CN112638426B

CN112638426B - Aromatic nitro-based linker, antibody conjugated drug containing linker and use of linker

Info

Publication number: CN112638426B
Application number: CN201980056464.5A
Authority: CN
Inventors: 周辛波; 王彦明; 李家国; 李松; 钟武; 樊士勇; 肖典; 肖军海; 郑志兵; 李行舟; 谢云德; 曹瑞源; 王晓奎
Original assignee: Academy of Military Medical Sciences AMMS of PLA
Current assignee: Academy of Military Medical Sciences AMMS of PLA
Priority date: 2018-09-21
Filing date: 2019-09-18
Publication date: 2023-06-16
Anticipated expiration: 2039-09-18
Also published as: WO2020057543A1; CN112638426A

Abstract

The linker shown in the formula I, an antibody coupling medicament containing the linker, application of the linker, a pharmaceutical composition containing the antibody coupling medicament, and application of the antibody coupling medicament in treating and/or preventing diseases.

Description

Aromatic nitro-based linker, antibody conjugated drug containing linker and use of linker

The present application is based on and claims priority from CN application number 201811106544.0, filing date 2018, 9, 21, the disclosure of which is incorporated herein by reference in its entirety.

Technical Field

The application belongs to the field of pharmaceutical chemistry, and in particular relates to an aromatic nitro-based linker, an antibody coupling drug containing the linker, application of the linker, a pharmaceutical composition containing the antibody coupling drug, and application of the antibody coupling drug in treating and/or preventing diseases.

Background

The antibody-drug conjugate (ADC) realizes the combination of the advantages of monoclonal antibody drug and small molecular cytotoxin, structurally comprises three components of antibody (antibody), cytotoxin (cytoxin) and linker (linker), and specifically targets tumors through the antibody, and then releases cytotoxin to further kill tumor cells. ADC overcomes the drug resistance problem of the monoclonal antibody drug to a certain extent. Since the effect of an ADC is primarily dependent on the cytotoxin carried by the antibody, antibody-mediated biological effects are not necessary, and even some degree of mutation of the antigen does not affect the efficacy of the ADC. For example, classical ADC drug Kadcyla, treatment of HER2 resistant to naked anti-Herceptin ⁺ Breast cancer can extend the overall survival by a further 6 months compared to standard therapies. Another clinical study showed that the incidence of grade 3 adverse reactions was reduced by 50 in the Kadcyla-treated group compared to the standard-therapy chemotherapeutic-treated groupPercent of the total weight of the composition. The ADC has the advantages of greatly improving therapeutic index compared with the conventional chemotherapeutics through attenuation and synergism. As a novel anti-tumor 'weapon', the ADC is expected to break through in the field of tumor disease treatment.

The success of an ADC depends not only on the optimization of each component, but also on the rational collocation and integration of the individual components. The three components of the ADC play different roles, and thus the ADC also has different requirements for the three components: the antibody of the ADC needs to meet the requirement that the antibody can specifically target tumors and has proper affinity and internalizable performance; the toxin of the ADC needs to meet the requirements of high toxicity, definite action mechanism, coupling and the like; the ADC has 2 points of most basic requirements for the linker: the first is to ensure the stability of the ADC in the blood circulation system, and the second is to ensure that the ADC can release toxins quickly and effectively after reaching the tumor.

As a junction between the antibody and the toxin in the ADC structure, the performance of the linker directly affects the potency, toxicity and pharmacokinetics of the final product. ADC is taken as a long-acting biological medicine, the administration period is usually 2-3 weeks/time, the linker simultaneously perfectly realizes two basic requirements of long-term stability of a circulatory system and quick release of a target tissue, and the design and construction process of the ADC have great difficulty.

According to the different toxin release mechanisms, the linkers of ADC can be divided into two categories, namely cleavable linkers and non-cleavable linkers, wherein the cleavable linkers can be further divided into enzyme-cleavable linkers and chemically-cleavable linkers. ADCs comprising cleavable linkers typically can be effective by releasing the free toxin, whereas ADCs comprising non-cleavable linkers are effective without cleavage of the covalent bond between the linker and the toxin. The enzyme cleavable linker has a relatively wider application range and higher drug release selectivity, and is now the most widely used tool in the process of ADC drug development.

The cleavage of the predominant enzymatically cleavable linker involves only cathepsin B and β -glucuronidase, both of which are ubiquitous non-tumor specific lysosomal enzymes that act selectively on dipeptide fragments or glucuronic acid groups, respectively, driving toxin release by catalyzing the cleavage process of the linker in the ADC. The most widely used enzyme cleavable linkers at this stage are the more mature dipeptide type linkers under investigation. Existing ADC release enzymes, such as cathepsin B or β -glucuronidase, which are commonly found in lysosomes of most cells of mammals, for those unavoidable ADC toxin off-targets, release enzymes in normal tissue lysosomes can degrade ADC and release cytotoxins of high killing power (e.g. MMAE) while causing toxicity to normal tissues; at the same time, the released nonionic free cytotoxins can further penetrate the cell membrane by bystander effect to cause systemic toxicity to surrounding normal tissues.

Although the targeting of ADC drugs is dramatically improved compared with traditional chemotherapeutics, the proportion of ADC drugs which can actually reach tumor tissues after administration is less than 1%. How to reduce the safety hazards caused by the ubiquitous and unavoidable off-target highly toxic cytotoxins, or become one of the core problems facing ADC development. The selectivity of the ADC enzymolysis drug release process to tumor tissues is improved, and the problems are hopeful to be solved or improved. The novel enzymolysis type connector only used for selectively cracking tumor tissues is designed, so that release of off-target cytotoxin in normal tissues or cells can be avoided or reduced, systematic toxicity generated in the ADC administration process can be reduced, and the therapeutic index of the ADC is further improved.

BRIEF SUMMARY OF THE PRESENT DISCLOSURE

The application relates to a compound shown in a formula I or a salt thereof,

wherein:

R ₁ is that

R ₂ Halogen (e.g. fluorine, chlorine, bromine, iodine) or

Wherein R is ₄ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, p is 0, 1, 2, 3 or 4;

R ₃ is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 An alkoxy group;

r is 0, 1, 2, 3 or 4;

ar is aryl, heteroaryl, arylo-heterocyclyl or heteroarylo-heterocyclyl, preferably five-or six-membered aryl or heteroaryl; preferably, the nitro group on Ar is in the conjugated position with Z in the aromatic system, and more preferably, when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to Z;

L ₁ Selected from: - (CH) ₂ ) _m -，-(CH ₂ ) _m O-，-(CH ₂ ) _m NH-，-(CH ₂ ) _m C(O)-，-(CH ₂ CH ₂ O) _n -，-(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -、-(CH ₂ ) _m -(CH ₂ CH ₂ O) _n -、-O-、-NH-、-S-、-NCH ₃ -、-NH(CH ₂ ) _m -、-C(O)-、

Wherein each m is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, each n is independently 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12, and m and n are not both 0; r is R ₅ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, q is 0, 1, 2, 3 or 4;

L ₂ selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-C(O)NH-CH[(CH ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6;

x is selected from: -C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-，-CH(CH ₃ )O-，-C(CH ₃ ) ₂ O-，-CH ₂ NH-，-CH(CH ₃ )NH-，-C(CH ₃ ) ₂ NH-, -S-, -S (O) -or-S (O) ₂ -；

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -。

In certain embodiments, in the compound of formula I, L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain embodiments, in the compound of formula I, L ₂ is-C (O) NH-CH [ (CH) ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-, wherein d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6.

In certain embodiments, the compounds of formula I have the structure of formula Ia,

Wherein: r is R ₁ 、R ₂ 、R ₃ 、r、L ₁ 、L ₂ X, Z are defined herein.

In certain embodiments, the compounds of formula I have the structure of formula Ib,

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₁ Is that

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₂ Is that

Wherein R is ₄ And p is as defined herein.

Wherein R is ₄ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, p is 0, 1, 2, 3 or 4; further preferably, R ₄ Is hydrogen, fluorine, chlorine, bromine, iodine, nitro, methyl, ethyl, methoxy or ethoxy, more preferably p is 0 or 1.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₄ Is hydrogen.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₄ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₄ Is nitro.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₄ Is methyl or ethyl.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₄ Methoxy or ethoxy.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, p is 0.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, p is 1.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, p is 2.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, p is 3.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, p is 4.

In certain embodiments, in the compounds of formula I, ar is a phenyl ring.

In certain embodiments, in the compounds of formula I, formula Ia, or formula Ib, the nitro group on Ar is para or ortho to Z.

In certain embodiments, in the compounds of formula I, formula Ia, or formula Ib, the nitro group on Ar is para to Z.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is hydrogen.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Methoxy or ethoxy.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is propoxy.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is methyl or ethyl.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₃ Is n-propyl or isopropyl.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 0 or 1.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 0.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 1.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 2.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 3.

In certain embodiments, in a compound of formula I, formula Ia, or formula Ib, r is 4.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ Is- (CH) ₂ ) _m -wherein m is as defined herein.

In certain embodiments, each m is independently 1 or 2 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each m is independently 3 or 4 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each m is independently 5 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each m is independently 6 or 7 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each m is independently 8 or 9 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each m is independently 10, 11, or 12 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ Is that

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ Is- (CH) ₂ ) _m O-，-(CH ₂ ) _m NH-or- (CH) ₂ ) _m C (O) -, wherein each m is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ Is- (CH) ₂ CH ₂ O) _n -，-(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -or- (CH) ₂ ) _m -(CH ₂ CH ₂ O) _n -wherein each m and n is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₁ is-O-, -NH-, -S-, -NCH ₃ -、-NH(CH ₂ ) _m -or-C (O) -, wherein m is defined as described herein.

Wherein R is ₅ And q is as defined herein.

In certain embodiments, each n in the compounds of formula I, formula Ia, or formula Ib is independently 1, 2, or 3.

In certain embodiments, each n in the compounds of formula I, formula Ia, or formula Ib is independently 4, 5, or 6.

In certain embodiments, each n in the compounds of formula I, formula Ia, or formula Ib is independently 7, 8, or 9.

In certain embodiments, each n in the compounds of formula I, formula Ia, or formula Ib is independently 10, 11, or 12.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is hydrogen.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is methyl or ethyl.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is n-propyl, isopropyl or n-butyl.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is nitro.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Methoxy or ethoxy.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, R ₅ Is propoxy.

In certain embodiments, in the compounds of formula I, formula Ia, or formula Ib, q is 1 or 2.

In certain embodiments, in the compounds of formula I, formula Ia, or formula Ib, q is 3 or 4.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; further preferably, each i is independently 1, 2 or 3.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-C (O) NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, formula I, formula IIIa or Ib, L ₂ is-NH (CH) ₂ ) _i C (O) -, wherein i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-C (O) (CH ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-NH (CH) ₂ ) _i O-, wherein i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-NH (CH) ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-or-N (CH) ₃ )(CH ₂ ) _i N(CH ₃ ) -wherein each i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-O (CH) ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-or-O (CH) ₂ ) _i S-, wherein each i is as defined herein.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ is-O-, -NH-, -S-, -S (O) -, -S (O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-or-C (O) -.

In certain embodiments, in a compound of formula I, formula Ia or formula Ib, L ₂ Is empty.

In certain embodiments, each I is independently 1, 2, or 3 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each I is independently 4, 5, or 6 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each I is independently 7, 8, or 9 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, each I is independently 10, 11, or 12 in a compound of formula I, formula Ia, or formula Ib.

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-C (O) -.

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-O-, -NH-or-NCH ₃ -。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-CH ₂ O-，-CH(CH ₃ ) O-, or-C (CH) ₃ ) ₂ O-。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-CH ₂ NH-，-CH(CH ₃ ) NH-or-C (CH) ₃ ) ₂ NH-。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, X is-S-, -S (O) -or-S (O) ₂ -。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, Z is-CH ₂ -。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, Z is-CH (CH) ₃ )-。

In certain embodiments, in the compounds of formula I, formula Ia or formula Ib, Z is-C (CH ₃ ) ₂ -。

In certain embodiments, the compound of formula I, formula Ia, or formula Ib is selected from:

the application also relates to application of the compound shown in the formula I or the formula Ia or the formula Ib or salt thereof in preparing an antibody coupling medicament.

The application also relates to a compound shown in a formula II or a salt thereof,

wherein:

R ₁ is that

r is 0, 1, 2, 3 or 4;

L ₁ selected from- (CH) ₂ ) _m -，-(CH ₂ ) _m O-，-(CH ₂ ) _m NH-，-(CH ₂ ) _m C(O)-，-(CH ₂ CH ₂ O) _n -，-(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -、-(CH ₂ ) _m -(CH ₂ CH ₂ O) _n -、-(CH ₂ CH ₂ O) _n -、-O-、-NH-、-S-、-NCH ₃ -、-NH(CH ₂ ) _m -、-C(O)-、

L ₂ selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-C(O)NH-CH[(CH ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-and-C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6;

x is selected from: -C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-，-CH(CH ₃ )O-，-C(CH ₃ ) ₂ O-，-CH ₂ NH-，-CH(CH ₃ )NH-，-C(CH ₃ ) ₂ NH-, -S-, -S (O) -and-S (O) ₂ -；

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -；

t is 0 or 1;

b is an active compound selected from a drug, cytotoxin, detection reagent, diagnostic reagent or targeting vector; preferably, B is a cytotoxin, an anti-tumor drug, an antiviral drug, an anti-infective drug or an immunomodulator drug; further preferred, B is a cytotoxin, such as a tubulin inhibitor, a DNA alkylating agent, a DNA intercalating agent, an enzyme inhibitor, an antimetabolite, a peptide or a nucleotide;

preferably, B is coupled to the carbonyl (i.e. site-wise) or Z group through an N atom or O atom in the active compound molecule.

In certain embodiments, in the compound of formula II, L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain embodiments, in the compound of formula II, L ₂ is-C (O) NH-CH [ (CH) ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-, wherein d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6.

In certain embodiments, the compound of formula II has the structure of formula IIa,

wherein: r is R ₁ 、R ₃ 、r、L ₁ 、L ₂ X, Z, B, t are defined herein;

in certain embodiments, the compound of formula II has a structure of formula IIb,

wherein: l (L) ₁ 、L ₂ The definition of B is as described in the application.

In certain embodiments, in a compound of formula II or formula IIa, R ₁ Is that

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is hydrogen.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Methoxy or ethoxy.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is propoxy.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is methyl or ethyl.

In certain embodiments, in a compound of formula II or formula IIa, R ₃ Is n-propyl or isopropyl.

In certain embodiments, in a compound of formula II or formula IIa, r is 0 or 1.

In certain embodiments, in a compound of formula II or formula IIa, r is 0.

In certain embodiments, in a compound of formula II or formula IIa, r is 1.

In certain embodiments, in a compound of formula II or formula IIa, r is 2.

In certain embodiments, in a compound of formula II or formula IIa, r is 3.

In certain embodiments, in a compound of formula II or formula IIa, r is 4.

In certain embodiments, in the compounds of formula II, ar is a phenyl ring.

In certain embodiments, in the compounds of formula II, the nitro group on Ar is para or ortho to Z.

In certain embodiments, in the compound of formula II, the nitro group on Ar is para to Z.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ Is- (CH) ₂ ) _m -wherein m is as defined herein.

In certain embodiments, each m is independently 1 or 2 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each m is independently 3 or 4 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each m is independently 5 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each m is independently 6 or 7 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each m is independently 8 or 9 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each m in a compound of formula II, formula IIa, or formula IIb is independently 10, 11, or 12.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ Is that

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ Is- (CH) ₂ ) _m O-，-(CH ₂ ) _m NH-or- (CH) ₂ ) _m C (O) -, wherein each m is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ Is- (CH) ₂ CH ₂ O) _n -，-(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -or- (CH) ₂ ) _m -(CH ₂ CH ₂ O) _n -wherein each m and n is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₁ is-O-, -NH-, -S-, -NCH ₃ -、-NH(CH ₂ ) _m -or-C (O) -, wherein m is defined as described herein.

Wherein R is ₅ And q is as defined herein.

In certain embodiments, each n in the compounds of formula II, formula IIa, or formula IIb is independently 1, 2, or 3.

In certain embodiments, each n in the compounds of formula II, formula IIa, or formula IIb is independently 4, 5, or 6.

In certain embodiments, each n in the compounds of formula II, formula IIa, or formula IIb is independently 7, 8, or 9.

In certain embodiments, each n in the compounds of formula II, formula IIa, or formula IIb is independently 10, 11, or 12.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is hydrogen.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is methyl or ethyl.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is n-propyl, isopropyl or n-butyl.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is nitro.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Methoxy or ethoxy.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, R ₅ Is propoxy.

In certain embodiments, in the compounds of formula II, formula IIa, or formula IIb, q is 1 or 2.

In certain embodiments, in the compounds of formula II, formula IIa, or formula IIb, q is 3 or 4.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; further preferably i is 1, 2 or 3.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-C (O) NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-NH (CH) ₂ ) _i C (O) -, wherein i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-C (O) (CH ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-NH (CH) ₂ ) _i O-, wherein i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-NH (CH) ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-or-N (CH) ₃ )(CH ₂ ) _i N(CH ₃ ) -wherein each i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-O (CH) ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-or-O (CH) ₂ ) _i S-, wherein each i is as defined herein.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ is-O-, -NH-, -S-, -S (O) -, -S (O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-or-C (O) -.

In certain embodiments, in a compound of formula II, formula IIa or formula IIb, L ₂ Is empty.

In certain embodiments, each i is independently 1, 2, or 3 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each i is independently 4, 5, or 6 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each i is independently 7, 8, or 9 in a compound of formula II, formula IIa, or formula IIb.

In certain embodiments, each i in a compound of formula II, formula IIa, or formula IIb is independently 10, 11, or 12.

In certain embodiments, in the compounds of formula II or formula IIa, X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

In certain embodiments, in a compound of formula II or formula IIa, X is-C (O) -.

In certain embodiments, in the compounds of formula II or formula IIa, X is-O-, -NH-or-NCH ₃ -。

In certain embodiments, in a compound of formula II or formula IIa, X is-CH ₂ O-，-CH(CH ₃ ) O-, or-C (CH) ₃ ) ₂ O-。

In certain embodiments, in a compound of formula II or formula IIa, X is-CH ₂ NH-，-CH(CH ₃ ) NH-or-C (CH) ₃ ) ₂ NH-。

In certain embodiments, in a compound of formula II or formula IIa, X is-S-, -S (O) -or-S (O) ₂ -。

In certain embodiments, in the compounds of formula II or formula IIa, Z is-CH ₂ -。

In certain embodiments, in the compound of formula II or formula IIa, Z is-CH (CH ₃ )-。

In certain embodiments, in the compound of formula II or formula IIa, Z is-C (CH ₃ ) ₂ -。

In certain embodiments, in a compound of formula II or formula IIa, t is 1.

In certain embodiments, in the compounds of formula II, formula IIa, or formula IIb, B is auristatin, monomethyl auristatin E (MMAE), maytansine (maytansine) or a derivative thereof (e.g., maytansinoids, DM1, DM3, DM 4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins, and derivatives thereof.

In certain embodiments, in the compounds of formula II, formula IIa, or formula IIb, B is monomethyl auristatin E (MMAE).

In certain embodiments, the compounds of formula II, formula IIa, or formula IIb have structures shown in IIIa and IIIb:

wherein B is coupled to a carbonyl group (i.e. site-x) or methylene group via an N atom or O atom in the active compound molecule, B, t is as defined herein.

In certain embodiments, the compound of formula II, formula IIa, or formula IIb is selected from:

the application also relates to application of the compound shown in the formula II, the formula IIa or the formula IIb or salt thereof in preparation of antibody coupling medicaments.

The application also relates to a compound shown in a formula IV or a salt thereof,

wherein: ar is aryl, heteroaryl, arylo-heterocyclyl or heteroarylo-heterocyclyl, preferably five-or six-membered aryl or heteroaryl; preferably, the nitro group on Ar is in the conjugated position with Z in the aromatic system, and more preferably, when Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar is para or ortho to Z;

r is 0, 1, 2, 3 or 4;

L ₂ selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-C(O)NH-CH[(CH ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 23, 4, 5 or 6;

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -；

t is 0 or 1;

a is a targeting compound selected from the group consisting of a protein, an antibody, a polypeptide, an enzyme, and a small molecule; preferably, a is coupled to site # through an S atom in the targeting compound molecule;

preferably, B is coupled to the carbonyl (i.e. site-wise) or Z group through an N atom or O atom in the active compound molecule;

a is a number between 0.5 and 8.5, for example a number between 0.8 and 5, a number between 1 and 4, a number between 2 and 6, a number between 3 and 7, a number between 4 and 8, a number between 3.5 and 8.5, a number between 3.5 and 4.5, or a number between 6.5 and 8.5, preferably a is about 4, 5, 6, 7 or 8.

In certain embodiments, in the compound of formula IV, L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

In certain embodiments, in the compound of formula IV, L ₂ is-C (O) NH-CH [ (CH) ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-, wherein d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6.

In certain embodiments, the compound of formula IV has a structure of formula IVa,

wherein L is ₁ 、L ₂ 、X、R ₃ R, Z, A, B, a, t are defined herein.

In certain embodiments, the compound of formula IV has a structure of formula IVb,

wherein L is ₁ 、L ₂ A, B, a, t are defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

In certain embodiments, in a compound of formula IV or formula IVa, R ₃ Is hydrogen.

In certain embodiments, in a compound of formula IV or formula IVa, R ₃ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, formula IV or formula IVaIn the compound, R ₃ Methoxy or ethoxy.

In certain embodiments, in a compound of formula IV or formula IVa, R ₃ Is propoxy.

In certain embodiments, in a compound of formula IV or formula IVa, R ₃ Is methyl or ethyl.

In certain embodiments, in a compound of formula IV or formula IVa, R ₃ Is n-propyl or isopropyl.

In certain embodiments, in a compound of formula IV or formula IVa, r is 0 or 1.

In certain embodiments, in a compound of formula IV or formula IVa, r is 0.

In certain embodiments, in a compound of formula IV or formula IVa, r is 1.

In certain embodiments, in a compound of formula IV or formula IVa, r is 2.

In certain embodiments, in a compound of formula IV or formula IVa, r is 3.

In certain embodiments, in a compound of formula IV or formula IVa, r is 4.

In certain embodiments, in the compounds of formula IV, ar is a phenyl ring.

In certain embodiments, in the compounds of formula IV, the nitro group on Ar is para or ortho to Z.

In certain embodiments, the nitro group on Ar is para to Z in the compounds of formula IV.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ Is- (CH) ₂ ) _m -wherein m is as defined herein.

In certain embodiments, each m is independently 1 or 2 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, each m is independently 3 or 4.

In certain embodiments, each m is independently 5 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, each m is independently 6 or 7.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, each m is independently 8 or 9.

In certain embodiments, each m is independently 10, 11, or 12 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ Is that

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ Is- (CH) ₂ ) _m O-，-(CH ₂ ) _m NH-or- (CH) ₂ ) _m C (O) -, wherein each m is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ Is- (CH) ₂ CH ₂ O) _n -，-(CH ₂ CH ₂ O) _n -(CH ₂ ) _m -or- (CH) ₂ ) _m -(CH ₂ CH ₂ O) _n -wherein each m and n is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₁ is-O-, -NH-, -S-, -NCH ₃ -、-NH(CH ₂ ) _m -or-C (O) -, wherein m is defined as described herein.

Wherein R is ₅ And q is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, each n is independently 1, 2, or 3.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, each n is independently 4, 5 or 6.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, each n is independently 7, 8 or 9.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, each n is independently 10, 11 or 12.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is hydrogen.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is fluorine, chlorine, bromine or iodine.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is methyl or ethyl.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is n-propyl, isopropyl or n-butyl.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is nitro.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Methoxy or ethoxy.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, R ₅ Is propoxy.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, q is 1 or 2.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, q is 3 or 4.

In certain embodiments, the compounds of formula IV or formula IVa or formula IVbIn the compound, L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; further preferably i is 1, 2 or 3.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-C (O) NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-NH (CH) ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-NH (CH) ₂ ) _i C (O) -, wherein i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-C (O) (CH ₂ ) _i NH-, wherein i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-NH (CH) ₂ ) _i O-, wherein i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-NH (CH) ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-or-N (CH) ₃ )(CH ₂ ) _i N(CH ₃ ) -wherein each i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-O (CH) ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-or-O (CH) ₂ ) _i S-, wherein each i is as defined herein.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ is-O-, -NH-, S-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-or-C (O) -.

In certain embodiments, in a compound of formula IV or formula IVa or formula IVb, L ₂ Is empty.

In certain embodiments, each i is independently 1, 2, or 3 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, each i is independently 4, 5, or 6 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, each i is independently 7, 8, or 9 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, each i is independently 10, 11, or 12 in a compound of formula IV or formula IVa or formula IVb.

In certain embodiments, in the compounds of formula IV or formula IVa, X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

In certain embodiments, in the compound of formula IV or formula IVa, X is-C (O) -.

In certain embodiments, in the compound of formula IV or formula IVa, X is-O-, -NH-or-NCH ₃ -。

In certain embodiments, in a compound of formula IV or formula IVa, X is-CH ₂ O-，-CH(CH ₃ ) O-, or-C (CH) ₃ ) ₂ O-。

In certain embodiments, in a compound of formula IV or formula IVa, X is-CH ₂ NH-，-CH(CH ₃ ) NH-or-C (CH) ₃ ) ₂ NH-。

In certain embodiments, in the compound of formula IV or formula IVa, X is-S-, -S (O) -or-S (O) ₂ -。

In certain embodiments, in the compound of formula IV or formula IVa, Z is-CH ₂ -。

In certain embodiments, in the compound of formula IV or formula IVa, Z is-CH (CH ₃ )-。

In certain embodiments, formula IV or formulaIn the compound shown in IVa, Z is-C (CH ₃ ) ₂ -。

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, t is 1.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, B is auristatin (MMAE), monomethyl auristatin E (MMAE), maytansine (maytansine) or a derivative thereof (e.g., maytansinoids, DM1, DM3, DM 4), paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, PBD (pyrrolobenzodiazepines) cytotoxins and derivatives thereof.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, B is monomethyl auristatin E (MMAE).

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, a is a monoclonal antibody having a thiol group as a coupling site, or a site-directed mutation or modification of a monoclonal antibody having a thiol group as a coupling site.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, a is selected from: anti-HER 2 humanized monoclonal antibody mil40, trastuzumab (HERCEPTIN), pertuzumab (PERJETA), cetuximab (ERBITUX), panitumumab (vecitux), rituximab (RITUXAN), alemtuzumab (CAMPATH), tiimumab (ZEVALIN), tositumumab (BEXXAR), ofatuzumab (arzero), bevacizumab (AVASTIN), ipilimumab (YERVOY), denomumab (XGEVA), pehmab (keyruda), nivolumab (Opdivo), avelumab (Bavencio), atezolizumab (Tecentriq), durvalumab (Imfinzi), sacitumumab, rovapuzumab, and biological analogs thereof.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, a is an anti-HER 2 humanized monoclonal antibody mil40.

In certain embodiments, in the compounds of formula IV or formula IVa or formula IVb, a is a number between 2 and 7, or a is a number between 3 and 6 or a number between 4 and 5, preferably a is about 4, 5, 6, 7 or 8.

In certain embodiments, the compound of formula IV or formula IVa or formula IVb has a structure of formula IV-1 or formula IV-2,

wherein B, a is defined herein, MAB is a monoclonal antibody, preferably anti-HER 2 humanized monoclonal antibody mil40.

In certain embodiments, the compound of formula IV or formula IVa or formula IVb is selected from:

wherein MAB is a monoclonal antibody, preferably an anti-HER 2 humanized monoclonal antibody mil40, a is defined herein, preferably about 4.

The present application also relates to pharmaceutical compositions comprising at least one compound described by formula IV, or a salt, solvate thereof, and one or more pharmaceutically acceptable carriers or excipients.

The application also relates to the use of a compound of formula IV or a salt thereof in the manufacture of a medicament for treating or lessening the severity of a disease or disorder.

The present application also relates to methods of treating or lessening the severity of a disease or disorder comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula IV or a salt thereof.

The present application also relates to compounds of formula IV or salts thereof for use in treating or lessening the severity of a disease or disorder.

The present application also relates to methods of diagnosing, preventing or treating a disease or condition comprising administering to a patient in need of such treatment a therapeutically effective amount of a compound of formula IV or a salt thereof.

In certain embodiments, the disease or disorder is selected from the group consisting of a tumor, an infectious disease, a hematological disease, a metabolic disease, and inflammation.

In certain embodiments, the tumor is selected from the group consisting of cancer, lymphoma, lymphoid tumor, blastoma, sarcoma, and leukemia.

In certain embodiments, the cancer is selected from: breast cancer (e.g., HER2 positive breast cancer); squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; peritoneal cancer; liver cancer; stomach cancer; gastrointestinal cancer; membranous adenocarcinoma; glioblastoma; cervical cancer; ovarian cancer; liver cancer; cancer of the shoulders; urethral cancer; hepatoma; breast cancer; intestinal cancer; colon cancer; rectal cancer; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; renal cancer or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma; multiple myeloma and B-cell lymphoma; brain cancer; gallbladder cancer; esophageal cancer; bile duct cancer; head and neck cancer and related metastases.

Definition of the definition

As used herein, the term "antibody" is a common immunoglobulin, a Y-shaped protein that the immune system uses to recognize and neutralize foreign objects (e.g., bacteria and viruses). Antibodies can specifically recognize unique portions of a foreign target (referred to as antigens) because each tip of a Y-protein antibody contains a site that can specifically recognize an antigen, and after binding to a specific antigen, antibodies can mediate a variety of related biological effects. Antibodies consist of two identical heavy chains and two identical light chains, each chain being linked by disulfide bonds formed by the thiol groups in the half-size amino acid residues. A "monoclonal antibody" is a monospecific antibody, all of which are composed of the same immune cells, all of which are clones of the sole parent cell, and thus all of which are identical.

As used herein, the term "cytotoxin" refers to molecules that are toxic to cancer cells after release from the cells. Toxins of particular interest in this application include methyl auristatin E (MMAE), auristatin, maytansinoids or derivatives thereof (e.g., maytansinoids, DM1, DM3, DM 4), calicheamicin, carcinomycin, doxorubicin, camptothecins or PBD cytotoxins and derivatives thereof.

As used herein, the term "linker" is a molecule having two reactive ends, one of which can be coupled to an antibody and the other end for coupling to an active compound, such as a cytotoxin. The antibody-conjugated end of the linker is typically a site that can be coupled through the thiol or lysine amine group of a cysteine on the antibody, and the conjugated end of the toxin of the linker is typically an active site that can be coupled through a thiol, amino, carboxyl or hydroxyl group on the toxin molecule, and when the term linker is used to describe a linker in conjugated form, it may no longer include one or two reactive end reaction sites (e.g., a leaving group for a thiol-reactive group, a leaving group for an amine-reactive group) as the linker has reacted with one or both of the antibody and the cytotoxin to form a covalent bond.

As used herein, the term "antibody-conjugated drug" or "ADC" is a product formed by coupling a multi-molecule (typically 1-8) cytotoxins to an antibody molecule, each via a linker. An antibody conjugated to one or more cytotoxins. Antibodies are typically monoclonal antibodies that are selective for specific antigens of cancer.

As used herein, the term "about" is understood to be within +/-20%, +/-18%, +/-15%, +/-12%, +/-10%, +/-9%, +/-8%, +/-7%, +/-6%, +/-5%, +/-4%, +/-3%, +/-2%, +/-1%, +/-0.5%, +/-0.4%, +/-0.3%, +/-0.2%, and +/-0.1%. Unless otherwise apparent from the context, all numbers provided herein are modified by the term "about".

Tumor disease types of interest for the antibody conjugated drugs described herein include, but are not limited to, cancer, breast cancer, lymphoma, lymphoid tumor, blastoma, sarcoma, and leukemia. More specific examples of such cancers include squamous cell carcinoma (e.g., epithelial squamous cell carcinoma); lung cancer, including small cell lung cancer, non-small cell lung cancer, adenocarcinoma of the lung, and squamous carcinoma of the lung; peritoneal cancer; liver cancer; gastric or gastric cancer, including gastrointestinal cancer; membranous adenocarcinoma; glioblastoma; cervical cancer; ovarian cancer; liver cancer; cancer of the shoulders; urethral cancer; hepatoma; breast cancer, including, for example, HER2 positive breast cancer; colon cancer; rectal cancer; colorectal cancer; endometrial or uterine cancers; salivary gland cancer; renal cancer or renal cancer; prostate cancer; vulvar cancer; thyroid cancer; liver cancer; anal cancer; penile cancer; melanoma; myeloma and B-cell lymphoma; brain cancer; head and neck cancer and related metastases.

The term "salt" refers to salts that retain the biological effectiveness and properties of a compound, which are undesirable for use in medicine for biological or other reasons. In many cases, the compounds disclosed herein are capable of forming acid and/or base salts by virtue of the presence of amino and/or completion groups or the like. Pharmaceutically acceptable acid addition salts may be composed of inorganic and organic acids. Inorganic acids from which salts may be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts may be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Pharmaceutically acceptable base addition salts may be composed of inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are the ammonium, potassium, sodium, calcium and magnesium salts. Organic bases from which salts can be derived include, for example, primary, secondary and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, particularly such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine and ethanolamine. Many such salts are known in the art, as described in W087/05297, johnston et al, published on 9.11/1987 (incorporated herein by reference in its entirety).

As used herein, C _l -C ₆ "1 and 6" in (a) refers to the number of carbon atoms in a given group. That is, the group may contain numbers of carbon atoms (inclusive) from "1" to "6". Thus, for example, "C ₁ -C ₄ Alkyl "or" C _1-4 Alkyl "refers to all alkyl groups having 1 to 4 carbon atomsI.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-。

The term "halogen" as used herein refers to any one of the radioactively stabilized atoms in column 7 of the periodic table of elements, e.g., fluorine, chlorine, bromine, iodine, etc., with chlorine being the most preferred.

As used herein, the term "aryl" refers to an unsaturated aromatic carbocyclyl of 5-14 carbon atoms having one single ring or two or more fused rings of a conjugated pi-electron system. The "aryl" group preferably has 5 to 10, 5 to 8 or 5 to 6 carbon atoms. Typical examples of "aryl" include, but are not limited to, phenyl, naphthyl, anthracenyl, and the like.

Herein, the term "heteroaryl" refers to an aryl group as defined herein wherein at least one ring member is a heteroatom selected from nitrogen, oxygen or sulfur. The "heteroaryl" preferably has 5-10, 5-8 or 5-6 ring members. Typical examples of "heteroaryl" include, but are not limited to, furyl, imidazolyl, thienyl, triazolyl, indolyl, tetrazolyl, pyridyl, pteridinyl, pyrimidinyl, triazolyl, quinolinyl, isoquinolinyl, quinazolinyl, quinoxalinyl, and the like.

Herein, the term "arylheterocyclyl" refers to a cyclic group having two or more fused rings, wherein two or more carbons are common to two adjacent rings, wherein at least one ring is aryl as defined herein, and at least one ring is heterocyclyl.

Herein, the term "heteroarylheterocyclyl" refers to a cyclic group having two or more fused rings, wherein two or more carbons are common to two adjacent rings, wherein at least one ring is heteroaryl as defined herein, and at least one ring is heterocyclyl.

Herein, the term "heterocyclyl" refers to a saturated or partially unsaturated cyclic hydrocarbon group having 3 to 12 ring members of a single ring or a bicyclic or multiple condensed rings (including condensed, bridged and spiro rings), and at least one ring member is a heteroatom selected from nitrogen, oxygen or sulfur. The "heterocyclyl" preferably has 3-10,3-8,5-8,3-6 or 5-6 ring members. Typical examples of "heterocyclyl" include, but are not limited to, tetrahydrofuranyl, tetrahydrothienyl, pyrrolidinyl, piperazinyl, thiazinyl, piperidinyl, morpholinyl, and the like.

Abbreviations/abbreviations

ADC (anti-drug conjugate): an antibody-conjugated drug;

DAR (Drug to antibody ratio): drug/antibody molar ratio;

DCM (Dichloromethane): dichloromethane;

DIPEA (N, N-dioropropylethynamine): diisopropylethylamine;

DMAC (Dimethylacetamide): n, N-dimethylacetamide;

DMF (N, N-dimethyl formamide): n, N-dimethylformamide;

DMSO (Dimethyl Sulphoxide): dimethyl sulfoxide;

EA (Ethyl acetate): ethyl acetate;

EDCI (1- (3-dimethylmineopyyl) -3-ethylcarbodiimide Hydrochloride): 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride;

HOBt (1-hydroxybenzyltriazole): 1-hydroxybenzotriazole;

HER2 (Human epidermal growth factor receptor 2): human epidermal growth factor receptor 2;

MAB (Monoclonal Antibody): monoclonal antibodies

MMAE (Monomethyl auristatin E): monomethyl auristatin E;

NAC (N-Acetyl-L-cysteine): n-acetylcysteine;

TCEP (Tris (2-carboxynyl) phosphine): tris (2-carboxyethyl) phosphine;

THF (Tetrahydrofuran): tetrahydrofuran;

tris (Tris (hydroxymethyl) aminomethane): tris (hydroxymethyl) aminomethane;

when the names of the compounds used herein are inconsistent with the chemical structural formula, the chemical structural formula is based.

The pharmaceutical compositions as described herein comprise a compound of formula iv herein, or a salt, solvate thereof, in association with a conventional pharmaceutically acceptable carrier or excipient. The pharmaceutical composition may be administered by, for example, oral or parenteral routes, such as intravenous injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, and the like.

As used herein, the term "effective amount" refers to an amount sufficient to achieve a desired therapeutic effect, e.g., an amount that achieves relief of symptoms associated with the disease to be treated.

It is further noted that the dosage and method of use of the compounds of the present application will depend upon a number of factors including the age, weight, sex, natural health, nutritional status of the patient, the strength of activity of the compound, the time of administration, the metabolic rate, the severity of the condition and the subjective judgment of the treating physician. The preferred dosage is between 0.01 and 100mg/kg body weight/day.

In certain embodiments, the synthetic routes for ADCs described herein are as follows:

wherein L is ₁ And a is as defined herein.

Advantageous technical effects of the present application

The antibody coupled drug provided by the application can be selectively cracked to release toxin under the anoxic condition, but the drug release process can not smoothly occur under the normoxic condition. The process of drug release shows high selectivity to hypoxia factors at both the enzyme level and the cellular level.

Drawings

FIG. 1 shows the amount of MMAE released by the L1-MMAE conjugates under nitroreductase at various time points under hypoxic and normoxic conditions;

FIG. 2 shows the amount of MMAE released by the L2-MMAE conjugate under nitroreductase at various time points under hypoxic and normoxic conditions;

FIG. 3 shows the amount of MMAE released in breast cancer cell line HCC1954 at various time points ADC-I and ADC-II under hypoxic and normoxic conditions;

FIG. 4 shows the amount of MMAE released in breast cancer cell line BT-474 by ADC-I and ADC-II at various time points under hypoxic and normoxic conditions;

FIG. 5 shows the amount of MMAE released in gastric cell line NCI-N87 at various time points for ADC-I and ADC-II under hypoxic and normoxic conditions.

Detailed Description

Embodiments of the present application will be described in detail below with reference to examples, but it will be understood by those skilled in the art that the following examples are only for illustration of the present application and should not be construed as limiting the scope of the present application. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

Example 1: preparation of linkers L1 and L2

1) Preparation of intermediate S3:

maleic anhydride (S1, 4.86g,49.55 mmol) was added to a solution of 6-aminocaproic acid (S2, 5.0g,38.11 mmol) in acetic acid (AcOH, 150 mL) and the resulting mixture was stirred at 120℃for 6 hours under reflux. After cooling to room temperature, the reaction mixture was poured into water, extracted with ethyl acetate (EA, 3X 20 mL), and the combined organic layers were washed with saturated brine and with anhydrous Na ₂ SO ₄ Drying and evaporating under reduced pressure to obtain crude product, and purifying the crude product by column chromatography to obtain target product S3 as white solid powder (5.92 g,74% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ11.98(br，1H)，7.01(s，2H)，3.39(m，2H)，2.17(t，J＝7.4Hz，2H)，1.51-1.44(m，2H)，1.24-1.17(m，2H)。MS(ESI)m/z：210.0[M-H] ^- 。

2) Preparation of intermediate S4:

s3 (4.66 g,22.0 mmol), 2,4, 6-trimethylpyridine (11.6 mL,88.0 mmol) and N-hydroxysuccinimide (NHS, 5.08g,44.0 mmol) were added to a solution of tetrahydrofuran (THF, 100 mL), cooled to 0deg.C, and trifluoroacetic anhydride (6.12 mL,44.0 mmol) was added dropwise over 30 minutes. The resulting mixture was stirred at room temperature for 1 hour, then concentrated under reduced pressure to remove the solvent, and the resulting residue was dissolved again in EA (200 mL). The obtained EA solution was washed 3 times with HCl solution (1 mol/L) and saturated brine, and the obtained organic phase was taken up in anhydrous Na ₂ SO ₄ Drying, concentrating under reduced pressure to remove EA to obtain a crude product, and purifying the crude product by column chromatography to obtain a target product S4 which is colorless oily liquid and is converted into white solid (4.71 g,69% yield) when the target product S4 is placed at a low temperature. ¹ H-NMR(400MHz，CDCl ₃ )：δ6.69(s，2H)，3.53(t，J＝7.3Hz，2H)，2.84(s，4H)，2.61(t，J＝7.4Hz，2H)，1.78(m，2H)，1.63(m，2H)，1.45-1.37(m，2H)。MS(ESI)m/z：309.4[M+H] ⁺ ；331.2[M+Na] ⁺ 。

3) Preparation of intermediate S7:

s5 (7.86 g,50.0 mmol) and S1 (4.90 g,50.0 mmol) were added to AcOH solution (100 mL), reacted at 120℃for 6 hours with stirring, cooled to room temperature, the reaction solution was poured into water, extracted with EA (3X 50 mL), the organic layers were combined, washed with saturated brine and then dried over anhydrous Na ₂ SO ₄ Drying and removal of the solvent under reduced pressure gave the crude product as a white solid (9.96 g,84% yield) which was used directly in the next step without purification. The above white solid (13.1 g,55 mmol), 2,4, 6-trimethylpyridine (26.4 mL,200 mmol) and NHS (23.0 g,200 mmol) were dissolved together in THF (250 mL), cooled to 0℃after all dissolution, and trifluoroacetic anhydride (27.8 mL,200 mmol) was added dropwise over 45 minutes,stirring was continued for 18 hours at room temperature, then chloroform (300 mL) and hydrochloric acid solution (1 mol/L,250 mL) were added to the reaction mixture and extraction was performed with dichloromethane (DCM, 3X 100 mL). The extracted organic layer was washed twice with hydrochloric acid (1N, 250 mL) and with Na ₂ SO ₄ After drying, DCM was removed by concentration under reduced pressure to give the crude product as a yellow solid. The yellow solid was slurried with diethyl ether (3X 200 mL) to give the desired product S7 as a white powdery solid (15 g,90% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ6.71(s，1H)，3.39(d，J＝7.2Hz，2H)，2.82(d，J＝7.3Hz，4H)，2.58(m，1H)，2.15(m，2H)，1.80(m，2H)，1.56(m，1H)，1.54(m，2H)，1.06(m，2H)。MS(ESI)m/z：352.6[M+NH ₄ ] ⁺ ；357.4[M+Na] ⁺ 。

4) Preparation of intermediate S9:

to a solution of S8 (1.0 g,5.52 mmol) in methanol (MeOH, 30 mL) was added concentrated H ₂ SO ₄ (2 mL) and refluxed with stirring at 75deg.C for 18 hours, then the solvent was evaporated under reduced pressure to give the crude product, which was further purified by column chromatography to give the desired product S9 as a white solid powder (920 mg,75% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.00(d，J＝8.4Hz，1H)，7.66(d，J＝1.4Hz，1H)，7.61(dd，J＝8.4Hz，1H)，3.84(s，3H)，2.45(s，3H)。MS(ESI)m/z：196.0[M+H] ⁺ ；213.0[M+NH ₄ ] ⁺ 。

5) Preparation of intermediate S10:

s9 (1.0 g,5.1 mmol), AIBN (catalytic amount, 50 mg) and N-bromosuccinimide (NBS, 1.0g,5.6 mmol) are dissolved in benzene (50 mL) and heated under reflux with stirring for 18 hours. After the reaction is finished, evaporating the solvent under reduced pressure to obtain a crude product, and further carrying out column chromatographyPurification gave the desired product S10 as a white solid powder (1.14 g,81% yield). ¹ H-NMR(400MHz，CDCl ₃ )：δ7.91(d，J＝8.4Hz，1H)，7.75(d，J＝2.0Hz，1H)，7.64(dd，J＝8.4Hz，1H)，5.50(s，2H)，3.94(s，1H)。MS(ESI)m/z：272.9[M+H] ⁺ 。

6) Preparation of intermediate S11:

to a solution of S10 (250 mg,0.91 mmol) in acetone/water (1:1 (v/v), 10 mL) was added Na ₂ CO ₃ (0.48 g,4.55 mmol) and heated to 65℃with stirring for 6 hours, then additional NaOH (0.18 g,5 eq.) was added and the reflux reaction continued for 1 hour. After the reaction was stopped, the acetone was removed by concentration under reduced pressure, and the pH of the solution was adjusted to about 2 with concentrated HCl and extracted multiple times with EA. The organic phases were combined, washed three times with saturated brine and dried over MgSO ₄ Drying is performed. The solvent was distilled off under reduced pressure to give a crude product, which was further purified by column chromatography to give the desired product S11 as a reddish brown flaky solid (140 mg,76% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ7.97(d，J＝8.4Hz，1H)，7.74(d，J＝2.0Hz，1H)，7.65(dd，J＝8.4Hz，1H)，5.59(s，1H)，4.63(d，J＝3.18.4Hz，2H)。MS(ESI)m/z：195.9[M-H] ^- 。

7) Preparation of intermediate S12:

s11 (500 mg,2.53 mmol), EDCI (0.73 g,3.8 mmol), HOBt (0.51 g,3.8 mmol) and DIPEA (0.49 g,3.8 mmol) were dissolved in DMF (15 mL) and the mixture solution was stirred at room temperature for 1 hour, then tert-butyl 2-amino-ethylamino-carboxylate (0.81 g,5.06 mmol) was added, the mixture was continued to be stirred at room temperature for 18 hours, after stopping the reaction, the solvent was removed by concentration under reduced pressure to give a crude product, which was further purified by column chromatography to give the target Product S12 was a yellow gel-like solid (760 mg,89% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.66(t，J＝5.6Hz，1H)，8.02(d，J＝8.4Hz，1H)，7.59(d，J＝8.4Hz，1H)，7.53(s，1H)，6.88(t，J＝11.5Hz，1H)，5.59(t，J＝5.6Hz，1H)，4.62(d，J＝5.6Hz，2H)，3.23(q，J＝6.2Hz，2H)，3.09(q，J＝6.2Hz，2H)，1.39(s，9H)。MS(ESI)m/z：340.2[M+H] ⁺ ；362.2[M+Na] ⁺ 。

8) Preparation of intermediate S13:

s12 (760 mg,2.24 mmol) was dissolved in 2mol/L HCl in EA (3 mL) and stirred at room temperature overnight. After the reaction, the solvent was removed under reduced pressure to give a crude product, which was further purified by column chromatography to give the desired product S13 as a pale yellow solid powder (0.58 g,94% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.94(t，J＝5.6Hz，1H)，8.04(d，J＝8.4Hz，1H)，7.90(br，1H)，7.67(s，1H)，7.62(d，J＝8.4Hz，1H)，5.69(br，1H)，4.63(s，2H)，3.49(q，J＝5.9Hz，2H)，2.95(t，J＝6.7Hz，2H)。MS(ESI)m/z：240.2[M+H] ⁺ ；262.2[M+Na] ⁺ 。

9) Preparation of intermediate S14:

to a solution of S13 (760 mg,2.24 mmol) in DMF (20 mL) was added S4 (1.18 g,3.82 mmol) and DIPEA (0.49 g,3.82 mmol). The mixture was stirred at room temperature overnight, then the solvent was evaporated under reduced pressure to give the crude product, and further purified by column chromatography to give the desired product S14 as a pale yellow glassy solid (1.14 g,81% yieldd). ¹ H-NMR(400MHz，DMSO-d6)：δ8.67(t，J＝5.6Hz，1H)，8.02(d，J＝8.4Hz，1H)，7.87(t，J＝5.4Hz，1H)，7.59(d，J＝9.4Hz，1H)，7.52(s，1H)，7.00(s，2H)，5.61(t，J＝5.7Hz，1H)，4.62(d，J＝5.6Hz，2H)，3.39(q，J＝7.2Hz，2H)，3.22(m，4H)，2.05(t，J＝7.5Hz，2H)，1.48(m，4H)，1.18(m，2H)。MS(ESI)m/z：433.6[M+H] ⁺ ；455.4[M+Na] ⁺ 。

10 Preparation of intermediate S15:

to a solution of S13 (530 mg,2.22 mmol) in DMF (20 mL) were added S7 (815 mg,2.44 mmol) and DIPEA (0.31 g,2.44 mmol). The mixture was stirred at room temperature overnight, then the solvent was evaporated under reduced pressure to give a crude product, which was further purified by column chromatography to give the desired product S15 as a pale yellow glassy solid (660 mg,65% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.65(t，J＝5.6Hz，1H)，8.02(d，1H)，7.77(t，J＝5.6Hz，1H)，7.59(d，J＝9.4Hz，1H)，7.51(s，1H)，7.01(s，2H)，5.59(t，J＝5.7Hz，1H)，4.62(d，J＝5.6Hz，2H)，3.25-3.18(m，6H)，3.22(m，4H)，2.00(tt，J＝3.4Hz，1H)，1.72(d，J＝10.6Hz，2H)，1.62(d，J＝10.6Hz，2H)，1.52(m，1H)，1.26(qd，J＝4.2Hz，2H)，0.89(qd，J＝3.4Hz，2H)。MS(ESI)m/z：459.5[M+H] ⁺ ；481.2[M+Na] ⁺ 。

11 Preparation of linker L1):

to a solution of S14 (1.10 g,2.54 mmol) in DMF (10 mL) was added bis (4-nitrophenyl) carbonate (1.55 mg,5.09 mmol) and DIPEA (0.66 g,5.09 mmol) and stirred overnight at room temperature. After the reaction, the crude product is obtained by concentrating under reduced pressure to remove the solvent, and the crude product is further purified by column chromatography to obtain the target product L1 as pale yellow glassy solid (1.38 g,97% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.75(t，J＝5.3Hz，1H)，8.34(dt，J＝9.2Hz，2H)，8.11(d，J＝8.1Hz，1H)，7.86(t，J＝5.7Hz，1H)，7.76(dd，J＝8.3Hz，1H)，7.71(d，J＝1.7Hz，1H)，7.61(dt，J＝9.2Hz，2H)，7.00(s，2H)，5.44(s，2H)，3.35(t，J＝6.6Hz，2H)，3.24(m，4H)，3.05(t，J＝7.4Hz，2H)，1.47(m，4H)，1.17(m，2H)。MS(ESI)m/z：598.4[M+H] ⁺ ；615.3[M+NH ₄ ] ⁺ 。

12 Preparation of linker L2):

to a solution of S15 (0.46 g,1.0 mmol) in DCM (10 mL) was added 4-nitrophenyl chloroformate (0.40 mg,2 mmol) and pyridine (5 mL) and stirred at room temperature overnight. After the completion of the reaction, the solid residue obtained by removing the solvent by concentration under reduced pressure was further purified by column chromatography to give the objective product L2 as a white solid powder (0.30 g,48% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.75(t,J＝5.2Hz,1H),8.34(d,J＝9.0Hz,2H),8.10(d,J＝8.4Hz,1H),7.77(m,2H),7.70(s,1H),7.60(d,J＝9.0Hz,2H),7.00(s,2H),5.43(s,2H),3.21(m,6H),2.00(tt,J＝12.3Hz,1H),1.72(d,J＝11.2Hz,2H),1.59(d,J＝11.8Hz,2H),1.48(m,1H),1.27(qd,J＝4.3Hz,2H),0.86(qd,J＝3.3Hz,2H)。MS(ESI)m/z：624.4[M+H] ⁺ ；646.4[M+Na] ⁺ 。

Example 2: preparation of linker-MMAE conjugates

1) Preparation of L1-MMAE:

l1 (73.24 mg,0.12 mmol), MMAE (80.0 mg,0.11mmol, from Concortis Biosystems) and HOBt (15.06 mg,0.11 mmol) were dissolved in DMF (6 mL) and DIPEA (28.80 mg,0.22 mmol) was added after all dissolution. The reaction was stirred at room temperature overnight, after the reaction was completed, it was poured into water (20 mL) and extracted with EA (3×20 mL). The EA organic layers were combined, washed with saturated brine and dried over anhydrous Na ₂ SO ₄ After drying, the solvent crude product was removed by concentration under reduced pressure. The crude product was further purified by column chromatography using 80:1 to 10: DCM/MeOH (v/v) of 1Elution was performed to give the target product L1-MMAE as a white solid powder (83 mg,64% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.70(br，1H))，8.44(t，J＝8.9Hz，0.5H)，8.19(d，J＝8.7Hz，0.5H)，8.04(dd，J＝8.4Hz，1H)，7.73-7.84(m，1.5H)，7.73-7.57(m，2.5H)，7.31-7.15(m，5H)，7.00(s，2H)，5.44-5.36(dd，J＝5.0Hz，1H)，5.31-5.12(m，2H)，4.76-4.65(br，1H)，4.54-4.24(m，3H)，4.00(m，2H)，3.79(m，0.5H)，3.58-3.46(m，1.5H)，3.38(m，2H)，3.24-3.12(m，12H)，2.97-2.81(m，4H)，2.41(d，J＝16.0Hz，1H)，2.25(m，1H)，2.14-1.99(m，5H)，1.80-1.74(br，3H)，1.52-1.43(m，5H)，1.29-1.09(m，7H)，1.05-0.94(m，6H)，0.88-0.75(m，18H)。HRMS(ESI)m/z：588.7847[M+2H] ²⁺ ；1198.6376[M+Na] ⁺ 。

2) Preparation of L2-MMAE:

l2 (36.50 mg,0.0585 mmol), MMAE (40.0 mg,0.0557 mmol) and HOBt (8.0 mg,0.0585 mmol) were dissolved in DMF (3 mL) and DIPEA (7.56 mg,0.0585 mmol) was added after all dissolution. The reaction was stirred at room temperature overnight, after the reaction was completed, it was poured into water (15 mL) and extracted with EA (3×30 mL). The EA organic layers were combined, washed with saturated brine and dried over anhydrous Na ₂ SO ₄ After drying, the solvent crude product was removed by concentration under reduced pressure. The crude product was further purified by column chromatography using 80:1 to 10:1 in DCM/MeOH (v/v) to give the desired product L2-MMAE as a white solid powder (40 mg,59% yield). ¹ H-NMR(400MHz，DMSO-d6)：δ8.29-7.98(m,2H),7.62-7.50(m,3H),7.37-7.30(m,4H),7.28-7.23(m,1H),6.69(s,2H),6.64-6.53(m,2H),5.64-5.46(m,0.5H),5.37-5.10(m,1.5H),4.96-4.91(m,1H),4.73-4.61(m,2H),4.23-3.99(m,4H),3.84-3.69(m,1H),3.62-3.28(m,14H),2.97-2.83(m,5H),2.47-2.21(m,3H),2.11-2.02(m,3H),1.89-1.68(m,9H),1.43-1.23(m,9H),1.02-0.67(m,24H)。HRMS(ESI)m/z：601.8398[M+2H] ²⁺ ；1224.6522[M+Na] ⁺ 。

Example 3: preparation of ADCs

1) Preparation of a common buffer salt solution:

buffer saline-1 (buffer-1): dissolving 3.11g of L-histidine in 1L of double distilled water, and regulating the pH value to about 5.50 (+ -0.05) by using medical glacial acetic acid after the L-histidine is completely dissolved; filtering with 0.22 μm filter membrane, sterilizing, bottling, and storing at 4deg.C for short term.

Buffer saline-2 (buffer-2): weighing 6.06g of TRIS base, dissolving 0.93g of EDTA 2Na, and then fixing the volume to 100mL; weighing 7.88g of TRIS and HCl, and 0.93g of EDTA and 2Na, dissolving, and fixing the volume to 100mL; adding TRIS & HCl solution to TRIS & base solution, intermodulation to ph=8.50 (±0.05); filtering with 0.22 μm filter membrane, sterilizing, bottling, and storing at 4deg.C for short term.

Buffer saline-3 (buffer-3): 1.715mL of medical glacial acetic acid is measured by a pipette, dissolved in 200mL of secondary distilled water, fully and uniformly mixed, filtered and sterilized by a filter membrane with the thickness of 0.22 mu m, bottled and stored at the temperature of 4 ℃ for a short period of time for standby.

2) Antibody coupling reaction:

(1) replacement of pharmaceutically acceptable antibodies: the anti-HER 2 humanized monoclonal antibody mil40 (purchased from Zhejiang Zhengpharmaceutical Co., ltd.) is used for coupling, and is a biological imitation of herceptin, and the initial preparation solution contains 0.616mg/mL histidine hydrochloride (monohydrate), 0.364mg/mL L-histidine, 22.727mg/mL trehalose, 100mg/mL Tween-20 and other pharmaceutical excipients, and in order to remove the interference of the excipients, the freeze-thawing antibody stock solution is firstly placed in room temperature to be slowly melted, and is replaced into a buffer solution-1 through a G25 dextran gel column. After completion of the displacement, concentration was carried out by ultrafiltration centrifugation (final concentration > 5 mg/mL), and the concentration was measured by an ultraviolet spectrophotometer.

(2) Preparation of coupling reaction liquid: according to the amount of the desired conjugated antibody (1 eq), the buffer-1 solution of the antibody was accurately removed using a pipette, and a certain amount of buffer-1 was added so that the antibody concentration was about 10mg/mL. The pH was adjusted to around 6-8 using buffer-2 and transferred to a clean reaction vial with a pipette.

(3) Reduction of antibodies: the reaction solution (100 rpm) in the vial was slowly stirred, 2 to 5eq of 2.87mg/mL TCEP. HCl solution was added, and after the addition was completed, the reaction solution was slowly stirred at room temperature and reacted for 60 to 180 minutes.

(4) Coupling of antibodies: calculating the volume of the organic solvent (DMAC or DMSO) to be added so as to account for 5-15% of the total volume; the mass of the small molecule load (linker-MMAE conjugate) that needs to be added is calculated at the same time, typically small molecule load needs to be slightly excessive (typically 8 eq), and the concentration of loaded organic solvent that needs to be added is calculated. After the loaded solution was precisely prepared, it was slowly added dropwise to the reduced antibody reaction solution. And continuously stirring slowly at room temperature, and reacting for 0.5-5 h according to specific coupling conditions.

(5) Termination of the reaction: after the reaction solution reached a predetermined coupling time, an excessive amount of a water-soluble small molecule N-acetylcysteine solution (1.63 mg/mL) containing a reducing thiol group was added, and the reaction was continued with slow stirring for 30 minutes.

(6) Primary purification of the product: after the end of the coupling termination reaction, adding buffer-3 to adjust back the pH of the reaction solution to about 5.50; filtering the obtained reaction liquid, performing primary purification by adopting a G25 sephadex column, collecting the effluent of the components in the front section (about 80%), performing ultrafiltration concentration again, performing sterile filtration, and performing sample split charging; and (3) storing other products at-80 ℃ for standby except that part of the reserved samples for product analysis are stored at 4 ℃ for a short period of time.

By adopting the method, L1-MMAE and L2-MMAE are respectively coupled to an anti-HER 2 humanized monoclonal antibody mil40 antibody to respectively prepare corresponding ADC-I and ADC-II:

ADC-I：

wherein: MAB is an antibody, a is about 4.

ADC-II：

/>

Wherein: MAB is an antibody, a is about 4.

The ADCs prepared in example 3 and their associated mass information are as follows:

example 4: enzymatic hydrolysis drug release performance and hypoxia selectivity evaluation of L1-MMEA conjugate

Following the procedure described in the relevant literature (bioconjug.chem.2006, 17, 831-840.) to a solution of N-acetyl-L-cysteine in PBS (NAC, 900 μl,0.31mg/mL, ph=7.4) was added a solution of L1-MMAE conjugate in DMSO (100 μl,2 mg/mL), after mixing, it was incubated in a water bath at 30 ℃ for 10 minutes after which HPLC detection showed complete conversion of L1-MMAE to NAC-L1-MMAE conjugate, which was used as stock without further purification.

To PBS buffer (390. Mu.L, 100mM, pH=7.4) was purged with nitrogen and NADPH (75. Mu.L, 20mM, purchased from ARK), NADPH-CYP reductase (10. Mu.L, 3mg, product number CYP004, purchased from Cypex Co.) was added rapidly. After incubating the mixture at 37℃for 10 minutes, a stock solution (25. Mu.L) of NAC-L1-MMAE conjugate was added, and then the incubation was continued in an anoxic incubator at 37℃ (0.1% O) ₂ ) Wherein the ratio of enzyme to substrate is 1:400. at the same time, a normoxic control group (20.0% O) ₂ ). 50. Mu.L of each sample was sampled at the subsequent time points (0 h, 0.25h, 0.5h, 1h, 2h, 4h, 6h, 12h, 24 h) and vortex quenched with cold acetonitrile (200. Mu.L) and then frozen at-80℃and 3 samples were run in parallel at each time point. After sampling was completed, all samples were melted at room temperature and centrifuged to remove proteins, and the supernatant was taken for LC/MS analysis. The result is that the content of the released MMAE was determined according to the standard curve method based on the AUC of the corresponding released MMAE at each time point. The amount of MMAE released by the nitroreductase enzyme from the L1-MMAE conjugate at various time points under hypoxic and normoxic conditions is shown in figure 1.

Tumors have a special microenvironment compared to normal tissues, where hypoxia is a ubiquitous feature of solid tumors. This example evaluates the enzymatic release profile of conjugate L1-MMAE and the selectivity of the release process for hypoxia. The reduction of the aryl nitro group in linker L1 depends on the nitroreductase NADPH-CYP reduction enzyme (E.C. 1.6.2.4), reduced NADPH, and the anoxic environment. As shown in FIG. 1, the substrate L1-MMAE can selectively release the carried cytotoxin MMAE under the action of nitroreductase in an anoxic environment, and the release amount of the MMAE is gradually increased with the time. In contrast, in the normoxic control group, the amount of release of MMAE was less, and the amount of release was not significantly increased over time. The test proves that the linker L1 cleavage and the enzymolysis drug release process initiated in the later stage have obvious selectivity to hypoxia.

Example 5: enzymatic drug release performance and hypoxia selectivity evaluation of L2-MMAE

This example further evaluates the enzymatic release profile of the conjugate L2-MMAE of linker L2 and cytotoxin MMAE. The procedure was performed with reference to the description in example 4. The amount of MMAE released by the nitroreductase from the L2-MMAE conjugate at various time points under hypoxic and normoxic conditions is shown in figure 2. The test results show that the L2-MMAE conjugate is similar to the L1-MMAE conjugate, the cleavage of the linker and the enzymolysis release process of the carried toxin MMAE have selectivity to hypoxia, the release amount of the MMAE is gradually increased along with the time extension, and the relative normoxic control group has only a small amount of MMAE falling off in a longer time. This example further demonstrates that linker L2 release has selectivity for hypoxia.

Example 6: evaluation of drug release hypoxia selectivity of ADCs in breast cancer cells HCC1954

This example evaluates the feasibility of drug release from ADCs at the cellular level and the selectivity of drug release process for hypoxia. The cell line used in this implementation was the antigen (HER 2) -positive human breast cancer cell line HCC1954 (purchased from ATCC). ADCs tested are ADC-I and ADC-II prepared in example 1. Culture medium for HCC1954 cells was composed of 10% Fetal Bovine Serum (FBS) and RPMI medium group containing 1% penicillin-streptomycin (PS) And (3) forming the finished product. At 37 ℃,5% CO ₂ Cells were cultured in flasks with 95% relative humidity. When the cells reach 80% -90% confluence, the cells are separated and inoculated. Will be 1.0X10 ⁶ The HCC1954 cells were seeded in T75 flasks at 37℃with 5% CO ₂ Incubated at 95% relative humidity for 2 days. HCC1954 cells were divided into blank and ADC dosing groups, where each dosing group included two replicates. The dosing groups were re-incubated with 15mL of HCC1954 cell culture medium described above containing approximately 100ng of ADC, and the blank group was not supplemented with test ADC. After the treatment is completed, the treatment is performed under oxygen deficiency (1%O) ₂ ) And normoxic (20% O) ₂ ) Cells were cultured for 12h, 24h and 48h. The medium was discarded, the cells were isolated with trypsin/EDTA, and trypsin was inactivated by adding medium containing excess serum, then centrifuged at 120g for 5 min, 10mL of medium of HCC1954 cells was added for mixing, and then an automatic cell counter (Nexcelom was used

Vision Cell Profiler) the number and viability of the cells were counted. The pellet was centrifuged and washed twice with ice-cold PBS (ph=7.4). The cell pellet was extracted by adding 6mL of cold methanol, and then the suspension was kept at-20 ℃ for 30 minutes and centrifuged at 13000g for 20 minutes. The supernatant was evaporated by blowing nitrogen. The resulting residue was redissolved in 600 μl of methanol containing the internal standard (is=100 nmol/L alprazolam). Samples were analyzed by LC-MS/MS and quantified. The amount of MMAE released in breast cancer cell line HCC1954 at various time points ADC-I and ADC-II under hypoxic and normoxic conditions is shown in figure 3.

The test results showed that both ADCs tested (ADC-I and ADC-II) showed significant hypoxia selectivity in the release of the carried toxin MMAE on the antigen (HER 2) -positive HCC1954 breast cancer cell line, under hypoxic conditions (0.1% O ₂ ) The amount of toxin released in the cells increases with time; in contrast, only a small amount of MMAE release was detected in cells cultured under normoxic conditions, and the amount of release was hardly increased with the time, as shown in fig. 3. This example demonstratesADC-I and ADC-II can smoothly release carried toxins in HCC1954 breast cancer cells, and the drug release process has selectivity for hypoxia.

Example 7: drug release hypoxia selectivity assessment of ADCs in breast cancer cells BT-474

This example further evaluates the release profile of ADCs in antigen (HER 2) -positive human breast cancer cell line BT-474 cells (purchased from ATCC) and the dependence of the release process on hypoxia. The ADCs tested for this implementation are ADC-I and ADC-II prepared in example 1. The procedure was carried out with reference to a similar description in example 6. BT-474 cells were cultured in DMEM medium containing 10% FBS, 1% PS and 0.01mg/mL insulin; at 37 ℃,5% CO ₂ Cells were cultured in flasks with 95% relative humidity. When the cells reach 80% -90% confluence, the cells are separated and inoculated. The number is about 2.0X10 ⁶ BT-474 cells were seeded in T75 flasks at 37℃with 5% CO ₂ Incubated at 95% relative humidity for 2 days. Cells were divided into a blank group and an ADC administration group, which contained two replicates. The cells were re-cultured with 15mL of the above BT-474 cell culture medium containing approximately 1500ng of ADC, respectively, and no test ADC was added to the blank. Respectively at hypoxia (1%O) ₂ ) And normoxic (20% O) ₂ ) Cells were cultured for 12h, 24h and 48h. Removing medium, separating cells with trypsin/EDTA, inactivating trypsin with medium containing excessive serum, centrifuging for 5 min at 120g, adding 10mL BT-474 cell culture medium, mixing, and then using an automatic cell counter (Nexcelom)

VisionCell Profiler) to calculate cell number and viability. The pellet was centrifuged and washed twice with cold PBS solution (ph=7.4). Adding 6mL of cold methanol to extract cell sediment; the suspension was then kept at-20℃for 30 minutes and centrifuged at 13000g for 20 minutes. The supernatant was evaporated by blowing nitrogen. The resulting residue was redissolved in 600 μl of methanol containing an internal standard (is=100 nmol/L alprazolam), and the samples were analyzed and quantified by LC-MS/MS. Under anoxic and normoxic conditions The amount of MMAE released by ADC-I and ADC-II in the breast cancer cell line BT-474 at different time points is shown in FIG. 4.

The test results show that the two ADCs (ADC-I and ADC-II) tested showed significant hypoxia selectivity in the release of the carried toxin MMAE on antigen (HER 2) -positive BT-474 breast cancer cell line, under hypoxic conditions (0.1% O ₂ ) The amount of toxin released in the cells increases with time; in contrast, only a small amount of MMAE release was detected in cells cultured under normoxic conditions, and the amount of release was hardly increased with the time. This example further demonstrates that ADC-I and ADC-II can release the carried toxin smoothly in BT-474 breast cancer cells, and that the drug release process is selective for hypoxia.

Example 8: drug release hypoxia selectivity assessment of ADCs in gastric cancer cells NCI-N87

This example further evaluates the drug release properties of this class of ADCs in antigen (HER 2) -positive human gastric cancer cell line NCI-N87 cells (purchased from ATCC) and the dependence of the drug release process on hypoxia. The ADCs tested for this implementation are ADC-I and ADC-II prepared in example 1. The procedure was carried out with reference to a similar description in example 6. NCI-N87 cells were cultured in DMEM medium containing 10% FBS, 1% PS and 0.01mg/mL insulin; at 37 ℃,5% CO ₂ Cells were cultured in flasks with 95% relative humidity. When the cells reach 50% -60% confluence, the cells are separated and inoculated. The number is about 3.0X10 ⁶ NCI-N87 cells of (A) were seeded in T75 flasks at 37℃with 5% CO ₂ Incubated for 3 days at 95% relative humidity. Cells were divided into a blank group and an ADCs dosing group, which contained two replicates. The cells were re-cultured with 15mL of NCI-N87 cell culture medium containing about 1000ng of ADC, and the control group was not supplemented with ADCs, and were subjected to hypoxia (1%O) ₂ ) And culturing the cells under normoxic conditions for 12h, 24h and 48h. Removing medium, separating cells with trypsin/EDTA, inactivating trypsin with medium containing excessive serum, centrifuging for 5 min at 120g, adding 10mL of NCI-N87 cell culture medium, mixing, and automatically counting cellsDevice (Nexcelom)

Vision Cell Profiler) to calculate cell number and viability. The pellet was centrifuged and washed twice with cold PBS solution (ph=7.4). Adding 6mL of cold methanol to extract cell sediment; the suspension was then kept at-20℃for 30 minutes and centrifuged at 13000g for 20 minutes. The supernatant was evaporated by blowing nitrogen. The resulting residue was redissolved in 600 μl of methanol containing an internal standard (is=100 nmol/L alprazolam), and the samples were analyzed and quantified by LC-MS/MS. The amount of MMAE released by ADC-I and ADC-II in gastric cell line NCI-N87 at various time points under hypoxic and normoxic conditions is shown in FIG. 5.

The test results show that the two ADCs (ADC-I and ADC-II) tested also show significant hypoxia selectivity during the release of toxin MMAE on antigen (HER 2) -positive NCI-N87 gastric cancer cell line, under hypoxic conditions (0.1% O ₂ ) The amount of toxin released in the cells increases with time; in contrast, only small amounts of MMAE release were detected in cells cultured under normoxic conditions, and their release amounts were hardly increased significantly with prolonged time. This example further demonstrates that ADC-I and ADC-II can release the carried toxin smoothly in NCI-N87 gastric cancer cells, and that the drug release process is selective for hypoxia.

Examples 6, 7 and 8 demonstrate that ADC-I and ADC-II can release carried toxins smoothly in various tumor cells, and the drug release process has selectivity for hypoxia.

Example 9: in vitro cytotoxicity evaluation of ADCs (ADC-I and ADC-II)

This example evaluates ADCs (ADC-I and ADC-II) under anaerobic conditions (0.1% O) ₂ ) Is a cell-free antigen. The antigens HER2 positive cell lines (BT-474, HCC1954 and NCI-N87) and HER2 negative cell lines MCF-7, MDA-MB-468 used in this example were purchased from ATCC. All cell lines were cultured in medium supplemented with 10% fetal bovine serum (cell lines BT-474, MCF-7 in DMEM medium, cell lines NCI-N87, HCC1954 and MDA-MB-468 in RPMI1640 medium; described below Medium is the same as herein) containing 5% CO at 37 ℃C ₂ Cells were passaged three times per week at a ratio of 1:4, each medium containing 10% FBS (heat-inactivated) and 1% penicillin/streptomycin. Oxygen concentration in the cell culture environment is determined by

CO ₂ Incubator (48R, #CO 48312044) was controlled.

Cells (3.3X10) ⁴ Individual cells/mL) were added to each well of a 384-well plate, after which 10 μl of compound solution (test drug was dissolved in PBS at ph=7.4, the initial concentration was about 250 μg/mL, and 10 solutions of the concentrations to be tested were diluted down with 10-fold dilution) were added to each well of the assay plate. The plates were incubated at 37℃with 5% CO ₂ ，0.1％O ₂ Incubate at 95% humidity for 7 days. The plates were then incubated at room temperature for about 10 minutes, and 40 μl of CTG reagent (Promega,

) Plates were incubated for 30 minutes at room temperature. Luminescence was detected using EnSpire Plate Reader and data analysis and IC were performed using Prism5for Windows (Graphpad software, inc., la Jolla, CA, USA) ₅₀ Is calculated by the computer. The test drug included two ADCs (ADC-I and ADC-II) and unconjugated naked antibody mil40 was set as a control.

The test results are shown in Table 1, where ADC-I and ADC-II have significant cytotoxicity in antigen-positive tumor cells, compared to antigen-negative cells, cytotoxicity (IC ₅₀ ) Can be generally improved by 300-5000 times. Cytotoxicity (IC compared to naked antibody ₅₀ ) The method has different degrees of improvement, and in addition, the maximum inhibition rate (Maxinhibit) has remarkable improvement. The two ADCs tested (ADC-I and ADC-II) only had a slight difference in linker structure, but the activities of both on the positive and negative test cells of each strain were essentially equivalent. This example further demonstrates the potential drug-forming properties of ADCs based on the linkers shown in the present application.

Table 1: in vitro cytotoxicity of ADCs under hypoxia

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present application and not for limiting the same; although the present application has been described in detail with reference to preferred embodiments, those of ordinary skill in the art will appreciate that: modifications may be made to the specific embodiments herein or equivalents may be substituted for part of the technical features; without departing from the spirit of the technical solutions of the present application, it should be covered in the scope of the technical solutions claimed in the present application.

Claims

1. A compound shown in a formula I or a salt thereof,

wherein:

R ₁ is that

R ₂ Is halogen or

r is 0, 1, 2, 3 or 4;

ar is a five-membered or six-membered aryl or heteroaryl group;

Wherein m is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and m and n are not both 0; r is R ₅ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, q is 0, 1, 2, 3 or 4;

L ₂ selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-C(O)NH-CH[(CH ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6;

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -。

2. The compound of claim 1, or a salt thereof, wherein the halogen is fluorine, chlorine, bromine, or iodine.

3. The compound of claim 1, or a salt thereof, wherein the nitro group on Ar is in a conjugated position with Z in the aromatic system.

4. A compound according to claim 3, wherein Ar is a six-membered aryl or heteroaryl group, the nitro group on Ar being para or ortho to Z.

5. The compound of claim 1, or a salt thereof, wherein L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

6. The compound or salt thereof according to claim 1, wherein the compound has a structure represented by formula Ia,

wherein: r is R ₁ 、R ₂ 、R ₃ 、r、L ₁ 、L ₂ X, Z are defined as in claim 1.

7. The compound or salt thereof according to claim 1, wherein the compound has a structure represented by formula Ib,

8. The compound of any one of claims 1 to 7, or a salt thereof, wherein:

R ₁ is that

9. The compound of any one of claims 1 to 7, or a salt thereof, wherein: r is R ₂ Is that

Wherein R is ₄ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, p is 0, 1, 2, 3 or 4.

10. The compound of claim 9, or a salt thereof, wherein R ₄ Is hydrogen, fluorine, chlorine, bromine, iodine, nitro, methyl, ethyl, methoxy or ethoxy.

11. The compound of claim 9, or a salt thereof, wherein p is 0 or 1.

12. A compound according to any one of claims 1 to 7, or a salt thereof, wherein Ar is a benzene ring.

13. The compound of claim 12, or a salt thereof, wherein the nitro group on Ar is para or ortho to Z.

14. The compound of any one of claims 1 to 7, or a salt thereof, wherein R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

15. The compound of any one of claims 1 to 7, or a salt thereof, wherein r is 0 or 1.

16. The compound of any one of claims 1 to 7, or a salt thereof, wherein L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

17. The compound of any one of claims 1 to 7, or a salt thereof, wherein L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

18. The compound of claim 17, or salt thereof, wherein i is 1, 2, or 3.

19. The compound according to any one of claims 1 to 7, wherein X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

20. The compound according to any one of claims 1 to 7, wherein Z is-CH ₂ -。

21. The compound of any one of claims 1 to 7, or a salt thereof, wherein the compound is selected from the group consisting of:

22. use of a compound according to any one of claims 1 to 21, or a salt thereof, in the manufacture of an antibody-conjugated drug.

23. A compound represented by formula II or a salt thereof,

wherein:

R ₁ is that

r is 0, 1, 2, 3 or 4;

ar is a five-membered or six-membered aryl or heteroaryl group;

Wherein m is 0, 1, 2, 3, 4, 5, 6, 7,8. 9, 10, 11 or 12, n is 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12, and m and n are not simultaneously 0; r is R ₅ Is hydrogen, fluorine, chlorine, bromine, iodine, C _1-4 Alkyl, nitro or C _1-4 Alkoxy, q is 0, 1, 2, 3 or 4;

L ₂ selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-C(O)NH-CH[(CH ₂ ) _d -NHC(O)-(CH ₂ CH ₂ O) _e -(CH ₂ ) _f -CH ₃ ]-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-and-C (O) -, or L ₂ Is empty, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12; d is 1, 2, 3, 4, 5 or 6; e is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12; f is 1, 2, 3, 4, 5 or 6;

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -；

t is 0 or 1;

b is an active compound selected from the group consisting of a drug, a detection reagent and a targeting vector.

24. The compound of claim 23, or salt thereof, wherein the nitro group on Ar is in a conjugated position with Z in the aromatic system.

25. The compound of claim 23, or a salt thereof, wherein and Ar is a six-membered aryl or heteroaryl, the nitro group on Ar being para or ortho to Z.

26. The compound of claim 23, or a salt thereof, wherein L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

27. The compound of claim 23, or a salt thereof, wherein B is a cytotoxin or diagnostic agent.

28. The compound of claim 23, or a salt thereof, wherein B is an anti-tumor drug, an anti-infective drug, or an immunomodulatory drug.

29. The compound of claim 23, or a salt thereof, wherein B is a tubulin inhibitor, a DNA alkylating agent, a DNA intercalating agent, an enzyme inhibitor, an antiviral drug, or an antimetabolite.

30. The compound of claim 23, or a salt thereof, wherein B is a peptide or nucleotide.

31. A compound or salt thereof according to claim 23, wherein preferably B is coupled to the carbonyl or Z group of the site x through an N atom or O atom in the active compound molecule.

32. The compound or salt thereof according to claim 23, wherein the compound has a structure represented by formula IIa,

wherein: r is R ₁ 、R ₃ 、r、L ₁ 、L ₂ X, Z, B, t are defined in claim 24.

33. The compound or salt thereof according to claim 23, wherein the compound has a structure represented by formula IIb,

wherein: l (L) ₁ 、L ₂ B is as defined in claim 24.

34. The compound of any one of claims 23 to 33, or a salt thereof, wherein: r is R ₁ Is that

35. The compound of any one of claims 23 to 33, or a salt thereof, wherein R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

36. The compound of any one of claims 23 to 33, or a salt thereof, wherein r is 0 or 1.

37. The compound of any one of claims 23 to 33, or a salt thereof, wherein Ar is a benzene ring.

38. The compound of claim 37, or a salt thereof, wherein the nitro group on Ar is para or ortho to Z.

39. The compound of any one of claims 23 to 33, or a salt thereof, wherein L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

40. The compound of any one of claims 23 to 33, or a salt thereof, wherein L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

41. The compound of claim 40, wherein i is 1, 2 or 3, or a salt thereof.

42. A compound or salt according to any one of claims 23 to 33 wherein X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

43. The compound of any one of claims 23 to 33, or a salt thereof, wherein Z is-CH ₂ -。

44. The compound of any one of claims 23 to 33, or a salt thereof, wherein t is 1.

45. The compound of any one of claims 23 to 33, or a salt thereof, wherein B is auristatin, monomethyl auristatin E, maytansinoid, DM1, DM3, DM4, paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, or PBD toxoid.

46. The compound of claim 45, wherein B is monomethyl auristatin E, or a salt thereof.

47. The compound of any one of claims 23 to 33, or a salt thereof, wherein the compound has the structure shown in tia and mb:

wherein B is coupled to a carbonyl or methylene group at a site through an N atom or an O atom in the active compound molecule, B, t being as defined in any one of claims 23 to 33.

48. The compound of any one of claims 23 to 33, or a salt thereof, wherein the compound is selected from the group consisting of:

49. use of a compound according to any one of claims 23 to 48, or a salt thereof, in the manufacture of an antibody-conjugated drug.

50. A compound represented by formula IV or a salt thereof,

wherein: ar is a five-membered or six-membered aryl or heteroaryl group;

r is 0, 1, 2, 3 or 4;

Z is-CH ₂ -，-CH(CH ₃ ) -or-C (CH) ₃ ) ₂ -；

t is 0 or 1;

a is a targeting compound selected from the group consisting of proteins and small molecules;

b is an active compound selected from a drug, a detection reagent or a targeting vector;

a is a number between 0.5 and 8.5.

51. The compound of claim 50, wherein the nitro group on Ar is in a conjugated position with Z in the aromatic system.

52. The compound of claim 50, wherein Ar is a six-membered aryl or heteroaryl, and the nitro group on Ar is para or ortho to Z.

53. The compound according to claim 50, wherein L ₂ Selected from: -C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C(O)-，-C(O)(CH ₂ ) _i NH-，-NH(CH ₂ ) _i O-，-NH(CH ₂ ) _i S-，-N(CH ₃ )(CH ₂ ) _i NH-，-N(CH ₃ )(CH ₂ ) _i N(CH ₃ )-，-O(CH ₂ ) _i NH-，-O(CH ₂ ) _i C(O)-，-O(CH ₂ ) _i O-，-O(CH ₂ ) _i S-，-O-，-NH-，-S-，-S(O)-，-S(O) ₂ -，-NCH ₃ -，-NH(CH ₂ ) ₂ NH-, -C (O) -, or L ₂ Is empty, wherein each i is independently 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12.

54. The compound of claim 50, wherein A is an antibody, polypeptide, or enzyme.

55. The compound of claim 50, wherein A is coupled to site # through an S atom in the targeting compound molecule.

56. The compound of claim 50, wherein B is a cytotoxin or a diagnostic agent.

57. The compound of claim 50, wherein B is an anti-tumor drug, an anti-infective drug, or an immunomodulator drug.

58. The compound of claim 50, wherein B is a tubulin inhibitor, a DNA alkylating agent, a DNA intercalating agent, an enzyme inhibitor, an antiviral agent, or an antimetabolite agent.

59. The compound of claim 50, wherein B is a peptide or nucleotide.

60. The compound of claim 50, wherein B is coupled to the carbonyl or Z group of the site through an N atom or O atom in the active compound molecule.

61. The compound of claim 50, wherein a is a number between 0.8 and 5, or a salt thereof.

62. The compound of claim 50, wherein a is a number between 1 and 4, or a salt thereof.

63. The compound of claim 50, wherein a is a number between 2 and 6.

64. The compound of claim 50, wherein a is a number between 3 and 7.

65. The compound of claim 50, wherein a is a number between 4 and 8.

66. The compound of claim 50, wherein a is a number between 3.5 and 8.5.

67. The compound of claim 50, wherein a is a number between 3.5 and 4.5.

68. The compound of claim 50, wherein a is a number between 6.5 and 8.5.

69. The compound of claim 50, wherein a is 4, 5, 6, 7 or 8, or a salt thereof.

70. The compound of any one of claims 50 to 69, or a salt thereof, wherein R ₃ Is hydrogen, fluorine, chlorine, bromine, iodine, methoxy, ethoxy, propoxy, methyl, ethyl, n-propyl or isopropyl.

71. The compound of any one of claims 50 to 69, or a salt thereof, wherein r is 0 or 1.

72. The compound of any one of claims 50 to 69, or a salt thereof, wherein Ar is a benzene ring.

73. The compound of claim 72, wherein the nitro group on Ar is para or ortho to Z.

74. The compound of any one of claims 50 to 69, or a salt thereof, wherein L ₁ Is- (CH) ₂ ) _m -or

Wherein m is 4, 5, 6 or 7.

75. The compound of any one of claims 50 to 69, or a salt thereof, wherein L ₂ is-C (O) NH (CH) ₂ ) _i NH-，-NH(CH ₂ ) _i NH-，-NH(CH ₂ ) _i C (O) -or-C (O) (CH ₂ ) _i NH-, wherein i is 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12.

76. The compound of claim 75, wherein i is 1, 2, or 3, or a salt thereof.

77. The compound according to any one of claim 50 to 69 or a salt thereof, wherein X is-C (O) -, -O-, -NH-, -NCH ₃ -，-CH ₂ O-, or-S-.

78. The compound according to any one of claims 50 to 69, or a salt thereof, wherein preferably Z is-CH ₂ -。

79. The compound of any one of claims 50 to 69, or a salt thereof, wherein t is 1.

80. The compound of any one of claims 50 to 69, or a salt thereof, wherein B is auristatin, monomethyl auristatin E, maytansinoid, DM1, DM3, DM4, paclitaxel, calicheamicin, duocarmycin, doxorubicin, camptothecin, or PBD toxoid.

81. The compound of claim 80, or a salt thereof, wherein B is monomethyl auristatin E.

82. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is a monoclonal antibody having a thiol group as a coupling site.

83. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is a monoclonal antibody with thiol-based coupling site directed mutation or modification.

84. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is selected from: anti-HER 2 humanized monoclonal antibodies mil40, trastuzumab, pertuzumab, cetuximab, panitumumab, rituximab, alemtuzumab, ibritumomab, tositumomab, ofatuzumab, bevacizumab, ipilimab, denouzumab, pemetuzumab, nivolumab, avelumab, atezolizumab, durvalumab, sacituzumab, rovapituzumab.

85. The compound of claim 84, or a salt thereof, wherein a is anti-HER 2 humanized monoclonal antibody mil40.

86. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is a number between 2 and 7.

87. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is a number between 3 and 6.

88. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is a number between 4 and 5.

89. The compound of any one of claims 50 to 69, or a salt thereof, wherein a is 4, 5, 6, 7 or 8.

90. The compound or salt thereof according to claim 50 to 69, wherein the compound has a structure represented by formula IVa,

wherein L is ₁ 、L ₂ 、X、R ₃ R, Z, A, B, a, t are defined in any one of claims 50 to 69.

91. The compound or salt thereof according to claim 50 to 69, wherein the compound has a structure represented by formula IVb,

wherein L is ₁ 、L ₂ A, B, a, t are defined in any one of claims 50 to 69.

92. The compound or salt thereof according to claim 50 to 69, wherein the compound has a structure represented by formula IV-1 or formula IV-2,

wherein B, a is defined as claimed in any one of claims 50 to 69 and MAB is a monoclonal antibody.

93. The compound of claim 92 or a salt thereof, wherein MAB is an anti-HER 2 humanized monoclonal antibody mil40.

94. The compound of any one of claims 50 to 69, or a salt thereof, wherein the compound is selected from the group consisting of

Wherein MAB is a monoclonal antibody and a is defined in any one of claims 50 to 69.

95. The compound of claim 94, or a salt thereof, wherein MAB is an anti-HER 2 humanized monoclonal antibody mil40.

96. A pharmaceutical composition comprising at least one compound of any one of claims 50 to 95, or a salt thereof, and one or more pharmaceutically acceptable carriers or excipients.

97. Use of a compound of any one of claims 50 to 95 or a salt thereof or a pharmaceutical composition of claim 96 in the manufacture of a medicament for treating or lessening the severity of a disease or disorder selected from the group consisting of a tumor, an infectious disease, a metabolic disease.

98. The use of claim 97, wherein the disease or disorder is cancer or inflammation.

99. The use of claim 97, wherein the disease or disorder is a blastoma, sarcoma, or hematological disease.

100. The use of claim 98, wherein the cancer is selected from the group consisting of: leukemia; breast cancer; squamous cell carcinoma; lung cancer; peritoneal cancer; liver cancer; gastrointestinal cancer; membranous adenocarcinoma; glioblastoma; cervical cancer; ovarian cancer; bladder cancer; urethral cancer; hepatoma; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; renal cancer; prostate cancer; vulvar cancer; thyroid cancer; anal cancer; penile cancer; melanoma; multiple myeloma; lymphomas; lymphoid neoplasms; brain cancer; gallbladder cancer; esophageal cancer; bile duct cancer; head and neck cancer and related metastases.

101. The use of claim 100, wherein the gastrointestinal cancer is gastric or intestinal cancer and the colorectal cancer is colon cancer or rectal cancer.

102. The use of claim 100, wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

103. The use of claim 100, wherein the breast cancer is HER2 positive breast cancer, the lung cancer is adenocarcinoma of the lung or squamous carcinoma of the lung, and the lymphoma is B-cell lymphoma.

104. The use of claim 100, wherein the squamous cell carcinoma is an epithelial squamous cell carcinoma.

105. A compound according to any one of claims 50 to 95 or a salt thereof or a pharmaceutical composition according to claim 96 for use in treating or lessening the severity of a disease or disorder selected from the group consisting of a tumour, an infectious disease, a metabolic disease.

106. The compound or salt thereof or the pharmaceutical composition of claim 105, wherein the disease or disorder is cancer or inflammation.

107. The compound or salt thereof or the pharmaceutical composition of claim 105, wherein the disease or disorder is a blastoma, a sarcoma, or a hematological disease.

108. The compound or salt thereof or the pharmaceutical composition of claim 106, wherein the cancer is selected from the group consisting of: leukemia; breast cancer; squamous cell carcinoma; lung cancer; peritoneal cancer; liver cancer; gastrointestinal cancer; membranous adenocarcinoma; glioblastoma; cervical cancer; ovarian cancer; bladder cancer; urethral cancer; hepatoma; colorectal cancer; endometrial cancer; uterine cancer; salivary gland cancer; renal cancer; prostate cancer; vulvar cancer; thyroid cancer; anal cancer; penile cancer; melanoma; multiple myeloma; lymphomas; lymphoid neoplasms; brain cancer; gallbladder cancer; esophageal cancer; bile duct cancer; head and neck cancer and related metastases.

109. The compound of claim 108, or a salt thereof, or the pharmaceutical composition, wherein the gastrointestinal cancer is gastric cancer or intestinal cancer and the colorectal cancer is colon cancer or rectal cancer.

110. The compound or salt thereof of claim 108 or the pharmaceutical composition, wherein the lung cancer is small cell lung cancer or non-small cell lung cancer.

111. The compound or salt thereof or the pharmaceutical composition of claim 108, wherein the breast cancer is HER2 positive breast cancer, the lung cancer is adenocarcinoma of the lung or squamous carcinoma of the lung, and the lymphoma is B-cell lymphoma.

112. The compound or salt thereof of claim 108 or the pharmaceutical composition, wherein the squamous cell carcinoma is epithelial squamous cell carcinoma.