WO2025153047A1 - Camptothecin derivative and preparation method therefor and use thereof - Google Patents

Abstract

The present invention relates to the technical field of medicinal chemistry. Disclosed are a camptothecin derivative and a preparation method therefor and the use thereof. The derivative is a compound as shown in formula (I) and/or a pharmaceutically acceptable salt thereof, wherein R₁ is selected from at least one of H, -NO₂, -NH₂, -OH and halogen; R₂ is selected from at least one of H, acyl and substituted acyl; and R₁ and R₂ are not H at the same time. The method for preparing the camptothecin derivative comprises the following steps: performing a substitution reaction on a compound as shown in formula (II) and a reactant to obtain a compound as shown in formula (III), wherein the reactant is a reactant containing nitro and/or a reactant containing substituted acyl, Ra is nitro and hydrogen, and Rb is selected from at least one of acyl, substituted acyl and hydrogen. The camptothecin derivative has a higher tumor cell inhibitory activity and better safety.

Description

Camptothecin derivatives and preparation methods and applications thereof

CROSS-REFERENCE TO RELATED APPLICATIONS

The present invention claims the benefit of Chinese patent application 202410077258.5 filed on January 18, 2024, the contents of which are incorporated herein by reference.

Technical Field

The present invention relates to the technical field of pharmaceutical chemistry, and in particular to a camptothecin derivative and a preparation method thereof, and application of the compound in the preparation of antibody-drug conjugates.

Background Art

The main function of DNA topoisomerase is to help unwind the DNA superhelical structure and promote the transcription and replication of DNA chains. Inhibition of topoisomerase activity causes a large amount of broken DNA to accumulate in tumor cells, inducing tumor cell death. Topoisomerases are divided into topoisomerase I (Topo I) and topoisomerase II (Topo II). Camptothecin and its derivatives are an important class of DNA topoisomerase I inhibitors. Camptothecin derivatives such as irinotecan and topotecan have been used clinically to treat malignant tumors.

Camptothecin was first isolated from the plant Camptotheca acuminata of the Davidiaceae family. It has strong cytotoxicity and has good therapeutic effects on malignant tumors such as digestive tract tumors (gastric cancer, colon cancer, rectal cancer), liver cancer, breast cancer, bladder cancer and leukemia. The main disadvantages of camptothecin are poor solubility and stability, high toxicity and a small safety window, which limits its clinical application.

Prodrug is a medicinal chemistry technology that expands the therapeutic window. One type of prodrug is antibody-drug conjugate (ADC), which can simultaneously improve the water solubility and therapeutic window of camptothecin derivatives through the high targeting and water solubility of antibodies and linkers to relevant antigens in tumor cells. If the linker is sufficiently stable, ADC can become a sustained-release prodrug, which also increases the therapeutic window. Camptothecin derivatives Deruxtecan and SN38, ADC-conjugated prodrugs formed by conjugation with multiple antibodies, have become high-quality solid tumor therapeutics.

Another type of prodrug is hypoxia activated prodrugs (HAP). Mitomycin C, as a prodrug relying on this activation mechanism, can be used to treat gastric cancer and pancreatic cancer. Tumor tissues are locally hypoxic because their vascular structure and metabolism are different from those of normal tissues. This is an important mechanism for tumors to develop resistance to chemotherapy, immunotherapy, and radiotherapy. Hypoxia upregulates various reductases such as nitroreductase. This special tumor environment can serve as an inducing mechanism for specific activation of chemotherapeutic drugs. Radiotherapy usually further upregulates reductases, so HAP can be used as a radiosensitizer. HAP, as a small molecule prodrug, is more convenient to use than ADC and can penetrate the blood-brain barrier to enter the central nervous system to treat brain tumors and brain metastases. HAP and its active metabolites can be used as small molecule toxins for ADC molecules.

It is known that camptothecin derivatives have low cell proliferation inhibition activity and require a higher dose/antibody ratio (DAR) as ADC toxins, which can easily cause ADC instability, resulting in higher production costs, greater synthesis difficulty and a lower safety window. Therefore, more active camptothecin derivatives are of great significance to the design and development of new ADCs.

Summary of the invention

The purpose of the present invention is to overcome the problems of narrow safety window and low activity of camptothecin derivatives in the prior art, and to provide a camptothecin derivative and its preparation method and application. The camptothecin derivative provided by the present invention has higher tumor cell inhibition activity and better safety, and has a larger safety window as an ADC toxin.

In order to achieve the above object, the present invention provides a camptothecin derivative in a first aspect, wherein the derivative is a compound represented by formula (I) and/or a pharmaceutically acceptable salt thereof:

Wherein, R ₁ is at least one selected from H, -NO ₂ , -NH ₂ , -OH and halogen; R ₂ is at least one selected from H, acyl and substituted acyl, and R ₁ and R ₂ are not H at the same time.

Preferably, the halogen is selected from at least one of F, Cl and Br.

Preferably, the _R1 is selected from at least one of H, _-NO2 and _-NH2 , and _R2 is hydrogen or a substituted acyl group.

Preferably, the substituted acyl group is at least one of a hydroxy substituted acyl group and/or an alkoxy substituted acyl group.

Preferably, the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group; further, it is an α-hydroxy-substituted acyl group containing a C1-C6 alkane, and more preferably, it is an α-hydroxyacetyl group; the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocycle and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and further preferably, it is an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and more preferably, it is a (1-methyl-2-nitro-1H-imidazol-5-yl)methoxyacyl group.

Preferably, the derivative is selected from:

At least one of .

Further preferably, the derivative is selected from:

At least one of .

The second aspect of the present invention provides a method for preparing a camptothecin derivative, the method comprising the following steps:

The compound represented by formula (II) and the reactant are subjected to a substitution reaction to obtain a compound represented by formula (III);

wherein the reactant is a reactant containing a nitro group and/or a reactant containing a substituted acyl group; Ra is a nitro group and hydrogen; Rb is at least one selected from an acyl group, a substituted acyl group and hydrogen, preferably a substituted acyl group and hydrogen, and Ra and Rb are not H at the same time; or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV); or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV);

(3) subjecting the compound represented by formula (IV) to a Sandmeyer reaction; or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV);

(3) subjecting the compound represented by formula (IV) to a Buchwald-Hartwig coupling reaction;

Preferably, before the substitution reaction, the compound represented by formula (II) is reacted with an amino protecting agent.

Further preferably, the amino protecting agent is at least one selected from benzyl chloroformate, di-tert-butyl dicarbonate and 9-fluorenylmethyl chloroformate.

Preferably, the reactant containing a nitro group is nitric acid.

Preferably, the substituted acyl group is a hydroxy substituted acyl group and/or an alkoxy substituted acyl group.

Preferably, the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group; further, it is an α-hydroxy-substituted acyl group containing a C1-C6 alkane, and more preferably, it is an α-hydroxyacetyl group; the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocycle and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and further preferably, it is an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring.

Further preferably, the reactant containing a substituted acyl group is selected from 2-hydroxyacetic acid, phenyl chloroformate and (1-methyl-2-nitro-1H-imidazol-5-yl)methyl chloroformate.

Preferably, in step (2), the reduction reaction comprises contacting the compound represented by formula (III) with a reducing agent.

More preferably, the reducing agent is tetrahydroxydiboron and/or sodium borohydride.

Preferably, when the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, the substitution reaction comprises:

The compound represented by formula (II) and a reactant containing a nitro group are subjected to a substitution reaction to obtain a compound represented by formula (V); and the compound represented by formula (V) and a reactant containing a substituted acyl group are subjected to a substitution reaction;

Further preferably, when the reactant is a reactant containing a nitro group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, temperature of -4-0°C, and time of 50-70 min.

When the reactant is a reactant containing a substituted acyl group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, temperature of 25-30° C., and time of 120-130 min.

The reduction reaction conditions at least meet the following requirements: inert gas protection, temperature of 20-25° C., and time of 4-10 min.

The third aspect of the present invention provides the use of the derivatives described in the first aspect and the derivatives prepared by the method described in the second aspect in the preparation of anti-tumor drugs.

Preferably, the anti-tumor drug is an antibody-drug conjugate.

Preferably, the anti-tumor drug is a hypoxia-activated prodrug.

Preferably, the anti-tumor drug is a small molecule active drug.

Further preferably, the tumor is selected from at least one of colon cancer, gastric cancer, breast cancer and lung cancer.

Further preferably, the tumor is an in situ lesion and/or a metastatic lesion.

Further preferably, the tumor is selected as a central tumor and/or a central metastatic lesion.

The fourth aspect of the present invention provides an antibody-drug conjugate, in which the derivative described in the first aspect and/or the derivative prepared by the method described in the second aspect is connected to the antibody via a linker and/or is directly coupled to the antibody.

Through the above technical scheme, the camptothecin derivatives provided by the present invention have high tumor cell inhibitory activity and great clinical use value. As ADC toxins, they can further expand the safety window of ADC drugs. Moreover, the activity difference before and after activation as a prodrug is large. Before activation, it will not cause harm to normal tissues. It will only be active after activation in tumor tissues, and has higher tumor cell inhibitory activity and better safety.

Preferably, the prodrug of camptothecin derivatives activated by nitroreductase has low activity and does not cause damage to normal tissues before activation, and only has high activity after activation in tumor tissues. Camptothecin derivatives with a large difference in activity before and after activation have great clinical value. Camptothecin derivatives can improve the therapeutic window and solubility and improve drugability by introducing water-soluble groups or preparing prodrugs.

BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings are used to provide a further understanding of the present invention and constitute a part of the specification. Together with the following specific embodiments, they are used to explain the present invention but do not constitute a limitation of the present invention. In the accompanying drawings:

FIG1 is a hydrogen spectrum of compound 1 prepared in Example 1 of the present invention;

FIG2 is a hydrogen spectrum of compound 2 prepared in Example 2 of the present invention;

FIG3 is a hydrogen spectrum of compound 3 obtained in Example 3 of the present invention;

FIG4 is a hydrogen spectrum of compound 4 obtained in Example 4 of the present invention;

FIG5 is a hydrogen spectrum of compound 5 obtained in Example 5 of the present invention;

FIG6 is a comparison chart of the weight changes of mice in Test Example 2 of the present invention;

FIG. 7 is a comparison diagram of tumor volume changes in Test Example 2 of the present invention.

DETAILED DESCRIPTION

The endpoints and any values of the ranges disclosed in this article are not limited to the precise ranges or values, and these ranges or values should be understood to include values close to these ranges or values. For numerical ranges, the endpoint values of each range, the endpoint values of each range and the individual point values, and the individual point values can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

In a first aspect, the present invention provides a camptothecin derivative, wherein the derivative is a compound represented by formula (I) and/or a pharmaceutically acceptable salt thereof:

Wherein, R ₁ is at least one selected from H, -NO ₂ , -NH ₂ , -OH and halogen; R ₂ is at least one selected from H, acyl and substituted acyl, and R ₁ and R ₂ are not H at the same time.

During the research process of camptothecin derivatives, the inventors found that camptothecin derivatives having the structure of formula (I) have higher cancer cell inhibitory activity and better safety, can inhibit the growth of tumor cells, and have a larger safety window as ADC toxins.

According to the present invention, "acceptable" means that a formulation component or active ingredient has no undue adverse effect on health for the general purpose of treatment.

According to the present invention, "pharmaceutically acceptable" refers to a substance, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., a substance that, when administered to a subject, does not cause undesirable biological effects or interact in a deleterious manner with any of its constituent components.

According to the present invention, "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant stimulation to the organism to which it is administered and does not cause the biological activity and properties of the compound to disappear. In certain specific aspects, the pharmaceutically acceptable salt is obtained by reacting the compound represented by formula (I) with an acid, such as an inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, phosphoric acid; an organic acid such as formic acid, acetic acid, propionic acid, oxalic acid, trifluoroacetic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, picric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, and an acidic amino acid such as aspartic acid and glutamic acid.

According to the present invention, the compound represented by formula (I) and its pharmaceutically acceptable salt can be prepared into various preparations, which contain the compound of the present invention or its pharmaceutically acceptable salt within a safe and effective amount and a pharmacologically acceptable excipient or carrier.

According to the present invention, pharmaceutically acceptable salts also include corresponding solvent addition forms or crystal forms, in particular solvates or multiple crystal forms. Solvates contain stoichiometric or non-stoichiometric solvents and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water during crystallization, or alcoholates are formed when the solvent is ethanol. Solvates of the compounds shown in formula (I) can be easily prepared or formed into corresponding solvates according to the method described in the present invention.

According to the present invention, the hydrate of the solvate of the compound shown in formula (I) is obtained by recrystallization from a mixed solvent of water/organic solvent, and the organic solvent used includes, but is not limited to, tetrahydrofuran, acetone, ethanol or methanol. In addition, the compound mentioned here can exist in non-solvated and solvated forms. In a word, for the compound and preparation method provided by the present invention, the solvated form is considered to be equivalent to the non-solvated form.

According to the present invention, the compound shown in formula (I) can be prepared into different forms, including but not limited to amorphous, powder and nanoparticle forms. In addition, the compound shown in formula (I) can be a single crystal or a polymorph. Polymorphs include different lattice arrangements of the same elements of the compound. Polymorphs usually have different X-ray diffraction spectra, infrared spectra, melting points, densities, hardnesses, crystal forms, optical and electrical properties, stability and solubility. Different influencing factors include recrystallization solvents, crystallization rates and storage temperatures that may cause a single crystal to be the leading polymorphic compound.

According to the present invention, the compound shown in formula (I) may have chiral centers and/or chiral axes, and therefore appear in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers and cis-trans isomers. Each chiral center or chiral axis will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included in the protection scope of the present invention. The compound shown in formula (I) disclosed in the present invention includes all isomeric forms corresponding to the compound.

According to the present invention, the compound may contain an unnatural ratio of atomic isotopes on one or more atoms constituting the compound. For example, the compound may be labeled with a radioactive isotope, such as deuterium ( ^2H ), tritium ( ^3H ) and C-14 ( ^14C ). For another example, a deuterated compound may be formed by replacing a hydrogen atom with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with undeuterated drugs, deuterated drugs generally have the advantages of reducing toxic side effects, increasing drug stability, enhancing therapeutic effects, and extending the half-life of drugs in vivo. All isotopic composition changes of the compounds of the present invention, whether radioactive or not, are included in the scope of the present invention. In the present invention, if not otherwise specified, the use of "or" or "and" means "and/or".

According to the present invention, the halogen is selected from at least one of F, Cl and Br; the inventors found that when R ₁ is F, Cl and Br, the corresponding compound has anti-tumor activity.

According to the present invention, when the R ₁ is selected from at least one of H, -NO ₂ and -NH ₂ , and R ₂ is hydrogen or substituted acyl, the corresponding compounds have good anti-tumor activity. The inventor unexpectedly found during the research that the prodrug activity of camptothecin derivatives containing nitro groups is low, but the camptothecin derivatives containing amino groups after activation by nitroreductase have higher activity, and the camptothecin derivatives with a large difference in activity before and after activation have great clinical use value. When the substituted acyl group is at least one of hydroxy substituted acyl and/or alkoxy substituted acyl, the activity of the camptothecin derivatives is good.

According to the present invention, when the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group, the side chain of the derivative contains an -OH group that is easy to connect, and has good cell activity, which is suitable as a small molecule toxin for ADC. Further preferably, when the α-hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group containing C1-C6 alkanes, at least one of the α-hydroxy-substituted acyl group containing C1-C6 straight-chain alkanes, the α-hydroxy-substituted acyl group containing C1-C6 branched alkanes, and the α-hydroxy-substituted acyl group containing cycloalkyl groups can be used. The α-hydroxyl group containing C1-C6 straight-chain alkanes, C1-C6 branched alkanes or cycloalkyl groups can retain the tumor cell inhibitory activity of camptothecin derivatives while optimizing the drugability and inhibitory activity of camptothecin derivatives; more preferably, when the α-hydroxy-substituted acyl group is an α-hydroxyacetyl group, the camptothecin derivative has higher tumor cell inhibitory activity and better safety, and has a larger safety window as an ADC toxin.

According to the present invention, the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocycle and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring. The inventors found during the research that when the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocycle and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, the alkoxy-substituted acyl group can be self-eliminated under the action of a reductase to generate a camptothecin derivative with better tumor cell inhibition activity. Further preferably, the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring. The inventors found that after the nitro group of the imidazole ring enters the tumor tissue, it is reduced to an amine group under the catalysis of nitroreductase to form an unstable intermediate, and then a metabolite with better activity is generated through a self-elimination reaction, which has better prodrug properties and better tumor cell inhibition activity. More preferably, the camptothecin derivative in which the alkoxy-substituted acyl group is (1-methyl-2-nitro-1H-imidazol-5-yl)methoxyacyl generates a more active metabolite exitecan through a self-elimination reaction, has better prodrug properties and better safety, and has a larger safety window as an ADC toxin.

According to the present invention, "halogen" (or halo) refers to fluorine, chlorine, bromine or iodine. The term "halo" (or "halogen substitution") that appears before the group name means that the group is partially or fully halogenated, that is, replaced by F, Cl, Br or I in any combination, preferably replaced by F or Cl.

According to the present invention, "cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic or polycyclic), if the carbocyclic ring contains at least one double bond, then the partially unsaturated cycloalkyl can be referred to as "cycloalkenyl", or if the carbocyclic ring contains at least one triple bond, then the partially unsaturated cycloalkyl can be referred to as "cycloalkynyl". Cycloalkyl can include monocyclic or polycyclic (e.g., with 2, 3 or 4 fused rings) groups and spirocycles. In some embodiments, cycloalkyl is monocyclic. In some embodiments, cycloalkyl is monocyclic or bicyclic. The ring-forming carbon atoms of cycloalkyl can be optionally oxidized to form oxo or sulfide groups. Cycloalkyl also includes cycloalkylene. In some embodiments, cycloalkyl contains 0, 1 or 2 double bonds. In some embodiments, cycloalkyl contains 1 or 2 double bonds (partially unsaturated cycloalkyl). In some embodiments, cycloalkyl can be fused with aryl, heteroaryl, cycloalkyl and heterocycloalkyl. In some embodiments, cycloalkyl can be fused with aryl, cycloalkyl and heterocycloalkyl. In some embodiments, cycloalkyl can be fused with aryl and heterocycloalkyl. In some embodiments, cycloalkyl can be fused with aryl and cycloalkyl. Examples of cycloalkyl include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinenyl, norcarbyl, bicyclo[1.1.1]pentanyl, bicyclo[2.1.1]hexanyl, etc.

According to the present invention, "heterocycloalkyl" refers to a non-aromatic ring or ring system, which may optionally contain one or more alkenylene groups as part of a ring structure, and which has at least one heteroatom ring member independently selected from boron, phosphorus, nitrogen, sulfur, oxygen and phosphorus. If the heterocycloalkyl contains at least one double bond, then the partially unsaturated heterocycloalkyl may be referred to as "heterocycloalkenyl", or if the heterocycloalkyl contains at least one triple bond, then the partially unsaturated heterocycloalkyl may be referred to as "heterocycloalkynyl". The heterocycloalkyl may include a monocyclic, bicyclic, spirocyclic or polycyclic (e.g., having two fused or bridged rings) ring system. In certain embodiments, the heterocycloalkyl is a monocyclic group having 1,2 or 3 heteroatoms independently selected from nitrogen, sulfur and oxygen. The ring-forming carbon atoms and heteroatoms of the heterocycloalkyl may be optionally oxidized to form oxo or sulfide ion groups or other oxidized bonds (e.g., C(O), S(O), C(S) or S(O)2, N-oxides, etc.), or nitrogen atoms may be quaternized. The heterocycloalkyl may be connected via ring-forming carbon atoms or ring-forming heteroatoms. In some embodiments, heterocycloalkyl contains 0 to 3 double bonds. In some embodiments, heterocycloalkyl contains 0 to 2 double bonds. The definition of heterocycloalkyl also includes a portion of an aromatic ring having one or more fused to the heterocycloalkyl ring (i.e., sharing a key with it), such as benzo derivatives of piperidine, morpholine, azacycloheptatriene or thienyl, etc. The heterocycloalkyl containing a fused aromatic ring can be connected via any ring-forming atoms, including the ring-forming atoms of the fused aromatic ring. Examples of heterocycloalkyl include, but are not limited to, azetidinyl, azepanyl, dihydrobenzofuranyl, dihydrofuranyl, dihydropyranyl, N-morpholinyl, 3-oxa-9-azaspiro[5.5]undecyl, 1-oxa-8-azaspiro[4.5]decyl, piperidinyl, piperazinyl, oxopiperazinyl, pyranyl, pyrrolidinyl, quinuclyl, tetrahydrofuranyl, tetrahydropyranyl, 1,2,3,4-tetrahydroquinolinyl, tropanediyl, 4,5,6,7-tetrahydrothiazolo[5,4-c]pyridinyl, 4,5,6,7-tetrahydro-1H-imidazole [4,5-c]pyridine, N-methylpiperidinyl, tetrahydroimidazolyl, pyrazolidinyl, butyrolactam, valerolactam, imidazolinone, hydantoin, dioxolane, phthalimide, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholinyl, thiomorpholinyl, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazinyl, pyranyl, pyridone, 3-pyrrolinyl, thiopyranyl, pyranone, tetrahydrothiophenyl, 2-azaspiro[3.3]heptyl, indolyl, and the like.

According to the present invention, "alkoxy" refers to an alkyl group bonded to the rest of the molecule via an ether oxygen atom. Representative alkoxy groups are those having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy and tert-butoxy. As used herein, "alkoxy" includes unsubstituted and substituted alkoxy groups, especially alkoxy groups substituted by one or more halogens. Preferred alkoxy groups are selected from _-OCH3 , _-OCF3 , _-CHF2O , _-CF3CH2O , -i- _PrO , -n-PrO, -i-BuO, -n-BuO or -t-BuO.

According to the present invention, the derivative is selected from:

The inventors have found that the compound having the above stereo configuration has tumor cell inhibition activity.

Preferably, the derivative is selected from:

When at least one of the above is present, it has higher tumor cell inhibitory activity and has great clinical value. As an ADC toxin, it can further expand the safety window of ADC drugs.

According to the present invention, a wedge-shaped solid key is used. and dotted wedge key To indicate the absolute configuration of a stereocenter, use a straight solid bond and straight dashed key To indicate the relative configuration of a stereocenter, use a wavy line Denotes a solid wedge bond or dotted wedge key Or use a wavy line Represents a straight solid bond or straight dashed key Unless otherwise stated, Indicates a single bond or a double bond.

In a second aspect, the present invention provides a method for preparing a camptothecin derivative, the method comprising the following steps:

The compound represented by formula (II) and the reactant are subjected to a substitution reaction to obtain a compound represented by formula (III);

wherein the reactant is a reactant containing a nitro group and/or a reactant containing a substituted acyl group; Ra is a nitro group and hydrogen; Rb is at least one selected from an acyl group, a substituted acyl group and hydrogen, preferably a substituted acyl group and hydrogen, and Ra and Rb are not H at the same time; or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV); or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV);

(3) subjecting the compound represented by formula (IV) to a Sandmeyer reaction; or

(1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III);

Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen;

(2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV);

(3) subjecting the compound represented by formula (IV) to a Buchwald-Hartwig coupling reaction;

The compound represented by formula (I) can be prepared by the above preparation method. The compound has high tumor cell inhibitory activity and great clinical value. As an ADC toxin, it can further expand the safety window of ADC drugs, and the preparation process is simple and easy to operate.

According to the present invention, before the substitution reaction, the compound shown in formula (II) is reacted with an amino protecting agent; after the amino group is protected, the amino group in the compound shown in formula (II) can be prevented from being substituted during the substitution process. Preferably, the amino protecting agent is selected from at least one of benzyl chloroformate, di-tert-butyl dicarbonate and 9-fluorenylmethyl chloroformate, and the corresponding amino protecting agent can be selected according to the substituent group and the reaction conditions. The amino protecting agent can also adopt other derivatives of the same type of benzyloxycarbonyl, tert-butyloxycarbonyl and fluorenylmethyloxycarbonyl according to different reaction conditions, or other amino protecting agents with the same effect, derivatives of methoxycarbonyl, allyloxycarbonyl or trityl. The inventors have found that methoxycarbonyl chloride can also be used to introduce methoxycarbonyl, allyl chloroformate to introduce allyloxycarbonyl, or trityl ether to introduce trityl to protect the amino group. The above-mentioned amino protecting agent can be deprotected according to different protecting groups using conditions such as catalytic hydrogenolysis, acidolysis cracking, Na/NH ₃ (liquid) reduction, Lewis acid, etc. After the substitution reaction is completed, the protection can be removed according to the removal conditions of the amino protecting agent, and then the corresponding substituent group can be introduced on the amino group. Alternatively, the compound represented by formula (II) can be subjected to substitution reaction with the reactant first, and then other groups can be introduced. If there are reactive groups on the compound that compete with the groups introduced later, the corresponding protecting agent can be selected to protect them before the substitution reaction.

According to the present invention, the reactant containing nitro groups is nitric acid; the nitric acid can be used alone or in combination with other reagents, and pure nitric acid, fuming nitric acid and concentrated nitric acid can be used as the reactant for nitro groups; concentrated nitric acid or fuming nitric acid mixed with concentrated sulfuric acid in a certain ratio can also be used as the reactant for nitro groups, and the ratio can be 1:3-6, or a mixing ratio well known to those skilled in the art; nitric acid acetic anhydride solution can also be used as the reactant for nitro groups, and the volume ratio of nitric acid to acetic anhydride is 1:50-70.

According to the present invention, when the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group, the side chain of the derivative contains an -OH group that is easy to connect, and has good cell activity, and is suitable as a small molecule toxin for ADC. Further preferably, when the α-hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group containing C1-C6 alkanes, at least one of an α-hydroxy-substituted acyl group containing C1-C6 straight-chain alkanes, an α-hydroxy-substituted acyl group containing C1-C6 branched alkanes, and an α-hydroxy-substituted acyl group containing cycloalkyl groups can be used. The α-hydroxyl group containing C1-C6 straight-chain alkanes, C1-C6 branched alkanes, or cycloalkyl groups can retain the tumor cell inhibitory activity of camptothecin derivatives while optimizing the drugability and inhibitory activity of camptothecin derivatives. The alkoxy substituted acyl group is an alkoxy substituted acyl group containing a heterocycle and/or an alkoxy substituted acyl group containing a nitro substituted imidazole ring. The inventors found during the research that when the alkoxy substituted acyl group is an alkoxy substituted acyl group containing a heterocycle and/or an alkoxy substituted acyl group containing a nitro substituted imidazole ring, the alkoxy substituted acyl group can be self-eliminated under the action of a reductase to generate a camptothecin derivative with better tumor cell inhibitory activity. More preferably, when the reactant containing the substituted acyl group is selected from 2-hydroxyacetic acid, phenyl chloroformate and chloroformic acid (1-methyl-2-nitro-1H-imidazol-5-yl) methyl ester, the synthetic route is short, the reaction conditions are simple, and the operation is easy. The synthesized compound has higher tumor cell inhibitory activity and better safety, and has a larger safety window as an ADC toxin.

According to the present invention, in step (2), the reduction reaction includes contacting the compound represented by formula (III) with a reducing agent; the reducing agent can be tetrahydroxydiboron, sodium borohydride, lithium aluminum tetrahydride or hydrogen gas alone, or different reducing agents can be selected according to the substituent groups. Catalysts such as 4,4'-bipyridine, Raney nickel (Raney Ni), palladium carbon, platinum carbon, etc. can also be added to the reduction reaction system to shorten the reaction process. Preferably, when the reducing agent is tetrahydroxydiboron and/or sodium borohydride, the reduction effect is better. When the reducing agent is tetrahydroxydiboron, 4,4'-bipyridine can be added as a catalyst to shorten the reduction reaction time.

According to the present invention, when the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, the substitution reaction comprises:

The compound represented by formula (II) and a reactant containing a nitro group are subjected to a substitution reaction to obtain a compound represented by formula (V); and the compound represented by formula (V) and a reactant containing a substituted acyl group are subjected to a substitution reaction;

The inventors have found that the above-mentioned substitution step can improve the yield, reduce the generation of by-products, and the reaction conditions are mild and easy to operate.

According to the present invention, when the reactant is a reactant containing a nitro group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, the inert gas is selected from xenon, argon or nitrogen, the temperature is -4-0°C, specifically -4°C, -3°C, -2°C, -1°C, 0°C, or any value between the above two values, the time is 50-70min, specifically 50min, 55min, 60min, 65min, 70min, or any value between the above two values;

When the reactant is a reactant containing a substituted acyl group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, the inert gas is selected from xenon, argon or nitrogen, the temperature is 25-30°C, specifically 25°C, 26°C, 27°C, 28°C, 29°C, 30°C, or any value between the above two values, and the time is 120-130min, specifically 120min, 121min, 122min, 123min, 124min, 125min, 126min, 127min, 128min, 129min, 130min, or any value between the above two values;

The conditions of the reduction reaction at least meet the following requirements: inert gas protection, the inert gas is selected from xenon, argon or nitrogen, the temperature is 20-25°C, specifically 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, or any value between the above two values, and the time is 4-10min, specifically 4min, 5min, 6min, 7min, 8min, 9min, 10min, or any value between the above two values.

The reaction solvent used in the above preparation method can be ethyl acetate (EA), dimethylformamide (DMF), dichloromethane (DCM) and 1,2-dichloroethane (DCE) or a reaction solvent known to those skilled in the art; after the above reaction is completed, the target compound is obtained by post-treatment. The post-treatment process includes quenching, extraction, washing and purification, wherein water can be selected as a quenching agent for the quenching reaction, and the amount is about 100-500mL, and the amount can be appropriately expanded or reduced according to the amount of the reactants; methanol, acetonitrile, tetrahydrofuran, ethyl acetate, dichloromethane and other solvents can be used for extraction, and the amount of the extractant is about 300-500mL, and the amount can be appropriately expanded or reduced according to the amount of the reactants; washing can be carried out by washing the extractant with saturated salt water, saturated sodium carbonate aqueous solution and saturated sodium bicarbonate aqueous solution, and after washing, anhydrous sodium sulfate or anhydrous magnesium sulfate and other reagents can be used for drying; before purification, a small amount of crude product solution is obtained by vacuum concentration and then purified by silica gel column, or directly purified by preparative liquid chromatography (pre-HPLC). In addition to the main reactants and reaction solvents, catalysts, reaction aids, etc. can be added to the reaction system to accelerate the reaction process and reduce the generation of by-products. Catalysts and reaction aids can be palladium carbon Pd/C, 4,4'-bipyridine, triethylamine (TEA or Et3N), N,N-diisopropylethylamine (DIEA) and 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), etc. During the reaction, a small amount of reactants can be extracted by syringe for thin layer chromatography (TLC) analysis or liquid chromatography-mass spectrometry (LC-MS) analysis, and the reaction can be quenched after the reaction is confirmed to be completed.

As a specific embodiment of the present invention, a specific method for introducing a halogen substituent is: converting an amine group into a halogen by a Sandmeyer reaction. Specifically, under the action of CuCN, the corresponding benzonitrile is obtained, and then treated with CuCl or CuBr to obtain the corresponding halogenated derivative. Alternatively, an iodinated derivative is obtained by heating with sodium iodide, and then treated with silver tetrafluoroborate to obtain a diazonium fluoroborate, and then heated to obtain a fluorinated derivative.

As a specific embodiment of the present invention, a specific method for introducing a hydroxyl substituent is as follows: a halogen atom-substituted compound and an amino compound are subjected to a Buchwald-Hartwig coupling reaction to obtain a substituted amino compound, and finally a hydroxyl-substituted compound in which Ra is a hydroxyl group is obtained by deprotection and hydrochloric acid acidification. Alternatively, an amino-substituted compound is mixed with nitrous acid and subjected to a Sandmeyer reaction to obtain a hydroxyl-substituted compound in which Ra is a hydroxyl group.

In addition to the above-mentioned preparation method, it is also possible to synthesize by combining the known techniques disclosed in the prior art with the method disclosed in the present invention. In addition, the solvent, temperature and other reaction conditions mentioned here can be changed according to different reactants. The starting materials for the synthesis of camptothecin derivatives can be synthesized or commercially available. The compounds of the present invention and other related compounds with different substituents can be synthesized using known techniques and raw materials. The general method of compound preparation can be changed by using appropriate reagents and the conditions of introducing different groups in the molecular formula provided here.

Illustratively, the compound represented by formula (I) can be prepared by the method of general reaction scheme 1-3.

The general reaction scheme 1 is as follows:

Using exitecan (compound 1A) as a starting material, the amino group is protected by an amino protecting agent di-tert-butyl dicarbonate ((Boc) ₂ O) to obtain compound 1B, which is then subjected to a substitution reaction with a reactant containing a nitro group, using a mixed acid of nitric acid (HNO ₃ ) and acetic anhydride (Ac ₂ O) as the reactant containing a nitro group to obtain compound 1C; after the substitution reaction with the reactant containing a nitro group is completed, the Boc protecting group of the amino group of compound 1C is removed by trifluoroacetic acid (TFA) to obtain compound 1; compound 1 is subjected to a substitution reaction with a reactant containing a substituted acyl group, using 2-hydroxyacetic acid as the reactant containing a substituted acyl group to obtain compound 2; compound 2 is subjected to a contact reaction with a reducing agent, using tetrahydroxydiboron as the reducing agent, to obtain compound 3;

The general reaction scheme 2 is as follows:

Compound 1C is directly contacted with a reducing agent, tetrahydroxydiboron, to obtain compound 1D, and the Boc protecting group of the amino group of compound 1D is removed by trifluoroacetic acid (TFA) to obtain compound 4;

The general reaction scheme 3 is as follows:

Using exitecan (compound 1A) as the starting material, compound 1F was obtained by reaction with phenyl chloroformate, and then coupled with (1-methyl-2-nitro-1H-imidazol-5-yl)methanol to obtain compound 5.

After the nitro group of imidazole in compound 5 enters the tumor tissue, it is reduced to an amine group under the catalysis of nitroreductase to form an unstable intermediate 5A, which then generates the active substance ixetemcan (compound 1A) through a self-elimination reaction;

After entering the tumor tissue, the 8-nitro group in compound 2 is reduced to an amine group under the catalysis of nitroreductase to generate the active substance compound 3;

After entering the tumor tissue, the 8-nitro group in compound 1 is reduced to an amine group under the catalysis of nitroreductase to generate the active substance compound 4;

According to the present invention, although the numerical ranges and parameters used to define the broader scope of the present invention are approximate values, the relevant numerical values in the specific embodiments have been presented as accurately as possible. However, any numerical value is inherently inevitably containing standard deviations due to individual test methods. Here, "about" usually means that the actual value is within plus or minus 10%, 5%, 1% or 0.5% of a specific value or range. Alternatively, the term "about" represents that the actual value falls within the acceptable standard error of the mean value, depending on the consideration of those skilled in the art. Except for the experimental examples, or unless otherwise explicitly stated, it should be understood that all ranges, quantities, values and percentages used herein (for example, to describe the amount of material, the length of time, temperature, operating conditions, quantitative ratios and other similar ones) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the attached claims are all approximate values and can be changed as needed. At least these numerical parameters should be understood as the indicated significant digits and the values obtained by using the general rounding method.

In the third aspect, the present invention provides the use of the derivatives described in the first aspect and the derivatives prepared by the method described in the second aspect in the preparation of anti-tumor drugs; the anti-tumor drugs can use the camptothecin derivatives as active ingredients of chemical drugs, or use the camptothecin derivatives as antibody-drug conjugates of small molecule toxins; preferably, the anti-tumor drug can better exert anti-tumor effects when it is an antibody-drug conjugate, wherein the antibody can be a monoclonal antibody, a bispecific antibody or a nanoantibody, specifically HER2, HER3, CD25, CD30 and c-MET, etc., or antibodies that are currently in public use. The camptothecin derivatives can also be used as sensitizers for radiotherapy for adjuvant therapy.

According to the present invention, preferably, the anti-tumor drug is a hypoxia activated prodrug (HAP); further preferably, the anti-tumor drug is a small molecule active drug. Hypoxia activated prodrug refers to a drug that can be activated and release drug molecules in a hypoxic environment. This type of drug is stable in a normoxic environment, but can be activated by a single electron reduction process in an oxygen-deficient environment, thereby exerting its cytotoxic effect. The main feature of hypoxia activated prodrugs is that they only play a role in killing tumor cells in an oxygen-deficient environment, thereby reducing side effects on normal tissues. When administering the compounds of the present invention, they can be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), or topically. Solid dosage forms for oral administration include capsules, tablets, pills, powders, and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starches, lactose, sucrose, glucose, mannitol, and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinyl pyrrolidone, sucrose, and acacia; (c) humectants, for example, glycerol; (d) disintegrants, for example, agar, calcium carbonate, potato starch or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) solubilizers, for example, paraffin; (f) absorption accelerators, for example, quaternary ammonium compounds; (g) wetting agents, for example, cetyl alcohol and glyceryl monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In the case of capsules, tablets and pills, the dosage forms may also comprise buffering agents.

According to the present invention, solid dosage forms such as tablets, pills, capsules, pills and granules can be prepared using coatings and shell materials, such as enteric coatings and other materials known in the art. They may contain opacifiers, and the release of the active compound or compounds in such compositions can be released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxes. If necessary, the active compound can also be formed into microcapsules with one or more of the above-mentioned excipients.

According to the present invention, the liquid dosage form for oral administration includes a pharmaceutically acceptable emulsion, solution, suspension, syrup or tincture. In addition to the active compound, the liquid dosage form may contain an inert diluent conventionally used in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oil, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or a mixture of these substances, etc.

According to the present invention, in addition to these inert diluents, the composition may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening agents, flavoring agents and perfumes.

According to the invention, in addition to the active compounds, suspensions may contain suspending agents such as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances and the like.

According to the present invention, the composition for parenteral injection can comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for redissolving into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

According to the present invention, the dosage form of the compounds of the present invention for topical administration includes ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under aseptic conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required if necessary.

According to the present invention, the compound can be administered alone or in combination with other pharmaceutically acceptable compounds or physical therapies such as radiotherapy, photodynamic therapy, and ablation therapy. When using a pharmaceutical composition, a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, wherein the dosage during administration is a pharmaceutically effective dosage, and for a person weighing 60 kg, the daily dosage is usually 1-2000 mg, preferably 50-1000 mg. Of course, the specific dosage should also take into account factors such as the route of administration and the patient's health status, which are all within the skill range of skilled physicians.

According to the present invention, the terms "treatment", "treatment process" or "therapy" as used herein include alleviating, inhibiting or improving the symptoms or conditions of a disease; inhibiting the occurrence of complications; improving or preventing potential metabolic syndrome; inhibiting the occurrence of a disease or symptom, such as controlling the development of a disease or condition; alleviating a disease or symptom; reducing a disease or symptom; alleviating complications caused by a disease or symptom, or preventing or treating signs caused by a disease or symptom. As used herein, a compound or pharmaceutical composition, after administration, can improve a disease, symptom or condition, especially improve its severity, delay the onset, slow the progression of the disease, or reduce the duration of the disease. Whether fixed or temporary administration, continuous administration or intermittent administration, can be attributed to or related to the administration.

According to the present invention, "active ingredient" refers to the compound shown in formula (I), and the pharmaceutically acceptable inorganic or organic salt of the compound shown in formula (I). The compound of the present invention may contain one or more asymmetric centers (chiral centers or chiral axes), and therefore appear in the form of racemates, racemic mixtures, single enantiomers, diastereoisomer compounds and single diastereomers. The asymmetric center that may exist depends on the properties of the various substituents on the molecule. Each such asymmetric center will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures and pure or partially pure compounds are included within the scope of the present invention. The present invention is meant to include all such isomeric forms of these compounds.

According to the present invention, the terms "compound", "composition", "agent" or "medicine or medicament" are used interchangeably herein and refer to a compound or composition that, when administered to a subject (human or animal), is capable of inducing a desired pharmaceutical and/or physiological response through local and/or systemic effects.

According to the present invention, the term "administered, administering or administration" refers to directly administering the compound or composition, or administering a prodrug, derivative, or analog of the active compound.

According to the present invention, "safe and effective amount" means: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound is determined based on the specific circumstances such as the age, condition, and course of treatment of the subject.

According to the present invention, "pharmaceutically acceptable excipients or carriers" refer to: one or more compatible solid or liquid fillers or gel substances, which are suitable for human use and must have sufficient purity and sufficiently low toxicity. "Compatibility" here means that the components in the composition can be mixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Some examples of pharmacologically acceptable excipients or carriers include cellulose and its derivatives (such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.), gelatin, talc, solid lubricants (such as stearic acid, magnesium stearate), calcium sulfate, vegetable oils (such as soybean oil, sesame oil, peanut oil, olive oil, etc.), polyols (such as propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifiers (such as ), wetting agents (such as sodium lauryl sulfate), colorants, flavoring agents, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

According to the present invention, the present invention provides a method for treating a disease using the compound, antibody-drug conjugate, pharmaceutical composition, or drug-mechanical composition of the present invention, including a combination of radiotherapy and chemotherapy, wherein the disease includes but is not limited to cancer or benign tumors.

According to the present invention, in some embodiments, a method for cancer treatment is provided, the method comprising administering to an individual in need an effective amount of any of the foregoing compounds, a pharmaceutical composition of an antibody-drug conjugate. In other embodiments, the cancer is a blood cancer and a solid tumor, including but not limited to leukemia, breast cancer, lung cancer, pancreatic cancer, colon cancer, bladder cancer, brain cancer, urothelial cancer, prostate cancer, liver cancer, ovarian cancer, head and neck cancer, gastric cancer, mesothelioma or all cancer metastases. According to the present invention, the tumor can be a central nervous system tumor, including a primary tumor and a tumor metastasized to the central nervous system, selected from at least one of colon cancer, gastric cancer, breast cancer and lung cancer. Preferably, the tumor is selected from an in situ lesion and/or a metastatic lesion. Further preferably, the tumor is selected as a central tumor and/or a central metastatic lesion. The inventors have found that the camptothecin derivatives provided by the present invention have high tumor cell inhibitory activity when used for the treatment of the above-mentioned diseases, and have great clinical use value. As ADC toxins, they can further expand the safety window of ADC drugs, and there is a large difference in activity before and after activation as prodrugs. Before activation, they will not cause damage to normal tissues, and will only exert their activity after activation in tumor tissues. They have higher tumor cell inhibitory activity and better safety.

In a fourth aspect, the present invention provides an antibody-drug conjugate, in which the derivative described in the first aspect and/or the derivative prepared by the method described in the second aspect are connected to the antibody through a linker and/or directly coupled to the antibody. The antibody in the above-mentioned antibody-drug conjugate can be a monoclonal antibody, a bispecific antibody or a nanoantibody, specifically HER2, HER3, CD25, CD30 and c-MET, etc., or an antibody currently in public use; the linker can be a cleavable linker or a non-cleavable linker, the non-cleavable linker is a maleamide-type linker and/or a thiol hexamethyleneamide-type linker; the cleavable linker is a hydrazone bond, a carbonate, a polypeptide, a disulfide bond-type linker, or other linkers currently in public use. The camptothecin derivative provided by the present invention contains different substituents in its structure with different activities, and a derivative that can be directly coupled to the antibody is selected or the linker is fixed to the antibody through antibody modification technology, and then the camptothecin derivative is coupled to the antibody.

Other terms in the present invention may be interpreted in a conventional manner in the art.

The present invention uses the following abbreviations: 1,2-dichloroethane (DCE); dichloromethane (DCM); methanol (MeOH); tetrahydrofuran (THF); dimethylformamide (DMF); triethylamine (TEA or Et ₃ N); trifluoroacetic acid (TFA); di-tert-butyl dicarbonate ((Boc) ₂ O); 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU); N,N-diisopropylethylamine (DIEA); palladium on carbon (Pd/C); acetic anhydride (Ac ₂ O); nuclear magnetic resonance (NMR); liquid chromatography-mass spectrometry (LC-MS); thin layer chromatography (TLC); preparative liquid chromatography (pre-HPLC). The present invention will be described in detail below through examples.

In the following examples, unless otherwise specified, the reagents or raw materials used were conventional biochemical reagent grade products, and Deruxtecan was purchased from Haoyuan Biopharmaceutical Technology Co., Ltd.

In the following examples, 1H-NMR (nuclear magnetic resonance hydrogen spectrum) was measured using a Varian Mercury 400 nuclear magnetic resonance instrument, and chemical shifts were expressed in δ (ppm); the silica gel used for separation was 200-300 mesh unless otherwise specified, and the ratios of the eluents were all by volume.

Unless otherwise specified, the room temperature in the following examples means 25±5°C.

Example 1: Synthesis of Compound 1

The specific process includes:

(1) Synthesis of Compound 1B

1 g of isotecan (compound 1A, 1.88 mmol) was dissolved in 20 mL of DMF, and 570 mg of triethylamine (Et ₃ N, 5.64 mmol) and 1 g of di-tert-butyl dicarbonate ((Boc) ₂ O, 4.59 mmol) were added and mixed under nitrogen protection, and the mixture was stirred at room temperature under nitrogen protection. After 2 hours, the reaction was complete by TLC detection, and the mixture was poured into 500 mL of water, and then extracted three times with 500 mL of ethyl acetate (EA). The combined organic phase was washed twice with saturated brine, dried with anhydrous sodium sulfate (Na ₂ SO ₄ ), filtered, concentrated and purified by silica gel column (eluent was MeOH:DCM in a volume ratio of 1:5) to obtain yellow oily compound 1B (900 mg, yield 90%). LC-MS (ESI, M+H) ⁺ =536.1.

(2) Synthesis of Compound 1C

At 0°C under nitrogen protection, 300 mg of compound 1B (0.56 mmol) was dissolved in a mixed acid of 0.15 mL of nitric acid and 9 mL of Ac ₂ O, and stirred at 0°C under nitrogen protection for 1 hour. After concentration under reduced pressure, the mixture was purified by silica gel column (the eluent was MeOH:DCM in a volume ratio of 1:5) to obtain compound 1C (160 mg, yield 49%) as a white solid (ESI, M+H) ⁺ =581.3.

(3) Synthesis of Compound 1

160 mg of compound 1C (0.28 mmol) was dissolved in 5 mL of DCM, 1 mL of TFA was added at 0°C under nitrogen protection, and the mixture was stirred for 1 hour at 0°C under nitrogen protection. After concentration under reduced pressure, the mixture was purified by preparative liquid chromatography (Pre-HPLC) to obtain yellow solid compound 1 (30 mg, yield 23%). LC-MS (ESI, M+H) ⁺ = 481.2. The corresponding hydrogen spectrum is shown in Figure 1.

¹ H-NMR (400MHz, DMSO-d ₆ )δ8.47(s,3H),7.73(d,J＝10.7Hz,1H),6.76(s,1H),5.72(s,1H),5.49(s,3 H),5.12(s,1H),3.09(s,2H),2.43(s,3H),2.26-1.99(m,4H),0.94(s,3H).

Example 2: Synthesis of Compound 2

The specific process includes:

At room temperature and under nitrogen protection, 68 mg of compound 1 (0.14 mmol), 32 mg of 2-hydroxyacetic acid (0.42 mmol) and 80 mg of HATU (0.21 mmol) were dissolved in 20 mL of DMF, 54 mg of DIEA (0.42 mmol) was added, and stirred for 2 hours at room temperature and under nitrogen protection. After reduced pressure concentration, the mixture was purified by Pre-HPLC to obtain compound 2 (17 mg, yield 22%) as a yellow solid. LC-MS (ESI, M+H) ⁺ =539.3. The corresponding hydrogen spectrum is shown in Figure 2.

¹ H-NMR (400MHz, DMSO-d ₆ )δ8.38(d,J＝8.9Hz,1H),7.61(d,J＝10.7Hz,1H),6.70(s,1H),5.58(s,1H),5.45(s,3H),5 .23(s,2H),3.94(s,2H),3.24–3.09(m,2H),2.40(s,3H),2.26–1.97(m,4H),0.92(s,3H).

Example 3: Synthesis of Compound 3

The specific process includes:

14 mg of compound 2 (0.02 mmol) was dissolved in 3 mL of DMF, 6.99 mg of tetrahydroxydiboron (0.07 mmol) and 0.41 mg of 4,4'-bipyridine were added at 25°C under nitrogen protection, mixed and stirred for 4 minutes, concentrated under reduced pressure, and purified by Pre-HPLC to obtain yellow solid compound 3 (6.7 mg, yield 45%). LC-MS (ESI, M+H) ⁺ = 509.1. The corresponding hydrogen spectrum is shown in Figure 3.

¹ H-NMR (400MHz, DMSO-d ₆ )δ8.35(d,J＝8.9Hz,1H),7.61(d,J＝11.0Hz,1H),6.98(s,1H),6.48(s,2H),5.49(s,2H),5.39(s,1H),5.31(s,1H),5. 08(d,J＝7.3Hz,2H),3.95(d,J＝5.8Hz,2H),3.18–3.04(m,2H),2.33(s,3H),2.04(dd,J＝7.4,2.1Hz,4H),0.89(s,3H).

Example 4: Synthesis of Compound 4

(1) Synthesis of Compound 1D

800 mg of compound 1C was dissolved in 10 mL of DMF, and then 372 mg of tetrahydroxydiboron (4.1 mmol) and 21.5 mg of 4,4'-bipyridine (0.13 mmol) were added. The mixture was stirred at 25°C for 5 minutes under nitrogen protection. After the reaction was completed, 100 mL of water was added to terminate the reaction, and yellow solid compound 1D (240 mg, yield 31%) was collected by filtration. LC-MS (ESI, M+H) ⁺ = 551.2.

(2) Synthesis of Compound 4

240 mg of compound 1D (0.43 mmol) was dissolved in 5 mL of DCM, 1 mL of TFA was added at 25°C under nitrogen protection, and the mixture was stirred for 1 hour at 25°C under nitrogen protection. After concentration under reduced pressure, the mixture was purified by preparative HPLC to obtain yellow compound 4 (40 mg, yield 20%). LC-MS (ESI, M+H) ⁺ = 451.2. The corresponding hydrogen spectrum is shown in FIG4 .

¹ H-NMR (400MHz, DMSO-d ₆ )δ8.41(s,3H),7.74(d,J＝11.0Hz,1H),7.07(s,2H),6.55(s,1H),5.59(s,1H),5.44(s,1H),5.38 (s,2H),5.02(s,1H),3.24(s,1H),3.06(t,J=13.8Hz,1H),2.38(s,3H),2.06(s,4H),0.92(s,3H).

Example 5: Synthesis of Compound 5

(1) Synthesis of Compound 1F

150 mg of exitecan (Compound 1A, 0.3 mmol) was dissolved in 40 mL of DMF, 3 mL of THF was added at 0°C under nitrogen protection to dissolve 107 mg of phenyl chloroformate (0.6 mmol) solution, 86 mg of Et ₃ N (0.86 mmol) was added, and the mixture was stirred at room temperature for 2 hours. 100 mL of water was added to terminate the reaction, and yellow solid Compound 1F (125 mg, yield 59%) was obtained by filtration and separation. LC-MS (ESI, M+H) ⁺ = 556.0.

(2) Synthesis of Compound 5

40 mg of compound 1F (0.07 mmol) was dissolved in 1 mL of DCE, and 22 mg of (1-methyl-2-nitro-1H-imidazol-5-yl)methanol (0.14 mmol) was added at 25°C under nitrogen protection. After stirring at 25°C for 5 minutes, the reaction temperature was raised to 85°C and stirring was continued for 16 hours under nitrogen protection. After purification by preparative HPLC, a white solid compound 5 (20.9 mg, yield 45%) was obtained. LC-MS (ESI, M+H) ⁺ = 619.0. The corresponding hydrogen spectrum is shown in Figure 5.

¹ H-NMR (400MHz, DMSO-d ₆ )δ8.23(s,1H),7.75(s,1H),7.29(s,2H),6.51(s,1H),5.32(d,J＝61.4Hz,7H),4.01(s,3H),3.28-3.20( m,1H),3.16-3.04(m,1H),2.37(s,3H),2.29-2.18(m,1H),2.18-2.06(m,1H),1.86(s,2H),0.87(s,3H).

Test Example 1

Cell antiproliferative activity assay

Camptothecin derivative compounds 1-5 prepared in Examples 1-5 of the present invention, wherein compounds 1 and 4 have similar structures, compounds 2 and 3 have similar structures, and compound 5 can be metabolized to generate exotecan (compound 1A) in tumor cells. The cell antiproliferative activity of camptothecin derivatives was determined by measuring the antiproliferative activity of compounds 1-5 and Deruxtecan against gastric cancer (MKN45), colon cancer (HCT116), breast cancer (MCF7) and lung cancer (A549) cells.

The specific testing process is as follows:

The culture dish containing MKN45, HCT116 cells and culture medium was placed in a 37°C, 5% _CO2 incubator for culture, cells with good growth were taken, the original culture medium was discarded, and the cells were resuspended and counted respectively by the culture medium; the cell suspension was added to a 96-well plate, with 4000 cells per well, and incubated in a 37°C, 5% _CO2 cell culture incubator for 24 hours; the drug dilution of compound 1-5 and the addition of the drug were cultured for 72 hours, and the ATP content of cancer cells in each well was measured by CellTiter-Lumi. ATP is the direct source of cell energy, so the proliferation and toxicity of cells can be directly reflected by detecting the ATP content in the cell. The Prism software calculates the _IC50 (half-inhibitory concentration) value by the ATP content value. The software uses the inhibition rate as the y value and the drug concentration as the x value for four-parameter curve fitting, and records the drug concentration value corresponding to the inhibition rate value between the maximum inhibition rate and the minimum inhibition rate (the software defaults to the _IC50 value). The _IC50 calculation results are shown in Table 1:

Table 1

NA indicates that the data is under testing and/or validation.

The culture dish containing MCF7, A549 cells and culture medium was placed in a 37°C, 5% _CO2 incubator for culture, cells in good growth state were taken, the original culture medium was discarded, and the cells were resuspended and counted respectively by the culture medium; the cell suspension was added to a 96-well plate, with 10,000 cells per well, and incubated in a 37°C, 5% _CO2 cell incubator for 24 hours; the drug of compound 1-5 was diluted and added to the drug for 72 hours, and then the reduction effect of the living cells in each well on the MTS tetrazolium compound was measured by adding the MTS substrate to generate a colored formazan dye soluble in the cell culture medium. The proliferation and toxicity state of the cells can be directly reflected by detecting the OD light absorption value at 490nm in the culture medium. The survival rate was obtained by calculating the ratio of the OD values of the drug-treated group and the control non-drug-treated group and multiplying it by 100%. The Prism software calculates the IC ₅₀ (half-inhibitory concentration) value based on the survival rate of each sample. The software uses the survival rate as the y value and the drug concentration as the x value to perform a four-parameter curve fit, and records the drug concentration value corresponding to the survival rate value between the maximum survival rate and the minimum survival rate (the software defaults to the IC ₅₀ value). The IC ₅₀ calculation results are shown in Table 2:

Table 2

NA indicates that the data is under testing and/or validation.

Compared with Deruxtecan, the nitro-containing compounds 1, 2 and 5 prepared by the present invention have very weak in vitro anti-proliferation activity against MKN45, HCT116, MCF7 and A549 cells, but the amino compound 3 with a similar structure to compound 1 has significantly better anti-proliferation activity in HCT116, MCF7 and A549 cells than Deruxtecan, so the nitro compounds 1, 2 and 5 can be used as prodrugs and converted into active amino compounds under the catalysis of nitroreductase in hypoxic tumor cells, thereby killing cancer cells. Active amino compounds contain -OH or _-NH2 groups that are easy to connect in the side chain and have strong cell activity, so they are suitable as small molecule toxins for ADC.

Test Example 2

Evaluation of the efficacy of the compounds prepared in Example 2 and Example 3 in the Balb/c Nude mouse HCT116 subcutaneous tumor model

HCT116 (human colon cancer cells, Shanghai Runnuo Biotechnology Co., Ltd.) cells were cultured in 5A medium containing 15wt% fetal bovine serum, 100 units/ml penicillin and 100 μg/ml streptomycin, and cultured continuously in a cell culture incubator at 37°C with 5vol% _CO2 , and passaged 2-3 times a week. When the cells grew to the logarithmic growth phase, the cells were collected and washed twice with serum-free medium, and finally resuspended with serum-free 5A for cell counting, and the cells were collected, counted and inoculated into 36 6-8 week old female Balb/c Nude mice (purchased from Shanghai Jihui Experimental Animal Breeding Co., Ltd., and raised in the animal room of Shanghai Runnuo Biotechnology Co., Ltd.).

Each Balb/c Nude mouse was subcutaneously inoculated with 0.1 mL of HCT116 tumor cell solution containing 50 wt% Matrigel (about 5.00 × 10 ⁶ cells/mouse) in the right armpit, and the day of inoculation was designated as day 0. When the mean tumor volume reached about 99 cubic millimeters, 24 mice were selected and randomly divided into groups according to tumor size and body weight using Excel, with 8 mice in each group. The drugs were administered on the day of grouping, and the dosage was calculated as follows:

The required dosage of compound 2 prepared in Example 2 is (average weight of mice = 30 g plus 30% loss): 25 mg; (2+6) mg/kg×0.03 kg/mouse×8 mice×10 times×130%=25 mg.

The required dosage of compound 3 prepared in Example 3 is (average weight of mice = 30 g plus 30% loss): 6 mg;

2mg/kg×0.03kg/animal×8animals×10 times×130%=6mg.

The first group (G1) was a vehicle control group (5wt% mannitol + 95wt% citrate buffer, pH 6.5), the second group was compound 2 prepared in Example 2 (6 mg/kg in the first week, 12 mg/kg in the second week), and the third group was compound 3 prepared in Example 3 (2 mg/kg in the first week, 1 mg/kg in the second week). Subcutaneous administration (volume 10 mg/kg body weight) was performed once a day for 5 consecutive days per week, followed by 2 days of rest, for a total of 2 weeks of administration and 4 weeks of observation.

The change in the weight of mice during the experiment is shown in Figure 6. It can be seen from Figure 6 that the weight of mice inoculated with compound 3 prepared in Example 3 dropped to the lowest value around the 7th day of inoculation, and although there was an increase thereafter, the weight was stable, and the survival period of the mice was significantly prolonged compared with the mice in the compound 2 prepared in Example 2 and the vehicle control group. The change in tumor volume during inoculation is shown in Figure 7. It can be seen from Figure 7 that compound 3 prepared in Example 3 can effectively inhibit the growth of tumor volume and prolong the survival period of mice.

The preferred embodiments of the present invention are described in detail above, but the present invention is not limited thereto. Within the technical concept of the present invention, the technical solution of the present invention can be subjected to a variety of simple modifications, including the combination of various technical features in any other suitable manner, and these simple modifications and combinations should also be regarded as the contents disclosed by the present invention and belong to the protection scope of the present invention.

Claims (10)

A camptothecin derivative, characterized in that the derivative is a compound represented by formula (I) and/or a pharmaceutically acceptable salt thereof:
Wherein, R ₁ is at least one selected from H, -NO ₂ , -NH ₂ , -OH and halogen; R ₂ is at least one selected from H, acyl and substituted acyl, and R ₁ and R ₂ are not H at the same time. The derivative according to claim 1, characterized in that the halogen is selected from at least one of F, Cl and Br; Preferably, the R ₁ is selected from at least one of H, -NO ₂ and -NH ₂ , and R ₂ is hydrogen or a substituted acyl group; Preferably, the substituted acyl group is at least one of a hydroxy substituted acyl group and/or an alkoxy substituted acyl group; Preferably, the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group; further, it is an α-hydroxy-substituted acyl group containing a C1-C6 alkane, and more preferably, it is an α-hydroxyacetyl group; the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocycle and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and further preferably, it is an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and more preferably, it is a (1-methyl-2-nitro-1H-imidazol-5-yl)methoxyacyl group. The derivative according to claim 1 or 2, characterized in that the derivative is selected from:

At least one of; Preferably, the derivative is selected from: At least one of . A method for preparing a camptothecin derivative, characterized in that the method comprises the following steps: The compound represented by formula (II) and the reactant are subjected to a substitution reaction to obtain a compound represented by formula (III); wherein the reactant is a reactant containing a nitro group and/or a reactant containing a substituted acyl group; Ra is a nitro group and hydrogen; Rb is at least one selected from an acyl group, a substituted acyl group and hydrogen, preferably a substituted acyl group and hydrogen, and Ra and Rb are not H at the same time; or (1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III); wherein the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen; (2) subjecting the compound represented by formula (III) to a reduction reaction to obtain the compound represented by formula (IV); or (1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III); Wherein, the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen; (2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV); (3) subjecting the compound represented by formula (IV) to a Sandmeyer reaction; or (1) subjecting the compound represented by formula (II) and a reactant to a substitution reaction to obtain a compound represented by formula (III); wherein the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, or a reactant containing a nitro group; Ra is a nitro group, and Rb is a substituted acyl group or hydrogen; (2) subjecting the compound represented by formula (III) to a reduction reaction to obtain a compound represented by formula (IV); (3) subjecting the compound represented by formula (IV) to a Buchwald-Hartwig coupling reaction;
The method according to claim 4, characterized in that before the substitution reaction, the compound represented by formula (II) is reacted with an amino protecting agent; Preferably, the amino protecting agent is selected from at least one of benzyl chloroformate, di-tert-butyl dicarbonate and 9-fluorenylmethyl chloroformate. The method according to claim 4, characterized in that the reactant containing a nitro group is nitric acid; The substituted acyl group is a hydroxy substituted acyl group and/or an alkoxy substituted acyl group; Preferably, the hydroxy-substituted acyl group is an α-hydroxy-substituted acyl group; further, it is an α-hydroxy-substituted acyl group containing a C1-C6 alkane, and more preferably, it is an α-hydroxyacetyl group; the alkoxy-substituted acyl group is an alkoxy-substituted acyl group containing a heterocyclic ring and/or an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring, and further preferably, it is an alkoxy-substituted acyl group containing a nitro-substituted imidazole ring; More preferably, the reactant containing a substituted acyl group is selected from 2-hydroxyacetic acid, phenyl chloroformate and (1-methyl-2-nitro-1H-imidazol-5-yl)methyl chloroformate. The method according to claim 4, characterized in that in step (2), the reduction reaction comprises contacting the compound represented by formula (III) with a reducing agent; Preferably, the reducing agent is tetrahydroxydiboron and/or sodium borohydride. The method according to any one of claims 4 to 7, characterized in that when the reactant is a mixture of a reactant containing a nitro group and a reactant containing a substituted acyl group, the substitution reaction comprises: The compound represented by formula (II) and a reactant containing a nitro group are subjected to a substitution reaction to obtain a compound represented by formula (V); and the compound represented by formula (V) and a reactant containing a substituted acyl group are subjected to a substitution reaction;
Preferably, when the reactant is a reactant containing a nitro group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, temperature of -4-0°C, time of 50-70 min; When the reactant is a reactant containing a substituted acyl group, the conditions of the substitution reaction at least meet the following requirements: inert gas protection, temperature of 25-30° C., and time of 120-130 min; The reduction reaction conditions at least meet the following requirements: inert gas protection, temperature of 20-25° C., and time of 4-10 min. Use of the derivative according to any one of claims 1 to 3 and the derivative prepared by the method according to any one of claims 4 to 8 in the preparation of anti-tumor drugs; Preferably, the anti-tumor drug is an antibody-drug conjugate; Preferably, the anti-tumor drug is a hypoxia-activated prodrug; Preferably, the anti-tumor drug is a small molecule active drug; Preferably, the tumor is selected from at least one of colon cancer, gastric cancer, breast cancer and lung cancer; Preferably, the tumor is an in situ lesion and/or a metastatic lesion; Preferably, the tumor is selected as a central tumor and/or a central metastatic lesion. An antibody-drug conjugate, characterized in that, in the antibody-drug conjugate, the derivative described in any one of claims 1 to 3 and/or the derivative prepared by the method described in any one of claims 4 to 8 is connected to the antibody through a linker and/or is directly coupled to the antibody.