CAMPTOTHECIN COMPOUND, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

Abstract

The present invention relates to a Camptothecin compound having anti-tumor activity, a preparation method therefor, and an application thereof. Specifically, the present invention relates to a compound as shown below or a pharmaceutically acceptable form thereof, a pharmaceutical composition thereof, a preparation method therefor, and a use thereof. The compound can be used as a drug for treating diseases in abnormal cell proliferation,

Claims

1. A compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof, wherein the compound has the structure shown below: ##STR00221## wherein, R.sub.1 and R.sub.2 are each independently selected from the group consisting of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxyl, C.sub.1-6 haloalkyl, hydroxyl, cyano and C.sub.3-6 cycloalkyl; or, R.sub.1 and R.sub.2 are connected with adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocyclic ring; R.sub.3 is hydrogen or is connected with an ortho-carbon atom of R.sub.1 to form a 6-membered carbocyclic ring; A is selected from one of ##STR00222## R.sub.4 is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocyclyl; R.sub.5 and R.sub.6 are each independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.1-6 alkoxyalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocyclyl, aryl and heteroaryl; or R.sub.5 and R.sub.6 are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic or heterocyclic ring; m=1 or 2.

2. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure of Formula (I): ##STR00223## in the above Formula (I), R.sup.x is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocyclyl; R.sup.y and R.sup.z are not hydrogen at the same time, and are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, C.sub.1-6 alkylaminoalkyl, C.sub.1-6 alkoxyalkyl, 3- to 6-membered heterocyclylalkyl and 3- to 6-membered heterocyclyl.

3. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure of Formula (II): ##STR00224## in the above Formula (II), A is selected from one of ##STR00225## R.sup.x is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocyclyl; R.sup.y and R.sup.z are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocyclyl, 3- to 6-membered heterocyclylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl and heteroaryl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered ring.

4. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure of Formula (III): ##STR00226## in the above Formula (III), A is selected from one of ##STR00227## R.sup.x is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocyclyl; R.sup.y and R.sup.z are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocyclyl, 3- to 6-membered heterocyclylalkyl, 4- to 6-membered heterocyclyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl and heteroaryl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered ring.

5. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure of Formula (IV): ##STR00228## in the above Formula (IV), R.sup.a and R.sup.b are independently selected from the group consisting of hydrogen, halogen, C.sub.1-6 alkyl, C.sub.1-6 hydroxyalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkoxy, C.sub.1-6 haloalkyl, hydroxyl and cyano; or R.sup.a and R.sup.b are connected with adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocyclic ring; R.sup.c and R.sup.d are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocyclyl, 3- to 6-membered heterocyclylalkyl, C.sub.2-6 alkenyl and C.sub.2-6 alkynyl, or R.sup.c and R.sup.d are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic or heterocyclic ring; R.sup.c is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl and C.sub.2-C.sub.5 heterocyclyl; q=0 or 1; when q=0, R.sup.c and R.sup.d are not hydrogen at the same time.

6. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure of Formula (V): ##STR00229## in the above Formula (V), R is selected from the group consisting of C.sub.3-6 cycloalkyl and C.sub.1-6 alkoxy; A is selected from one of ##STR00230## R.sup.x is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.3-6 cycloalkyl and 3- to 6-membered heterocyclyl; R.sup.y and R.sup.z are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.3-6 cycloalkyl, 3- to 6-membered heterocyclyl, 3- to 6-membered heterocyclylalkyl, C.sub.2-6 alkenyl, C.sub.2-6 alkynyl, aryl and heteroaryl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered ring.

7. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, wherein the compound has the structure as follows: ##STR00231## ##STR00232## ##STR00233## ##STR00234## ##STR00235## ##STR00236## ##STR00237## ##STR00238## ##STR00239## ##STR00240## ##STR00241## ##STR00242## ##STR00243## ##STR00244## ##STR00245## ##STR00246## ##STR00247## ##STR00248## ##STR00249## ##STR00250## ##STR00251## ##STR00252## ##STR00253## ##STR00254## ##STR00255## ##STR00256## ##STR00257## ##STR00258## ##STR00259## ##STR00260##

8. A compound represented by Formula (VI) or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof, wherein the compound has the structure as follows:
M-L-E-DFormula (VI) wherein, M is a linker moiety of an antibody or antigen-binding fragment thereof; L is a linker connecting the linker moiety M and E; E is a structural fragment connecting L and D; D is a structural fragment of a cytotoxic drug wherein the cytotoxic drug is selected from the compound according to claim 1.

9. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein the compound has the structure as shown below: ##STR00261## ##STR00262## ##STR00263## ##STR00264## ##STR00265## ##STR00266## ##STR00267##

10. A pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, and one or more pharmaceutically acceptable carriers.

11. A kit product, which comprises: a) at least one of the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, or a pharmaceutical composition comprising the same and one or more pharmaceutically acceptable carriers, which is used as a first therapeutic agent; b) optionally at least one additional therapeutic agent as a second therapeutic agent, or a pharmaceutical composition containing the additional therapeutic agent as a second pharmaceutical composition; and c) optionally a packaging and/or instruction for use.

12. (canceled)

13. A method for treating abnormal cell proliferation, comprising a step of administering to an individual in need thereof a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 1, or a pharmaceutical composition comprising the same and one or more pharmaceutically acceptable carriers.

14. (canceled)

15. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 2, wherein R.sup.x is selected from the group consisting of hydrogen and C.sub.1-6 alkyl.

16. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 2, wherein R.sup.x is hydrogen.

17. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 2, wherein R.sup.y and R.sup.z are not hydrogen at the same time, and are independently selected from the group consisting of hydrogen ##STR00268## dimethylaminomethylene, morpholinomethylene and methoxymethylene.

18. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 2, wherein R.sup.y is hydrogen and R.sup.z is selected from the group consisting of ##STR00269## dimethylaminomethylene, morpholinomethylene and methoxymethylene.

19. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 3, wherein R.sup.x is selected from the group consisting of hydrogen and C.sub.1-6 alkyl.

20. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 3, wherein R.sup.y and R.sup.z are independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.3-6 cycloalkyl and C.sub.2-6 alkenyl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered cycloalkyl.

21. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 3, wherein R.sup.y is selected from the group consisting of hydrogen and C.sub.1-6 alkyl, R.sup.z is selected from the group consisting of hydrogen, C.sub.1-6 alkyl and C.sub.3-6 cycloalkyl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered cycloalkyl.

22. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 4, wherein R.sup.x is hydrogen.

23. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 4, wherein R.sup.y and R.sup.z are each independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.3-6 cycloalkyl and vinyl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered ring.

24. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 4, wherein R.sup.y is hydrogen, R.sup.z is selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.3-6 cycloalkyl and vinyl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

25. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.a and R.sup.b are independently selected from the group consisting of hydrogen, halogen and C.sub.1-6 alkyl, or R.sup.a and R.sup.b are connected with adjacent carbon atoms to form a 5- to 6-membered oxygen-containing heterocyclic ring.

26. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.a and R.sup.b are independently selected from the group consisting of hydrogen, fluorine, chlorine and methyl, or R.sup.a and R.sup.b together with the benzene ring connected thereto form ##STR00270## wherein Z is selected from the group consisting of CH.sub.2, CD.sub.2, CH.sub.2CH.sub.2 and CF.sub.2.

27. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.a is methyl, R.sup.b is fluorine, or R.sup.a and R.sup.b together with the benzene ring connected thereto form ##STR00271##

28. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.c and R.sup.d are independently selected from the group consisting of hydrogen, ##STR00272## C.sub.1-6 alkoxyalkyl and C.sub.1-6 alkylaminoalkyl, or R.sup.c and R.sup.d are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

29. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.c is hydrogen, R.sup.d is selected from the group consisting of hydrogen, ##STR00273## methoxyethyl and cyclopropyl, or R.sup.c and R.sup.d are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

30. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 5, wherein R.sup.c is selected from the group consisting of hydrogen and C.sub.1-6 alkyl.

31. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 6, wherein R is selected from the group consisting of methyl, methoxy and cyclopropyl.

32. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 6, wherein R.sup.y and R.sup.z are each independently selected from the group consisting of hydrogen, C.sub.1-6 alkyl, C.sub.1-6 haloalkyl, C.sub.1-6 alkoxyalkyl, C.sub.1-6 alkylaminoalkyl, C.sub.3-6 cycloalkyl and vinyl, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

33. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 6, wherein both R.sup.y and R.sup.z are hydrogen, or R.sup.y and R.sup.z are connected with adjacent carbon atoms to form a 3- to 6-membered carbocyclic ring.

34. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 6, wherein R.sup.x is selected from the group consisting of hydrogen and C.sub.1-6 alkyl.

35. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 6, wherein R.sup.x is hydrogen.

36. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein M is selected from the group consisting of the following structures: ##STR00274##

37. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein M is selected from the group consisting of the following structures: ##STR00275##

38. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein L is a divalent structure constituted of one or more selected from the group consisting of: C.sub.1-6 alkylene, N(R), carbonyl, O, Val, Cit, Phe, Lys, D-Val, Leu, Gly, Ala, Asn, Val-Cit, Val-Ala, Val-Lys, Val-Lys(Ac), Phe-Lys, Phe-Lys(Ac), D-Val-Leu-Lys, Gly-Gly-Arg, Ala-Ala-Asn, Ala-Ala-Ala, Val-Lys-Ala, Gly-Gly-Gly, Gly-Gly-Phe-Gly, Gly-Gly-Gly-Gly-Gly, ##STR00276## ##STR00277## wherein R represents hydrogen, C.sub.1-6 alkyl or (CH.sub.2CH.sub.2O).sub.r-containing alkyl; r is an integer selected from 1 to 10; s is an integer selected from 1 to 10.

39. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein L is selected from the group consisting of the following structures: ##STR00278##

40. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein L is selected from the following structure: ##STR00279##

41. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein E is selected from the group consisting of single bond, NHCH.sub.2, ##STR00280##

42. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein E is NHCH.sub.2.

43. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein the cytotoxic drug is selected from the group consisting of ##STR00281## ##STR00282## ##STR00283## ##STR00284## ##STR00285## ##STR00286## ##STR00287## ##STR00288## ##STR00289## ##STR00290## ##STR00291## ##STR00292## ##STR00293## ##STR00294## ##STR00295## ##STR00296## ##STR00297## ##STR00298## ##STR00299## ##STR00300## ##STR00301## ##STR00302## ##STR00303## ##STR00304## ##STR00305## ##STR00306## ##STR00307## ##STR00308##

44. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein D is a structural fragment formed by removing a hydrogen atom from the cytotoxic drug.

45. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 43, wherein D is a structural fragment formed by removing a hydrogen atom from the cytotoxic drug.

46. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein D is selected from the group consisting of the following structures: ##STR00309## ##STR00310## ##STR00311## ##STR00312##

47. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein D is selected from the group consisting of the following structures: ##STR00313##

48. The compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, wherein the compound is selected from: ##STR00314##

49. A pharmaceutical composition, which comprises the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, and one or more pharmaceutically acceptable carriers.

50. A kit product, which comprises: a) at least one of the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, or a pharmaceutical composition comprising the same and one or more pharmaceutically acceptable carriers, which is used as a first therapeutic agent; b) optionally at least one additional therapeutic agent as a second therapeutic agent, or a pharmaceutical composition containing the additional therapeutic agent as a second pharmaceutical composition; and c) optionally a packaging and/or instruction for use.

51. A method for treating abnormal cell proliferation, comprising a step of administering to an individual in need thereof a therapeutically effective amount of the compound or a pharmaceutically acceptable salt, ester, stereoisomer, polymorph, solvate, nitrogen oxide, isotope-labeled product, metabolite or prodrug thereof according to claim 8, or a pharmaceutical composition comprising the same and one or more pharmaceutically acceptable carriers.

Description

SPECIFIC MODELS FOR CARRYING OUT THE INVENTION

[0530] The present invention is further illustrated through the description of specific embodiments, but this is not a limitation to the present invention. Those skilled in the art can make various modifications or improvements according to the teaching of the present invention without departing from the basic idea and scope of the present invention.

[0531] Abbreviations in the present invention have the following meanings:

TABLE-US-00001 Abbre- viation Full name Abbreviation of the of the of the reagents reagents reagents Full name of the reagents NBS N- HATU N,N,N,N-Tetramethyl-O-(7- Bromosuccinimide azabenzotriazol-1-yl)urea hexafluorophosphate DMF- N,N- DIPEA Diisopropylethylamine DMA Dimethylformamide dimethylacetal THF Tetrahydrofuran DMSO Dimethyl sulfoxide DMF N,N- Pd/C Palladium on carbon Dimethylformamide LCMS Combination of Py Pyridine liquid chromatography and mass spectrometry FBS Fetal bovine serum CCK8 2-(2-Methoxy-4-nitrophenyl)- 3-(4-nitrophenyl)-5-(2,4- disulfobenzene)- 2H-tetrazole monosodium salt

[0532] The structures of the compounds described in the following Examples were confirmed by nuclear magnetic resonance (.sup.1H NMR) or mass spectrometry (MS).

[0533] The instrument for nuclear magnetic resonance (.sup.1H NMR) was Bruker 400 MHz nuclear magnetic resonance instrument; hexadeuteriodimethylsulfoxide (DMSO-d.sub.6); the internal standard substance was tetramethylsilane (TMS).

[0534] Abbreviations in nuclear magnetic resonance (NMR) spectra in Examples were shown below.

[0535] s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet, br: broad, J: coupling constant, Hz: Hertz, DMSO-d.sub.6: deuterated dimethylsulfoxide. The ? values were expressed in ppm.

[0536] The instrument for mass spectrometer (MS) was Agilent (ESI) mass spectrometer, Agilent 6120B.

Example 1

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

[0537] ##STR00180##

[0538] The compound 2-hydroxypent-3-ynoic acid (4.29 mg, 37.63 ?mol) was dissolved in DMF (1 mL), added with HATU (21.46 mg, 56.44 ?mol), SM1-1 (10.00 mg, 22.94 ?mol) and DIPEA (7.29 mg, 56.44 ?mol), and reacted at 25? C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high performance liquid chromatography (conditions as follows) to obtain the title compounds 1-1-A (3.24 mg) and 1-1-B (3.98 mg).

[0539] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0540] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00002 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 30 70 28 18 90 10 28

[0541] Retention time: 1-1-A: 10.8 min; 1-1-B: 11.1 min.

[0542] The structural characterization data of 1-1-A were as follows:

[0543] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.58 (d, J=8.4 Hz, 1H), 7.78 (d, J=11.2 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 6.17 (d, J=6.0 Hz, 1H), 5.58-5.47 (m, 1H), 5.42 (s, 2H), 5.23 (s, 2H), 4.73-4.64 (m, 1H), 3.25-3.06 (m, 2H), 2.39 (s, 3H), 2.27-2.05 (m, 2H), 1.96-1.77 (m, 5H), 0.87 (t, J=7.2 Hz, 3H).

[0544] ESI-MS (m/z): 532.2 [M+H].sup.+.

[0545] The structural characterization data of 1-1-B were as follows:

[0546] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.62 (d, J=8.8 Hz, 1H), 7.78 (d, J=10.8 Hz, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 6.19 (d, J=6.0 Hz, 1H), 5.58-5.47 (m, 1H), 5.42 (s, 2H), 5.21 (d, J=4.8 Hz, 2H), 4.73-4.65 (m, 1H), 3.27-3.06 (m, 2H), 2.39 (s, 3H), 2.27-2.05 (m, 2H), 1.93-1.80 (m, 2H), 1.80 (d, J=2.0 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H).

[0547] ESI-MS (m/z): 532.2 [M+H].sup.+.

Example 2

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

[0548] ##STR00181##

[0549] The compound ethylene glycolic acid (9.61 mg, 94.07 ?mol) was dissolved in DMF (2 mL), added with HATU (44.70 mg, 117.58 ?mol), compound SM1-1 (25.00 mg, 0.047 mmol) and DIPEA (24.30 mg, 188.13 ?mol), and reacted at 25? C. for 2 hours. The reaction solution was concentrated under reduced pressure, and the concentrate was directly purified by preparative high performance liquid chromatography to obtain the title compounds 1-7-A (4.00 mg) and 1-7-B (1.38 mg).

[0550] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0551] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00003 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 30 70 28 2 30 70 28 18 90 10 28

[0552] Retention time: 1-7-A: 8.7 min; 1-7-B: 9.1 min.

[0553] The structural characterization data of 1-7-A were as follows:

[0554] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.51 (s, J=8.8 Hz, 1H), 7.76 (s, J=10.8 Hz, 1H), 7.29 (s, 1H), 6.52 (s, 1H), 6.15-6.04 (m, 1H), 5.58-5.49 (m, 1H), 5.42 (s, 2H), 5.39 (s, 1H), 5.24-5.01 (m, 3H), 4.54 (s, J=4.9 Hz, 1H), 3.24-3.05 (m, 2H), 2.37 (s, 3H), 2.16 (s, 2H), 1.91-1.79 (m, 2H), 0.87 (t, J=7.2 Hz, 3H).

[0555] ESI-MS (m/z): 520.1 [M+H].sup.+.

[0556] The structural characterization data of 1-7-B were as follows:

[0557] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.48 (s, 1H), 7.78 (s, 1H), 7.30 (s, 1H), 6.12-5.92 (m, 1H), 5.54-5.47 (m, 1H), 5.42 (s, 2H), 5.37 (dt, J=1.7 Hz, 1H), 5.18 (s, 2H), 5.16-5.14 (m, 1H), 4.5-4.52 (m, 1H), 3.22-3.08 (m, 2H), 2.38 (s, 3H), 2.25-2.16 (m, 1H), 2.16-2.06 (m, 1H), 1.93-1.79 (m, 2H), 0.87 (t, J=7.2 Hz, 3H).

[0558] ESI-MS (m/z): 520.1 [M+H].sup.+.

Example 3

N-((1S,9S)-5-chloro-9-ethyl-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 and N-((1R,9S)-5-chloro-9-ethyl-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

[0559] ##STR00182## ##STR00183## ##STR00184##

Step 1: Synthesis of 1-chloro-3-bromo-2-methyl-5-nitrobenzene

[0560] Compound 2-1-01 (5.00 g, 29.14 mmol) was dissolved in n-heptane (25 mL) at 25? C., added with concentrated sulfuric acid (25 mL), heated to 50? C., added with NBS in batches at 50? C. (6.22 g, 34.97 mmol), reacted at 50? C. for 2 hours, the reaction was monitored by thin layer chromatography (ethyl acetate:petroleum ether=1:10), the reaction solution was cooled to room temperature and added into ice water in dropwise manner, extracted with toluene, the organic phases were combined, washed with sodium sulfite solution, water, and saturated brine, dried over anhydrous sodium sulfate, concentrated under reduced pressure, the crude product was purified by preparative high performance liquid chromatography, and the fractions was lyophilized to obtain 4.88 g of the title compound.

[0561] Chromatographic column: C18 ODS 45 mm?450 mm?8.0 ?m

[0562] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00004 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 60 40 60 10 60 40 60 40 100 0 60

Step 2: Synthesis of 3-chloro-5-bromo-4-methylaniline

[0563] At 25? C., compound 2-1-02 (4.88 g, 19.48 mmol) was dissolved in ethyl acetate (100 mL), added with platinum on carbon (2.00 g, 19.48 mmol, content 5%), subjected to hydrogen replacement, and reacted under the protection of hydrogen balloon at 60? C. for 4 hours, and the reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to obtain 3.68 g of the crude title compound, which was directly used in the next reaction without further purification.

Step 3: Synthesis of N-(3-chloro-5-bromo-4-methylphenyl)acetamide

[0564] At 20? C., compound 2-1-03 (3.63 g, 14.82 mmol) was dissolved in ethyl acetate (70 mL), added with triethylamine (4.50 g, 44.45 mmol) and acetic anhydride (2.27 g, 22.23 mmol), reacted at 20? C. for 20 hours, and the reaction was monitored by LCMS. The reaction solution was added with water, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain a crude product, which was slurried with a mixed solvent of ethyl acetate:petroleum ether=1:5 to obtain 2.86 g of the title compound.

Step 4: Synthesis of (Z)-4-(5-acetamido-3-chloro-2-methylphenyl)but-3-enoic acid

[0565] At 20? C., compound 2-1-04 (1.80 g, 6.86 mmol) was dissolved in THF (20 mL) and water (5 mL), added with vinylacetic acid (708.31 mg, 8.23 mmol), DIPEA (1.95 g, 15.08 mmol), and tris(o-methylphenyl)phosphorus (62.60 mg, 0.20 mmol), the reaction system was subjected to nitrogen replacement, then heated to 70? C. and reacted for 5 hours, and the reaction was monitored by LCMS. The reaction solution was added with 1N sodium hydroxide solution to adjust pH to 8, and ethyl acetate was added for extraction. The aqueous phase was adjusted to pH=3 with 1N hydrochloric acid, extracted with ethyl acetate, and the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.82 g of the title compound, which was directly used in the next reaction.

Step 5: Synthesis of 4-(5-acetamido-3-chloro-2-methylphenyl)butanoic acid

[0566] At 20? C., compound 2-1-05 (2.60 g, 9.71 mmol) was dissolved in THF (50 mL), added with Pd/C (0.52 g, content 10%), the system was subjected to hydrogen replacement, then reacted under the protection of hydrogen balloon at 40? C. for 2 hours, and the reaction was monitored by LCMS.

[0567] The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 2.43 g of the title compound, which was directly used in the next reaction without further purification.

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

[0568] Compound 2-1-06 (2.43 g, 9.01 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5? C., added dropwise with trifluoroacetic anhydride (3.78 g, 18.02 mmol, 2.50 mL), reacted at 5? C. for 4 hours, and the reaction was monitored by LCMS. The reaction solution was added to water, adjusted with ION sodium hydroxide to pH=9, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and purified by flash silica gel column (ethyl acetate: petroleum ether=0-20%) to obtain 1.53 g of the title compound.

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

[0569] At 5? C., potassium tert-butoxide (1.50 g, 13.37 mmol) was dissolved in THF (16 mL) and tert-butanol (4 mL), added dropwise with compound 2-1-07 (1.53 g, 6.08 mmol) in THF solution (16 mL), then added dropwise with amyl nitrite (1.14 g, 9.73 mmol) after 10 minutes, and reacted at 5? C. for 1 hour, the reaction was monitored by LCMS. The reaction solution was adjusted to pH=5 with 1N hydrochloric acid, extracted with ethyl acetate, the combined organic phase was dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was slurried with methyl tert-butyl ether to obtain 1.20 g of the title compound.

Step 8: N-(7-Amino-3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0570] At 20? C., compound 2-1-08 (0.50 g, 1.78 mmol) was dissolved in methanol (8 mL) and 2N hydrochloric acid (8 mL), added with Pd/C (0.15 g, content 10%), the system was subjected to hydrogen replacement, and reacted at 5? C. for 2 hours under the protection of a hydrogen balloon, and the reaction was monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure to obtain 0.52 g of the hydrochloride salt of the title compound, which was directly used in the next reaction without further purification.

Step 9: Synthesis of N,N-(3-chloro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1,7-diyl)diacetamide

[0571] At 20? C., compound 2-1-09 (0.52 g, 1.70 mmol) was dissolved in pyridine (5 mL), added with acetic anhydride (2 mL), reacted at 20? C. for 2 hours, and the reaction was monitored by LCMS. The reaction solution was added to water, extracted with ethyl acetate, the organic phases were washed with water, combined, dried over anhydrous sodium sulfate, concentrated under reduced pressure, and the concentrate was purified by flash silica gel column (ethyl acetate:petroleum ether=0-30%) to obtain 0.22 g of the title compound.

Step 10: Synthesis of N-(8-amino-6-chloro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)acetamide

[0572] At 20? C., compound 2-1-10 (0.45 g, 1.46 mmol) was dissolved in methanol (16 mL), added with 2N hydrochloric acid (16 mL), heated to 60? C. and reacted for 2 hours, and the reaction was monitored by LCMS. The reaction solution was cooled, added with saturated sodium bicarbonate solution to adjust pH=8, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain 0.23 g of the title compound, which was used directly in the next step without further purification.

Step 11: Synthesis of N-((9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10, 13,15-hexahydro-1H,12H-benzopyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)acetamide

[0573] Compound 2-1-11 (0.23 g, 0.78 mmol) was dissolved in toluene (10 mL), added with (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (0.23 g, 0.87 mmol), p-toluenesulfonic acid (26.73 mg, 0.16 mmol), heated to 140? C. and reacted for 5 hours, and the reaction was monitored by LCMS. The reaction solution was concentrated, and the crude product was purified by flash silica gel column (methanol:dichloromethane=0-10%) to obtain 0.15 g of the title compound.

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

[0574] Compound 2-1-12 (40.00 mg, 0.08 mmol) was added into concentrated hydrochloric acid (1 mL), heated to 100? C. and reacted for 5 hours, and the reaction was monitored by LCMS. The reaction solution was filtered, the filtrate was purified by preparative high performance liquid chromatography, and the fractions were lyophilized to obtain 12.00 mg of the trifluoroacetic acid salt of the title compound 2-23.

[0575] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0576] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

TABLE-US-00005 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 5 95 28 2 5 95 28 18 50 50 28

[0577] The structural characterization data were as follows:

[0578] ESI-MS (m/z): 452.1[M+H].sup.+.

Step 13: Synthesis of 2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-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 and 2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-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

[0579] At 25? C., the trifluoroacetic acid salt of compound 2-23 (40.00 mg, 81.91 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with 2-((tert-butyldiphenylsilyl)oxy)acetic acid (30.91 mg, 98.29 ?mol), HATU (62.25 mg, 163.81 ?mol) and N,N-diisopropylethylamine (42.34 mg, 327.63 ?mol), reacted at 25? C. for 0.5 hours, the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was added with water, extracted with dichloromethane/methanol (v/v=10/1), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol=20:1) to obtain two isomers, according to the Rf values, the two isomers were named 2-1-13-A (15.00 mg, Rf value was 0.3) and 2-1-13-B (12.00 mg, Rf value was 0.35).

Step 14: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-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 and N-((1R,9S)-5-chloro-9-ethyl-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

[0580] At 25? C., 2-1-13-A (15.00 mg) and 2-1-13-B (12.00 mg) were dissolved in tetrahydrofuran (1 mL) in two reaction flasks, respectively, added dropwise with a mixed solution of tetrabutyl ammonium fluoride (1M in tetrahydrofuran)/glacial acetic acid (v/v=13/1) (50 uL), reacted at 25? C. for 0.5 hours, and the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography, and the fractions were lyophilized to obtain the title compounds 2-1-A (6.94 mg) and 2-1-B (4.00 mg).

[0581] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0582] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00006 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 3 20 80 28 18 90 10 28

[0583] The structural characterization data of 2-1-A were as follows:

[0584] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.43 (d, J=8.8 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.65-5.36 (m, 4H), 5.21 (q, J=19.0 Hz, 2H), 3.95 (d, J=5.7 Hz, 2H), 3.26-3.11 (m, 2H), 2.53 (s, 3H), 2.30-2.08 (m, 2H), 1.94-1.79 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0585] ESI-MS (m/z): 510.1[M+H].sup.+.

[0586] The structural characterization data of 2-1-B were as follows:

[0587] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.45 (d, J=8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.64-5.35 (m, 4H), 5.19 (q, J=19.0 Hz, 2H), 3.97 (d, J=5.2 Hz, 2H), 3.27-3.10 (m, 2H), 2.51 (s, 3H), 2.27-2.10 (m, 2H), 1.93-1.80 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).

[0588] ESI-MS (m/z): 510.1[M+H].sup.+.

Example 4

[0589] ##STR00185##

[0590] At 25? C., the hydrochloride salt of 2-23 (30.00 mg, 61.43 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with (S)-2-((tert-butyldiphenylsilyl) oxy)propionic acid (24.21 mg, 73.72 ?mol), HATU (35.01 mg, 92.14 ?mol) and N,N-diisopropylethylamine (23.82 mg, 184.29 ?mol), reacted at 25? C. for 1 hour, and the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was added with water, extracted with dichloromethane/methanol (v/v=10/1), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol=15:1) to obtain two isomers, and according to the Rf values, the two isomers were named 2-7-01-A (6.00 mg, Rf value was 0.35) and 2-7-01-B (6.00 mg, Rf value was 0.40).

[0591] At 25? C., 2-7-01-A (6.00 mg, 7.87 ?mol) and 2-7-01-B (6.00 mg, 7.87 ?mol) were dissolved in anhydrous THF (1 mL) in two reaction flasks, respectively, added dropwise with a mixed solution of tetrabutylammonium fluoride (1M in tetrahydrofuran)/glacial acetic acid (v/v=13/1) (50 ?L), reacted at 25? C. for 0.5 hours, and the reaction was monitored with LCMS. When the reaction was completed, the reaction solution was purified by preparative high performance liquid chromatography, and the fractions were lyophilized to obtain the title compounds 2-7-A (2.50 mg) and 2-7-B (3.00 mg).

[0592] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0593] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00007 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 15 85 28 16 90 10 28

[0594] The structural characterization data of 2-7-A were as follows:

[0595] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.38 (d, J=8.8 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.56-5.47 (m, 2H), 5.42 (s, 2H), 5.26-5.11 (m, 2H), 4.17-4.06 (m, 1H), 3.27-3.10 (m, 2H), 2.52 (s, 3H), 2.27-2.08 (m, 2H), 1.86 (tt, J=14.1, 7.3 Hz, 2H), 1.30 (d, J=6.7 Hz, 3H), 0.87 (t, J=7.3 Hz, 3H).

[0596] ESI-MS (m/z): 524.2 [M+H].sup.+.

[0597] The structural characterization data of 2-7-B were as follows:

[0598] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.48 (d, J=9.1 Hz, 1H), 8.12 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.67 (d, J=4.8 Hz, 1H), 5.55 (dd, J=14.6, 7.3 Hz, 1H), 5.43 (s, 2H), 5.24 (d, J=19.0 Hz, 1H), 5.03 (d, J=19.0 Hz, 1H), 4.21-4.08 (m, 1H), 3.28-3.08 (m, 2H), 2.51 (s, 3H), 2.16 (d, J=6.2 Hz, 2H), 1.94-1.82 (m, 2H), 1.42 (d, J=6.8 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H).

[0599] ESI-MS (m/z): 524.2 [M+H].sup.+.

Example 5

(2S)N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2S)N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2R)N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2R)N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide

[0600] ##STR00186##

Step 1: Synthesis of (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropylacetamide and (2S)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropylacetamide and (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropylacetamide and (2R)-2-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropylacetamide

[0601] At 25? C., the hydrochloride salt of 2-23 (30.00 mg, 61.43 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with 2-((tert-butyldiphenylsilyl)oxy)-2-cyclopropylacetic acid (26.13 mg, 73.72 ?mol), HATU (35.01 mg, 92.14 ?mol) and N,N-diisopropylethylamine (23.82 mg, 184.29 ?mol), reacted at 25? C. for 1 hour, the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was added with water, extracted with dichloromethane/methanol (v/v=10/1), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol=15:1) to obtain two isomers, according to the Rf values, the two isomers were named 2-12-01-A (8.00 mg, Rf value was 0.35) and 2-12-01-B (10.00 mg, Rf value was 0.40).

Step 2: Synthesis of (2S)N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2S)N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2R)N-((1S,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide and (2R)N-((1R,9S)-5-chloro-9-ethyl-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-cyclopropyl-2-hydroxyacetamide

[0602] At 25? C., 2-12-01-A (8.00 mg, 10.15 ?mol) and 2-12-01-B (10.00 mg, 12.68 ?mol) were dissolved in anhydrous THF (1 mL) in two reaction flasks, respectively, added dropwise with a mixed solution of tetrabutylammonium fluoride (1M in tetrahydrofuran)/glacial acetic acid (v/v=13/1) (50 ?L), reacted at 25? C. for 0.5 hours, and the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography. The reaction using 2-12-01-A as the raw material gave two isomeric products after the separation, and the corresponding fractions were lyophilized respectively to obtain compound 2-12-A (0.77 mg), and 2-12-B (1.03 mg); while the reaction using 2-12-01-B as the raw material gave two isomeric products after the separation, and the corresponding fractions were lyophilized respectively to obtain compound 2-12-C(2.50 mg), and 2-12-D (1.00 mg). The purification conditions for 2-12-A/2-12-B were as follows:

[0603] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0604] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00008 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

[0605] Peak retention time: 2-12-A: 10.0-11.0 min, 2-12-B: 11.0-12.5 min

[0606] The purification conditions for 2-12-C/2-12-D were as follows:

[0607] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0608] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00009 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

[0609] Peak retention time: 2-12-C: 10.6-11.4 min, 2-12-D: 11.4-12.5 min

[0610] The structural characterization data of 2-12-A were as follows:

[0611] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.38 (d, J=8.9 Hz, 1H), 8.15 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63-5.53 (m, 1H), 5.51 (d, J=5.1 Hz, 1H), 5.42 (s, 2H), 5.28 (d, J=19.2 Hz, 1H), 5.16 (d, J=19.1 Hz, 1H), 3.60 (t, J=5.6 Hz, 1H), 3.28-3.11 (m, 2H), 2.52 (s, 3H), 2.22-2.10 (m, 2H), 1.86 (tt, J=14.1, 7.3 Hz, 2H), 1.23 (d, J=4.9 Hz, 1H), 0.87 (t, J=7.2 Hz, 3H), 0.56-0.36 (m, 4H).

[0612] ESI-MS (m/z): 550.2 [M+H].sup.+.

[0613] The structural characterization data of 2-12-B were as follows:

[0614] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.36 (d, J=8.7 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.57-5.48 (m, 1H), 5.42 (s, 2H), 5.40 (d, J=5.4 Hz, 1H), 5.26 (d, J=19.3 Hz, 1H), 5.18 (d, J=19.0 Hz, 1H), 3.65-3.60 (m, 1H), 3.26-3.12 (m, 2H), 2.52 (s, 3H), 2.26-2.09 (m, 2H), 1.86 (tt, J=14.1, 7.2 Hz, 2H), 1.19-1.08 (m, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.51-0.27 (m, 4H).

[0615] ESI-MS (m/z): 550.2 [M+H].sup.+.

[0616] The structural characterization data of 2-12-C were as follows:

[0617] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.42 (d, J=9.0 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.56 (dd, J=14.8, 6.8 Hz, 1H), 5.52 (d, J=5.2 Hz, 1H), 5.42 (s, 2H), 5.29 (d, J=19.2 Hz, 1H), 5.16 (d, J=19.1 Hz, 1H), 3.62-3.58 (m, 1H), 3.27-3.08 (m, 2H), 2.51 (s, 3H), 2.27-2.08 (m, 2H), 1.87 (tt, J=14.0, 7.2 Hz, 2H), 1.25 (dd, J=13.2, 6.8 Hz, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.64-0.28 (m, 4H).

[0618] ESI-MS (m/z): 550.1 [M+H].sup.+.

[0619] The structural characterization data of 2-12-D were as follows:

[0620] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.38 (d, J=8.7 Hz, 1H), 8.16 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.59-5.49 (m, 1H), 5.43 (d, J=5.3 Hz, 1H), 5.43 (s, 2H), 5.26 (d, J=19.1 Hz, 1H), 5.17 (d, J=19.0 Hz, 1H), 3.64 (t, J=5.8 Hz, 1H), 3.17 (dd, J=15.6, 8.3 Hz, 2H), 2.27-2.07 (m, 2H), 1.94-1.79 (m, 2H), 1.19-1.06 (m, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.49-0.28 (m, 4H).

[0621] ESI-MS (m/z): 550.1 [M+H].sup.+.

Example 6

N-((1S,9S)-5-chloro-9-ethyl-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-hydroxy-2-methylpropylamine and N-((1R,9S)-5-chloro-9-ethyl-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-hydroxy-2-methylpropylamine

[0622] ##STR00187##

[0623] At 25? C., the hydrochloride salt of 2-23 (30.00 mg, 61.43 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with 2-((tert-butyldimethylsilyl)oxy)-2-methylpropionic acid (16.10 mg, 73.72 ?mol), HATU (35.01 mg, 92.14 ?mol) and N,N-diisopropylethylamine (23.82 mg, 184.29 ?mol), reacted at 25? C. for 1 hour, the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was concentrated, and the crude product was purified and separated by preparative high-performance liquid chromatography to obtain two isomers. The fractions were lyophilized respectively, and the two isomers were named 2-17-A (2.65 mg) and 2-17-B (2.69 mg).

[0624] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0625] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00010 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

[0626] The peak retention time: 2-17-A: 9.5-10.2 min, and 2-17-B: 10.4-10.6 min.

[0627] The structural characterization data of 2-17-A were as follows:

[0628] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.36 (d, J=9.1 Hz, 1H), 8.13 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.55-5.45 (m, 2H), 5.42 (s, 2H), 5.28 (d, J=19.0 Hz, 1H), 5.05 (d, J=19.0 Hz, 1H), 3.27-3.11 (m, 2H), 2.51 (s, 1H), 2.24-2.10 (m, 2H), 1.86 (tt, J=14.0, 7.2 Hz, 2H), 1.46 (s, 3H), 1.35 (s, 3H), 0.87 (t, J=7.3 Hz, 3H).

[0629] MS m/z (ESI): 538.2 [M+H].sup.+.

[0630] The structural characterization data of 2-17-B were as follows:

[0631] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.40 (d, J=9.2 Hz, 1H), 8.13 (s, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.60-5.46 (m, 2H), 5.42 (s, 2H), 5.29 (d, J=19.0 Hz, 1H), 5.02 (d, J=19.0 Hz, 1H), 3.28-3.08 (m, 2H), 2.50 (s, 3H), 2.22-2.10 (m, 2H), 1.87 (tt, J=14.2, 7.2 Hz, 2H), 1.47 (s, 3H), 1.35 (s, 3H), 0.88 (t, J=7.3 Hz, 3H).

[0632] MS m/z (ESI): 538.2 [M+H].sup.+.

Example 7

N-((1S,9S)-5-chloro-9-ethyl-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-hydroxycyclopropane-1-carboxamide and N-((1R,9S)-5-chloro-9-ethyl-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-hydroxycyclopropane-1-carboxamide

[0633] ##STR00188##

Step 1: Synthesis of 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-5-chloro-9-ethyl-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 and 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-5-chloro-9-ethyl-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

[0634] At 25? C., the hydrochloride salt of 2-23 (30.00 mg, 61.43 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with 1-((tert-butyldiphenylsilyl)oxy) cyclopropane-1-carboxylic acid (25.10 mg, 73.72 ?mol), HATU (35.01 mg, 92.14 ?mol) and N,N-diisopropylethylamine (23.82 mg, 184.29 ?mol), reacted at 25? C. for 1 hour, the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was added with water, extracted with dichloromethane/methanol (v/v=10/1), the organic phases were combined, dried over anhydrous sodium sulfate, and concentrated under reduced pressure, and the crude product was purified and separated by preparative thin-layer chromatography (dichloromethane:methanol=15:1) to obtain two isomers, according to the Rf values, the two isomers were named 2-20-01-A (4.00 mg, Rf value was 0.30) and 2-20-01-B (4.00 mg, Rf value was 0.35).

Step 2: Synthesis of N-((1S,9S)-5-chloro-9-ethyl-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-hydroxycyclopropane-1-carboxamide and N-((1R,9S)-5-chloro-9-ethyl-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-hydroxycyclopropane-1-carboxamide

[0635] At 25? C., 2-20-01-A (4.00 mg, 5.17 ?mol) and 2-20-01-B (4.00 mg, 5.17 ?mol) were dissolved in anhydrous THF (1 mL) in two reaction flasks respectively, added dropwise with a mixed solution of tetrabutylammonium fluoride (1M in tetrahydrofuran)/glacial acetic acid (v/v=13/1) (50 ?L), reacted at 25? C. for 0.5 hours, and the reaction was monitored by LCMS. When the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography, and the fractions were lyophilized to obtain the title compounds 2-20-A (0.71 mg) and 2-20-B (1.05 mg).

[0636] The purification conditions for 2-20-A were as follows:

[0637] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0638] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00011 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 15 85 28 16 90 10 28

[0639] The purification conditions for 2-20-B were as follows:

[0640] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0641] Mobile phase A: acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00012 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 20 28 18 80 20 28

[0642] The structural characterization data of 2-20-A were as follows:

[0643] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.58 (d, J=9.0 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 6.30 (s, 1H), 5.55 (dd, J=13.2, 8.2 Hz, 1H), 5.43 (s, 2H), 5.26 (d, J=19.0 Hz, 1H), 5.10 (d, J=19.0 Hz, 1H), 3.29-3.09 (m, 2H), 2.52 (s, 3H), 2.31-2.15 (m, 2H), 1.93-1.80 (m, 2H), 1.25-1.14 (m, 2H), 0.98-0.90 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0644] ESI-MS (m/z): 536.2 [M+H].sup.+.

[0645] The structural characterization data of 2-20-B were as follows:

[0646] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.63 (d, J=9.0 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 6.35 (s, 1H), 5.55 (dd, J=13.5, 8.6 Hz, 1H), 5.43 (s, 2H), 5.29 (d, J=19.1 Hz, 1H), 5.08 (d, J=19.1 Hz, 1H), 3.28-3.10 (m, 2H), 2.51 (s, 3H), 2.30-2.14 (m, 2H), 1.93-1.81 (m, 2H), 1.26-1.14 (m, 2H), 1.02-0.90 (m, 2H), 0.89 (d, J=10.9 Hz, 3H).

[0647] ESI-MS (m/z): 536.2 [M+H].sup.+.

Example 8

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

[0648] ##STR00189## ##STR00190##

Step 1: Synthesis of 3-bromo-4-chloro-5-fluoroaniline

[0649] Compound 3-1-01 (2.00 g, 10.53 mmol) was dissolved in N,N-dimethylformamide (30 mL), then slowly added with N-chlorosuccinimide (1.69 g, 12.63 mmol); after the addition, the reaction was carried out at room temperature for 16 hours and monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-25%) to obtain 0.95 g of the title compound.

[0650] The structural characterization data were as follows:

[0651] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 6.77 (dd, J=2.5, 1.4 Hz, 1H), 6.51 (dd, J=11.7, 2.5 Hz, 1H), 5.84 (s, 2H).

Step 2: Synthesis of N-(3-bromo-4-chloro-5-fluorophenyl)acetamide

[0652] Compound 3-1-02 (0.95 g, 4.23 mmol) was dissolved in ethyl acetate (20 mL), and added with acetic anhydride (648.13 mg, 6.35 mmol) under nitrogen protection; after the addition, the temperature was raised to 50? C., the reaction was carried out for 15 hours and monitored by LCMS. The reaction solution was quenched with methanol (5 mL), and directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-40%) to obtain 1.01 g of the title compound.

[0653] The structural characterization data were as follows:

[0654] ESI-MS (m/z): 265.9[M+H].sup.+.

Step 3: Synthesis of (E)-4-(5-acetylamino-2-chloro-3-fluorophenyl)-3-butenoic acid

[0655] Compound 3-1-03 and 3-butenoic acid (387.65 mg, 4.50 mmol) were dissolved in a mixed solvent of 1,4-dioxane (24 mL) and water (8 mL), then added with N,N-diisopropylethylamine (1.45 g, 11.26 mmol), tris(o-methylphenyl)phosphine (114.21 mg, 375.24 ?mol) and palladium acetate (42.12 mg, 187.62 ?mol); after the addition, the reaction system was subjected to nitrogen replacement three times, and heated up to 100? C. to conduct the reaction for 16 hours under nitrogen atmosphere, and the reaction was monitored by LCMS. After the reaction solution was cooled to room temperature, 1N aqueous sodium hydroxide solution (60 mL) and ethyl acetate (50 mL) were added, shaken and layered. After the lower aqueous phase was separated, the pH was adjusted to about 3 with 4 mol/L hydrochloric acid aqueous solution, then subjected to extraction with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

[0656] The structural characterization data were as follows:

[0657] ESI-MS (m/z): 272.0[M+H].sup.+.

Step 4: Synthesis of 4-(5-acetylamino-2-chloro-3-fluorophenyl)butanoic acid

[0658] The crude product of compound 3-1-04 (1.00 g, 3.68 mmol) was dissolved in tetrahydrofuran (15 mL), then added with 10% palladium on carbon (0.10 g), after the addition, the reaction system was subjected to replacement three times with hydrogen balloon, the reaction was carried out under hydrogen atmosphere for 4 hours and monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.00 g of a crude product of the title compound.

[0659] The structural characterization data were as follows:

[0660] ESI-MS (m/z): 274.0[M+H].sup.+.

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

[0661] The crude product of compound 3-1-05 (1.00 g, 3.65 mmol) was dissolved in trifluoroacetic acid (5 mL), cooled to 5? C., then slowly added with trifluoroacetic anhydride (3.84 g, 18.27 mmol, 2.54 mL); after the addition, the reaction was carried out at 5? C. for 2 hours and monitored by LCMS. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column, to obtain 0.43 g of the title compound.

[0662] The structural characterization data were as follows:

[0663] ESI-MS (m/z): 256.1[M+H].sup.+.

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

[0664] Tetrahydrofuran (16 mL) and tert-butanol (4 mL) were added to a reaction flask, cooled to 5? C. in an ice bath, added with potassium tert-butoxide (415.18 mg, 3.70 mmol), then compound 3-1-06 (0.43 mg, 1.68 mmol) was dissolved in tetrahydrofuran (1 mL) and added slowly thereto, followed by an addition of isoamyl nitrite (315.24 mg, 2.69 mmol) after 10 minutes, and after the addition, the reaction was carried out at 5? C. for 1 hour, the reaction was monitored by LCMS. After the reaction solution was quenched with saturated ammonium chloride aqueous solution, it was extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and then filtered, and the filtrate was concentrated under reduced pressure to obtain 455.00 mg of a crude product of the title compound.

[0665] The structural characterization data were as follows:

[0666] ESI-MS (m/z): 285.0[M+H].sup.+.

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

[0667] The crude product of compound 3-1-07 (0.40 g, 1.41 mmol) was dissolved in methanol (10 mL), then added with 3 mol/L aqueous hydrochloric acid (1 mL) and 10% palladium on carbon (40.00 mg), after the addition, the reaction system was subjected to replacement three times with a hydrogen balloon, the reaction was carried out at room temperature under hydrogen atmosphere for 1 hour and monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 0.43 g of a crude hydrochloride of the title compound.

[0668] The structural characterization data were as follows:

[0669] ESI-MS (m/z): 271.0[M+H].sup.+.

Step 8: Synthesis of (9H-fluoren-9-yl)methyl(8-acetamide-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0670] The crude hydrochloride of compound 3-1-08 (0.43 g, 1.19 mmol) was dissolved in 1,4-dioxane (15 mL), then added with sodium bicarbonate (400.35 mg, 4.77 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (481.81 mg, 1.43 mmol), after the addition, the reaction was carried out under stirring at room temperature for 2 hours and monitored by LCMS. The reaction solution was poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid in water=20%-100%) to obtain 301.00 mg of the title compound.

[0671] The structural characterization data were as follows:

[0672] ESI-MS (m/z): 493.2[M+H].sup.+.

[0673] Step 9: Synthesis of (9H-fluoren-9-yl)methyl(8-amino-5-chloro-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate Compound 3-1-09 (300.00 mg, 608.61 ?mol) was dissolved in dioxane (5 mL), added with 12 mol/L concentrated hydrochloric acid (1 mL), after the addition, the temperature was raised to 60? C., the reaction was carried out for 2 hours and monitored by LCMS. The reaction solution was poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate:petroleum ether=0-50%) to obtain 198.00 mg of the title compound.

[0674] The structural characterization data were as follows:

[0675] ESI-MS (m/z): 451.1[M+H].sup.+.

Step 10: Synthesis of (9H-fluoren-9-yl)methyl((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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)carbamate

[0676] (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (138.72 mg, 526.96 ?mol) and compound 3-1-10 (198.00 mg, 439.13 ?mol) were added in toluene (10 mL), and then added with p-toluenesulfonic acid (75.53 mg, 439.13 ?mol), after the addition, the temperature was raised to 140? C., the reaction was carried out for 4 hours, the reaction solution was directly evaporated to dryness at 140? C. under reduced pressure to obtain a crude product, and the crude product was purified by flash silica gel column (methanol:dichloromethane=0-5%) to obtain 256.00 mg of the title compound.

[0677] The structural characterization data were as follows:

[0678] ESI-MS (m/z): 678.1[M+H].sup.+.

Step 11: Synthesis of (1S,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinoline-10,13-dione and (1R,9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinoline-10,13-dione

[0679] Compound 3-1-11 (201.18 mg, 296.67 ?mol) was dissolved in N,N-dimethylformamide (4 mL), then added with diethylamine (108.49 mg, 1.48 mmol), after the addition, the reaction was carried out at room temperature for 0.5 hours, and monitored by LCMS. After the reaction solution was distilled under reduced pressure to remove ethylenediamine, the pH was adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain the title compounds 3-1-A (44.00 mg), 3-1-B (43.00 mg).

[0680] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0681] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00013 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 10 90 28 3 10 90 28 18 70 30 28

[0682] 3-1-A (6 min LCMS, the earlier peak with retention time of 1.276 min)

[0683] The structural characterization data were as follows:

[0684] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.00 (d, J=10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (d, J=19.3 Hz, 1H), 5.44 (s, 2H), 5.38 (d, J=19.3 Hz, 1H), 4.43-4.38 (m, 1H), 3.28-3.10 (m, 2H), 2.22-2.12 (m, 1H), 2.12-2.02 (m, 1H), 1.93-1.80 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0685] ESI-MS (m/z): 456.1[M+H].sup.+.

[0686] 3-1-B (6 min LCMS, the later peak with a retention time of 1.300 min)

[0687] The structural characterization data thereof were as follows:

[0688] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.98 (d, J=10.3 Hz, 1H), 7.32 (s, 1H), 5.61 (d, J=19.4 Hz, 1H), 5.44 (s, 2H), 5.32 (d, J=19.4 Hz, 1H), 4.44-4.36 (m, 1H), 3.33-3.25 (m, 1H), 3.22-3.11 (m, 1H), 2.23-2.13 (m, 1H), 2.11-2.03 (m, 1H), 1.96-1.82 (m, 2H), 0.89 (t, J=7.3 Hz, 3H).

[0689] ESI-MS (m/z): 456.1[M+H].sup.+.

[0690] 6 min LCMS conditions:

[0691] Chromatographic column: Waters SunFire C18 OBD 4.6 mm?50 mm?5.0 ?m

[0692] Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00014 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 9

N-((1S,9S)-4-Chloro-9-ethyl-5-fluoro-9-hydroxy-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 and N-((1R,9S)-4-Chloro-9-ethyl-5-fluoro-9-hydroxy-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

[0693] ##STR00191##

Step 1: Synthesis of (S)-10-benzyl-23-(2-(methylsulfonyl)pyrimidin-5-yl)-6,9,12,15,18-pentoxo-3-oxo-5,8,11,14,17-pentaazoctadecane-22-yne-carboxylic acid

[0694] Compound 3-4-01 (30.00 mg, 70.00 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added with 2,5-dioxopyrrolidin-1-yl 6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl-amide (28.00 mg, 77.00 ?mol), and reacted at room temperature for 1 hour, and the reaction was monitored by LCMS. The reaction solution was directly purified by preparative high performance liquid chromatography, and the fractions was lyophilized to obtain the title compound 3-4-03 (20.00 mg).

[0695] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0696] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00015 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0697] The structural characterization data were as follows:

[0698] ESI-MS (m/z): 691.0[M+18]+.

Step 2: Synthesis of N((S)-10-benzyl-1-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl-amide and N((S)-10-benzyl-1-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hex-5-ynyl-amide

[0699] Compound 3-1-A (36.00 mg, 79.70 ?mol) and compound 3-4-03 (64.43 mg, 95.64 ?mol) in single configuration were dissolved in N,N-dimethylformamide (2 mL), then added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 ?mol) and triethylamine (24.19 mg, 239.10 ?mol), after the addition, the reaction was carried out at room temperature for 1 hour and monitored by LCMS. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-A (51.00 mg) in single configuration.

[0700] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0701] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00016 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

[0702] The structural characterization data were as follows:

[0703] ESI-MS (m/z): 1111.0[M+H].sup.+.

[0704] Compound 3-1-B (36.00 mg, 79.70 ?mol) and compound 3-4-03 (64.43 mg, 95.64 ?mol) in single configuration were dissolved in N,N-dimethylformamide (2 mL), then added with 4-(4,6-dimethoxytriazin-2-yl)-4-methylmorpholine hydrochloride (46.98 mg, 159.40 ?mol) and triethylamine (24.19 mg, 239.10 ?mol), after the addition, the reaction was carried out at room temperature for 1 hour and monitored by LCMS. The reaction solution was directly purified by high performance liquid chromatography to obtain the title compound 3-4-04-B (52.00 mg) in single configuration.

[0705] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0706] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00017 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

[0707] The structural characterization data were as follows:

[0708] ESI-MS (m/z): 1111.0[M+H].sup.+.

Step 3: Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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 and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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

[0709] Compound 3-4-04-A (40.00 mg, 35.99 ?mol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then added with 4 mol/L ethyl acetate hydrochloride (1 mL), after the addition, the reaction was carried out at room temperature for 0.5 hour and monitored by LCMS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by high performance liquid chromatography to obtain the title compound 3-4-A (4.75 mg) in single configuration.

[0710] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0711] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00018 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

[0712] The structural characterization data were as follows:

[0713] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.50 (d, J=8.9 Hz, 1H), 8.05 (d, J=10.3 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.67-5.60 (m, 1H), 5.49 (t, J=5.8 Hz, 1H), 5.43 (s, 2H), 5.21 (s, 2H), 3.96 (d, J=5.8 Hz, 2H), 3.32-3.22 (m, 2H), 2.28-2.15 (m, 2H), 1.93-1.80 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0714] ESI-MS (m/z): 514.0[M+H].sup.+.

[0715] Compound 3-4-04-B (40.00 mg, 35.99 ?mol) was weighed and dissolved in a mixed solvent of dichloromethane (2 mL) and methanol (1 mL), then added with 4 mol/L ethyl acetate hydrochloride (1 mL), after the addition, the reaction was carried out at room temperature for 0.5 hour and monitored by LCMS. The reaction solution was directly concentrated to dryness under reduced pressure to obtain a crude product, and the crude product was purified by high performance liquid chromatography to obtain the title compound 3-4-B (8.24 mg) in single configuration.

[0716] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0717] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00019 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 15 85 28 3 15 85 28 18 90 10 28

[0718] The structural characterization data were as follows:

[0719] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.52 (d, J=9.0 Hz, 1H), 8.05 (d, J=10.3 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.68-5.58 (m, 1H), 5.53 (t, J=5.8 Hz, 1H), 5.43 (d, J=2.9 Hz, 2H), 5.20 (d, J=7.3 Hz, 2H), 3.97 (d, J=5.7 Hz, 2H), 3.31-3.21 (m, 2H), 2.26-2.15 (m, 2H), 1.92-1.82 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0720] ESI-MS (m/z): 514.0[M+H].sup.+.

Example 10

(S)N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0721] ##STR00192## ##STR00193##

Step 1: Synthesis of 1-(4-fluoro-3-methylphenyl)-3-(isopropylamino)propan-1I-one

[0722] At 20? C., compound 4-12-01 (500.00 mg, 3.29 mmol), formaldehyde solution (2.5 mL, 37%) and isopropylamine (388.46 mg, 6.57 mmol) were dissolved in isopropanol (5 mL), added dropwise with concentrated hydrochloric acid (2.5 mL) at 0? C., the reaction solution was stirred at 100? C. for 16 hours and monitored by LCMS. The reaction solution was concentrated under reduced pressure to obtain a crude product, which was purified by preparative high performance liquid chromatography, and the fractions were lyophilized to obtain 200.00 mg of the title compound.

[0723] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0724] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00020 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0725] The structural characterization data were as follows:

[0726] ESI-MS (m/z): 224.1 [M+H].sup.+.

Step 2: Synthesis of 1-(4-fluoro-5-methyl-2-nitrophenyl)-3-(isopropylamino)propan-1-one

[0727] At 0? C., compound 4-12-02 (100.00 mg, 0.49 mmol) was added into concentrated sulfuric acid (0.5 mL), then added with potassium nitrate (54.34 mg, 0.54 mmol), and the reaction was reacted at 0? C. for 1 hour and monitored by LCMS. The reaction solution was poured into ice water, and purified by reverse phase column (acetonitrile: 0.05% formic acid water=0-30%) to obtain 90.00 mg of the title compound.

[0728] The structural characterization data were as follows:

[0729] ESI-MS (m/z): 269.0 [M+H].sup.+.

Step 3: Synthesis of 1-(2-amino-4-fluoro-5-methylphenyl)-3-(isopropylamino)propan-1-one

[0730] At 25? C., compound 4-12-03 (200.00 mg, 0.75 mmol) was added into methanol (20.0 mL), then added with 10% palladium on carbon (10.00 mg), the reaction solution was subjected to hydrogen replacement, reacted at 20? C. for 16 hours under hydrogen atmosphere, the reaction was monitored by LCMS. The reaction solution was filtered and concentrated under reduced pressure to obtain 183.00 mg of the title compound.

[0731] The structural characterization data were as follows:

[0732] ESI-MS (m/z): 239.1 [M+H].sup.+.

Step 4: Synthesis of (S)-4-ethyl-8-fluoro-4-hydroxy-11-(2-(isopropylamino)ethyl)-9-methyl-1,12-dihydro-14H-pyrano[3,4:6,7]indolizino[1,2-b]quinoline-3,14(4H)-dione

[0733] At 25? C., compound 4-12-04 (50.00 mg, 0.21 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (55.23 mg, 0.21 mmol) were added into toluene (3 mL), then added with p-toluenesulfonic acid (3.61 mg, 0.02 mmol), and the reaction solution was reacted at 130? C. for 4 hours and monitored by LCMS. The reaction solution was concentrated under reduced pressure, the crude product was purified by preparative high-performance liquid chromatography, and the fractions were lyophilized to obtain 2.00 mg of a trifluoroacetic acid salt of the title compound.

[0734] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0735] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

TABLE-US-00021 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 8 92 28 2.00 8 92 28 18.00 60 40 28

[0736] The structural characterization data were as follows:

[0737] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.58 (s, 2H), 8.22 (d, J=8.1 Hz, 1H), 7.97 (d, J=10.7 Hz, 1H), 7.35 (s, 1H), 6.59 (s, 1H), 5.47 (s, 2H), 5.41 (s, 2H), 3.58-3.45 (m, 3H), 3.31-3.23 (m, 2H), 2.56 (s, 3H), 1.98-1.80 (m, 2H), 1.26 (d, J=6.3 Hz, 6H), 0.89 (t, J=7.3 Hz, 3H).

[0738] ESI-MS (m/z): 466.2 [M+H].sup.+

Step 5: Synthesis of (S)-2-((tert-butyldiphenylsilyl)oxy)-N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyranol[3,4:6,7]indolizinol[1,2-b]quinolin-11-yl)ethyl)-N-isopropylacetamide

[0739] Compound 4-12-05 (22.00 mg, 47.26 ?mol) and 2-((tert-butyldiphenylsilyl)oxy)acetic acid (16.35 mg, 51.99 ?mol) were dissolved in N,N-dimethylformamide (1 mL), then added with HATU (21.55 mg, 56.71 ?mol) and N,N-diisopropylethylamine (18.32 mg, 141.78 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS. The reaction solution was directly purified by a C18 reverse-phase column (acetonitrile: 0.05% aqueous formic acid=30%-100%) to obtain 18.00 mg of the title compound.

[0740] The structural characterization data were as follows:

[0741] ESI-MS (m/z): 762.3[M+H].sup.+.

Step 6: Synthesis of (S)N-(2-(4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-11-yl)ethyl)-2-hydroxy-N-isopropylacetamide

[0742] Compound 4-12-06 (18.00 mg, 23.62 ?mol) was dissolved in N,N-dimethylformamide (1 mL), then added with potassium fluoride (6.86 mg, 118.12 ?mol), then heated to 50? C. and reacted for 1 hour, and the reaction was monitored by LCMS. The reaction solution was directly purified by high performance liquid chromatography to obtain 1.53 mg of the title compound.

[0743] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0744] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00022 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 15 85 28 18.00 90 10 28

[0745] The structural characterization data were as follows:

[0746] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.53 (d, J=8.2 Hz, 1H), 7.91 (d, J=10.8 Hz, 1H), 7.32 (s, 1H), 6.55 (s, 1H), 5.44 (d, J=13.8 Hz, 4H), 4.72 (t, J=5.5 Hz, 1H), 4.21 (d, J=5.5 Hz, 2H), 3.99-3.90 (m, 1H), 3.54-3.38 (m, 4H), 2.54 (s, 3H), 1.92-1.83 (m, 2H), 1.17 (dd, J=6.6, 3.1 Hz, 6H), 0.87 (t, J=7.3 Hz, 3H).

[0747] ESI-MS (m/z): 524.2[M+H].sup.+.

Example 11

(S)N-((4-ethyl-8-fluoro-4-hydroxy-9-methyl-3,14-dioxo-3,4,12,14-tetrahydro-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-11-yl)methyl)-1-hydroxycyclopropane-carboxamide

[0748] ##STR00194##

[0749] Raw material (S)-11-(aminomethyl)-4-ethyl-8-fluoro-4-hydroxy-9-methyl-1H-pyrano[3,4:6,7]indolizino[1,2-b]quinolin-3,14(4H,12H)-dione (4-10-01, prepared according to the method disclosed in WO2020219287, 30.00 mg, 67.00 ?mol), and 1-hydroxycyclopropane-carboxylic acid (7.56 mg, 0.074 mmol) were dissolved in DMF (1 mL), added with HBTU (34.30 mg, 0.14 mmol) and diisopropylethylamine (26.09 mg, 0.20 mmol) under stirring, and reacted at room temperature for 4 hours. Water and ethyl acetate were added and stirred, and allowed to stand to separate the layers, and the organic phase was washed with saturated brine and concentrated under reduced pressure. The concentrate was purified by preparative thin-layer chromatography (dichloromethane:methanol=20:1), and then purified by preparative high-performance liquid chromatography to obtain 1.20 mg of solid.

[0750] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0751] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00023 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 20 80 28 2.00 20 80 28 18.00 80 20 28

[0752] The structural characterization data were as follows:

[0753] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.96 (t, J=6.0 Hz, 1H), 8.51 (d, J=8.0 Hz, 1H), 7.90 (d, J=10.8 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 6.30 (s, 2H), 6.30 (s, 1H), 5.52 (s, 2H), 5.44 (s, 2H), 4.84 (d, J=6.0 Hz, 2H), 2.51 (s, 3H), 1.91-1.81 (m, 2H), 1.01 (dd, J=7.2, 4.1 Hz, 2H), 0.87 (t, J=7.3 Hz, 3H), 0.83 (t, J=3.6 Hz, 2H).

[0754] ESI-MS (m/z): 494.1[M+1]+.

Example 12

(1S,9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione and (1R,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione

[0755] ##STR00195## ##STR00196## ##STR00197##

Step 1: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid

[0756] N-(3-Bromo-5-fluoro-4-methylphenyl)acetamide (2.00 g, 8.13 mmol) and 2-methyl-3-butenoic acid (976.44 mg, 9.75 mmol) were weighed and dissolved in a mixed solvent of 1,4-dioxane (15 mL) and water (5 mL), then added with tris(o-methylphenyl)phosphine (247.37 mg, 812.76 ?mol), palladium acetate (91.24 mg, 406.38 ?mol) and N,N-diisopropylethylamine (2.31 g, 17.88 mmol), after the addition, the reaction system was subjected to nitrogen replacement three times, the temperature was raised to 80? C. and the reaction was carried out for 3 hours under nitrogen atmosphere and monitored by LCMS. After the reaction solution was cooled to room temperature, 1 mol/L sodium hydroxide aqueous solution (60 mL) and ethyl acetate (50 mL) were added and shaken to separated layers. After separating the lower layer of aqueous phase, the pH was adjusted to about 3 with 4 mol/L hydrochloric acid aqueous solution, then extraction was carried out with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated under reduced pressure to obtain 1.90 g of the title compound.

[0757] The structural characterization data were as follows:

[0758] ESI-MS (m/z): 266.1[M+H].sup.+.

Step 2: Synthesis of 4-(5-acetylamino-3-fluoro-2-methylphenyl)-2-methylbutanoic acid

[0759] (E)-4-(5-Acetylamino-3-fluoro-2-methylphenyl)-2-methyl-3-butenoic acid (1.90 g, 7.16 mmol) was dissolved in methanol (40 mL), added with 10% palladium on carbon (0.15 g) under the protection of nitrogen, then the reaction system was subjected to replacement three times with a hydrogen balloon, the reaction was carried out under hydrogen atmosphere for 2 hours and monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.51 g of the title compound.

[0760] The structural characterization data were as follows:

[0761] ESI-MS (m/z): 268.1[M+H].sup.+.

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

[0762] 4-(5-Acetylamino-3-fluoro-2-methylphenyl)-2-methylbutanoic acid (1.50 g, 5.61 mmol) was weighed and dissolved in trifluoroacetic acid (20 mL), cooled to 5? C., then added dropwise with trifluoroacetic anhydride (2.36 g, 11.22 mmol). After the addition, the reaction was carried out at 5? C. for 2 hours and monitored by LCMS. The reaction solution was slowly poured into saturated aqueous sodium bicarbonate solution, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate:petroleum ether=0-30%) to obtain 1.05 g of the title compound.

[0763] The structural characterization data were as follows:

[0764] ESI-MS (m/z): 250.1[M+H].sup.+.

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

[0765] N-(3-Fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (0.55 g, 2.21 mmol) was weighed and dissolved in acetic acid (8 mL), then added with bromine (387.85 mg, 2.43 mmol), after the addition, the temperature was raised to 50? C., the reaction was carried out for 15 hours and monitored by LCMS. The reaction solution was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-30%) to obtain 461.00 mg of the title compound.

[0766] The structural characterization data were as follows:

[0767] ESI-MS (m/z): 328.0[M+H].sup.+.

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

[0768] N-(7-Bromo-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (460.00 mg, 1.40 mmol) was weighed and dissolved in N,N-dimethylformamide (10 mL), then added with sodium azide (273.37 mg, 4.21 mmol), after the addition, the reaction was carried out at room temperature for 1 hour and monitored by LCMS. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, and filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-50%) to obtain 347.00 mg of the title compound.

[0769] The structural characterization data were as follows:

[0770] ESI-MS (m/z): 291.1[M+H].sup.+.

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

[0771] N-(7-Azido-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (347.00 mg, 1.20 mmol) was weighed and dissolved in tetrahydrofuran (10 mL), added with 10% palladium on carbon (30.00 mg) under nitrogen protection, then the reaction system was subjected to replacement three times with a hydrogen balloon, and the reaction was carried out for 2 hours under hydrogen atmosphere and monitored by LCMS. The reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain a crude product, which was purified by a C18 reverse-phase column (acetonitrile: 0.05% formic acid aqueous solution=0%-30%) to obtain 205.00 mg of the title compound.

[0772] The structural characterization data were as follows:

[0773] ESI-MS (m/z): 265.1[M+H].sup.+.

Step 7: Synthesis of (9H-fluoren-9-yl)methyl(8-acetylamino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0774] N-(7-Amino-3-fluoro-4,7-dimethyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (200.00 mg, 756.73 ?mol) was weighed and dissolved in a mixed solvent of 1,4-dioxane (6 mL) and water (3 mL), then added with sodium bicarbonate (254.28 mg, 3.03 mmol) and 9-fluorenylmethyl-N-succinimide carbonate (650.55 mg, 1.14 mmol), after the addition, the reaction was carried out under stirring at room temperature for 2 hours and monitored by LCMS. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by a C18 reverse phase column (acetonitrile: 0.05% aqueous formic acid=20%-80%) to obtain 301.00 mg of the title compound.

[0775] The structural characterization data were as follows:

[0776] ESI-MS (m/z): 487.0[M+H].sup.+.

Step 8: Synthesis of (9H-fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0777] (9H-Fluoren-9-yl)methyl(8-acetylamino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (101.00 mg, 207.59 ?mol) was dissolved in 1,4-dioxane (5 mL), then added with 3 mol/L hydrochloric acid aqueous solution (5 mL), the temperature was raised to 50? C., the reaction was carried out for 15 hours and monitored by LCMS. The reaction solution was slowly poured into saturated aqueous sodium bicarbonate solution, then extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (methanol:dichloromethane=0%-5%) to obtain 71.00 mg of the title compound.

[0778] The structural characterization data were as follows:

[0779] ESI-MS (m/z): 445.2[M+H].sup.+.

Step 9: Synthesis of (9H-fluoren-9-yl)methyl((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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)carbamate

[0780] (9H-Fluoren-9-yl)methyl(8-amino-6-fluoro-2,5-dimethyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (35.00 mg, 132.96 ?mol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolazin-3,6,10(4H)-trione (49.25 mg, 110.80 ?mol) were added into toluene (3 mL), then added with p-toluenesulfonic acid (19.08 mg, 110.80 ?mol), the temperature was raised to 140? C. and the reaction was carried out for 4 hours. The liquid reaction solution was directly evaporated to dryness under reduced pressure at 140? C. to obtain a crude product. The crude product was purified by a C18 reverse phase column (acetonitrile: 0.05% aqueous formic acid=20%-80%) to obtain 21.00 mg of the title compound.

[0781] The structural characterization data were as follows:

[0782] ESI-MS (m/z): 672.2[M+H].sup.+.

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

[0783] (9H-Fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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) carbamate (21.00 mg, 31.26 ?mol) was dissolved in N,N-dimethylformamide (1 mL), then added with diethylamine (0.2 mL), the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS. After the reaction solution was distilled under reduced pressure to remove ethylenediamine, the pH was adjusted to 2-3 with 1 mol/L hydrochloric acid aqueous solution, and the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers 5-13-A (1.30 mg) and 5-13-B (1.68 mg).

[0784] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0785] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00024 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 3.00 10 90 28 18.00 90 10 28

[0786] The structural characterization data of 5-13-A (6 min LCMS, the earlier peak with retention time of 1.373 min) were as follows:

[0787] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.88 (d, J=10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J=3.5 Hz, 2H), 5.46 (d, J=2.5 Hz, 2H), 3.25-3.17 (m, 2H), 2.41 (s, 3H), 2.38-2.28 (m, 2H), 1.91-1.84 (m, 2H), 1.79 (s, 3H)), 0.88 (t, J=7.3 Hz, 3H).

[0788] ESI-MS (m/z): 450.2[M+H].sup.+.

[0789] The structural characterization data of 5-13-B (6 min LCMS, the later peak with a retention time of 1.523 min) were as follows:

[0790] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.76 (d, J=10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J=19.8 Hz, 1H), 5.50-5.40 (m, 3H), 3.26-3.17 (m, 1H), 3.08-2.96 (m, 1H), 2.38 (s, 3H), 2.19-2.11 (m, 1H), 2.04 (td, J=13.0, 5.1 Hz, 1H), 1.91-1.79 (m, 2H), 1.34 (s, 3H), 0.87 (t, J=7.3 Hz, 3H).

[0791] ESI-MS (m/z): 450.2[M+H].sup.+.

[0792] 6 min LCMS conditions:

[0793] Chromatographic column: Waters SunFire C18 OBD 4.6 mm?50 mm?5.0 ?m

[0794] Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00025 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 13

(1S,9S)-1-(Aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione and (1R,9S)-1-(aminomethyl)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione

[0795] ##STR00198## ##STR00199##

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

[0796] Compound 5-13-04 (1.00 g, 4.25 mmol) was dissolved in N,N-dimethylformamide dimethyl acetal (10 mL), then heated to 120? C. and reacted for 3 hours, and the reaction was monitored by LCMS. After the reaction solution was cooled to room temperature, it was directly evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=20%-100%) to obtain 891.00 mg of the title compound.

[0797] The structural characterization data were as follows:

[0798] ESI-MS (m/z): 291.1[M+H].sup.+.

Step 2: Synthesis of N-(7-(aminomethylene)-3-fluoro-4-methyl-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0799] Compound 5-7-01 (0.89 g, 3.07 mmol) was dissolved in ethanol (25 mL), then added with ammonium acetate (2.36 g, 30.65 mmol), after the addition, the reaction was carried out at room temperature for 16 hours and monitored by LCMS. The reaction solution was evaporated to dryness under reduced pressure, then added with dichloromethane (30 mL) and water (20 mL), stirred and allowed to stand to separate the organic phase, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 785.00 mg of the title compound.

[0800] The structural characterization data were as follows:

[0801] ESI-MS (m/z): 263.1I[M+H].sup.+.

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

[0802] Compound 5-7-02 (0.80 g, 3.05 mmol) was dissolved in ethanol (200 mL), then added with 10% palladium on carbon (0.40 mg) and concentrated hydrochloric acid (0.2 mL), and the reaction system was subjected to the replacement three times with a hydrogen balloon, and then the reaction was carried out at room temperature under hydrogen atmosphere for 3 hours and monitored by LCMS. The reaction solution was directly filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 905.00 mg of a hydrochloride of the title compound.

[0803] The structural characterization data were as follows:

[0804] ESI-MS (m/z): 265.1[M+H].sup.+.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl ((8-acetamido-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)carbamate

[0805] The hydrochloride salt of compound 5-7-03 (0.90 g, 2.99 mmol) was dissolved in 1,4-dioxane (20 mL), then added with sodium bicarbonate (1.01 g, 11.97 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.21 g, 3.59 mmol), after the addition, the reaction was carried out under stirring at room temperature for 1 hour and monitored by LCMS. The reaction solution was poured into water, then extracted with ethyl acetate, the organic phases were combined, washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate:petroleum ether=0-50%) to obtain 1.30 g of the title compound.

[0806] The structural characterization data were as follows:

[0807] ESI-MS (m/z): 487.1[M+H].sup.+.

Step 5: Synthesis of (9H-fluoren-9-yl)methyl ((8-amino-6-fluoro-5-methyl-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)methyl)carbamate

[0808] Compound 5-7-04 (0.80 g, 1.64 mmol) was dissolved in 1,4-dioxane (20 mL), and added with 3 mol/L hydrochloric acid aqueous solution (20 mL) under nitrogen protection. After addition, the temperature was raised to 60? C., the reaction was carried out for 15 hours and monitored by LCMS. The reaction solution was slowly poured into water, then extracted with ethyl acetate, the organic phases were combined and washed with saturated brine, dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product. The crude product was purified by flash silica gel column (ethyl acetate:petroleum ether=0-40%) to obtain 561.00 mg of the title compound.

[0809] The structural characterization data were as follows:

[0810] ESI-MS (m/z): 445.1[M+H].sup.+.

Step 6: Synthesis of (9H-fluoren-9-yl)methyl (((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)methyl)carbamate

[0811] (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizin-3,6,10(4H)-trione (597.00 mg, 2.27 mmol) and compound 5-7-05 (840.00 mg, 1.89 mmol) were added to toluene (60 mL), then added with p-toluenesulfonic acid (325.00 mg, 1.89 mmol). After the addition, the temperature was raised to 140? C. and the reaction was carried out for 4 hours, then the reaction solution was directly evaporated to dryness under reduced pressure at 140? C. to obtain a crude product. The crude product was purified by flash silica gel column (methanol:dichloromethane=0-5%) to obtain 563.00 mg of the title compound.

[0812] The structural characterization data were as follows:

[0813] ESI-MS (m/z): 672.2[M+H].sup.+.

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

[0814] Compound 5-7-06 (454.00 mg, 675.89 ?mol) was dissolved in N,N-dimethylformamide (5 mL), then added with diethylamine (1 mL), and reacted at room temperature for 0.5 hours. The reaction was monitored by LCMS. After ethylenediamine was evaporated from the reaction solution under reduced pressure, the pH was adjusted to 2-3 with formic acid, and the reaction solution was directly purified by preparative high-performance liquid chromatography, and the fractions were lyophilized to obtain the title compounds 5-7-A (32.00 mg) and 5-7-B (56.00 mg).

[0815] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0816] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00026 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 10 90 28 3 10 90 28 18 90 10 28

[0817] The structural characterization data of 5-7-A (6 min LCMS, the earlier peak with retention time of 1.488 min) were as follows:

[0818] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.88 (d, J=10.6 Hz, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.60 (d, J=3.5 Hz, 2H), 5.46 (d, J=2.5 Hz, 2H), 3.25-3.17 (m, 2H), 2.41 (s, 3H), 2.38-2.28 (m, 2H), 1.91-1.84 (m, 2H), 1.79 (s, 3H)), 0.88 (t, J=7.3 Hz, 3H).

[0819] ESI-MS (m/z): 450.2[M+H].sup.+.

[0820] The structural characterization data of 5-7-B (6 min LCMS, the later peak with a retention time of 1.596 min) are as follows:

[0821] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 7.76 (d, J=10.8 Hz, 1H), 7.30 (s, 1H), 6.52 (s, 1H), 5.73 (d, J=19.8 Hz, 1H), 5.50-5.40 (m, 3H), 3.26-3.17 (m, 1H), 3.08-2.96 (m, 1H), 2.38 (s, 3H), 2.19-2.11 (m, 1H), 2.04 (td, J=13.0, 5.1 Hz, 1H), 1.91-1.79 (m, 2H), 1.34 (s, 3H), 0.87 (t, J=7.3 Hz, 3H).

[0822] ESI-MS (m/z): 450.2[M+H].sup.+.

[0823] 6 min LCMS conditions:

[0824] Chromatographic column: Waters SunFire C18 OBD 4.6 mm?50 mm?5.0 ?m

[0825] Mobile phase A: 0.05% acetonitrile; mobile phase B: water (0.05% formic acid)

TABLE-US-00027 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 90 10 2 4.2 10 90 2 5.7 10 90 2 5.71 90 10 2 6.70 90 10 2

Example 14

N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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-hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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-hydroxycyclopropane-1-carboxamide

[0826] ##STR00200##

[0827] Step 1: Synthesis of 1-((tert-butyldiphenylsilyl)oxy)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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 or 1-((tert-butyldiphenylsilyl)oxy)-N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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

[0828] Compound 5-13-A (10.00 mg, 22.25 ?mol) in single configuration and 1-((tert-butyldiphenylsilyl)oxy)cyclopropane-carboxylic acid (11.36 mg, 33.37 ?mol) were dissolved in N,N-dimethylformamide (1 mL), then added with HATU (12.68 mg, 33.37 ?mol) and N,N-diisopropylethylamine (8.63 mg, 66.74 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hour and monitored by LCMS. The reaction solution was directly purified by C18 reverse phase column (acetonitrile: 0.05% formic acid aqueous solution=30%-100%) to obtain the title compound 5-16-01-A (7 mg) in single configuration.

[0829] The structural characterization data were as follows:

[0830] ESI-MS (m/z): 772.3 [M+H].sup.+.

[0831] Step 2: Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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-hydroxycyclopropane-1-carboxamide or N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-1,4-dimethyl-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-hydroxycyclopropane-1-carboxamide Compound 5-16-01-A (7.00 mg, 9.07 ?mol) was dissolved in N,N-dimethylformamide (1 mL), then added with potassium fluoride (2.63 mg, 45.34 ?mol), and then the temperature was raised to 50? C., the reaction was carried out for 1 hour and monitored by LCMS. The reaction solution was directly purified by high performance liquid chromatography to obtain a single stereoisomer of the title compound 5-16-A (1.73 mg).

[0832] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0833] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00028 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 18.00 90 10 28

[0834] The structural characterization data were as follows:

[0835] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.26 (s, 1H), 7.78 (d, J=10.8 Hz, 1H), 7.30 (s, 1H), 6.56 (s, 1H), 6.52 (s, 1H), 5.52 (d, J=19.3 Hz, 1H), 5.43 (d, J=4.4 Hz, 2H), 4.94 (d, J=19.2 Hz, 1H), 3.30-3.24 (m, 1H), 3.11-3.00 (m, 1H), 2.95-2.84 (m, 1H), 2.39 (s, 3H), 1.98-1.80 (m, 3H), 1.62 (s, 3H), 0.87 (t, J=7.9 Hz, 3H).

[0836] ESI-MS (m/z): 534.2[M+H].sup.+.

Example 15

N-((10S)-10-Benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4;6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide or N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-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-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide

[0837] ##STR00201##

Step 1: Separation and Purification of (9S)-1-amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzopyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione

[0838] Compound 2-23 (16.00 mg) was purified by preparative high-performance liquid chromatography, and two diastereoisomers were separated under the following purification conditions to obtain 5.10 mg of a trifluoroacetic acid salt of 2-23-A (retention time 9.85 min) and 7.12 mg of a trifluoroacetic acid salt of 2-23-B (retention time 10.62 min).

[0839] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0840] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

TABLE-US-00029 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 5 95 28 2 5 95 28 18 50 50 28

[0841] The structural characterization data were as follows:

[0842] 2-23-A:

[0843] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.59 (s, 1H), 5.78-5.63 (m, 1H), 5.50-5.36 (m, 3H), 5.10-5.06 (m, 1H), 3.20-3.04 (m, 2H), 2.56 (s, 3H), 2.26-2.13 (m, 2H), 1.93-1.79 (m, 2H), 0.88 (t, J=7.2 Hz, 3H).

[0844] ESI-MS (m/z): 452.1[M+H].sup.+.

[0845] 2-23-B:

[0846] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.42 (s, 3H), 8.27 (s, 1H), 7.36 (s, 1H), 6.58 (s, 1H), 5.78-5.63 (m, 1H), 5.50-5.36 (m, 3H), 5.10-5.06 (m, 1H), 3.20-3.04 (m, 2H), 2.55 (s, 3H), 2.26-2.13 (m, 2H), 1.93-1.79 (m, 2H), 0.88 (t, J=7.2 Hz, 3H).

[0847] ESI-MS (m/z): 452.0[M+H].sup.+.

Step 2: Synthesis of N-((10S)-10-benzyl-1-(((1S,9S)-5-chloro-9-ethyl-9-hydroxy-4-methyl-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyrano[3,4;6,7]indolizino[1,2-b]quinolin-1-yl)amino)-1,6,9,12,15-pentaoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide or N-((10S)-10-benzyl-1-(((1R,9S)-5-chloro-9-ethyl-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-pentoxo-3-oxa-5,8,11,14-tetraazahexadecan-16-yl)-6-(2-(methylsulfonyl)pyrimidin-5-yl)hexadecanamide

[0848] At 25? C., the trifluoroacetic acid salt of 2-23-A (34.71 mg, 61.43 ?mol) was dissolved in N,N-dimethylformamide (1 mL), added sequentially with 3-4-03 (49.66 mg, 73.72 ?mol), HATU (35.01 mg, 92.14 ?mol) and N,N-diisopropylethylamine (23.82 mg, 184.29 ?mol), reacted at 25? C. for 0.5 hours, and the reaction was monitored by LCMS, when the reaction was completed, the reaction solution was purified by preparative high-performance liquid chromatography (conditions were as follows), and the fractions were lyophilized to obtain 11.04 mg of the title compound D-L-15 with a retention time of 7.5 min.

[0849] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0850] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00030 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 30 70 28 3 30 70 28 18 90 10 28

[0851] The structural characterization data were as follows:

[0852] D-L-15:

[0853] ESI-MS (m/z): 1107.3[M+H].sup.+.

Example 16

N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-cyclopropyl-2-hydroxyacetamide

[0854] ##STR00202##

Step 1: Synthesis of N-((9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-cyclopropyl-2-hydroxyacetamide

[0855] The formate salt of single stereoisomer compound 3-1-A (50 mg, 109.68 ?mol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 ?mol) were dissolved in N,N-dimethylformamide (2 mL), then added with HATU (7.57 mg, 219.36 ?mol) and N,N-diisopropylethylamine (42.53 mg, 329.04 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound (3-12-A: 12.96 mg, 3-12-B: 13.56 mg).

[0856] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0857] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00031 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[0858] The structural characterization data of 3-12-A (6 min LCMS, the earlier peak) were as follows:

[0859] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.44 (d, J=9.0 Hz, 1H), 8.05 (d, J=10.2 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (q, J=6.7 Hz, 1H), 5.52 (d, J=5.1 Hz, 1H), 5.42 (s, 2H), 5.24 (q, J=19.2 Hz, 2H), 3.61 (dd, J=6.2, 5.1 Hz, 1H), 3.32-3.21 (m, 2H), 2.19 (q, J=6.5 Hz, 2H), 1.92-1.80 (m, 2H), 1.26-1.20 (m, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.57-0.34 (m, 4H).

[0860] ESI-MS (m/z): 554.0[M+H].sup.+.

[0861] The structural characterization data of 3-12-B (6 min LCMS, the later peak) were as follows:

[0862] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.43 (d, J=8.7 Hz, 1H), 8.06 (d, J=10.3 Hz, 1H), 7.33 (s, 1H), 5.57 (q, J=6.7 Hz, 1H), 5.43 (s, 2H), 5.30-5.17 (m, 2H), 3.64 (d, J=6.2 Hz, 1H), 3.29 (q, J=6.7 Hz, 2H), 2.28-2.13 (m, 2H), 1.93-1.78 (m, 2H), 1.18-1.08 (m, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.50-0.29 (m, 4H).

[0863] ESI-MS (m/z): 554.0[M+H].sup.+.

[0864] The formate salt of another single stereoisomer compound 3-1-B (50 mg, 109.68 ?mol) and 2-cyclopropyl-2-hydroxyacetic acid (25.47 mg, 219.36 ?mol) were dissolved in N,N-dimethylformamide (2 mL), then added with HATU (7.57 mg, 219.36 ?mol) and N,N-diisopropylethylamine (42.53 mg, 329.04 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (3-12-C: 20.19 mg, 3-12-D: 18.33 mg).

[0865] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0866] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00032 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 90 28 3.00 30 90 28 18.00 90 10 28

[0867] The structural characterization data of 3-12-C(6 min LCMS, the earlier peak) were as follows:

[0868] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.47 (d, J=9.0 Hz, 1H), 8.06 (d, J=10.3 Hz, 1H), 7.33 (s, 1H), 6.54 (s, 1H), 5.62 (q, J=6.5 Hz, 1H), 5.53 (d, J=5.1 Hz, 1H), 5.43 (s, 2H), 5.32-5.16 (m, 2H), 3.61 (dd, J=6.3, 5.1 Hz, 1H), 3.32-3.22 (m, 2H), 2.19 (q, J=6.5 Hz, 2H), 1.92-1.80 (m, 2H), 1.28-1.20 (m, 1H), 0.87 (t, J=7.3 Hz, 3H), 0.54-0.35 (m, 4H).

[0869] ESI-MS (m/z): 554.0[M+H].sup.+.

[0870] The structural characterization data of 3-12-D (6 min LCMS, the later peak) were as follows:

[0871] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.44 (d, J=8.8 Hz, 1H), 8.05 (d, J=10.2 Hz, 1H), 7.34 (s, 1H), 6.55 (s, 1H), 5.58 (q, J=6.7 Hz, 1H), 5.45 (d, J=5.2 Hz, 1H), 5.43 (s, 2H), 5.31-5.14 (m, 2H), 3.65 (t, J=5.7 Hz, 1H), 3.33-3.21 (m, 2H), 2.28-2.13 (m, 2H), 1.95-1.80 (m, 2H), 1.16-1.09 (m, 1H), 0.88 (t, J=7.3 Hz, 3H), 0.46-0.31 (m, 4H).

[0872] ESI-MS (m/z): 554.0[M+H].sup.+.

Example 17

Preparation of (S)N-((1S,9S)-4-Chloro-9-ethyl-5-fluoro-9-hydroxy-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-hydroxypropylamine and (S)N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-hydroxypropylamine

[0873] ##STR00203##

[0874] At 25? C., (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13(1H,9H)-dione (80.0 mg, 175.5 ?mol) and L-lactic acid (31.6 mg, 351.0 ?mol) were dissolved in DMF (3 mL), then added with HATU (121.1 mg, 351.0 ?mol) and DIPEA (68.0 mg, 526.5 ?mol), and reacted at room temperature for 2 hour; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain compound 3-7-A (6.1 mg, yield 12%) and compound 3-7-B (9.6 mg, yield 20%).

[0875] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0876] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00033 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 70 24 2.00 30 70 24 18.00 90 10 24

[0877] The structural characterization data of compound 3-7-A (6 min LC-MS, the earlier peak with a retention time of 2.49 min) were as follows:

[0878] MS m/z (ESI): 528.2 [M+H].sup.+

[0879] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.55 (d, J=9.2 Hz, 1H), 8.06 (d, J=10.4 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 6.67 (d, J=4.8 Hz, 1H), 5.65-5.59 (m, 1H), 5.43 (s, 2H), 5.29-5.21 (m, 1H), 5.14-5.10 (m, 1H), 4.15-4.10 (m, 1H), 3.27-3.20 (m, 1H), 2.22-2.15 (m, 2H), 1.92-1.81 (m, 2H), 1.41 (d, J=6.8 Hz, 3H), 1.30-1.23 (m, 1H), 0.89-0.85 (t, J=7.2 Hz, 3H).

[0880] The structural characterization data of compound 3-7-B (6 min LC-MS, the later peak with a retention time of 2.50 min) were as follows:

[0881] MS m/z (ESI): 528.2 [M+H].sup.+

[0882] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.46 (d, J=8.8 Hz, 1H), 8.06 (d, J=10.4 Hz, 1H), 7.33 (s, 1H), 6.56 (s, 1H), 5.60-5.53 (ms, 1H), 5.51 (d, J=5.2 Hz, 1H), 5.43 (s, 2H), 5.27-5.14 (m, 2H), 4.16-4.08 (m, 1H), 3.28-3.22 (m, 1H), 2.22-2.19 (m, 2H), 1.92-1.81 (m, 2H), 1.49-1.39 (m, 1H), 1.29 (d, J=6.8 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H).

Example 18

Synthesis of N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide and N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl-1-hydroxycyclopropylcarboxamide

[0883] ##STR00204##

[0884] At 25? C., (9S)-1-amino-4-chloro-9-ethyl-5-fluoro-9-hydroxy-2,3,12,15-tetrahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13(1H,9H)-dione (80 mg, 175.49 ?mol) and 1-hydroxycyclopropanecarboxylic acid (35.83 mg, 350.98 ?mol) were dissolved in DMF (2 mL), then added with HATU (121.14 mg, 350.98 ?mol) and DIPEA (68.04 mg, 526.47 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hour and monitored by HPLC-MS; the reaction solution was concentrated to dryness and directly purified by preparative high-performance liquid chromatography to obtain the title compounds 3-17-A (5.3 mg) and 3-17-B (3.5 mg).

[0885] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0886] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00034 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[0887] The structural characterization data were as follows:

[0888] The structural characterization data of 3-17-A (6 min LCMS, the earlier peak with a retention time of 2.657 min) were as follows:

[0889] ESI-MS (m/z): 540.0[M+H].sup.+.

[0890] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.69 (d, J=8.8 Hz, 1H), 8.06 (d, J=10.2 Hz, 1H), 7.35 (s, 1H), 6.57 (s, 1H), 6.32 (s, 1H), 5.62 (s, 1H), 5.45 (s, 2H), 5.34-5.24 (m, 1H), 5.20-5.10 (m, 1H), 2.26 (s, 2H), 2.00 (s, 1H), 1.88 (s, 2H), 1.47-1.12 (m, 8H), 1.01-0.80 (m, 6H).

[0891] The structural characterization data of 3-17-A (6 min LCMS, the later peak with a retention time of 2.724 min) were as follows:

[0892] ESI-MS (m/z): 540.0[M+H].sup.+.

[0893] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.69 (d, J=8.8 Hz, 1H), 8.06 (d, J=10.2 Hz, 1H), 7.35 (s, 1H), 6.57 (s, 1H), 6.32 (s, 1H), 5.62 (s, 1H), 5.45 (s, 2H), 5.34-5.24 (m, 1H), 5.20-5.10 (m, 1H), 2.26 (s, 2H), 2.00 (s, 1H), 1.88 (s, 2H), 1.47-1.12 (m, 8H), 1.01-0.80 (m, 6H).

Example 19

Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0894] ##STR00205## ##STR00206##

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

[0895] N-(4-Chloro-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (570 mg, 2.23 mmol) and cyclopropylboronic acid (574.56 mg, 6.69 mmol) were dissolved in 1,4-dioxane, then added with dichlorodi-tert-butyl-(4-dimethylaminophenyl)phosphine palladium (II) (480 mg, 677.97 ?mol) and cesium carbonate (2.17 g, 6.69 mmol); under nitrogen protection, the reaction was carried out in microwaves at 115? C. for 2 hours and monitored by LCMS; the reaction solution was diluted with ethyl acetate and filtered, the filtrate was extracted with ethyl acetate (30 ml*3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered, the filtrate was evaporated to dryness under reduced pressure, and the resultant crude product was purified by column chromatography silica gel column (PE:EA=1:4) to obtain 550 mg of the title compound.

[0896] The structural characterization data were as follows:

[0897] ESI-MS (m/z): 262.1[M+H].sup.+.

Step 2: Synthesis of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide

[0898] Tetrahydrofuran (30 mL) and tert-butanol (10 mL) were added to a reaction flask, cooled to 5? C. in an ice bath, then added with potassium tert-butoxide (945 mg, 8.42 mmol), then N-(4-chloro-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.0 g, 3.83 mmol) was dissolved in tetrahydrofuran (1 mL) and slowly added thereto in dropwise manner, followed by an addition of isoamyl nitrite (718 mg, 6.12 mmol) after 10 minutes, after the addition, the reaction was carried out at 5? C. for 1 hour and monitored by LCMS; the reaction solution was quenched with saturated ammonium chloride aqueous solution (50 mL), extracted with ethyl acetate (40 ml*3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and then filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.2 g of a crude product of the title compound.

[0899] The structural characterization data were as follows:

[0900] ESI-MS (m/z): 291.1[M+H].sup.+.

Step 3: Synthesis of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide hydrochloride

[0901] The crude product of N-(4-cyclopropyl-3-fluoro-7-(hydroxyimino)-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (1.2 g, 1.41 mmol) was dissolved in methanol (7.5 mL) and tetrahydrofuran (7.5 mL), then added with 1 mol/L hydrochloric acid aqueous solution (7.5 mL) and 10% palladium on carbon (450 mg), after the addition, the reaction system was subjected to replacement three times with a hydrogen balloon, and the reaction was carried out at room temperature under hydrogen atmosphere for 1 hour and monitored by LCMS; the reaction solution was filtered, and the filtrate was concentrated to dryness under reduced pressure to obtain 1.05 g of a crude product.

[0902] The structural characterization data were as follows:

[0903] ESI-MS (m/z): 277.1[M+H].sup.+.

Step 4: Synthesis of (9H-fluoren-9-yl)methyl(8-acetamide-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0904] Crude product of N-(7-amino-4-cyclopropyl-3-fluoro-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide hydrochloride (1.05 g, 3.80 mmol) was dissolved in 1,4-dioxane (10 mL), then added with sodium bicarbonate (1.3 g, 15.20 mmol), water (10 mL) and 9-fluorenylmethyl-N-succinimidyl carbonate (1.54 g, 4.56 mmol), after the addition, the reaction was carried out under stirring at room temperature for 2 hours and monitored by LCMS; the reaction solution was poured into water (50 mL), and then extracted with ethyl acetate (40 mL*3), the organic phases were combined, washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by C18 reverse phase column to obtain 2.0 g of the title compound.

[0905] The structural characterization data were as follows:

[0906] ESI-MS (m/z): 499.2[M+H].sup.+.

Step 5: Synthesis of (9H-fluoren-9-yl)methyl(8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[0907] (9H-Fluoren-9-yl)methyl(8-acetamido-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (2.0 g, 3.21 mmol, 80%) was dissolved in dioxane (20 mL), then added with 12 mol/L concentrated hydrochloric acid (5 mL), after the addition, the temperature was raised to 70? C., the reaction was carried out for 2 hours and monitored by LCMS; the reaction solution was poured into water (40 mL), then extracted with ethyl acetate (30 mL*3), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by column chromatography silica gel column (PE:EA=2:1) to obtain 740 mg of the title compound.

[0908] The structural characterization data were as follows:

[0909] ESI-MS (m/z): 457.3[M+H].sup.+.

Step 6: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2,3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate

[0910] (S)-4-Ethyl-4-hydroxyl-7,8-dihydro-1H-pyrano[3,4-f]indolizin-3,6,10(4H)-trione (442 mg, 1.68 mmol) and (9H-fluoren-9-yl)methyl(8-amino-5-cyclopropyl-6-fluoro-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (640 mg, 1.40 mmol) were added to toluene (30 mL), and then added with p-toluenesulfonic acid (242 mg, 1.40 mmol), after the addition, the temperature was raised to 135? C., the reaction was carried out for 2 hours. The reaction solution was directly evaporated to dryness at 140? C. under reduced pressure to obtain a crude product; and the crude product was purified by column chromatography silica gel column (DCM:MeOH=33:1) to obtain 1.02 g of the title compound.

[0911] The structural characterization data were as follows:

[0912] ESI-MS (m/z): 684.1[M+H].sup.+.

Step 7: Synthesis of (1S,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9, 12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione (Compound 5-29-1) and (1R,9S)-1-amino-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-1,2,3,9, 12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione

[0913] (9H-Fluoren-9-yl)methyl ((9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-2, 3,9,10,13,15-hexahydro-1H,12H-benzo[de]pyran[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)carbamate (1.02 g, 1.49 mmol) were dissolved in N,N-dimethylformamide (15 mL), then added with diethylamine (5 ml), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was distilled under reduced pressure to remove ethylenediamine, then adjusted to pH=2-3 with 1 mol/L hydrochloric acid aqueous solution, the reaction solution was then directly purified by preparative high-performance liquid chromatography to obtain two isomers of the title compound (5-22-7-A: 60 mg; 5-22-7-B: 55 mg).

[0914] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0915] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00035 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 3.00 10 90 28 18.00 70 30 28

[0916] The structural characterization data of 5-22-7-A (6 min LCMS, the earlier peak with a retention time of 2.28 min) were as follows:

[0917] ESI-MS (m/z): 462.2[M+H].sup.+.

[0918] The structural characterization data of 5-22-7-B (6 min LCMS, the later peak with a retention time of 2.35 min) were as follows:

[0919] ESI-MS (m/z): 462.2[M+H].sup.+.

Step 8: Synthesis of N-((1S,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2, 3,9,10,12,13,15-tetrahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide and N-((1R,9S)-4-cyclopropyl-9-ethyl-5-fluoro-9-hydroxy-10,13-dioxo-1,2,3,9,10,12, 13,15-tetrahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxyacetamide

[0920] At 25? C., single stereoisomer compound 5-28-7-A (40 mg, 86 ?mol) and glycolic acid (8 mg, 104 ?mol) were dissolved in DMF (2 mL), then added with HATU (40 mg, 104 ?mol) and DIPEA (36 mg, 258 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hr and monitored by LCMS; the reaction solution was directly purified by preparative high-performance liquid chromatography to obtain 12.5 mg of compound 5-22-A.

[0921] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0922] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00036 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0923] The structural characterization data were as follows:

[0924] The structural characterization data of 5-22-A (6 min LCMS, the earlier peak with a retention time of 2.540 min) were as follows:

[0925] ESI-MS (m/z): 520.0[M+H].sup.+.

[0926] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.46 (d, J=8.9 Hz, 1H), 7.74 (d, J=11.9 Hz, 1H), 7.30 (s, 1H), 6.53 (s, 1H), 5.64-5.56 (m, 1H), 5.49 (t, J=5.8 Hz, 1H), 5.42 (s, 2H), 5.19 (s, 2H), 3.96 (d, J=5.7 Hz, 2H), 2.25-2.10 (m, 2H), 2.04-1.79 (m, 4H), 1.23 (s, 2H), 1.15-1.05 (m, 2H), 0.87 (t, J=7.2 Hz, 3H), 0.80-0.70 (m, 2H).

[0927] At 25? C., single stereoisomer compound 5-28-7-B (30 mg, 65 ?mol) and glycolic acid (6 mg, 78 ?mol) were dissolved in DMF (2 mL), then added with HATU (40 mg, 104 ?mol) and DIPEA (17 mg, 130 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hr and monitored by LCMS; the reaction solution was concentrated to dryness and directly purified by preparative high-performance liquid chromatography to obtain 13.83 mg of compound 5-22-B.

[0928] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0929] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00037 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[0930] The structural characterization data were as follows:

[0931] The structural characterization data of 5-22-B (6 min LCMS, the later peak with a retention time of 2.612 min) were as follows:

[0932] ESI-MS (m/z): 520.0[M+H].sup.+.

[0933] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.49 (d, J=8.9 Hz, 1H), 7.74 (d, J=11.9 Hz, 1H), 7.31 (s, 1H), 6.53 (s, 1H), 5.60 (s, 1H), 5.51 (t, J=5.9 Hz, 1H), 5.43 (s, 2H), 5.25-5.13 (m, 2H), 3.97 (d, J=5.8 Hz, 2H), 2.18 (s, 2H), 2.04-1.91 (m, 4H), 1.90-1.80 (m, 1H), 1.23 (s, 6H), 1.15-1.05 (m, 2H), 0.87 (t, J=7.2 Hz, 4H), 0.80-0.70 (m, 2H).

Example 20

Synthesis of (R)-3-(dimethylamino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide and (S)-3-(dimethylamino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide

[0934] ##STR00207##

Step 1: Synthesis of (9H-fluoro-9-yl)methyl ((S)-3-(((15,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate and (9H-fluoro-9-yl)methyl ((R)-3-(((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)amino)-2-hydroxy-3-oxopropyl)carbamate

[0935] At 25? C., (1S,9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13(1H,9H)-dione mesylate (72 mg, 166 ?mol) and 3-((((9H-fluoren-9-yl)methoxy)carbonyl)amino)-2-hydroxypropionic acid (65 mg, 199 ?mol) were dissolved in DMF (2 mL), then added with HATU (95 mg, 250 ?mol) and DIPEA (65 mg, 498 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was concentrated to dryness and directly purified by preparative high-performance liquid chromatography to obtain the title compounds (Compound 1-10-1-A, 24 mg; and Compound 1-10-1-B, 28 mg).

[0936] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0937] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00038 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[0938] The structural characterization data of compound 1-10-1-A (6 min LCMS, the later peak with a retention time of 3.283 min) were as follows:

[0939] ESI-MS (m/z): 745.4[M+H].sup.+.

[0940] The structural characterization data of compound 1-10-1-B (6 min LCMS, the later peak with a retention time of 3.465 min) were as follows:

[0941] ESI-MS (m/z): 745.4[M+H].sup.+.

Step 2: Synthesis of (R)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide and (S)-3-amino-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide

[0942] At 25? C., compound 1-10-1-B (28 mg, 37 ?mol) was dissolved in DMF (2 mL), then added with diethylamine (1 mL), after the addition, the reaction was carried out at room temperature for 1.0 hour; the reaction solution was concentrated to dryness to obtain 28 mg of a crude product (compound 1-10-2-B), which was directly used in the next reaction.

[0943] At 25? C., compound 1-10-1-A (24 mg, 33 ?mol) was dissolved in DMF (2 mL), then added with diethylamine (1 mL), after the addition, the reaction was carried out at room temperature for 1.0 hour; the reaction solution was concentrated to dryness to obtain 24 mg of a crude product (compound 1-10-2-A), which was directly used in the next reaction.

[0944] The structural characterization data were as follows:

[0945] ESI-MS (m/z): 523.2[M+H].sup.+.

Step 3: Synthesis of (R)-3-(dimethylamino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide and (S)-3-(dimethylamino)-N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-1-yl)-2-hydroxypropionamide

[0946] At 25? C., compound 1-10-2-B (28 mg, 37 ?mol, 70%) was dissolved in methanol (2 mL), then added with formaldehyde solution (1 mL), after the addition, the reaction was carried out at room temperature for 16.0 hours, then sodium cyanoborohydride (7.07 mg, 96.45 ?mol) was then added and reacted at room temperature for 1.0 hour; the reaction was monitored by LCMS; the reaction solution was concentrated to dryness, and directly purified by preparative high performance liquid chromatography to obtain 1.3 mg of the title compound (Compound 1-10B).

[0947] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0948] Mobile phase A: methanol; mobile phase B: water (0.05% formic acid)

TABLE-US-00039 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[0949] The structural characterization data of compound 1-10-B (6 min LCMS, the later peak with a retention time of 1.937 min) were as follows:

[0950] ESI-MS (m/z): 551.2[M+H].sup.+.

[0951] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.48 (d, J=8.6 Hz, 1H), 8.31 (s, 2H), 7.80 (d, J=11.0 Hz, 1H), 7.31 (s, 1H), 6.55 (s, 1H), 5.54 (s, 1H), 5.43 (s, 2H), 5.34 (d, J=19.2 Hz, 1H), 5.19 (d, J=19.1 Hz, 1H), 4.09-4.06 (m, 1H), 3.20-3.15 (m, 2H), 2.59-2.53 (m, 1H), 2.45-2.42 (m, 1H), 2.42-2.38 (s, 3H), 2.23-2.19 (d, J=7.0 Hz, 1H), 2.13 (s, 6H), 2.12-2.08 (m, 1H), 2.02-1.95 (m, 1H), 1.90-1.85 (m, 2H), 1.23 (s, 2H), 0.88 (d, J=7.2 Hz, 3H).

[0952] At 25? C., compound 1-10-2-A (24 mg, 33 ?mol, 70%) was dissolved in methanol (2 mL), then added with formaldehyde solution (1 mL), after the addition, the reaction was carried out at room temperature for 16.0 hours, then sodium cyanoborohydride (6.06 mg, 96.45 ?mol) was added and reacted at room temperature for 1.0 hour; the reaction was monitored by LCMS; the reaction solution was concentrated to dryness and directly purified by preparative high performance liquid chromatography to obtain 4.44 mg of the title compound (Compound 1-10-A).

[0953] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0954] Mobile phase A: methanol; mobile phase B: water (0.05% formic acid)

TABLE-US-00040 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 15 85 28 2.00 15 85 28 18.00 90 10 28

[0955] The structural characterization data of compound 1-10-A (6 min LCMS, the earlier peak with a retention time of 1.920 min) were as follows:

[0956] ESI-MS (m/z): 551.2[M+H].sup.+.

[0957] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.58 (d, J=9.0 Hz, 1H), 8.28 (s, 1H), 7.84 (d, J=10.9 Hz, 1H), 7.37 (s, 1H), 6.61 (s, 1H), 5.66-5.59 (m, 1H), 5.49 (s, 2H), 5.35 (d, J=19.1 Hz, 1H), 5.17 (d, J=18.9 Hz, 1H), 4.23-4.16 (m, 1H), 3.23 (d, J=7.8 Hz, 2H), 2.75-2.67 (m, 2H), 2.45 (s, 3H), 2.30 (s, 6H), 2.27-2.17 (m, 2H), 2.14-1.99 (m, 1H), 1.98-1.87 (m, 2H), 1.30 (s, 2H), 0.93 (t, J=7.3 Hz, 3H).

Example 21

(S)-14-(2-(Cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolano[4, 5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-8,11(10H,13H)-dione (Compound 4-14)

[0958] ##STR00208## ##STR00209##

Step 1: Synthesis of 2-nitro-4,5-methylenedioxyacetophenone

[0959] Compound 4-14-1 (10.0 g, 60.92 mmol) was dissolved in nitromethane (100 mL), then slowly added dropwise with concentrated nitric acid (26 mL) under stirring, and reacted at room temperature for 2 hours. The reaction was monitored by TLC, showing a small amount of starting material remained, and the product was obvious. The reaction solution was neutralized by slowly adding saturated aqueous sodium bicarbonate solution in dropwise manner, extraction was carried out three times by adding dichloromethane, the organic phases were combined, washed with saturated brine three times, then dried, and concentrated to obtain a crude product, which was purified by silica gel column (eluent: 0-20% ethyl acetate/petroleum ether) to obtain 9.8 g of the title compound.

Step 2: Synthesis of 6-amino-3,4-methylenedioxyacetophenone

[0960] Compound 4-14-2 (2.0 g, 9.56 mmol) was dissolved in ethyl acetate (20 mL), then added with 10% palladium on carbon (0.2 g), subjected to hydrogen replacement and reacted under hydrogen protection with stirring for 4 hours. After filtration, the filtrate was concentrated under reduced pressure to obtain 1.7 g of a crude product of the title compound.

Step 3: Synthesis of 6-acetamido-3,4-methylenedioxyacetophenone

[0961] Compound 4-14-3 (1.7 g, 9.49 mmol) was dissolved in acetic anhydride (17 mL), and reacted under stirring for 1 hour. After the solvent was evaporated under reduced pressure, water was added and stirred, the solid was filtered, washed with water, and dried in vacuum to obtain 2.08 g of a crude product of the title compound.

Step 4: Synthesis of (E)-N-(6-(3-(dimethylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

[0962] Compound 4-14-4 (1.88 g, 8.50 mmol) was dissolved in DMF-DMA (30 mL), heated to 120? C. and reacted for 2 hours. The solvent was evaporated under reduced pressure to obtain 2.33 g of a crude product of the title compound.

[0963] The structural characterization data were as follows:

[0964] ESI-MS (m/z): 277.2[M+1]+.

Step 5: Synthesis of (E)-N-(6-(3-(cyclopropylamino)acryloyl)benzo[d][1,3]dioxol-5-yl)acetamide

[0965] Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), then added dropwise with cyclopropylamine (413.3 mg, 7.24 mmol), heated to 50? C. and reacted for 16 hours. The solvent was evaporated under reduced pressure to obtain 208 mg of a crude product of the title compound.

[0966] The structural characterization data were as follows:

[0967] ESI-MS (m/z): 289.2[M+1]+.

Step 6: Synthesis of N-(6-(3-(cyclopropylamino)propionyl)benzo[d][1,3]dioxol-5-yl)acetamide

[0968] Compound 4-14-6 (208 mg, 0.72 mmol) was dissolved in glacial acetic acid (4 mL), then added with sodium borohydride (13.65 mg, 0.36 mmol) under stirring in an ice-water bath, then warmed to room temperature and reacted under stirring for 3 hours. The solvent was evaporated under reduced pressure to obtain 209 mg of a crude product of the title compound.

[0969] The structural characterization data were as follows:

[0970] ESI-MS (m/z): 291.1[M+1]+.

Step 7: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-acetamidobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[0971] Compound 4-14-7 (200 mg, 0.69 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), then added with 9-fluorenylmethyl-N-succinimido carbonate (395 mg, 0.68 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) under stirring, and reacted at room temperature for 2 hours. Water and ethyl acetate were added under stirring, allowed to stand for liquid separation, the organic phase was washed with saturated brine, dried and concentrated, and purified by silica gel column (eluent: 30% ethyl acetate/petroleum ether) to obtain 350 mg of the title compound.

Step 8: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-aminobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(cyclopropyl)carbamate

[0972] Compound 4-14-8 (350 mg, 0.68 mmol) was dissolved in 1,4-dioxane (10 mL), then added dropwise with 3N aqueous hydrochloric acid (10 mL), heated to 60? C. and reacted under stirring for 16 hours. Water and ethyl acetate were added and stirred, allowed to stand for liquid separation, the organic phase was washed with water, dried and concentrated, and purified by silica gel column (eluent: 33% ethyl acetate/petroleum ether) to obtain 218 mg of the title compound.

Step 9: Synthesis of (S)-(9H-fluoren-9-yl)methylcyclopropyl (2-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3,4]:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)carbamate

[0973] Compound 4-14-9 (40 mg, 0.085 mmol) and (S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizin-3,6,10(4H)-trione (24.62 mg, 0.094 mmol) were dissolved in toluene (1 mL), then added with p-toluenesulfonic acid (2.93 mg, 0.017 mmol), heated to 120? C. and reacted for 4 hours. After concentration under reduced pressure, 59 mg of a crude product of the title compound was obtained.

[0974] The structural characterization data were as follows:

[0975] ESI-MS (m/z): 698.1[M+1]+.

Step 10: Synthesis of (S)-14-(2-(cyclopropylamino)ethyl)-7-ethyl-7-hydroxy-7H-[1,3]dioxolano[4,5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-8,11(10H,13H)-dione

[0976] Compound 4-14-10 (59 mg, 0.085 mmol) was dissolved in DMF (1 mL), then added dropwise with diethylamine (0.5 mL), and reacted under stirring for 1 hour. The reaction solution was distilled under reduced pressure to remove diethylamine, acidified by adding dropwise with 3N hydrochloric acid, purified by HPLC (purification conditions were as follows), and lyophilized to obtain 12.66 mg of a trifluoroacetic acid salt of the title compound.

[0977] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0978] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

TABLE-US-00041 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

[0979] The structural characterization data were as follows:

[0980] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.73 (s, 2H), 7.67 (s, 1H), 7.57 (s, 1H), 7.26 (s, 1H), 6.54 (s, 1H), 6.33 (s, 2H), 5.44 (s, 2H), 5.34 (s, 2H), 3.40 (s, 4H), 2.82 (s, 1H), 1.91-1.81 (m, 2H), 0.87 (t, J=7.2 Hz, 5H), 0.79 (d, J=7.4 Hz, 2H).

[0981] ESI-MS (m/z): 476.1[M+1]+.

Example 22

(S)-7-Ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxolano[4,5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-8,11(10H,13H)-dione (Compound 4-15)

[0982] ##STR00210## ##STR00211##

Step 1: Synthesis of (E)-N-(6-(3-((2-methoxyethyl)amino)acryloyl)benzo[d][1,3]dioxol-5-yl) acetamide

[0983] Compound 4-14-5 (200 mg, 0.72 mmol) was dissolved in ethanol (5 mL), then added dropwise with 2-methoxyethylamine (543.7 mg, 7.24 mmol), heated to 50? C. and reacted for 16 hours. The reaction was monitored by LCMS, showing a small amount of starting material remained, and the product was obvious. The solvent was evaporated under reduced pressure to obtain 221 mg of the title compound, which was directly used in the next reaction.

[0984] The structural characterization data were as follows:

[0985] ESI-MS (m/z): 307.1[M+1]+

Step 2: Synthesis of N-(6-(3-((2-methoxyethyl)amino)propionyl)benzo[d][1,3]dioxol-5-yl) acetamide

[0986] Compound 4-15-1 (200 mg, 0.65 mmol) was dissolved in glacial acetic acid (4 mL), then added with sodium borohydride (12.35 mg, 0.33 mmol) under stirring in an ice-water bath, warmed to room temperature and reacted under stirring for 3 hours. The reaction was monitored by LCMS, showing the starting material disappeared and the product was obvious. The solvent was evaporated under reduced pressure to obtain 200 mg of the title compound, which was directly used in the next reaction.

[0987] ESI-MS (m/z): 309.1[M+1]+

Step 3: Synthesis of (9H-fluoren-9-yl)methyl (3-(6-acetamidobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[0988] The crude product of compound 4-15-2 (200 mg, 0.65 mmol) was dissolved in 1,4-dioxane (20 mL) and water (20 mL), then added with 9-fluorenylmethyl-N-succinylimino carbonate (372 mg, 0.65 mmol) and sodium bicarbonate (231.5 mg, 2.76 mmol) under stirring, and reacted at room temperature for 2 hours. The reaction was monitored by TLC, showing the starting material disappeared, and the product was obvious. Water and ethyl acetate were added, stirred, allowed to stand for liquid separation, the organic phase was washed with saturated brine, dried and concentrated, and purified by silica gel column (eluent: 50% ethyl acetate/petroleum ether) to obtain 180 mg of the title compound.

Step 4: Synthesis of ((9H-fluoren-9-yl)methyl (3-(6-aminobenzo[d][1,3]dioxol-5-yl)-3-oxopropyl)(2-methoxyethyl)carbamate

[0989] Compound 4-15-3 (180 mg, 0.68 mmol) was dissolved in 1,4-dioxane (5 mL), added dropwise with 3N aqueous hydrochloric acid (5 mL), heated to 60? C. and reacted under stirring for 16 hours. Water and ethyl acetate were added and stirred, allowed to stand for liquid separation, the organic phase was washed with water, dried and concentrated, and purified by silica gel column (eluent: 45% ethyl acetate/petroleum ether) to obtain 132 mg of the title compound.

Step 5: Synthesis of (S)-(9H-fluoren-9-yl)methyl (2-(7-ethyl-7-hydroxy-8,11-dioxo-8,10,11,13-tetrahydro-7H-[1,3]dioxolano[4,5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-14-yl)ethyl)(2-methoxyethyl)carbamate

[0990] Compound 4-15-4 (130 mg, 0.266 mmol) and rac-(4S)-4-ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizin-3,6,10-trione (70.05 mg, 0.266 mmol) were dissolved in toluene (4 mL), then added with p-toluenesulfonic acid (9.16 mg, 0.053 mmol), heated to 120? C. and reacted for 4 hours. The reaction was monitored by LCMS, showing the starting material disappeared and the product was obvious. After concentration under reduced pressure, 190 mg of a crude product of the title compound was obtained.

[0991] The structural characterization data were as follows:

[0992] ESI-MS (m/z): 716.1[M+1]+

Step 6: Synthesis of (S)-7-ethyl-7-hydroxy-14-(2-((2-methoxyethyl)amino)ethyl)-7H-[1,3]dioxolano[4,5-g]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-8,11(10H,13H)-dione

[0993] Compound 4-15-5 (190 mg, 0.265 mmol) was dissolved in DMF (3 mL), then added dropwise with diethylamine (2 mL), and reacted under stirring for 1 hour. The reaction was monitored by LCMS, showing the starting material disappeared and the product was obvious. The reaction solution was distilled under reduced pressure to remove diethylamine, acidified by adding dropwise with 3N hydrochloric acid, purified, and lyophilized to obtain 99.28 mg of the title compound.

[0994] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[0995] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00042 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0 20 80 28 2 20 80 28 18 80 20 28

[0996] The structural characterization data were as follows:

[0997] .sup.1H NMR (400 MHz, DMSO-d.sub.6): ? 8.68 (s, 2H), 7.68 (s, 1H), 7.56 (s, 1H), 7.26 (s, 1H), 6.52 (s, 1H), 6.32 (s, 2H), 5.44 (s, 2H), 5.31 (s, 2H), 3.65-3.58 (m, 2H), 3.42 (d, J=10.2 Hz, 2H), 3.36 (s, 3H), 3.22 (d, J=4.0 Hz, 4H), 1.94-1.80 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[0998] ESI-MS (m/z): 494.2[M+1]+

Example 23

N-((1S,9S)-9-Ethyl-5-fluoro-9-hydroxy-4-methoxy-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 and N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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

[0999] ##STR00212## ##STR00213## ##STR00214##

Step 1: Synthesis of N-(3-bromo-5-fluoro-4-methoxyphenyl)acetamide

[1000] 3-Bromo-5-fluoro-4-methoxy-aniline (1.7 g, 7.73 mmol) was dissolved in tetrahydrofuran (30 mL), then added with triethylamine (2.35 g, 23.18 mmol) and acetic anhydride (1.18 g, 11.59 mmol), after the addition, the temperature was raised to 50? C., the reaction was stirred for 4 hours and monitored by LCMS; after the reaction solution was cooled to room temperature, it was diluted with ethyl acetate (50 mL), then washed with water (30 mL) and saturated brine (30 mL) once each, the organic phase was separated and dried over anhydrous sodium sulfate, filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was slurried (with petroleum ether: ethyl acetate=5:1) and purified to obtain 1.1 g of the title compound.

[1001] The structural characterization data were as follows:

[1002] ESI-MS (m/z): 262.0[M+H].sup.+.

Step 2: Synthesis of (E)-4-(5-acetylamino-3-fluoro-2-methoxyphenyl)-3-butenoic acid

[1003] N-(3-Bromo-5-fluoro-4-methoxyphenyl)acetamide (1.1 g, 4.20 mmol) and 3-butenoic acid (397.47 mg, 4.62 mmol) were dissolved in 1,4-dioxahexane (20 mL) and water (5 mL), then added with triethylamine (1.27 g, 12.59 mmol), tris(o-methylphenyl)phosphine (127.75 mg, 419.73 ?mol) and palladium acetate (47.12 mg, 209.89 ?mol), after the addition, the reaction system was subjected to nitrogen replacement three times, heated to 100? C. and reacted for 4 hours under nitrogen atmosphere; the reaction was monitored by LCMS; after the reaction solution was cooled to room temperature, 1 mol/L sodium hydroxide aqueous solution (50 mL) and ethyl acetate (50 mL) were added and shaken for liquid separation. The lower aqueous phase was separated and adjusted to pH of about 3 with 4 mol/L hydrochloric acid aqueous solution, and then extracted with ethyl acetate (40 mL*2), the organic phases were combined and washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 1.1 g of a crude product of the title compound.

[1004] The structural characterization data were as follows:

[1005] ESI-MS (m/z): 268.1 [M+H].sup.+.

Step 3: Synthesis of 4-(5-acetylamino-3-fluoro-2-methoxyphenyl)butanoic acid

[1006] The crude product of (E)-4-(5-acetylamino-3-fluoro-2-methoxyphenyl)-3-butenoic acid (1.1 g, 4.12 mmol) was dissolved in methanol (20 mL), then added with 10% palladium on carbon (100 mg), after the addition, the reaction system was subjected to hydrogen replacement three times with a hydrogen balloon, and the reaction was carried out for 4 hours under hydrogen atmosphere and monitored by LCMS; the reaction solution was filtered, the filtrate was concentrated under reduced pressure to dryness to obtain 1.05 g of a crude product of the title compound.

[1007] The structural characterization data were as follows:

[1008] ESI-MS (m/z): 270.1[M+H].sup.+.

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

[1009] The crude product of 4-(5-acetylamino-3-fluoro-2-methoxyphenyl)butanoic acid (1.1 g, 4.09 mmol) was dissolved in trifluoroacetic acid (10 mL), cooled to 5? C., then added slowly with trifluoroacetic anhydride (4.29 g, 20.43 mmol), after the addition, the reaction system was naturally warmed to room temperature and reacted for 2 hours; the reaction was monitored by LCMS; the reaction solution was slowly poured into water (60 mL), and then extracted with ethyl acetate (40 mL*3), the organic phases were combined, washed with saturated aqueous sodium bicarbonate until neutral, and then washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, and filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain a crude product, which was purified by flash silica gel column (ethyl acetate:petroleum ether=0-40%) to obtain 503 mg of the title compound.

[1010] The structural characterization data were as follows:

[1011] ESI-MS (m/z): 252.1 [M+H].sup.+.

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

[1012] Tetrahydrofuran (15 mL) and tert-butanol (4 mL) were added to a reaction flask, cooled to 5? C. in an ice bath, added with potassium tert-butoxide (491.26 mg, 4.38 mmol), then N-(3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (500 mg, 1.99 mmol) was dissolved in tetrahydrofuran (5 mL) and slowly added dropwise thereto, followed by an addition of isoamyl nitrite (373.01 mg, 3.18 mmol) after 10 minutes, after the addition, the reaction was carried out at 5? C. for 1 hour and monitored by LCMS; the reaction solution was quenched with saturated ammonium chloride aqueous solution (50 mL), extracted with ethyl acetate (40*2), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, then filtered, and the filtrate was evaporated to dryness under reduced pressure to obtain 550 mg of a crude product of the title compound.

[1013] The structural characterization data were as follows:

[1014] ESI-MS (m/z): 281.1[M+H].sup.+.

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

[1015] The crude product of N-(3-fluoro-7-(hydroxyimino)-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (520 mg, 1.86 mmol) was dissolved in a mixed solution of methanol (10 mL) and tetrahydrofuran (10 mL), then added with 1 mol/L aqueous hydrochloric acid (3.71 mL) and 10% palladium on carbon (50 mg), after the addition, the reaction system was subjected to hydrogen replacement three times with a hydrogen ball, and the reaction was carried out at room temperature under hydrogen atmosphere for 1 hour and monitored by LCMS; the reaction solution was filtered, and the filtrate was concentrated under reduced pressure to dryness to obtain 551 mg of a crude hydrochloride of the title compound.

[1016] The structural characterization data were as follows:

[1017] ESI-MS (m/z): 267.1[M+H].sup.+.

Step 7: Synthesis of (9H-fluoren-9-yl)methyl (8-acetamide-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1018] The crude hydrochloride of N-(7-amino-3-fluoro-4-methoxy-8-oxo-5,6,7,8-tetrahydronaphthalen-1-yl)acetamide (550 mg, 1.64 mmol) was dissolved in 1,4-dioxane (15 mL), then added with sodium bicarbonate (549.45 mg, 6.54 mmol), water (5 mL) and 9-fluorenylmethyl-N-succinimido carbonate (1.12 g, 1.96 mmol), after the addition, the reaction was carried out under stirring at room temperature for 2 hours and monitored by LCMS; the reaction solution was poured into water (50 mL), and then extracted with ethyl acetate (40 mL*2), the organic phases were combined, washed with saturated brine (40 mL), dried over anhydrous sodium sulfate, then filtered, and the filtrate was dried under reduced pressure to obtain a crude product. The crude product was purified by C18 (acetonitrile/0.05% formic acid aqueous solution, 20% acetonitrile-100% acetonitrile) reverse phase column to obtain 410 mg of the title compound.

[1019] The structural characterization data were as follows:

[1020] ESI-MS (m/z): 489.1[M+H].sup.+.

Step 8: Synthesis of (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate

[1021] (9H-Fluoren-9-yl)methyl (8-acetamide-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydro naphthalen-2-yl)carbamate (410 mg, 839.29 ?mol) was dissolved in dioxane (10 mL), and added with 12 mol/L concentrated hydrochloric acid (2 mL), after the addition, the temperature was raised to 70? C., the reaction was carried out for 2 hours and monitored by LCMS; the reaction solution was poured into water (30 mL), then extracted with ethyl acetate (30 mL*2), the organic phases were combined, washed with saturated brine (30 mL), dried over anhydrous sodium sulfate, then filtered, and the filtrate was dried under reduced pressure to obtain a crude product, which was purified by flash silica gel (ethyl acetate:petroleum ether=0-60%) to obtain 351 mg of the title compound.

[1022] The structural characterization data were as follows:

[1023] ESI-MS (m/z): 447.1[M+H].sup.+.

Step 9: Synthesis of (9H-fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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)carbamate

[1024] (S)-4-Ethyl-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizin-3,6,10(4H)-trione (247.64 mg, 940.71 ?mol) and (9H-fluoren-9-yl)methyl (8-amino-6-fluoro-5-methoxy-1-oxo-1,2,3,4-tetrahydronaphthalen-2-yl)carbamate (350 mg, 783.93 ?mol) were added to toluene (15 mL), and then added with p-toluenesulfonic acid (134.84 mg, 783.93 ?mol). After the addition, the temperature was raised to 135? C., the reaction was carried out for 4 hours. The reaction solution was directly evaporated to dryness at 135? C. under reduced pressure to obtain a crude product; and the crude product was purified by flash silica gel (methanol:dichloromethane=0-6%) column to obtain 358 mg of the title compound.

[1025] The structural characterization data were as follows:

[1026] ESI-MS (m/z): 674.2[M+H].sup.+.

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

[1027] (9H-Fluoren-9-yl)methyl ((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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)carbamate (358 mg, 531.41 ?mol) was dissolved in N,N-dimethylformamide (4 mL), then added with diethylamine (0.4 mL), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was evaporated to dryness under reduced pressure to obtain a crude product, which was slurried in ethyl acetate for purification to obtain 220 mg of the title compound.

[1028] The structural characterization data were as follows:

[1029] ESI-MS (m/z): 452.1 [M+H].sup.+.

Step 11: Synthesis of N-((1S,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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 and N-((1R,9S)-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-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

[1030] (9S)-1-Amino-9-ethyl-5-fluoro-9-hydroxy-4-methoxy-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione (50 mg, 110.76 ?mol) and 2-hydroxyacetic acid (16.85 mg, 221.51 ?mol) were dissolved in N,N-dimethylformamide (2 mL), then added with HATU (84.17 mg, 221.51 ?mol) and N,N-diisopropylethylamine (42.94 mg, 332.27 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers (5-34-A: 6.22 mg, 5-34-B: 9.81 mg).

[1031] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[1032] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

TABLE-US-00043 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 10 90 28 2.00 10 90 28 18.00 90 10 28

[1033] The structural characterization data of 5-34-A wre as follows:

[1034] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.53 (d, J=9.0 Hz, 1H), 7.89 (d, J=12.3 Hz, 1H), 7.33 (s, 1H), 5.58 (q, J=7.5, 7.0 Hz, 1H), 5.42 (d, J=2.1 Hz, 2H), 5.25-5.09 (m, 2H), 3.98 (s, 2H), 3.96 (d, J=1.1 Hz, 3H), 3.30-3.10 (m, 2H), 2.15 (q, J=7.4 Hz, 2H), 1.93-1.80 (m, 2H), 0.87 (t, J=7.3 Hz, 3H).

[1035] ESI-MS (m/z): 510.2[M+H].sup.+.

[1036] The structural characterization data of 5-34-B were as follows:

[1037] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.55 (d, J=9.0 Hz, 1H), 7.90 (d, J=12.3 Hz, 1H), 7.34 (s, 1H), 5.59 (q, J=7.4, 6.8 Hz, 1H), 5.43 (s, 2H), 5.25-5.11 (m, 2H), 3.99 (s, 2H), 3.96 (d, J=1.1 Hz, 3H), 3.30-3.13 (m, 2H), 2.15 (q, J=6.4 Hz, 2H), 1.93-1.82 (m, 2H), 0.88 (t, J=7.3 Hz, 3H).

[1038] ESI-MS (m/z): 510.2[M+H].sup.+.

Example 24

(2R)N-((1S,9S)-5-Chloro-9-ethyl-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-hydroxypropylamine and (2R)N-((1R,9S)-5-chloro-9-ethyl-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-hydroxypropylamine

[1039] ##STR00215##

Step 1: Synthesis of (2R)N-((1S,9S)-5-chloro-9-ethyl-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-hydroxypropylamine and (2R)N-((1R,9S)-5-chloro-9-ethyl-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-hydroxypropylamine

[1040] (9S)-1-Amino-5-chloro-9-ethyl-9-hydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H,13H-benzo[de]pyrano[3,4:6,7]indolizino[1,2-b]quinolin-10,13-dione (50 mg, 110.64 ?mol) and (2R)-2-hydroxypropane acid (19.93 mg, 221.29 ?mol) were dissolved in N,N-dimethylformamide (2 mL), then added with HATU (84.09 mg, 221.29 ?mol) and N,N-diisopropylethylamine (42.90 mg, 331.93 ?mol), after the addition, the reaction was carried out at room temperature for 0.5 hours and monitored by LCMS; the reaction solution was directly purified by preparative high performance liquid chromatography to obtain two isomers of the title compound (2-27-A: 5.73 mg, 2-27-B: 7.59 mg)

[1041] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[1042] Mobile phase A: acetonitrile; mobile phase B: water (0.05% trifluoroacetic acid)

TABLE-US-00044 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 90 28 2.00 30 90 28 18.00 90 10 28

[1043] The structural characterization data of 2-27-A were as follows:

[1044] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.45 (d, J=9.1 Hz, 1H), 8.14 (s, 1H), 7.30 (s, 1H), 6.54 (s, 1H), 5.63 (s, 1H), 5.56 (q, J=8.0 Hz, 1H), 5.42 (s, 2H), 5.25 (d, J=19.0 Hz, 1H), 5.08 (d, J=19.0 Hz, 1H), 4.13 (q, J=6.7 Hz, 1H), 3.27-3.12 (m, 2H), 2.51 (s, 3H), 2.23-2.13 (m, 2H), 1.92-1.80 (m, 2H), 1.41 (d, J=6.8 Hz, 3H), 0.87 (t, J=7.3 Hz, 3H).

[1045] ESI-MS (m/z): 524.2[M+H].sup.+.

[1046] The structural characterization data of 2-27-B were as follows:

[1047] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.40 (d, J=8.9 Hz, 1H), 8.15 (s, 1H), 7.31 (s, 1H), 6.54 (s, 1H), 5.55-5.49 (m, 1H), 5.43 (d, J=2.2 Hz, 2H), 5.18 (q, J=19.0 Hz, 2H), 4.13 (q, J=6.6 Hz, 1H), 3.24-3.12 (m, 2H), 2.51 (s, 3H), 2.22-2.10 (m, 2H), 1.92-1.82 (m, 2H), 1.30 (d, J=6.7 Hz, 3H), 0.88 (t, J=7.3 Hz, 3H).

[1048] ESI-MS (m/z): 524.2[M+H].sup.+.

Example 25

Preparation of (R)N-((1S,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-hydroxypropylamine and (R)N-((1R,9S)-4-chloro-9-ethyl-5-fluoro-9-hydroxy-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-hydroxypropylamine

[1049] ##STR00216##

[1050] At 25? C., 3-1-A (20.0 mg, 43.9 ?mol) and D-lactic acid (7.90 mg, 87.8 ?mol) were dissolved in DMF (1.0 mL), then added with HATU (33.4 mg, 87.8 ?mol) and DIPEA (17.0 mg, 131.6 ?mol), reacted at room temperature for 2 hours; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative high performance liquid chromatography to obtain compound 3-26-A (15.4 mg, yield 64%).

[1051] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[1052] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1053] Retention time: 5.3-6.2 min

TABLE-US-00045 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 70 28 2.00 30 70 28 18.00 90 10 28

[1054] The structural characterization data were as follows:

[1055] MS m/z (ESI): 528.2 [M+H].sup.+

[1056] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.52 (d, J=9.2 Hz, 1H), 8.05 (d, J=10.0 Hz, 1H), 7.33 (s, 1H), 6.55 (s, 1H), 5.62-5.59 (m, 2H), 5.43 (s, 2H), 5.28-5.10 (m, 2H), 4.13-4.11 (m, 1H), 3.41-3.38 (m, 1H), 3.28-3.22 (m, 1H), 2.20-2.18 (m, 2H), 1.92-1.80 (m, 2H), 1.40 (d, J=6.8 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H).

[1057] At 25? C., 3-1-B (20.0 mg, 43.9 ?mol) and D-lactic acid (7.90 mg, 87.8 ?mol) were dissolved in DMF (1.0 mL), then added with HATU (33.4 mg, 87.8 ?mol) and DIPEA (17.0 mg, 131.6 ?mol), reacted at room temperature for 2 hours; the reaction solution was concentrated to remove most of the DMF, and the residue was purified by preparative high performance liquid chromatography to obtain compound 3-26-B (4.8 mg, yield 20%).

[1058] Chromatographic column: SunFire Prep C18 OBD 19 mm?150 mm?5.0 ?m

[1059] Mobile phase A: acetonitrile; Mobile phase B: water (0.05% formic acid)

[1060] Retention time: 7.5-8.5 min

TABLE-US-00046 Time Mobile phase Mobile phase Flow rate [min] A [%] B [%] [mL/min] 0.00 30 70 28 4.00 30 70 28 20.00 90 10 28

[1061] The structural characterization data were as follows:

[1062] MS m/z (ESI): 528.2 [M+H].sup.+

[1063] .sup.1H NMR (400 MHz, DMSO-d.sub.6) ? 8.48 (d, J=8.8 Hz, 1H), 8.06 (d, J=10.4 Hz, 1H), 7.34 (s, 1H), 6.57 (br, 1H), 5.61-5.55 (m, 1H), 5.48-5.39 (m, 2H), 5.27-5.16 (m, 2H), 4.15-4.10 (m, 1H), 3.32-3.21 (m, 3H), 2.23-2.16 (m, 2H), 1.92-1.81 (m, 2H), 1.30 (d, J=6.4 Hz, 3H), 0.87 (t, J=7.2 Hz, 3H).

[1064] Biological Evaluation

[1065] 1. Tumor Cell Proliferation Inhibition Test

[1066] 1. Inhibitory Effect of Compounds on HT29 Cell Proliferation

[1067] (1) Cell plating: First, tumor cells HT29 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 1.

TABLE-US-00047 TABLE 1 Source of tumor cells Cell name Tumor type Source HT29 Human colon Cell Bank of Chinese cancer cells Academy of Sciences

[1068] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1069] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-?S) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 2.

[1070] (2) Data results

TABLE-US-00048 TABLE 2 Inhibitory activity on HT29 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 11.27 Example 1 (1-1-A) 1.28 Example 1 (1-1-B) 1.54 Example 3 (2-1-A) 5.03 Example 3 (2-2-B) 7.52 Example 4 (2-7-A) 1.56 Example 4 (2-7-B) 5.44 Example 5 (2-12-A) 3.01 Example 5 (2-12-B) 2.34 Example 5 (2-12-C) 1.49 Example 5 (2-12-D) 3.29 Example 6 (2-17-A) 1.24 Example 6 (2-17-B) 1.59 Example 7 (2-20-A) 1.29 Example 7 (2-20-B) 4.03 Example 8 (3-1-A) 0.16 Example 8 (3-1-B) 0.71 Example 9 (3-4-A) 5.55 Example 9 (3-4-B) 4.98 Example 10 (4-12) 4.79 Example 11 (4-10) 0.52 Example 12 (5-13-A) 0.88 Example 13 (5-7-A) 3.13 Example 13 (5-7-B) 4.66 Example 14 (5-16-A) 3.87 Example 20 (1-10-A) 61.60 Example 20 (1-10-B) 10.26 Example 21 (4-14) 0.56 Example 22 (4-15) 5.20

[1071] The detection results showed that the compounds of the present invention in Table 2 had potent inhibitory effect on the proliferation of HT29 colon cancer cells.

[1072] 2. Inhibitory effect of compounds on A549 cell proliferation

[1073] (1) Cell plating: First, tumor cells A549 were cultured in the corresponding medium, then digested with trypsin, centrifuged and resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 3.

TABLE-US-00049 TABLE 3 Source of tumor cells Cell name Tumor type Source A549 Human lung Cell Bank of Chinese cancer cells Academy of Sciences

[1074] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1075] In vitro cell activity detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 4.

[1076] (2) Data results

TABLE-US-00050 TABLE 4 Inhibitory activity on A549 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 94.18 Example 1 (1-1-A) 66.56 Example 4 (2-7-A) 6.97 Example 4 (2-7-B) 16.39 Example 5 (2-12-A) 6.96 Example 5 (2-12-B) 5.52 Example 5 (2-12-C) 6.64 Example 7 (2-20-A) 8.14 Example 7 (2-20-B) 23.06 Example 11 (4-10) 68.48

[1077] The detection results showed that the compounds of the present invention in Table 4 had significant inhibitory effect on the proliferation of A549 human lung cancer cells.

[1078] 3. Inhibitory effect of compounds on HCC-1806 cell proliferation

[1079] (1) Cell plating: First, tumor cells HCC1806 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 5.

TABLE-US-00051 TABLE 5 Source of tumor cells Cell name Tumor type Source HCC1806 Human breast squamous Cell Bank of Chinese carcinoma cells Academy of Sciences

[1080] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1081] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 6.

[1082] (2) Data results

TABLE-US-00052 TABLE 6 Inhibitory activity on HCC1806 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 3.03 Example 3 (2-1-A) 1.43 Example 4 (2-7-A) 1.25 Example 5 (2-12-A) 1.40 Example 5 (2-12-B) 1.07 Example 5 (2-12-C) 2.08 Example 12 (5-13-A) 0.96 Example 16 (3-12-A) 0.16 Example 16 (3-12-B) 1.76 Example 16 (3-12-C) 1.91 Example 16 (3-12-D) 1.53 Example 17 (3-7-B) 1.94 Example 18 (3-17-A) 1.70 Example 18 (3-17-B) 2.26 Example 19 (5-22-A) 2.57 Example 19 (5-22-B) 3.71 Example 24 (2-27-A) 4.55 Example 24 (2-27-B) 3.93 Example 25 (3-26-A) 1.27 Example 25 (3-26-B) 4.93

[1083] The detection results showed that the compounds of the present invention in Table 6 had significant inhibitory effect on the proliferation of HCC1806 human breast squamous carcinoma cells.

[1084] 4. Inhibitory effect of compounds on SKOV-3 cell proliferation

[1085] (1) Cell plating: First, tumor cells SKOV-3 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 7.

TABLE-US-00053 TABLE 7 Source of tumor cells Cell name Tumor type Source SKOV-3 Human ovarian Nanjing Cobioer cancer cells Biosciences

[1086] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1087] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 8.

[1088] (2) Data results

TABLE-US-00054 TABLE 8 Inhibitory activity on SKOV-3 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 17.14 Example 1 (1-1-A) 7.81 Example 3 (2-1-A) 7.12 Example 4 (2-7-A) 8.64 Example 4 (2-7-B) 9.47 Example 5 (2-12-A) 6.85 Example 5 (2-12-B) 4.99 Example 5 (2-12-C) 6.52 Example 7 (2-20-A) 6.79 Example 12 (5-13-A) 2.09 Example 21 (4-14) 2.16

[1089] The detection results showed that the compounds of the present invention in Table 8 had significant inhibitory effect on the proliferation of SKOV-3 human ovarian cancer cells.

[1090] 5. Inhibitory effect of compounds on NCIH358 cell proliferation

[1091] (1) Cell plating: First, tumor cells NCIH358 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 9.

TABLE-US-00055 TABLE 9 Source of tumor cells Cell name Tumor type Source NCI-H358 Human non-small cell Nanjing Cobioer lung cancer cells Biosciences

[1092] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1093] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 10.

[1094] (2) Data results

TABLE-US-00056 TABLE 10 Inhibitory activity on NCI-H358 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 58.84 Example 1 (1-1-A) 65.22 Example 2 (1-7-A) 11.38 Example 2 (1-7-B) 51.52 Example 3 (2-1-A) 68.62 Example 4 (2-7-A) 35.42 Example 4 (2-7-B) 51.06 Example 5 (2-12-A) 17.65 Example 5 (2-12-B) 15.76 Example 5 (2-12-C) 23.08 Example 7 (2-20-A) 18.56 Example 12 (5-13-A) 7.01 Example 16 (3-12-A) 15.45 Example 16 (3-12-B) 19.77 Example 16 (3-12-C) 28.29 Example 16 (3-12-D) 20.75 Example 17 (3-7-A) 49.38 Example 17 (3-7-B) 22.22 Example 18 (3-17-A) 13.34 Example 18 (3-17-B) 37.34 Example 19 (5-22-A) 29.94 Example 19 (5-22-B) 61.36 Example 21 (4-14) 11.98 Example 22 (4-15) 17.76 Example 24 (2-27-A) 56.46 Example 24 (2-27-B) 69.12 Example 25 (3-26-A) 31.64

[1095] The detection results showed that the compounds of the present invention in Table 10 had a significant inhibitory effect on the proliferation of NCIH358 human non-small cell lung cancer cells.

[1096] 6. Inhibitory effect of compounds on NCIN87 cell proliferation

[1097] (1) Cell plating: First, tumor cells NCIN87 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 11.

TABLE-US-00057 TABLE 11 Source of tumor cells Cell name Tumor type Source NCI-N87 Human gastric cancer ATCC cells

[1098] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1099] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 12.

[1100] (2) Data results

TABLE-US-00058 TABLE 12 Inhibitory activity on NCI-N87 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 7.18 Example 1 (1-1-A) 4.58 Example 2 (1-7-A) 2.70 Example 2 (1-7-B) 5.48 Example 4 (2-7-A) 3.76 Example 5 (2-12-A) 4.91 Example 5 (2-12-B) 4.97 Example 5 (2-12-C) 4.51 Example 7 (2-20-A) 4.54 Example 8 (3-1-A) 0.37 Example 8 (3-1-B) 1.34 Example 9 (3-4-A) 4.38 Example 9 (3-4-B) 6.51 Example 11 (4-10) 2.29 Example 12 (5-13-A) 2.33 Example 13 (5-7-A) 6.03 Example 13 (5-7-B) 7.31 Example 16 (3-12-A) 0.44 Example 16 (3-12-B) 3.45 Example 16 (3-12-C) 3.99 Example 16 (3-12-D) 3.03 Example 17 (3-7-A) 5.32 Example 17 (3-7-B) 4.54 Example 18 (3-17-A) 3.01 Example 18 (3-17-B) 4.19 Example 19 (5-22-A) 3.35 Example 19 (5-22-B) 5.51 Example 24 (2-27-A) 8.32 Example 24 (2-27-B) 14.67 Example 25 (3-26-A) 1.23 Example 25 (3-26-B) 25.34

[1101] The detection results showed that the compounds of the present invention in Table 12 had significant inhibitory effect on the proliferation of NCIN87 human gastric cancer cells.

[1102] 7. Inhibitory effect of compounds on Hela cell proliferation

[1103] (1) Cell plating: First, tumor cells Hela were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 13.

TABLE-US-00059 TABLE 13 Source of tumor cells Cell name Tumor type Source Hela Human cervical Nanjing Cobioer cancer cells Biosciences

[1104] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1105] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 14.

[1106] (2) Data results

TABLE-US-00060 TABLE 14 Inhibitory activity on Hela cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 17.57 Example 1 (1-1-A) 4.57 Example 1 (1-1-B) 3.65 Example 3 (2-1-A) 16.98 Example 4 (2-7-A) 13.77 Example 5 (2-12-A) 13.25 Example 5 (2-12-B) 16.68 Example 7 (2-20-A) 11.17 Example 11 (4-10) 3.04

[1107] The detection results showed that the compounds of the present invention in Table 14 had significant inhibitory effect on the proliferation of Hela human cervical cancer cells.

[1108] 8. Inhibitory effect of compounds on HCC70 cell proliferation

[1109] (1) Cell plating: First, tumor cells HCC70 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 15.

TABLE-US-00061 TABLE 15 Source of tumor cells Cell name Tumor type Source HCC70 Human breast Nanjing Cobioer cancer cells Biosciences

[1110] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1111] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 16.

[1112] (2) Data results

TABLE-US-00062 TABLE 16 Inhibitory activity on HCC70 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 226.10 Example 1 (1-1-A) 224.85 Example 4 (2-7-A) 150.70 Example 4 (2-7-B) 192.20 Example 5 (2-12-A) 170.93 Example 5 (2-12-B) 176.16 Example 5 (2-12-C) 210.68 Example 7 (2-20-A) 104.99 Example 12 (5-13-A) 36.65

[1113] The detection results showed that the compounds of the present invention in Table 16 had significant inhibitory effect on the proliferation of HCC70 human breast cancer cells. 9. Inhibitory effect of compounds on MDA-MB-231 cell proliferation

[1114] (1) Cell plating: First, tumor cells MDA-MB-231 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 17.

TABLE-US-00063 TABLE 17 Source of tumor cells Cell name Tumor type Source MDA-MB-231 Human breast Nanjing Cobioer cancer cells Biosciences

[1115] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1116] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 18.

[1117] (2) Data results

TABLE-US-00064 TABLE 18 Inhibitory activity on MDA-MB-231 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 381.70 Example 1 (1-1-A) 363.60 Example 3 (2-1-A) 299.70 Example 4 (2-7-A) 123.50 Example 5 (2-12-A) 123.50 Example 9 (3-4-A) 284.80 Example 12 (5-13-A) 332.60 Example 13 (5-7-B) 172.40 Example 20 (1-10-A) 97.57 Example 20 (1-10-B) 27.24 Example 22 (4-15) 175.40

[1118] The detection results showed that the compounds of the present invention in Table 18 had significant inhibitory effect on the proliferation of MDA-MB-231 human breast cancer cells.

[1119] 10. Inhibitory effect of compounds on Jeko-1 cell proliferation

[1120] (1) Cell plating: First, tumor cells Jeko-1 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 19.

TABLE-US-00065 TABLE 19 Source of tumor cells Cell name Tumor type Source Jeko-1 Human mantle cell lymphoma ATCC

[1121] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1122] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 20.

[1123] (2) Data results

TABLE-US-00066 TABLE 20 Inhibitory activity on Jeko-1 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 1.25 Example 1 (1-1-A) 1.33 Example 2 (1-7-A) 0.15 Example 2 (1-7-B) 0.66 Example 4 (2-7-A) 0.28 Example 9 (3-4-A) 0.27 Example 9 (3-4-B) 0.15 Example 11 (4-10) 0.45 Example 13 (5-7-A) 0.16 Example 13 (5-7-B) 0.18 Example 21 (4-14) 0.34 Example 22 (4-15) 0.11

[1124] The detection results showed that the compounds of the present invention in Table 20 had significant inhibitory effect on the proliferation of Jeko-1 human mantle cell lymphoma cells.

[1125] 11. Inhibitory effect of compounds on MDA-MB-453 cell proliferation

[1126] (1) Cell plating: First, tumor cells MDA-MB-453 were cultured in corresponding medium, then digested with trypsin, centrifuged, resuspended, and counted, and the cells were adjusted to an appropriate concentration for plating. The source of the tumor cells was listed in Table 21.

TABLE-US-00067 TABLE 21 Source of tumor cells Cell name Tumor type Source MDA-MB-453 Human breast cancer Concortis cells

[1127] Co-incubation of the compound of the present invention and tumor cells: after the cells adhered to the wall, the medium was removed, and the diluted bioactive molecule (the compound of the present invention) was added to the wells of the above plate, and incubated for 72 h.

[1128] In vitro cell viability detection: After the incubation, 50 ?L of Cell Counting-Lite? 2.0 reagent (Vazyme/Novazyme) was added to each well, shaken and mixed well in the dark, reacted for 10 minutes, and then the detection was carried out with a microplate reader (manufacturer: BMG, Model: PHERAStar-FS) for reading. The background RLU was obtained by using cell-free medium (with Cell Counting-Lite?), and the vehicle RLU was obtained by using cell-containing medium (with Cell Counting-Lite?). Cell inhibition rate=1?(sample RLU?background RLU)/(solvent RLU?background RLU)?100%. According to the four-parameter model fitting curve, the half maximal inhibitory concentration (IC50) of the compound was calculated, and the detection results were shown in Table 22.

[1129] (2) Data results

TABLE-US-00068 TABLE 22 Inhibitory activity on MDA-MB-453 cell proliferation Name IC.sub.50 (nM) Comparative Compound 1 6.99 Example 16 (3-12-A) 0.47 Example 16 (3-12-B) 4.32 Example 16 (3-12-C) 5.52 Example 16 (3-12-D) 3.98 Example 17 (3-7-A) 6.23 Example 17 (3-7-B) 4.87 Example 18 (3-17-A) 3.32 Example 18 (3-17-B) 6.16 Example 19 (5-22-A) 7.34 Example 19 (5-22-B) 5.67 Example 24 (2-27-A) 11.83 Example 25 (3-26-A) 3.95

[1130] The detection results showed that the compounds of the present invention in Table 22 had significant inhibitory effect on the proliferation of MDA-MB-453 human breast cancer cells.

[1131] The structures of Comparative Compound 1 and Comparative Compound 2 were as follows:

##STR00217##

[1132] 2. Antibody conjugation test

[1133] The preparation and conjugation of ADC D-L-15 sample was as follows:

[1134] 1.036 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg/mL) was diluted with 0.1M edetate disodium solution (pH 7.6), then adjusted with 1M Na.sub.2HPO.sub.4 solution to pH of 7.6, added with 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) in an amount 2.4 times the amount of substance, mixed well and allowed to stand at room temperature for 90 minutes. The above solution system was added with Compound D-L-15 dissolved in dimethyl sulfoxide in an amount 5 times the amount of substance, mixed well, allowed to stand at room temperature for 2 hours, and the buffer was replaced with 10 mM histidine buffer solution at pH 6.0 by using NAP-5 gel column (Cytiva), then sucrose and Tween-20 were added and mixed well to obtain the antibody drug conjugate ADC D-L-15 (1.77 mL, 8.60 mg/mL).

##STR00218##

[1135] The molecular weight of ADC D-L-15 was determined by LC-MS, and the calculated drug to antibody ratio, i.e., DAR value, was 4.11, as shown in Table 23 and Table 24.

TABLE-US-00069 TABLE 23 Measured molecular weight of ADC D-L-15 Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Measured 23356.98 24384.43 N/A N/A N/A value HC Measured 50172.24 51199.41 52226.65 53253.93 N/A value

TABLE-US-00070 TABLE 24 DAR values for ADC D-L-15 Name Maximum signal intensity Ratio DAR LC-DAR0 3266.67 0.453 4.11 LC-DAR1 3948.06 0.547 HC-DAR0 551.28 0.175 HC-DAR1 1141.65 0.362 HC-DAR2 774.39 0.246 HC-DAR3 685.97 0.218 HC-DAR4 0.00 0.000

[1136] Chromatographic determination conditions: [1137] Liquid chromatography column: Thermo MAbPac RP 3.0*100 mm; [1138] Mobile phase A: 0.1% FA/H.sub.2O; Mobile phase B: 0.1% FA/ACN; [1139] Flow rate: 0.25 mL/min; Sample chamber temperature: 8? C.; Column temperature: 60? C.; Injection volume: 2 ?L;

TABLE-US-00071 Time (min) 2 20 22 25 26 30 Mobile phase A (vol. %) 75 60 5 5 75 75 Mobile phase B (vol. %) 25 40 95 95 25 25

[1140] Mass spectrometry conditions: [1141] Mass spectrometer model: AB Sciex Triple TOF 5600+; [1142] GS1 35; GS2 35; CUR 30; TEM 350; ISVF 5500; DP 250; CE 10; Accumulation time 0.5 s; m/z 600-4000; Time bins to sum 40.

[1143] The preparation and conjugation of ADC 3-4-04-A sample was as follows:

[1144] 0.518 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg/mL) was diluted with 0.1M edetate disodium solution (pH 7.6), then adjusted to pH7.6 with 1M Na.sub.2HPO.sub.4 solution, added with 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) in an amount 5.5 times the amount of substance, mixed well and allowed to stand at room temperature for 90 minutes. The above solution system was added with Compound 3-4-04-A dissolved in dimethyl sulfoxide in an amount 10 times the amount of substance, mixed well and allowed to stand at room temperature for 2 hours; the buffer was replaced with 10 mM histidine buffer solution at pH 6.0 by using NAP-5 gel column (Cytiva), then sucrose and Tween-20 were added and mixed well to obtain the antibody-drug conjugate ADC 3-4-04-A (1.50 mL, 5.60 mg/mL).

##STR00219##

[1145] The molecular weight of ADC 3-4-04-A was determined by LC-MS, and the calculated drug to antibody ratio, i.e., DAR value, was 7.47, as shown in Table 25 and Table 26. The chromatographic determination conditions were the same as that of ADC D-L-15.

TABLE-US-00072 TABLE 25 Measured molecular weight of ADC 3-4-04-A Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Measured value 23356.79 24388.20 N/A N/A N/A HC Measured value 50171.57 51199.68 52233.48 53265.97 54297.82

TABLE-US-00073 TABLE 26 DAR values for ADC 3-4-04-A Name Maximum signal intensity Ratio DAR LC-DAR0 223.44 0.061 7.47 LC-DAR1 3458.32 0.939 HC-DAR0 122.34 0.068 HC-DAR1 0.09 0.000 HC-DAR2 1.11 0.001 HC-DAR3 1682.87 0.932 HC-DAR4 0.18 0.000

[1146] The preparation and conjugation of ADC 3-4-04-B sample was as follows:

[1147] 0.518 mL of hIgG antibody (anti-chicken lysozyme antibody, 19.3 mg/mL) was diluted with 0.1M edetate disodium solution (pH 7.6), then adjusted to pH7.6 with 1M Na.sub.2HPO.sub.4 solution, added with 10 mM TCEP (tris(2-carboxyethyl)phosphine) solution (pH 7.6) in an amount 5.5 times the amount of substance, mixed well and allowed to stand at room temperature for 90 minutes. The above solution system was added with Compound 3-4-04-B dissolved in dimethyl sulfoxide in an amount of 10 times the amount of substance, mixed well and allowed to stand at room temperature for 2 hours, and the buffer was replaced with 10 mM histidine buffer solution at pH 6.0 by using NAP-5 gel column (Cytiva), then sucrose and Tween-20 were added and mixed well to obtain the antibody-drug conjugate ADC 3-4-04-B (1.50 mL, 5.60 mg/mL).

##STR00220##

[1148] The molecular weight of ADC 3-4-04-B was determined by LC-MS, and the calculated drug to antibody ratio, i.e., DAR value, was 8.04, as shown in Table 27 and Table 28. The chromatographic determination conditions were the same as that of ADC D-L-15.

TABLE-US-00074 TABLE 27 Measured molecular weight of ADC 3-4-04-B Peptide chain mAb DAR1 DAR2 DAR3 DAR4 LC Measured value 23356.32 24388.01 N/A N/A N/A HC Measured value 50166.19 51196.05 52232.67 53265.82 54296.20

TABLE-US-00075 TABLE 28 DAR values for ADC 3-4-04-B Name Maximum signal intensity Ratio DAR LC-DAR0 0.02 0.000 8.04 LC-DAR1 3735.97 1.000 HC-DAR0 0.68 0.000 HC-DAR1 0.12 0.000 HC-DAR2 0.05 0.000 HC-DAR3 1867.41 0.980 HC-DAR4 36.69 0.019

[1149] The above experiments prove that the cytotoxic drug-linker compound of the present invention could be successfully conjugated with an antibody to obtain an antibody drug conjugate.

[1150] Although the specific implementation of the present invention has been described in detail, those skilled in the art will understand that, according to all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the protection scope of the present invention. The full scope of the present invention is given by the appended claims and any equivalents thereof.

CAMPTOTHECIN COMPOUND, PREPARATION METHOD THEREFOR, AND APPLICATION THEREOF

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A61K45/06

HUMAN NECESSITIES

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A61K47/6843

HUMAN NECESSITIES

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C07D491/22

CHEMISTRY; METALLURGY

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A61K47/68037

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A61P35/00

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C07D491/22

CHEMISTRY; METALLURGY

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A61K47/68

HUMAN NECESSITIES

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A61P35/00

HUMAN NECESSITIES

Abstract

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