temperature naturally and slurried to precipitate a white solid, and filtered. The filter cake was collected and dried with an oil pump to give the title product 19f (57 mg, 99.2% yield).

Step 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-tetraazahexadec-16-yl)carbamate 19g

[0511] Compound 1b (30 mg, 63.6 μmol) was added to a reaction flask, and 1 mL of a 10% (v/v) solution of methanol in dichloromethane was added. The system was purged with argon three times, and the mixture was cooled to 0-5° C. in an ice-water bath, followed by addition of a drop of triethylamine. The mixture was stirred until compound 1 b was dissolved. Compound 19f (57 mg, 83.1 μmol) was dissolved in 1 mL of a 10% (v/v) solution of methanol in dichloromethane, and then the above reaction mixture was added dropwise, followed by addition of 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride (26 mg, 93.9 μmol). The reaction mixture was warmed to room temperature and stirred for 1 h. 10 mL of dichloromethane and 5 mL of water were added, and the resulting mixture was stirred for 5 min, and let stand to form layers. The organic phase was collected, and the aqueous phase was extracted with dichloromethane (10 mL×3). The organic phases were combined, 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 with developing solvent system B to give the title product 19g (56 mg, 79.8% yield).

[0512] MS m/z (ESI): 1103.1 [M+1].

Step 8

(S)-2-(2-(2-aminoacetylamino)acetylamino)-N-(2-((((S)-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)-3-phenylpropanamide 19h

[0513] Compound 19g (4.6 mg, 4.16 μmol) was dissolved in 1.5 mL of dichloromethane, and 0.75 mL of diethylamine was added. The reaction mixture was stirred at room temperature for 1.6 h and concentrated under reduced pressure. 2 mL of toluene was added, followed by concentration under reduced pressure; the procedures were repeated twice. The residue was slurried with 3 mL of n-hexane, and the upper n-hexane layer was removed; the procedures were repeated three times. The slurry was concentrated under reduced pressure to give the crude title product 19h (4.0 mg), which was directly used in the next step without purification.

Step 9

(1 r,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-1H,12H-benzo[de]pyrano[3′,4′:6,7]indolizino[1,2-b]quinolin-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-pentaaza-tetrahexadec-46-yl)-4-((2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)methyl)cyclohexane-1-carboxamide 19

[0514] Crude 19h (4.0 mg) was dissolved in 1 mL of N,N-dimethylformamide, and compound 17g (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 mixture was stirred at room temperature for 40 min and filtered. The filtrate was purified by high performance liquid chromatography (separation conditions: chromatography column: XBridge Prep C18 OBD 5 μm 19×250 mm; mobile phase: A-water (10 mmol of NH.sub.4OAc), B-acetonitrile, gradient elution, flow rate: 18 mL/min). The corresponding fractions were collected and concentrated under reduced pressure to give the title product 19 (2.1 mg, 32.4% yield).

[0515] .sup.1H NMR (400 MHz, DMSO-d.sub.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 1-20 (Reference Example)

[0516] ##STR00101##

[0517] The title compound 20 was prepared as disclosed in “Example 58 on page 163 of the specification of Patent CN104755494A”.

[0518] The following antibodies can be prepared using a conventional preparation method for antibodies, and can be obtained by, for example, vector construction, transfection of eukaryotic cells such as HEK293 cells (Life Technologies Cat. No. 11625019), and expression purification.

[0519] The following is the sequence of trastuzumab:

TABLE-US-00004 Light chain SEQ ID NO: 1 DIQMTQSPSSLSASVGDRVTITCRASQDVNTAVAWYQQKPGKAPKLLIY SASFLYSGVPSRFSGSRSGTDFTLTISSLQPEDFATYYCQQHYTTPPTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Heavy chain SEQ ID NO: 2 EVQLVESGGGLVQPGGSLRLSCAASGFNIKDTYIHWVRQAPGKGLEWVA RIYPTNGYTRYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCSR WGGDGFYAMDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGC LVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSL GTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFL FPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKP REEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAK GQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPEN NYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQ KSLSLSPGK

[0520] The following is the sequence of pertuzumab:

TABLE-US-00005 Light chain SEQ ID NO: 3 DIQMTQSPSSLSASVGDRVTITCKASQDVSIGVAWYQQKPGKAPKLLIY SASYRYTGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYYIYPYTF GQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQ WKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEV THQGLSSPVTKSFNRGEC Heavy chain SEQ ID NO: 4 EVQLVESGGGLVQPGGSLRLSCAASGFTFTDYTMDWVRQAPGKGLEWVA DVNPNSGGSIYNQRFKGRFTLSVDRSKNTLYLQMNSLRAEDTAVYYCAR NLGPSFYFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

[0521] The following is the sequence of B7H3 antibody 1F9DS:

TABLE-US-00006 Light chain SEQ ID NO: 5 DTVVTQEPSFSVSPGGTVTLTCGLSSGSVSTSHYPSWYQQTPGQAPRML IYNTNTRSSGVPDRFSGSILGNKAALTITGAQADDESDYYCAIHVDRDI WVFGGGTKLTVLGQPKANPTVTLFPPSSEELQANKATLVCLISDFYPGA VTVAWKADGSPVKAGVETTKPSKQSNNKYAASSYLSLTPEQWKSHRSYS CQVTHEGSTVEKTVAPTEC Heavy chain SEQ ID NO: 6 QVQLVQSGGGVVQPGTSLRLSCAASGFIFSSSAMHWVRQAPGKGLEWVA VISYDGSNKYYVDSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAR SARLYASFDYWGQGALVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCL VKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLG TQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLF PPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKG QPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENN YKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQK SLSLSPGK

Example 1-21. ADC-1

[0522] ##STR00102##

[0523] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

[0524] Compound 10-shorter retention time compound (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, and the solution was added to the above 2.0 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-1 of FADC-1 general formula in PBS buffer (5.0 mg/mL, 1.1 mL), which was then stored at 4° C.

[0525] Mean calculated by UV-HPLC: n=5.09.

Example 1-22. ADC-2

[0526] ##STR00103##

[0527] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

[0528] Compound 10-longer retention time compound (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, and the solution was added to the above 2.0 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-2 of FADC-1 general formula in PBS buffer (4.95 mg/mL, 1.1 mL), which was then stored at 4° C.

[0529] Mean calculated by UV-HPLC: n=7.39.

Example 1-23. ADC-3

[0530] ##STR00104##

[0531] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 2.5 mL, 9.96 mg/mL, 0.168 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL, 0.82 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 2.0 mL of the solution was collected for use in the next step.

[0532] Compound 8 (2.1 mg, 2.02 μmol) was dissolved in 0.10 mL of DMSO, and the solution was added to the above 2.0 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-3 of FADC-3 general formula in PBS buffer (5.24 mg/mL, 1.1 mL), which was then stored at 4° C.

[0533] Mean calculated by UV-HPLC: n=7.36.

Example 1-24. ADC-4

[0534] ##STR00105##

[0535] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 3.74 mL, 13.38 mg/mL, 0.338 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 6.7 mg/mL, and 1.3 mL of the solution was collected for use in the next step.

[0536] Compound 9-shorter retention time compound 9-A (1.0 mg, 0.93 μmol) was dissolved in 0.10 mL of DMSO, and the solution was added to the above 1.3 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-4 of FADC-4A general formula in PBS buffer (1.72 mg/mL, 2.36 mL), which was then stored at 4° C.

[0537] Mean calculated by UV-HPLC: n=7.39.

Example 1-25. ADC-5

[0538] ##STR00106##

[0539] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 3.0 mL, 6.70 mg/mL, 0.136 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.067 mL, 0.67 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath, and 0.614 mL of the solution was collected for use in the next step.

[0540] Compound 9-shorter retention time compound 9-A (0.5 mg, 0.42 μmol) was dissolved in 0.031 mL of DMSO, and the solution was added to the above 0.614 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-5 of FADC-4A general formula in PBS buffer (3.08 mg/mL, 0.82 mL), which was then stored at 4° C.

[0541] Mean calculated by UV-HPLC: n=3.16.

Example 1-26. ADC-6

[0542] ##STR00107##

[0543] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 3.74 mL, 13.38 mg/mL, 0.338 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 6.7 mg/mL, and 0.75 mL of the solution was collected for use in the next step.

[0544] Compound 9-longer retention time compound 9-B (0.68 mg, 0.63 μmol) was dissolved in 0.10 mL of DMSO, and the solution was added to the above 0.75 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-6 of FADC-4B general formula in PBS buffer (1.78 mg/mL, 1.78 mL), which was then stored at 4° C.

[0545] Mean calculated by UV-HPLC: n=3.94.

Example 1-27. ADC-7

[0546] ##STR00108##

[0547] To antibody pertuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 1.0 mL of the solution was collected for use in the next step.

[0548] Compound 8 (0.65 mg, 0.6 μmol) was dissolved in 0.1 mL of DMSO, and the solution was added to the above 1.0 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-7 of FADC-7 general formula in PBS buffer (1.42 mg/mL, 2.15 mL), which was then stored at 4° C.

[0549] Mean calculated by UV-HPLC: n=6.91.

Example 1-28. ADC-8

[0550] ##STR00109##

[0551] To antibody pertuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 1.6 mL of the solution was collected for use in the next step.

[0552] Compound 10-shorter retention time compound (1.04 mg, 1.0 μmol) was dissolved in 0.1 mL of DMSO, and the solution was added to the above 1.6 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-8 of FADC-8 general formula in PBS buffer (2.14 mg/mL, 2.31 mL), which was then stored at 4° C.

[0553] Mean calculated by UV-HPLC: n=6.58.

Example 1-29. ADC-9

[0554] ##STR00110##

[0555] To antibody pertuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 5.0 mL, 10 mg/mL, 0.338 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.173 mL, 1.73 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath and diluted to 5.0 mg/mL, and 0.8 mL of the solution was collected for use in the next step.

[0556] Compound 9-shorter retention time compound 9-A (0.55 mg, 0.5 μmol) was dissolved in 0.1 mL of DMSO, and the solution was added to the above 0.8 mL of the solution. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-9 of FADC-9A general formula in PBS buffer (2.27 mg/mL, 1.11 mL), which was then stored at 4° C.

[0557] Mean calculated by UV-HPLC: n=3.16.

Example 1-30. ADC-10

[0558] ##STR00111##

[0559] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.574 mL, 38.78 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 19.76 μL, 197.6 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0560] Compound 14-shorter retention time compound (0.64 mg, 588 nmol) was dissolved in 40 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-10 of FADC-10 general formula in PBS buffer (5.48 mg/mL, 1.03 mL), which was then stored at 4° C.

[0561] Mean calculated by UV-Vis: n=6.25.

Example 1-31. ADC-11

[0562] ##STR00112##

[0563] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.646 mL, 43.64 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 22.24 μL, 222.4 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0564] Compound 14-longer retention time compound (0.72 mg, 662 nmol) was dissolved in 40 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-11 of FADC-10 general formula in PBS buffer (2.13 mg/mL, 1.87 mL), which was then stored at 4° C.

[0565] Mean calculated by UV-Vis: n=7.03.

Example 1-32. ADC-12

[0566] ##STR00113##

[0567] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.726 mL, 49.05 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 25.0 μL, 250.0 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0568] Compound 15 (0.81 mg, 754 nmol) was dissolved in 40 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-12 of FADC-12 general formula in PBS buffer (3.34 mg/mL, 1.45 mL), which was then stored at 4° C.

[0569] Mean calculated by UV-Vis: n=6.93.

Example 1-33. ADC-13

[0570] ##STR00114##

[0571] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.287 mL, 19.39 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 9.88 μL, 98.8 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0572] Compound 16 (0.32 mg, 294 nmol) was dissolved in 20 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-13 of FADC-13 general formula in PBS buffer (2.37 mg/mL, 0.88 mL), which was then stored at 4° C.

[0573] Mean calculated by UV-Vis: n=6.53.

Example 1-34. ADC-14

[0574] ##STR00115##

[0575] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.592 mL, 40.0 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0576] Compound 17 (0.92 mg, 598 nmol) was dissolved in 40 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-14 of FADC-14 general formula in PBS buffer (0.30 mg/mL, 12.0 mL), which was then stored at 4° C.

[0577] Mean calculated by UV-Vis: n=7.61.

Example 1-35. ADC-15

[0578] ##STR00116##

[0579] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.592 mL, 40.0 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 20.38 μL, 203.8 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0580] Compound 18 (0.93 mg, 599 nmol) was dissolved in 40 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-15 of FADC-15 general formula in PBS buffer (0.32 mg/mL, 11.8 mL), which was then stored at 4° C.

[0581] Mean calculated by UV-Vis: n=7.89.

Example 1-36. ADC-16

[0582] ##STR00117##

[0583] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.53 mL, 35.8 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 18.25 μL, 182.5 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0584] Compound 19 (0.83 mg, 534 nmol) was dissolved in 35 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-16 of FADC-16 general formula in PBS buffer (0.32 mg/mL, 12.0 mL), which was then stored at 4° C.

[0585] Mean calculated by UV-Vis: n=7.43.

Example 1-37. ADC-17

[0586] ##STR00118##

[0587] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 2.0 mL, 135.12 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 43.2 μL, 432 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0588] Compound 9-shorter retention time compound 9-A (2.22 mg, 2067 nmol) was dissolved in 175 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-17 of FADC-4A general formula in PBS buffer (1.32 mg/mL, 12.0 mL), which was then stored at 4° C.

[0589] Mean calculated by UV-Vis: n=5.42.

Example 1-38. ADC-18 (Reference Example)

[0590] ##STR00119##

[0591] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 1.5 mL, 101.3 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0592] Compound 20 (2.0 mg, 1934 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-18 of FADC-18 general formula in PBS buffer (0.79 mg/mL, 13.0 mL), which was then stored at 4° C.

[0593] Mean calculated by UV-Vis: n=7.23.

Example 1-39. ADC-19

[0594] ##STR00120##

[0595] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 1.36 mL, 91.9 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 46.9 μL, 469 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0596] Compound 9-shorter retention time compound 9-A (2.0 mg, 1862 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-19 of FADC-4A general formula in PBS buffer (0.73 mg/mL, 13.0 mL), which was then stored at 4° C.

[0597] Mean calculated by UV-Vis: n=6.26.

Example 1-40. ADC-20

[0598] ##STR00121##

[0599] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 1.5 mL, 101.3 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 51.7 μL, 517 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0600] Compound 10-longer retention time compound (2.0 mg, 1815 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-20 of FADC-1 general formula in PBS buffer (0.73 mg/mL, 13.0 mL), which was then stored at 4° C.

[0601] Mean calculated by UV-Vis: n=7.43.

Example 1-41. ADC-21 (Reference Example)

[0602] ##STR00122##

[0603] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 1.86 mL, 125.4 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 63.9 μL, 639 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0604] Compound 20 (2.07 mg, 2001 nmol) was dissolved in 150 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-21 of FADC-18 general formula in PBS buffer (2.91 mg/mL, 4.44 mL), which was then stored at 4° C.

[0605] Mean calculated by UV-Vis: n=7.23.

Example 1-42. ADC-22

[0606] ##STR00123##

[0607] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 1.88 mL, 127.2 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 64.9 μL, 649 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0608] Compound 9-shorter retention time compound 9-A (2.1 mg, 1955 nmol) was dissolved in 150 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-22 of FADC-4A general formula in PBS buffer (3.56 mg/mL, 3.98 mL), which was then stored at 4° C.

[0609] Mean calculated by UV-Vis: n=6.79.

Example 1-43. ADC-23 (Reference Example)

[0610] ##STR00124##

[0611] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 345 mL, 23.31 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.89 mL, 118.9 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3.5 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0612] Compound 20 (362 mg, 350 μmol) was dissolved in 7.12 mL of MeCN and 3.56 mL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was purified by desalting through ultrafiltration membranes with 2% (v/v) MeCN and 1% (v/v) DMSO-containing aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5) and aqueous succinic acid buffer (0.01 M aqueous succinic acid buffer at pH 5.3) successively. Then sucrose was added at 60 mg/mL, and tween 20 at 0.2 mg/mL. The mixture was bottled and lyophilized to give a lyophilized powder sample of exemplary product ADC-23 of FADC-18 general formula, which was then stored at 4° C. Mean calculated by UV-Vis: n=7.05.

Example 1-44. ADC-24

[0613] ##STR00125##

[0614] To antibody trastuzumab in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 332 mL, 22.43 μmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 11.44 mL, 114.4 μmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3.5 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0615] Compound 9-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 the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was purified by desalting through ultrafiltration membranes with 4% (v/v) MeCN and 2% (v/v) DMSO-containing aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5) and aqueous succinic acid buffer (0.01 M aqueous succinic acid buffer at pH 5.3) successively. Then sucrose was added at 60 mg/mL, and tween 20 at 0.2 mg/mL. The mixture was bottled and lyophilized to give a lyophilized powder sample of exemplary product ADC-24 of FADC-4A general formula, which was then stored at 4° C.

[0616] Mean calculated by UV-Vis: n=7.07.

Example 1-45. ADC-25

[0617] ##STR00126##

[0618] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 2.14 mL, 144.60 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 73.7 μL, 740 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0619] Compound 9-shorter retention time compound 9-A (3.0 mg, 2793 nmol) was dissolved in 150 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-25 of FADC-25 general formula in PBS buffer (1.28 mg/mL, 13.0 mL), which was then stored at 4° C.

[0620] Mean calculated by UV-Vis: n=6.87.

Example 1-46. ADC-26 (Reference Example)

[0621] ##STR00127##

[0622] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0623] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-26 of FADC-26 general formula in PBS buffer (1.61 mg/mL, 4.0 mL), which was then stored at 4° C.

[0624] Mean calculated by UV-Vis: n=6.15.

Example 1-47. ADC-27

[0625] ##STR00128##

[0626] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0627] Compound 9-shorter retention time compound 9-A (1.02 mg, 950 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-27 of FADC-25 general formula in PBS buffer (1.94 mg/mL, 3.5 mL), which was then stored at 4° C.

[0628] Mean calculated by UV-Vis: n=6.11.

Example 1-48. ADC-28 (Reference Example)

[0629] ##STR00129##

[0630] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 2.36 mL, 159.47 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 81.3 μL, 810 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0631] Compound 20 (3.0 mg, 2901 nmol) was dissolved in 150 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-28 of FADC-26 general formula in PBS buffer (1.29 mg/mL, 13.0 mL), which was then stored at 4° C.

[0632] Mean calculated by UV-Vis: n=7.46.

Example 1-49. ADC-29

[0633] ##STR00130##

[0634] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.80 mL, 50.06 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 28.6 μL, 290 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0635] Compound 9-shorter retention time compound 9-A (1.29 mg, 1201 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-29 of FADC-25 general formula in PBS buffer (2.63 mg/mL, 2.4 mL), which was then stored at 4° C.

[0636] Mean calculated by UV-Vis: n=7.24.

Example 1-50. ADC-30 (Reference Example)

[0637] ##STR00131##

[0638] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.86 mL, 58.4 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 29.1 μL, 290 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0639] Compound 20 (1.0 mg, 967 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-30 of FADC-26 general formula in PBS buffer (1.61 mg/mL, 4.0 mL), which was then stored at 4° C.

[0640] Mean calculated by UV-Vis: n=6.15.

Example 1-51. ADC-31

[0641] ##STR00132##

[0642] To antibody B7H3 antibody 1F9DS in aqueous PBS buffer (0.05 M aqueous PBS buffer at pH 6.5, 10.0 mg/mL, 0.89 mL, 60.14 nmol) was added at 37° C. a prepared aqueous solution of tris(2-carboxyethyl)phosphine (TCEP) (10 mM, 30.1 μL, 300 nmol). The reaction mixture was shaken on a water bath shaker at 37° C. for 3 h before the reaction was terminated. The reaction mixture was cooled to 25° C. in a water bath.

[0643] Compound 8 (1.0 mg, 943 nmol) was dissolved in 100 μL of DMSO, and the solution was added to the above reaction mixture. The reaction mixture was shaken on a water bath shaker at 25° C. for 3 h before the reaction was terminated. The reaction mixture was desalted and purified through a Sephadex G25 gel column (elution phase: 0.05 M aqueous PBS buffer at pH 6.5, containing 0.001 M EDTA) to give exemplary product ADC-31 of FADC-31 general formula in PBS buffer (1.47 mg/mL, 4.5 mL), which was then stored at 4° C.

[0644] Mean calculated by UV-Vis: n=6.33.

Example 1-52. ADC-32

[0645] ##STR00133##

[0646] At 25° C., in a buffer containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA (pH 5.6), 5.0 g of trastuzumab stock solution (34.44 nmol; trastuzumab was diluted with 20 mM histidine-hydrochloric acid buffer to a final concentration of 15 mg/mL) and 34.64 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 120.84 μmol) were stirred in a water bath at a constant temperature for 3 h to give a solution of intermediate I.

[0647] Compound 9-shorter retention time compound 9-A (406.2 mg, 378.17 μmol) was dissolved in 9.98 mL of DMSO to give a solution of compound 9-A in DMSO. 23.42 mL of DMSO was added to the above solution of intermediate I in advance, and then the above solution of compound 9-A in DMSO was added to the solution of intermediate I to which DMSO had been added in advance. The reaction mixture was stirred in a water bath at 25° C. for 1 h, quenched with cysteine, and filtered. At 25° C., the reaction mixture was subjected to buffer exchange by 10-fold and 16-fold volume equal-volume ultrafiltration through an ultrafiltration membrane (30 kd) with a 10% (v/v) DMSO-containing aqueous buffer of 20 mM histidine-hydrochloric acid and 2.5 mM EDTA (pH=6.0), and an aqueous buffer of 10 mM histidine-hydrochloric acid (pH=5.5), successively, to remove small molecules and residual solvents, and exemplary product ADC-32 of FADC-4A general formula was obtained.

[0648] Mean calculated by HIC: n=6.0.

Example 1-53. ADC-33

[0649] ##STR00134##

[0650] At 37° C., in a buffer containing 20 mM histidine-hydrochloric acid and 2.5 mM EDTA (pH 6.0), 8.0 g of trastuzumab stock solution (55.11 nmol; trastuzumab was diluted with 20 mM histidine buffer to a final concentration of 15 mg/mL) and 123.95 mg of tris(2-carboxyethyl)phosphine hydrochloride (reducing agent TCEP, Sigma, 432.41 μmol) were stirred in a water bath at a constant temperature for 5 h to give a solution of intermediate I.

[0651] Compound 9-shorter retention time compound 9-A (754.7 mg, 702.63 μmol) was dissolved in 15.99 mL of DMSO to give a solution of compound 9-A in DMSO. 37.34 mL of DMSO was added to the above solution of intermediate I in advance, and then the above solution of compound 9-A in DMSO was added to the solution of intermediate I to which DMSO had been added in advance. The reaction mixture was stirred in a water bath at 37° C. for 1 h before the reaction was terminated, and the reaction mixture was filtered. At 25° C., the reaction mixture was subjected to buffer exchange by 8-fold and 16-fold volume equal-volume ultrafiltration through an ultrafiltration membrane (30 kd) with a 10% (v/v) DMSO-containing aqueous buffer of 20 mM histidine-hydrochloric acid and 2.5 mM EDTA (pH=6.0), and an aqueous buffer of 10 mM histidine-hydrochloric acid (pH=6.0), successively, to remove small molecules and residual solvents, and exemplary product ADC-33 of FADC-4A general formula was obtained.

[0652] Mean calculated by HIC: n=7.15.

[0653] Drug Loading Analysis of ADC Stock Solution

[0654] Purpose and Principle of Experiment

[0655] An ADC is an antibody cross-linked drug. Its mechanism for treating diseases is to transport toxin molecules into cells depending on the targeting ability of the antibody and kill the cells. The drug loading plays a decisive role in the drug efficacy. The drug loading in the ADC stock solution was determined by ultraviolet-visible spectrophotometry (UV-Vis) or hydrophobic interaction chromatography (HIC).

[0656] 1. Ultraviolet-Visible Spectrophotometry

[0657] Cuvettes containing sodium succinate buffer were placed into the reference cell and sample cell, and the absorbance of the solvent blank was subtracted. Then, a cuvette containing test solution was placed into the sample cell, and the absorbances at 280 nm and 370 nm were determined.

[0658] Calculation: the loading in the ADC stock solution was determined by ultraviolet spectrophotometry (instrument: Thermo nanodrop 2000 ultraviolet spectrophotometer), based on the principle that the total absorbance value of the ADC stock solution at a certain wavelength was the sum of the absorbance values of the cytotoxic drug and the monoclonal antibody at that wavelength, namely:

A.sub.280 nm=ε.sub.mab-280bC.sub.mab+ε.sub.Drug-280bC.sub.Drug  (1)

[0659] ε.sub.Drug-280: the mean molar extinction coefficient of the drug at 280 nm was 5100;

[0660] C.sub.Drug: the concentration of the drug;

[0661] ε.sub.mab-280: the mean molar extinction coefficient of the trastuzumab stock solution or the pertuzumab stock solution at 280 nm was 214,600;

[0662] C.sub.mab: the concentration of the monoclonal antibody stock solution of trastuzumab or pertuzumab;

[0663] b: the optical path length was 1 cm.

[0664] Similarly, an equation for the total absorbance value of the sample at 370 nm can be given as:

A.sub.370 nm=ε.sub.mab-370bC.sub.mab+ε.sub.Drug-370bC.sub.Drug  (2)

[0665] ε.sub.Drug-370: the mean molar extinction coefficient of the drug at 370 nm was 19,000;

[0666] C.sub.Drug: the concentration of the drug;

[0667] ε.sub.mab-370: the extinction coefficient of the monoclonal antibody stock solution of trastuzumab or pertuzumab at 370 nm was 0;

[0668] C.sub.mab: the concentration of the monoclonal antibody stock solution of trastuzumab;

[0669] b: the optical path length was 1 cm.

[0670] The drug loading can be calculated using both equations (1) and (2) as well as the extinction coefficients of the monoclonal antibody and the drug at both wavelengths and their concentrations.

[0671] Drug loading n=C.sub.Drug/C.sub.mab

[0672] 2. Hydrophobic Interaction Chromatography

[0673] 2.1 HPLC Analysis was Carried Out Under the Following Measuring Conditions:

[0674] HPLC system: Agilent high performance liquid chromatograph HPLC system

[0675] Detector: DAD detector (measurement wavelength: 280 nm)

[0676] Chromatography column: TSKgel Butyl-NPR (4.6 mm×100 mm, 2.5 μm)

[0677] Column temperature: 30° C.

[0678] Flow rate: 0.5 mL/min

[0679] Sample chamber temperature: 4° C.

[0680] Mobile phase A: a pH 7.00 aqueous solution containing 1.5 M ammonium sulfate

[0681] ((NH.sub.4).sub.2SO.sub.4) and 20 mM disodium phosphate (Na.sub.2HPO.sub.4).

[0682] Mobile phase B: a pH 7.00 mixed solution containing 75% 20 mM disodium phosphate (Na.sub.2HPO.sub.4) and 25% isopropanol.

[0683] Gradient program: 0.0% B to 0.0% B (0 mM to 3.0 min), 0.0% B to 100.0% B (3.0 min to 30.0 mM), 100.0% B to 100.0% B (30.0 min to 33.0 min), 0.0% B to 0.0% B (33.1 min to 40.0 min),

[0684] Sample injection amount: 50 μg

[0685] 2.2 Data Analysis

[0686] Based on the current data, because of the characteristics of the column, based on the difference in salt concentrations, the antibody drug conjugate was eluted in ascending order according to the number of bound drugs. Therefore, the distribution of the number of conjugated drugs was obtained by measuring the area of each peak. According to the elution order, the peaks were DO (antibody with no drug linker bound), D2, D4, D6 and D8. The drug loading DAR (n) was calculated as:

TABLE-US-00007 TABLE 1 Drug loading calculation by hydrophobic interaction chromatography Number of Name linked drugs Weighted peak area percentage D 0 0 Peak area of D 0 × number of linked drugs D 2 2 Peak area of D 2 × number of linked drugs D 4 4 Peak area of D 4 × number of linked drugs D 6 6 Peak area of D 6 × number of linked drugs D 8 8 Peak area of D 8 × number of linked drugs Drug loading n = Σ(weighted peak areas)/100

Biological Evaluation

Test Example 1-1: Test for Inhibition of In Vitro Proliferation of Tumor Cells by Compounds Disclosed Herein

[0687] I. Purpose

[0688] This experiment is intended to determine the inhibitory activity of the pharmaceutical compounds of the present disclosure against the in vitro proliferation of U87MG cells (Cell Bank, Chinese Academy of Sciences, Cat #TCHu138) and SK-BR-3 tumor cells (human breast cancer cells, ATCC, Cat #HTB-30). The cells were treated in vitro with a compound at different concentrations. After 6 days of culture, the proliferation of the cells was determined using a CTG (CellTiter-Glo® Luminescent Cell Viability Assay, Promega, Cat #G7573) reagent, and the in vitro activity of the compound was evaluated according to the IC.sub.50 value.

[0689] II. Method

[0690] The method for testing the inhibition of the in vitro proliferation of U87MG cells was described below as an example for the method for assaying for the inhibitory activity of the compounds of the present disclosure against the in vitro proliferation of tumor cells. The method was also applicable to, but not limited to, the test for the inhibitory activity against the in vitro proliferation of other tumor cells.

[0691] 1. Cell culture: U87MG and SK-BR-3 cells were cultured in a 10% FBS-containing EMEM medium (GE, Cat #SH30024.01) and a 10% FBS-containing McCoy's 5A medium (Gibco, Cat #16600-108), respectively.

[0692] 2. Cell preparation: U87MG and SK-BR-3 cells growing at log phase were collected, washed once with PBS (phosphate buffered saline, Shanghai BasalMedia Technologies Co., Ltd.), and then digested with 2-3 mL of trypsin (0.25% Trypsin-EDTA (1×), Gibico, Life Technologies) for 2-3 min; after being fully digested, the cells were eluted with 10-15 mL of a cell culture medium; the eluate was centrifuged at 1000 rpm for 5 min, and the supernatant was discarded; then the cells were resuspended in 10-20 mL of a cell culture medium to form single-cell suspensions.

[0693] 3. Cell plating: the U87MG and SK-BR-3 single-cell suspensions were each well mixed, and their cell densities were adjusted with a cell culture medium to 2.75×10.sup.3 cells/mL and 8.25×10.sup.3 cells/mL, respectively; the density-adjusted cell suspensions were each well mixed and added to a 96-well cell culture plate at 180 μL/well. To each of the peripheral wells of the 96-well plates was added 200 μL of media only. The plates were incubated in an incubator (37° C., 5% CO.sub.2) for 24 h.

[0694] 4. Compound preparation: the compound was dissolved in DMSO (dimethyl sulfoxide, Shanghai Titan Scientific Co., Ltd.) to prepare a stock solution at an initial concentration of 10 mM.

[0695] Small-molecule compounds were at an initial concentration of 500 nM. Drug formulation was performed as follows.

[0696] Different test samples at concentration 100 μM (30 μL) were added to the first column of a 96-well U-bottom plate, and 20 μL of DMSO was added to each well of the second column through the eleventh column. The samples in the first column (10 μL) were added to the 20 μL of DMSO in the second column, and the mixtures were well mixed. The mixtures (10 μL) were added to the third column, and so on to the tenth column. The drugs in the plate (5 μL per well) were transferred to EMEM media (95 μL), and the mixtures were well mixed for later use.

[0697] ADCs were prepared at an initial concentration of 10 nM or 500 nM as follows.

[0698] Different test samples at concentration 100 nM or 5 μM (100 μL) were added to the first column of a 96-well plate, and 100 μL of PBS was added to each well of the second column through the eleventh column. The samples in the first column (50 μL) were added to the 100 μL of PBS in the second column, and the mixtures were well mixed. The mixtures (50 μL) were added to the third column, and so on, by 3-fold dilution, to the tenth column.

[0699] 5. Sample adding: the test samples prepared at different concentrations (20 μL) were added to the culture plate, with two duplicate wells set for each sample. The plate was incubated in an incubator (37° C., 5% CO.sub.2) for 6 days.

[0700] 6. Color developing: the 96-well cell culture plate was taken out, and 90 μL of CTG solution was added to each well, followed by 10 mM of incubation at room temperature.

[0701] 7. Plate reading: the 96-well cell culture plate was taken out and tested in a microplate reader (BMG labtech, PHERAstar FS) for chemiluminescence.

[0702] III. Data Analysis

[0703] Data were processed and analyzed using Microsoft Excel and Graphpad Prism 5. The experimental results are shown in the Table 2 below.

TABLE-US-00008 TABLE 2 IC.sub.50 values of the small-molecule fragments of the present disclosure in inhibiting in vitro proliferation of SK-BR-3 cells and U87 cells IC.sub.50 (nM) Compound No. SK-BR-3 U87 1 0.12 0.23 2-shorter retention time 2-B 0.33 0.86 2-longer retention time 2-A 8.11 2.31 3-shorter retention time 0.36 0.83 3-longer retention time 1.67 2.98 4 1.9  / 5 / 4.81 6 / 1.83 7 / 1.95

[0704] Conclusion: The small-molecule fragments of the present disclosure have significant inhibitory activity against the proliferation of SK-BR-3 cells and U87 cells, and the chiral centers have certain influence on the inhibitory activity of the compounds.

Test Example 1-2: Test for Inhibition of In Vitro Proliferation of HER2-Targeted Tumor Cells by Antibody Drug Conjugates of the Present Disclosure

[0705] This experiment was intended to determine the inhibitory activity of the HER2-targeting antibody drug conjugates of the present disclosure against the in vitro proliferation of SK-BR-3 (human breast cancer cells, ATCC, Cat #HTB-30) and MDA-MB-468 (human breast cancer cells, ATCC, Cat #HTB-132). The cells were treated in vitro with a compound at different concentrations. After 6 days of culture, the proliferation of cells was determined using CTG reagents, and the in vitro activity of the compound was evaluated according to IC.sub.50 value.

[0706] The test method of Test Example 1 was followed. SK-BR-3 and MDA-MB-468 cells were used in the tests, and the cell culture media were a 10% FBS-containing McCoy's 5A medium (Gibco, Cat #16600-108), a 10% FBS-containing EMEM medium (GE, Cat #SH30024.01), and a 10% FBS-containing L-15 medium (ThermoFisher, Cat #11415-114). The cell densities of the three strains of cells were adjusted to 8.33×10.sup.3 cells/mL, 8.33×10.sup.3 cells/mL and 1.39×10.sup.4 cells/mL with cell culture media, respectively, and the density-adjusted cell suspensions were well mixed and added to 96-well cell culture plates at 180 μL/well. The related compounds were tested, and the results are shown in Table 3 below.

TABLE-US-00009 TABLE 3 IC.sub.50 values of the antibody drug conjugates of the present disclosure in inhibiting in vitro proliferation of tumor cells with HER2 targets IC.sub.50 (nM) Compound No. SK-BR-3 MDA-MB-468 ADC-3 0.43 >50 ADC-4 0.30 >50 ADC-6 0.48 >50 ADC-7 0.14 >50 ADC-9 0.95 >50 ADC-10 1.36 >50 ADC-11 0.73 >50 ADC-12 0.82 >50 ADC-13 0.47 >50 ADC-14 0.53 >50 ADC-15 0.38 >50 ADC-16 0.49 >50 ADC-17 0.37 >50

[0707] Conclusion: the HER2-targeting antibody drug conjugates of the present disclosure showed significant inhibitory activity against the proliferation of HER2-positive SK-BR-3 cells while showed weak inhibitory activity against the proliferation of HER2-negative MDA-MB-468 cells, exhibiting good selectivity.

Test Example 1-3: Plasma Stability Test for Her2-ADC

[0708] Samples ADC-19, ADC-18 and ADC-20, human plasma, monkey plasma (Shanghai Medicilon Inc.) and a solution of 1% BSA (Sigma) in PBS (Sangon Biotech (Shanghai)) were each filtered through a 0.22 μm filter for sterilization. ADC-19, ADC-18 and ADC-20 were each added to the sterile plasma or 1% BSA solution in PBS at a final concentration of 200 μg/mL. The resulting mixture was incubated in a cell incubator at 37° C. The day of incubation was defined as day 0. Samples were taken on days 7, 14 and 21 and tested for the free toxin.

[0709] 25 μL of samples were taken out to a 96-well plate; 50 μL of an internal standard working solution (100 ng/mL camptothecin solution in acetonitrile) and 150 μL of acetonitrile were added; the mixture was vortexed for 5 min and centrifuged for 10 min (4000 rpm), and 5 μL of the resulting samples were used for LC/MS/MS (Applied Biosystems, USA) analysis.

[0710] The results showed that: ADC-19 was fairly stable in both human and monkey plasma, as well as a solution of 1% BSA in PBS, with a release rate of free toxin of no more than 2.1% at the highest, and tended to be stable on day 14, see FIG. 1A.

[0711] ADC-18 was poorly stable in human and monkey plasma with release rates of free toxin of 14.5% and 8.10% at the highest, respectively. It was relatively stable in 1% BSA solution in PBS, see FIG. 1B.

[0712] ADC-20 was poorly stable in human plasma, monkey plasma, and a solution of 1% BSA in PBS, with release rates of free toxin of 21.7%, 29.7% and 21.7% at the highest, respectively. It was in a degraded state in the solution of 1% BSA in PBS, see FIG. 1C.

Test Example 1-4: Evaluation of Drug Efficacy on JIMT-1 Tumor-Bearing Mice

[0713] I. Purpose

[0714] With nu/nu nude mice used as test animals, Her2-ADC antibodies T-DM1, ADC-21 and

[0715] ADC-24 were administered by intraperitoneal injection, and then their efficacy was evaluated in trastuzumab (Herceptin)-resistant human breast cancer cell strain JIMT-1 xenograft tumor nude mice.

[0716] II. Test Drugs and Materials

[0717] 1. Test drugs

[0718] T-DM1 (prepared by reference US20050169933)

[0719] ADC-21: 3 mg/kg

[0720] ADC-21: 10 mg/kg

[0721] ADC-24: 3 mg/kg

[0722] ADC-24: 10 mg/kg

[0723] Blank control (Blank): PBS

[0724] 2. Preparation method: all prepared by dilution in PBS.

[0725] 3. Test animals

[0726] nu/nu nude mice, purchased from Beijing Vital River.

[0727] III. Method

[0728] Mice were inoculated subcutaneously on the right flank with JIMT-1 cells (Nanjing Cobioer) (5×10.sup.6 cells/mouse, with 50% artificial basement membrane). After 8 days, the tumors grew to a size of 203.09±11.94 mm.sup.3, and the animals were randomized into 6 groups of 8 (d1).

[0729] The drug was administrated by intraperitoneal injection for a total of 2 times. The tumor volumes and body weights were measured twice a week and the results were recorded. Excel 2003 statistical software was used. The mean values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT; and the inter-group difference P-values were calculated as TTEST.

[0730] Tumor volume (V) was calculated as: V=1/2×L.sub.long×L.sub.short.sup.2

[0731] Relative volume (RTV)=V.sub.T/V.sub.0

[0732] Tumor inhibition rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

[0733] Where V.sub.0 and V.sub.T are the tumor volumes at the beginning and end of the experiment, respectively. C.sub.RTV and T.sub.RTV are the relative tumor volumes of the blank group (Vehicle, PBS) and the experimental groups, respectively, at the end of the experiment.

[0734] IV. Results

[0735] The experimental results are shown in FIG. 2. The experiment ended after two intraperitoneal injections were performed and then observation was performed for 34 days. T-DM1 (10 mg/kg) had no inhibition effect on tumors; 3 mg/kg of ADC-21 had the tumor inhibition rate of 46.22% (P<0.01); 10 mg/kg of ADC-21 had the tumor inhibition rate of 56.77% (P<0.001); 3 mg/kg of ADC-24 had the tumor inhibition rate of 62.77% (P<0.001); 10 mg/kg of ADC-24 had the tumor inhibition rate of 76.32% (P<0.001). Under the condition of the same dosage, the tumor inhibition effect of the ADC-24 was significantly better than that of the ADC-21.

Test Example 1-5: Evaluation of Drug Efficacy on SK-BR-3 Tumor-Bearing Mice

[0736] I. Purpose

[0737] With nu/nu nude mice used as test animals, Her2-ADC antibodies ADC-21 and ADC-22 were administered by intraperitoneal injection, and then their efficacy was evaluated in human breast cancer cell SK-BR-3 xenograft tumor nude mice.

[0738] II. Test Drugs and Materials

[0739] 1. Test drugs

[0740] ADC-21: 1 mg/kg

[0741] ADC-21:6 mg/kg

[0742] ADC-22: 1 mg/kg

[0743] ADC-22: 6 mg/kg

[0744] Blank control (Blank): PBS.

[0745] 2. Preparation method: all prepared by dilution in PBS.

[0746] 3. Test animals

[0747] nu/nu nude mice, purchased from Beijing Vital River.

[0748] III. Method

[0749] Mice were inoculated subcutaneously on the right flank with SK-BR-3 cells (ATCC) (5×10.sup.6 cells/mouse, with 50% artificial basement membrane). After 20 days, the tumors grew to a size of 153.34±11.73 mm.sup.3, and the animals were randomized into 5 groups of 8 (d0).

[0750] The drug was administrated by intraperitoneal injection for a total of 1 time. The tumor volumes and body weights were measured twice a week and the results were recorded. Excel 2003 statistical software was used. The mean values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT; and the inter-group difference P-values were calculated as TTEST.

[0751] Tumor volume (V) was calculated as: V=1/2×L.sub.long×L.sub.short.sup.2

[0752] Relative volume (RTV)=V.sub.T/V.sub.0

[0753] Tumor inhibition rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

[0754] Where V.sub.0 and V.sub.T are the tumor volumes at the beginning and end of the experiment, respectively. C.sub.RTV and T.sub.RTV are the relative tumor volumes of the blank control group and the experimental groups, respectively, at the end of the experiment.

[0755] IV. Results

[0756] The experimental results are shown in FIG. 3. The experiment ended after one intraperitoneal injection was performed and then observation was performed for 28 days. 1 mg/kg of ADC-21 had the tumor inhibition rate of 15.01%; 6 mg/kg of ADC-21 had the tumor inhibition rate of 77.4%, and had significant difference compared with that of the blank control (P<0.001). 1 mg/kg of ADC-22 had the tumor inhibition rate of 19.82%; 6 mg/kg of ADC-22 had the tumor inhibition rate of 98.38% (P<0.001). Under the condition of the same dosage of 6 mg/kg, the tumor inhibition effect of the ADC-22 was significantly better than that of the ADC-21.

Test Example 1-6: Plasma Stability

[0757] Sample ADC-25 was well mixed with each of human plasma, monkey plasma and 1% BSA solution in PBS at a final concentration of 100 μg/mL, and then the mixtures were sterilized by filtration and incubated in a 37° C. water bath. The day of incubation was defined as day 0. Samples were taken on days 7, 14 and 21 and tested for the free toxin. Samples at different time points were taken out, left to cool to room temperature, and well mixed by vortexing. 25 μL of each of the samples was added to a 96-well plate, followed by the addition of 50 μL of an internal standard working solution (100 ng/mL camptothecin solution in acetonitrile) and 150 μL of acetonitrile. The resulting mixtures were vortexed for 5 min and centrifuged at 4000 rpm for 10 min. 5 μL of the supernatant was collected for LC/MS/MS analysis.

[0758] The results showed that: ADC-25 was fairly stable in both human and monkey plasma, as well as a solution of 1% BSA in PBS, with a release rate of free toxin of no more than 2% at the highest, and tended to be stable on day 14, see FIG. 4.

Test Example 1-7: Evaluation of Efficacy of ADCs Against Human Brain Astrocytoma U87MG Nude Mouse Xenograft Tumor

[0759] I. Purpose

[0760] With BALB/cA-nude mice used as test animals, the efficacy of the ADC compounds of the present disclosure against human brain astrocytoma U87MG nude mouse xenograft tumors was evaluated in this experiment.

[0761] II. Test Drugs and Materials

[0762] 1. Test drugs

[0763] ADC-27 (3 mg/kg)

[0764] ADC-26 (3 mg/kg)

[0765] Blank control (Blank): PBS buffer at pH 7.4.

[0766] 2. Preparation method: PBS buffer at pH 7.4.

[0767] 3. Test animals

[0768] BALB/cA-nude mice: purchased from Shanghai Jiesijie Laboratory Animal Co., Ltd.

[0769] III. Method

[0770] Six to seven-week-old laboratory female BALB/cA-nude mice were inoculated subcutaneously with human brain astrocytoma U87MG cells (human brain astrocytoma, Cell Bank, Chinese Academy of Sciences, Cat #TCHu138). On the tenth day after inoculation of the cells, the animals were randomly grouped (DO), 8 animals per group, administered by intraperitoneal injection once a week for a total of 3 times, and tumor volumes and body weights were measured 2-3 times per week and the data were recorded.

[0771] Tumor volume (V) was calculated as follows:

V=1/2×a×b.sup.2

[0772] where a and b represent length and width, respectively.

[0773] Relative volume (RTV)=V.sub.T/V.sub.0

[0774] Tumor inhibition rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

[0775] Where V.sub.0 and V.sub.T are the tumor volumes at the beginning and end of the experiment, respectively. C.sub.RTV and T.sub.RTV are the relative tumor volumes of the control group (Blank) and the experimental groups, respectively, at the end of the experiment.

[0776] IV. Results

[0777] Intraperitoneal injection (i.p.) administration was given once a week for a total of 3 times; after observation for 22 days, 3 mg/kg of ADC-27 had the tumor inhibition rate of 63.3% (P<0.0001), and 3 mg/kg of ADC-26 had the inhibition rate of 49.1%. ADC-27 showed a stronger anti-tumor efficacy than ADC-26.

[0778] The weights of all animals in each group were normal in the administration process, and the ADCs had no obvious toxic or side effect. The results are shown in Table 4 and FIG. 5. The detected antibodies could effectively inhibit the growth of U87MG xenograft tumor in tumor-bearing nude mice, and showed dose dependency.

TABLE-US-00010 TABLE 4 Efficacy of administered antibodies against human brain astrocytoma U87MG nude mouse xenograft tumors (D22) Relative Tumor tumor inhibition Mean tumor volume (mm.sup.3) volume rate (%) Group Day 0 SEM Day 22 SEM Day 22 SEM Day 22 Blank 167.06 17.74 2906.96 327.6 17.76 1.63 — control ADC-27 167.07 16.06 1172.48 80.27 7.55 0.95 63.3*** 3 mpk ADC-26 167.73 17.63 1561.03 303.37 8.83 1.17 491*** 3 mpk ***indicates P < 0.001

Test Example 1-8: Evaluation of Efficacy of ADCs Against Pleural Effusion Metastatic Human Pharyngeal Cancer Cell Detroit 562 Nude Mouse Xenograft Tumors

[0779] I. Purpose

[0780] With BALB/cA-nude mice used as test animals, and the efficacy of the ADC compounds of the present disclosure against pleural effusion metastatic human pharyngeal cancer cell Detroit 562 nude mouse xenograft tumors was evaluated in this experiment.

[0781] II. Test Drugs and Materials

[0782] 1. Test drugs

[0783] ADC-29 (3 mg/kg)

[0784] ADC-28 (3 mg/kg)

[0785] Negative control ADC (3 mg/kg): antibody drug conjugate formed by coupling non-B7H3-targeted antibodies to compound 20.

[0786] 2. Preparation method: all prepared by dilution in PBS.

[0787] 3. Test animals

[0788] BALB/cA-nude mice: purchased from Changzhou Cavens Laboratory Animal Co., Ltd.

[0789] III. Method

[0790] Six to seven-week-old laboratory female BALB/cA-nude mice were inoculated subcutaneously with pleural effusion metastatic human pharyngeal cancer cells Detroit 562 cells (ATCC, Cat #ATCC® CCL-138™). On the tenth day after inoculation of the cells, the animals were randomly grouped (DO), 8 animals per group, administered by intraperitoneal injection once a week for a total of 3 times, and tumor volumes and body weights were measured 2-3 times per week and the data were recorded. Tumor volume (V) was calculated as follows:

V=1/2×a×b.sup.2

[0791] where a and b represent length and width, respectively.

[0792] Relative volume (RTV)=V.sub.T/V.sub.0

[0793] Tumor inhibition rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

[0794] Where V.sub.0 and V.sub.T are the tumor volumes at the beginning and end of the experiment, respectively. C.sub.RTV and T.sub.RTV are the relative tumor volumes of the control group (negative control) and the experimental groups, respectively, at the end of the experiment.

[0795] IV. Results

[0796] The drugs were administered by intraperitoneal injection once a week, and a total of 3 injections were administered. On day 28 of observation, tumor inhibition rates of the test ADCs ADC-29 3 mg/kg (3 mpk) and ADC-28 3 mg/kg mg/kg (3 mpk) reached 72.27% (P<0.001) and 56.2% (P<0.001), respectively. ADC-29 showed a stronger anti-tumor efficacy than ADC-28.

[0797] The weights of all animals in each group were normal in the administration process, and the ADCs had no obvious toxic or side effect. The results are shown in Table 5 and FIG. 6. The detected antibodies could effectively inhibit the growth of Detroit 562 xenograft tumor in tumor-bearing nude mice, and showed dose dependency.

TABLE-US-00011 TABLE 5 Efficacy of administered antibodies against Detroit 562 xenograft tumors in tumor-bearing nude mice (D28) Relative Tumor Mean tumor Mean tumor tumor inhibition volume (mm.sup.3) volume (mm.sup.3) volume rate (%) Group Day 0 SEM Day 28 SEM Day 28 SEM Day 28 Negative 182.70 6.79 1317.99 223.20 7.47 1.46 — control ADC-29 182.59 6.50  381.48 105.76 2.07 0.58 72.27*** 3 mpk ADC-28 182.57 6.92  578.07 160.13 3.43 1.09 56.2*** 3 mpk ***indicates P < 0.001

Test Example 1-9: Evaluation of Drug Efficacy on U87-MG Tumor-Bearing Mice

[0798] I. Purpose

[0799] With Balb/c nude mice used as test animals, the efficacy of the B7H3-antibody drug conjugate after intraperitoneal injection was evaluated on a human glioblastoma cell U87MG xenograft tumor model of the mice.

[0800] II. Test drugs and materials

[0801] 1. Test drugs

[0802] ADC-30 1 mg/kg

[0803] ADC-30 3 mg/kg

[0804] ADC-31 1 mg/kg

[0805] ADC-31 3 mg/kg

[0806] Blank control (Blank): PBS

[0807] 2. Preparation method: all prepared by dilution in PBS.

[0808] 3. Test animals

[0809] BALB/cA-nude mice: purchased from Shanghai Slac Laboratory Animal Co., Ltd.

[0810] III. Method

[0811] Mice were inoculated subcutaneously on the right flank with U87MG cells (human brain astrocytoma, Cell Bank, Chinese Academy of Sciences, Cat #TCHu138) (2.5×10.sup.6 cells/mouse). After 14 days, the tumors grew to a size of 167.49 mm.sup.3, and the animals were randomized into 5 groups of 8 (d1).

[0812] The drug was administrated by intraperitoneal injection once a week, and a total of 3 injections were given. The tumor volumes and body weights were measured twice a week and the results were recorded.

[0813] Excel 2003 statistical software was used. The mean values were calculated as avg; the SD values were calculated as STDEV; the SEM values were calculated as STDEV/SQRT; and the inter-group difference P-values were calculated as TTEST.

[0814] Tumor volume (V) was calculated as: V=1/2×L.sub.long×L.sub.short.sup.2

[0815] Relative volume (RTV)=V.sub.T/V.sub.0

[0816] Tumor inhibition rate (%)=(C.sub.RTV−T.sub.RTV)/C.sub.RTV (%)

[0817] Where V.sub.0 and V.sub.T are the tumor volumes at the beginning and end of the experiment, respectively. C.sub.RTV and T.sub.RTV are the relative tumor volumes of the blank control group (Vehicle) and the experimental groups, respectively, at the end of the experiment.

[0818] IV. Results

[0819] The experimental results are shown in FIG. 7. The drugs were administrated by intraperitoneal injection once a week, and a total of 3 injections were administered. On day 18 of observation, tumor inhibition rates of test ADCs ADC-30 1 mg/kg, ADC-30 3 mg/kg, ADC-31 1 mg/kg and ADC-31 3 mg/kg reached 0.31%, 45.23% (P<0.0001), 39.22% (P<0.01) and 80.24% (P<0.0001), respectively. Under the condition of the same dosage, the tumor inhibition effect of the ADC-31 was significantly better than that of the ADC-30.

[0820] II. Formulation

[0821] The equipment used in the formulation preparation and determination and the calculation method for results are shown below.

[0822] SEC molecular exclusion chromatography:

[0823] This is a method for analyzing the separation of absolute by the relative relationship between the pore size of the gel pores and the size of the polymer sample molecule coil. SEC % (SEC monomer content percentage)=A monomer/A total×100% (A monomer represents the peak area of the main peak monomer in the sample, and A total represents the sum of all peak areas).

[0824] Instrument for SEC measurement: Agilent 1260; column: waters, XBrige BEH200 Å SEC (300×7.8 mm 3.5 μm).

[0825] CE capillary gel electrophoresis:

[0826] This is a method of moving the gel into a capillary as a supporting medium for electrophoresis and separating according to the molecular weight of the sample under a certain voltage.

[0827] Reduced CE purity percentage=A main peak/A total×100% (A main peak represents the light chain main peak area+the heavy chain main peak area, and A total represents the sum of all peak areas).

[0828] Instrument for CE measurement: Beckman model plus800

[0829] Turbidity measurement:

[0830] The degree to which light is hindered from passing through a water layer indicates the ability of the water layer to scatter and absorb light. It is related not only to the suspended matter content but also to the particle composition, size and shape as well as the reflection performance of its surface. In comparing the absorption values of the same protein sample at the same concentration at the same wavelength (within near ultraviolet and visible wavelength regions), the greater the absorption value is, the greater the turbidity is, the more the protein molecules in the sample tend to aggregate. The instrument for measurement was a multifunctional microplate reader (Molecular Devices M5). Samples in the same volume were added to a 96-well plate and the absorbance values were read.

[0831] Osmotic pressure measurement:

[0832] The freezing point method was used for measuring the osmotic pressure. The freezing point of a solution is measured by using a high-sensitivity temperature-sensing element on the basis of the proportional relation between the freezing point depression value and the molar concentration of the solution, and then converted into the osmotic pressure through electric quantity. Manufacturer of instrument: Loser, model: OM815.

[0833] Protein concentration determination:

[0834] Because the toxin in the antibody drug conjugate absorbed light at 280 nm, the protein concentration was corrected using the following formulas:

A.sub.280=C.sub.d×ε.sub.280d+C.sub.mab×ε.sub.280mab

A.sub.370=C.sub.d×ε.sub.370d

where Cd represents the toxin concentration, Cmab represents the protein concentration, 6280d represents the extinction coefficient of the toxin at 280 nm, ε280mab represents the extinction coefficient of the protein at 280 nm, 6370d represents the extinction coefficient of the toxin at 370 nm, ε280mab=1.49 mg-1*cm-1*mL, ε280d=5000 (molar extinction coefficient of toxin at 280 nm)/1074.13 (molecular weight of toxin)=4.65 mg-1*cm-1*mL, and ε370d=19000 (molar extinction coefficient of toxin at 370 nm)/1074.13 (molecular weight of toxin)=17.69 mg-1*cm-1*mL; the above extinction coefficients were mass extinction coefficients.

[0835] Instrument for protein concentration measurement: ultraviolet-visible spectrophotometer; model: Nano Drop oneC; optical path length: 1 mm.

[0836] Illustratively, a molecular weight of the naked antibody is 145.181 kDa, and a molecular weight of the toxin is 1074 Da; if the mean DAR value is 5.7, the calculated ADC molecular weight will be 151.317 kDa, which is 1.042 times that of the naked antibody. Illustratively, if the ADC DAR value is 5.7 and the concentration of the naked antibody protein is 20.00 mg/mL, the ADC concentration will be 20.84 mg/mL.

Example 2-1. Screening for Buffer Systems and pH Values for Formulations

[0837] 20 mg/mL (protein concentration based on naked antibody) ADC-33 formulations (containing no saccharide, surfactant and other buffers except for the following buffer systems) were prepared using the following buffer systems:

[0838] 1) 10 mM histidine-sodium acetate, pH 5.0

[0839] 2) 10 mM histidine-sodium acetate, pH 5.5

[0840] 3) 10 mM histidine-sodium acetate, pH 6.0

[0841] 4) 10 mM histidine-sodium acetate, pH 6.5

[0842] 5) 10 mM histidine-histidine hydrochloride, pH 5.5

[0843] 6) 10 mM succinic acid-sodium succinate, pH 5.5

[0844] 7) 10 mM citric acid-sodium citrate, pH 5.5

[0845] Samples were taken for a high-temperature stability study (40° C.) and a shaking study (25° C., 300 rpm). The shaken sample contained 0.1 mg/mL polysorbate 80 (PS80), and the other samples contained 0.4 mg/mL PS80. Each formulation was filtered, and a container was filled with it, plugged and capped. The samples were subjected to the forced degradation experiments described above and examined for appearance, SEC and turbidity.

[0846] The results are shown in Table 6. The results show that where histidine-sodium acetate was used as the buffer, the appearances of the samples gradually worsened, the turbidities increased, and the SEC monomer proportion decreased, as the pH value increased within pH 5.0-6.5. At pH 5.5, the sample containing 10 mM histidine-sodium acetate and the sample containing 10 mM histidine hydrochloride were similar in appearance and SEC %, and were superior to those containing 10 mM succinic acid-sodium succinate or 10 mM citric acid-sodium citrate. Surprisingly, the formulation containing 10 mM succinic acid-sodium succinate was significantly superior in turbidity to the other formulations after the shaking study.

TABLE-US-00012 TABLE 6 pH and buffer system stability results Turbidity No. Conditions Appearance A550 SEC % 1 D 0 Clear and transparent 0.051 95.9 40° C. D 12 Slightly opalescent 0.058 92.0 Shaking D 12 Slightly opalescent 0.055 95.0 2 D 0 Clear and transparent 0.050 95.8 40° C. D 12 Slightly opalescent 0.061 91.6 Shaking D 12 Slightly opalescent 0.056 93.3 3 D 0 Clear and transparent 0.051 95.4 40° C. D 12 Slightly opalescent 0.061 91.0 Shaking D 12 Slightly opalescent 0.060 91.3 4 D 0 Clear and transparent 0.050 94.4 40° C. D 12 A large amount of particles 0.085 89.3 like a haze of smoke Shaking D 12 Slightly opalescent 0.058 87.4 5 D 0 Clear and transparent 0.051 95.5 40° C. D 12 Slightly opalescent 0.065 90.9 Shaking D 12 Slightly opalescent 0.060 93.1 6 D 0 Clear and transparent 0.053 95.7 40° C. D 12 Slightly opalescent, 0.055 89.7 fine particles Shaking D 12 Slightly opalescent 0.053 91.5 7 D 0 Clear and transparent 0.057 95.5 40° C. D 12 Opalescent 0.063 88.8 Shaking D 12 Opalescent 0.059 89.3 Note: in the table, “D” indicates day; for example, D 12 indicates 12 days, and D 0 indicates the start of the experiment; the same applies below.

Example 2-2. Screening for Surfactant Species

[0847] 20 mg/mL (protein concentration based on naked antibody) ADC-32 formulations containing different polysorbate surfactants and each containing 10 mM succinic acid-sodium succinate (pH 5.0) buffer and 80 mg/mL sucrose were prepared. Each formulation was filtered, and a container was filled with it, plugged and capped. The samples were subjected to a high-temperature stability study (40° C.) and a shaking study (25° C., 300 rpm) and examined for appearance, SEC and reduced CE.

[0848] The results are shown in Table 7. There were no significant differences in appearance, SEC and reduced CE between the PS20 (polysorbate 20)-containing formulations and the PS80-containing formulations under different conditions, which indicates that both PS20 and PS80 were able to stabilize ADC-32 effectively.

TABLE-US-00013 TABLE 7 Results of the screening for polysorbate species Poly- sorbate Reduced species Conditions Appearance SEC % CE % PS80 D 0 Clear and transparent 99.1 98.7 0.2 Shaking D 15 Clear and transparent 97.2 98.4 mg/mL 40° C. D 15 Clear and transparent 95.4 98.3 PS20 D 0 Clear and transparent 99.0 98.7 0.2 Shaking D 15 Clear and transparent 97.7 98.7 mg/mL 40° C. D 15 Clear and transparent 95.4 98.2

Examples 2-3. Screening for Surfactant Concentrations

[0849] Samples containing 0, 0.1, 0.2, 0.4 and 0.6 mg/mL PS80 were prepared. Each sample also contained 10 mM succinic acid-sodium succinate (pH 5.0), 80 mg/mL sucrose and 20 mg/mL (protein concentration based on naked antibody) ADC-32. The samples were filtered to remove bacteria and particles and diluted with 0.9% NaCl such that the ADC-32 concentration was 0.2 mg/mL. The dilutions were examined for appearance and insoluble microparticles.

[0850] The results are shown in Table 8. Where the PS80 concentration in the formulation was at 0-0.2 mg/mL, as the concentration increased, the increase in the insoluble microparticles in the dilution became smaller and there were fewer and fewer visible particles. Where the concentration was at 0.2-0.6 mg/mL, there were no differences in insoluble microparticles between the dilutions and the appearances were good.

TABLE-US-00014 TABLE 8 Results of the screening for PS80 concentrations Insoluble PS80 microparticles/100 mL concen- ≥2 ≥10 ≥25 tration Time Appearance μm μm μm 0 mg/mL 0 h Clear and transparent, 8892 458 33 occasional particles 4° C. 24 h Distinct particles 39408 4317 100 0.1 mg/mL 0 h Clear and transparent, 5267 525 17 PS80 occasional particles 4° C. 24 h Clear and transparent, 13342 508 0 occasional particles 0.2 mg/mL 0 h Clear and transparent 2733 308 17 PS80 4° C. 24 h Clear and transparent 4050 283 17 0.4 mg/mL 0 h Clear and transparent 1367 175 17 PS80 4° C. 24 h Clear and transparent 3267 433 8 0.6 mg/mL 0 h Clear and transparent 2933 267 0 PS80 4° C. 24 h Clear and transparent 4167 192 0

Examples 2-4. Screening for Sugar Species

[0851] Different samples containing 80 mg/mL sucrose, trehalose and mannitol were prepared. Each sample also contained 10 mM succinic acid-sodium succinate (pH 5.0), 0.2 mg/mL PS80 and 20 mg/mL (protein concentration based on naked antibody) ADC-32. The samples were filtered to remove bacteria and particles. Each formulation was filtered, and a container was filled with it, plugged and capped. The samples were subjected to a high-temperature stability study (40° C.) and a −35° C./4° C. freezing/thawing cycle study and examined for appearance, SEC and reduced CE.

[0852] The results are shown in Table 9. Under freezing/thawing conditions, sucrose was superior to trehalose and mannitol in appearance. Sucrose and trehalose were superior to mannitol in SEC results. At 40° C., sucrose was slightly superior to trehalose in reduced CE results.

TABLE-US-00015 TABLE 9 Results of the screening for saccharide species Saccharide Reduced species Conditions Appearance SEC % CE % 80 mg/mL D 0 Clear and transparent 99.23 97.44 sucrose 5 freezing/ Clear and transparent 98.88 97.54 thawing cycles 40° C. D 17 Clear and transparent 95.05 98.04 80 mg/mL D 0 Clear and transparent 99.23 97.33 trehalose 5 freezing/ A small amount of fine 98.83 98.36 thawing cycles particles 40° C. D 17 Clear and transparent 95.38 96.50 80 mg/mL D 0 Clear and transparent 99.20 97.55 mannitol 5 freezing/ Distinct fine particles 90.24 96.72 thawing cycles 40° C. D 17 Clear and transparent 95.29 97.28

Examples 2-5. Screening for pH, Antibody Drug Conjugate Concentrations and Polysorbates

[0853] With 10 mM succinic acid-sodium succinate as the buffer and 80 mg/mL sucrose as the stabilizer, formulas were designed with respect to the pH, ADC-32 protein concentration (based on the naked antibody) and the polysorbate concentration, as shown in Table 10. A high-temperature stability study (40° C.), a lighting study (4° C., 4500 Lx) and a −35° C./4° C. freezing/thawing cycle study were conducted, and the results were statistically analyzed using the least square method with SEC and reduced CE as evaluation indexes. The results are shown in Table 11 and FIG. 8. The formulations of the present disclosure are shown to be highly pH-dependent. Under lighting, shaking and 40° C. conditions, as the pH increased, there was a greater decrease in SEC; similarly, under lighting conditions, as the pH increased, reduced CE increased, and there was a greater decrease in the monomer peak, which suggests that the formulations of the present disclosure should be at low pH values to have improved stability. Under shaking conditions, as the PS80 concentration increased, there was a downward trend toward SEC. There were no differences in the effects PS80 had on SEC and reduced CE under the other conditions. As the ADC-32 protein concentration increased, there was a greater decrease in the SEC monomer peak at 40° C. There were no differences in the effects the ADC-32 protein concentration had on SEC and reduced CE under the other conditions.

TABLE-US-00016 TABLE 10 Formula designs for screening experiments ADC-32 protein concentration PS80 (mg/mL, based on naked Formula No. pH (10.sup.−4 g/mL) antibody) 1 5.0 1.7 10 2 5.2 1 30 3 5.5 3 10 4 4.8 2 30 5 4.8 1 20 6 5.5 1 10 7 5.5 2 30 8 4.8 3 16 9 5.5 2 20 10 5.2 3 30 11 5.2 2 20 12 5.2 2 20

TABLE-US-00017 TABLE 11 Results of the screening for formulas Re- SEC ΔSEC duced ΔReduced No. Conditions Appearance % % CE % CE % 1 D0 Clear and transparent 99.2 0.0 99.0 0.0 Lighting D10 Clear and transparent, 88.9 10.3 91.5 7.5 yellowish Shaking D15 Clear and transparent 97.8 1.4 99.0 0.0 40° C. D15 Clear and transparent 96.0 3.2 97.9 1.1 2 D0 Clear and transparent 98.9 0.0 98.7 0.0 Lighting D10 Clear and transparent, 79.4 19.5 90.5 8.2 yellowish Shaking D15 Clear and transparent 96.3 2.6 99.1 −0.4 40° C. D15 Clear and transparent 94.7 4.2 97.9 0.7 3 D0 Clear and transparent 98.9 0.0 98.7 0.0 Lighting D10 Clear and transparent, 77.2 21.7 87.2 11.5 yellowish Shaking D15 Clear and transparent 96.9 2.0 97.5 1.2 40° C. D15 Clear and transparent 95.1 3.8 98.2 0.5 4 D0 Clear and transparent 99.0 0.0 98.7 0.0 Lighting D10 Clear and transparent, 86.3 12.7 91.4 7.3 yellowish Shaking D15 Clear and transparent 97.7 1.3 98.8 −0.1 40° C. D15 Clear and transparent 95.3 3.7 98.1 0.6 5 D0 Clear and transparent 99.1 0.0 98.7 0.0 Lighting D10 Clear and transparent, 90.0 9.1 92.1 6.7 yellowish Shaking D15 Clear and transparent 97.5 1.6 98.4 0.4 40° C. D15 Clear and transparent 95.7 3.4 98.0 0.7 6 D0 Clear and transparent 98.9 0.0 98.9 0.0 Lighting D10 Clear and transparent, 76.2 22.8 86.9 12.0 yellowish Shaking D15 Clear and transparent 95.0 4.0 98.5 0.4 40° C. D15 Clear and transparent 95.2 3.7 98.1 0.8 7 D0 Clear and transparent 98.7 0.0 98.3 0.0 Lighting D10 Clear and transparent, 68.2 30.5 86.8 11.4 yellowish Shaking D15 Clear and transparent 96.1 2.6 98.9 −0.6 40° C. D15 Clear and transparent 94.3 4.4 97.7 0.6 8 D0 Clear and transparent 99.2 0.0 99.0 0.0 Lighting D10 Clear and transparent, 88.3 10.9 91.8 7.2 yellowish Shaking D15 Clear and transparent 98.1 1.0 98.9 0.1 40° C. D15 A small amount of 95.8 3.4 98.5 0.5 small particles 9 D0 Clear and transparent 98.9 0.0 97.6 0.0 Lighting D10 Clear and transparent, 73.8 25.1 87.1 10.5 yellowish Shaking D15 Clear and transparent 96.3 2.6 97.7 −0.1 40° C. D15 Clear and transparent 94.7 4.1 97.1 0.5 10 D0 Clear and transparent 98.9 0.0 98.1 0.0 Lighting D10 Clear and transparent, 80.9 18.0 90.4 7.7 yellowish Shaking D15 Clear and transparent 97.3 1.6 99.0 −1.0 40° C. D15 Clear and transparent 94.8 4.2 98.5 −0.5 11 D0 Clear and transparent 99.1 0.0 98.7 0.0 Lighting D10 Clear and transparent, 80.5 18.6 88.7 10.0 yellowish Shaking D15 Clear and transparent 97.2 1.9 98.4 0.3 40° C. D15 Clear and transparent 95.4 3.7 98.3 0.3 12 D0 Clear and transparent 99.0 0.0 98.7 0.0 Lighting D10 Clear and transparent, 78.2 20.8 88.4 10.3 yellowish Shaking D15 Clear and transparent 97.1 1.9 99.1 −0.4 40° C. CD15 Clear and transparent 95.4 3.6 97.9 0.8

Examples 2-6. Lyophilization Process Development

[0854] A 20 mg/mL (protein concentration based on naked antibody) ADC-33 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the lyophilization process parameters 1 (see Table 12). The lyophilization sample was in the form of a white powder cake with a flat surface and contained 1.05% water. The bottom of the powder cake was slightly depressed in the center. The sample was reconstituted with water for injection, and the pH value was measured. The results are shown in Table 13. The ionic strength (≥10 mM) was high enough for buffering. The formula included 80 mg/mL sucrose, which could meet the iso-osmotic requirement.

TABLE-US-00018 TABLE 12 Lyophilization process parameters 1 Lyophilization Temperature Time Retention Degree of process setting setting time vacuum parameter (° C.) (min) (h) (Pa) Pre-freezing 5 10 1 N/A Pre-freezing −45 50 2 N/A Primary drying −25 120 40 10 Secondary drying 25 60 8 1

TABLE-US-00019 TABLE 13 Final formula pH and osmolality results Osmotic pH of pH of pressure of stock pH after buffer stock solution solution reconstitution 5.00 298 mosm 5.06 5.06

[0855] A 20 mg/mL (protein concentration based on naked antibody) ADC-33 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the lyophilization process parameters 2 (see Table 14). The surface of the powder cake was flat and showed no wrinkles and depressions. 0.89% water was contained. The lyophilization process could meet the requirement for product quality.

TABLE-US-00020 TABLE 14 Lyophilization process parameters 2 Temperature Time Retention Degree of Lyophilization setting setting time vacuum process parameter (° C.) (min) (h) (Pa) Pre-freezing −5 10 1 N/A Pre-freezing −45 40 2 N/A Primary drying −20 120 35 10 Secondary drying 25 60 8 1

[0856] A 20 mg/mL (protein concentration based on naked antibody) ADC-32 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered, bottled and then lyophilized according to the lyophilization process parameters 3 (see Table 15). The surface of the powder cake was flat and showed no depressions. The bottom edge of the powder cake slightly shrank. 1.17% water was contained. The lyophilization process could substantially meet the requirement for product quality.

TABLE-US-00021 TABLE 15 Lyophilization process parameters 3 Temperature Time Retention Degree of Lyophilization setting setting time vacuum process parameter (° C.) (min) (h) (Pa) Pre-freezing −5 10 1 N/A Pre-freezing −45 40 2 N/A Primary drying −15 120 30 10 Secondary drying 25 60 9 1

Examples 2-7. Long-Term Stability Data

[0857] A 20 mg/mL (protein concentration based on naked antibody) ADC-32 stock solution containing 10 mM succinic acid-sodium succinate pH 5.0, 80 mg/mL sucrose and 0.2 mg/mL PS80 was prepared, filtered and bottled, and then pilot batches of finished products of the stock solution were formulated according to the lyophilization process parameters 2, subjected to long-term storage at 2-8° C., reconstituted and then tested. The results are shown in Table 16. Compared to the products on DO, those under 2-8° C. M3 conditions showed SEC, reduced CE and free toxin levels within acceptable ranges (SEC %≥93%; reduced CE %≥95%; free toxin level ≤330 ppm). The SEC slightly decreased by 0.5%, while both the reduced CE and free toxin level remained unchanged.

TABLE-US-00022 TABLE 16 Long-term stability data results Batch Reduced No. Conditions SEC % CE % Free toxin P1927 D 0 97.6 99.3 <5 ppm 2~8° C. M 3 97.2 99.2 <5 ppm P1929 D 0 97.8 99.3 <5 ppm 2~8° C. M 3 97.2 99.2 <5 ppm Note: in the table, “M” represents month; for example, M 3 represents 3 months.