CN117715913A

CN117715913A - Immunoconjugates and methods

Info

Publication number: CN117715913A
Application number: CN202280050890.XA
Authority: CN
Inventors: 韩晓军; 苏维·图拉·马留卡·奥尔; 凯文·杜安·邦克; 黄琴华; 金伯利·费舍尔
Original assignee: Zeno Management Inc
Current assignee: Yimi Rumei Co ltd
Priority date: 2021-07-19
Filing date: 2022-07-15
Publication date: 2024-03-15

Abstract

The present invention provides immunoconjugates of formula (I), ab- [ S-L ¹ ‑L ² ‑L ³ ‑L ⁴ ‑L ⁵ ‑L ⁶ ‑L ⁷ ‑D] _n (I) The immunoconjugate of formula (I) comprises a linking group for linking the antibody targeting ligand (Ab) to the drug (D). Embodiments of such immunoconjugates can be used to deliver the drug to a selected cell or tissue, for example, for treating cancer.

Description

Immunoconjugates and methods

Incorporation by reference of any priority application

Any and all applications identified in the application data sheet filed with the present application for which foreign or domestic priority claims are hereby expressly incorporated by reference under 37cfr 1.57.

Background

Technical Field

The present application relates to conjugates comprising a linking group for linking an antibody targeting ligand to a cell killing moiety (such as a drug), methods of making such conjugates, and methods of using such conjugates to deliver a cell killing moiety to a selected cell or tissue, for example, for treating or inhibiting cancer.

Description of the invention

Many antibody-drug conjugates (ADCs) have been developed for medical use. See, e.g., nejamoghaddam, M.et al, "anti-body-Drug Conjugates: possibilities and Challenges", avicenna J Med Biotech (1), 3-23 (2019). Antibodies in ADCs act as targeting agents to deliver drugs to selected cells or tissues, such as cancer cells or tumors. In the united states, the united states Food and Drug Administration (FDA) has approved several ADC formulations, including oantituzumab (inotuzumab ozogamicin) (trade name BESPONSA), gemtuzumab ozogamicin (gemtuzumab ozogamicin) (trade name MYLOTARG), veltuximab (brentuximab vedotin) (trade name ADCETRIS), and enmeltuzumab (ado-trastuzumab emtansine) (trade name KADCYLA).

Us patent 10,155,821 discloses ADCs in which an anti-tumor compound is conjugated to an anti-HER 2 antibody via a linker. See also U.S. patent publications 2020/0385486 and 2019/007780. Trastuzumab (Trastuzumab deruxtecan) is one example of an ADC in which an anti-HER 2 antibody (trastuzumab) is attached to an anti-tumor compound (delutecan) via a cleavable maleimide tetrapeptide linker. The FDA has approved a formulation called desicatuzumab (fam-trastuzumab deruxtecan-nxki) (trade name ENHERTU) for the treatment of adult patients with unresectable or metastatic HER2 positive breast cancer who have received two or more previous anti-HER 2 based regimens in a metastatic environment. Figure 1 shows the manner in which a linker is thought to link an antibody (mAb) to a drug moiety.

FDA approval represents a milestone in ongoing therapeutic ADC development. However, there remains a need for improved ADCs to help address the long felt need for additional options for treating cancer and/or delivering therapeutic payloads to selected cells or tissues.

Disclosure of Invention

Some embodiments provide an immunoconjugate of formula (I) comprising an antibody or antigen binding fragment (Ab) and a drug moiety (D) and a linker connecting the Ab to D. In one embodiment, the immunoconjugate of formula (I) comprises a drug moiety of formula (II).

One embodiment provides an immunoconjugate having formula (I),

Ab-[S-L ¹ -L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D] _n

(I)

wherein:

ab is an antibody or antigen binding fragment;

L ¹ is that

L ² Is absent from,Or->

Z ¹ And Z ² Each independently is hydrogen, halogen, NO ₂ 、-O-(C ₁ -C ₆ Alkyl) or C ₁ -C ₆ An alkyl group;

L ³ is- (CH) ₂ )n ¹ -C (=o) -or- (CH) ₂ CH ₂ O)n ¹ -(CH ₂ )n ¹ C(＝O)-；

n ¹ Independently an integer from 0 to 12;

L ⁴ is a tetrapeptide residue;

L ⁵ is absent or- [ NH (CH) ₂ )n ² ]n ³ -；

n ² An integer of 0 to 6;

n ³ an integer of 0 to 2;

L ⁶ is absent or not present

L ⁷ Is absent from,

D is a drug moiety; and is also provided with

n is an integer from 1 to 10.

In one embodiment, D in formula (I) is a drug moiety of formula (II) having the structure:

wherein:

R ¹ and R is ² Each independently selected from the group consisting of hydrogen, halogen, -CN, -OR ⁵ 、-NR ⁵ R ⁶ Substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl), substituted or unsubstituted-O- (C) ₁ -C ₆ Haloalkyl) - [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Or substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-such that R ¹ And R is ² Taken together to form a ring;

R ³ is hydrogen or substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ ；

R ⁴ Is hydrogen, substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² Or substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² ；

X ¹ is-O-, -S (O) _n6 ) -, -NH-, -O- (c=o) -, -NH- (c=o) -O-, -NH- (c=o) -NH-, or-NH-S (O) _n6 )-；

X ² is-OR ⁹ 、-SR ⁹ or-NHR ⁹ ；

R ⁵ And R is ⁶ Each independently is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ ；

m is 1 or 2;

n ⁴ and n ⁵ Each independently 0, 1 or 2, provided that n ⁴ And n ⁵ Are not 0;

n ⁶ 0, 1 or 2;

each Y is independently H or halogen;

each p is independently 1, 2, 3, 4, 5, or 6;

each q is independently 0, 1, 2, 3, 4, 5, or 6;

each t is independently 1, 2, 3, 4, 5, or 6;

R ⁷ is H, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ ；

R ⁸ Is substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ Substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ Or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₂ -X ³ ；

R ⁹ Is H, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ Provided that exactly R ⁷ And R is ⁹ One of them is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ The method comprises the steps of carrying out a first treatment on the surface of the And is also provided with

Each X is ³ independently-H, -OH, -SH or-NH ₂ 。

One embodiment provides a compound of formula (IV), or a pharmaceutically acceptable salt thereof, having the structure:

wherein:

R ³ is hydrogen or substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ ；

X ² is-OH, -SH or-NR ⁵ R ⁶ ；

R ⁵ And R is ⁶ Each independently is hydrogenHalogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ ；

R ⁷ Is H, -COR ⁸ 、-CO ₂ R ⁸ Or- (CO) -NHR ⁸ ；

R ⁸ Is substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _P O(CY ₂ )q] _t CY ₃ ；

m is 1 or 2;

n ⁶ 0, 1 or 2; and is also provided with

Each Y is independently H or halogen;

Each p is independently 1, 2, 3, 4, 5, or 6;

each q is independently 0, 1, 2, 3, 4, 5, or 6; and is also provided with

Each t is independently 1, 2, 3, 4, 5, or 6;

provided that formula (IV) does not represent delutidine or isatecan.

One embodiment provides a pharmaceutical composition comprising an immunoconjugate as described herein, a pharmaceutical compound as described herein, or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

One embodiment provides a method for treating cancer or tumor comprising administering to a subject having cancer or tumor an effective amount of an immunoconjugate as described herein, a pharmaceutical compound as described herein or a pharmaceutically active salt thereof, or a pharmaceutical composition as described herein.

One embodiment provides the use of an effective amount of an immunoconjugate as described herein, a pharmaceutical compound as described herein or a pharmaceutically active salt thereof, or a pharmaceutical composition as described herein, in the manufacture of a medicament for treating cancer or tumor.

Some embodiments provide a conjugate of formula (III) comprising a functional group M1, a drug moiety (D), and a linker connecting Mi to D. In one embodiment, the conjugate of formula (III) comprises a drug moiety of formula (II).

One embodiment provides a conjugate having formula (III),

Mi-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D

(III)

wherein:

mi is

L ² Is absent from,

n ¹ Independently an integer from 0 to 12;

L ⁴ is a tetrapeptide residue;

L ⁵ is absent or- [ NH (CH) ₂ )n ² ]n ³ -；

n ² An integer of 0 to 6;

n ³ an integer of 0 to 2;

L ⁶ is absent or not present

And is also provided with

L ⁷ Is absent from,

D is a drug moiety.

One embodiment provides a method of preparing an immunoconjugate, the method comprising: reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with a conjugate as described herein under reaction conditions effective to form an immunoconjugate as described herein.

Preferred alternatives include:

1. an antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

a VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 1;

a VHCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 2; and

a VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO 3; and

b) A light chain comprising:

VLCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 8;

a VLCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of AAS; and

VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 10;

wherein the antibody or antigen binding fragment thereof specifically binds to an extracellular domain of a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1).

2. The antibody or antigen-binding fragment of alternative 1, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 5, 7, 12 or 14.

3. One or more nucleic acids encoding the antibody or antigen binding fragment thereof according to any one of alternatives 1 or 2, such as an antibody or antigen binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to the nucleic acid sequence set forth in any one of SEQ ID NOs 4, 6, 11 or 13.

4. A host cell comprising one or more nucleic acids according to alternative 3.

5. The immunoconjugate according to any one of the preceding embodiments, wherein Ab is the antibody or antigen binding fragment according to claim 1 or 2 alternatively.

6. An antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

a VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 15;

a VHCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 16; and

a VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 17; and

b) A light chain comprising:

VLCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 22;

VLCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAY; and

VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 24;

7. The antibody or antigen-binding fragment of alternative 6, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 19, 21, 26 or 28.

8. One or more nucleic acids encoding an antibody or antigen binding fragment thereof according to any one of alternatives 6 or 7, such as an antibody or antigen binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 18, 20, 25 or 27.

9. A host cell comprising one or more nucleic acids according to alternative 8.

10. The immunoconjugate according to any one of the preceding embodiments, wherein Ab is an antibody or antigen binding fragment according to alternative 6 or 7.

11. An antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

a VHCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 29;

a VHCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 30; and

A VHCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 31; and

b) A light chain comprising:

VLCDR 1 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 36;

VLCDR 2 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of DAS; and

VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 38;

wherein the antibody or antigen binding fragment specifically binds to an extracellular domain of a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1).

12. The antibody or antigen-binding fragment of alternative 11, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 33, 35, 40 or 42.

13. One or more nucleic acids encoding an antibody or antigen binding fragment thereof according to any one of alternatives 11 or 12, such as an antibody or antigen binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 32, 34, 39 or 41.

14. A host cell comprising one or more nucleic acids according to alternative 13.

15. The immunoconjugate according to any one of the preceding embodiments, wherein Ab is an antibody or antigen binding fragment according to alternative 11 or 12.

16. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 6 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 13.

17. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of alternative 16, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 7 and the light chain comprises the polypeptide sequence of SEQ ID No. 14.

18. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of any one of alternatives 16 or 17, wherein the antibody or binding fragment thereof is conjugated to a molecule.

19. The antibody or binding fragment thereof of alternative 18, or a composition comprising the antibody or binding fragment thereof, wherein the molecule is a drug, toxin, or cytokine.

20. The immunoconjugate of any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to any one of alternatives 16 or 17.

21. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of alternatives 16 to 19, such as an immunoconjugate according to alternative 20, the method comprising administering the antibody or binding fragment thereof or the composition according to any one of alternatives 16 to 19 to a subject in need thereof, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

22. The antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of alternatives 16 to 19, such as the immunoconjugate according to alternative 20, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.

23. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 20 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 27.

24. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of alternative 23, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 21 and the light chain comprises the polypeptide sequence of SEQ ID No. 28.

25. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of any one of alternatives 23 or 24, wherein the antibody or binding fragment thereof is conjugated to a molecule.

26. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of alternative 25, wherein the molecule is a drug, toxin, or cytokine.

27. The immunoconjugate of any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to any one of alternatives 23 or 24.

28. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof according to any one of claims 23 to 26 or a composition comprising the antibody or binding fragment thereof, such as an immunoconjugate according to claim 27, the method comprising administering the antibody or binding fragment thereof according to any one of claims 23 to 26 or the composition to a subject in need thereof, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

29. The antibody or binding fragment thereof or the composition comprising the antibody or binding fragment thereof according to any one of alternatives 23 to 26, such as the immunoconjugate according to alternative 27, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.

30. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 34 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 41.

31. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of alternative 30, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 35 and the light chain comprises the polypeptide sequence of SEQ ID No. 42.

32. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of any one of alternatives 30 or 31, wherein the antibody or binding fragment thereof is conjugated to a molecule.

33. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof of alternative embodiment 32, wherein the molecule is a drug, toxin or cytokine.

34. The immunoconjugate of any one of claims 1 to 25, wherein the antibody or binding fragment thereof is an antibody or binding fragment thereof according to any one of alternatives 30 or 31.

35. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof according to any one of alternatives 30 to 33 or a composition comprising the antibody or binding fragment thereof, such as an immunoconjugate according to alternative 34, the method comprising administering an antibody or binding fragment thereof according to any one of alternatives 30 to 33 or the composition to a subject in need thereof, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

36. The antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of alternatives 30 to 33, such as the immunoconjugate according to alternative 34, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.

Drawings

Fig. 1 shows a detrastuzumab antibody-drug conjugate.

FIG. 2 shows a reaction scheme for preparing a compound of formula (IV). IV-2 is formed from IV-1 under acidic or basic hydrolysis conditions. The Fischer-Tropsch reaction of IV-2 and IV-3 gives IV-4. The protecting group (Pg) in IV-4 is removed to give IV-5. Alkylation, esterification or amidation of IV-5 to give IV.

FIG. 3 shows a reaction scheme for preparing a compound of formula (IV-1 a). The Hertz reaction between IV-6 and IV-7 gives IV-8. The IV-8 is hydrogenated to obtain IV-9, which is hydrolyzed to obtain IV-10. Intramolecular Fu Lide-Krafft reaction of IV-10 gives IV-11a. Alpha alkylation of IV-11a gives IV-12, which is treated with alpha-hydroxylation or alpha-amination conditions to give IV-1a (when n) ⁵ When=0).

FIG. 4 shows a reaction scheme for preparing a compound of formula (IV-1 b). Dehydrogenation of IV-11b yields IV-13. The Michael reaction of IV-13 followed by dehydrogenation gives IV-14.IV-14 Michael reaction with oxygen-containing or Nitrogen-containing nucleophiles to give IV-1b (when n ⁵ When=1).

Fig. 5 shows a reaction scheme for preparing a conjugate of formula (III), which includes attaching a linking moiety to a compound of formula (IV). The reaction of IIIA-1 and IIIA-2 under heating provides IIIA-3. Reaction of IIIA-3 with N-hydroxysuccinimide gives IIIA-4. The reaction of IIIA-4 and IIIA-5 gives IIIA-6.IIIA-6 and IIIA-7 are reacted under amide coupling conditions to yield IIIA-8.

FIG. 6 shows a reaction scheme for preparing IIIA-13. The reaction of IIIA-9 with N-hydroxysuccinimide (IIIA-10) gives IIIA-11, which is reacted with IIIA-12 to give IIIA-13.

FIG. 7 shows a reaction scheme for preparing IIIA-5. IIIA-14 and Pb (OAc) ₄ The reaction of (C) gives IIIA-15. IIIA-15 and IIIA-16 were treated in the presence of NaOH to give IIIA-17. Treatment of IIIA-17 with DBU gives IIIA-18, which is coupled to IIIA-19 to IIIA-20. Hydrogenation of IIIA-20 gives IIIA-5.

FIG. 8 shows a reaction scheme for preparing IIIB-4. The reaction of IIIA-4 and IIIB-1 in the presence of a base yields IIIB-2, which is reacted with 4-aminobenzyl alcohol and EEDQ to produce IIIB-3. Treatment of IIIB-3 with 4-nitrophenyl chloroformate gives IIIB-4.

FIG. 9 shows a reaction scheme for preparing IIIB-13. Reaction of IIIB-5 with DHP yields IIIB-6.IIIB-6 was reacted with oxalyl chloride and catalytic DMF to produce IIIB-7. The reaction of IIIB-7 and IIIA-7 gives IIIB-8, which is reacted with 4-nitrophenyl chloroformate to give IIIB-9. Reaction of IIIB-9 and IIIB-10 in the presence of a base gives IIIB-11. Treatment of IIIB-11 with TFA gave IIIB-12. The combination of IIIB-12 and IIIB-4 in the presence of a base gives IIIB-13.

FIG. 10 shows a reaction scheme for preparing IIIC-7. Reaction of IIIC-1 with Pb (OAc) 4 produces IIIC-2 which is reacted with IIIC-3 in ZnCl ₂ In the presence of (C) to give IIIC-4. Treatment of IIIC-4 with DBU gives IIIC-5. Amide coupling of IIIC-5 and IIIA-3 gives IIIC-6, which is reacted with HF-pyridine to give IIIC-7.

Fig. 11 shows a reaction scheme for preparing an immunoconjugate of formula (I), comprising attaching an Ab to a conjugate of formula (III). The michael reaction of the thiol group of cysteine from the antibody with the maleimide in formula III yields formula I.

FIG. 12 shows a reaction scheme for preparing compounds 1-14.

FIG. 13 shows a reaction scheme for preparing compounds 2-30.

FIG. 14 shows a reaction scheme for preparing compounds 3-34 and 3-35.

FIG. 15 shows a reaction scheme for preparing compounds 4-36 and 4-37.

FIG. 16 shows a reaction scheme for preparing compounds 5-47 and 5-48.

FIG. 17 shows a reaction scheme for preparing compounds 3-34 and 3-35.

FIG. 18 shows a reaction scheme for preparing compounds 7-59 and 7-60.

FIG. 19 shows a reaction scheme for preparing compounds 8-71 and 8-72.

FIG. 20 shows a reaction scheme for preparing compounds 9-74.

FIG. 21 shows a reaction scheme for preparing compounds 11-80.

FIG. 22 shows a reaction scheme for preparing compounds 13-84 and 13-85.

FIG. 23 shows a reaction scheme for preparing compounds 14-91 and 14-92.

FIG. 24 shows a reaction scheme for preparing compounds 15-94.

FIG. 25 shows a reaction scheme for preparing compounds 18-112.

FIG. 26 shows a reaction scheme for preparing compounds 21-120.

FIG. 27 shows measurement results of cell-binding saturation data for anti-ROR-1 antibodies produced by the methods described herein. The ROR-1 positive cell line JeKo-1 was incubated with the anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890 in a titration series as compared to the positive control antibody UC 961. Cells were washed, stained with secondary antibodies, and cell binding saturation was detected by flow cytometry and reported as Mean Fluorescence Intensity (MFI).

FIG. 28 shows ROR-1 receptor internalization data for anti-ROR-1 antibodies ATX-875, ATX-P-885, ATX-P-890. The ROR-1 positive cell lines JeKo-1 and MDA-MB-468 were incubated under supersaturated conditions with the anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890 and the positive control antibody UC961 in order to bind all available ROR-1 receptors. Cells were washed and incubated at 37 ℃ at 4 different time points (30 min, 1 hour, 2 hours and 4 hours) and then internalization was stopped by placing the cells in ice. Receptor internalization was determined by flow cytometry and reported as percent receptor internalization relative to zero hours.

FIGS. 29A-29D show the cell binning data for the anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890. Cell-sorting assays were performed to assess whether ATX-P-875, ATX-P-885 and ATX-P-890 bound the same epitope on the ROR-1 receptor as the control antibodies UC961 and 4A 5. (29A) depicts a staining pattern of antibodies binding to the same epitope. (29B) depicts a staining pattern of antibodies binding to different epitopes. ATX-P-875, ATX-P-885 and ATX-P-890 were incubated with different amounts of ROR-1_ + MDA-MB-468, respectively. Next, the anti-ROR-1 antibody was fluorescently labeled with a secondary antibody. Finally, MDA-MB-468 cells coated with anti-ROR-1 antibody were incubated with saturating doses of fluorescently labeled UC961 (29C) or 4A5 (29D) and analyzed by flow cytometry, and ATX-P-875, ATX-P-885, and ATX-P-890 antibody signals were compared to the UC961 or 4A5 signals.

FIG. 30 shows the AC-SINS data for anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890. Antibody developability was assessed by performing an AC-SINS assay and evaluating the potential for self-interactions. Rituximab (Rituximab) and Infliximab (Infliximab) were used as controls to demonstrate low drift and high drift, respectively. The results of the determination of ATX-P-875, ATX-P-885 and ATX-P-890 fall within the ranges determined for the control antibodies.

FIG. 31 shows the biochemical binning results by SPR of the anti-ROR 1 antibodies ATX-P-875, ATX-P-885, ATX-P-890 compared to control anti-ROR 1 antibodies UC961 (ATX-P-453) and 4a 5.

Figure 32 shows linker and payload combinations conjugated to 3 unique antibodies (mAb a=atx-P-875, mAb b=atx-P-885, mAb c=atx-P-890). Antibodies mAb A (ATX-P-875), mAb B (ATX-P-885) and mAb C (ATX-P-890) were conjugated to 6 separate new linkers/payloads (18-112, 19-113, 20-114, 21-120, 22-121, 23-122) to create antibody-drug conjugates (ADCs).

FIG. 33 shows a CTG assay in which ROR+ (JeKo-1) cells were incubated with ADC produced as described in FIG. 32 at successive three-fold dilutions and cell viability was assessed after 72 hours.

FIG. 34 shows the nucleotide and amino acid sequences of the anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890.

Detailed Description

Definition of the definition

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. All patents, applications, published applications, and other publications cited herein are incorporated by reference in their entirety unless otherwise indicated. Where there are multiple definitions for terms herein, the definitions in this section control unless otherwise indicated.

As used herein, a "conjugate" is a compound that comprises two or more substances (such as antibodies, linker moieties, and/or drug moieties) that are joined together by chemical bonds. Examples of conjugates include antibody-drug conjugates (which may optionally include a linker moiety), drug-linker conjugates, and antibody-linker conjugates. An "immunoconjugate" is a conjugate comprising an immune substance, such as an antibody.

As used herein, an "antibody" (Ab) is a protein prepared by the immune system or a synthetic variant thereof that binds to a specific site on a cell or tissue. An "antigen binding fragment" (Fab) is a portion of an antibody that binds to a specific antigen. Monoclonal antibodies are a class of synthetic antibodies. In cancer treatment, monoclonal antibodies may kill cancer cells directly, they may block the development of tumor vessels, or they may help the immune system kill cancer cells.

Whenever a group is described as "optionally substituted," the group may be unsubstituted or substituted with one or more of the indicated substituents. Also, when a group is described as "unsubstituted or substituted," if substituted, the substituents may be selected from one or more of the indicated substituents. If no substituent is indicated, it is intended that the indicated "optionally substituted" or "substituted" group may be substituted with one or more groups selected, individually and independently, from alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkenyl, aryl, heteroaryl, heterocyclyl, aryl (alkyl), cycloalkyl (alkyl), heteroaryl (alkyl), heterocyclyl (alkyl), hydroxy, alkoxy, acyl, cyano, halo, thiocarbonyl, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amido, N-amido, S-sulfonamido, N-sulfonamido, C-carboxy, O-carboxy, nitro, sulfinyl, haloalkyl, haloalkoxy, amino, monosubstituted amino groups, disubstituted amino groups, monosubstituted amine (alkyl) and disubstituted amine (alkyl).

As used herein, "C _a To C _b "wherein" a "and" b "are integers indicating the number of carbon atoms in the group. The indicated groups may contain from "a" to "b" (inclusive) carbon atoms. Thus, for example, "C ₁ To C ₄ Alkyl "groups refer to all alkyl groups having 1 to 4 carbons, i.e., CH ₃ -、CH ₃ CH ₂ -、CH ₃ CH ₂ CH ₂ -、(CH ₃ ) ₂ CH-、CH ₃ CH ₂ CH ₂ CH ₂ -、CH ₃ CH ₂ CH(CH ₃ ) -and (CH) ₃ ) ₃ C-. If "a" and "b" are not specified, then the broadest scope recited in these definitions is assumed.

If two "R" groups are described as "taken together," the R groups and the atoms to which they are attached may form a cycloalkyl, cycloalkenyl, aryl, heteroaryl, or heterocyclic ring. For example, but not limited to, if R is ortho to the benzene ring ¹ And R is ² The substituents are represented by-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² "taken together" to form a ring means-O- (CR) ⁵ R ⁶ ) _m -O-at R ¹ And R is ² Covalently bonded in position to the benzene ring to form a heterocycle:

as used herein, the term "alkyl" refers to a fully saturated aliphatic hydrocarbon group. The alkyl moiety may be branched or straight chain. Examples of branched alkyl groups include, but are not limited to, isopropyl, sec-butyl, tert-butyl, and the like. Examples of straight-chain alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, and the like. An alkyl group may have 1 to 30 carbon atoms (whenever it occurs herein, a numerical range such as "1 to 30" means each integer within the given range; for example, "1 to 30 carbon atoms" means an alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, although the definition of the invention also covers the term "alkyl" which occurs without a numerical range specified). The alkyl group may also be a medium size alkyl group having 1 to 12 carbon atoms. The alkyl group may also be a lower alkyl group having 1 to 6 carbon atoms. The alkyl group may be substituted or unsubstituted. Unless the context indicates otherwise, the alkyl group is generally monovalent. For example, one skilled in the art recognizes that C ₁ -C ₆ Alkyl groups are divalent in the formula: - (C) ₁ -C ₆ Alkyl) -X ² 。

As used herein, the term "alkylene" refers to a divalent fully saturated straight chain aliphatic hydrocarbon group. Examples of alkylene groups include, but are not limited to, methylene, ethylene, propylene, butylene, pentylene, hexylene, heptylene, and octylene. Alkylene groups can be usedRepresenting the number of carbon atoms followed by "". For example, a->Representing sub-regionsAnd (3) ethyl. An alkylene group may have from 1 to 30 carbon atoms (whenever it occurs herein, a numerical range such as "1 to 30" means each integer within the given range; e.g., "1 to 30 carbon atoms" means an alkyl group may consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 30 carbon atoms, although the definition of the invention also covers the term "alkylene" which occurs without a numerical range specified). The alkylene group may also be a medium-sized alkyl group having 1 to 12 carbon atoms. The alkylene group may also be a lower alkyl group having 1 to 4 carbon atoms. The alkylene group may be substituted or unsubstituted. For example, lower alkylene groups may be formed by replacing one or more hydrogens of the lower alkylene group and/or by replacing with a C3-6 monocyclic cycloalkyl group (e.g., ) To replace two hydrogens on the same carbon.

The term "alkenyl" as used herein refers to a monovalent straight or branched chain group of two to twenty carbon atoms containing one or more carbon double bonds and includes, but is not limited to, 1-propenyl, 2-methyl-1-propenyl, 1-butenyl, 2-butenyl, and the like. The alkenyl group may be unsubstituted or substituted.

The term "alkynyl" as used herein refers to a monovalent straight or branched chain group of two to twenty carbon atoms containing one or more carbon triple bonds, including but not limited to 1-propynyl, 1-butynyl, 2-butynyl, and the like. Alkynyl groups may be unsubstituted or substituted.

As used herein, the term "halogen atom" or "halogen" means any of the radioactively stable atoms in column 7 of the periodic table of elements, such as fluorine, chlorine, bromine and iodine.

As used herein, "haloalkyl" refers to an alkyl group in which one or more of the hydrogen atoms is replaced with a halogen (e.g., monohaloalkyl, dihaloalkyl, trihaloalkyl, and polyhaloalkyl). Such groups include, but are not limited to, chloromethyl, fluoromethyl, difluoromethyl, trifluoromethyl, 1-chloro-2-fluoromethyl, 2-fluoroisobutyl and pentafluoroethyl. Haloalkyl groups may be substituted or unsubstituted.

As used herein, "haloalkenyl" refers to alkenyl groups (e.g., monohaloalkenyl, dihaloalkenyl, trihaloalkenyl, and polyhaloalkenyl) in which one or more of the hydrogen atoms are replaced with halogen.

As used herein, "haloalkynyl" refers to an alkynyl group in which one or more of the hydrogen atoms are replaced with halogen (e.g., monohaloalkynyl, dihalynyl, trihaloalkynyl, and polyhaloalkynyl).

As used herein, "haloalkoxy" refers to an alkoxy group (e.g., monohaloalkoxy, dihaloalkoxy, and trihaloalkoxy) in which one or more of the hydrogen atoms are replaced with halogen. Such groups include, but are not limited to, chloromethoxy, fluoromethoxy, difluoromethoxy, trifluoromethoxy, 1-chloro-2-fluoromethoxy and 2-fluoroisobutoxy. Haloalkoxy groups may be substituted or unsubstituted.

Where the number of substituents (e.g., haloalkyl, haloalkenyl, haloalkynyl) is not specified, one or more substituents may be present. For example, "haloalkyl" may include one or more of the same or different halogens. As another example, "C ₁ To C ₃ Alkoxyphenyl "may include one or more of the same or different alkoxy groups containing one, two or three atoms.

As used herein, free radical refers to a species having a single unpaired electron such that the species containing the free radical can be covalently bonded to another species. Thus, in this context, the radicals are not necessarily free radicals. Instead, free radicals refer to specific portions of larger molecules. The term "radical" may be used interchangeably with the term "group".

The term "pharmaceutically acceptable salt" refers to a salt of a compound that does not cause significant irritation to the organism to which it is applied and does not abrogate the biological activity and properties of the compound. In some embodiments, the salt is an acid addition salt of a compound. Pharmaceutical salts can be prepared by reacting a compound with an inorganic acid (such as a hydrohalic acid (e.g., hydrochloric acid or hydrogenBromate), sulfuric acid, nitric acid, and phosphoric acid (such as 2, 3-dihydroxypropyl dihydrogen phosphate). Pharmaceutical salts may also be obtained by reacting a compound with an organic acid such as an aliphatic or aromatic carboxylic or sulfonic acid (e.g., formic acid, acetic acid, succinic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, nicotinic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, benzoic acid, salicylic acid, 2-oxoglutarate or naphthalenesulfonic acid). Pharmaceutical salts can also be obtained by reacting a compound with a base to form a salt, such as an ammonium salt, an alkali metal salt (such as a sodium, potassium or lithium salt), an alkaline earth metal salt (such as a calcium or magnesium salt), a carbonate, a bicarbonate, an organic base (such as dicyclohexylamine, N-methyl-D-glucamine, tris (hydroxymethyl) methylamine, C ₁ -C ₇ Salts of alkylamines, cyclohexylamines, triethanolamine, ethylenediamine) and salts formed by reaction with amino acids such as arginine and lysine. For the compounds of formula (I), those skilled in the art understand that when the salt is formed from a nitrogen-based group (e.g., NH ₂ ) The nitrogen-based group may associate with a positive charge (e.g., NH ₂ Can be changed into NH ₃ ⁺ ) And the positive charge may be formed by a negatively charged counterion (such as Cl ^- ) Balance.

It will be appreciated that in any of the compounds described herein having one or more chiral centers, each center may independently be in the R-or S-configuration or mixtures thereof, if absolute stereochemistry is not explicitly indicated. Thus, the compounds provided herein can be enantiomerically pure enantiomerically enriched racemic mixtures or diastereomerically pure diastereomerically enriched stereoisomeric mixtures. Furthermore, it should be understood that in any of the compounds described herein having one or more double bonds that produce a geometric isomer that may be defined as E or Z, each double bond may independently be E or Z or a mixture thereof. Also, it should be understood that in any of the compounds described, all tautomeric forms are also intended to be included.

It is to be understood that where the compounds disclosed herein have an valency less than full, they are filled with hydrogen or isotopes thereof, such as hydrogen-1 (protium) and hydrogen-2 (deuterium).

It is understood that the compounds described herein may be isotopically labeled. Substitution with isotopes such as deuterium may afford certain therapeutic advantages resulting from increased metabolic stability, such as increased in vivo half-life or reduced dosage requirements, for example. Each chemical element as represented in the structure of the compound may comprise any isotope of the element. For example, in the structure of a compound, the presence of a hydrogen atom in the compound may be explicitly disclosed or understood. At any position of the compound where a hydrogen atom may be present, the hydrogen atom may be any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Thus, unless the context clearly indicates otherwise, reference to a compound herein encompasses all possible isotopic forms.

It is to be understood that the methods and combinations described herein include crystalline forms (also referred to as polymorphs, which include different crystal packing arrangements of the same elemental composition of the compound), amorphous phases, salts, solvates, and hydrates. In some embodiments, the compounds described herein are present in solvated form with pharmaceutically acceptable solvents (such as water, ethanol, and the like). In other embodiments, the compounds described herein exist in unsolvated forms. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and can form during the crystallization process with pharmaceutically acceptable solvents (such as water, ethanol, etc.). The hydrate forms when the solvent is water or the alkoxide forms when the solvent is an alcohol. Furthermore, the compounds provided herein may exist in unsolvated forms as well as solvated forms. In general, solvated forms are considered equivalent to unsolvated forms useful for the purposes of the compounds and methods provided herein.

For the range values provided, it is understood that each intervening value, between the upper and lower limit of that range, is encompassed within the embodiments.

Terms and phrases used in this application, and particularly in the appended claims, and variations thereof, should be construed to be open ended, and not limiting, unless otherwise specifically noted. For the foregoing examples, the term "including" should be construed as "including but not limited to", etc.; as used herein, the term 'comprising' is synonymous with 'comprising', 'containing' or 'characterized as' and is inclusive or open-ended, and does not exclude additional, unrecited elements or method steps; the term 'having' should be interpreted as 'having at least'; the term 'comprising' should be interpreted as 'including but not limited to'; the term 'example' is used to provide an illustrative example of the item in question, rather than an exhaustive or limiting list thereof; and the use of terms such as 'preferably', 'preferred', 'desired' and 'expected' and terms of similar semantics should not be construed to imply that certain features are critical, essential or even important to the structure or function, but are merely intended to highlight alternative or additional features that may or may not be utilized in a particular embodiment. Furthermore, the term "comprising" should be interpreted as synonymous with the phrase "having at least" or "comprising at least". The term "comprising" when used in the context of a compound, composition or device means that the compound, composition or device contains at least the recited features or components, but may also contain additional features or components.

For substantially any plural and/or singular terms used herein, those having skill in the art can translate from the plural to the singular and/or from the singular to the plural as is appropriate to the context and/or application. For clarity, various singular/plural permutations may be explicitly stated herein. The indefinite article "a" or "an" does not exclude a plurality. The mere fact that certain measures are recited in mutually different dependent claims does not indicate that a combination of these measures cannot be used to advantage. Any reference signs in the claims shall not be construed as limiting the scope.

Compounds of formula (I)

Various embodiments disclosed herein relate to a compound of formula (IV), or a pharmaceutically acceptable salt thereof, having the structure:

in various embodiments, R in formula (IV) ¹ And R is ² Each independently selected from the group consisting of hydrogen, halogen, -CN, -OR ⁵ 、-NR ⁵ R ⁶ Substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl), substituted or unsubstituted-O- (C) ₁ -C ₆ Haloalkyl) - [ (CY) ₂ ) _p O(CY ₂ )q] _t CY ₃ Or substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-such that R ¹ And R is ² Taken together to form a ring. In one embodiment, R ¹ And R is ² At least one of which is hydrogen. In one embodiment, R ¹ And R is ² At least one of which is halogen. For example, in one embodiment, R ¹ And R is ² At least one of which is fluorine. In one embodiment, R ¹ And R is ² At least one of them is-CN. In one embodiment, R ¹ And R is ² At least one of them is-OR ⁵ Wherein R is ⁵ Is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . For example, in one embodiment, R ¹ And R is ² At least one of which is methoxy.

In one embodiment, R in formula (IV) ¹ And R is ² At least one of them is-NR ⁵ R ⁶ Wherein R is ⁵ And R is ⁶ Each independently is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . In one embodiment, R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ An alkyl group. For example, in one embodiment, R ¹ And R is ² At least one of which is methyl. In one embodiment, R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. For example, in one embodiment, R ¹ And R is ² At least one of them is difluoromethyl. In one embodiment, R ¹ And R is ² At least one of them is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). For example, in one embodiment, R ¹ And R is ² At least one of which is methoxy. In one embodiment, R ¹ And R is ² At least one of which is- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . In one embodiment, R ¹ And R is ² Is a substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² Taken together to form a ring, wherein-O- (CR) ⁵ R ⁶ ) _m The end of the-O-is at R of formula (IV) ¹ And R is ² Covalently bonded in position to the benzene ring to form a heterocycle.

In one embodiment, R in formula (IV) ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of which is halogen. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are hydrogen. In one embodiment, R ¹ And R is ² Neither is hydrogen.

In one embodimentIn the formula (IV), R is ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently halogen. In one embodiment, R ¹ And R is ² Neither is halogen.

In one embodiment, R in formula (IV) ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Alkyl, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Alkyl, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted C ₁ -C ₆ An alkyl group.

In one embodiment, R in formula (IV) ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Haloalkyl, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted C ₁ -C ₆ A haloalkyl group.

In one embodiment, R in formula (IV) ¹ And R is ² One of them being substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Is a substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² Taken together to form a ring. In various embodiments, R ¹ And R is ² Each independently selected from the group consisting of hydrogen, fluorine, methoxy, methyl, difluoromethyl and-O- (CH) ₂ ) -O-such that R ¹ And R is ² Taken together to form a ring.

In various embodiments, R in formula (IV) ³ Is hydrogen or substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independently H or halogen. In one embodiment, R ³ Is hydrogen. In one embodiment, R ³ Is substituted or unsubstituted C ₁ -C ₆ An alkyl group. For example, in one embodiment, R ³ Is methyl. In one embodiment, R ³ Is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ³ Is- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independently H or halogen.

In various embodiments, R in formula (IV) ⁴ Is hydrogen, substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² Wherein X is ² is-OH, -SH or-NR ⁵ R ⁶ . In one embodiment, R ⁴ Is hydrogen. In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkenyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² 。

In various embodiments, X in formula (IV) ¹ is-O-, -S (O) _n6 ) -, -NH-, -O- (c=o) -, -NH- (c=o) -O-, -NH- (c=o) -NH-, or-NH-S (O) _n6 ) -, where n ⁶ 0, 1 or 2. In one embodiment, X ¹ is-O-. In one embodiment, X ¹ is-S (O) _n6 ) -. In one embodiment, X ¹ is-NH-. In one embodiment, X ¹ is-O- (c=o) -. In one embodiment, X ¹ is-NH- (c=o) -. In one embodiment, X ¹ is-NH- (C=O) -O-. In one embodiment, X ¹ is-NH- (C=O) -NH-. In one embodiment, X ¹ is-NH-S (O) _n6 )-。

In various embodiments, X in formula (IV) ² is-OH, -SH or-NR ⁵ R ⁶ Wherein R is ⁵ And R is ⁶ Each independently is hydrogen, halogen or substituted or unsubstituted C ₁ -C ₆ Alkyl group、C ₁ -C ₆ Haloalkyl, - [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . In one embodiment, X ² is-OH. In one embodiment, X ² is-SH. In one embodiment, X ² is-NR ⁵ R ⁶ 。

In various embodiments, R in formula (IV) ⁵ And R is ⁶ Each independently is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independently H or halogen, and the variables p, q, and t are as described elsewhere herein. In one embodiment, R ⁵ And R is ⁶ Each independently is hydrogen or substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ⁵ And R is ⁶ Both are hydrogen. In one embodiment, R ⁵ And R is ⁶ Each independently is a substituted or unsubstituted C ₁ -C ₆ An alkyl group.

In various embodiments, R in formula (IV) ⁷ Is H, -COR ⁸ 、-CO ₂ R ⁸ Or- (CO) -NHR ⁸ Wherein R is ⁸ Described elsewhere herein. In one embodiment, R ⁷ H. In one embodiment, R ⁷ is-COR ⁸ . In one embodiment, R ⁷ is-CO ₂ R ⁸ . In one embodiment, R ⁷ Is- (CO) -NHR ⁸ 。

In various embodiments, R in formula (IV) ⁸ Is substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein the variables p, q, t and Y are described elsewhere herein. In one embodiment, R ⁸ Is substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ⁸ Is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ⁸ Is- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ 。

In various embodiments, m in formula (IV) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

In various embodiments, n in formula (IV) ⁴ And n ⁵ Each independently 0, 1 or 2, provided that n ⁴ And n ⁵ Neither is 0. In one embodiment, n ⁴ And n ⁵ Both are 1. In one embodiment, n ⁴ Is 0 and n ⁵ 1. In one embodiment, n ⁴ Is 0 and n ⁵ 2. In one embodiment, n ⁴ Is 1 and n ⁵ Is 0. In one embodiment, n ⁴ Is 2 and n ⁵ Is 0.

In various embodiments, n in formula (IV) ⁶ 0, 1 or 2. In one embodiment, n ⁶ 0, in which case X ¹ is-S-or-NH-S-. In one embodiment, n ⁶ 1, in which case X ¹ is-S (=o) -or-NH-S (=o) -. In one embodiment, n ⁶ 2, in which case X ¹ is-S (=O) ₂ -or-NH-S (=o) ₂ -。

In various embodiments, each Y in formula (IV) is independently H or halogen. In one embodiment, each Y is hydrogen. In one embodiment, -CY ₂ Is CH ₂ . In one embodiment, -CY ₃ Is CH ₃ . In one embodiment, -CY ₃ For CHF ₂ . In one embodiment, -CH ₂ F is CH ₃ . In one embodiment, -CY ₃ Is CF (CF) ₃ 。

In various embodiments, each p in formula (IV) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, p is 1. In one embodiment, p is 2.

In various embodiments, each q in formula (IV) is independently 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q is 1. In one embodiment, q is 2.

In various embodiments, each t in formula (IV) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, t is 1. In one embodiment, p is t.

In various embodiments, formula (IV) does not represent delutinkang or isatecan.

In various embodiments, the compound of formula (IV) is represented by formula (IVa):

in formula (IVa), the variables are the same as defined elsewhere herein for formula (IV).

In various embodiments, the compound of formula (IV) is represented by formula (IVb):

in formula (IVb), the variables are the same as defined elsewhere herein for formula (IV).

In various embodiments, the compound of formula (IV) is represented by formula (IVc):

in formula (IVc), the variables are the same as defined elsewhere herein for formula (IV).

In various embodiments, the compound of formula (IV) is represented by a structure selected from the group consisting of:

conjugate(s)

Various embodiments disclosed herein relate to conjugates of formula (III) having the following structure:

Mi-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D

(III)

In various embodiments, mi in formula (III) isD is a drug moiety, and-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -a linker connecting Mi to D. />

In various embodiments, L in formula (III) ² Is absent from,Or->Wherein Z is ¹ And Z ² Each independently is hydrogen, halogen, NO ₂ 、-O-(C ₁ -C ₆ Alkyl) or C ₁ -C ₆ An alkyl group. In one embodiment, L in formula (III) ² Is absent. In one embodiment, L in formula (III) ² Is->In a real worldIn embodiments, L in formula (III) ² Is->

In various embodiments, Z in formula (III) ¹ And Z ² Each independently is hydrogen, halogen, NO ₂ 、-O-(C ₁ -C ₆ Alkyl) or C ₁ -C ₆ An alkyl group. In one embodiment, Z ¹ And Z ² At least one of which is hydrogen. In one embodiment, Z ¹ And Z ² At least one of which is halogen. In one embodiment, Z ¹ And Z ² At least one of which is NO ₂ . In one embodiment, Z ¹ And Z ² At least one of them is-O- (C) ₁ -C ₆ Alkyl). For example, in one embodiment, Z ¹ And Z ² At least one of which is methoxy. In one embodiment, Z ¹ And Z ² At least one of which is C ₁ -C ₆ An alkyl group. For example, in one embodiment, Z ¹ And Z ² At least one of which is methyl.

In various embodiments, L in formula (III) ³ Is- (CH) ₂ )n ¹ -C (=o) -or- (CH) ₂ CH ₂ O)n ¹ -(CH ₂ )n ¹ C (=o) -, wherein n ¹ Independently an integer from 0 to 12. In one embodiment, L ³ Is- (CH) ₂ )n ¹ -C (=o) -. For example, in one embodiment, L ³ is-C (=o) -. In one embodiment, L ³ Is- (CH) ₂ CH ₂ O)n ¹ -(CH ₂ )n ¹ C (=o) -. For example, in one embodiment, L ³ is-CH ₂ C (=o) -. In embodiments, n ¹ Is an integer of 1 to 12, such as 1 to 6 or 1 to 3.

In various embodiments, L in formula (III) ⁴ Are tetrapeptide residues. For example, in one embodiment, L ⁴ Is selected from GGFG (gly-gly-phe-gly), EGGF (glu-gly-gly-phe), SGGF (ser-gly-gly-phe) and KGGF(lys-gly-gly-phe).

In various embodiments, L in formula (III) ⁵ Is absent or- [ NH (CH) ₂ )n ² ]n ³ -, where n ² Is an integer of 0 to 6 and n ³ Is an integer of 0 to 2. In one embodiment, L ⁵ Is absent. In one embodiment, L ⁵ Is- [ NH (CH) ₂ )n ² ]n ³ -. For example, in one embodiment, L ⁵ is-NH-. In another embodiment, L ⁵ is-NHCH ₂ -。

In various embodiments, L in formula (III) ⁶ Is absent or not presentIn one embodiment, L ⁶ Is absent. In another embodiment, L ⁶ Is->

In various embodiments, L in formula (III) ⁷ Is absent from,In one embodiment, L ⁷ Is absent. In one embodiment, L ⁷ Is->In one embodiment, L ⁷ Is thatIn one embodiment, L ⁷ Is->In one embodiment, L ⁷ Is thatIn various embodiments, formula (la)D in the conjugate of (III) is a drug moiety as described herein (e.g., according to the heading "drug moiety" below). In one embodiment, D is a cytotoxic anticancer drug moiety.

In various embodiments, the conjugate of formula (III) is represented by a structure selected from the group consisting of ¹ And Z ² Each independently selected from hydrogen, fluorine, chlorine, -NO ₂ and-OCH ₃ ：

Drug fraction

In various embodiments, D in the immunoconjugate of formula (I) or the conjugate of formula (III) is a drug moiety. The drug moiety may be any compound of formula (IV) as described herein (e.g., as described above under the heading "compound") suitably modified such that linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -connected to D. For example, in various embodiments, drug moiety D is a compound of formula (II) having the structure:

those skilled in the art will appreciate that the compound of formula (II) is represented by formula (II) via R ⁴ (when defined as including X) ² And thus includes R ⁹ When) or via R ⁷ To the linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -, as described below.

In various embodiments, R in formula (II) ¹ And R is ² Each independently selected from the group consisting of hydrogen, halogen, -CN, -OR ⁵ 、-NR ⁵ R ⁶ Substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl), substituted or unsubstituted-O- (C) ₁ -C ₆ Haloalkyl) - [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Or substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-such that R ¹ And R is ² Taken together to form a ring. In one embodiment, R ¹ And R is ² At least one of which is hydrogen. In one embodiment, R ¹ And R is ² At least one of which is halogen. For example, in one embodiment, R ¹ And R is ² At least one of which is fluorine. In one embodiment, R ¹ And R is ² At least one of them is-CN. In one embodiment, R ¹ And R is ² At least one of them is-OR ⁵ Wherein R is ⁵ Is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . For example, in one embodiment, R ¹ And R is ² At least one of which is methoxy.

In one embodiment, R in formula (II) ¹ And R is ² At least one of them is-NR ⁵ R ⁶ Wherein R is ⁵ And R is ⁶ Each independently is hydrogen, halogenPlain, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . In one embodiment, R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ An alkyl group. For example, in one embodiment, R ¹ And R is ² At least one of which is methyl. In one embodiment, R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. For example, in one embodiment, R ¹ And R is ² At least one of them is difluoromethyl. In one embodiment, R ¹ And R is ² At least one of them is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). For example, in one embodiment, R ¹ And R is ² At least one of which is methoxy. In one embodiment, R ¹ And R is ² At least one of which is- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ . In one embodiment, R ¹ And R is ² Is a substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² Taken together to form a ring, wherein-O- (CR) ⁵ R ⁶ ) _m The end of the-O-is at R of formula (IV) ¹ And R is ² Covalently bonded in position to the benzene ring to form a heterocycle.

In one embodiment, R in formula (II) ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of which is halogen. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of them is hydrogen, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are hydrogen. In one embodiment, R ¹ And R is ² Neither is hydrogen.

In one embodiment, R in formula (II) ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of which is halogen, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently halogen. In one embodiment, R ¹ And R is ² Neither is halogen.

In one embodiment, R in formula (II) ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Alkyl, and R ¹ And R is ² The other of (2) is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Alkyl, and R ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted C ₁ -C ₆ An alkyl group.

In one embodiment, R in formula (II) ¹ And R is ² One of which is substituted or unsubstituted C ₁ -C ₆ Haloalkyl, andand R is ¹ And R is ² The other of (C) is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted C ₁ -C ₆ A haloalkyl group.

In one embodiment, R in formula (II) ¹ And R is ² One of them being substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Both are independently substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Not being substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl). In one embodiment, R ¹ And R is ² Is a substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² Taken together to form a ring. In various embodiments, R ¹ And R is ² Each independently selected from the group consisting of hydrogen, fluorine, methoxy, methyl, difluoromethyl and-O- (CH) ₂ ) -O-such that R ¹ And R is ² Taken together to form a ring.

In various embodiments, R in formula (II) ³ Is hydrogen or substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independently H or halogen. In one embodiment, R ³ Is hydrogen. In one embodiment, R ³ Is substituted or unsubstituted C ₁ -C ₆ An alkyl group. For example, in one embodiment, R ³ Is methyl. In one embodiment, R ³ Is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group. In one embodiment, R ³ Is- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independentlyAnd is H or halogen.

In various embodiments, R in formula (II) ⁴ Is hydrogen, substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² Wherein X is ² is-OR ⁹ 、-SR ⁹ or-NHR ⁹ And R is ⁹ Is H, absent, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ . In one embodiment, R ⁴ Is hydrogen, in which case X ² Is absent and the compound of formula (II) is bound via R ⁷ To the linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -, as described in more detail below.

In other embodiments, when R ⁴ Comprises X ² And R is ⁹ Is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ When the compound of formula (II) is bound via R ⁴ To the linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -. In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkenyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² . In one embodiment, R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² . Wherein R is ⁴ Comprises X ² In each such embodiment of (2), R is provided ⁹ Is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ Thus providing for the routing of the compound of formula (II) through R ⁴ To the linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -an option.

In various embodiments, X in formula (II) ¹ is-O-, -S (O) _n6 ) -, -NH-, -O- (c=o) -, -NH- (c=o) -O-, -NH- (c=o) -NH-, or-NH-S (O) _n6 ) -, where n ⁶ 0, 1 or 2. In one embodiment, X ¹ is-O-. In one embodiment, X ¹ is-S (O) _n6 ) -. In one embodiment, X ¹ is-NH-. In one embodiment, X ¹ is-O- (c=o) -. In one embodiment, X ¹ is-NH- (c=o) -. In one embodiment, X ¹ is-NH- (C=O) -O-. In one embodiment, X ¹ is-NH- (C=O) -NH-. In one embodiment, X ¹ is-NH-S (O) _n6 )-。

In various embodiments, R in formula (II) ⁵ And R is ⁶ Each independently is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Wherein each Y is independently H or halogen, and the variables p, q, and t are as described elsewhere herein. In one embodiment, R ⁵ And R is ⁶ Each independently is hydrogen or substituted or unsubstituted C ₁ -C ₆ An alkyl group. In one embodiment, R ⁵ And R is ⁶ Both are hydrogen. In one embodiment, R ⁵ And R is ⁶ Each independently is a substituted or unsubstituted C ₁ -C ₆ An alkyl group.

In various embodiments, R in formula (II) ⁷ Is H, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ Wherein each R is ⁸ Independently substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ Substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ Or- [ (CY) ₂ ) _P O(CY ₂ ) _q ] _t CY ₂ -X ³ . In one embodiment, R ⁷ H. In one embodiment, R ⁷ is-COR ⁸ . In one embodiment, R ⁷ is-CO ₂ R ⁸ . In one embodiment, R ⁷ Is- (CO) -NHR ⁸ . Those skilled in the art will appreciate that when R ⁷ Is H, -COR ⁸ 、-CO ₂ R ⁸ Or- (CO) -NHR ⁸ When a compound of formula (II) is attached to linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ Connection via R ⁴ As described elsewhere herein.

In various embodiments, each R in formula (II) ⁸ Independently substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ Substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ Or- [ (CY) ₂ ) _P O(CY ₂ )q] _t CY ₂ -X ³ Wherein X is ³ is-H, -OH, -SH or-NH ₂ . In one embodiment, each R ⁸ Independently substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ . In one embodiment, each R ⁸ Independently substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ . In one embodiment, each R ⁸ Independently is- [ (CY) ₂ ) _p O(CY ₂ )q] _t CY ₂ -X ³ 。

In various embodiments, X in formula (II) ² is-OR ⁹ 、-SR ⁹ or-NHR ⁹ Wherein R is ⁹ Is H, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ . In one embodiment, X ² is-OR ⁹ . In one embodiment, X ² is-SR ⁹ . In one embodiment, X ² is-NHR ⁹ 。

In various embodiments, R in formula (II) ⁹ Is H, -COR ⁸ 、-CO ₂ R ⁸ 、-(CO)-NHR ⁸ 、L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ Wherein R is ⁸ Is substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ Substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ Or- [ (CY 2) _p O(CY ₂ ) _q ] _t CY ₂ -X ³ . In one embodiment, R ⁹ H. In one embodiment, R ⁹ is-COR ⁸ . In one embodiment, R ⁹ is-CO ₂ R ⁸ . In one embodiment, R ⁹ Is- (CO) -NHR ⁸ . Those skilled in the art will appreciate that when R ⁹ Is H, -COR ⁸ 、-CO ₂ R ⁸ Or- (CO) -NHR ⁸ When a compound of formula (II) is attached to linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ Connection via R ⁷ As described elsewhere herein.

In various embodiments, R in formula (II) ⁹ Is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ . In one embodiment, R ⁹ Is L ⁴ . In one embodiment, R ⁹ Is L ⁵ . In one embodiment, R ⁹ Is L ⁶ . In one embodiment, R ⁹ Is L ⁷ . Those skilled in the art will appreciate that when R ⁹ Is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ When a compound of formula (II) is attached to linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ Connection via R ⁴ As described elsewhere herein. In one embodiment, exactly R ⁷ And R is ⁹ One of them is L ⁴ 、L ⁵ 、L ⁶ Or L ⁷ In this case, a single covalent bond connects drug D to linker-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -, thereby connecting to Mi.

In various embodiments, each X in formula (II) ³ Independently is-H, -OH, -SH or-NH 2. In one embodiment, X ³ is-H. In one embodiment, X ³ is-OH. In one embodiment, X ³ is-SH. In one embodiment, X ³ is-NH ₂ 。

In various embodiments, m in formula (II) is 1 or 2. In one embodiment, m is 1. In another embodiment, m is 2.

In various embodiments, n in formula (II) ⁴ And n ⁵ Each independently 0, 1 or 2, provided that n ⁴ And n ⁵ Neither is 0. In one embodiment, n ⁴ And n ⁵ Both are 1. In one embodiment, n ⁴ Is 0 and n ⁵ 1. In one embodiment, n ⁴ Is 0 and n ⁵ 2. In one embodiment, n ⁴ Is 1 and n ⁵ Is 0. In one embodiment, n ⁴ Is 2 and n ⁵ Is 0.

In various embodiments, n in formula (II) ⁶ 0, 1 or 2. In one embodiment, n ⁶ 0, in which case X ¹ is-S-or-NH-S-. In one embodiment, n ⁶ 1, in which case X ¹ is-S (=o) -or-NH-S (=o) -. In one embodiment, n ⁶ 2, in which case X ¹ is-S (=O) ₂ -or-NH-S (=o) ₂ -。

In various embodiments, each Y in formula (II) is independently H or halogen. In one embodiment, each Y is hydrogen. In one embodiment, -CY ₂ Is CH ₂ . In one embodiment, -CY ₃ Is CH ₃ . In one embodiment, -CY ₃ For CHF ₂ . At the position ofIn one embodiment, -CH ₂ F is CH ₃ . In one embodiment, -CY ₃ Is CF (CF) ₃ 。

In various embodiments, each p in formula (II) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, p is 1. In one embodiment, p is 2.

In various embodiments, each q in formula (II) is independently 0, 1, 2, 3, 4, 5, or 6. In one embodiment, q is 1. In one embodiment, q is 2.

In various embodiments, each t in formula (II) is independently 1, 2, 3, 4, 5, or 6. In one embodiment, t is 1. In one embodiment, p is t.

In various embodiments, formula (II) does not represent delutinkang or isatecan.

Immunoconjugates

Various embodiments disclosed herein relate to immunoconjugates of formula (I) having the following structure:

Ab-[S-L ¹ -L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D] _n

(I)

in various embodiments, L in formula (III) ¹ Is L ¹ Is that

In various embodiments, L in formula (III) ² Is absent from,Or->Wherein Z is ¹ And Z ² Each independently is hydrogen, halogen, NO ₂ 、-O-(C ₁ -C ₆ Alkyl) or C ₁ -C ₆ An alkyl group. In one embodiment, L in formula (III) ² Is not in existence of. In one embodiment, L in formula (III) ² Is->In one embodiment, L in formula (III) ² Is->

In various embodiments, L in formula (III) ⁴ Are tetrapeptide residues. For example, in one embodiment, L ⁴ Is a tetrapeptide selected from GGFG (gly-gly-phy-gly), EGGF (glu-gly-gly-phy), SGGF (ser-gly-gly-phy) and KGGF (lys-gly-gly-phy).

In various embodiments, L in formula (III) ⁷ Is absent from,In one embodiment, L ⁷ Is absent. In one embodiment, L ⁷ Is->In one embodiment, L ⁷ Is thatIn one embodiment, L ⁷ Is->In one embodiment, L ⁷ Is->

In various embodiments, D in the immunoconjugate of formula (I) is a drug moiety as described herein (e.g., according to the heading "drug moiety" above). In one embodiment, D is a cytotoxic anticancer drug moiety. In one embodiment, the drug moiety is irinotecan.

In various embodiments, ab in formula (III) is an antibody or antigen binding fragment. In one embodiment, the Ab specifically binds to a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1). In one embodiment, ab binds to the surface of cancer cells. In one embodiment, ab is an anti-HER 2 antibody.

In various embodiments, the immunoconjugate of formula (I) is represented by a structure selected from the group consisting of ¹ And Z ² Each independently selected from hydrogen, fluorine, chlorine, -NO ₂ and-OCH ₃ ：

Pharmaceutical composition

Some embodiments described herein relate to pharmaceutical compositions that may include an effective amount of one or more compounds described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

The term "pharmaceutical composition" refers to a mixture of one or more compounds and/or salts disclosed herein with other chemical components (such as diluents or carriers). The pharmaceutical compositions facilitate administration of the compounds to organisms. Pharmaceutical compositions may also be obtained by reacting a compound with an inorganic or organic acid, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. The pharmaceutical compositions will generally be formulated according to the particular intended route of administration.

The term "physiologically acceptable" defines a carrier, diluent or excipient that does not abrogate the biological activity and properties of the compound and does not cause significant injury or harm to the animal to which the composition is intended to be delivered.

As used herein, "vector" refers to a compound that facilitates the incorporation of the compound into a cell or tissue. For example, but not limited to, dimethyl sulfoxide (DMSO) is a common carrier that facilitates uptake of many organic compounds into cells or tissues of a subject.

As used herein, "diluent" refers to an ingredient in a pharmaceutical composition that does not have significant pharmaceutical activity but may be pharmaceutically necessary or desirable. For example, diluents may be used to increase the volume of a powerful drug product that is too small in mass to be manufactured and/or administered. It may also be a dissolved liquid for a pharmaceutical product to be administered by injection, ingestion or inhalation. A common form of diluent in the art is an aqueous buffer solution such as, but not limited to, phosphate buffered saline that mimics the pH and isotonicity of human blood.

As used herein, "excipient" refers to a substantially inert substance added to a pharmaceutical composition to provide the composition with, but not limited to, volume, consistency, stability, binding capacity, lubrication, disintegration capacity, and the like. For example, stabilizers such as antioxidants and metal chelators are excipients. In one embodiment, the pharmaceutical composition comprises an antioxidant and/or a metal chelator. "diluent" is a type of excipient.

The pharmaceutical compositions described herein may be administered to a human patient per se, or into a composition, wherein the pharmaceutical composition is admixed with other active ingredients (as in combination therapy), or with a carrier, diluent, excipient, or combination thereof. The correct formulation depends on the route of administration selected. Techniques for formulating and administering the compounds described herein are known to those skilled in the art.

The pharmaceutical compositions disclosed herein may be manufactured in a manner known per se, for example by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or tabletting processes. In addition, the active ingredient is contained in an amount effective to achieve its intended use. Many of the compounds used in the pharmaceutical combinations disclosed herein may be provided as salts with pharmaceutically compatible counterions.

There are a variety of techniques in the art for administering compounds, salts, and/or compositions including, but not limited to, oral, rectal, pulmonary, topical, aerosol, injection, infusion, and parenteral delivery (including intramuscular, subcutaneous, intravenous, intramedullary injections, intrathecal, direct intraventricular, intraperitoneal, intranasal, and intraocular injections). In some embodiments, the compound of formula (I), or a pharmaceutically acceptable salt thereof, may be administered orally.

The compounds, salts and/or compositions may also be administered in a topical manner rather than a systemic manner, for example, via direct injection or implantation of the compounds into the affected area by way of a depot or sustained release formulation. Furthermore, the compounds may be administered in targeted drug delivery systems, for example, in targeted ligand coated liposomes for a specific cell or tissue type. Liposomes will target and be selectively taken up by the target cell or tissue.

The composition may, if desired, be present in a package or dispenser device which may include one or more unit dosage forms containing the active ingredient. The package may for example comprise a metal or plastic foil, such as a blister package. The package or dispenser device may be accompanied by instructions for administration. The package or dispenser may also be accompanied by a notice associated with the container format as prescribed by a government agency regulating the manufacture, use or sale of pharmaceuticals, which notice reflects approval by the agency of the format of the pharmaceutical for human or veterinary administration. For example, such notification may be a label approved by the U.S. food and drug administration for prescription drugs or an approved product insert. Compositions that may comprise compounds and/or salts formulated in compatible pharmaceutical carriers as described herein may also be prepared, placed in a suitable container, and labeled for use in treating the indicated condition.

Use and method of treatment

Some embodiments described herein relate to a method for treating a cancer or tumor described herein, which method may include administering an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) to a subject having a cancer or tumor. Other embodiments described herein relate to the use of an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) in the manufacture of a medicament for treating a cancer or tumor described herein. Other embodiments described herein relate to an effective amount of a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) or a pharmaceutical composition comprising a compound described herein (e.g., an immunoconjugate compound of formula (I), a pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof) for treating a cancer or tumor described herein.

Examples of cancers and tumors include, but are not limited to: lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell carcinoma, peritoneal cancer, liver cancer, hepatocellular carcinoma, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulval cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

As used herein, "subject" refers to an animal, which is the subject of treatment, observation or experiment. "animals" include cold and warm-blooded vertebrates and invertebrates such as fish, shellfish, reptiles and in particular mammals. "mammal" includes, but is not limited to, mice, rats, rabbits, guinea pigs, dogs, cats, sheep, goats, cows, horses, primates (e.g., monkeys, chimpanzees, and apes), and particularly humans. In some embodiments, the subject may be a human. In some embodiments, the subject may be a child and/or infant, e.g., a febrile child or infant. In other embodiments, the subject may be an adult.

As used herein, the terms "treatment" and "therapy" do not necessarily mean to completely cure or eliminate a disease or disorder. Any degree of alleviation of any undesired sign or symptom of a disease or disorder may be considered treatment and/or therapy. In addition, the treatment may include an action that may worsen the overall health sensation or appearance of the subject.

The terms "therapeutically effective amount" and "effective amount" are used to indicate the amount of active compound or agent that elicits the biological or medicinal response being indicated. For example, a therapeutically effective amount of a compound, salt or composition may be an amount required to prevent, reduce or ameliorate symptoms of a disease or disorder, or to prolong survival of a subject being treated. The response may occur in a tissue, system, animal or human and includes alleviation of the signs or symptoms of the disease or condition being treated. Determination of an effective amount is well within the ability of those skilled in the art, given the disclosure provided herein. The therapeutically effective amount (required to be a dose) of the compounds disclosed herein will depend on the route of administration, the type of animal being treated (including humans), and the physical characteristics of the particular animal under consideration. The dose may be modulated to achieve the desired effect, but will depend on the following factors: such as weight, diet, concurrent medication, and other factors as will be appreciated by those skilled in the medical arts.

For example, an effective amount of a compound is an amount that produces the following effects: (a) reduction, alleviation or disappearance of one or more symptoms caused by cancer, (b) reduction in tumor size, (c) tumor elimination, and/or (d) stabilization of long-term disease of the tumor (growth arrest). In the treatment of lung cancer (such as non-small cell lung cancer), a therapeutically effective amount is an amount that reduces or eliminates cough, shortness of breath, and/or pain.

The amount of immunoconjugate compound of formula (I), pharmaceutical compound of formula (IV), or pharmaceutically acceptable salt thereof, required for treatment will vary not only with the particular compound or salt selected, but also with the route of administration, the nature and/or symptoms of the disease or disorder being treated, and the age and condition of the patient, and will ultimately be at the discretion of the attendant physician or clinician. In the case of administration of pharmaceutically acceptable salts, the dosage can be calculated as the free base. As will be appreciated by those of skill in the art, in certain instances, it may be necessary to administer the compounds disclosed herein in amounts exceeding or even well exceeding the dosage ranges described herein in order to effectively and invasively treat a particularly aggressive disease or condition.

Generally, however, suitable dosages will generally be in the range of about 0.05mg/kg to about 10 mg/kg. For example, a suitable dose may be in the range of about 0.10mg/kg body weight/day to about 7.5mg/kg body weight/day, such as about 0.15mg/kg body weight/day to about 5.0mg/kg body weight/day, about 0.2mg/kg body weight/day to 4.0mg/kg body weight/day, or any amount therebetween. The compounds may be administered in unit dosage form; for example, comprising 1mg to 500mg, 10mg to 100mg, 5mg to 50mg or any amount therebetween of active ingredient per unit dosage form.

The required dose may conveniently be presented in a single dose form or in divided dose forms administered at appropriate intervals (e.g. in sub-dose forms of two, three, four or more times daily). The sub-dose itself may be further divided into, for example, a plurality of discrete loosely spaced applications.

As will be apparent to those skilled in the art, the available in vivo dosage to be administered and the particular mode of administration will vary depending upon the age, weight, severity of affliction, the species of mammal being treated, the particular compounds employed, and the particular use for which these compounds are employed. Determination of an effective dosage level (i.e., the dosage level necessary to achieve the desired result) can be accomplished by one of ordinary skill in the art using conventional methods, such as, for example, human clinical trials, in vivo studies, and in vitro studies. For example, the useful dosage of the immunoconjugate compound of formula (I), the pharmaceutical compound of formula (IV), or a pharmaceutically acceptable salt thereof can be determined by comparing their in vitro and in vivo activities in an animal model. Such comparison can be accomplished by comparison with established drugs such as cisplatin and/or gemcitabine.

The dosage and interval may be adjusted individually to provide a plasma level of the active moiety sufficient to maintain a regulatory effect or Minimum Effective Concentration (MEC). The MEC of each compound will vary but can be estimated from in vivo data and/or in vitro data. The dosage necessary to achieve MEC will depend on the individual characteristics and route of administration. However, HPLC assays or bioassays can be used to determine plasma concentrations. The MEC value may also be used to determine the inter-dose time. The composition should be administered using a regimen that maintains plasma levels between 10% and 90%, preferably between 30% and 90%, most preferably between 50% and 90% higher than MEC. In the case of topical administration or selective ingestion, the effective local concentration of the drug may not be correlated with plasma concentration.

It should be noted that in the event of a condition arising from toxicity or organ dysfunction, the attending physician will know how and when to terminate, interrupt or adjust administration. Conversely, in cases where the clinical response is inadequate (eliminating toxicity), the attending physician will also know to adjust the treatment to a higher level. The magnitude of the dosage administered in the management of the disorder of interest will vary depending on the severity of the disease or condition to be treated and the route of administration. For example, the severity of a disease or disorder can be assessed in part by standard prognostic assessment methods. Furthermore, the dosage and possibly the frequency of dosage will also vary depending on the age, weight and response of the individual patient. Procedures comparable to those discussed above may be used in veterinary medicine.

The efficacy and toxicity of the compounds, salts, and compositions disclosed herein can be assessed using known methods. For example, the toxicology of a particular compound or subset of compounds (sharing certain chemical moieties) may be established by determining its in vitro toxicity to a cell line, such as a mammalian and preferably human cell line. The results of such studies generally predict toxicity in animals (such as mammals or more particularly humans). Alternatively, known methods can be used to determine toxicity of a particular compound in an animal model (such as mouse, rat, rabbit, dog, or monkey). Several accepted methods (such as in vitro methods, animal models, or human clinical trials) can be used to establish the efficacy of a particular compound. In selecting a model to determine efficacy, the skilled artisan can follow current techniques to select an appropriate model, dose, route of administration, and/or regimen.

Synthesis

The pharmaceutical compounds of formula (IV), or pharmaceutically acceptable salts thereof, may be prepared by the skilled artisan in a variety of ways using known techniques, as guided by the detailed teachings herein provided. For example, in one embodiment, the pharmaceutical compound of formula (IV) is prepared according to the general scheme shown in fig. 2-4.

The conjugates of formula (III) can be prepared by the skilled artisan in a variety of ways using known techniques, as guided by the detailed teachings herein provided. For example, in one embodiment, the conjugates of formula (II) are prepared according to the general scheme shown in fig. 5-10. Although illustrated with a particular connector and payload, one skilled in the art will appreciate that other connectors and/or payloads may be used in a similar manner.

Immunoconjugates of formula (I) can be prepared by the skilled artisan in a variety of ways using known techniques, as guided by the detailed teachings herein provided. For example, in one embodiment, the immunoconjugate of formula (I) is prepared according to the general scheme shown in fig. 11. In one embodiment, a method of preparing an immunoconjugate as described herein comprises reacting an effective amount of a thiol-functionalized antibody or antigen binding fragment with a conjugate as described herein under reaction conditions effective to form the immunoconjugate. In one embodiment, the method further comprises reducing the antibody or antigen-binding fragment under reducing conditions effective to form a thiol-functionalized antibody or antigen-binding fragment.

Examples

Additional embodiments are disclosed in more detail in the examples below, which are not intended to limit the scope of the claims in any way.

Example 1

N- ((1S, 8S) -8-ethyl-4-fluoro-8-hydroxy-3-methyl-9, 12-dioxo-1,2,8,9,12,14-hexahydro-11H-cyclopenta [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acetamide (1-14) (FIG. 12)

1-14 (stereochemistry at optionally designated carbon)

1-bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2)：

2-fluoro-1-methyl-4-nitrobenzene (1-1) (25.0 g,322mmol,1.0 eq.) in heptane (250 mL) and H ₂ SO ₄ The mixture in (250 mL) was heated at 70 ℃. N-bromosuccinimide (68.84 g,386.78mmol,1.2 eq.) was then added in portions to the above mixture at 70 ℃. The resulting red suspension was stirred at 70℃for 15h. TLC (petroleum ether/ethyl acetate=10/1, r _f =0.6) shows that a new main spot is formed. The reaction mixture was poured into ice water (1L) and extracted with ethyl acetate (3X 500 mL). The combined organic phases were washed with brine (500 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluting with petroleum ether to give 1-bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2) (17.0 g,20% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 8.27(t,J＝1.79Hz,1H),7.89(dd,J＝8.76,2.21Hz,1H),2.44(d,J＝2.50Hz,3H)。

3-bromo-5-fluoro-4-methylaniline (1-3)：

To a solution of bromo-3-fluoro-2-methyl-5-nitrobenzene (1-2) (15.0 g,64.1mmol,1.0 eq.) in ethanol (750 mL) and water (150 mL) was added iron powder (10.7 g,192mmol,3.0 eq.) and NH ₄ Cl (6.86 g,128mmol,2.0 eq.). The suspension was stirred at 80℃for 2h. TLC (petroleum ether/ethyl acetate=10/1, r _f =0.3) shows that a new main spot is formed. After cooling to 25 ℃, the reaction mixture was filtered through a celite pad, washing with ethanol (500 mL). The combined filtrates were concentrated to dryness, and the residue was purified by silica gel column chromatography eluting with 9% ethyl acetate/petroleum ether to give 3-bromo-5-fluoro-4-methylaniline (1-3) (8.0 g,55% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 6.67-6.73(m,1H),6.34(dd,J＝10.97,2.27Hz,1H),3.65(br s,2H),2.20(d,J＝2.15Hz,3H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-111.86。

N- (3-bromo-5-fluoro-4-methylphenyl) acetamide (1-4)：

To a mixture of 3-bromo-5-fluoro-4-methylaniline (1-3) (8.00 g,39.2mmol,1.0 eq.) in ethyl acetate (100 mL) was added triethylamine (8.13 g,80.4mmol,11.2mL,2.05 eq.) and acetic anhydride (5.20 g,51.0mmol,4.77mL,1.3 eq.). The reaction mixture was stirred at 25 ℃ for 3h. TLC (petroleum ether/ethyl acetate=3/1, r _f =0.25) shows that a new main spot is formed. The reaction mixture was quenched with water and extracted with ethyl acetate (3×50 mL). The combined organic phases were washed with brine (100 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give N- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4) (8.8 g,91% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 7.59(d,J＝1.53Hz,1H),7.41(dd,J＝11.55,2.02Hz,1H),2.26(d,J＝2.20Hz,3H),2.11(s,3H)。

(E) -3- (5-acetamido-3-fluoro-2-methylphenyl) acrylic acid tert-butyl ester (1-5)：

To N- (3-bromo-5-fluoro-4-methyl)To an orange solution of dioxane (100 mL) was added N-cyclohexyl-N-methylcyclohexylamine (6.99 g,35.8mmol,7.58mL,1.1 eq.) and Pd (t-Bu), followed by (1-4) (8.00 g,32.5mmol,1.0 eq.) and tert-butyl acrylate (4.58 g,35.8mmol,5.19mL,1.1 eq.) ₃ P) ₂ (831 mg,1.63mmol,0.05 eq.). The reaction mixture was stirred under nitrogen at 100 ℃ for 16h. LCMS (retention time=0.797 min) showed formation of the desired product. The reaction mixture was quenched with water (100 mL) and extracted with ethyl acetate (3X 100 mL). The combined organic layers were washed with brine (200 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated to dryness under reduced pressure. The residue was purified by silica gel column chromatography eluting with 16% ethyl acetate/petroleum ether to give tert-butyl (E) -3- (5-acetamido-3-fluoro-2-methylphenyl) acrylate (1-5) (10.0 g,94% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 10.06(s,1H),7.72(d,J＝15.77Hz,1H),7.61(s,1H),7.51(dd,J＝11.98,1.47Hz,1H),6.22(d,J＝15.77Hz,1H),2.20(d,J＝1.59Hz,3H),2.04(s,3H)1.49(s,9H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-115.02。LCMS(ESI+)m/z：C ₁₆ H ₂₁ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 294.1, found: 294.0.

3- (5-Acetylamino-3-fluoro-2-methylphenyl) propionic acid tert-butyl ester (1-6)：

To a solution of tert-butyl (E) -3- (5-acetamido-3-fluoro-2-methylphenyl) acrylate (1-5) (2.80 g,9.55mmol,1.0 eq.) in dichloromethane (20 mL) and methanol (20 mL) was added Pd/C (10 wt%) (1.01 g,0.1 eq.) under an argon atmosphere. The suspension was degassed in vacuo and used with H ₂ Purging three times. The mixture is put in H ₂ (15 psi) at 25℃for 12h. TLC (petroleum ether/ethyl acetate=3/1, r _f =0.2) shows that a new main spot is formed. Exchange of H with argon ₂ After the atmosphere, it was filtered through a pad of celite, and the filter cake was washed with methanol (100 mL). The combined filtrates were concentrated under reduced pressure to give tert-butyl 3- (5-acetamido-3-fluoro-2-methylphenyl) propionate (1-6) (2.6 g,83% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 7.33(dd,J＝11.80,2.02Hz,1H),7.06(s,1H),2.88(t,J＝7.64Hz,2H),2.49(t,J＝7.70Hz,2H),2.17(d,J＝1.96Hz,3H),2.10(s,3H),1.41(s,9H)。

3- (5-Acetylamino-3-fluoro-2-methylphenyl) propionic acid (1-7)：

To a solution of tert-butyl 3- (5-acetamido-3-fluoro-2-methylphenyl) propionate (1-6) (2.60 g,8.80mmol,1.0 eq.) in dichloromethane (30 mL) was added trifluoroacetic acid (10 mL) at 25 ℃. The reaction mixture was stirred at 25 ℃ for 12h. TLC (petroleum ether/ethyl acetate=3/1, r _f =0.01) shows that a new main spot is formed. The reaction mixture was concentrated to give 3- (5-acetamido-3-fluoro-2-methylphenyl) propionic acid (1-7) (2.1 g,89% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 7.35(dd,J＝11.86,1.83Hz,1H),7.04(s,1H),2.91(t,J＝7.76Hz,2H),2.55(t,J＝7.83Hz,2H),2.17(d,J＝1.83Hz,3H),2.10(s,3H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-117.33,-77.77。

N- (6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide (1-8)：

To a solution of 3- (5-acetamido-3-fluoro-2-methyl-phenyl) propionic acid (1-7) (2.10 g,8.78mmol,1.0 eq.) in trifluoroacetic acid (7 mL) was added trifluoroacetic anhydride (7.37 g,35.1mmol,4.88mL,4.0 eq.). The reaction mixture was stirred at 60℃for 15h. LCMS showed formation of the desired product. The reaction mixture was treated with 50% acetonitrile/H ₂ O solution (100 mL) was diluted and the pH was adjusted to about 7 by adding 25% aqueous NaOH at 0deg.C. The mixture was extracted with ethyl acetate (3X 100 mL). The combined organic layers were washed with brine (100 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give N- (6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide (1-8) (1.5 g,69% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 8.02(d,J＝12.57Hz,1H),3.01-3.08(m,2H),2.72-2.79(m,2H),2.18-2.23(m,6H)。LCMS(ESI+)m/z：C ₁₂ H ₁₃ FNO ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 222.1, found: 222.0.

n- (6-fluoro-2-oximino-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide(1-9)：

A solution of potassium tert-butoxide (1.19 g,10.58mmol,1.3 eq.) in tetrahydrofuran (12 mL), ethanol (2.4 mL) and butanol (2.4 mL) was stirred at 0deg.C for 0.5h. Isoamyl nitrite (1.43 g,12.2mmol,1.64mL,1.5 eq) and N- (6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide (1-8) (1.80 g,8.14mmol,1.0 eq) were added to the above mixture. The reaction mixture was stirred at 20℃for 3h. TLC (petroleum ether/ethyl acetate=1/1, r _f =0.4) shows that a new main spot is formed. The resulting red suspension was cooled to 0 ℃ and quenched with 1N hydrochloric acid solution (50 mL) and extracted with ethyl acetate (3×50 mL). The combined organic layers were washed with brine (50 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give N- (6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide (1-9) (2.0 g,88% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 12.81(s,1H),10.33(s,1H),8.03(d,J＝12.57Hz,1H),3.71(s,2H),2.20(s,3H),2.16(s,3H)。

N- (2-amino-6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide hydrochloride (1- -10)：

To a solution of N- (6-fluoro-2-hydroxyimino-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide (1-9) (2.00 g,6.98mmol,1 eq.) in methanol (100 mL) was added Pd/C (10 wt%) (1.48 g,0.2 eq.) and hydrochloric acid (12M, 1.74mL,3 eq.) under nitrogen. The suspension was degassed in vacuo and used with H ₂ Purging three times. The mixture is put in H ₂ (15 psi) at 20℃for 3h. TLC (petroleum ether/ethyl acetate=1/1, r _f =0) shows that a new main spot is formed. Exchange of H with Nitrogen ₂ After atmosphere, it was filtered through a pad of celite and washed with methanol (200 mL). The combined filtrates were concentrated to dryness to give N- (2-amino-6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide hydrochloride (1-10) (1.5 g,71% yield). This material was used directly in the next step without further purification. ¹ H NMR(400MHz,DMSO-D ₆ )δppm 9.99(s,1H),8.02(d,J＝12.59Hz,1H),4.33(br s,1H),3.98(br s,2H),3.50(dd,J＝16.99,8.19Hz,1H),3.00(dd,J＝17.18,4.46Hz,1H),2.21(s,3H),2.18(s,3H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-102.81。

N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl) acetamide (1-11)：

To a mixture of N- (2-amino-6-fluoro-7-methyl-3-oxo-2, 3-dihydro-1H-inden-4-yl) acetamide hydrochloride (1-10) (1.50 g,6.35mmol,1.0 eq.) in dichloromethane (45 mL) was added triethylamine (1.93 g,19.0mmol,2.65mL,3.0 eq.) and acetic anhydride (778 mg,7.62mmol,0.714mL,1.2 eq.). The reaction mixture was stirred at 25 ℃ for 2h. TLC (petroleum ether/ethyl acetate=1/1, r _f =0.1) shows that a new main spot is formed. The reaction mixture was quenched with water (50 mL) and extracted with dichloromethane (3×50 mL). The combined organic layers were washed with brine (50 mL), and dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a red gum.

The gum was dissolved in HCl/MeOH (20 mL, 4M) and stirred at 25℃for 2h. LCMS showed formation of the desired product. The resulting red suspension was concentrated to give a red residue. The residue was diluted with methanol (2 mL) and purified by preparative HPLC (column: phenomenex luna C, 80X 40mm X3 um; mobile phase: [ water (HCl) -ACN)]The method comprises the steps of carrying out a first treatment on the surface of the B%:28% -48%,7 min) to give N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl) acetamide (1-11) (0.70 g,47% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 6.52(d,J＝11.56Hz,1H),4.48(dd,J＝8.23,5.01Hz,1H),3.44(dd,J＝17.05,8.23Hz,1H),2.83(dd,J＝17.11,4.95Hz,1H),2.11(d,J＝1.07Hz,3H),2.02(s,3H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-107.29。 ¹³ CNMR(100MHz,CD ₃ OD)：201.9,172.1,167.9,165.4,153.8,153.7,143.1,143.0,117.0,112.6,112.4,101.6,101.3,55.5,31.8,20.8,8.0。LCMS(ESI+)m/z：C ₁₂ H ₁₄ FN ₂ O ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 237.1, found: 237.0.

(R) -N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl]Acetamide (1-12-P1) And (S) -N-7-amino-5-fluoro-4-methyl-1-oxo-2, 3-diHydrogen-1H-inden-2-yl]Acetamide (1-12-P2)：

N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl) acetamide (1-11) (0.70 g,2.96 mmol) was dissolved in MeOH with NH ₄ OH neutralization and separation by chiral SFC to obtain (R) -N-7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl ]Acetamide (1-12-P1) (Compound 1-12-P1 may be the opposite enantiomer to the depicted enantiomer) (200 mg,28% yield) and (S) -N-7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl]Acetamide (1-12-P2) (Compounds 1-12-P2 may be the opposite enantiomer to the depicted enantiomer) (180 mg,26% yield). Note that: stereochemistry is arbitrarily specified. SFC separation method: column: DAICEL CHIRALPAK AD (250 mm. Times.30 mm,10 um); mobile phase: [0.1% NH ₃ H ₂ O IPA]The method comprises the steps of carrying out a first treatment on the surface of the B%:30% -30%,12min, SFC (1-12-P1, rt= 3.012 min) and SFC (1-12-P2, rt= 3.270 min).

Spectrum of 1-12-P1: ¹ H NMR(400MHz,CD ₃ OD)δppm 2.01(s,3H)2.05(s,3H)2.74(dd,J＝16.81,5.01Hz,1H)3.40(br d,J＝8.70Hz,1H)4.50(dd,J＝8.23,5.01Hz,1H)6.26(d,J＝12.40Hz,1H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-108.05。LCMS(ESI+)m/z：C ₁₂ H ₁₄ FN ₂ O ₂ [ MH of (V)] ⁺ Calculated values: 237.10, found: 237.0.

spectrum of 12-P2: ¹ H NMR(400MHz,CD ₃ OD)δppm 2.01(s,3H)2.05(s,3H)2.74(dd,J＝17.11,4.95Hz,1H)3.40(br d,J＝8.82Hz,1H)4.50(dd,J＝8.29,5.07Hz,1H)6.26(d,J＝12.40Hz,1H)。 ¹⁹ F NMR(400MHz,CDCl ₃ )δppm-108.03。LCMS(ESI+)m/z：C ₁₂ H ₁₄ FN ₂ O ₂ [ MH of (V)] ⁺ Calculated values: 237.10, found: 237.1.

n- ((1S, 8S) -8-ethyl-4-fluoro-8-hydroxy-3-methyl-9, 12-dioxo-1,2,8,9,12,14-hexahydro- - 11H-cyclopenta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamides (1-14)

N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl) acetamide (1-11)) (50 mg,0.21 mmol), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3, 4-f)]A mixture of indolizine-3, 6,10 (4H) -trione (1-13) (111.4 mg,0.42 mmol), tsOH (36.2 mg,0.21 mmol) in xylene (2 mL) was heated at 140℃for 12H. All volatiles were removed under high vacuum and the residue was first chromatographed, followed by chiral SFC separation to give 1-14a and 1-14b. LCMS (esi+) m/z: c (C) ₂₅ H ₂₃ FN ₃ O ₅ [ MH of (V)] ⁺ Calculated values: 464.2, found: 464.3.

example 2

N- ((1S, 9S) -9-ethyl-9-hydroxy-10, 13-dioxo-1,2,9,10,13,15-hexahydro-12H-cyclopenta [ de ] [1,3] dioxolo [4,5-g ] pyrano [3',4':6,7] indolizino [1,2-b ] quinolin-1-yl) acetamide (2-30)

(FIG. 13)

2-30 (stereochemistry at optionally designated carbon)

2-bromo-6-methoxy-4-nitrophenol (2-16)：

To a solution of 2-methoxy-4-nitrophenol (2-15) (50.0 g, 298 mmol,1.0 eq.) in glacial acetic acid (500 mL) at 20deg.C was slowly added bromine (52.0 g,325mmol,16.8mL,1.1 eq.) using a dropping funnel. The resulting mixture was stirred at 20℃for 2h. TLC (petroleum ether: ethyl acetate=2:1) indicated that the starting material was consumed and a major spot with lower polarity formed. The reaction mixture was slowly poured into water (1.5L) while stirring, and the resulting mixture was stirred at 20 ℃ for an additional 10min. The mixture was filtered and concentrated to dryness under reduced pressure. The residue was triturated with water (2X 500 mL) and the resulting product collected by filtration and dried in a vacuum oven to give 2-bromo-6-methoxy-4-nitrophenol (2-16) (60.0 g,73 yield). ¹ H NMR(400MHz,CDCl ₃ ):δppm 8.13(d,J＝2.4Hz,1H),7.74(d,J＝2.4Hz,1H),6.61(s,1H),4.03(s,3H)。LCMS(ESI-)m/z：C ₇ H ₅ BrNO ₄ ^- [ of ]MH] ^- Calculated values: 245.9, found: 245.9.

3-bromo-5-nitrobenzene-1, 2-diol (2-17)：

To 2-bromo-6-methoxy-4-nitrophenol (2-16) (30.0 g,121mmol,1 eq.) in CH at 0deg.C ₂ Cl ₂ To a solution of (1.5L) was added boron tribromide (45.4 g,181mmol,17.5mL,1.5 eq.). The reaction mixture was warmed to 30 ℃ and stirred for 15h. TLC (petroleum ether: ethyl acetate=1:1) indicated that the starting material was consumed and a new major spot with higher polarity formed. The reaction mixture was quenched with methanol (200 mL) and concentrated under reduced pressure to give 3-bromo-5-nitrobenzene-1, 2-diol (2-17) (26.9 g,95% yield). ¹ H NMR(400MHz,DMSO-D ₆ ):δppm10.94(s,2H),7.87(d,J＝2.8Hz,1H),7.62(d,J＝2.8Hz,1H)。

4-bromo-6-nitro-1, 3-benzodioxole (2-18)：

To a solution of 3-bromo-5-nitrobenzene-1, 2-diol (2-17) (30.0 g,128mmol,1.0 eq.) in N, N-dimethylformamide (1L) was added Cs at 20deg.C ₂ CO ₃ (125 g,385mmol,3 eq.) and diiodomethane (54.9 g,205mmol,16.6mL,1.6 eq.). The reaction mixture was stirred at 100℃for 12h. TLC (petroleum ether: ethyl acetate=3:1) indicated that the starting material was consumed and a new major spot with lower polarity formed. The reaction mixture was cooled to 20 ℃, poured into ice water (1.5L), and extracted with ethyl acetate (2×1.5L). The combined organic layers were washed with brine (2X 1L), dried over anhydrous Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give a brown oil. The oil was triturated with toluene (20 mL) and the resulting product collected by filtration and dried in a vacuum oven to give 4-bromo-6-nitro-1, 3-benzodioxole (2-18) (17 g,48% yield). ¹ H NMR(400MHz,CDCl ₃ ):δppm 8.06(d,J＝2.0Hz,1H),7.63(d,J＝2.4Hz,1H),6.23(s,2H)。

7-bromobenzo [ d ]][1,3]Dioxol-5-amine (2-19)：

To 4-bromo-6-nitro-1, 3-benzodioxole (2-18) (10.0 g,40.6mmol,1.0 eq.) in ethyl acetate at 20deg.CTo a solution of alcohol (500 mL) and water (100 mL) was added iron (6.81 g,122mmol,3.0 eq.) and NH ₄ Cl (4.35 g,81.3mmol,2.0 eq.). The mixture was stirred at 80℃for 2h. TLC (petroleum ether: ethyl acetate=1:1) indicated that the starting material was consumed and a new major spot with lower polarity formed. After the reaction mixture was cooled to 20 ℃, it was filtered through a celite pad, washing with ethanol (500 mL). The filtrate was concentrated under reduced pressure and crushed ice was added. The resulting product was collected by filtration, washed with water, and dried in a vacuum oven to give 7-bromobenzo [ d ]][1,3]Dioxol-5-amine (2-19) (7.5 g,76% yield). ¹ H NMR(400MHz,CDCl ₃ ):δppm 6.29(s,1H),6.21(s,1H),5.94(s,2H),3.51(s,2H)。

N- (7-bromobenzo [ d ])][1,3]Dioxol-5-yl) acetamides (2-20)：

To a solution of 7-bromo-1, 3-benzodioxol-5-amine (2-19) (6.00 g,27.78mmol,1.0 eq.) in ethyl acetate (50 mL) was added acetic anhydride (3.40 g,33.33mmol,3.12mL,1.2 eq.) and triethylamine (8.43 g,83.3mmol,11.6mL,3.0 eq.) at 20 ℃ and the resulting mixture was stirred for 10h. TLC (ethyl acetate: methanol=4:1) indicated that the starting material was consumed and a new major spot with higher polarity formed. The reaction mixture was purified by adding saturated NaHCO at 0deg.C ₃ (50 mL) was quenched and extracted with ethyl acetate (2X 50 mL). The combined organic layers were taken up over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (eluting with petroleum ether/ethyl acetate=1/3 to 1/4) to give N- (7-bromobenzo [ d ]][1,3]Dioxol-5-yl) acetamide (2-20) (5.5 g,69% yield). ¹ H NMR(400MHz,CDCl ₃ ):δppm 7.19(s,1H),7.13(d,J＝1.6Hz,1H),7.02(d,J＝2.0Hz,1H),6.02(s,2H),2.15(s,3H)。LCMS(ESI+)m/z：C ₉ H ₉ BrNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 258.0, found: 257.9.

(E) -3- (6-acetamido-1, 3-benzodioxol-4-yl) acrylic acid tert-butyl ester (2-21)：

To N- (7-bromo-1, 3-benzo) under nitrogen atmosphereTo a solution of dioxol-5-yl) acetamide (2-20) (2.00 g,7.75mmol,1.0 eq.) and tert-butyl acrylate (1.09 g,8.52mmol,1.24mL,1.1 eq.) in dioxane (20 mL) was added N-cyclohexyl-N-methyl-cyclohexylamine (1.67 g,8.52mmol,1.81mL,1.1 eq.) and bis (tri-tert-butylphosphine) palladium (0) (198 mg,0.387mmol,0.05 eq.). The mixture was stirred at 100℃for 5h. TLC (petroleum ether: ethyl acetate=2:1, r _f =0.4) and LCMS showed starting material was consumed and the desired product formed. After cooling to room temperature, the reaction mixture was filtered and the filtrate was concentrated to give the product, which was then triturated with ethyl acetate (40 mL) at 20 ℃ for 10min, then filtered and the filtrate was concentrated under reduced pressure to give tert-butyl (E) -3- (6-acetamido-1, 3-benzodioxol-4-yl) acrylate (2-21) (2.0 g,76% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 7.44(d,J＝16.09Hz,1H)7.23(d,J＝1.91Hz,1H)7.05(br s,1H)6.86(d,J＝1.91Hz,1H)6.55(d,J＝15.97Hz,1H)6.06(s,2H)2.17(s,3H)1.53(s,9H)。LCMS(ESI+)m/z：C ₁₆ H ₂₀ NO ₅ -C ₄ H ₉ ( ⁱ Bu)+H ^]+ [ M-56 of] ⁺ Calculated values: 249.1, found: 249.9.

3- (6-acetamido-1, 3-Benzodioxol-4-yl) propionic acid tert-butyl ester (2-22)：

To a suspension of Pd/C (2.90 g,2.46mmol,10% purity, 0.5 eq.) in MeOH (20 mL) was added a solution of tert-butyl (E) -3- (6-acetamido-1, 3-benzodioxol-4-yl) acrylate (2-21) (1.50 g,4.91mmol,1.0 eq.) in MeOH (20 mL) at 20deg.C. The mixture was hydrogenated at 20℃under a hydrogen atmosphere at 15psi for 2h. TLC (petroleum ether/ethyl acetate=1/1, r _f =0.48) shows that the raw material was consumed and a new spot formed. Exchange of H with Nitrogen ₂ After the atmosphere, the reaction mixture was filtered, and the filtrate was concentrated to give tert-butyl 3- (6-acetamido-1, 3-benzodioxol-4-yl) propionate (2-22) (1.4 g,83% yield). It was used in the next step without further purification. ¹ H NMR(400MHz,CDCl ₃ )δppm 7.09(d,J＝1.96Hz,1H),6.98(br s,1H),6.64(s,1H),5.94(s,2H),2.83(t,J＝7.76Hz,2H),2.51-2.59(m,2H),2.14(s,3H),1.43(s,9H)。LCMS(ESI+)m/z：C ₁₆ H ₂₁ NO ₅ Na ⁺ [ M+Na ]] ⁺ Calculated values: 330.1, found: 330.1.

3- (6-acetamido-1, 3-Benzodioxol-4-yl) propionic acid (2-23)：

To tert-butyl 3- (6-acetamido-1, 3-benzodioxol-4-yl) propionate (2-22) (1.40 g,4.56mmol,1.0 eq.) at 20℃in CH ₂ Cl ₂ To a solution of (15 mL) was added TFA (3 mL) and the mixture was stirred for 2h. TLC (petroleum ether: ethyl acetate=0:1, r _f =0.48) shows that the raw material was consumed and a new spot formed. The reaction mixture was concentrated under reduced pressure, and the residue was purified by column chromatography (eluting with petroleum ether/ethyl acetate=1/0 to 1/9) to give 3- (6-acetamido-1, 3-benzodioxol-4-yl) propionic acid (2-23) (0.85 g,71% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 7.08(d,J＝1.91Hz,1H),6.76(d,J＝1.79Hz,1H),5.93(s,2H),2.80-2.88(m,2H),2.57-2.65(m,2H),2.07(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₄ NO ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 252.1, found: 251.9.

n- (6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxol-5-yl) acetamides (2- 24)：

To a solution of 3- (6-acetamido-1, 3-benzodioxol-4-yl) propionic acid (2-23) (16.0 g,63.7mmol,1.0 eq.) in TFA (64 mL) was added TFAA (53.5 g,255mmol,35.4mL,4 eq.) dropwise at 20deg.C. The resulting solution was heated to 60 ℃ for 10 hours. TLC (petroleum ether: ethyl acetate=2:1, r _f =0.40) and LCMS indicated the reaction was complete. After cooling to room temperature, the reaction mixture was poured into a solution of acetonitrile (100 mL) and water (100 mL). After cooling to 0 ℃, the pH of the mixture was adjusted to 7 with 25% aqueous sodium hydroxide (150 mL) and water (100 mL) was added. The resulting mixture was extracted with ethyl acetate (500 mL. Times.3). The combined organic layers were washed with brine (800 mL), dried over anhydrous Na ₂ SO ₄ Drying and passingFiltered and concentrated under reduced pressure. The residue was triturated with ethyl acetate (100 mL), filtered and the filter cake collected to give N- (6-oxo-7, 8-dihydro-cyclopenta [ g)][1,3]Benzodioxol-5-yl) acetamide (2-24) (10.4 g,59% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm10.47(s,1H),7.85(s,1H),6.15(s,2H),2.84-3.04(m,2H),2.60-2.74(m,2H),2.17(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₂ NO ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 234.1, found: 234.0.

n- (7-oximino-6-oxo-8H-cyclopenta [ g ]][1,3]Benzodioxol-5-yl) acetamides (2-25)：

A solution of potassium tert-butoxide (1.0M, 45.0mL,2.1 eq.) in THF (41.6 mL), etOH (6.7 mL) and n-BuOH (6.7 mL) was stirred at 0deg.C for 30min. Isoamyl nitrite (3.01 g,25.7mmol,3.46mL,1.2 eq.) and N- (6-oxo-7, 8-dihydro cyclopenta [ g) at 0deg.C][1,3]Benzodioxol-5-yl) acetamide (2-24) (5.00 g,21.4mmol,1.0 eq.) was added to the above solution. The resulting mixture was stirred at 20℃for 3h. LCMS showed formation of the desired product. The reaction mixture was quenched with 1.0N hydrochloric acid (200 mL), and ethyl acetate (200 mL) was added. The resulting precipitate was collected by filtration. The filtrate was extracted with ethyl acetate (3×500 mL), the combined organic layers were washed with brine, dried over sodium sulfate, filtered and concentrated under reduced pressure. Combining the product with the filter cake to give N- (7-oximino-6-oxo-8H-cyclopenta [ g ] ][1,3]Benzodioxol-5-yl) acetamide (2-25) (5.6 g,99% yield) ¹ H NMR(400MHz,DMSO-D ₆ )δppm 12.71(s,1H),10.53(s,1H),7.89(s,1H),6.19(s,2H),3.63(s,2H),2.17(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₁ N ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 263.1, found: 263.0.

n- (7-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxol-5-yl) ethyl Amide hydrochloride (2-26)

To N- (7-oximino-6-oxo-8H-cyclopenta [ g ]][1,3]Benzodioxol-5-yl) acetamide (2-25) (550 mg,2.10mmol,1.0 eq.) and HCl (12M, 0.26mL,1.5 eq.) were added Pd/C (10 wt.%) (300 mg) to a mixture of MeOH (50 mL). The reaction mixture was purged three times with hydrogen and stirred at 20 ℃ under a 15psi hydrogen atmosphere for 2h. LCMS indicated completion of the reaction. Exchange of H with Nitrogen ₂ After the atmosphere, the reaction mixture was filtered through a celite pad, and the filtrate was concentrated under reduced pressure to give N- (7-amino-6-oxo-7, 8-dihydro-cyclopenta [ g)][1,3]Benzodioxol-5-yl) acetamide hydrochloride (2-26) (500 mg,83% yield). It was used in the next step without further purification. ¹ H NMR(400MHz,DMSO-D ₆ )δppm 10.09(br s,1H),9.05(br s,2H),8.82(br s,2H),7.85(s,1H),6.23-6.70(m,2H),3.98-4.39(m,2H),3.35-3.48(m,1H),2.16(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₃ N ₂ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 249.1, found: 249.0.

n- (7-acetamido-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxole-5- Base) acetamide (2-27)：

At 20℃to N- (7-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ] ][1,3]To a solution of benzodioxol-5-yl) acetamide hydrochloride (2-26) (4.50 g,15.8mmol,1.0 eq.) in DCM (150 mL) was added TEA (4.80 g,47.4mmol,6.60mL,3.0 eq.), acetic anhydride (1.94 g,19.0mmol,1.78mL,1.2 eq.) and the mixture stirred for 3h. TLC (ethyl acetate, R) _f =0.40) and LCMS indicated the reaction was complete. The mixture was diluted with DCM (500 mL) and washed with water (100 mL). The organic layer was treated with anhydrous Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure. The residue was purified by column chromatography (eluting with petroleum ether/ethyl acetate=2/1 to 0/1,5% THE) to give N- (7-acetamido-6-oxo-7, 8-dihydro-cyclopenta [ g)][1,3]Benzodioxol-5-yl) acetamide (2-27) (2.55 g,50% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 10.35(s,1H),8.53(d,J＝7.51Hz,1H),7.87(s,1H),6.13-6.19(m,2H),4.30-4.37(m,1H),3.17-3.32(m,1H),2.79(dd,J＝16.75,5.07Hz,1H),2.14(s,3H),1.85(s,3H)。LCMS(ESI+)m/z：C ₁₄ H ₁₅ N ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 291.1, found: 291.0.

n- (5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxol-7-yl) ethyl Amide (2-28)：

At 20℃to N- (7-acetamido-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxol-5-yl) acetamide (2-27) (2.55 g,8.78mmol,1.0 eq.) was added to a solution of MeOH (110 mL) in HCl/MeOH (4M, 110mL,50 eq.) and the solution stirred for 3h. LCMS indicated completion of the reaction. The reaction mixture was concentrated under reduced pressure, the residue was dissolved in MeOH (10 mL) and taken up with saturated NaHCO ₃ The pH was adjusted to 7 and then extracted with ethyl acetate (20 mL. Times.3). The combined organic phases were taken up in anhydrous Na ₂ SO ₄ Drying, filtering and concentrating under reduced pressure to obtain N- (5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g)][1,3]Benzodioxol-7-yl) acetamide (2-28) (2.0 g,91% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.40(d,J＝7.89Hz,1H),6.21(s,1H),5.97(d,J＝15.79Hz,2H),4.35(td,J＝8.17,5.37Hz,1H),3.20(dt,J＝16.55,8.17Hz,1H),2.62(dd,J＝16.66,5.26Hz,1H),1.84(s,3H)。 ¹³ CNMR(100MHz,DMSO-D ₆ )：δ199.6,169.1,154.3,145.6,133.4,127.7,111.3,101.3,93.5,54.5,28.7,22.3。LCMS(ESI+)m/z：C ₁₂ H ₁₃ N ₂ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 249.1, found: 249.0.

n- [ (7R) -5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxole-7- Base group]Acetamide (2-29-P1) and N- [ (7S) -5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzom two Oxacyclopenten-7-yl]Acetamide (2-29-P2)：

N- (5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ] [1,3] benzodioxol-7-yl) acetamide (2-28) (1.0 g) was dissolved in MeOH and isolated by chiral SFC to give N- [ (7R) -5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ] [1,3] benzodioxol-7-yl ] acetamide (2-29-P1) (compound 22-9-P1 may be the opposite enantiomer of the depicted enantiomer) (250 mg,24% yield) and N- [ (7S) -5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ] [1,3] benzodioxol-7-yl ] acetamide (2-29-P2) (compound 2-29-P2 may be the opposite enantiomer of the depicted enantiomer) (250 mg,24% yield).

SFC separation method: column (column: phenomenex-Cellulose-2 (250 mm. Times.30 mm,10 um); mobile phase: [ Neu-ETOH ]; B%:40% -40%,5 min). Compounds (2-29-P1, rt= 2.719 min) and (2-29-P2, rt=2.942 min) were isolated by chiral SFC. Note that: the stereochemistry of 2-29-P1 and 2-29-P2 is arbitrarily specified.

Spectrum of 2-29-P1: ¹ H NMR(400MHz,DMSO-D6)δppm 8.30(br d,J＝7.95Hz,1H),6.59(s,2H),6.17(s,1H),5.96(d,J＝15.89Hz,2H),4.36(td,J＝8.16,5.32Hz,1H),3.16-3.25(m,1H),2.61(dd,J＝16.69,5.20Hz,1H),1.84(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₃ N ₂ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 249.1, found: 248.9.

spectrum of 2-29-P2: ¹ H NMR(400MHz,DMSO-D6)δppm 8.30(br d,J＝7.95Hz,1H),6.59(s,2H),6.17(s,1H),5.96(d,J＝15.89Hz,2H),4.36(td,J＝8.19,5.26Hz,1H),3.21(dd,J＝16.69,8.50Hz,1H),2.61(dd,J＝16.69,5.20Hz,1H),1.85(s,3H)。LCMS(ESI+)m/z：C ₁₂ H ₁₃ N ₂ C ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 249.1, found: 248.9.

n- [ (7R) -5-amino-6-oxo-7, 8-dihydro-cyclopenta [ g ]][1,3]Benzodioxol-7-yl]Acetamide (2-29-P1) (30 mg,0.12 mmol), (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3, 4-f)]A mixture of indolizine-3, 6,10 (4H) -trione (1-13) (63.6 mg,0.24 mmol), tsOH (20.7 mg,0.12 mmol) in xylene (2 mL) was heated at 140℃for 12H. All volatiles were removed under high vacuum and the residue was chromatographed to give 2-30.LCMS (esi+) m/z: c (C) ₂₅ H ₂₂ N ₃ O ₇ [ MH of (V)] ⁺ Calculated values: 476.5, found: 476.4。

Example 3

(1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (3-34)

And (1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (3-35)

(FIG. 14)

(stereochemistry at optionally designated carbon)

(S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2, 3-dihydrobenzo [ de ]]Pyrano [3',4':6,7]Indolizine And [1,2-b ]]Quinoline-1,10,13 (9H, 12H, 15H) -trione (3-32)：

To a solution of (1S, 9S) -1-amino-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 ] quinoline-10, 13-dione methanesulfonate (100 mg,0.230mmol,1 eq.) in methanol (20 mL) at 25℃was added basic resin (300 mg) and the mixture stirred under ultrasound for 30min. The mixture was filtered and the filtrate was concentrated to give the free base.

To a solution of the free base obtained above in methanol (2 mL) was added 3, 5-di-tert-butyl-1, 2-benzoquinone (101 mg,0.459mmol,2 eq.). The reaction mixture was stirred at 60℃for 10min. The color of the reaction mixture changed from red to dark yellow. The reaction mixture was stirred at 25 ℃ for a further 3h. TLC (ethyl acetate: methanol=8:1) indicated about 10% unreacted starting material remained and a new spot formed. By adding oxalic acid (2M tetrahydrofuran/H at 25 ℃C ₂ O=3:1 solution, 0.5 mL) quench the reaction mixture and stir the mixture for 3h. The reaction mixture was diluted with tetrahydrofuran (5 mL), filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by preparative HPLC (under HCl conditions) to give (S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2, 3-dihydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-1,10,13 (9H, 12H, 15H) -trione (3-32) (70 mg,25% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.94(d,J＝10.6Hz,1H),7.35(s,1H),5.44(s,2H),5.39(s,2H),3.55-3.47(m,2H),3.09(t,J＝7.0Hz,2H),2.46(s,3H),1.92-1.79(m,2H),0.91-0.85(m,3H)。LCMS(ESI)m/z：C ₂₄ H ₂ OFN ₂ O ₅ ⁺ [ M+H of (H) ⁺ ]Calculated values: 435.1, found: 435.2.

preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: HCl/H ₂ O＝0.040％v/v；B：CH ₃ CN

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220nm and 254nm

(9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3', 4':6,7]indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-33)：

To the resulting (S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2, 3-dihydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-1,10,13 (9H, 12H, 15H) -trione (3-32) (70 mg,0.16mmol,1.0 eq.) in methanol (2 mL) was added AcOH (27.6 mg,0.460mmol, 26.3. Mu.L, 10 eq.) and NaBH ₃ CN (20.1 mg,0.322mmol,2.0 eq.). The mixture was stirred at 0℃for 10min and then at 20℃for 12h. TLC (ethyl acetate: methanol=8:1) indicated that the starting material was consumed and a new major spot formed. The reaction mixture was heated at 25 DEG C Department H ₂ O (0.5 mL) was quenched and concentrated under reduced pressure. The residue was purified by preparative HPLC to give (9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (33) (20 mg,29% yield).

Chiral SFC isolation of compound 3-33 gives (1 r,9 s) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 h,13 h-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13-dione (3-34) (compound 3-34 may be the opposite diastereomer of the depicted diastereomer (2.9 mg,4% yield) (peak 1) in SFC at 1.429min and (1 s,9 s) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10 h,13 h-benzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13-dione (3-35) (compound 3-35 may be the opposite diastereomer of the depicted) (peak 4.9 mg,7% yield) at 1.429min is the specified stereochemical at 1.534% of SFC.

(1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Spectrum of quinoline-10, 13-dione (3-34): ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.73(dd,J＝10.8,1.8Hz,7.31(s,1H),6.50(s,1H),5.97(d,J＝16.0Hz,1H),5.42(s,2H),5.41(d,J＝19.2Hz,1H),5.28(d,J＝18.6Hz,1H),5.12-5.17(m,1H),3.20-3.26(m,1H),2.98-3.05(m,1H),2.31-2.35(m,1H),2.33(s,3H),1.95-2.00(m,1H),1.84-1.90(m,2H),0.88(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(400MHz,DMSO-D ₆ )：δppm-111.8(s,1F)。LCMS(ESI)m/z：C ₂₄ H ₂₂ FN ₂ O ₅ ⁺ [ MH of (V) ⁺ ]Calculated values: 437.1, found: 437.1.

(1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-1,2,3,9,12,15-hexahydro-10H, 13H-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Spectrum of quinoline-10, 13-dione (3-35): ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.73(dd,J＝10.8,1.8Hz,7.31(s,1H),6.50(s,1H),5.97(d,J＝16.0Hz,1H),5.42(s,2H),5.41(d,J＝19.2Hz,1H),5.28(d,J＝18.6Hz,1H),5.12-5.17(m,1H),3.20-3.26(m,1H),2.98-3.05(m,1H),2.31-2.35(m,1H),2.33(s,3H),1.95-2.00(m,1H),1.84-1.90(m,2H),0.88(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(400MHz,DMSO-D ₆ )：δppm-111.8(s,1F)。LCMS(ESI)m/z：C ₂₄ H ₂₂ FN ₂ O ₅ ⁺ [ MH of (V) ⁺ ]Calculated values: 437.1, found: 437.1.

preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: HCl/H ₂ O＝0.040％v/v；B：CH ₃ CN

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220nm and 254nm

SFC separation method：

Instrument: waters SFC150AP preparation SFC

Column: DAICEL CHIRALCEL OD (250 mm. Times.30 mm,10 um)

Mobile phase: a is CO2, B is EtOH

Gradient: b% = 45% isocratic elution mode

Flow rate: 70g/min

Wavelength: 220nm

Column temperature: 35 DEG C

System backpressure: 120 bar

Example 4

(1R, 8S) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-36) and (1S, 8S) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-37) (FIG. 15)

* The stereochemistry at the carbon is arbitrarily specified.

N- ((8S) -8-ethyl-4-fluoro-8-hydroxy-3-methyl-9, 12-dioxo-2,8,9,11,12,14-hexahydro-1H-) Cyclopenta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamides (1-14)

To N- (7-amino-5-fluoro-4-methyl-1-oxo-2, 3-dihydro-1H-inden-2-yl) acetamide (100 mg,0.423mmol,1.0 eq.) (1-11) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f at 140 ℃]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (223 mg,0.847mmol,2.0 eq.) in xylene (10 mL) was added 4-methylbenzenesulfonic acid (29.1 mg,0.169mmol,0.4 eq.) and the mixture was stirred in a 40mL sealed tube at 140℃for 36H. It was cooled to room temperature and concentrated under reduced pressure, and the residue was purified by silica gel column chromatography eluting with 9% meoh in dichloromethane to give N- ((8S) -8-ethyl-4-fluoro-8-hydroxy-3-methyl-9, 12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamide (1-14) (8.50 mg,4.3% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.65(d,J＝8.11Hz,1H),7.72(d,J＝11.56Hz,1H),7.34(s,1H),6.51(d,J＝2.27Hz,1H),5.95(br d,J＝5.01Hz,1H),5.43(s,2H),5.09-5.13(m,2H),3.82-3.93(m,1H),3.30(br s,1H),2.38(s,3H),1.94(d,J＝2.86Hz,3H),1.87(br d,J＝7.39Hz,2H),0.87(br d,J＝4.41Hz,3H)。LCMS(ESI+)m/z：C ₂₅ H ₂₃ FN ₃ O ₅ [ MH of (V)] ⁺ Calculated values: 464.1, found: 464.2.

(1R, 8S) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta-e [de]Pyrano [3',4':6,7 ]Indolazino [1,2-b ]]Quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-36) and (1S, 8S) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta [ de ]]Pyrano [3', 4':6,7]indolazino [1,2-b ]]Quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-37)：

A solution of N- ((8S) -8-ethyl-4-fluoro-8-hydroxy-3-methyl-9, 12-dioxo-2,8,9,11,12,14-hexahydro-1H-cyclopenta [ de ] pyrano [3',4':6,7] indolizino [1,2-H ] quinolin-1-yl) acetamide (1-14) (150 mg,0.323mmol,1.0 eq.) in 6N aqueous HCl (15 mL) was stirred in a sealed tube at 60℃for 15H. After cooling to 25 ℃, the reaction mixture was concentrated under reduced pressure, the residue was diluted with methanol (5 mL) and purified by preparative HPLC to give (1 r,8 s) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta [ de ] -pyrano [3',4':6,7] indolizino [1,2-H ] quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-36) (5.0 mg,3.3% yield) (compound 4-36 may be the opposite enantiomer of the depicted enantiomer) and (1 s,8 s) -1-amino-8-ethyl-4-fluoro-8-hydroxy-3-methyl-11, 14-dihydro-1H-cyclopenta [ de ] pyrano [3',4' ] 6,7] indolizino [1,2-H ] quinoline-9, 12 (2H, 8H) -dione hydrochloride (4-36) (compound 4-36 may be the opposite enantiomer) (compound 4-37.0 mg, 4.37% yield). Note that: stereochemistry is arbitrarily specified.

Preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: HCl/H ₂ O＝0.040％v/v；B：CH ₃ CN

Column: phenomenex luna C18 80×40mm×3um

Flow rate: 40mL/min

Monitoring wavelength: 220 and 254nm

Spectra of 4-36: ¹ H NMR(400MHz,D ₂ O)δppm 7.33-7.40(m,2H),5.65(br d,J＝5.72Hz,1H),5.44-5.52(m,1H),5.33(br d,J＝17.17Hz,2H),5.18-5.26(m,1H),4.00(br dd,J＝18.06,7.81Hz,1H),3.45(br d,J＝18.84Hz,1H),2.30(s,3H),1.90(q,J＝7.27Hz,2H),0.87(t,J＝7.27Hz,3H)。 ¹⁹ F NMR(376MHz,D ₂ O)δppm-106.41。LCMS(ESI+)m/z：C ₂₃ H ₂₁ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 422.1, found: 422.0.SFC (rt= 1.208 min).

Spectra of 4-37: NMR (400 MHz, D) ₂ O)δppm 7.43(s,1H),7.24(br d,J＝11.26Hz,1H),5.58(br dd,J＝8.00,3.25Hz,1H),5.54(d,J＝16.26Hz,1H),5.37-5.45(m,2H),5.22(br d,J＝19.01Hz,1H),4.05(br dd,J＝18.01,8.13Hz,1H),3.52-3.61(m,1H),2.36(s,3H),1.95(q,J＝7.30Hz,2H),0.93(t,J＝7.38Hz,3H)。 ¹⁹ F NMR(376MHz,D2O)δppm-106.53。LCMS(ESI+)m/z：C ₂₃ H ₂₁ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 422.1, found: 422.0.SFC (rt=1.252 min).

Example 5

Synthesis of (1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (5-47) and (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (5-48)

* The stereochemistry at the carbon is arbitrarily specified.

3-bromo-5-fluoro-4-methyl-aniline (1-3)：

To a solution of 1-bromo-3-fluoro-2-methyl-5-nitro-benzene (1-2) (100 g,427mmol,1.0 eq.) in ethyl acetate (1.50L) was added Pt/C (10 wt%, 10.0 g) under an argon atmosphere. The resulting suspension was degassed in vacuo and then treated with H ₂ After three purges, it was purged at H ₂ (15 psi) at 60℃for 4h. Then exchange H with argon ₂ The combined reaction mixture was filtered through a pad of celite under an atmosphere, and the filter cake was washed with ethyl acetate (10.0L). (eighteen additional reactions were set up as described above and all nineteen reaction mixtures were combined). Concentrating the combined filtrates under reduced pressure to give 3-bromo-5-fluoro-4-methyl-aniline(1-3) (1.8 kg, purity 71%,77% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 6.79(s,1H),6.50(dd,J＝11.74,2.08Hz,1H),2.10(d,J＝2.08Hz,3H)。 ¹⁹ F NMR(376MHz,CD ₃ OD)δ＝-112.13。LCMS(ESI+)m/z：C ₇ H ₈ BrFN ⁺ [ MH of (V)] ⁺ Calculated values: 205.0, found: 205.8.

n- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4)：

To a solution of 3-bromo-5-fluoro-4-methyl-aniline (1-3) (300 g,1.47mol,1.0 eq.) in ethyl acetate (4.50L) was added triethylamine (420 ml,3.02mol,2.1 eq.) and acetic anhydride (178 ml,1.91mol,1.3 eq.) and the mixture was stirred at 15 ℃ for 12h. (another five vials were set up as described above and all six reaction mixtures were combined). The combined mixture was treated with saturated NH ₄ The Cl solution (15.0L) was quenched and extracted with ethyl acetate (3X 5.0L). The combined organic layers were washed with brine (8.0L), and dried over Na ₂ SO ₄ Dried, concentrated, and the residue was dissolved in dichloromethane (2.00L) and purified by silica gel column chromatography eluting with 15% ethyl acetate/petroleum ether to give N- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4) (1.2 kg,55% yield). ¹ H NMR(400MHz,CD ₃ OD)δ＝7.59(t,J＝1.5Hz,1H),7.41(dd,J＝2.0,11.6Hz,1H),2.26(d,J＝2.2Hz,3H),2.11(s,3H)。 ¹⁹ F NMR(376MHz,CD ₃ OD)δ＝-112.95。LCMS(ESI+)m/z：C ₉ H ₁₀ BrFNO ⁺ [ MH of (V)] ⁺ Calculated values: 247.0, found: 247.8.

(E) -4- (5-acetamido-3-fluoro-2-methyl-phenyl) but-3-enoic acid (5-38)：

At N ₂ To a mixture of N- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4) (200 g, 803 mmol,1.0 eq.) in tetrahydrofuran (1.00L) and water (200 mL) was added diisopropylethylamine (56 mL,3.25mol,4.0 eq.), triorthophenylphosphine (49.5 g,163mmol,0.20 eq.), but-3-enoic acid (168 g,1.95mol,2.4 eq.) and Pd (OAc) ₂ (18.2 g,81.3mmol,0.10 eq.) and the reaction mixture was stirred at 75℃for 16h. (five are set as described above)Additional reactions and all six reaction mixtures were combined). The combined reaction mixtures were diluted with water (2.0L), adjusted to ph=2 by adding 3N HCl, the mixture was filtered through a celite pad, and the filter cake was washed with ethyl acetate (4.0L). The mixture was extracted with water (4.0L) and the aqueous phase was extracted with ethyl acetate (3X 1.80L). The combined organic layers were washed with brine (4.0L), dried over anhydrous Na ₂ SO ₄ Drying, filtration, concentration and purification of the residue by silica gel column chromatography eluting with 70% ethyl acetate/petroleum ether gave (E) -4- (5-acetamido-3-fluoro-2-methyl-phenyl) but-3-enoic acid (5-38) (400 g,49% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝12.32(br s,1H),10.02(s,1H),7.48(ddd,J＝1.6,8.3,12.0Hz,1H),7.39(s,1H),7.08-6.98(m,1H),6.94-6.83(m,1H),6.75-6.50(m,1H),6.13(td,J＝7.2,15.7Hz,1H),5.69(d,J＝15.5Hz,1H),3.59(ddd,J＝2.5,4.1,6.5Hz,1H),3.56-3.46(m,1H),3.31-3.21(m,1H),2.16-2.06(m,3H),2.05-1.99(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δ＝-115.3。LCMS(ESI+)m/z：C ₁₃ H ₁₅ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 252.1, found: 252.0.

4- (5-acetamido-3-fluoro-2-methyl-phenyl) butanoic acid (5-39)：

Pd/C (30.0 g,39.8mmol,10 wt%, 0.10 eq) was added to a solution of (E) -4- (5-acetamido-3-fluoro-2-methyl-phenyl) but-3-enoic acid (5-38) (100 g, 390 mmol,1.0 eq) in methanol (1.50L) under an argon atmosphere. The resulting suspension was degassed in vacuo and used with H ₂ Purge three times and at H ₂ (15 psi) at 35℃for 12h. (three additional reactions were set up as described above and all four reaction mixtures were combined). In the substitution of H with argon ₂ After the atmosphere, the combined mixture was filtered through a celite pad and the filter cake was washed with methanol (8L). The combined filtrates were concentrated under reduced pressure to give 4- (5-acetamido-3-fluoro-2-methyl-phenyl) butanoic acid (5-39) (330 g,82% yield), which was used directly in the next step without purification. ¹ H NMR(400MHz,DMSO-d ₆ )δ＝12.77-11.04(m,1H),9.98(s,1H),7.42(dd,J＝1.7,12.2Hz,1H),7.04(s,1H),2.59-2.53(m,2H),2.27(t,J＝7.2Hz,2H),2.09(d,J＝1.8Hz,3H),2.01(s,3H),1.71(quin,J＝7.5Hz,2H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δ＝-115.64。LCMS(ESI+)m/z：C ₁₃ H ₁₇ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 254.1, found: 254.0.

n- (7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-40)：

At 0 ℃, at N ₂ To a solution of 4- (5-acetamido-3-fluoro-2-methyl-phenyl) butanoic acid (5-39) (110 g,434mmol,1.0 eq.) in trifluoroacetic acid (330 mL) was added trifluoroacetic anhydride (121 mL,869mmol,2.0 eq.) and the mixture stirred at 15 ℃ for 15h. (two additional reactions were set up as described above and all three reaction mixtures were combined). The combined reaction mixtures were poured into 50% acetonitrile in water (6.0L) at 0 ℃ and stirred at 0 ℃ for 0.5h. The resulting suspension was adjusted to ph=7 with 25% naoh aqueous solution at 0 ℃. The mixture was filtered and the residue was washed with water (1.00L), methyl tert-butyl ester (2.00L) and then dried under high vacuum. The residue was triturated with methyl tert-butyl ester (600 mL) and filtered to give N- (7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-40) (300 g,88% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝12.18(s,1H),8.28(d,J＝13.2Hz,1H),2.89(t,J＝6.1Hz,2H),2.68-2.60(m,2H),2.18-2.08(m,6H),1.99(quin,J＝6.4Hz,2H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δ＝-103.89。LCMS(ESI+)m/z：C ₁₃ H ₁₅ FNO ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 236.1, found: 236.0.

n- (7-fluoro-3-hydroxy-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-41)：

To a solution of N- (7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-40) (75.0 g,319mmol,1.0 eq.) in methanol (1.20L) was added a solution of KOH (53.7 g,956mmol,3 eq.) and (diacetoxyiodo) benzene (113 g,351mmol,1.1 eq.) in methanol (600 mL) under argon at 0deg.C, and the mixture was stirred at 15deg.C for 3h. (three additional reactions were set up as described above and all four were used)The reaction mixtures were combined). The combined mixture was adjusted to pH 4 by the addition of 1N HCl, concentrated at 35℃to remove most of the methanol, and the mixture was extracted with dichloromethane (3X 1.0L). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Dried, filtered, and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=5/1 to 4/1) to give N- (7-fluoro-3-hydroxy-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-41) (210 g,66% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝11.92(s,1H),8.26(d,J＝13.1Hz,1H),5.90-4.94(m,1H),4.28(dd,J＝5.0,12.7Hz,1H),3.07-2.97(m,1H),2.96-2.84(m,1H),2.29-2.20(m,1H),2.16(s,3H),2.10(d,J＝1.2Hz,3H),1.95-1.82(m,1H)。 ¹⁹ F NMR(377MHz,DMSO-d ₆ )δ＝-104.3。LCMS(ESI+)m/z：C ₁₃ H ₁₅ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 252.1, found: 252.0.

n- (3-allyloxy-7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-42)：

Ag was added to a solution of N- (7-fluoro-3-hydroxy-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-41) (70.0 g,279mmol,1.0 eq.) in acetonitrile (1.40L) at 15deg.C ₂ A solution of O (129 g,557mmol,2.0 eq.) and 3-iodoprop-1-ene (140 g,836mmol,76.3mL,3.0 eq.) in acetonitrile (280 mL) and the mixture stirred at 40℃for 5h. (two additional reactions were set up as described above and all three reaction mixtures were combined). The combined mixture was filtered through a pad of celite and the filter cake was washed with dichloromethane (5.00L). The combined filtrates were concentrated and the residue was purified by silica gel chromatography (petroleum ether/ethyl acetate=9/1-4/1) to give the material. This material was triturated with methyl tert-butyl ester (500 mL) and filtered to give N- (3-allyloxy-7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-42) (198g, 81% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝11.81(s,1H),8.27(d,J＝13.1Hz,1H),5.93(tdd,J＝5.3,10.5,17.2Hz,1H),5.29(qd,J＝1.8,17.2Hz,1H),5.16(dd,J＝1.7,10.5Hz,1H),4.32-4.06(m,3H),3.12-2.83(m,2H),2.32-2.23(m,1H),2.17(s,3H),2.11(d,J＝1.5Hz,3H),2.09-1.98(m,1H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δ＝-104.2。LCMS(ESI+)m/z：Cl ₆ H ₁₉ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 292.1, found: 291.9.

n- [ 7-fluoro-8-methyl-4-oxo-3- (2-oxoethoxy) tetrahydronaphthalen-5-yl]Acetamide (5-43)：

A mixture of N- (3-allyloxy-7-fluoro-8-methyl-4-oxo-tetrahydronaphthalen-5-yl) acetamide (5-42) (42.0 g,144mmol,1.0 eq.) in dichloromethane (840 mL) and methanol (420 mL) was cooled to-70 ℃, ozone (6.92 g,144mmol,1.0 eq.) was bubbled into the mixture for 60min, followed by O ₂ Bubbling for 30min. Then, methylthiomethane (26.5 ml,360mmol,2.5 eq.) was added to the mixture at-70 ℃ and warmed to 15 ℃ and stirred at 15 ℃ for 1h. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixtures were quenched with water (5.0L) and extracted with dichloromethane (3X 1.0L). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtering and concentrating to obtain N- [ 7-fluoro-8-methyl-4-oxo-3- (2-oxoethoxy) tetrahydronaphthalen-5-yl]Acetamide (5-43) (168 g,59% yield) which was used directly in the next step without further purification. ¹ H NMR(400MHz,CDCl ₃ )δ＝11.94(br s,1H),9.80(s,1H),8.44(d,J＝12.7Hz,1H),4.55-4.30(m,1H),3.85-3.69(m,2H),3.51-3.48(m,1H),3.18-3.03(m,1H),2.96-2.78(m,1H),2.52-2.34(m,1H),2.26-2.21(m,3H),2.14(br dd,J＝1.7,3.9Hz,3H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δ＝-100.7。LCMS(ESI+)m/z：C ₁₅ H ₁₇ FNO ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 294.1, found: 293.9.

n- [ 7-fluoro-3- (2-hydroxyethoxy) -8-methyl-4-oxo-tetrahydronaphthalen-5-yl]Acetamide (5-44)：

To N- [ 7-fluoro-8-methyl-4-oxo-3- (2-oxoethoxy) tetrahydronaphthalen-5-yl at 0deg.C]Acetamide (5-43) (42 g,85.9mmol,60% purity, 1.0 eq.) in THF (850 mL) and H ₂ NaBH was added in portions to a solution of O (425 mL) ₄ (975mg，25.8mmol，0.30 eq.) the mixture was stirred at 0 ℃ for 10min and quenched with cold water (2.0L). (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixtures were extracted with dichloromethane (3×1.0L), the organic layer was washed with brine, and dried over Na ₂ SO ₄ Drying, filtration, concentration and purification of the residue by silica gel chromatography (petroleum ether/ethyl acetate=1/1-1/3) gives N- [ 7-fluoro-3- (2-hydroxyethoxy) -8-methyl-4-oxo-tetrahydronaphthalen-5-yl]Acetamide (5-44) (70 g,61% yield). ¹ H NMR(400MHz,CDCl ₃ )δ＝11.96(br s,1H),8.45(d,J＝12.7Hz,1H),4.06(dd,J＝4.6,12.0Hz,1H),4.01-3.62(m,4H),3.11(td,J＝4.6,17.6Hz,1H),3.03-2.79(m,2H),2.45-2.36(m,1H),2.24(s,3H),2.15(d,J＝1.5Hz,4H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δ＝-100.9。LCMS(ESI+)m/z：C ₁₅ H ₁₉ FNO ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 296.1, found: 296.1.

8-amino-6-fluoro-2- (2-hydroxyethoxy) -5-methyl-tetralin-1-one (5-45)：

To N- [ 7-fluoro-3- (2-hydroxyethoxy) -8-methyl-4-oxo-tetrahydronaphthalen-5-yl under argon]To a solution of acetamide (5-44) (21.0 g,71.0mmol,1.0 eq.) in methanol (400 mL) was added HCl (2N, 630mL,18 eq.) and the mixture was stirred at 15℃for 18h and saturated NaHCO was added ₃ The pH is adjusted to be 7 to 8. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixtures were extracted with ethyl acetate (3×2.0L), the organic layer was washed with brine, and dried over Na ₂ SO ₄ Dried, filtered and concentrated. The residue was triturated with methyl tert-butyl ester/dichloromethane (1:2, 300 mL) and filtered to give 8-amino-6-fluoro-2- (2-hydroxyethoxy) -5-methyl-tetrahydronaphthalen-1-one (5-45) (42 g,81% yield). ¹ H NMR(400MHz,CD ₃ OD)δ＝6.30(d,J＝12.3Hz,1H),4.06(dd,J＝4.4,11.2Hz,1H),3.83-3.67(m,4H),3.04(td,J＝4.9,17.5Hz,1H),2.85-2.73(m,1H),2.40-2.30(m,1H),2.09-1.93(m,4H)。 ¹⁹ F NMR(376MHz,CD ₃ OD)δ＝-108.7。LCMS(ESI+)m/z：C ₁₃ H ₁₇ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 254.1, found: 254.1.

(9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [de]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-46)：

To 8-amino-6-fluoro-2- (2-hydroxyethoxy) -5-methyl-3, 4-dihydronaphthalen-1 (2H) -one (5-45) (10.5 g,41.4mmol,1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f under argon at 120 ℃ ]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (12.0 g,45.6mmol,1.1 eq.) in toluene (525 mL) was added o-cresol (31.5 mL,303mmol,7.3 eq.) and 4-methylbenzenesulfonic acid pyridin-1-ium (1.56 g,6.23mmol,0.15 eq.) and the mixture was stirred at 120℃for 13H. (three additional reactions were set up as described above and the four reaction mixtures were combined). The combined reaction mixtures were concentrated under reduced pressure and the residue was purified by column chromatography on silica gel eluting with 13% methanol in ethyl acetate to give (9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-46) (34.2 g,43% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.58(dd,J＝10.8,1.91Hz,1H),7.23(d,J＝6.8Hz,1H),6.49(s,1H),5.40(s,2H),4.95-5.24(m,2H),4.88(dt,J＝8.4,4.4Hz,1H),4.69-4.81(m,1H),3.78-3.91(m,1H),3.56-3.76(m,3H),3.10(d,J＝16.4Hz,1H),2.77-2.92(m,1H),2.33-2.45(m,1H),2.21(s,3H),1.83-2.04(m,3H),0.77-1.00(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.57。LCMS(ESI+)m/z：C ₂₆ H ₂₆ FN ₂ O ₆ ⁺ [ M+H of (H)] ⁺ Calculated values: 481.2, found: 481.0.

(1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [de]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-47) and (1S, 9S) -9- Ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4': 6,7]indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-48)：

(9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-46) (31.0 g,64.3mmol,1.0 eq.) was prepared by chiral SFC (instrument: waters SFC350 preparation SFC; column: DAICEF CHIRAFCEF OD (250 mm. Times.50 mm,10 um); mobile phase: A was CO) ₂ B is MeOH; gradient: b% = 60% isocratic elution mode; flow rate: 200g/min; wavelength: 220nm; column temperature: 40 ℃; system backpressure: 100 bar) to give (1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-47) (13.5 g,43% yield) (compound 5-47 may be the opposite enantiomer to the depicted enantiomer) and (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-48) (10.0 g,32% yield) (compound 5-48 may be the opposite enantiomer to that depicted). Note that: stereochemistry is arbitrarily specified.

Spectrum 5-47: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.64(d,J＝10.88Hz,1H),7.25(s,1H),6.50(s,1H),5.13-5.52(m,4H),4.95(br dd,J＝8.25,3.75Hz,1H),4.74(br t,J＝5.00Hz,1H),3.84(dt,J＝9.72,4.96Hz,1H),3.59-3.76(m,3H),3.10-3.21(m,1H),2.84-2.99(m,1H),2.23-2.46(m,4H),2.04(m,1H),1.87(m,2H),0.88(t,J＝7.32Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.59。LCMS(ESI+)m/z：C ₂₆ H ₂₆ FN ₂ O ₆ ⁺ [ M+H of (H)] ⁺ Calculated values: 481.2, found: 481.0. chiral SFC: rt=1.48 min.

Spectrum 5-48: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.74(d,J＝10.97Hz,1H),7.30(s,1H),6.51(s,1H),5.31-5.46(m,4H),5.03(dd,J＝8.11,3.70Hz,1H),4.74(t,J＝5.36Hz,1H),3.81-3.89(m,1H),3.59-3.75(m,3H),3.21(dt,J＝17.02,5.38Hz,1H),2.94-3.07(m,1H),2.31-2.45(m,4H),2.08(m,1H),1.88(dt,J＝13.23,6.62Hz,2H),0.89(t,J＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.53。LCMS(ESI+)m/z：C ₂₆ H ₂₆ FN ₂ O ₆ ⁺ [ M+H of (H)] ⁺ Calculated values: 481.2, found: 481.0. chiral SFC: rt=1.61 min.

Example 6

Synthesis of (1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (3-34) and (1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (3-35) (FIG. 17)

* The stereochemistry at the carbon is arbitrarily specified.

2- (allyloxy) -8-amino-6-fluoro-5-methyl-3, 4-dihydronaphthalen-1 (2H) -one (6-49)：

To a mixture of N- (7- (allyloxy) -3-fluoro-4-methyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (5-42) (600 mg,2.06mmol,1.0 eq.) in methanol (6.0 mL) was added HCl/methanol (6.0 mL,4M,11.65 eq.) under argon, the mixture was stirred at 25℃for 2h, cooled to 0℃and purified by the addition of saturated NaHCO ₃ The pH was adjusted to 8. It was extracted with dichloromethane (3×40 mL), the combined organic layers were washed with brine, and dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by silica gel column chromatography eluting with 10% ethyl acetate/petroleum ether gave 2- (allyloxy) -8-amino-6-fluoro-5-methyl-3, 4-dihydronaphthalen-1 (2H) -one (6-49) (418 mg,81% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm6.24-6.68(m,2H),6.20(d,J＝11.62Hz,1H),5.91-6.03(m,1H),5.33(dq,J＝17.24,1.63Hz,1H),5.20(dq,J＝10.38,1.35Hz,1H),4.36(m,1H),4.17(m,1H),3.97(dd,J＝10.15,4.16Hz,1H),3.01(dt,J＝17.45,5.33Hz,1H),2.75(m,1H),2.27(dq,J＝13.17,5.02Hz,1H),2.09-2.19(m,1H),2.04(d,J＝1.71Hz,3H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δppm-106.28。LCMS(ESI+)m/z：C ₁₄ H ₁₇ FNO ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 250.1, found: 250.1.

(9S) -1- (allyloxy) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Piirae-type pyridine Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (6-50)：

To 2- (allyloxy) -8-amino-6-fluoro-5-methyl-3, 4-dihydronaphthalen-1 (1H) -one (6-49) (200 mg, 0.803 mmol,1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f under argon at 120 ℃]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (232 mg,0.883mmol,1.1 eq.) in toluene (10 mL) was added o-cresol (0.319 mL,5.86mmol,7.3 eq.) and 4-methylbenzenesulfonic acid pyridin-1-ium (30.2 mg,0.120mmol,0.15 eq.) and the mixture was stirred at 120℃for 32H, concentrated under reduced pressure and the residue was purified by silica gel column chromatography eluting with 55% ethyl acetate/petroleum ether to give (9S) -1- (allyloxy) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (6-50) (100 mg,26% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 7.58-7.67(m,2H),6.03-6.16(m,1H),5.74(dd,J＝16.26,1.83Hz,1H),5.39-5.52(m,2H),5.21-5.36(m,3H),4.94(m,1H),4.38-4.47(m,1H),4.26(m,1H),3.87(d,J＝19.32Hz,1H),3.23-3.36(m,1H),2.99(br t,J＝13.02Hz,1H),2.45-2.57(m,1H),2.40(br s,3H)2.11-2.25(m,1H),1.82-1.98(m,2H),1.04(td,J＝7.37,2.87Hz,3H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δppm-110.35。LCMS(ESI+)m/z：C ₂₇ H ₂₆ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 477.2, found: 477.2.

(9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] ]Pyrano [3', 4':6,7]indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-33)：

At 25℃under argon, to (9S) -1- (allyloxy) -9-Ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (6-50) (300 mg,0.63mmol,1.0 eq.) was added ZnCl to a mixture of tetrahydrofuran (15 mL) ₂ (110 mg,0.82mmol,1.3 eq.) and Pd (PPh) added after 0.25h ₃ ) ₄ (58.0 mg,0.157mmol,0.25 eq.) and 0.25h later Bu was added ₃ SnH (3.33 mL,12.6mmol,20 eq.) and the mixture was stirred at 50deg.C for 1.5h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography eluting with 5% methanol/ethyl acetate to give a residue which was further purified by preparative HPLC to give (9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-33) (90.0 mg,32% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.72(d,J＝10.97Hz,1H),7.30(d,J＝2.27Hz,1H),6.50(d,J＝0.72Hz,1H),5.98(dd,J＝5.90,2.92Hz,1H),5.36-5.46(m,3H),5.23-5.32(m,1H),5.14(dt,J＝9.60,4.86Hz,1H),3.23(dt,J＝16.90,4.60Hz,2H),2.96-3.06(m,1H),2.35(s,3H),1.94-2.06(m,1H),1.80-1.93(m,2H),0.88(td,J＝7.30,1.49Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.78。LCMS(ESI+)m/z：C ₂₄ H ₂₂ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 437.2, found: 437.0.

preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: H2O; b: meOH (MeOH)

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220 and 254nm

(1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano-s [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-34) and (1S, 9S) -9-ethyl-5-fluoro- 1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline- 10,13 (1H, 9H) -diketones (3-35)：

(9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-33) (90.0 mg,0.206mmol,1.0 eq.) was prepared by chiral SFC (instrument: waters SFC80 preparation SFC; column: DAICEL CHIRALCEL OD (250 mm. Times.30 mm,10 um); mobile phase: A was CO) ₂ B is EtOH; gradient: b% = 55% isocratic elution mode; flow rate: 60g/min; wavelength: 220nm; column temperature: 40 ℃; system backpressure: 100 bar) to give (1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-34) (26.1 mg,29% yield) (compound 3-34 may be the opposite enantiomer to the depicted enantiomer) and (1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (3-35) (25.1 mg,28% yield) (compound 3-35 can be the opposite enantiomer to that depicted). Note that: the stereochemistry of the two products is arbitrarily specified.

Spectra of 3-34: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.73(d,J＝10.97Hz,1H),7.30(s,1H),6.50(s,1H),5.98(d,J＝5.84Hz,1H),5.37-5.47(m,3H),5.26-5.35(m,1H),5.10-5.20(m,1H),3.20-3.30(m,2H),2.97-3.07(m,1H),2.35(s,3H),1.95-2.05(m,1H),1.78-1.94(m,2H),0.88(t,J＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.78。LCMS(ESI+)m/z：C ₂₄ H ₂₂ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 437.2, found: 437.0. chiral SFC: rt=1.43 min.

Spectra of 3-35: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.73(d,.J＝11.09Hz,1H),7.30(s,1H),6.50(s,1H),5.97(d,J＝5.84Hz,1H),5.34-5.48(m,3H),5.21-5.32(m,1H),5.09-5.20(m,1H),3.20-3.24(m,2H),2.96-3.08(m,1H),2.35(s,3H),1.99(d,J＝9.54Hz,1H),1.80-1.93(m,2H),0.89(t,J＝7.09Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.80。LCMS(ESI+)m/z：C ₂₄ H ₂₂ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 437.2, found: 437.0. chiral SFC: rt=1.54 min.

Example 7

(1S, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-11, 14 (2H, 10H) -dione methanesulfonate (7-59) and (1R, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-11, 14 (2H, 10H) -dione methanesulfonate (7-60) (FIG. 18)

* The stereochemistry at the carbon is arbitrarily specified.

5- (5-acetamido-3-fluoro-2-methylphenyl) pent-4-enoic acid (7-51)：

At N ₂ N- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4) (50.0 g,203mmol,1.0 eq.) in THF (250 mL) and H under an atmosphere ₂ To a solution of O (50.0 mL) was added pent-4-enoic acid (49.9 mL,487mmol,2.4 eq.), diisopropylethylamine (155 mL,894mmol,4.4 eq.), triorthophenylphosphine (12.4 g,40.6mmol,0.20 eq.) and palladium (II) diacetate (4.56 g,20.3mmol,0.10 eq.) and the mixture stirred at 75℃for 24h and cooled to 30 ℃. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined mixture was filtered through a pad of celite and the filter cake was washed with water (2.0L) and ethyl acetate (3.0L). The mixture was adjusted to pH 4-5 with HCl solution (5N), extracted with ethyl acetate (3X 800 mL) and the organic layer was dried over Na ₂ SO ₄ Dried, concentrated, and the residue purified by flash column chromatography on silica gel eluting with 30% ethyl acetate/petroleum etherTo give 5- (5-acetamido-3-fluoro-2-methylphenyl) pent-4-enoic acid (7-51) (0.20 kg,83% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝12.19(br s,1H),9.99(s,1H),7.51-7.28(m,1H),7.00(s,1H),6.17-5.36(m,2H),3.35-3.24(m,2H),3.19-2.93(m,2H),2.07(s,3H),2.01(s,3H) ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-115.96。

5- (5-acetamido-3-fluoro-2-methylphenyl) pentanoic acid (7-52)：

Pd/C (25.0 g,170mmol,10 wt%, 1.0 eq) was added to a mixture of 5- (5-acetamido-3-fluoro-2-methylphenyl) pent-4-enoic acid (7-51) (50.0 g,170mmol,90% purity, 1.0 eq) in methanol (500 mL) under an argon atmosphere. After the mixture was evacuated and H was used ₂ After three fills, it is then heated to 25℃at H ₂ (15 Psi) for 12h. (three additional reactions were set up as described above and all four reaction mixtures were combined). In the substitution of H with argon ₂ After the atmosphere, the mixture was filtered through a celite pad, the filter cake was washed with methanol (6.00L), and the filtrate was concentrated to give 5- (5-acetamido-3-fluoro-2-methylphenyl) pentanoic acid (7-52) (190 g,94% yield), which was used directly in the next step without further purification. ¹ H NMR(400MHz,DMSO-d ₆ )δ＝9.97(s,1H),7.41(dd,J＝1.7,12.2Hz,1H),7.03(s,1H),2.54(br t,J＝7.4Hz,2H),2.23(t,J＝7.0Hz,2H),2.08(d,J＝1.7Hz,3H),2.01(s,3H),1.59-1.43(m,4H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δppm-115.806。LCMS(ESI+)m/z：C ₁₄ H ₁₉ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 268.1, found: 268.0.

n- (3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-1-yl) acetamide (7-53)：

A mixture of 5- (5-acetamido-3-fluoro-2-methylphenyl) pentanoic acid (7-52) (38.0 g,142mmol,1.0 eq.) in polyphosphoric acid (500 mL) was stirred at 110deg.C for 3h. (four additional reactions were set up as described above and all five reaction mixtures were combined). The combined reaction mixture was slowly poured into stirring ice water (10.0L) with saturated NaHCO ₃ The pH of the solution was adjusted to 7 with ethyl acetate (3X 1.00)L) extracting the mixture. The combined organic layers were washed with brine, dried over anhydrous Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 15% ethyl acetate/petroleum ether gives N- (3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Rotalin-1-yl) acetamide (7-53) (91 g,64% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝9.83(s,1H),7.19(d,J＝11.6Hz,1H),2.69(t,J＝6.3Hz,2H),2.58-2.52(m,2H),2.16(d,J＝2.0Hz,3H),1.95(s,3H),1.74-1.58(m,4H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.49。LCMS(ESI+)m/z：C ₁₄ H ₁₇ FNO ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 250.12, found: 250.0.

(Z) -N- (3-fluoro-8- (oximino) -4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rota-1-yl Acetamide (7-54)：

To a mixture of potassium tert-butoxide (1M in tetrahydrofuran, 191mL,2.1 eq.) in tetrahydrofuran (194 mL), ethanol (31.4 mL) and N-butanol (31.4 mL) was added N- (2-fluoro-1-methyl-5-oxo-6, 7,8, 9-tetrahydrobenzo [ 7) at 0deg.C]The rota-n-4-yl) acetamide (7-53) (22.7 g,90.9mmol,1.0 eq.) and isoamyl nitrite (14.8 ml,109mmol,1.2 eq.) and the mixture was stirred at 20℃for 3h. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixture was cooled to 0 ℃, quenched with 0.5N hydrochloric acid (1.00L) and extracted with ethyl acetate (3×300 mL), the organic layer was washed with brine, dried over sodium sulfate, filtered, and concentrated under reduced pressure. The residue was triturated with methyl tert-butyl ester (500 mL) and filtered to give (Z) -N- (3-fluoro-8- (oximino) -4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [ 7) ]Rota-n-1-yl) acetamide (7-54) (80.0 g,88% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝12.69-11.95(m,1H),10.10(s,1H),7.28(d,J＝11.6Hz,1H),2.68(br t,J＝6.6Hz,2H),2.57(t,J＝6.8Hz,2H),2.17(d,J＝1.8Hz,3H),1.96(s,3H),1.78-1.75(m,2H) ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-107.85,-109.67,-110.39。LCMS(ESI+)m/z：C ₁₄ H ₁₆ FN ₂ O ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 279.1, found: 279.1.

n- (8-amino-3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotalin-1-yl) acetamides Hydrochloride (7-55)：

At N ₂ To N- [ (5E) -2-fluoro-5-oximino-1-methyl-6, 7,8, 9-tetrahydrobenzo [7 ] under an atmosphere]Rotaen-4-yl]Acetamide (7-54) (20.0 g,75.7mmol,1.0 eq.) to a mixture of methanol (240 mL) was added hydrochloric acid (12M, 18.9mL,3.0 eq.) and Pd/C (4.00 g,75.7mmol,10 wt%, 1.0 eq.), the mixture was evacuated and treated with H ₂ Filling three times, and at H ₂ (15 Psi) at 20℃for 3h. (three additional reactions were set up as described above and all four reaction mixtures were combined). In the substitution of H with argon ₂ After the atmosphere, the reaction mixtures were combined, diluted with methanol (800 mL), filtered through a celite pad, the filter cake was washed with methanol (10.0L) and the combined filtrate was concentrated to dryness under reduced pressure. The residue was triturated with methyl tert-butyl ether (800 mL) and filtered to give N- (8-amino-3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Rota-n-1-yl) acetamide hydrochloride (7-55) (75 g,98% yield). ¹ H NMR showed the material to contain about 25% of the Ac-removal by-product. ¹ H NMR(400MHz,DMSO-d ₆ )δ＝10.34(s,1H),8.75-8.40(m,3H),7.31-7.20(m,1H),4.44-4.21(m,1H),2.98(dt,J＝6.1,7.9Hz,1H),2.85-2.75(m,1H),2.47(br s,1H),2.27-2.11(m,3H),2.10-1.94(m,3H),1.94-1.50(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-107.88,-110.63。LCMS(ESI+)m/z：C ₁₄ H ₁₆ FN ₂ O ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 265.1, found: 265.0.

n, N' - (3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotaene-1, 8-diyl) diacetyl Amine (7-56)：

To N- (5-amino-2-fluoro-1-methyl-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]To a suspension of the rota-n-4-yl) acetamide hydrochloride (7-55) (25.0 g,87.2mmol,1.0 eq.) in methylene chloride (225 mL) was added triethylamine (36.4 mL,262mmol,3.0 eq.) and acetic anhydride (9.80 mL,105mmol,1.2 eq.) and the mixture was stirred at 20℃for 3h. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixture was treated with saturated NH ₄ Cl solution (2.00L) was extracted, the aqueous phase was extracted with ethyl acetate (3X 500 mL) and the combined organic layers were extracted with Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 30% ethyl acetate/petroleum ether gives N, N' - (3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Decene-1, 8-diyl) diacetamide (7-56) (54.0 g,53% yield) and N- (4-amino-2-fluoro-1-methyl-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Rota-n-5-yl) acetamide (7-57) (9.00 g,10% yield).

Spectrum 7-56: : ¹ H NMR(400MHz,DMSO-d ₆ )δ＝9.61(br s,1H),8.69(br d,J＝4.5Hz,1H),7.69(d,J＝12.2Hz,1H),4.56-4.39(m,1H),2.93(br dd,J＝7.0,12.3Hz,1H),2.43(br dd,J＝10.3,14.7Hz,1H),2.14(d,J＝1.2Hz,3H),2.01(s,4H),1.93(s,4H),1.81-1.61(m,2H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.86。LCMS(ESI+)m/z：C ₁₆ H ₂₀ FN ₂ O ₃ ⁺ [ M+H of (H) ] ⁺ Calculated values: 307.1, found: 307.1.

spectrum 7-57: ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.25(br d,J＝7.1Hz,1H),6.45-6.33(m,3H),4.57(td,J＝6.7,11.5Hz,1H),2.97-2.86(m,1H),2.81-2.70(m,1H),2.07-1.97(m,4H),1.95-1.88(m,1H),1.84(s,3H),1.70-1.59(m,1H),1.57-1.42(m,1H)。 ¹⁹ F NMR(376MHz,DMSO)δppm-110.23。LCMS(ESI+)m/z：C ₁₄ H ₁₈ FN ₂ O ₃ ⁺ [ M+H of (H)] ⁺ Calculated values: 265.1, found: 265.1.

n- (4-amino-2-fluoro-1-methyl-5-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Rotaen-6-yl) acetamides (7-57)：

To N, N' - (3-fluoro-4-methyl-9-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]To a mixture of rotaene-1, 8-diyl-diacetamide (7-56) (18.0 g,61.6mmol,1.0 eq.) in methanol (180 mL) was added hydrochloric acid/methanol (4 m,180mL,11.7 eq.) andthe mixture was stirred at 25℃for 2h. (three additional reactions were set up as described above and all four reaction mixtures were combined). The combined reaction mixtures were concentrated under reduced pressure and taken up in saturated NaHCO ₃ The residue was extracted with solution (2.00L) and ethyl acetate/methanol (10/1, 3X 800 mL). The combined organic phases were washed with brine, dried over anhydrous Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 30% ethyl acetate/petroleum ether gives N- (4-amino-2-fluoro-1-methyl-6, 7,8, 9-tetrahydro-5H-benzo [ 7)]Rota-n-5-yl) acetamide (7-57) (25 g,54% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δ＝8.25(br d,J＝7.1Hz,1H),6.45-6.33(m,3H),4.57(td,J＝6.7,11.5Hz,1H),2.97-2.86(m,1H),2.81-2.70(m,1H),2.07-1.97(m,4H),1.95-1.88(m,1H),1.84(s,3H),1.70-1.59(m,1H),1.57-1.42(m,1H)。 ¹⁹ F NMR(376MHz,CDCl ³ )δppm-111.22。LCMS(ESI+)m/z：C ₁₄ H ₁₈ FN ₂ O ₃ ⁺ [ M+H of (H)] ⁺ Calculated values: 265.1, found: 265.1.

n- ((10S) -10-ethyl-6-fluoro-10-hydroxy-5-methyl-11, 14-dioxo-2,3,4,10,11,13,14, 16-octahydro-1H-cyclohepta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamide (7-58)：

To N- (4-amino-2-fluoro-1-methyl-5-oxo-6, 7,8, 9-tetrahydro-5H-benzo [7 ]]Decen-6-yl) acetamide (7-57) (230 mg, 0.87mmol, 1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (458 mg,1.74mmol,2.0 eq.) in toluene (23 mL) was added 4-methylbenzenesulfonic acid pyridin-1-ium (87.4 mg,0.348mmol,0.4 eq.) and the mixture was stirred in a sealed tube at 120℃for 16H. The reaction mixture was concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel eluting with 50% ethyl acetate/petroleum ether to give N- ((10S) -10-ethyl-6-fluoro-10-hydroxy-5-methyl-11, 14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamide (7-58) (240 mg,54% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δ＝8.71(br d,J＝6.2Hz,1H),7.73(d,J＝10.5Hz,1H),7.29(s,1H),6.52(d,J＝7.4Hz,1H),5.62-5.50(m,1H),5.46-5.31(m,3H),5.22-5.11(m,1H),3.27-3.20(m,2H),3.17(d,J＝4.8Hz,1H),2.42(s,3H),2.31-2.20(m,1H),2.14-2.02(m,2H),1.97(s,3H),1.84-1.60(m,2H),0.87(q,J＝7.4Hz,3H)。LCMS(ESI+)m/z：C ₂₇ H ₂₇ FN ₃ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 492.2, found: 492.1.

(1S, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [de]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-11, 14 (2H, 10H) -dione mesylate (7-59) and (1R, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [ de ]]Pyrans (pyrans) And [3',4':6,7]Indolazino [1,2-b ]]Quinoline-11, 14 (2H, 10H) -dione methanesulfonate (7-60)：

To N- ((10S) -10-ethyl-6-fluoro-10-hydroxy-5-methyl-11, 14-dioxo-2,3,4,10,11,13,14,16-octahydro-1H-cyclohepta [ de ] under argon]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamide (7-58) (160 mg,0.101mmol,1.0 eq.) in a mixture of 2-methoxyethanol (0.8 mL) methylcyclohexane (0.8 mL), H ₂ O (0.75 mL) and methanesulfonic acid (0.25 mL), the mixture was stirred at 100deg.C for 8H, concentrated, and the residue was purified by preparative HPLC to give (1S, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-11, 14 (2H, 10H) -dione methanesulfonate (7-59) (5.5 mg,2.4% yield) (compound 7-59 may be the opposite enantiomer to that depicted) and (1R, 10S) -1-amino-10-ethyl-6-fluoro-10-hydroxy-5-methyl-3,4,13,16-tetrahydro-1H-cyclohepta [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-11, 14 (2H, 10H) -dione methanesulfonate (7-60) (6.4 mg,3.6% yield) (compound 7-60 may be the opposite enantiomer to the depicted enantiomer). Note that: stereochemistry is arbitrarily specified.

Preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：MeOH

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220nm and 254nm

Spectrum 7-59: ¹ H NMR(400MHz,D ₂ O)δppm 7.38-7.49(m,2H),5.52-5.62(m,1H),5.35-5.46(m,3H),5.16(t,J＝4.4Hz,1H),3.40-3.51(m,1H),2.99-3.13(m,1H),2.77(s,6H),2.48-2.57(m,2H),2.38(d,J＝1.6Hz,3H),2.22-2.33(m,1H),2.06-2.20(m,1H),1.94(q,J＝7.5Hz,2H),0.90(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,D ₂ O)δppm-111.53。LCMS(ESI+)m/z：C ₂₅ H ₂₅ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 450.2, found: 450.0.

spectrum 7-60: ¹ H NMR(400MHz,D ₂ O)δppm 7.40-7.51(m,2H),5.44-5.60(m,2H),5.33-5.43(m,2H),5.09-5.14(m,1H),3.37-3.48(m,1H),3.01-3.14(m,1H),2.77(s,3H),2.43-2.54(m,1H),2.39(s,3H),2.27-2.35(m,1H),2.06-2.23(m,2H),1.95(q,J＝7.6Hz,2H),0.92(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,D ₂ O)δppm-109.21。LCMS(ESI+)m/z：C ₂₅ H ₂₅ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 450.2, found: 450.0.

example 8

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-71) and (1R, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-72) (FIG. 19)

* The stereochemistry at the carbon is arbitrarily specified.

4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbut-3-enoic acid (8-61)：

To a mixture of N- (3-bromo-5-fluoro-4-methyl-phenyl) acetamide (1-4) (10.0 g,40.6mmol,1.0 eq.) and 2-methylbut-3-enoic acid (13.0 g,130mmol,3.2 eq.) in tetrahydrofuran (40 mL) and water (10 mL) under nitrogen was added N-ethyl-N, N-diisopropylamine (38.2 mL,219mmol,5.4 eq.), triorthophenylphosphine (2.47 g,8.13mmol,0.2 eq.) and diacetoxy palladium (912 mg,4.06mmol,0.1 eq.) and the mixture was stirred at 75℃for 5h, quenched with water (50 mL) and the pH was adjusted to about 3 by the addition of 3N hydrochloric acid at 0 ℃. It was filtered through a pad of celite and the filtrate was extracted with ethyl acetate (3×150 mL), the organic layer was washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration, and purification of the residue by flash column chromatography on silica gel eluting with 50% ethyl acetate/petroleum ether gave 4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbut-3-enoic acid (8-61) (6.00 g,56% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 12.38(s,1H),10.02(d,J＝4.0Hz,1H),7.46-7.52(m,1H),6.99-7.38(m,1H),6.65-6.69(m,1H),6.17(dd,J＝15.6,8.0Hz,1H),3.49(d,J＝7.2Hz,1H),2.11(dd,J＝13.2,1.6Hz,3H),2.02(d,J＝4.8Hz,3H),1.24-1.88(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-115.68。LCMS(ESI+)m/z：C ₁₄ H ₁₇ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 266.1, found: 266.1.

4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbutanoic acid (8-62)：

Pd/C (10 wt%) (2.40 g,0.12 eq.) was added to a mixture of 4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbut-3-enoic acid (1) (5.00 g,18.8mmol,1.0 eq.) in methanol (20 mL) under nitrogen, the suspension was degassed in vacuo and H ₂ Purging three times and at 25℃at H ₂ Stirring for 10h at (15 psi). In the presence of argonChange H ₂ After the atmosphere, it was filtered through a pad of celite, and the filter cake was washed with methanol (300 mL). The combined filtrates were concentrated under reduced pressure to give 4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbutanoic acid (8-62) (4.4 g,87% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 10.0(s,1H),7.42(dd,J＝12.4,1.6Hz,1H),7.04(s,1H),2.52-2.58(m,2H),2.24(t,J＝6.8Hz,2H),2.08(d,J＝1.6Hz,3H),2.01(s,3H),1.40-1.65(m,4H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-125.77。LCMS(ESI+)m/z：C ₁₄ H ₁₉ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 268.1, found: 268.1.

n- (7-fluoro-3, 8-dimethyl-4-oxotetralin-5-yl) acetamide (8-63)：

To a mixture of 4- (5-acetamido-3-fluoro-2-methylphenyl) -2-methylbutanoic acid (8-62) (5.00 g,18.7mmol,1.0 eq.) in trifluoroacetic acid (10 mL) was added trifluoroacetic anhydride (5.20 mL,37.4mmol,2.0 eq.) at 0deg.C, the mixture was stirred at 0deg.C for 2h, quenched with ice water (100 mL), and the pH was adjusted to about 7 by adding 25% aqueous NaOH at 0deg.C. It was extracted with ethyl acetate (3X 200 mL), the combined organic layers were washed with brine, and dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 8% ethyl acetate/petroleum ether gave N- (7-fluoro-3, 8-dimethyl-4-oxotetralin-5-yl) acetamide (8-63) (3.50 g,75% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm12.13(s,1H),8.28(d,J＝13.2Hz,1H),2.93-3.04(m,1H),2.81-2.92(m,1H),2.63-2.75(m,1H),2.07-2.18(m,7H),1.74(qd,J＝12.0,4.8Hz,1H),1.15(d,J＝6.4Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-104.64。LCMS(ESI+)m/z：C ₁₄ H ₁₇ FNO ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 250.1, found: 250.1.

1- (8-acetamido-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) hydrazine-1, 2-dimethyl Di-tert-butyl ester of acid (8-64)：

At 0℃under nitrogen, to N- (7-fluoro-3,to a mixture of 8-dimethyl-4-oxotetralin-5-yl) acetamide (8-63) (35.1 g,140mmol,1.0 eq.) in toluene (700 mL) was added dropwise sodium bis (trimethylsilyl) amide (309 mL,1M,2.2 eq.) and the mixture was cooled to-40℃and a solution of di-tert-butyl diazene-1, 2-dicarboxylate (42.1 g,183mmol,1.3 eq.) in toluene (350 mL) was added dropwise. The reaction mixture was warmed to 25 ℃, stirred at 25 ℃ for 4h, cooled to 0 ℃, diluted with water (1L) and extracted with ethyl acetate (3×500 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 20% ethyl acetate/petroleum ether gave di-tert-butyl 1- (8-acetamido-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) hydrazine-1, 2-dicarboxylate (8-64) (41.0 g,60% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 11.61-11.84(m,1H),8.26(d,J＝12.8Hz,1H),7.96-8.15(m,1H),2.96-3.16(m,2H),2.68-2.84(m,1H),2.12(s,4H),2.08(s,3H),1.35-1.46(m,21H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-105.29。LCMS(ESI+)m/z：C ₂₄ H ₃₅ FN ₃ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 480.2, found: 502 (MS+Na).

N- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2- (prop-2-ylidene) hydrazino) -5,6,7, 8-tetrahydronaphthalen-1-yl) Acetamide (8-66)：

To a solution of di-tert-butyl 1- (8-acetamido-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) hydrazine-1, 2-dicarboxylate (8-64) (41.0 g,85.5mmol,1.0 eq.) in dichloromethane (820 mL) was added trifluoroacetic acid (410 mL), the mixture was stirred at 25 ℃ for 1h, acetone (480 mL) was added and the mixture was stirred at 25 ℃ for a further 0.5h. This was concentrated to give N- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2- (prop-2-ylidene) hydrazino) -5,6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-66) (18.1 g,66% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm11.90(s,1H),8.31(d,J＝12.8Hz,1H),3.05-3.14(m,1H),2.90-3.02(m,1H),2.11-2.19(m,7H),2.05-2.08(m,2H),1.98(d,J＝2.0Hz,6H),1.34(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-75.04。LCMS(ESI+)m/z：C ₁₇ H ₂₃ FN ₃ O ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 320.1, found: 320.1.

n- (7-amino-3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-67)：

To a mixture of N- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2- (prop-2-ylidene) hydrazino) -5,6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-66) (15.1 g,47.3mmol,1.0 eq.) in acetic acid (302 mL) was added zinc powder (40.8 g,624mmol,13.2 eq.) in portions, the mixture was stirred at 20℃for 2h, filtered and the filtrate concentrated under reduced pressure to give N- (7-amino-3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-67) (23.8 g, crude product) which was used directly in the next step without further purification. ¹ H NMR(400MHz,DMSO-D ₆ )δppm 11.56(s,1H),8.26-8.30(m,1H),3.01-3.09(m,2H),2.12-2.19(m,10H),1.43(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-73.57。LCMS(ESI+)m/z：C ₁₄ H ₁₈ FN ₂ O ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 265.1, found: 265.1.

n, N' - (3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalene-1, 7-diyl) diacetic acid amide (8-68)：

To a mixture of N- (7-amino-3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-67) (23.8 g,73.3mmol,1.0 eq.) in dichloromethane (414 mL) was added acetic anhydride (8.28 mL,88.0mmol,1.2 eq.) and triethylamine (30.6 mL,220mmol,3.0 eq.) and the mixture stirred at 25℃for 12h, quenched with water (500 mL) and extracted with dichloromethane (3X 200 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 70% ethyl acetate/petroleum ether gave N, N' - (3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalene-1, 7-diyl) diacetamide (8-68) (7.5 g,51% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm11.81(s,1H),8.20-8.37(m,2H),2.94-3.03(m,1H),2.77-2.88(m,1H),2.65(m,1H),2.06-2.18(m,6H),1.85(m,1H),1.79(s,3H),1.32(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-105.03。

N- (8-amino-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) acetamide (8-69)：

To a mixture of N, N' - (3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalene-1, 7-diyl) diacetamide (8-68) (7.50 g,24.4mmol,1.0 eq.) in methanol (105 mL) was added HCl/MeOH (105 mL, 4M), the mixture was stirred at 25℃for 2h, concentrated under reduced pressure, the residue was diluted with dichloromethane (300 mL) and saturated NaHCO ₃ Aqueous (2X 200 mL) extraction. The organic layer was washed with brine, dried over Na ₂ SO ₄ Dried, filtered and concentrated under reduced pressure to give N- (8-amino-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) acetamide (8-69) (5.50 g,89% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.91(s,1H),7.39(s,2H),6.36(d,J＝12.4Hz,1H),2.78-2.89(m,1H),2.67-2.75(m,2H),1.97(d,J＝1.20Hz,3H),1.83-1.88(m,1H),1.80(s,3H),1.27(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-108.38。LCMS(ESI+)m/z：C ₁₄ H ₁₈ FN ₂ O ₂ ⁺ [ MH of (V)] ⁺ Calculated values: 265.1, found: 265.1.

n- ((9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13, 15-octahydro-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamides (8-70)：

To N- (8-amino-6-fluoro-2, 5-dimethyl-1-oxo-1, 2,3, 4-tetrahydronaphthalen-2-yl) acetamide (500 mg,1.89mmol,1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f under argon at 120 ℃]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (547 mg,2.08mmol,1.1 eq.) in toluene (25 mL) was added pyridine 4-methylbenzenesulfonate (71.3 mg,0.283mmol,0.15 eq.) and o-cresol (1.44 mL,13.8mmol,7.3 eq.) and the mixture was stirred at 130℃for 13H, and the water formed was removed with a Dean-Stark trap. Concentrating under reduced pressure, and purifying the residue by flash column chromatography on silica gel eluting with 7% methanol in dichloromethane to give To N- ((9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) acetamide (8-70) (362 mg,39% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.78(d,J＝11.2Hz,1H),7.30(s,1H),6.53(d,J＝10.0Hz,1H),5.25-5.54(m,4H),4.81-4.90(m,1H),3.24-3.29(m,1H),2.86-3.11(m,3H),2.39(s,3H),1.95(d,J＝3.6Hz,3H),1.84-1.89(m,2H),1.50(d,J＝4.8Hz,3H),0.87(d,J＝5.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.94。LCMS(ESI+)m/z：C ₂₇ H ₂₇ FN ₃ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 492.1, found: 492.2

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Piirae-type pyridine Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-71) and (1R, 9S) -1- Amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indoles and their use Pyrazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-72)：

A mixture of N- ((9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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) acetamide (8-70) (600 mg,1.22mmol,1.0 eq.) in dioxane (6 mL) and concentrated hydrochloric acid (6 mL, 12M) was stirred under argon at 100deg.C for 24h in a sealed tube. It was concentrated under reduced pressure and the residue was purified by preparative HPLC to give (1 s,9 s) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1 h,9 h) -dione hydrochloride (8-71) (61.0 mg,10% yield) and (1 r,9 s) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1 h,9 h) -dione hydrochloride (8-72) (85.0 mg,14% yield). Note that: stereochemistry is arbitrarily specified.

Preparative HPLC conditions：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: HCl/H ₂ O＝0.040％v/v；B：ACN

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220nm and 254nm

Spectrum 8-71: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.97(s,3H),7.89(d,J＝10.8Hz,1H),7.36(s,1H),5.73-5.81(m,1H),5.56-5.65(m,1H),5.41-5.49(m,2H),3.22(d,J＝4.4Hz,2H),2.34-2.44(m,5H),1.82-1.94(m,5H),0.88(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.24。LCMS(ESI+)m/z：C ₂₅ H ₂₅ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 450.1, found: 450.1

Spectrum 8-72: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.98(s,3H),7.89(d,J＝10.8Hz,1H),7.36(s,1H),5.71-5.79(m,1H),5.57-5.64(m,1H),5.46(s,2H),3.18-3.26(m,2H),2.36-2.44(m,5H),1.81-1.94(m,5H),0.87(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.22。LCMS(ESI+)m/z：C ₂₅ H ₂₅ FN ₃ O ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 450.1, found: 450.1

Example 9

N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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-hydroxyacetamide (9-74) (FIG. 20)

Acetic acid 2- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1, 2,3,9, 10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) amino) -2-oxo Ethyl ester (9-73)：

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]A mixture of quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-71) (40.0 mg,0.089mmol,1.0 eq.) and N-ethyl-N-isopropyl-2-amine (46.5. Mu.L, 0.266mmol,3.0 eq.) in N, N-dimethylformamide (2 mL) was stirred at 25℃for 0.5h, cooled to 0℃and 2-chloro-2-oxoethyl acetate (14.6 mg, 11.5. Mu.L, 0.106mmol,1.2 eq.) was added dropwise over 5 min. It was warmed to 25 ℃ and stirred at 25 ℃ for 3h, concentrated under reduced pressure, and the residue was purified by flash column chromatography on silica gel eluting with chloroform/methanol/water=7/3/1 to give acetic acid 2- (((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]) ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) amino) -2-oxoethyl ester (9-73) (30.0 mg,61% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.84(s,1H),7.78(d,J＝10.85Hz,1H),7.30(s,1H),6.51(s,1H),5.35-5.49(m,3H),5.13(d,J＝19.07Hz,1H),4.66(d,J＝14.66Hz,1H),4.50(d,J＝14.66Hz,1H),2.93-3.09(m,3H),2.39(s,3H),1.99(s,3H),1.83-1.95(m,3H),1.55(s,3H),0.88(t,J＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.87。LCMS(ESI+)m/z：C ₂₉ H ₂₉ FN ₃ O ₇ ⁺ [ MH of (V)] ⁺ Calculated values: 550.2, found: 550.2.

n- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12, 13, 15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) -2-hydroxyacetamides (9) 74)：

To acetic acid 2- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de) under argon at 25 ℃]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) amino) -2-oxoethyl ester (9-73) (30.0 mg,0.055mmol,1.0 eq.) in tetrahydroTo a mixture of furan (1 mL) was added 1N aqueous NaOH (0.235 mL,4.3 eq). The mixture was stirred at 25 ℃ for 2h, quenched with 1N aqueous hydrochloric acid (0.2793 ml,5.0 eq), concentrated under reduced pressure, and the residue was purified by flash column chromatography on silica gel eluting with chloroform/methanol/water=7/3/1 to give N- ((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydro-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) -2-hydroxyacetamide (9-74) (13.5 mg,50% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.31(s,1H),7.77(d,J＝10.85Hz,1H),7.29(s,1H),6.50(s,1H),5.34-5.54(m,4H),4.91(d,J＝19.07Hz,1H),3.77-4.01(m,2H),3.22-3.31(m,1H),2.87-3.13(m,2H),2.39(s,3H),1.77-2.01(m,3H),1.58(s,3H),0.87(t,7＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.92。LCMS(ESI+)m/z：C ₂₇ H ₂₇ FN ₃ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 508.2, found: 508.1.

example 10

N- ((1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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-hydroxyacetamide (10-75)

Examples 10 (10-75) were prepared in a similar manner to example 9 using 8-72 instead of 8-71.

¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.31(s,1H),7.77(d,J＝10.85Hz,1H),7.29(s,1H),6.50(s,1H),5.34-5.54(m,4H),4.91(d,J＝19.07Hz,1H),3.77-4.01(m,2H),3.22-3.31(m,1H),2.87-3.13(m,2H),2.39(s,3H),1.77-2.01(m,3H),1.58(s,3H),0.87(t,J＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.88。LCMS(ESI+)m/z：C ₂₇ H ₂₇ FN ₃ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 508.2, found: 508.1.

example 11

N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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) -4-hydroxybutyramide hydrochloride (11-80) (FIG. 21)

4- ((tert-Butyldiphenylsilyl) oxy) butanoic acid (11-77)：

To a mixture of sodium 4-hydroxybutyrate (11-76) (2.10 g,16.6mmol,1.0 eq.) and imidazole (1.70 g,25.0mmol,1.5 eq.) in N, N-dimethylformamide (31.5 mL) was added tert-butylchlorodiphenylsilane (5.49 g,19.9mmol,1.2 eq.) under argon, the mixture was stirred at 25℃for 1h, poured into ice water (60 mL) and extracted with ethyl acetate (3X 50 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 30% ethyl acetate/petroleum ether afforded 4- ((tert-butyldiphenylsilyl) oxy) butanoic acid (11-77) (2.60 g,41% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.61-7.63(m,6H),7.46(dd,J＝7.2,4.4Hz,4H),3.72(t,J＝6.4Hz,2H),2.65(t,J＝7.2Hz,2H),1.87(t,J＝6.8Hz,2H),1.00(s,9H)。LCMS(ESI-)m/z：C ₂₀ H ₂₅ O ₃ Si ^- [ MH of (V)] ^- Calculated values: 341.2, found: 341.2.

4- ((tert-Butyldiphenylsilyl) oxy) butanoyl chloride (11-78)：

Oxalyl dichloride (1.05 g,8.24mmol,2.0 eq.) and N, N-dimethylformamide (9.02 mg,0.03 eq.) are added to a mixture of 4- ((tert-butyldiphenylsilyl) oxy) butanoic acid (11-77) (1.50 g,4.10mmol,1.0 eq.) in dichloromethane (75 mL) at 0deg.C under nitrogen, the mixture is warmed to 25deg.C, stirred for 2h at 25deg.C, and concentrated under reduced pressure to give 4- ((tert-butyldiphenylsilyl) oxy) butanoyl chloride (11-78 (1.50 g, crude), which was used directly in the next step without purification. ¹ H NMR(400MHz,CDCl ₃ )δppm 7.63-7.71(m,6H),7.42-7.48(m,4H),3.72(dt,J＝11.6,6.0Hz,2H),2.96-3.13(m,1H),2.63(t,7＝7.6Hz,1H),1.91-2.01(m,2H),1.07(s,9H)。

4- ((tert-butyldiphenylsilyl) oxy) -N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl Phenyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]An indolizino [1 ] group of which the amino group is as defined in, 2-b]quinolin-1-yl) butanamide (11-79)：

To (1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-71) (100 mg,0.206mmol,1.0 eq.) and trimethylamine (166 mg, 229. Mu.L, 1.65mmol,8.0 eq.) were added to a mixture of dichloromethane (5 mL) 4- ((tert-butyldiphenylsilyl) oxy) butyryl chloride (11-78) (303 mg,0.839mmol,2.4 eq.) the mixture was stirred at 25℃for 1h, filtered, the filtrate concentrated and the residue purified by flash column chromatography on silica gel eluting with 60% dichloromethane/ethyl acetate to give 4- ((tert-butyldiphenylsilyl) oxy) -N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ] ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) butanamide (11-79) (30.0 mg,17% yield). LCMS (esi+) m/z: c (C) ₄₅ H ₄₉ FN ₃ O ₆ Si ⁺ [ MH of (V)] ⁺ Calculated values: 774.3, found: 774.2.

n- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12, 13, 15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) -4-hydroxybutyramide hydrochloride Salt (11-80)：

To 4- ((tert-butyldiphenylsilyl) oxy) -N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ] under nitrogen at 25 ℃]Pyrano [3',4':6,7]An indolizino [1 ] group of which the amino group is as defined in,2-b]quinolin-1-yl) butanamide (11-79) (30.0 mg,0.039mmol,1.0 eq.) HCl/dioxane (0.5 mL, 8M) was added dropwise to a stirred mixture of dioxane (0.15 mL), the mixture was stirred at 25℃for 1h, and the filter cake was filtered and collected. The material was triturated with methyl tert-butyl ether (2 mL) at 25℃for 30min, collected by filtration and dried under vacuum to give N- ((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) -4-hydroxybutyramide hydrochloride (11-80) (10.2 mg,46% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.61(s,1H),7.78(d,J＝10.8Hz,1H),7.31(s,1H),5.35-5.48(m,3H),4.87(d,J＝18.8Hz,1H),3.37(t,J＝6.4Hz,3H),3.22-3.32(m,1H),2.86-3.11(m,3H),2.39(s,3H),2.25-2.33(m,2H),1.79-1.95(m,3H),1.56-1.66(m,2H),1.51(s,3H),0.88(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.98。LCMS(ESI+)m/z：C ₂₉ H ₃₁ FN ₃ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 536.2, found: 536.2.

example 12

N- ((1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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) -4-hydroxybutyramide hydrochloride (12-81)

Examples 12 (12-81) were prepared in a similar manner to example 11 using 8-72 instead of 8-71. ¹ H NMR(400MHz,DMSO-D6)δppm 8.60(s,1H),7.78(d,J＝10.8Hz,1H),7.30(s,1H),5.31-5.50(m,3H),4.86(d,J＝18.8Hz,1H),3.36(t,J＝6.4Hz,2H),3.23-3.31(m,1H),2.78-3.15(m,3H),2.39(s,3H),2.26-2.34(m,2H),1.75-1.99(m,3H),1.60(quin,J＝6.8Hz,2H),1.50(s,3H),0.86(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D6)δppm-111.95。LCMS(ESI+)m/z：C ₂₉ H ₃₁ FN ₃ O ₆ ⁺ [ MH of (V)]+calculated value: 536.2, found value：536.1。

Example 13

(1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3,4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (13-84) and (1R, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3,4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (13-85) (FIG. 22)

* The stereochemistry at the carbon is arbitrarily specified.

N- (3-fluoro-7-hydroxy-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (13-82)：

To a mixture of N- (3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (8-63) (1.00 g,4.01mmol,1.0 eq.) and cesium carbonate (261 mg, 0.803 mmol,0.2 eq.) in dimethyl sulfoxide (16 mL) at 25deg.C was added triethyl phosphite (1.33 g,8.02mmol,1.38mL,2.0 eq.) and the reaction mixture was evacuated and taken with O ₂ Three times filled and at 25℃at O ₂ Stirring was carried out for 24h at (15 psi). After the oxygen atmosphere was replaced with nitrogen, the mixture was diluted with water (80 mL) and extracted with ethyl acetate (2×40 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 15% ethyl acetate/petroleum ether gave N- (3-fluoro-7-hydroxy-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (12-82) (620 mg,58% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 11.97(s,1H),8.29(d,J＝13.08Hz,1H),5.47(s,1H),2.94-3.04(m,1H),2.78-2.88(m,1H),2.17(s,3H),2.11(d,J＝1.59Hz,3H),1.96-2.09(m,2H),1.28(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-104.38。LCMS(ESI+)m/z：C ₁₄ H ₁₇ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 266.1, found: 266.0.

8-amino-6-fluoro-2-hydroxy-2, 5-dimethyl-3, 4-dihydronaphthalen-1 (2H) -one (12-83)：

To a mixture of N- (3-fluoro-7-hydroxy-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (12-82) (300 mg,1.13mmol,1.0 eq.) in methanol (6 mL) was added dropwise HCl/MeOH (6 mL, 4M) at 25℃and the mixture stirred at 25℃for 1.5h, cooled to 0℃and saturated NaHCO was added at 0℃ ₃ The pH was adjusted to 7. It was extracted with dichloromethane (3X 10 mL) and the combined organic layers were dried over Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave 8-amino-6-fluoro-2-hydroxy-2, 5-dimethyl-3, 4-dihydro-naphthalen-1 (2H) -one (13-83) (250 mg,99% yield), which was used directly in the next step without further purification. ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.41(br s,2H),6.37(d,J＝12.59Hz,1H),5.10(s,1H),2.81-2.92(m,1H),2.63-2.75(m,1H),1.89-2.01(m,5H),1.22(s,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-108.05。

(1S, 9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrans (pyrans) And [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13-dione (13-84) and (1R, 9S) -9-ethyl-5-fluoro-1, 9- Dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline- 10,13 (1H, 9H) -diketones (13-85)：

To 8-amino-6-fluoro-2-hydroxy-2, 5-dimethyl-3, 4-dihydronaphthalen-1 (2H) -one (13-83) (54.0 mg,241umol,1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f under argon at 120 ℃]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (95.5 mg,0.362mmol,1.5 eq.) in xylene (5.4 mL) was added 4-methylbenzenesulfonic acid (24.9 mg,0.145mmol,0.6 eq.) and the mixture was stirred in a sealed tube at 120℃for 24H. It was concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel eluting with 60% tetrahydrofuran/petroleum ether to give a residue which was further separated by preparative HPLC and lyophilized to give (1 s,9 s) -9-ethyl-5-fluoro-1, 9-dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (13-84) (70.1 mg,13% yield) (Compound 13-84 can be the opposite enantiomer to the depicted enantiomer) and (1) R9S) -9-ethyl-5-fluoro-1, 9-dihydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (13-85) (73.3 mg,13% yield) (compound 13-85 can be the opposite enantiomer to that depicted). Note that: stereochemistry is arbitrarily specified.

Preparative HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：CH ₃ OH

Column: phenomenex Luna 80X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220 and 254nm

Spectra of 13-84: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.76(d,J＝10.88Hz,1H),7.31(s,1H),6.51(s,1H),5.79(br s,1H),5.43(s,4H),3.25(br d,J＝2.57Hz,1H),2.96-3.09(m,1H),2.38(s,3H),2.24(br dd,J＝13.08,3.06Hz,1H),2.11(td,J＝13.24,5.69Hz,1H),1.87(m,2H),1.45(s,3H),0.88(t,J＝7.34Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )5ppm-112.10。LCMS(ESI+)m/z：C ₂₅ H ₂₄ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 451.1, found: 451.1.SFC: rt=1.338 min.

Spectra of 13-85: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.77(d,J＝11.00Hz,1H),7.31(s,1H),6.50(s,1H),5.80(br s,1H),5.44(s,4H),3.25(br s,1H),2.96-3.10(m,1H),2.38(s,3H),2.24(br dd,J＝12.65,3.24Hz,1H),2.12(td,J＝13.14,5.38Hz,1H),1.78-1.93(m,2H),1.44(s,3H),0.87(t,J＝7.27Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-112.07。LCMS(ESI+)m/z：C ₂₅ H ₂₄ FN ₂ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 451.1, found: 451.1.SFC: rt=1.453 min.

Example 14

(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (14-91) and (1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione (14-92) (FIG. 23)

* The stereochemistry at the carbon is arbitrarily specified.

N- (7- (allyloxy) -3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14- 86)：

N- (3-fluoro-7-hydroxy-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydro-naphthalen-1-yl) acetamide (13-82) (500 mg,1.88mmol,1.0 eq.) and Ag under argon at 20deg.C ₂ O (4.37 g,18.8mmol,10 eq.) to a mixture of acetonitrile (10 mL) was added 3-iodoprop-1-ene (6.33 g,3.44mL,37.7mmol,20 eq.) and the mixture was stirred at 40℃for 12h and cooled to 25 ℃. (three additional reactions were set up as described above and the four reaction mixtures were combined). The combined reaction mixture was filtered through celite pad and the filter cake was washed with dichloromethane (200 mL), the combined filtrates were concentrated and the residue was purified by flash column chromatography on silica gel eluting with 5% ethyl acetate/petroleum ether to give N- (7- (allyloxy) -3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-86) (1.30 g,54% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 11.82(s,1H),8.28(d,J＝13.13Hz,1H),5.70-5.83(m,1H),5.14(m,1H),5.01(m,1H),3.99(m,1H),3.79(m,1H),3.00(m,1H),2.77-2.88(m,1H),2.36(dt,J＝14.07,5.22Hz,1H),2.17(s,3H),2.11(d,J＝1.63Hz,3H),2.03(m,1H),1.30-1.38(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-104.04。LCMS(ESI+)m/z：C ₁₇ H ₂₁ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 306.1, found: 306.1.

n- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2-oxoethoxy) -5,6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-87)：

N- (7- (allyloxy) -3-fluoro-4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydro-naphthalen-1-yl) acetamide (14-86) (1.30 g,4.17mmol,1.0 eq.) was bubbled with ozone in a stirred mixture of dichloromethane (26 mL) and methanol (13 mL) at-78deg.C for more than 5min, followed by the addition of dimethyl sulfane (648 mg,0.766mL,10.4mmol,2.5 eq.) the reaction mixture was warmed to 25deg.C and stirred at 25deg.C for 1h, quenched with water (40 mL) and extracted with dichloromethane (3X 40 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration and concentration under reduced pressure gave N- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2-oxoethoxy) -5,6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-87) (1.20 g, crude), which was used directly in the next reaction without further purification.

N- (3-fluoro-7- (2-hydroxyethoxy) -4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-88)：

To N- (3-fluoro-4, 7-dimethyl-8-oxo-7- (2-oxoethoxy) -5,6,7, 8-tetrahydro-naphthalen-1-yl) acetamide (14-87) (1.20 g,2.73mmol,1.0 eq.) in tetrahydrofuran (30 mL) and H at 0deg.C ₂ To a mixture of O (15 mL) was added sodium tetrahydroboride (51.7 mg,1.37mmol,0.5 eq.) in portions, the mixture was stirred at 0deg.C for 10min, warmed to 25deg.C and stirred at 25deg.C for 20min, quenched with water (30 mL) and extracted with dichloromethane (3X 40 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 40% ethyl acetate/petroleum ether gave N- (3-fluoro-7- (2-hydroxyethoxy) -4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-88) (800 mg,61% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 12.07(br s,1H),8.46(d,J＝12.76Hz,1H),3.63-3.76(m,2H),3.53-3.61(m,1H),3.44-3.52(m,1H),3.03-3.23(m,1H),2.74-2.92(m,1H),2.35-2.46(m,1H),2.22-2.26(m,3H),2.12(br s,3H),2.03-2.11(m,1H),1.37-1.48(m,3H)。 ¹⁹ F NMR(376MHz,CDCl ₃ )δppm-101.12。LCMS(ESI+)m/z：C ₁₆ H ₂₁ FNO ₄ ⁺ [ MH of (V)] ⁺ Calculated values: 310.1, found: 310.1.

8-amino-6-fluoro-2- (2-hydroxyethoxy) -2, 5-dimethyl-3, 4-dihydronaphthalen-1 (2H) -one (14-89)：

To a solution of N- (3-fluoro-7- (2-hydroxyethoxy) -4, 7-dimethyl-8-oxo-5, 6,7, 8-tetrahydronaphthalen-1-yl) acetamide (14-88) (200 mg,581umol,1.0 eq.) in methanol (8 mL) was added 2N hydrochloric acid (8 mL) at 20 ℃ under argon and the mixture was stirred at 60 ℃ for 1h and cooled to 0 ℃. (three additional reactions were set up as described above and the four reaction mixtures were combined). The combined reaction mixture was cooled to 0 ℃ by addition of saturated NaHCO ₃ The pH was adjusted to 8, warmed to 25℃and extracted with dichloromethane (2X 100 mL). The combined organic layers were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration under reduced pressure, and purification of the residue by flash column chromatography on silica gel eluting with 40% ethyl acetate/petroleum ether gave 8-amino-6-fluoro-2- (2-hydroxyethoxy) -2, 5-dimethyl-3, 4-dihydronaphthalen-1 (2H) -one (14-89) (310 mg,45% yield). ¹ H NMR(400MHz,CD ₃ OD)δppm 6.30(d,J＝12.26Hz,1H),3.49-3.61(m,3H),3.39(m,1H),3.01-3.10(m,1H),2.69-2.79(m,1H),2.32(m,1H),2.00(d,J＝2.25Hz,4H),1.37(s,3H)。 ¹⁹ F NMR(376MHz,CD ₃ OD)δppm-108.91。LCMS(ESI+)m/z：C ₁₄ H ₁₉ FNO ₃ ⁺ [ MH of (V)] ⁺ Calculated values: 268.1, found: 268.1.

(9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzene And [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-90) ：

Under the condition of argon gas, the mixture is heated,to 8-amino-6-fluoro-2- (2-hydroxyethoxy) -2, 5-dimethyl-3, 4-dihydro-naphthalen-1 (2H) -one (14-89) (150 mg,0.561mmol,1.0 eq.) and (S) -4-ethyl-4-hydroxy-7, 8-dihydro-1H-pyrano [3,4-f at 120 ℃]To a mixture of indolizine-3, 6,10 (4H) -trione (1-13) (162 mg, 0.611 mmol,1.1 eq.) in toluene (7 mL) was added o-cresol (443 mg,0.426mL,4.10mmol,7.3 eq.) and pyridine 4-methylbenzenesulfonate (21.1 mg,0.084mmol,0.15 eq.) and the mixture stirred in a sealed tube at 120℃for 32H and cooled to 25 ℃. (an additional reaction was set up as described above and the two reaction mixtures were combined). The combined reaction mixtures were concentrated under reduced pressure and the residue was purified by flash column chromatography on silica gel eluting with ethyl acetate to give (9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-90) (210 mg,22% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.76(dd,J＝10.85,3.46Hz,1H),7.30(s,1H),6.51(d,J＝2.15Hz,1H),5.32-5.62(m,4H),4.74(q,J＝5.21Hz,1H),3.58-3.71(m,3H),2.92-3.06(m,1H),2.14-2.42(m,6H),1.81-1.91(m,2H),1.53(br d,J＝6.79Hz,3H),0.81-0.94(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.89。LCMS(ESI+)m/z：C ₂₇ H ₂₈ FN ₂ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 495.2, found: 495.2.

(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydro Benzo [ de ]]Pyrano [3',4':6,7 ]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-91) and (1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxy-ethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Piirae-type pyridine Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-92)：

(9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-90) (210 mg,0.424 mmol) was dissolved in methanol and purified by chiral SFC (instrument: wat)Preparation of ers SFC150 sfc. column: DAICEF CHIRAFPAK AD (250 mm. Times.30 mm,10 um); mobile phase: a is CO ₂ B is ethanol; gradient: b% = 50% isocratic elution mode; flow rate: 70g/min; wavelength: 220nm; column temperature: 40 ℃; system backpressure: 120 bar) to give (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-91) (65.1 mg,31% yield) (compound 14-91 may be the opposite enantiomer to that depicted) and (1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (14-92) (62.5 mg,30% yield) (compound 14-92 may be the opposite enantiomer to that depicted). Note that: stereochemistry is arbitrarily specified.

Spectra of 14-91: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.77(br d,J＝10.88Hz,1H),7.30(s,1H),5.30-5.63(m,4H),3.64(br s,3H),3.28-3.31,(m,2H),2.99(br s,1H),2.38(br s,3H),2.33(br s,1H),2.20(br s,1H),1.76-1.93(m,2H),1.53(br s,3H),0.81-0.92(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.87。LCMS(ESI+)m/z：C ₂₇ H ₂₈ FN ₂ O ₆ [ MH of (V)] ⁺ Calculated values: 495.2, found: 495.1. chiral SFC: rt=1.715 min.

14-92 spectra: ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.77(br d,J＝10.88Hz,1H),7.31(s,1H),5.33-5.57(m,4H),3.65(br s,3H),3.28-3.30,(m,2H),2.95(br s,1H),2.38(br s,3H),2.33(br s,1H),2.17(br s,1H),1.76-1.93(m,2H),1.53(br s,3H),0.84-0.92(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-111.90。LCMS(ESI+)m/z：C ₂₇ H ₂₈ FN ₂ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 495.2, found: 495.3. chiral SFC: rt= 1.948min.

Example 15

(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- ((2-hydroxyethyl) amino) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione hydrochloride (15-94) (FIG. 24)

(1S, 9S) -1- ((2- ((tert-butyldimethylsilyl) oxy) ethyl) amino) -9-ethyl-5-fluoro-9-hydroxy Phenyl-4-methyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -diketones (15-93)：

(1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydro-benzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]A mixture of quinoline-10, 13 (1H, 9H) -dione methanesulfonate (irinotecan) (100 mg,0.188mmol,1.0 eq.) and triethylamine (19.0 mg,0.188mmol,1.0 eq.) in methanol (2 mL) was stirred at 25℃for 0.5h, followed by the addition of 2- ((tert-butyldimethylsilyl) oxy) acetaldehyde (32.8 mg,0.188mmol,1.0 eq.). After stirring the reaction mixture at 25 ℃ for 1h, sodium cyanoborohydride (17.7 mg,0.282mmol,1.5 eq.) was added and stirred at 25 ℃ for a further 2h. Quench it by adding water (0.2 mL), dilute with dichloromethane (5 mL), and go through Na ₂ SO ₄ Dried, filtered, concentrated under reduced pressure, and the residue purified by preparative HPLC to give (1 h,9 h) -1- ((2- ((tert-butyldimethyl-silyl) oxy) ethyl) amino) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de)]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (15-93) (25.0 mg,22% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 7.74(d,J＝11.13Hz,1H),7.30(s,1H),6.51(s,1H),5.34-5.47(m,4H),4.30(br s,1H),3.69(t,J＝5.93Hz,2H),3.15-3.24(m,1H),2.96-3.07(m,1H),2.87(br d,J＝5.14Hz,1H),2.73-2.82(m,1H),2.37(s,3H),2.22(dd,J＝13.57,4.89Hz,1H),1.99-2.14(m,2H),1.87(dt,J＝17.36,7.09Hz,2H),0.87(t,J＝7.34Hz,3H),0.82(s,9H),0.03(d,J＝3.18Hz,6H). ¹⁹ F NMR(400MHz,DMSO-D ₆ )δppm-111.83。LCMS(ESI+)m/z：C ₃₂ H ₄₁ FN ₃ O ₅ Si ⁺ [ MH of (V)] ⁺ Calculated values: 594.2, found: 594.2.

HPLC method：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a:10mM NH ₄ HCO ₃ /H ₂ O；B：CH ₃ CN

Column: waters Xbridge BEH C18 100×30mm×10um

Flow rate: 25mL/min

Monitoring wavelength: 220 and 254nm

(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- ((2-hydroxyethyl) amino) -4-methyl-2,3,12,15-tetrahydrobenzene And [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione hydrochloride (15-94)

To (1S, 9S) -1- ((2- ((tert-butyldimethylsilyl) oxy) ethyl) amino) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-2,3,12,15-tetrahydrobenzo [ de ] under nitrogen at 25 DEG C]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (15-93) (25 mg,0.042mmol,1.0 eq.) was added dropwise HCl/MeOH (2.5 mL, 4M) to a mixture of methanol (0.5 mL), the mixture was stirred at 25℃for 1h, filtered, collected and dried under vacuum to give (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- ((2-hydroxyethyl) amino) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione hydrochloride (15-94) (18.0 mg,87% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.81-9.14(m,2H),7.89(d,J＝10.85Hz,1H),7.35(s,1H),6.56(s,1H),5.54-5.67(m,1H),5.37-5.50(m,3H),5.28(br d,J＝0.95Hz,1H),5.10(br s,1H),3.71(br s,2H),3.09-3.26(m,3H),2.79(br d,J＝13.95Hz,1H),2.41(s,3H),2.09-2.25(m,1H),1.77-1.96(m,2H),0.88(t,J＝7.33Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-D ₆ )δppm-110.92。LCMS(ESI+)m/z：C ₂₆ H ₂₇ FN ₃ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 480.2, found: 480.2.

example 16

(1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- ((2-hydroxyethyl) amino) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione hydrochloride (16-95)

Examples 16 (16-95) were prepared in a similar manner to example 15 using 8-71 instead of irinotecan. ¹ H NMR(400MHz,CD ₃ OD)δppm 7.82(d,J＝10.49Hz,1H),7.68(s,1H),5.71-5.78(m,1H),5.57-5.65(m,2H),5.41(d,J＝16.33Hz,1H),3.74-3.84(m,2H),3.35(br s,2H),3.20-3.28(m,2H),2.74(dt,J＝14.16,5.80Hz,1H),2.39-2.53(m,4H),2.09(s,3H),1.98(qd,J＝7.17,3.99Hz,2H),1.02(t,J＝7.33Hz,3H)。 ¹⁹ FNMR(376MHz,CD ₃ OD)δppm-111.87。LCMS(ESI+)m/z：C ₂₇ H ₂₉ FN ₃ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 494.2, found: 494.1.

example 17

(1R, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- ((2-hydroxyethyl) amino) -1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ] pyrano [3',4':6,7] indolizino [1,2-b ] quinoline-10, 13 (1H, 9H) -dione hydrochloride (17-96)

Examples 17 (17-96) were prepared in a similar manner to example 15 using 8-72 instead of irinotecan. ¹ H NMR(400MHz,CD ₃ OD)δppm 7.82(d,J＝10.39Hz,1H),7.68(s,1H),5.73-5.81(m,1H),5.63(d,J＝3.30Hz,1H),5.58(s,1H),5.42(d,J＝16.26Hz,1H),3.74-3.87(m,2H),3.34(br s,2H),3.22-3.28(m,2H),2.70-2.82(m,1H),2.36-2.56(m,4H),2.10(s,3H),1.89-2.04(m,2H),1.01(t,J＝7.34Hz,3H)。 ¹⁹ F NMR(376MHz,CD ₃ OD)δppm-111.90。LCMS(ESI+)m/z：C ₂₇ H ₂₉ FN ₃ O ₅ ⁺ [ MH of (V)] ⁺ Calculated values: 494.2, found: 494.1.

example 18

(S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) acetamido) -N- (2- (((2- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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-oxoethoxy) methyl) amino) -2-oxoethyl) -3-phenylpropionamide (18-112) (FIG. 25)

2, 5-Dioxopyrrolidin-1-yl 2- (2- (((benzyloxy) carbonyl) amino) acetamido) acetate (18-98)：

To 2- [ [2- (benzyloxycarbonylamino) acetyl]Amino group]To a mixture of acetic acid (18-97) (55.0 g,206mmol,1.0 eq.) in acetonitrile (550 mL) was added 1-hydroxypyrrolidine-2, 5-dione (26.1 g,227mmol,1.1 eq.) and N ¹ - ((ethylimino) methylene) -N ³ ,N ³ Dimethylpropane-1, 3-diamine hydrochloride (47.5 g,247mmol,1.2 eq.) the mixture was stirred at 25 ℃ for 4h, cooled to-10 ℃ and a white mass formed. The suspension was diluted with water (50 mL), stirred, filtered, and the filter cake dried in vacuo to give 2, 5-dioxopyrrolidin-1-yl 2- (2- (((benzyloxy) carbonyl) amino) acetamido) acetate (18-98) (85.0 g,175mmol,85% yield) which was used directly in the next step without purification. ¹ H NMR(400MHz,DMSO-d ₆ )δppm 2.81(s,4H),2.93-3.12(m,2H),3.69(d,J＝6.11Hz,2H),4.27(d,J＝5.87Hz,2H),5.04(s,2H),7.24-7.45(m,5H),7.57(t,J＝6.11Hz,1H),8.58(t,J＝5.87Hz,1H)。LCMS(ESI+)m/z：C ₁₆ H ₁₈ N ₃ O ₇ ⁺ [ MH of (V)] ⁺ Calculated values: 364.1, found: 364.1.

(S) -11-benzyl-3, 6, 9-trioxo-1-phenyl-2-oxa-4, 7, 10-triazadodecane-12-oic acid (18- 99)：

To 2, 5-Dioxopyrrolidin-1-yl 2- (2- (((benzyloxy) carbonyl) amino) acetamido) acetate (18-98) (85.0 g,175mmol,1.0 eq.) in acetonitrile (425 mL) and H ₂ To a mixture of O (425 mL) was added (2S) -2-amino-3-phenyl-propionic acid (34.7 g,210mmol,1.2 eq.) and trimethylamine (26.8 mL,193mmol,1.1 eq.) and the mixture was stirred at 25℃for 2h, cooled to 0℃and hydrochloric acid (12M, 16.2mL,1.1 eq.) was added and stirred at 0℃for 6h. The resulting suspension was warmed to 25 ℃, filtered and the filter cake was dried in vacuo to give (S) -11-benzyl-3, 6, 9-trioxo-1-phenyl-2-oxa-4, 7, 10-triazadodecane-12-oic acid (18-99) (50.0 g,108mmol,62% yield) which was used directly in the next step without further purification. ¹ H NMR(400MHz,DMSO-d ₆ )δppm 2.59(s,1H),2.88(dd,J＝13.77,9.00Hz,1H),3.04-3.11(m,1H),3.60-3.79(m,4H),4.43(td,J＝8.43,5.19Hz,1H),5.03(s,2H),7.15-7.40(m,10H),7.50(t,J＝6.02Hz,1H),8.04(br t,J＝5.60Hz,1H),8.16(d,J＝8.11Hz,1H),12.72(br d,J＝5.01Hz,1H)。LCMS(ESI+)m/z：C ₂₁ H ₂₄ N ₃ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 414.2, found: 414.2.

methyl acetate (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) ester (18-101)：

To 2- [ [2- (9H-fluoren-9-ylmethoxycarbonylamino) acetyl]Amino group]To a mixture of tetrahydrofuran (1.5L) and acetic acid (300 mL) was added lead tetraacetate (150 g,338mmol,1.2 eq.) acetic acid (18-100) (100 g,282mmol,1.0 eq.) and the mixture was stirred at 50℃for 36h, cooled to 25℃and filtered. The filtrate was washed twice with trisodium citrate dihydrate aqueous solution (2.5L, 20 wt%) and the organic phase was concentrated to about 2.0L, water (2.5L) was added and the mixture was stirred at 5 ℃ for 2h. The precipitate formed was filtered, washed with a mixture of cold (5 ℃) tetrahydrofuran and water (3:10, 1.2L) and dried in vacuo to give methyl acetate (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) ester (18-101) (100 g,86% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 2.09(s,3H),3.90(d,J＝5.01Hz,2H),4.19-4.28(m,1H),4.41-4.52(m,2H),5.25(d,J＝6.97Hz,2H),5.41-5.60(m,1H),7.17-7.27(m,1H),7.29-7.36(m,2H),7.41(t,J＝7.40Hz,2H),7.55-7.64(m,2H),7.77(d,J＝7.58Hz,2H)。LCMS(ESI+)m/z：C ₂₀ H ₂₀ N ₂ O ₅ Na ⁺ MNA of [ MNA ] of (A)] ⁺ Calculated values: 391.1, found: 391.1.

1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-oic acid benzyl ester (18- 102)：

To a mixture of acetic acid (2- ((((9H-fluoren-9-yl) methoxy) carbonyl) amino) acetamido) methyl ester (18-101) (80.0 g,173mmol,1.0 eq.) in 1, 2-dimethoxyethane (1.5L) was added benzyl 2-hydroxyacetate (57.7 g,49.3mL,347mmol,2.0 eq.) and cooled to 0deg.C and acetic acid (5.22 g,86.8mmol,4.97mL,0.5 eq.) was added. The mixture was stirred at 0deg.C for 1h, sodium hydroxide (10N, 17.3mL,1.0 eq) was added dropwise and stirred at 0deg.C for 1h, water (1.2L) was added and stirred at 0deg.C for 2h. The white precipitate formed was filtered, washed with cold (5 ℃) 1, 2-dimethoxyethane: water (1:2, 500 mL) and methyl tert-butyl ester (500 mL) and dried under vacuum at 40℃to give benzyl 1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-carboxylate (18-102) (60.0 g,69% yield). ¹ H NMR(400MHz,CDCl ₃ )δppm 3.82(d,J＝5.26Hz,2H),4.16-4.29(m,3H),4.45(d,J＝6.80Hz,2H),4.83(d,J＝7.02Hz,2H),5.16(s,2H),5.45(t,J＝5.59Hz,1H),7.08(s,1H),7.28-7.48(m,9H),7.59(d,J＝7.23Hz,2H),7.77(d,J＝7.45Hz,2H)。LCMS(ESI+)m/z：C ₂₇ H ₂₆ N ₂ O ₆ Na ⁺ MNA of [ MNA ] of (A)] ⁺ Calculated values: 497.2, found: 497.1.

benzyl 2- ((2-aminoacetylamino) methoxy) acetate HOBT salt (18-103)：

To a mixture of benzyl 1- (9H-fluoren-9-yl) -3, 6-dioxo-2, 9-dioxa-4, 7-diazaundec-11-carboxylate (18-102) (60.0 g,126mmol,1.0 eq.) in acetonitrile (1.08L) was added DBU (9.53 mL,63.2mmol,0.5 eq.). The mixture was stirred at 25 ℃Stirred for 4h, cooled to 0deg.C, HOBt (34.1 g,252mmol,2.0 eq.) was added and stirred for 1.5h at 0deg.C. It was warmed to 25 ℃, filtered, the filter cake washed with cold (5 ℃) acetonitrile (200 mL) and dried under vacuum to give benzyl 2- ((2-aminoacetamido) methoxy) acetate HOBt (18-103) (46.0 g,84% yield). ¹ H NMR(400MHz,METHANOL-d4)δppm 3.68(s,2H),4.21(s,2H),4.79(s,2H),5.18(s,2H),7.22-7.42(m,7H),7.62-7.74(m,2H)。LCMS(ESI-)m/z：C ₁₈ H ₃₃ N ₅ O ₆ ⁺ [ MH of (V)]-calculating a value: 269.1, found: 269.0.

(S) -11-benzyl-3, 6,9,12, 15-pentoxy-1-phenyl-2, 18-dioxa-4,7,10,13,16-pentaaza Eicosane-20-acid benzyl ester (18-105)：

To a mixture of benzyl 2- ((2-aminoacetylamino) methoxy) acetate HOBt (18-103) (46.0 g,118mmol,1.0 eq.) in acetonitrile (500 mL) at 0deg.C was added (S) -11-benzyl-3, 6, 9-trioxo-1-phenyl-2-oxa-4, 7, 10-triazadodecane-12-oic acid (18-104) (45.0 g,108mmol,0.9 eq.) and N ¹ - ((ethylimino) methylene) -N ³ ,N ³ Dimethylpropane-1, 3-diamine hydrochloride (25.5 g,133mmol,1.1 eq.). The mixture was stirred at 0deg.C for 3.5h, water (200 mL) was added and stirred at 0deg.C for 2h. The precipitate formed was filtered, washed with cold (5 ℃) acetonitrile: water (1:2, 201 mL) and dried in vacuo to give (S) -11-benzyl-3, 6,9,12, 15-pentoxy-1-phenyl-2, 18-dioxa-4,7,10,13,16-pentaazaeicosane-20-oic acid benzyl ester (18-105) (30.0 g,34% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 2.79(dd,J＝13.67,9.92Hz,1H),3.06(dd,J＝13.67,4.41Hz,1H),3.56-3.66(m,3H),3.69-3.81(m,3H),4.15(s,2H),4.51(td,J＝8.76,4.52Hz,1H),4.63(d,J＝6.62Hz,2H),5.03(s,2H)5.13-5.17(m,2H),7.03-7.46(m,15H),7.49-7.55(m,1H),8.00-8.10(m,1H),8.18(d,J＝7.94Hz,1H),8.36(t,J＝5.62Hz,1H),8.62(t,J＝6.73Hz,1H)。

(S) -16-amino-10-benzyl-6, 9,12, 15-tetraoxo-3-oxa-5, 8,11, 14-tetraazahexadecane-1- Acid (18-106)：

At 25℃under argon, to (S) -11-benzyl-benzyl 3,6,9,12, 15-pentoxy-1-phenyl-2, 18-dioxa-4,7,10,13,16-pentaazaeicosane-20-carboxylate (18-105) (10.0 g,15.4mmol,1.0 eq.) in tetrahydrofuran (210 mL) and H ₂ Pd/C (10 wt%) (4.40 g,1.2 eq.) was added to a mixture of O (140 mL), and the suspension was evacuated and treated with H ₂ Three times filled and at 25℃at H ₂ Stirring for 2.5h at (15 psi). In the substitution of H with argon ₂ While in atmosphere, it was filtered through a pad of celite and the filter cake was washed with water (80 mL) and ethanol (150 mL). The filtrate was concentrated to about 30mL, ethanol (120 mL) was added, and the mixture was stirred at 0 ℃ for 2h. The precipitate formed was collected by filtration, washed with ethanol (30 mL) and dried in vacuo to give (S) -16-amino-10-benzyl-6, 9,12, 15-tetraoxo-3-oxa-5, 8,11, 14-tetraazahexadecane-1-acid (18-106) (4.00 g,58% yield). ¹ H NMR(400MHz,D ₂ O)δppm 2.88-3.26(m,2H),3.64-4.16(m,8H),4.55-4.71(m,3H),7.11-7.50(m,5H)。LCMS(ESI-)m/z：C ₁₈ H ₂₄ N ₅ O ₇ ^- [ MH of (V)] ^- Calculated values: 422.2, found: 422.1.

(E) -4- ((4- (2-carboxyethyl) phenyl) amino) -4-oxobut-2-enoic acid (18-108)：

To a mixture of furan-2, 5-dione (4.16 g,42.3mmol,1.0 eq.) in diethyl ether (42 mL) was added a solution of 3- (4-aminophenyl) propionic acid (18-107) (7.00 g,42.3mmol,1.0 eq.) and 2, 6-lutidine (4.58 g,4.98mL,42.3mmol,1.0 eq.) in tetrahydrofuran (70 mL) at 25 ℃. The reaction mixture was stirred at 65 ℃ for 0.5h, cooled to 25 ℃, filtered, the filter cake washed with methyl tert-butyl ether (150 mL) and dried in vacuo to give (E) -4- ((4- (2-carboxyethyl) phenyl) amino) -4-oxobut-2-enoic acid (18-108) (11.0 g,98% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 12.12-13.09(m,2H),10.37(s,1H),7.52(d,J＝8.4Hz,2H),7.18(d,J＝8.4Hz,2H),6.46(d,J＝12.0Hz,1H),6.30(d,J＝12.0Hz,1H),2.78(t,J＝7.6Hz,2H),2.52(s,2H)。LCMS(ESI-)m/z：C ₁₃ H ₁₂ NO ₅ ^- [ M-H of] ^- Calculated values: 262.0, found: 261.9.

3- (4- (2, 5-dioxo-2)5-dihydro-1H-pyrrol-1-yl) phenyl propionic acid (18-109)：

To a mixture of (E) -4- ((4- (2-carboxyethyl) phenyl) amino) -4-oxobut-2-enoic acid (18-108) (11.0 g,41.7mmol,1.0 eq.) in acetic anhydride (100 mL) under nitrogen was added potassium acetate (2.26 g,22.9mmol,0.55 eq.). The suspension was evacuated and filled three times with nitrogen, stirred at 145 ℃ for 0.5h, cooled to 20 ℃ and concentrated under reduced pressure. The residue was diluted with ethyl acetate (300 mL), washed with brine, and taken up in Na ₂ SO ₄ Drying, filtration, concentration and purification of the residue by flash column chromatography on silica gel eluting with 33% -100% ethyl acetate/petroleum ether containing 20% tetrahydrofuran afforded 3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propanoic acid (18-109) (3.60 g,29% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 12.16(s,1H),7.33(d,J＝8.4Hz,2H),7.20-7.24(m,2H),7.16(s,2H),2.86(t,J＝7.6Hz,2H),2.57(t,J＝7.6Hz,2H)。LCMS(ESI-)m/z：C ₁₃ H ₁₀ NO ₄ ^- [ M-H of] ^- Calculated values: 244.0, found: 243.9.

3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propanoic acid 2, 5-dioxopyrrolidin-1-yl Esters (18-110)：

To a mixture of 3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionic acid (18-109) (3.60 g,14.6mmol,1.0 eq.) in acetonitrile (72 mL) was added 1-hydroxypyrrolidine-2, 5-dione (1.77 g,15.4mmol,1.05 eq.) and N, N' -methane-diyl dicyclohexylamine (3.18 g,15.4mmol,1.05 eq.). The reaction mixture was stirred at 25 ℃ for 3H, filtered and the filtrate containing 2, 5-dioxopyrrolidin-1-yl 3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionate (18-110) was used directly in the next step without work-up and purification. LCMS (esi+) m/z: c (C) ₁₇ H ₁₅ N ₂ O ₆ ⁺ [ MH of (V)] ⁺ Calculated values: 343.1, found: 343.1.

(S) -10-benzyl-20- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) -6,9,12,15, 18-pentoxy-3-oxa-5, 8,11,14, 17-pentanitrogenHeteroeicosane-1-carboxylic acid (18-111)：

To a mixture of 2, 5-dioxopyrrolidin-1-yl 3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionate (18-110) (1.62 g,4.72mmol,1.0 eq.) in acetonitrile (80 mL) was added (S) -16-amino-10-benzyl-6, 9,12, 15-tetraoxo-3-oxa-5, 8,11, 14-tetraazahexadecane-1-oic acid (18-106) (2.00 g,4.72mmol,1.0 eq.) and N-ethyl-N-isopropyl-propan-2-amine (0.188 mL,3.78mmol,0.8 eq.) at 25℃in H ₂ O (20 mL). The reaction mixture was stirred at 25 ℃ for 12H, filtered, concentrated, and the residue was purified by preparative HPLC to give (S) -10-benzyl-20- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) -6,9,12,15,18-pent-oxy-3-oxa-5, 8,11,14, 17-pentaazaeicosane-1-acid (18-111) (0.78 g,24% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.53-8.64(m,1H),8.31(t,J＝5.6Hz,1H),8.23(t,J＝5.6Hz,1H),8.16(d,J＝8.0Hz,1H),8.08(t,J＝5.2Hz,1H),7.14-7.38(m,11H),4.61(d,J＝6.8Hz,2H),4.44-4.55(m,1H),3.97(s,2H),3.64-3.80(m,5H),3.63(d,J＝5.6Hz,1H),3.06(dd,J＝13.6,4.4Hz,1H),2.78-2.89(m,3H),2.53-2.60(m,1H)。LCMS(ESI-)m/z：C ₃₁ H ₃₃ N ₆ O ₁₀ ^- [ MH of (V)] ^- Calculated values: 649.2, found: 649.3.

preparative HPLC conditions：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：ACN

Column: agela DuraShell C18 250×70mm×10um

Flow rate: 130mL/min

Monitoring wavelength: 220nm and 254nm

(S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) ethyl) amino) Amido) acetamido) -N- (2- (((2- (((1 s,9 s) -9-ethyl-5-fluoro-9-hydroxy-1, 4-di-N-methyl) amino)Methyl-10, 13-dioxy Substituted-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) Amino) -2-oxoethoxy methyl) amino) -2-oxoethyl) -3-phenylpropionamide (18-112)：

(S) -10-benzyl-20- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) -6,9,12,15,18-pent-oxy-3-oxa-5, 8,11,14, 17-penta-azaeicosane-1-oic acid (18-111) (246 mg,0.378mmol,1.0 eq), (1S, 9S) -1-amino-9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-2,3,12,15-tetrahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]A mixture of quinoline-10, 13 (1H, 9H) -dione hydrochloride (8-71) (170 mg,0.378mmol,1.0 eq.) 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine-4-ium tetrafluoroborate (124 mg,0.378mmol,1.0 eq.) and 4-methylmorpholine (0.166 mL,1.51mmol,4.0 eq.) in N, N-dimethylformamide (5 mL) was stirred at 25℃for 1h. The mixture was filtered and the filtrate was purified by preparative HPLC to give (S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) propanoylamino) acetamido) -N- (2- (((2- (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-10, 13-dioxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]) ]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinolin-1-yl) amino) -2-oxoethoxy methyl) amino) -2-oxoethyl) -3-phenylpropionamide (18-112) (130 mg,30% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.39(s,1H),8.32-8.37(m,1H),8.12-8.22(m,2H),8.04(t,J＝5.6Hz,1H),7.78(d,J＝10.8Hz,1H),7.26-7.34(m,3H),7.18-7.26(m,6H),7.16(s,2H),6.51(s,1H),5.28-5.50(m,3H),4.92(d,J＝18.8Hz,1H),4.67(d,J＝6.8Hz,2H),4.51(d,J＝4.0Hz,1H),3.90-4.05(m,2H),3.64-3.83(m,5H),3.62(d,J＝5.6Hz,1H),3.21-3.27(m,1H),3.00-3.13(m,2H),2.89-2.99(m,1H),2.76-2.87(m,3H),2.46(s,3H),2.38(s,3H),1.75-2.01(m,3H),1.58(s,3H),0.87(t,7＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.87。LCMS(ESI+)m/z：C ₅₆ H ₅₇ FN ₉ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1082.4, found: 1082.3.

preparative HPLC stripPiece：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：ACN

Column: phenomnex C18X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220 and 254nm

Example 19

N- ((S) -10-benzyl-1- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) benzamide (19-113)

Examples 19 (19-113) were prepared in a similar manner to examples 18 (18-112). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.87(t,J＝5.6Hz,1H),8.69(t,J＝6.4Hz,1H),8.32-8.41(m,2H),8.10-8.19(m,2H),7.90(d,J＝7.6Hz,1H),7.83-7.86(m,1H),7.78(d,J＝10.8Hz,1H),7.55-7.61(m,1H),7.48-7.53(m,1H),7.31(s,1H),7.18-7.26(m,6H),7.13-7.18(m,1H),6.51(s,1H),5.30-5.47(m,3H),4.92(d,J＝18.8Hz,1H),4.67(d,J＝7.2Hz,2H),4.51(dd,J＝8.0,4.4Hz,1H),3.84-4.04(m,4H),3.71-3.83(m,3H),3.54-3.68(m,1H),3.25(s,1H),2.88-3.15(m,3H),2.82(dd,J＝13.6,9.6Hz,1H),2.38(s,3H),1.73-2.04(m,3H),1.58(s,3H),0.87(t,7＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.87。LCMS(ESI+)m/z：C ₅₄ H ₅₃ FN ₉ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1054.3, found: 1054.3.

example 20

N- ((S) -10-benzyl-1- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1, 4-dimethyl-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) -1,6,9,12,15-pent-oxy-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide (20-114)

Examples 20 (20-114) were prepared in a similar manner to examples 18 (18-112). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.69(t,J＝6.4Hz,1H),8.39(s,1H),8.34(,J＝5.6Hz,1H),8.14(d,J＝8.0Hz,1H),8.04(t,J＝5.6Hz,1H),7.99(t,J＝5.6Hz,1H),7.78(d,J＝10.8Hz,1H),7.31(s,1H),7.20-7.28(m,4H),7.14-7.20(m,1H),6.98(s,2H),6.51(s,1H),5.35-5.48(m,3H),4.92(d,J＝18.8Hz,1H),4.67(d,J＝6.8Hz,2H),4.46-4.54(m,1H),3.90-4.04(m,2H),3.70-3.85(m,3H),3.55-3.67(m,3H),3.33-3.38(m,2H),3.26(d,J＝2.0Hz,1H),2.87-3.11(m,3H),2.81(dd,J＝13.6,9.6Hz,1H),4.46-4.54(m,1H),3.90-4.04(m,2H),3.70-3.85(m,3H),3.55-3.67(m,3H),3.33-3.38(m,2H),3.26(d,J＝2.0Hz,1H),2.87-3.11(m,3H),2.81(dd,J＝13.6,9.6Hz,1H),2.39(s,3H),2.08(t,J＝7.6Hz,2H),1.76-2.01(m,3H),1.58(s,3H),1.40-1.51(m,4H),1.12-1.23(m,2H),0.87(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.88。LCMS(ESI+)m/z：C ₅₃ H ₅₉ FN ₉ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1048.4, found: 1048.5.

example 21

(S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) acetamido) -N- (2- (((2- (((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) oxy) ethoxy) methyl) amino) -2-oxoethyl) -3-phenylpropionamide (21-120) (FIG. 26)

(2S) -2- [ [2- [ [2- [3- [4- (2, 5-dioxopyrrol-1-yl) phenyl ]]Propionylamino group]Acetyl group]Amino group] Acetyl group]Amino group]-3-phenyl-propionic acid (21-116)：

(S) -2- (2- (2-Aminoacetamido) acetamido) -3-phenylpropionic acid (21-115) (5.03 g,14.6mmol,1.0 eq.) and N-ethyl-N, N-diisopropylamine (1.52 g,11.7mmol,2.05mL,0.8 eq.) in H ₂ A mixture of O (25 mL) was added dropwise to a mixture of 2, 5-dioxopyrrolidin-1-yl 3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionate (18-110) (4.10 g,14.6mmol,1.0 eq.) in acetonitrile (75 mL). The reaction mixture was stirred for 5h at 25 ℃, filtered and the filtrate was purified by preparative HPLC to give (2S) -2- [ [2- [ [2- [3- [4- (2, 5-dioxopyrrol-1-yl) phenyl ] ]Propionylamino group]Acetyl group]Amino group]Acetyl group]Amino group]-3-phenyl-propionic acid (21-116) (3.32 g,40% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.21(t,J＝5.6Hz,1H),8.07(t,J＝5.6Hz,1H),7.87(d,J＝7.6Hz,1H),7.32(d,J＝8.4Hz,2H),7.06-7.27(m,9H),4.29(d,J＝5.2Hz,1H),3.61-3.77(m,4H),3.19(s,1H),3.03(d,J＝4.8Hz,1H),2.86(t,J＝8.0Hz,3H),2.67(s,1H)。LCMS(ESI-)m/z：C ₂₆ H ₂₅ N ₄ O ₇ ^- [ MH of (V)] ⁺ Calculated values: 505.1, found: 505.2.

preparative HPLC conditions：

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：ACN

Column: phenomnex C18X 250X 100mm 10u

Flow rate: 260mL/min

Monitoring wavelength: 220nm and 254nm

(9H-fluoren-9-yl) methyl (2- (((2- (((1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-4-methyl-10, 13-dio-ne) Oxo-1,2,3,9,10,12,13,15-octahydrobenzo [ de ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-1- Group) oxy) ethoxy) methyl) amino) -2-oxoethyl carbamate (21-118)：

To (1S, 9S) -9-ethyl-5-fluoro-9-hydroxy-1- (2-hydroxyethoxy) -4-methyl-2,3,12,15-tetrahydrobenzo [ de ] at 25 ]]Pyrano [3',4':6,7]Indolazino [1,2-b ]]Quinoline-10, 13 (1H, 9H) -dione (5-48) (1.25 g,2.60mmol,1.0 eq.) in a mixture of dichloromethane (37 mL) scandium (III) triflate (50.5 mg,0.260mmol,0.1 eq.) and acetic acid [ [2- (9H-fluoren-9-ylmethoxycarbonylamino) acetyl were added in three portions over 1.5H]Amino group]Methyl ester (21-117) (1.44 g,3.90mmol,1.5 eq.). The reaction mixture was stirred at 30℃for 36h, quenched with water (100 mL) and extracted with dichloromethane (3X 100 mL). The combined organic phases were washed with brine, dried over Na ₂ SO ₄ Drying, filtration, concentration and purification of the residue by flash column chromatography on silica gel eluting with 10% -100% dichloromethane/ethyl acetate gives (9H-fluoren-9-yl) methyl (2- (((1 s,9 s) -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]Indolazino [1,2-b ]]Quinolin-1-yl-oxy) ethoxy) methyl) -2-oxoethyl carbamate (21-118) (1.00 g,45% yield). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.90(br t,J＝6.60Hz,1H),7.80-7.91(m,2H),7.73(d,J＝11.13Hz,1H),7.56-7.63(m,2H),7.23-7.44(m,6H),6.53(s,1H),5.32-5.53(m,3H),4.96(br dd,J＝9.60,4.10Hz,1H),4.62-4.83(m,2H),4.44-4.59(m,1H),4.20-4.32(m,1H),4.05-4.19(m,3H),3.91-4.00(m,1H),3.57-3.78(m,5H),3.14-3.28(m,1H),2.86-3.08(m,1H),2.34(s,3H),1.85(br dd,J＝7.03,4.46Hz,3H),0.78-0.94(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.56。LCMS(ESI+)m/z：C ₄₄ H ₄₂ FN ₄ O ₉ ⁺ [ MH of (V)] ⁺ Calculated values: 789.3, found: 789.3.

2-amino-N- ((2- (((1 s,9 s) -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]Indolazino [1,2-b ]]Quinolin-1-yl) oxy) ethoxy Methyl) acetamide (21-119)：

To (9H-fluoren-9-yl) methyl (2- (((2- (((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]Indolazino [1,2-b ]]Quinolin-1-yl) oxy) ethoxy methyl) amino) -2-oxoethyl carbamate (21-118) (1.00 g,1.27mmol,1.0 eq.) piperidine (0.125 mL,1.27mmol,1.0 eq.) was added to a mixture of N, N-dimethylformamide (20 mL). The reaction mixture was stirred for 1h at 0deg.C, filtered and the filtrate was purified by preparative HPLC to give 2-amino-N- ((2- (((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]Indolazino [1,2-b ]]Quinolin-1-yl) oxy) ethoxy methyl) acetamide (21-119) (360 mg,45% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.78(t,J＝6.8Hz,1H),7.66-7.81(m,1H),7.32(s,1H),6.52(s,1H),5.35-5.46(m,3H),5.01(dd,J＝9.2,3.6Hz,1H),4.59-4.83(m,2H),3.92-4.03(m,1H),3.77(m,1H),3.59-3.71(m,2H),3.20-3.29(m,2H),3.16(s,2H),2.92-3.05(m,1H),2.59-2.84(m,1H),2.29-2.41(m,3H),1.72-2.09(m,3H),0.78-0.97(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.54。LCMS(ESI+)m/z：C ₂₉ H ₃₂ FN ₄ O ₇ ⁺ [ MH of (V)] ⁺ Calculated values: 567.2, found: 567.3.

preparative HPLC conditions

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a:10mM NH ₄ HCO ₃ /H ₂ O；B：ACN

Column: phenomnex C18X 30mm X3 um

Flow rate: 25mL/min

Monitoring wavelength: 220nm and 254nm

(S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) ethyl) amino) Amido) acetamido) -N- (2- (((2- (((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]Indolazino [1,2-b ]]Quinolin-1-yl) oxy Group) ethoxy) methyl) amino) -2-oxoethyl) -3-phenylpropionamide (21-120)：

(2S) -2- [ [2- [ [2- [3- [4- (2, 5-dioxopyrrol-1-yl) phenyl ] phenyl]Propionylamino group]Acetyl group]Amino group]Acetyl group]Amino group]-3-phenyl-propionic acid (21-116) (160 mg,0.317mmol,1.0 eq.) 2-amino-N- ((2- (((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]Indolazino [1,2-b ]]A mixture of quinolin-1-yl) oxy ethoxy methyl) acetamide (21-119) (180 mg,317umol,1.0 eq.) 4- (4, 6-dimethoxy-1, 3, 5-triazin-2-yl) -4-methylmorpholine-4-ium tetrafluoroborate (104 mg,0.317mmol,1.0 eq.) and 4-methylmorpholine (0.139 mL,1.27mmol,4.0 eq.) in N, N-dimethylformamide (3.6 mL) was stirred at 25℃for 1H, filtration and purification of the filtrate by preparative HPLC gave (S) -2- (2- (2- (3- (4- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) phenyl) propionylamino) acetamido) -N- (2- (((2- (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]Indolazino [1,2-b ]]Quinolin-1-yl-oxy) ethoxy) methyl) -2-oxoethyl) -3-phenyl-propionamide (21-120) (80.0 mg,23% yield). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.69(t,J＝6.8Hz,1H),8.28(t,J＝5.6Hz,1H),8.19(t,J＝5.6Hz,1H),8.10(d,J＝8.0Hz,1H),8.03(t,J＝5.6Hz,1H),7.76(d,J＝11.2Hz,1H),7.26-7.35(m,3H),7.13-7.25(m,8H),6.52(s,1H),5.34-5.53(m,4H),5.03(dd,J＝8.8,4.00Hz,1H),4.63-4.81(m,2H),4.41-4.57(m,1H),3.90-4.03(m,1H),3.74-3.84(m,3H),3.54-3.74(m,6H),3.16-3.30(m,2H),2.93-3.07(m,2H),2.73-2.88(m,3H),2.42-2.48(m,3H),2.37(s,3H),1.80-2.03(m,3H),0.87(t,J＝7.2Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.53。LCMS(ESI+)m/z：C ₅₅ H ₅₆ FN ₈ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1055.4, found: 1055.3.

preparative HPLC conditions

Instrument: gilson 281 semi-preparative HPLC system

Mobile phase: a: h ₂ O；B：ACN

Column: phenomnex C18X 30mm X3 um

Flow rate: 60mL/min

Monitoring wavelength: 220nm and 254nm

Example 22

N- ((S) -10-benzyl-1- (((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) oxy) -6,9,12, 15-tetraoxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -3- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) benzamide (22-121)

Examples 22 (22-121) were prepared in a similar manner to examples 21 (21-120). ¹ H NMR(400MHz,DMSO-d ₆ )δppm 8.86(t,J＝5.60Hz,1H),8.67(br t,J＝6.56Hz,1H),8.29(br t,J＝5.66Hz,1H),8.05-8.17(m,2H),7.89(d,J＝7.99Hz,1H),7.83(s,1H),7.75(d,J＝10.97Hz,1H),7.54-7.61(m,1H),7.47-7.53(m,1H),7.33(s,1H),7.11-7.25(m,7H),6.52(s,1H),5.34-5.48(m,4H),5.03(br dd,J＝8.82,3.58Hz,1H),4.63-4.79(m,2H),4.44-4.54(m,1H),3.96(br dd,J＝7.03,4.17Hz,1H),3.88(br d,J＝5.72Hz,2H),3.57-3.81(m,7H),3.19-3.27(m,1H),2.93-3.08(m,2H),2.78(br dd,J＝13.65,9.83Hz,1H),2.45(br s,1H),2.36(s,3H),1.81-2.02(m,3H),0.83-0.91(m,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.54。LCMS(ESI+)m/z：C ₅₃ H ₅₂ FN ₈ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1027.3, found: 1027.3.

example 23

N- ((S) -10-benzyl-1- (((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) oxy) -6,9,12, 15-tetraoxo-3-oxa-5, 8,11, 14-tetraazahexadecan-16-yl) -6- (2, 5-dioxo-2, 5-dihydro-1H-pyrrol-1-yl) hexanamide (23-122)

Examples 23 (23-122) were prepared in a similar manner to examples 21 (21-120). ¹ H NMR(400MHz,DMSO-D ₆ )δppm 8.63-8.74(m,1H),8.28(t,J＝5.63Hz,1H),8.09(d,J＝8.13Hz,1H),8.04(t,J＝5.63Hz,1H),7.98(t,J＝5.63Hz,1H),7.77(d,J＝11.01Hz,1H),7.34(s,1H),7.11-7.26(m,5H),6.98(s,2H),6.52(s,1H),5.31-5.58(m,4H),5.05(br dd,J＝9.13,4.00Hz,1H),4.61-4.82(m,2H),4.40-4.55(m,1H),3.90-4.04(m,1H),3.73-3.84(m,3H),3.60-3.73(m,5H),3.53-3.60(m,1H),3.33-3.37(m,2H),3.21-3.27(m,1H),2.94-3.08(m,2H),2.77(dd,J＝13.70,9.82Hz,1H),2.46-2.48(m,1H),2.38(s,3H),2.05-2.12(m,2H),1.82-2.01(m,3H),1.45(dq,J＝14.13,7.00Hz,4H),1.10-1.22(m,2H),0.87(t,J＝7.32Hz,3H)。 ¹⁹ F NMR(376MHz,DMSO-d ₆ )δppm-111.54。LCMS(ESI+)m/z：C ₅₂ H ₅₈ FN ₈ O ₁₃ ⁺ [ MH of (V)] ⁺ Calculated values: 1021.4, found: 1021.4.

example 24

CTG assay (Jeko-1 and MDA-MB-468)

CTG assay is a method to determine the number of living cells in culture based on the quantification of ATP present (an indicator of metabolically active cells). Cell assays require the addition of a single reagent, cell Titer Glo, in which cells are lysed and a luminescent signal is generated. The luminescent signal is proportional to the amount of ATP present. The amount of ATP is proportional to the number of cells present in the culture. For our assays, cells of Jeko-1 or MDA-MB-468 were ensured to be in log phase. Cells were transferred to 96 wells and treated with compounds at three-fold serial dilutions (10-point dilutions) from 1 μm to 0.0000508 μm for 72h according to manufacturer's instructions, usingLuminescent cell viability assay (Promega) cell viability was analyzed. The percentage of viable cells in each compound concentration was determined by normalization with the luminescence of the vehicle control and plotted as percent viability versus dose response curve by nonlinear fitting in GraphPad Prism software. Compound IC ₅₀ Calculated as the concentration of compound that killed 50% of the cells. Representative assay results are summarized in table 1.

Example 25

Human hepatocyte clearance (HHEP CL)

Human hepatocytes (from 10 mixed sex human donors at final concentration of 0.5X10) ⁶ Individual cells/ml) in Williams' E medium was incubated with test compound (0.90% acetonitrile and 0.10% DMSO, final concentration 1 mM) and positive control (7-ethoxycoumarin, 7-hydroxycoumarin, 0.90% acetonitrile and 0.10% DMSO, final concentration 3 mM) for 90min at 5% CO ₂ And 95% humidity in the incubator at 37 ℃ with continuous shaking at about 600 rpm. The total incubation volume was 200. Mu.l. Samples (25 mL) were taken at T0, 15, 30, 60 and 90min and added to ice-cold stop solution in the middle of the above timeThe solution (acetonitrile with 200ng/mL of tosylamide and labetalol as internal standard) (125 μl) was vortexed at 500rpm for 10min and centrifuged at 3220 Xg for 20min at 4 ℃. The assay plates were sealed and stored at 4 ℃ until LCMS analysis. Viability of hepatocytes at pre-incubation was determined to be 84.5%. Representative assay results are summarized in table 1.

Example 26

Human liver microsome clearance (HLM CL)

Working solutions were prepared by adding 5 μl of compound and control stock solution (10 mM in dimethyl sulfoxide, DMSO) to 495 μl Acetonitrile (ACN) (intermediate solution concentration: 100 μΜ,99% can and 1% DMSO). Microsomal working solutions of appropriate concentration were prepared in 100mM potassium phosphate buffer. After pre-incubating the reaction plate containing the mixture of compound and microsomes for 10min at 37 ℃, 98ml of 2mM NADPH and 2mM MgCl were added ₂ The solution was allowed to start the reaction. The final concentration of incubation medium was as follows: microsome-0.5 mg protein/ml, test compound/control compound-1 mM, NADPH-1mM, mgCl ₂ -1mM, acetonitrile 0.99%, DMSO 0.01%. Incubation was performed at 37℃for 60 min. Samples were taken at T0, T5, T15, T30, T45 and T60, added to ice-cold stop solution (acetonitrile with 200ng/mL of tosylamide and labetalol as internal standard) (125 μl) in the middle of the above time, shaken for 10min and centrifuged at 4000rpm for 20min at 4 ℃. The assay plate was analyzed by LCMS. Representative assay results are summarized in table 1.

Human liver microsomal clearance assay metabolism was assessed by the cytochrome P450 system (phase I enzyme). These enzymes oxidize the substrate by incorporating oxygen atoms into the hydrocarbon, resulting in the introduction of hydroxyl groups, or N-O-dealkylation and S-dealkylation of the substrate and the formation of more polar products that are easier to clean. Human hepatocyte clearance assays more widely measure the overall cellular metabolism (phase I and phase II enzyme pathways) of test compounds. Phase II enzymes catalyze conjugation reactions of xenobiotic metabolites and charged species (such as glutathione, sulfate, glycine or glucuronic acid) to form more polar compounds that are easier to scavenge.

Payloads with higher intrinsic clearance may provide better therapeutic index due to their potentially lower systemic plasma exposure. (Maderna, A.; doroski, M; subramannyam, C.; porte, A.; leverett, C.A.; vetelin, B.C.; chen, Z.; risley, H.; parris, K.; pandit, J.; varghese, A.H.; shanker, S.; song, C.; sukuru, S.C.; farley, K.A.; wagenaar, M.M.; shapiro, M.J.; musto, S.; lam, M-H.; loganzo, F.; O' Donnel, C.J.; discovery of cytotoxic dolastatin analog with N-terminal modifications "Journal Medicinal Chemistry,2014,57,10527-10543). In table 1, payloads with higher intrinsic clearance may have improved safety profiles because payloads with potential toxicity to healthy cells are rapidly removed from plasma, reducing their chances of interacting with healthy cells.

Example 27

PAMPA (parallel artificial membrane permeability measurement)

PAMPA is a method of determining the permeability of a substance from a donor compartment through an artificial membrane into an acceptor compartment through lipid infusion. See ottavanni, g.; martel, s.; carrupt, P-A. "Parallel Artificial Membrane Permeability Assay: A New Membrane for the Fast Prediction of Passive Human Skin Permeability", journal of Medicinal Chemistry,2006,49 (13), 3948-3954. A multi-well microtiter plate was used for the donor and the membrane/receptor compartment was placed on top; the entire assembly is commonly referred to as a "sandwich". At the beginning of the test, the drug was added to the donor compartment and the acceptor compartment was free of drug. After an incubation period, which may include agitation, the interlayers are separated and the amount of drug in each chamber is measured. The mass balance allows calculation of the drug remaining in the membrane.

PAMPA was performed by Pion Inc using GIT-0 lipids and 5. Mu.M donor solution in pH 5.0 and pH7.4PRISMA buffer (containing 0.05% DMSO). Higher PAMPA data is associated with better bystander killing. (Ogitani Y.; hagihara K.; oidate, M.; naito, H.; agatsuma T.; bystander killing effect of DS-8201a,a novel anti-human epidermal growth factor receptor anti-drug conjugate, in tumors with human epidermal growth factor receptor 2heterogeneity"Cancer Science,2016,107 (7), 1039-1046).

Representative assay results are summarized in table 1. Higher permeability is important because it means that the likelihood of "bystander killing" is greater. That is, once the payload neutralizes the tumor cells, the more permeable payload is more likely to escape the neutralized tumor cells and then intercalate into adjacent tumor cells. Once reached, it can neutralize the tumor cells, escape, become embedded in another adjacent tumor cell, and repeat the process.

TABLE 1

Dxd: delutidine; HHEP Cl: intrinsic clearance of human liver cells, mL/min/Kg; HHEP t _1/2 Hlm: human liver microsome clearance rate, mL/min/Kg. ND: not determined.

Example 28

By employing antibody development activities, novel and diverse anti-ROR-1 specific monoclonal antibodies were developed to bind to multiple regions of the ROR-1 extracellular domain (ECD) using three strategies: (1) immunization of mice in cohort 1 with full length ROR-1 ECD; (2) Mice from cohort 2 and cohort 3 were immunized with ROR-1 IgG-like domain; and (3) immunization of mice in cohort 4 with a short region of the human IgG-like sequence of ROR-1. After immunization of mice, monoclonal antibodies are produced using conventional methods. Briefly, unique variable heavy and light chain pairs from hybridoma and phage display activities were cloned into vectors designed to express full length antibodies as IgG in HEK293 cells under the control of the CMV promoter. Antibody expression vectors were complexed with polyethylenimine and transfected into HEK293 cultures. After shaking in 293 cell medium for 5 days at 37 ℃, the antibodies were captured on agarose based protein a resin. After several rigorous washes, the antibodies were eluted in glycine solution at pH 3, neutralized with Hepes at pH 9, and buffer exchanged into PBS.

Several monoclonal antibodies were developed using these methods and additional screening was performed on the antibodies produced to assess the specific characteristics of the antibodies. In order to fully evaluate the characteristics of the new antibodies, several assays were performed. First, confirmation of antibody binding to ROR-1 epitopes was confirmed biochemically as well as in ROR-1 positive cell lines. The specificity of binding was assessed biochemically by screening for binding to human ROR-2 protein, rodent ROR-1 protein, and in a cell-based assay. Further screening parameters include antibody internalization, epitope binning against known anti-ROR-1 antibodies (UC 961 and 4a 5), binding to human ROR-1 Ig-like domains, thermal drift, analysis to evaluate self-interactions with affinity capture self-interaction nanoparticle spectrometry (AC-SINS).

Example 29

Cell-binding saturation assays were developed to evaluate the extent to which the anti-ROR-1 antibodies developed in example 28 bind to endogenously expressed extracellular ROR-1 proteins on cell lines. More specifically, the anti-ROR-1 monoclonal antibodies developed in example E, such as ATX-P-875, ATX-P-885 and ATX-P-890, were analyzed in a cell binding assay. Briefly, two ROR-1 positive cell lines, jeKo-1 and MDA-MB-468, were incubated in titration series concentrations for each antibody construct. Cells were then washed and secondary antibody stained and detected by flow cytometry. The Mean Fluorescence (MFI) was determined by analysis on a cytometer software. The binding of ATX-P-875, ATX-P-885 and ATX-P-890 was compared with the cell binding saturation data of monoclonal anti-ROR-1 antibody UC 961. (see FIG. 27). As shown in FIG. 27, the cell-binding saturation of antibodies ATX-P-875, ATX-P-885 and ATX-P-890 was comparable to that of UC961, although higher concentrations of ATX-P-875 were required to achieve saturation than UC 961. ATX-P-890 and ATX-P-885 are good or improved, respectively, compared to UC-961 in the concentration required to reach binding saturation. Equivalent saturation to UC961 suggests that the anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890 have similar affinities for human ROR-1 targets as the clinically approved antibody UC-961.

Example 30

After measuring the saturation concentration (74 nM) in the binding assay, the anti-ROR-1 antibodies developed herein (ATX-P-875, P-885, P-890) were evaluated for their ability to internalize ROR-1 receptors on human ROR-1 positive cells (JeKo-1 and MDA-MB-468). Briefly, ROR positive cell lines are incubated with antibodies under supersaturated conditions in order to bind to all available ROR-1 receptors. Excess antibody was washed away and cells were incubated at 37 ℃ for the indicated amount of time for a four hour time period. At the end of each time point, internalization was stopped by placing an aliquot of cells on ice. Antibodies retained on the surface were detected using labeled secondary antibodies and flow cytometry. Percent internalization was calculated based on time zero, and it was assumed that 100% of the available receptors were on the cell surface at time zero. The results in FIG. 28 show that all antibodies reduced ROR-1 receptor internalization on JeKo-1 and MDA-MB-468 cells by at least 75% within 4 hours. Unexpectedly, in MDA-MB-468, internalization of the two anti-ROR-1 antibodies (ATX-P-875 and ATX-P-890) was improved relative to the clinically used UC961 anti-ROR-1 antibody, providing evidence that the ATX-P-875 and ATX-P-890 antibodies have improved ability to internalize ROR-1 receptors from the surface of a solid tumor.

Example 31

Cell binning was also used to determine whether monoclonal antibodies ATX-P-875, ATX-P-885 and ATX-P-890 bind the same epitope as conventionally known anti-ROR-1 binding monoclonal antibodies UC961 and 4A5 (control). In step 1 of the cell-sorting experiment, ATX-P-875, ATX-P-885 and ATX-P-890 monoclonal antibodies were incubated with different amounts of ROR-1 expressing cells (MDA-MB-468), respectively. In step 2, a fluorescently labeled secondary antibody recognizing the new antibody is incubated with the sample. Finally, in step 3, ROR-1 expressing cells coated with ATX-P-875, ATX-P-885 and ATX-P-890 were incubated with saturated doses of the labeled UC961 (Dy 650-UC 961) or 4A5 antibody (PE 4A 5) and analyzed by flow cytometry. The UC961 and 4A5 staining signals were then compared to the new antibody staining signals to determine if the ATX-P-875, ATX-P-885, and ATX-P-890 antibodies bound the same epitope as the known ROR-1 binding antibodies UC961 and 4A 5. FIG. 29A is a chart showing the staining expected if ATX-P-875, ATX-P-885 and ATX-P-890 antibodies bind the same epitope as the UC961 and 4A5 antibodies, below. FIG. 29B shows a predicted pattern if ATX-P-875, ATX-P-885 and ATX-P-890 antibodies bind to epitopes on ROR-1 different from the UC961 or 4A5 antibodies. Briefly, if the same epitope is bound, increased new antibody concentration will block the binding of the pre-labeled competitor antibody, thereby reducing the signal of the competitor antibody at higher concentrations. In the case of antibodies binding to different epitopes, each antibody (neo-antibody and competing antibody) will increase staining with increasing dose, as there is no competition for binding to the receptor. Cell-binning data obtained in MDA-MB-468 cells indicated that ATX-P-885 significantly bound the same epitope as UC961, and that both ATX-P-875 and ATX-P-890 significantly bound the same epitope as 4A 5. (see fig. 29C, 29D, and 30). The ability of the antibodies developed herein to bind different ROR-1 epitopes provides the opportunity to modulate targets in a variety of ways.

Example 32

Biochemical binning of anti-ROR-1 antibodies (ATX-P-875, P-885, P-890) by SPR was also evaluated compared to control anti-ROR-1 antibodies UC961 and 4a 5. In these experiments, 10ug/ml purified Hu/Cy/Rh ROR1-His clone protein was covalently coupled to HC30M chip. Separate dilutions of each antibody were injected onto the chip at 10pg/mL and binding was assessed by cartera SPR. Unexpectedly, the data indicated that there were 3 different binding epitopes between ATX-P-875, ATX-P-885 and ATX-P-890, with ATX-885 being the only antibody conferring partial blocking to the UC961 antibody (see FIG. 31). Cell sorting only assessed the ability of the antibodies to block UC961 or 4a5 (two clinically used ROR-1 antibodies). Biochemical SPR evaluation also tested the ability of antibodies to block each other and found that ATX-P-875 was able to block the binding of 4a5 and ATX-P-885, but still was unable to block UC961.

Example 33

The antibody profiles for ATX-P-875, ATX-P-885 and ATX-P-890 are summarized in FIG. 30 and tables 2-7, as compared to UC961. Initial assessment of antibody developability was performed by AC-SINS to evaluate the potential of self-interactions (fig. 30). The control antibodies Adalimumab (Adalimumab) and rituximab showed the expected low drift and infliximab showed the expected high drift. The anti-ROR-1 antibodies ATX-P-875, ATX-P-885 and ATX-P-890 developed herein were consistent with control antibodies that did not exhibit significant self-interactions and were unlikely to pose significant developability risks. In addition, in additional experiments, the binding properties of monoclonal antibodies ATX-P-875, ATX-P-885 and ATX-P-890 were compared to the binding properties of UC961. Tables 2-5 provide this antibody characterization data compared to the known ROR-1 binding antibody UC961, including the results listed for biochemical binding to purified protein and measurement by SPR (tables 2-5), cell binding to the ROR-1 positive cell line JeKo-1 and MDA-MB-468 (EC 50) (table 6) and cell internalization (internalization%) (table 7). Of particular note, it is believed that the reduced affinity of ATX-P-875 (KD: 1.09E-08) may provide unexpected therapeutic benefits as compared to UC961 and other anti-ROR-1 antibodies (ATX-P-885 and ATX-P-890). It is expected that by binding less tightly to the ROR-1 epitope, the ATX-P-885 antibody can penetrate further into the tumor to reach more distant cells expressing the ROR-1 target.

Table 2: human/cynomolgus monkey/rhesus monkey ROR-1 binding

Table 3: mouse ROR-1 binding

Table 4: rat ROR-1 binding

Table 5: human ROR-2 binding

Table 6: summary of cell binding

Table 7: summary of cell binding

Example 34

The synthesis of immunoconjugates was accomplished as described in this example. Antibodies were prepared as described in example 28 and suspended in PBS at pH 7.2 at the following protein concentrations: mAb A was 12.44mg/ml, mAb B was 13.29mg/ml, and mAb C was 14.90mg/ml. For reduction and conjugation calculations, a molecular weight of 150000Da was used for all antibodies.

Each antibody for reduction was prepared by adding 5% v/v of 500mM Tris,25mM pH 8.5 EDTA followed by TCEP (6 eq, 10mM stock of TCEP in water) and the mixture was kept at 20 ℃ for 2h.

After adding DMA and gently mixing with the reduced protein solution described above to reach the final 10% v/v during conjugation, a toxin-linker stock solution (12 eq, 50mM in DMA) was added and gently mixed. Bioconjugation was performed at 20 ℃ for about 16-20 h overnight; this was done in 2h, and the extended time allowed for maleimide ring opening.

Crude conjugate buffer was exchanged to PBS at pH7.4 using gravity fed NAP 25 (small scale) or flowing HiPrep G25 (large scale), the column was prepared and operated according to the manufacturer's instructions. To remove residual toxins, a slurry of 100mg/ml activated carbon (Sigma/C9157) in PBS at pH7.4 was prepared and added to achieve a 1mg carbon to 1mg starting antibody mass. It was gently mixed for 2h sufficient to keep the carbon in suspension. Then, the carbon was removed by centrifugation at 4000 g. Polysorbate 20 (PS 20) was added from a 10% w/v stock solution in PBS ph7.4 to reach the final 0.02% PS20 w/v in the product. The ADC was final filtered through a properly sized 0.2pm PES filter (chromatography direct/FIL-S-PES-022-13-100-S) under class A laminar flow. The final product was analyzed as follows: monomers and [ ADC ] mg/ml were analyzed by SEC HPLC, average DAR by PLRP, residual toxin by RP-HPLC, and endotoxin by Endosafe kinetic chromogenic substrate.

The analytical procedure was performed on HPLC instrument Agilent 1100 or 1260 using the protocols set out below and in tables 8 and 9.

SEC-HPLC-monomer content and ADC concentration (mg/mL)

Column: TOSOH TSKgel G3000SWXL 7.8mm 30cm 5pm particles (MERCK 808541) in combination with a safety protection column (MERCK 822858) with a GFC 3000X 3mm cartridge (Phenomnex)

Buffer solution: 0.2M phosphate 0.25M KCl 10%IPA pH 6.95.+ -. 0.1

Gradient: isocratic at 25℃at 0.5ml/min

Sample loadings of about 10 μg, monomer and concentration were determined from the 214nm signal. Antibody-based calibration curves report [ ADC ] mg/mL based on peak integral report monomer.

PLRP-HPLC-DAR assay

Column: PLRP-S2.1 mm. Times.5 cm,5pm (Agilent PL 912-1502)

Mobile phase a:0.1% v/v TFA/water

Mobile phase B:0.1% v/v TFA/ACN.

Gradient: 50℃at 1ml/min

Sample loading was 2 μg and analyzed at 214 nm.

TABLE 8

Time	B
		0.00	30
2.00	30
		10.00	41
11.50	90
		15.50	90
16.50	30
		20.00	30

Residual toxins

Column2.6μm C8LC column 50X 4.6mm, (Phenomex 00B-4497-E0) mobile phase A0.05% TFA/water

Mobile phase B0.05% tfa/ACN

Gradient: 60 ℃ at 2ml/min

TABLE 9

Time	B
		0.00	5
8.00	95
		8.10	100
9.00	100
		9.10	5
10.00	5

Analysis sample preparation: samples (50. Mu.l, ADC or PBS/PS20 matrix) were diluted with 2. Mu.l 5M NaCl, 150. Mu.l cold MeOH (from-20 ℃ C. Freezer), incubated for 30min at-20 ℃ C. And centrifuged at 21,000g for 30min at 4 ℃. The supernatant (125. Mu.l) was then extracted and mixed with 125. Mu.l WFI, 100. Mu.l of which was injected onto a Kinetex column. The data were analyzed at 214nm and residual toxin in the sample was estimated from an external calibration curve of the relevant toxin linker. The amount of bound toxin was determined using the ADC concentration and the calculated DAR, and the results were expressed as percent free versus free and bound.

Example 35

The new ROR-1 antibody-drug conjugates (ADCs) were evaluated by CTG assay in a similar manner as described in example 24 for screening payloads. A total of 3 unique antibodies (mAb a=875, mAb b=885, mAb c=890) were conjugated with 6 separate new linkers/payloads (18-112, 19-113, 20-114, 21-120, 22-121, and 23-122) (fig. 32). Briefly, ROR+ (JeKo-1/MDA-MB-468) or ROR- (Ramos) cells were transferred to 96 wells and treated with ADC for 72h at a three-fold serial dilution (10-point dilution) from 1mM to 0.0000508 mM. By usingLuminescent cell viability assay (Promega) cell viability was analyzed according to the manufacturer's instructions (fig. 33). The percentage of viable cells at each ADC concentration was determined by normalization with the luminescence of the vehicle control and plotted as percent viability versus dose response curve by nonlinear fitting in GraphPad Prism software. The IC50 of each ADC was calculated as the concentration of compound that killed 50% of the cells. Representative assay results are summarized in table 10. In the ROR+ cell line, ADCs containing 22-121, 21-120, and 23-122 achieved about 4-fold increase in potency compared to VLS-101. In addition, in vitro data demonstrated that mAb A (ATX-P-875) was slightly more potent than the other 2 ROR-1 targeting antibodies B and C (ATX-P-885 and ATX-P-895, respectively).

Table 10：

Furthermore, although the foregoing has been described in some detail by way of illustration and example for purposes of clarity and understanding, it will be understood by those skilled in the art that many and various modifications may be made without departing from the spirit of the disclosure. Therefore, it should be clearly understood that the forms disclosed herein are illustrative only and are not intended to limit the scope of the present disclosure, but rather to cover all modifications and alternatives falling within the true scope and spirit of the present disclosure.

Sequence listing

SEQ ID NO:1

ATX-P-875 VH CDR1(Kabat)

GFTFSNAW

SEQ ID NO:2

ATX-P-875 VH CDR2(Kabat)

IKSKTDGGTT

SEQ ID NO:3

ATX-P-875 VH CDR3(Kabat)

TTGPDDLDY

SEQ ID NO:4

ATX-P-875 VH nt

GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCASEQ

SEQ ID NO:5

ATX-P-875 VH AA

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSS

SEQ ID NO:6

ATX-P-875 HC IgG1-Fc nt

GAGGTGCAGCTGGTGGAGTCCGGGGGAGGCCTGGTCAAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACTTTCAGTAACGCCTGGATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTTGGCCGTATTAAAAGCAAAACTGATGGTGGGACAACAGACTACGCTGCACCCGTGAAAGGCAGATTCACCATCTCAAGAGATGATTCAAAAAACACGCTCTATCTGCAAATGAACAGCCTGAAAACCGAGGACACAGCCGTGTATTACTGTACCACAGGCCCTGACGATCTTGACTACTGGGGCCAGGGAACCCCGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCCCCAATCGAGAAAACCATTTCCAAGGCCAAAGGTCAACCAAGAGAACCCCAGGTGTATACTCTTCCGCCTTCTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCCGTCCTGGATAGCGACGGCTCATTTTTTCTGTATAGCAAACTGACTGTAGATAAATCACGGTGGCAGCAGGGCAATGTATTCAGTTGCTCCGTTATGCATGAAGCGTTACATAATCACTACACGCAGAAATCTCTTAGTCTTTCACCCGGT

SEQ ID NO:7

ATX-P-875 HC IgG1-Fc AA

EVQLVESGGGLVKPGGSLRLSCAASGFTFSNAWMSWVRQAPGKGLEWVGRIKSKTDGGTTDYAAPVKGRFTISRDDSKNTLYLQMNSLKTEDTAVYYCTTGPDDLDYWGQGTPVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:8

ATX-P-875 VL CDR1(Kabat)

QSISSY

ATX-P-875 VL CDR2(Kabat)

AAS

SEQ ID NO:10

ATX-P-875 VL CDR3(Kabat)

QQYDNLPIT

SEQ ID NO:11

ATX-P-875 VL nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

SEQ ID NO:12

ATX-P-875 VL AA

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIK

SEQ ID NO:13

ATX-P-875κLC nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGCGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGCCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAATCTCCCGATCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

SEQ ID NO:14

ATX-P-875κLC AA

DIQMTQSPSSLSASVGDRVTITCRASQSISSYLNWYQQKPGKAPKLLIYAASSLQSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYDNLPITFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWK VDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:15

ATX-P-885 VH CDR1(Kabat)

GGSFSGYY

SEQ ID NO:16

ATX-P-885 VH CDR2(Kabat)

INHSGST

SEQ ID NO:17

ATX-P-885 VH CDR3(Kabat)

AREGVYEDY

SEQ ID NO:18

ATX-P-885 VH nt

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO:19

ATX-P-885 VH AA

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSS

SEQ ID NO:20

ATX-P-885 HC IgG1-Fc nt

CAGGTGCAGCTACAGCAGTGGGGCGCAGGACTGTTGAAGCCTTCGGAGACCCTGTCCCTCACCTGCGCTGTCTATGGTGGGTCCTTCAGTGGTTACTACTGGAGCTGGATCCGCCAGCCCCCAGGGAAGGGGCTGGAGTGGATTGGGGAAATCAATCATAGTGGAAGCACCAACTACAACCCGTCCCTCAAGAGTCGAGTCACCATATCAGTAGACACGTCCAAGAACCAGTTCTCCCTGAAGCTGAGCTCTGTGACCGCCGCGGACACGGCTGTATATTACTGTGCGAGAGAGGGTGTCTACGAGGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCCCCAATCGAGAAAACCATTTCCAAGGCCAAAGGTCAACCAAGAGAACCCCAGGTGTATACTCTTCCGCCTTCTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCCGTCCTGGATAGCGACGGCTCATTTTTTCT

SEQ ID NO:21

ATX-P-885 HC IgG1-Fc AA

QVQLQQWGAGLLKPSETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIGEINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCAREGVYEDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:22

ATX-P-885 VL CDR1(Kabat)

QSVSNY

ATX-P-885 VL CDR2(Kabat)

DAY

SEQ ID NO:24

ATX-P-885 VL CDR3(Kabat)

QQRSNWPLT

SEQ ID NO:25

ATX-P-885 VL nt

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGA GCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAA

SEQ ID NO:26

ATX-P-885 VL AA

EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARF

SGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIK

SEQ ID NO:27

ATX-P-885κLC nt

GAAATTGTGTTGACACAGTCTCCAGCCACCCTGTCTTTGTCTCCAGGGGAAAGAGCCACCCTCTCCTGCAGGGCCAGTCAGAGTGTTAGCAACTACTTAGCCTGGTACCAACAGAAACCTGGCCAGGCTCCCAGGCTCCTCATCTATGATGCCTACAACAGGGCCACTGGCATCCCAGCCAGGTTCAGTGGCAGTGGGTCTGGGACAGACTTCACTCTCACCATCAGCAGCCTAGAGCCTGAAGATTTTGCAGTTTATTACTGTCAGCAGCGTAGCAACTGGCCTCTCACCTTCGGCCAAGGGACACGACTGGAGATTAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

SEQ ID NO:28

ATX-P-885κLC AA

EIVLTQSPATLSLSPGERATLSCRASQSVSNYLAWYQQKPGQAPRLLIYDAYNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTRLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

SEQ ID NO:29

ATX-P-890 VH CDR1(Kabat)

GYTFTGYY

SEQ ID NO:30

ATX-P-890 VH CDR2(Kabat)

INPNSGGT

SEQ ID NO:31

ATX-P-890 VH CDR3(Kabat)

VRDQVQLERFDS

SEQ ID NO:32

ATX-P-890 VH nt

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCA

SEQ ID NO:33

ATX-P-890 VH AA

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSS

SEQ ID NO:34

ATX-P-890 HC IgG1-Fc nt

CAGGTGCAGCTGGTGCAGTCTGGGGCTGAGGTGAAGAAGCCTGGGGCCTCAGTGAAGGTCTCCTGCAAGGCTTCTGGATACACCTTCACCGGCTACTATATGCACTGGGTGCGACAGGCCCCTGGACAAGGGCTTGAGTGGATGGGATGGATCAACCCTAACAGTGGTGGCACAAACTATGCACAGAAGTTTCAGGGCAGGGTCACCATGACCAGGGACACGTCCATCAGCACAGCCTACATGGAGCTGAGCAGGCTGAGATCTGACGACACGGCCGTGTATTACTGTGTGAGAGATCAGGTACAACTGGAACGGTTCGACTCCTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGCTAGCACTAAAGGGCCTTCTGTATTTCCCTTGGCCCCGTCCAGCAAATCGACCTCGGGAGGGACAGCCGCCCTGGGTTGCCTTGTGAAAGATTATTTCCCTGAGCCAGTTACCGTAAGTTGGAACAGTGGGGCGCTGACAAGTGGTGTGCACACGTTTCCTGCCGTCCTGCAATCATCGGGCTTGTATAGCCTCAGCTCTGTGGTCACTGTCCCAAGTTCATCGCTGGGCACTCAGACGTATATTTGCAATGTGAACCACAAACCTTCAAATACAAAAGTGGATAAACGCGTAGAACCGAAATCGTGTGATAAAACTCACACATGCCCGCCATGCCCGGCACCTGAACTGCTTGGTGGTCCCAGCGTGTTCCTGTTCCCGCCGAAGCCTAAAGATACTCTAATGATCAGCCGTACGCCAGAGGTGACATGTGTCGTGGTTGACGTGTCCCACGAAGATCCCGAAGTTAAGTTCAATTGGTATGTTGATGGTGTAGAGGTACACAATGCTAAGACTAAACCTCGCGAGGAGCAGTACAATTCGACCTATCGTGTCGTGAGCGTTCTGACCGTCCTTCACCAAGATTGGCTTAACGGCAAAGAATATAAGTGCAAGGTAAGCAATAAAGCACTTCCGGCCCCAATCGAGAAAACCATTTCCAAGGCCAAAGGTCAACCAAGAGAACCCCAGGTGTATACTCTTCCGCCTTCTCGTGAGGAAATGACTAAAAATCAAGTATCCCTTACGTGTCTGGTTAAAGGTTTTTATCCTAGCGATATTGCTGTTGAATGGGAATCGAACGGTCAGCCGGAGAATAATTATAAAACAACGCCACCCGTCCTGGATAGCGACGGCTCATTTTTTCTGTATAGCAAACTGACTGTAGATAAATCACGGTGGCAGCAGGGCAATGTATTCAGTTG CTCCGTTATGCATGAAGCGTTACATAATCACTACACGCAGAAATCTCTTAGTCTTTCACCCGGT

SEQ ID NO:35

ATX-P-890 HC IgG1-Fc AA

QVQLVQSGAEVKKPGASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMGWINPNSGGTNYAQKFQGRVTMTRDTSISTAYMELSRLRSDDTAVYYCVRDQVQLERFDSWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

SEQ ID NO:36

ATX-P-890 VL CDR1(Kabat)

QDISNY

ATX-P-890 VL CDR2(Kabat)

DAS

SEQ ID NO:38

ATX-P-890 VL CDR3(Kabat)

QQYDNLPPT

SEQ ID NO:39

ATX-P-890 VL nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCT GATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAA

SEQ ID NO:40

ATX-P-890 VL AA

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIK

SEQ ID NO:41

ATX-P-890κLC nt

GACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCAGGCGAGTCAGGACATTAGCAACTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTACGATGCATCCAATTTGGAAACAGGGGTCCCATCAAGGTTCAGTGGAAGTGGATCTGGGACAGATTTTACTTTCACCATCAGCAGCCTGCAGCCTGAAGATATTGCAACATATTACTGTCAACAGTATGATAATCTCCCTCCCACTTTCGGCCCTGGGACCAAGGTGGAAATCAAACGTACGGTAGCTGCCCCTTCAGTTTTTATCTTTCCGCCGTCTGACGAGCAGTTAAAATCCGGGACCGCTTCTGTAGTTTGCCTGCTGAATAATTTTTATCCGCGTGAGGCTAAAGTACAATGGAAAGTCGACAATGCTTTGCAGTCGGGAAATTCACAGGAAAGTGTTACGGAGCAGGATTCTAAAGATTCCACATATTCACTCAGCTCCACCCTTACACTGAGCAAAGCCGACTATGAAAAACATAAAGTTTACGCATGTGAGGTGACGCACCAAGGATTATCCAGTCCGGTCACAAAATCGTTTAACCGCGGTGAGTGT

SEQ ID NO:42

ATX-P-890κLC AA

DIQMTQSPSSLSASVGDRVTITCQASQDISNYLNWYQQKPGKAPKLLIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQYDNLPPTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

1. An immunoconjugate having formula (I),

Ab-[S-L ¹ -L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D] _n

(I)

wherein:

ab is an antibody or antigen binding fragment;

L ¹ is that

L ² Is absent from,

n ¹ Independently an integer from 0 to 12;

L ⁴ is a tetrapeptide residue;

L ⁵ is absent or- [ NH (CH) ₂ )n ² ]n ³ -；

n ² An integer of 0 to 6;

n ³ an integer of 0 to 2;

L ⁶ is absent or not present

L ⁷ Is absent from,

D is a drug moiety; and is also provided with

n is an integer from 1 to 10.

2. The immunoconjugate according to claim 1, wherein L ² Is absent.

3. The immunoconjugate according to claim 1, wherein L ² Is that

4. The immunoconjugate according to claim 1, wherein L ² Is that

5. The immunoconjugate according to any one of claims 1 to 4, wherein Z ¹ And Z ² At least one of which is hydrogen.

6. The immunoconjugate according to any one of claims 1 to 4, wherein Z ¹ And Z ² At least one of which is halogen.

7. The immunoconjugate according to any one of claims 1 to 4, wherein Z ¹ And Z ² At least one of which is NO ₂ 。

8. The immunoconjugate according to any one of claims 1 to 4, wherein Z ¹ And Z ² At least one of (a)The one is-O- (C) ₁ -C ₆ Alkyl).

9. The immunoconjugate according to any one of claims 1 to 4, wherein Z ¹ And Z ² At least one of which is C ₁ -C ₆ An alkyl group.

10. The immunoconjugate according to any one of claims 1 to 9, wherein L ³ Is- (CH) ₂ )n ¹ -C(＝O)-。

11. The immunoconjugate according to any one of claims 1 to 9, wherein L ³ Is- (CH) ₂ CH ₂ O)n ¹ -(CH ₂ )n ¹ C(＝O)-。

12. The immunoconjugate according to any one of claims 1 to 11, wherein L ⁴ Is gly-gly-phe-gly (GGFG).

13. The immunoconjugate according to any one of claims 1 to 11, wherein L ⁵ Is absent.

14. The immunoconjugate according to any one of claims 1 to 11, wherein L ⁵ Is- [ NH (CH) ₂ )n ² ]n ³ -。

15. The immunoconjugate according to any one of claims 1 to 14, wherein L ⁶ Is absent.

16. The immunoconjugate according to any one of claims 1 to 14, wherein L ⁶ Is that

17. The immunoconjugate according to any one of claims 1 to 16, wherein L ⁷ Is absent.

18. The immunoconjugate according to any one of claims 1 to 16, wherein L ⁷ Is that

19. The immunoconjugate according to any one of claims 1 to 16, wherein L ⁷ Is that

20. The immunoconjugate according to any one of claims 1 to 16, wherein L ⁷ Is that

21. The immunoconjugate according to any one of claims 1 to 20, wherein D is a drug moiety of formula (II) having the structure:

wherein:

X ² is-OR ⁹ 、-SR ⁹ or-NHR ⁹ ；

R ⁵ And R is ⁶ Each independently is hydrogen, halogen, substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ ；

m is 1 or 2;

n ⁶ 0, 1 or 2;

each Y is independently H or halogen;

each p is independently 1, 2, 3, 4, 5, or 6;

each q is independently 0, 1, 2, 3, 4, 5, or 6;

each t is independently 1, 2, 3, 4, 5, or 6;

Each X is ³ independently-H, -OH, -SH or-NH ₂ 。

22. The immunoconjugate according to claim 21, wherein the drug moiety is isatecan.

23. The immunoconjugate according to any one of claims 1 to 22, wherein Ab specifically binds to a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1).

24. The immunoconjugate according to any one of claims 1 to 23, wherein Ab binds to the surface of a cancer cell.

25. The immunoconjugate according to claim 1, wherein formula (I) is selected from

Wherein Z is ¹ And Z ² Each independently selected from hydrogen, fluorine, chlorine, -NO ₂ and-OCH ₃ 。

26. A compound of formula (IV) or a pharmaceutically acceptable salt thereof, having the structure:

wherein:

R ⁴ Is hydrogen, substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Haloalkyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkenyl) -X ² Substituted or unsubstituted- (C) ₁ -C ₆ Halogenated alkenyl groups)-X ² Substituted or unsubstituted- (C) ₁ -C ₆ Alkynyl) -X ² Or substituted or unsubstituted- (C) ₁ -C ₆ Haloalkynyl) -X ² ；

X ² is-OH, -SH or-NR ⁵ R ⁶ ；

R ⁷ Is H, -COR ⁸ 、-CO ₂ R ⁸ Or- (CO) -NHR ⁸ ；

R ⁸ Is substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl or- [ (CY) ₂ ) _P O(CY ₂ ) _q ] _t CY ₃ ；

m is 1 or 2;

n ⁶ 0, 1 or 2; and is also provided with

Each Y is independently H or halogen;

each p is independently 1, 2, 3, 4, 5, or 6;

each q is independently 0, 1, 2, 3, 4, 5, or 6; and is also provided with

Each t is independently 1, 2, 3, 4, 5, or 6;

provided that formula (IV) does not represent delutinkang or isatidine.

27. The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² At least one of which is hydrogen.

28. The compound according to claim 26 or 27, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² At least one of which is halogen.

29. The compound according to any one of claims 26 to 28, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ An alkyl group.

30. The compound according to any one of claims 26 to 29, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² At least one of which is substituted or unsubstituted C ₁ -C ₆ A haloalkyl group.

31. The compound according to any one of claims 26 to 30, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² At least one of them is a substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl).

32. The compound according to any one of claims 26 to 31, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² Is a substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-, such that R ¹ And R is ² Taken together to form a ring.

33. The compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein R ¹ And R is ² Each independently selected from the group consisting of hydrogen, fluorine, methoxy, methyl, difluoromethyl and-O- (CH) ₂ ) -O-such that R ¹ And R is ² Taken together to form a ring.

34. The compound according to any one of claims 26 to 33, or a pharmaceutically acceptable salt thereof, wherein R ³ Is hydrogen.

35. The compound according to any one of claims 26 to 33, or a pharmaceutically acceptable salt thereof, wherein R ³ Is substituted or unsubstituted C ₁ -C ₆ An alkyl group.

36. The compound of claim 35, or a pharmaceutically acceptable salt thereof, wherein R ³ Is methyl.

37. The compound according to any one of claims 26 to 36, or a pharmaceutically acceptable salt thereof, wherein R ⁴ Is hydrogen.

38. The compound according to any one of claims 26 to 36, or a pharmaceutically acceptable salt thereof, wherein R ⁴ Is substituted or unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² 。

39. The compound of claim 38, or a pharmaceutically acceptable salt thereof, wherein the- (C ₁ -C ₆ Alkyl) -X ² Is branched.

40. The compound according to claim 38 or 39, or a pharmaceutically acceptable salt thereof, wherein R ⁴ Is unsubstituted- (C) ₁ -C ₆ Alkyl) -X ² 。

41. A compound according to any one of claims 26 to 40, or a pharmaceutically acceptable salt thereof, wherein X ¹ is-O-.

42. A compound according to any one of claims 26 to 40, or a pharmaceutically acceptable salt thereof, wherein X ¹ is-NH-.

43. A compound according to any one of claims 26 to 40, or a pharmaceutically acceptable salt thereof, wherein X ¹ is-NH- (c=o) -.

44. A compound according to any one of claims 26 to 43, or a pharmaceutically acceptable salt thereof, wherein X ² is-OH.

45. The compound of any one of claims 26 to 44, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (IV) is represented by formula (Iva):

46. the compound of any one of claims 26 to 44, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (IV) is represented by formula (Ivb):

47. The compound of any one of claims 26 to 44, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (IV) is represented by formula (Ivc):

48. the compound of claim 26, or a pharmaceutically acceptable salt thereof, wherein the compound of formula (IV) is

49. A pharmaceutical composition comprising the immunoconjugate of any one of claims 1 to 25, the compound of any one of claims 26 to 48, or a pharmaceutically active salt thereof, and a pharmaceutically acceptable carrier, diluent, excipient, or combination thereof.

50. A method for treating cancer or a tumor, the method comprising administering to a subject suffering from the cancer or the tumor an effective amount of an immunoconjugate of any one of claims 1 to 25, a compound of any one of claims 26 to 48, or a pharmaceutically active salt thereof, or a pharmaceutical composition of claim 49.

51. The method of claim 50, wherein the cancer or the tumor is selected from lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell cancer, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulval cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

52. Use of an effective amount of an immunoconjugate according to any one of claims 1 to 25, a compound according to any one of claims 26 to 48, or a pharmaceutically active salt thereof, or a pharmaceutical composition according to claim 49, in the manufacture of a medicament for the treatment of cancer or tumor.

53. The method of claim 52, wherein the cancer or the tumor is selected from lung cancer, urothelial cancer, colorectal cancer, prostate cancer, ovarian cancer, pancreatic cancer, breast cancer, bladder cancer, gastric cancer, gastrointestinal stromal tumor, cervical cancer, esophageal cancer, squamous cell cancer, peritoneal cancer, liver cancer, hepatocellular cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, uterine cancer, salivary gland cancer, renal cancer, vulval cancer, thyroid cancer, penile cancer, leukemia, malignant lymphoma, plasmacytoma, myeloma, or sarcoma.

54. A conjugate having the formula (III),

Mi-L ² -L ³ -L ⁴ -L ⁵ -L ⁶ -L ⁷ -D

(III)

wherein:

mi is

L ² Is absent from,

n ¹ Independently an integer of 0 to 12；

L ⁴ Is a tetrapeptide residue;

L ⁵ is absent or- [ NH (CH) ₂ )n ² ]n ³ -；

n ² An integer of 0 to 6;

n ³ an integer of 0 to 2;

L ⁶ Is absent or not present

L ⁷ Is absent from,And is also provided with

D is a drug moiety.

55. The conjugate according to claim 54, wherein L ² Is absent.

56. The conjugate according to claim 54, wherein L ² Is that

57. The conjugate according to claim 54, wherein L ² Is that

58. The conjugate of any one of claims 54 to 57, wherein Z ¹ And Z ² At least one of which is hydrogen.

59. The conjugate of any one of claims 54 to 57, wherein Z ¹ And Z ² At least one of which is halogen.

60. The conjugate of any one of claims 54 to 57, wherein Z ¹ And Z ² At least one of which is NO ₂ 。

61. The conjugate of any one of claims 54 to 57, wherein Z ¹ And Z ² At least one of them is-O- (C) ₁ -C ₆ Alkyl).

62. The conjugate of any one of claims 54 to 57, wherein Z ¹ And Z ² At least one of which is C ₁ -C ₆ An alkyl group.

63. The conjugate of any one of claims 54 to 62, wherein L ³ Is- (CH) ₂ )n ¹ -C(＝O)-。

64. The conjugate of any one of claims 54 to 62, wherein L ³ Is- (CH) ₂ CH ₂ O)n ¹ -(CH ₂ )n ¹ C(＝O)-。

65. The conjugate of any one of claims 54 to 64, wherein L ⁴ Is gly-gly-phe-gly (GGFG).

66. The conjugate of any one of claims 54 to 65, wherein L ⁵ Is absent.

67. The conjugate of any one of claims 54 to 65, wherein L ⁵ Is- [ NH (CH) ₂ )n ² ]n ³ -。

68. The conjugate of any one of claims 54 to 67, wherein L ⁶ Is absent.

69. The conjugate of any one of claims 54 to 67, wherein L ⁶ Is that

70. The conjugate of any one of claims 54 to 69, wherein L ⁷ Is absent.

71. The conjugate of any one of claims 54 to 69, wherein L ⁷ Is that

72. The conjugate of any one of claims 54 to 69, wherein L ⁷ Is that

73. The conjugate of any one of claims 54 to 69, wherein L ⁷ Is that

74. The conjugate of any one of claims 54 to 73, wherein D is a compound of formula (II) having the structure:

wherein:

R ¹ and R is ² Each independently selected from the group consisting of hydrogen, halogen, -CN, -OR ⁵ 、-NR ⁵ R ⁶ Substituted or unsubstituted C ₁ -C ₆ Alkyl, substituted or unsubstituted C ₁ -C ₆ Haloalkyl, substituted or unsubstituted-O- (C) ₁ -C ₆ Alkyl group) Substituted or unsubstituted-O- (C) ₁ -C ₆ Haloalkyl) - [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₃ Or substituted or unsubstituted-O- (CR) ⁵ R ⁶ ) _m -O-such that R ¹ And R is ² Taken together to form a ring;

X ² is-OR ⁹ 、-SR ⁹ or-NHR ⁹ ；

m is 1 or 2;

n ⁶ is 0, 1 or 2

Each Y is independently H or halogen;

each p is independently 1, 2, 3, 4, 5, or 6;

each q is independently 0, 1, 2, 3, 4, 5, or 6;

each t is independently 1, 2, 3, 4, 5, or 6;

Each R ⁸ Independently substituted or unsubstituted C ₁ -C ₆ alkyl-X ³ Substituted or unsubstituted C ₁ -C ₆ Haloalkyl group X ³ Or- [ (CY) ₂ ) _p O(CY ₂ ) _q ] _t CY ₂ -X ³ ；

Each X is ³ independently-H, -OH, -SH or-NH ₂ 。

75. The conjugate of any one of claims 54 to 74, wherein formula (III) is selected from

76. A method of preparing an immunoconjugate, the method comprising: reacting an effective amount of a thiol-functionalized antibody or antigen-binding fragment with the conjugate of any one of claims 54 to 75 under reaction conditions effective to form an immunoconjugate of any one of claims 1 to 25.

77. The method of claim 76, further comprising reducing the antibody or antigen-binding fragment under reducing conditions effective to form the thiol-functionalized antibody or antigen-binding fragment.

78. An antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

b) A light chain comprising:

79. The antibody or antigen-binding fragment of claim 78, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 5, 7, 12 or 14.

80. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof according to any one of claims 78 or 79, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 4, 6, 11 or 13.

81. A host cell comprising one or more nucleic acids of claim 80.

82. The immunoconjugate according to any one of claims 1 to 25, wherein Ab is the antibody or antigen binding fragment of claim 78 or 79.

83. An antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

b) A light chain comprising:

VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 24; wherein the antibody or antigen binding fragment thereof specifically binds to an extracellular domain of a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1).

84. The antibody or antigen-binding fragment of claim 83, comprising an amino acid sequence that has at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 19, 21, 26 or 28.

85. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof according to any one of claims 83 or 84, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 18, 20, 25 or 27.

86. A host cell comprising one or more nucleic acids of claim 85.

87. The immunoconjugate according to any one of claims 1 to 25, wherein Ab is the antibody or antigen binding fragment of claim 83 or 84.

88. An antibody or antigen-binding fragment thereof, comprising:

a) A heavy chain comprising:

b) A light chain comprising:

VLCDR 3 comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of SEQ ID NO. 38; wherein the antibody or antigen binding fragment specifically binds to an extracellular domain of a human receptor tyrosine kinase, such as orphan receptor 1 (ROR 1).

89. The antibody or antigen-binding fragment of claim 88, comprising an amino acid sequence having at least 95% sequence identity to the amino acid sequence of any one of SEQ ID NOs 33, 35, 40 or 42.

90. One or more nucleic acids encoding the antibody or antigen-binding fragment thereof of any one of claims 88 or 89, such as an antibody or antigen-binding fragment thereof encoded by one or more nucleic acids comprising a sequence having at least 95% sequence identity to a nucleic acid sequence set forth in any one of SEQ ID NOs 32, 34, 39 or 41.

91. A host cell comprising one or more nucleic acids of claim 90.

92. The immunoconjugate of any one of claims 1 to 25, wherein Ab is the antibody or antigen binding fragment of claim 88 or 89.

93. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 6 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 13.

94. The antibody or binding fragment thereof of claim 93, or a composition comprising the antibody or binding fragment thereof, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 7 and the light chain comprises the polypeptide sequence of SEQ ID No. 14.

95. The antibody or binding fragment thereof of any one of claims 93 or 94, or a composition comprising the antibody or binding fragment thereof, wherein the antibody or binding fragment thereof is conjugated to a molecule.

96. The antibody or binding fragment thereof of claim 95, or a composition comprising said antibody or binding fragment thereof, wherein said molecule is a drug, toxin, or cytokine.

97. The immunoconjugate of any one of claims 1 to 25, wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof of any one of claims 93 or 94.

98. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of claims 93 to 96, such as an immunoconjugate of claim 97, the method comprising administering the antibody or binding fragment thereof or the composition according to any one of claims 93 to 96 to a subject in need thereof, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

99. The antibody or binding fragment thereof or composition comprising the antibody or binding fragment thereof according to any one of claims 93 to 96, such as an immunoconjugate according to claim 97, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.

100. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 20 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 27.

101. The antibody or binding fragment thereof of claim 100, or a composition comprising the antibody or binding fragment thereof, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 21 and the light chain comprises the polypeptide sequence of SEQ ID No. 28.

102. The antibody or binding fragment thereof of any one of claims 100 or 101, or a composition comprising the antibody or binding fragment thereof, wherein the antibody or binding fragment thereof is conjugated to a molecule.

103. The antibody or binding fragment thereof of claim 102, or a composition comprising the antibody or binding fragment thereof, wherein the molecule is a drug, toxin, or cytokine.

104. The immunoconjugate according to any one of claims 1 to 25, wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof according to any one of claims 100 or 101.

105. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof according to any one of claims 100 to 103 or a composition comprising the antibody or binding fragment thereof, such as the immunoconjugate of claim 104, the method comprising administering the antibody or binding fragment thereof or the composition according to any one of claims 100 to 103 to a subject in need thereof, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

106. The antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of claims 100 to 103, such as the immunoconjugate according to claim 104, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.

107. An antibody or binding fragment thereof, or a composition comprising said antibody or binding fragment thereof, wherein said heavy chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 34 and said light chain is encoded by the nucleic acid sequence set forth in SEQ ID No. 41.

108. The antibody or binding fragment thereof of claim 107, or a composition comprising the antibody or binding fragment thereof, wherein the heavy chain of the antibody comprises the polypeptide sequence of SEQ ID No. 35 and the light chain comprises the polypeptide sequence of SEQ ID No. 42.

109. The antibody or binding fragment thereof of any one of claims 107 or 108, or a composition comprising the antibody or binding fragment thereof, wherein the antibody or binding fragment thereof is conjugated to a molecule.

110. The antibody or binding fragment thereof of claim 109, or a composition comprising the antibody or binding fragment thereof, wherein the molecule is a drug, toxin, or cytokine.

111. The immunoconjugate according to any one of claims 1 to 25, wherein the antibody or binding fragment thereof is the antibody or binding fragment thereof according to any one of claims 107 or 108.

112. A method of inhibiting or treating a disease, such as cancer, using an antibody or binding fragment thereof according to any one of claims 107 to 110 or a composition comprising the antibody or binding fragment thereof, such as the immunoconjugate of claim 111, the method comprising administering to a subject in need thereof the antibody or binding fragment thereof or the composition according to any one of claims 107 to 110, optionally selecting a subject receiving treatment for the disease, such as cancer, and/or optionally determining inhibition of the disease, such as cancer, after administration of the antibody or binding fragment thereof.

113. The antibody or binding fragment thereof or a composition comprising the antibody or binding fragment thereof according to any one of claims 107 to 110, such as the immunoconjugate according to claim 111, for use as a medicament, such as for the purpose of inhibiting or treating a disease such as cancer.