Novel camptothecin derivatives, compositions containing same and uses thereof

Abstract

The present invention provides a class of camptothecin derivatives, compositions containing the same, and uses thereof. Specifically, the present invention provides a compound represented by formula (1) and a method for preparing the same, as well as the use of the compound represented by formula (1) and its optical isomers, crystal forms, and pharmaceutically acceptable salts in the preparation of a drug for treating cancer.

Description

[0001] This application claims priority to Chinese Patent Application No. 2020114637044, filed on December 11, 2020. This application incorporates the entire text of the aforementioned Chinese Patent Application. Technical Field

[0002] The present invention relates to the field of medicinal chemistry, and more particularly to a class of novel camptothecin derivatives, compositions containing the same and uses of the compounds. Background Art

[0003] DNA topoisomerases are located in the cell nucleus and act on DNA, thereby participating in cell replication, transcription, and mitosis. The primary function of topoisomerases is to decompose the supercoiled structure of DNA. Topoisomerases are divided into topoisomerase I (TopoI) and topoisomerase II (TopoII). Inhibition of topoisomerases leads to the accumulation of large amounts of broken DNA in tumor cells, inducing tumor cell death. DNA topoisomerase I inhibitors, including camptothecin and its derivatives, are clinically used to treat malignant tumors.

[0004] Camptothecin was first isolated from the plant Camptotheca acuminata of the Davidiaceae family. It has strong cytotoxicity and is effective against malignant tumors such as gastrointestinal 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, and high toxicity, which limits its clinical application. Camptothecin derivatives can increase their water solubility by introducing water-soluble groups or preparing prodrugs, thereby improving their drugability. Several camptothecin derivatives with significantly improved solubility have been approved for marketing, such as topotecan and its carbamate prodrug irinotecan.

[0005] In addition to being used as chemotherapy drugs to treat tumors, camptothecin derivatives are also used to couple with antibodies as small molecule toxins (payloads) for antibody-drug conjugates (ADCs). ADCs couple antibodies and small molecule toxins, combining the specificity of antibodies for binding to tumor cell surface antigens with the high activity of cytotoxic drugs in inhibiting and killing tumor cells. Compared with traditional chemotherapy drugs, ADCs can kill tumor cells more accurately and reduce the impact on normal cells. In recent years, ADCs using camptothecin derivatives as small molecule toxins have made great progress. DS-8201a (Trastuzumab Deruxtecan) is an ADC developed by Daiichi Sankyo of Japan and has been approved for marketing. This ADC uses the camptothecin derivative Deruxtecan as a small molecule toxin, and a GGFG tetrapeptide that can be hydrolyzed by cathepsin B and a self-cleaving structure as a linker.

[0006]

[0007] However, ADCs using camptothecin derivatives as small molecule toxins generally require a large drug-to-antibody ratio (DAR), are difficult to manufacture, and can easily lead to ADC instability. Therefore, novel, more active camptothecin derivatives offer broad application prospects as anti-tumor drugs or small molecule toxins for ADCs. The present invention provides a novel class of camptothecin derivatives that significantly enhance cellular activity compared to known compounds, such as Deruxtecan, which is of great significance for the development of new anti-tumor drugs and ADCs. Summary of the Invention

[0008] The present invention provides a class of camptothecin derivative compounds represented by general formula (1), or their optical isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates:

[0009]

[0010] In the general formula (1):

[0011] m is an integer of 0, 1 or 2;

[0012] X is selected from -O-, -S-, -S(O)-, -S(O2)- or -N(R 4 )-;

[0013] R 1 and R 2 is independently selected from H, halogen, OH, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, C3-6 cycloalkyl, NH2, NO2 or CN, or R 1 and R 2 Together with the connected benzene ring, it forms

[0014] R 3 Selected from C1-6 alkyl, C2-6 alkenyl, C1-3 alkoxy substituted C1-3 alkyl or C1-6 haloalkyl;

[0015] R 4 Selected from H, C1-6 alkyl or C1-6 haloalkyl;

[0016] R 5 Selected from H, C1-6 alkyl or C3-6 cycloalkyl;

[0017] R 6 and R 7 are independently selected from H, C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl, or R 6 and R 7 Together with the carbon atom to which it is connected, it forms a C3-6 cycloalkyl group or a 4-7 membered heterocycloalkyl group, or R6 and R 5 Connected to form a 5-7 membered lactam ring, R 7 Selected from H, C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl;

[0018] R 8 Selected from OH or NR 9 R 10 , R 9 and R 10 independently selected from H, C1-6 alkyl or C3-6 cycloalkyl; or R 9 and R 10 Together with the nitrogen atom to which it is attached, it forms a 4-7 membered heterocycloalkyl group, which is unsubstituted or substituted by 1-3 groups selected from the following groups: C1-6 alkyl, halogen, OH, CN or NH2.

[0019] In another preferred embodiment, wherein in the formula (1), R 8 For OH.

[0020] In another preferred embodiment, wherein in the formula (1), R 1 and R 2 R is independently selected from H, halogen, OH, Me, Et, OMe, OEt, CF3, NH2, NO2 or CN; 1 and R ２ R is independently preferably H, F, Cl, Me, Et, OMe, OEt or CF3; 1 and R 2 More preferably, R is independently H, F, Me, Et or OMe; 1 and R 2 More preferably, each is independently F or Me; or R 1 and R 2 Together with the connected benzene ring, it forms

[0021] In another preferred embodiment, wherein in the formula (1), R 3 Selected from Me, Et, R 3 Preferably, Et, R 3 More preferred is Et.

[0022] In another preferred embodiment, in the formula (1), X is selected from -O-, -S-, -S(O)-, -S(O2)-, -N(H)- or -N(Me)-; X is preferably -O-, -S-, -S(O)-, -S(O2)- or -N(Me)-; X is more preferably -S-.

[0023] In another preferred embodiment, wherein in the formula (1), R 5 Selected from H, Me, Et or R 5 Preferably H or Me; R 5 More preferably, it is H.

[0024] In another preferred embodiment, wherein in the formula (1), R 6 and R 7 Independently selected from H, Me, Et, CHF2, CF3, CH2CF3, R 6 and R 7 are independently preferably H, Me, CF3, R 6 and R 7 More preferably, each is independently H or R 6 and R 7 More preferably, each is independently H; R 6 and R 7 Independently more preferably

[0025] In another preferred embodiment, wherein in the formula (1), Selected from Preferably More preferably

[0026] In another preferred embodiment, wherein in the formula (1), R 8 For OH, Selected from Preferably More preferably

[0027] In some embodiments of the present invention, a camptothecin derivative compound, or a pharmaceutically acceptable salt thereof, is provided, wherein the compound has one of the following structures:

[0028]

[0029]

[0030] In some embodiments of the present invention, a camptothecin derivative compound, or a pharmaceutically acceptable salt thereof, is provided, wherein the compound has one of the following structures:

[0031]

[0032] In some embodiments of the present invention, the compound, isomer or pharmaceutically acceptable salt of formula (1) has one of the following structures:

[0033]

[0034]

[0035]

[0036]

[0037] In some embodiments of the present invention, the compound, isomer or pharmaceutically acceptable salt of formula (1) has one of the following structures:

[0038]

[0039]

[0040]

[0041] In some embodiments of the present invention, the present invention provides an antibody-drug conjugate, wherein the antibody-drug conjugate has one of the following structures:

[0042]

[0043]

[0044]

[0045] Wherein Ab represents a monoclonal antibody, preferably an anti-her2 antibody, more preferably trastuzumab; n is a value within the range of 2-8, preferably 4-8, more preferably 7-8; for example, 7.2, 7.3.

[0046] One object of the present invention is to provide the use of the compound of the present invention, or its optical isomers, crystal forms, pharmaceutically acceptable salts, hydrates or solvates as small molecule toxins in the preparation of antibody-drug conjugates (ADCs).

[0047] Another object of the present invention is to provide a pharmaceutical composition comprising a pharmaceutically acceptable excipient or carrier and the compound of the present invention, or its optical isomers, or pharmaceutically acceptable inorganic or organic salts as active ingredients.

[0048] Another object of the present invention is to provide the use of the compound of the present invention, or its optical isomers, pharmaceutically acceptable inorganic or organic salts, or the pharmaceutical composition for preparing drugs for treating tumors and other related diseases.

[0049] Another object of the present invention is to provide the use of the compound of the present invention, or its optical isomers, or pharmaceutically acceptable inorganic or organic salts for the preparation of a drug for treating tumors and other related diseases.

[0050] Another object of the present invention is to provide an antibody-drug conjugate (ADC), characterized in that the antibody-drug conjugate comprises an antibody, a small molecule toxin and a linker; the small molecule toxin is the compound described in the present invention; and the linker links the antibody and the small molecule toxin through a covalent bond.

[0051] It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed.

[0052] Synthesis of compounds

[0053] The following specifically describes the preparation methods of the compound of general formula (1) of the present invention, but these specific methods do not constitute any limitation to the present invention.

[0054] The compounds of formula (1) described above can be synthesized using standard synthetic techniques or known techniques in combination with the methods described herein. In addition, the solvents, temperatures and other reaction conditions mentioned herein can be varied. The starting materials used in the synthesis of the compounds can be synthesized or obtained from commercial sources. The compounds described herein and other related compounds having different substituents can be synthesized using known techniques and starting materials, including those found in March, ADVANCED ORGANIC CHEMISTRY 4 th Ed., (Wiley 1992); Carey and Sundberg, ADVANCED ORGANIC CHEMISTRY 4 th Ed., Vols.A and B (Plenum 2000, 2001), Green and Wuts, PROTECTIVE GROUPS IN ORGANIC SYNTHESIS3 rd Ed., (Wiley 1999). The general methods for the preparation of compounds can be modified by using appropriate reagents and conditions to introduce various groups into the formulae provided herein.

[0055] In one aspect, the compounds described herein are prepared according to methods known in the art. However, the conditions of the methods, such as reactants, solvents, bases, amounts of the compounds used, reaction temperatures, and reaction times, are not limited to the following explanations. The compounds of the present invention can also be conveniently prepared by optionally combining various synthetic methods described in this specification or known in the art, and such combinations can be easily performed by those skilled in the art. In one aspect, the present invention also provides a method for preparing the compound of formula (1), wherein the compound of formula (1) can be prepared using the following general reaction scheme 1.

[0056] General reaction scheme 1

[0057]

[0058]

[0059] Among them, R 1 、R 2 、R 3 、R 5 、R 6 、R 7 、R 8 and X are defined as above.

[0060] Compound A1 is used as the starting material, and a nucleophilic substitution reaction is performed to obtain compound A2. The nitro group is reduced under iron powder and hydrochloric acid conditions and hydrolyzed to obtain compound A3. A Friedel-Crafts acylation reaction is performed to obtain compound A4. Compound A4 is acetylated to obtain compound A5. Compound A5 is subjected to nitrosation, reduction and acetylation to obtain compound A6. The acetyl group on the aniline of compound A6 is removed to obtain the intermediate compound A7.

[0061] Using compound B1 as the starting material, compound B2 is obtained by bromination, compound B2 reacts with chiral amino acid to obtain compound B3, compound B3 undergoes affinity substitution reaction to obtain compound B4, cyano group is reduced to obtain compound B5, compound B5 undergoes diazotization and nucleophilic reaction to obtain compound B6, and finally ring closure is obtained to obtain intermediate compound B7.

[0062] Intermediate compound A7 and intermediate compound B7 undergo ring closure to obtain intermediate compound B8, which is then deacetylating to obtain compound B9. Finally, the target compound of the present invention is obtained through amino group modification or functional group transformation.

[0063] Further forms of compounds

[0064] "Pharmaceutically acceptable" as used herein 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 does not cause undesirable biological effects or interact in a deleterious manner with any of its components when administered to a subject.

[0065] The term "pharmaceutically acceptable salt" refers to a form of a compound that does not cause significant irritation to an organism to which it is administered and does not abrogate the biological activity and properties of the compound. In certain specific aspects, the pharmaceutically acceptable salt is obtained by reacting the compound of formula (1) with an acid, such as an inorganic acid such as hydrochloric acid, hydrobromic acid, hydrofluoric acid, sulfuric acid, phosphoric acid, nitric acid, phosphoric acid, 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.

[0066] It should be understood that references to pharmaceutically acceptable salts include solvent-added forms or crystallized forms, particularly solvates or polymorphs. Solvates contain stoichiometric or non-stoichiometric amounts of solvent and are selectively formed during crystallization with pharmaceutically acceptable solvents such as water, ethanol, etc. Hydrates are formed when the solvent is water, or alcoholates are formed when the solvent is ethanol. Solvates of compounds of formula (1) are conveniently prepared or formed according to the methods described herein. For example, hydrates of compounds of formula (1) are conveniently prepared by recrystallization from a mixed solvent of water / organic solvent, using organic solvents including, but not limited to, tetrahydrofuran, acetone, ethanol or methanol. In addition, the compounds mentioned herein can exist in unsolvated and solvated forms. In general, for the purposes of the compounds and methods provided herein, the solvated forms are considered to be equivalent to the unsolvated forms.

[0067] In other embodiments, the compound of formula (1) is prepared in different forms, including but not limited to, amorphous, crushed and nano-particle forms. In addition, the compound of formula (1) includes crystalline forms and can also be polymorphic. Polymorphs include different lattice arrangements of the same elemental composition of the compound. Polymorphs generally have different X-ray diffraction spectra, infrared spectra, melting points, density, hardness, crystal form, optical and electrical properties, stability and solubility. Different factors such as recrystallization solvent, crystallization rate and storage temperature may cause a single crystalline form to dominate.

[0068] In another aspect, compounds of formula (1) may have chiral centers and / or axial chirality and thus occur as racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers, and cis-trans isomers. Each chiral center or axial chirality will independently produce two optical isomers, and all possible optical isomers and diastereomeric mixtures as well as pure or partially purified compounds are included within the scope of the present invention. The present invention is intended to include all such isomeric forms of these compounds.

[0069] The compounds of the present invention may contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute the compound. For example, the compounds may be labeled with radioactive isotopes, such as tritium ( 3 H), iodine-125 ( 125 I) and C-14( 14 C). For example, deuterated compounds can be formed by replacing hydrogen atoms with heavy hydrogen. The bond formed by deuterium and carbon is stronger than the bond formed by ordinary hydrogen and carbon. Compared with non-deuterated drugs, deuterated drugs generally have advantages such as reduced toxic side effects, increased drug stability, enhanced efficacy, and prolonged drug half-life in vivo. All isotopic variations of the compounds of the present invention, whether radioactive or not, are included within the scope of this invention.

[0070] the term

[0071] Unless otherwise indicated, the terms used in this application, including the specification and claims, are defined as follows. It should be noted that, throughout the specification and the appended claims, the singular forms "a," "an," and "an" include plural referents unless the context clearly indicates otherwise. Conventional methods, such as mass spectrometry, nuclear magnetic resonance, HPLC, protein chemistry, biochemistry, recombinant DNA technology, and pharmacology, are employed unless otherwise indicated. Throughout this application, the use of "or" or "and" means "and / or," unless otherwise indicated.

[0072] Unless otherwise specified, "alkyl" refers to a saturated aliphatic hydrocarbon group, including straight and branched groups of 1 to 6 carbon atoms. Preferred are lower alkyl groups containing 1 to 4 carbon atoms, such as methyl, ethyl, propyl, 2-propyl, n-butyl, isobutyl, tert-butyl. As used herein, "alkyl" includes unsubstituted and substituted alkyl groups, especially alkyl groups substituted with one or more halogens. Preferred alkyl groups are selected from CH3, CH3CH2, CF3, CHF2, CF3CH2, CF3(CH3)CH, i Pr, n Pr, i Bu, n Bu or t Bu.

[0073] Unless otherwise specified, "alkylene" refers to a divalent alkyl group as defined above. Examples of alkylene groups include, but are not limited to, methylene and ethylene.

[0074] Unless otherwise specified, "alkenyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon double bond, including straight or branched chain groups of 1 to 14 carbon atoms. Preferably, the lower alkenyl group contains 1 to 4 carbon atoms, such as vinyl, 1-propenyl, 1-butenyl or 2-methylpropenyl.

[0075] Unless otherwise specified, "alkynyl" refers to an unsaturated aliphatic hydrocarbon group containing a carbon-carbon triple bond, including straight and branched chain groups of 1 to 14 carbon atoms. Preferably, the lower alkynyl group contains 1 to 4 carbon atoms, such as ethynyl, 1-propynyl or 1-butynyl.

[0076] Unless otherwise specified, "cycloalkyl" refers to a non-aromatic hydrocarbon ring system (monocyclic, bicyclic or polycyclic). If the carbocyclic ring contains at least one double bond, the partially unsaturated cycloalkyl group may be referred to as a "cycloalkenyl group", or if the carbocyclic ring contains at least one triple bond, the partially unsaturated cycloalkyl group may be referred to as a "cycloalkynyl group". Cycloalkyl groups may include monocyclic or polycyclic (e.g., having 2, 3, or 4 fused rings) groups and spirocycles. In some embodiments, the cycloalkyl group is monocyclic. In some embodiments, the cycloalkyl group is monocyclic or bicyclic. The ring-forming carbon atoms of the cycloalkyl group may optionally be oxidized to form an oxo or sulfide group. Cycloalkyl groups also include cycloalkylene groups. In some embodiments, the cycloalkyl group contains 0, 1, or 2 double bonds. In some embodiments, the cycloalkyl group contains 1 or 2 double bonds (partially unsaturated cycloalkyl groups). In some embodiments, the cycloalkyl group may be fused with an aryl group, a heteroaryl group, a cycloalkyl group, and a heterocycloalkyl group. In some embodiments, the cycloalkyl group may be fused with an aryl group, a cycloalkyl group, and a heterocycloalkyl group. In some embodiments, cycloalkyl groups can be fused with aryl groups and heterocycloalkyl groups. In some embodiments, cycloalkyl groups can be fused with aryl groups and cycloalkyl groups. Examples of cycloalkyl groups include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, cyclohexadienyl, cycloheptatrienyl, norbornyl, norpinyl, norcaryl, bicyclo[1.1.1]pentanyl, bicyclo[2.1.1]hexanyl, and the like.

[0077] Unless otherwise specified, "alkoxy" refers to an alkyl group bonded to the rest of the molecule through 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 those substituted with 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.

[0078] Unless otherwise specified, "aryl" refers to a hydrocarbon aromatic group. Aryl is monocyclic or polycyclic, for example, a monocyclic aryl ring fused to one or more carbocyclic aromatic groups. Examples of aryl include, but are not limited to, phenyl, naphthyl, and phenanthrenyl.

[0079] Unless otherwise specified, "heterocycloalkyl" refers to a non-aromatic ring or ring system that may optionally contain one or more alkenylene groups as part of the ring structure, having 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, the partially unsaturated heterocycloalkyl may be referred to as a "heterocycloalkenyl", or if the heterocycloalkyl contains at least one triple bond, the partially unsaturated heterocycloalkyl may be referred to as a "heterocycloalkynyl". The heterocycloalkyl may include a monocyclic, bicyclic, spirocyclic or polycyclic (e.g., having two fused or bridged rings) ring system. In some 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 groups or other oxidized bonds (e.g., C(O), S(O), C(S) or S(O) 2, N-oxides, etc.), or the nitrogen atom 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 parts with one or more aromatic rings fused to the heterocycloalkyl ring (i.e., sharing a key with it), such as benzo derivatives of piperidine, morpholine, azacycloheptatriene or thienyl. The heterocycloalkyl containing fused aromatic rings can be connected via any ring-forming atoms, including the ring-forming atoms of the fused aromatic ring. Examples of heterocycloalkyl groups 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 ... oxazolo[4,5-c]pyridine, N-methylpiperidinyl, tetrahydroimidazolyl, pyrazolidinyl, butyrolactamyl, valerolactamyl, imidazolinyl, hydantoinyl, dioxolane, phthalimide, pyrimidine-2,4(1H,3H)-dione, 1,4-dioxane, morpholinyl, thiomorpholinyl, thiomorpholine-S-oxide, thiomorpholine-S,S-oxide, piperazinyl, pyranyl, pyridonyl, 3-pyrrolinyl, thiopyranyl, pyranonyl, tetrahydrothiophenyl, 2-azaspiro[3.3]heptanyl, indolinyl,

[0080] Unless otherwise specified, "halogen" (or halo) refers to fluorine, chlorine, bromine or iodine. The term "halo" (or "halogen substituted") appearing before the name of a group indicates that the group is partially or fully halogenated, that is, substituted by F, Cl, Br or I in any combination, preferably substituted by F or Cl.

[0081] "Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where said event or circumstance occurs and instances where it does not.

[0082] The substituent "-O-CH2-O-" refers to the substituent in which two oxygen atoms are connected to two adjacent carbon atoms of a heterocycloalkyl, aryl or heteroaryl group, for example:

[0083] When the number of a linking group is 0, such as -(CH2)0-, it means that the linking group is a single bond.

[0084] When one of the variables is selected from a chemical bond, it means that the two groups it connects are directly connected. For example, when L in XLY represents a chemical bond, it means that the structure is actually XY.

[0085] Unless otherwise specified, the key is a solid wedge ( ) and wedge-shaped dashed key ( ) represents the absolute configuration of a stereocenter, with a straight solid bond ( ) and straight dashed bond ( ) indicates the relative configuration of the stereocenter, and a wavy line ( ) represents a wedge-shaped solid bond ( ) or a dotted wedge key ( ), or with a wavy line ( ) represents a straight solid bond ( ) or a straight dashed key ( ).

[0086] Unless otherwise stated, Indicates a single bond or a double bond.

[0087] Specific pharmaceutical and medical terms

[0088] The term "acceptable," as used herein, means that a prescribed ingredient or active ingredient has no undue adverse effect on health and well-being for the general purpose of treatment.

[0089] The terms "treat," "treatment," or "therapy" as used herein include alleviating, inhibiting, or ameliorating the symptoms of a disease or condition; inhibiting the development of complications; ameliorating or preventing underlying metabolic syndrome; inhibiting the development of a disease or symptom, such as controlling the progression of a disease or condition; alleviating a disease or symptom; causing a regression of 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, upon administration, can improve a disease, symptom, or condition, particularly by improving its severity, delaying its onset, slowing its progression, or reducing its duration. Whether the administration is fixed or temporary, continuous or intermittent, the circumstances attributable to or related to the administration can be explained.

[0090] "Active ingredient" refers to the compound of formula (1), as well as pharmaceutically acceptable inorganic or organic salts of the compound of formula (1). The compounds of the present invention may contain one or more asymmetric centers (chiral centers or axial chirality) and therefore appear in the form of racemates, racemic mixtures, single enantiomers, diastereomeric compounds and single diastereomers. The asymmetric centers that may exist depend 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 as well as 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.

[0091] 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), induces a desired pharmaceutical and / or physiological response through local and / or systemic action.

[0092] The term "administered," "administering," or "administration" as used herein refers to the direct administration of the compound or composition, or the administration of a prodrug, derivative, or analog of the active compound.

[0093] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the numerical values ​​of the specific examples are presented herein as precisely as possible. However, any numerical value inherently and inevitably contains standard deviations resulting from individual testing methods. As used herein, "about" generally refers to the actual value being within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range. Alternatively, the term "about" means that the actual value falls within an acceptable standard error of the mean, as determined by one skilled in the art. Except in the experimental examples, or unless otherwise expressly indicated, all ranges, amounts, values, and percentages used herein (e.g., to describe material amounts, time periods, temperatures, operating conditions, quantitative ratios, and the like) are to be understood as modified by the word "about." Therefore, unless otherwise indicated, the numerical parameters disclosed in this specification and the appended claims are approximate and may be modified as needed. At a minimum, these numerical parameters should be understood to include the number of significant digits indicated and to include normal rounding.

[0094] Unless otherwise defined in this specification, the scientific and technical terms used herein have the same meanings as commonly understood by those skilled in the art. In addition, unless otherwise defined in this specification, singular terms used in this specification include the plural form of the term, and plural terms also include the singular form of the term, unless otherwise defined in the context.

[0095] Therapeutic uses

[0096] The present invention provides methods of treating diseases including, but not limited to, cancer using the compounds, antibody-drug conjugates, or pharmaceutical compositions of the present invention.

[0097] In some embodiments, a method for treating cancer is provided, comprising administering to a subject in need thereof an effective amount of any of the aforementioned compounds, antibody-drug conjugates, or pharmaceutical compositions. In other embodiments, the cancer is a blood cancer or 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.

[0098] Route of administration

[0099] The compounds of the present invention and their pharmaceutically acceptable salts can be formulated into various formulations containing a safe and effective amount of the compounds of the present invention or their pharmaceutically acceptable salts and a pharmacologically acceptable excipient or carrier. "Safe and effective amount" means an amount of the compound sufficient to significantly improve the condition without causing serious side effects. The safe and effective amount of the compound will be determined based on the patient's age, condition, and duration of treatment, among other factors.

[0100] "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 Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.

[0101] The compounds of the present invention may be administered orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), or topically.

[0102] 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 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.

[0103] Solid dosage forms such as tablets, dragees, 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 opacifying agents, and the release of the active compound or compounds in such compositions can be delayed in a certain portion of the digestive tract. Examples of useful encapsulating components are polymeric substances and waxes. If desired, the active compound can also be microencapsulated with one or more of the above-mentioned excipients.

[0104] Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. 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, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1,3-butylene glycol, dimethylformamide and oils, in particular cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances.

[0105] Besides such inert diluents, the composition may also include adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring, and perfuming agents.

[0106] Suspensions, in addition to the active compounds, may contain suspending agents such as, for example, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances.

[0107] Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and non-aqueous carriers, diluents, solvents or excipients include water, ethanol, polyols and suitable mixtures thereof.

[0108] Dosage forms for topical administration of the compounds of this invention include ointments, powders, patches, sprays and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants that may be required.

[0109] The compounds of the present invention can be administered alone or in combination with other pharmaceutically acceptable compounds. When using a pharmaceutical composition, a safe and effective amount of the compounds of the present invention is applied to a mammal (e.g., a human) in need of treatment, wherein the dosage is a pharmaceutically effective dosage. For a 60 kg human, the daily dosage is generally 1 to 2000 mg, preferably 50 to 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, all of which are within the skill of a skilled physician.

[0110] The features described above, or in the embodiments, may be combined in any combination. All features disclosed in this specification may be used in any combination, and each feature disclosed in this specification may be replaced by any alternative feature that serves the same, equivalent, or similar purpose. Therefore, unless otherwise specified, the features disclosed are merely general examples of equivalent or similar features. BRIEF DESCRIPTION OF THE DRAWINGS

[0111] Figure 1This is the anti-tumor activity result of Example 11 of the present invention in mice. DETAILED DESCRIPTION

[0112] The following description will elaborate on various specific aspects, characteristics, and advantages of the above-mentioned compounds, methods, and pharmaceutical compositions so that the present invention will be readily apparent. It should be understood that the following detailed description and examples describe specific embodiments and are provided for reference only. After reading the present description, those skilled in the art may make various changes or modifications to the present invention, and such equivalents are within the scope of the present invention.

[0113] In all embodiments, 1 H-NMR was recorded on a Varian Mercury 400 nuclear magnetic resonance instrument, and chemical shifts are expressed in δ (ppm). Silica gel used for separation was 200-300 mesh unless otherwise specified, and the eluent ratios were by volume.

[0114] The present invention uses the following abbreviations: room temperature (RT, rt); aqueous solution (aq.); petroleum ether (PE); ethyl acetate (EA); dichloromethane (DCM); 1,4-dioxane; methanol (MeOH); methyl tert-butyl ether (MTBE); ethanol (EtOH); tetrahydrofuran (THF); dimethylformamide (DMF); N-methylpyrrolidone (NMP); dimethyl sulfoxide (DMSO); triethylamine (TEA); diisopropylethylamine (DIPEA); 4-dimethylaminopyridine (DMAP); carbon tetrachloride (CCl4); palladium on carbon (Pd / C); Eaton's reagent (7.7 wt% phosphorus pentoxide in methanesulfonic acid); iron powder (Fe); zinc powder (Zn); Ranyi nickel (Ranyi Ni); acetyl chloride (AcCl); acetic acid (AcOH); acetic anhydride (Ac2O); m-chloroperbenzoic acid (m-CPBA); n-butyl nitrite (n-BuNO); sodium nitrite (NaNO2); sodium hydride (NaH); magnesium sulfate (MgSO4); N-bromosuccinimide (NBS); p-toluenesulfonic acid monohydrate (TsOH.H2O); sodium carbonate (Na2CO3); potassium carbonate (K2CO3); equivalent (eq); gram / milligram (g / mg); mole / millimole (mol / mmol); liter / milliliter (L / mL); minute (min(s)); hour (h, hr, hrs); nitrogen (N2); nuclear magnetic resonance (NMR); liquid chromatography-mass spectrometry (LC-MS); thin layer chromatography (TLC); preparative liquid chromatography (pre-HPLC).

[0115] Preparation Example 1 Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-yl)acetamide (A7-a)

[0116]

[0117] Step 1: Synthesis of methyl 3-((3-fluoro-2-methyl-5-nitrophenyl)thio)propionate

[0118]

[0119] A1-a (1.73 g, 10 mmol, 1 eq), methyl 3-mercaptopropionate (1.8 g, 15 mmol, 1.5 eq), and NMP (10 mL) were added to a 50 mL three-necked flask. After the system dissolved, potassium carbonate (2 g, 15 mmol, 1.5 eq) was added and stirred at 60°C under argon for 8 h. After cooling to room temperature, the system was diluted with water (30 mL). The precipitated solid was filtered, washed with water, and the filter cake was separated by column chromatography (PE / EA = 1 / 12-1 / 7) to obtain a yellow solid A2-a (1.55 g, 56.8% yield). LC-MS: 274.2 [M+H] + .

[0120] Step 2: Synthesis of 3-((3-fluoro-2-methyl-5-aminophenyl)thio)propanoic acid

[0121]

[0122] A2-a (1.55 g, 5.67 mmol, 1 eq), iron powder (1.27 g, 22.69 mmol, 4 eq), ethanol (80 mL), and aqueous ammonium chloride solution (2.8 M, 28 mL, 5 eq) were added to a 250 mL three-necked flask and stirred at 90°C under argon for 16 h. After cooling to room temperature, the mixture was filtered through celite and the solid was washed with ethanol. The filtrate was concentrated, and the crude product was diluted with water (30 mL) and extracted with EA (30 mL x 2). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to give the crude product (1.5 g, equivalent yield). LC-MS: 244.3 [M+H] + .

[0123] The crude product (1.5 g, 5.67 mmol, 1 eq) and 1,4-dioxane (15 mL) were added to a 50 mL three-necked flask. After the system dissolved, concentrated hydrochloric acid (37%, 10 mL) was added and stirred at 65°C under argon for 4 h. After cooling to room temperature, 3N sodium carbonate solution was added to adjust the pH to 5. The mixture was extracted with EA (30 mL x 2). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated. The crude product was slurried (EA / PE = 1 / 5) to obtain a white solid A3-a (985 mg, two-step yield 75.8%). LC-MS: 228.2 [MH] - .

[0124] Step 3: Synthesis of 5-amino-7-fluoro-8-methylthiochroman-4-one

[0125]

[0126] A3-a (985 mg, 4.3 mmol, 1 eq) and Eaton's Reagent (15 mL) were added to a 50 mL three-necked flask and stirred at 60°C under argon for 1 h. After cooling to room temperature, the reaction solution was poured into ice water and the pH was adjusted to 8 with 3N sodium carbonate solution. The mixture was extracted with EA (30 mL x 2). The organic phase was washed with saturated brine, dried over sodium sulfate, and concentrated to afford crude product A4-a (1.05 g, equivalent yield). LC-MS: 212.3 [M+H] + .

[0127] Step 4: Synthesis of N-(7-fluoro-8-methyl-4-oxothiochroman-5-yl)acetamide

[0128]

[0129] A4-a (1.05 g, 4.3 mmol, 1 eq), DMAP (52.5 mg, 0.43 mmol, 0.1 eq), and DCM (15 mL) were added to a 50 mL three-necked flask. Acetyl chloride (674 mg, 8.6 mmol, 2 eq) and triethylamine (869 mg, 8.6 mmol, 2 eq) were added sequentially under an ice bath. The system was allowed to return to room temperature and stirred for 1 h, indicating complete reaction. Water (20 mL) was added to quench the reaction, and the layers were separated. The aqueous phase was extracted with DCM (20 mL x 2). The organic phases were combined, washed with saturated brine, dried, concentrated, and purified by column chromatography (EA / PE = 1 / 1) to afford A5-a (910 mg, 89% yield over two steps) as a yellow solid. LC-MS: 254.3 [M+H] + .

[0130] Step 5: Synthesis of N,N′-(7-fluoro-8-methyl-4-oxothiochroman-3,5-diyl)diethylamide

[0131]

[0132] Potassium tert-butoxide (191 mg, 1.7 mmol, 1.1 eq) and anhydrous THF (5 mL) were added to a 50 mL three-necked flask. A5-a (375 mg, 1.48 mmol, 1 eq) and n-butyl nitrite (190 mg, 1.85 mmol, 1.25 eq) were then added sequentially at -20°C. The system was warmed to 5°C and stirred for 2 h. The starting materials reacted completely. MTBE (15 mL) was added to dilute the system and filtered. The solid was dissolved in acetic acid (5 mL), and zinc powder (200 mg, 3.1 mmol, 2.1 eq) was added. After stirring at room temperature for 5 min, acetic anhydride (1 mL) was added. Stirring was continued for 2 h, and the system was washed with MeOH / DCM (3 / 30 mL). The system was concentrated, and the crude product was separated by column chromatography (EA / DCM = 1 / 10-1 / 5) to afford A6-a (175 mg, 41%) as a light brown solid. LC-MS: 311.1 [M+H] + .

[0133] Step 6: Synthesis of N-(5-amino-7-fluoro-8-methyl-4-oxothiochroman-3-yl)acetamide

[0134]

[0135] A6-a (175 mg, 0.56 mmol, 1 eq) and methanol / 1,4-dioxane (4 / 8 mL) were added to a 50 mL three-necked flask. After the system dissolved, concentrated hydrochloric acid (37%, 4 mL) was added and stirred at 40°C under argon for 2 h. After cooling to room temperature, 3N sodium carbonate solution was added to adjust the pH to 8. The mixture was filtered and the solid was dried to obtain A7-a (137 mg, 91% yield). LC-MS: 269.2 [M+H] + .

[0136] Similar to the synthesis of A7-a, the intermediates listed in the following table can be obtained:

[0137] Table 1. Intermediates A7-b to A7-y

[0138]

[0139]

[0140] Preparation Example 2 Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione (B7-a)

[0141]

[0142] Step 1: Synthesis of ethyl 2-bromo-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2′-[1,3]dioxolane]-7-yl)acetate

[0143]

[0144] B1-a (3 g, 9.9 mmol, 1 eq) was dissolved in DMF (75 mL), and NBS (1.5 g, 12 mmol, 1.2 eq) and m-CPBA (170 mg, 1 mmol, 0.1 eq) were added. The reaction was stirred at room temperature overnight. The system was poured into 500 mL of ice water, filtered, and the solid was washed with water and dried to obtain a gray solid B2-a (3.8 g, equivalent yield). LC-MS: 383.2 [M+H] + .

[0145] Step 2: Synthesis of 1-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2′-[1,3]dioxolane]-7-yl)-2-ethoxy-2-oxoethyl p-toluenesulfonate-D-proline ester

[0146]

[0147] B2-a (1 g, 2.6 mmol, 1 eq), sodium p-toluenesulfonyl-D-proline (1 g, 3.9 mmol, 1.5 eq), and K2CO3 (362 mg, 2.6 mmol, 1 eq) were dissolved in DMF (20 mL). The reaction was stirred at 65°C for 2 h under argon protection. The raw materials reacted completely, and the system was diluted with water (100 mL). The mixture was extracted with EA (100 mL*3). The organic phases were combined, washed twice with water and saturated brine, dried, concentrated, and separated by column chromatography (EA / DCM=1 / 6) to give B3-a (1.1 g, yield 74%) as a white solid. LC-MS: 572.2 [M+H] + .

[0148] Step 3: Synthesis of (S)-2-(6-cyano-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2′-[1,3]dioxolane-7-yl)-1-ethoxy-4-fluoro-1-oxobutyl-2-yl p-toluenesulfonate-D-proline ester

[0149]

[0150] B3-a (1 g, 1.7 mmol, 1 eq) was dissolved in DMF (20 mL) and NaH (101 mg, 60%, 2.5 mmol, 1.5 eq) was added under ice-cooling. The mixture was allowed to return to room temperature and stirred for 1 h. 2-Fluoroiodoethane (1.5 g, 8.6 mmol, 5 eq) was added under ice-cooling. The mixture was allowed to return to room temperature and stirred overnight. After the reaction was completed, the system was poured into ice-water (100 mL) and extracted with EA (100 mL*3). The organic phases were combined, washed twice with water and saturated brine, dried, concentrated, and separated by column chromatography (EA / DCM = 1 / 6) to obtain 743 mg of the crude product. Pre-HPLC analysis gave B4-a (150 mg, 70% de, 15% yield) as a white solid. LC-MS: 618.2 [M+H] + .

[0151] Step 4: Synthesis of (S)-2-(6-(acetylaminomethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2′-[1,3]dioxolane]-7-yl)-1-ethoxy-4-fluoro-1-oxobutyl-2-yl p-toluenesulfonate-D-proline ester

[0152]

[0153] Raney Ni (600 mg, 50% aqueous solution) was added to a 50 mL three-necked flask and washed three times with HOAc. Under argon protection, a solution of B4-a (150 mg, 0.24 mmol, 1 eq) in Ac2O / HOAc (4 / 1 mL) was added. The system was replaced with hydrogen three times and reacted at 65°C for 3 h. The mixture was filtered and washed with AcOH. The filtrate was concentrated and separated by column chromatography (MeOH / DCM = 1 / 20) to give B5-a (130 mg, yield 83%) as a colorless oily liquid. LC-MS: 664.2 [M+H] + .

[0154] Step 5: Synthesis of (S)-2-(6-(acetoxymethyl)-5-oxo-2,3-dihydro-5H-spiro[indolizine-1,2′-[1,3]dioxolane]-7-yl)-1-ethoxy-4-fluoro-1-oxobutyl-2-yl p-toluenesulfonate-D-proline ester

[0155]

[0156] B5-a (130 mg, 0.2 mmol, 1 eq) was dissolved in Ac2O / HOAc (3 / 1 mL) and NaNO2 (68 mg, 1 mmol, 5 eq) was added under ice-cooling. The mixture was returned to room temperature and stirred for 1 h. The reaction was complete and filtered. The solid was washed with AcOH. The filtrate was concentrated and CCl4 (15 mL) was added. The mixture was refluxed and stirred overnight. The system was washed with water and saturated brine, dried and concentrated. Column chromatography (MeOH / DCM = 1 / 20) was used to separate 90 mg of a colorless oily liquid. Pre-HPLC prepared B6-a (90 mg, 69% yield) as a white solid. LC-MS: 665.2 [M+H] + .

[0157] Step 6: Synthesis of (S)-4-(2-fluoroethyl)-4-hydroxy-7,8-dihydro-1H-pyrano[3,4-f]indolizine-3,6,10(4H)-trione

[0158]

[0159] B6-a (90 mg, 0.14 mmol, 1 eq) was dissolved in EtOH (3 mL), and 1N aqueous sodium carbonate solution (1 mL) was added. After stirring at room temperature for 1 h, the starting material disappeared. After concentration at room temperature, the system was lyophilized. The crude product was dissolved in 85% aqueous TFA solution (5 mL) and stirred at 85 degrees for 1 h. After the reaction was complete, the system was concentrated. The crude product was separated by Pre-HPLC as a white solid B7-a (7 mg, yield 17%, de 68%). LC-MS: 282.2 [M+H] + .

[0160] Similar to the synthesis of B7-a, the intermediates listed in the following table can be obtained:

[0161] Table 2. Intermediates B7-b to B7-d

[0162]

[0163]

[0164] Example 1: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3′,4′:6,7]indolizine[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione (Compound 1)

[0165]

[0166] Step 1: Synthesis of N-((9S)-9-ethyl-5-fluoro-9-hydroxy-4-methyl-10,13-dioxo-1,2,9,10,13,15-hexahydro-12H-pyrano[3′,4′:6,7]indolizine[1,2-b]thiopyrano[4,3,2-de]quinolin-1-yl)acetamide

[0167]

[0168] A7-a (108 mg, 0.4 mmol, 1 eq), B7-b (156 mg, 0.6 mmol, 1.5 eq), p-toluenesulfonic acid monohydrate (45 mg, 0.24 mmol, 0.6 eq), anhydrous magnesium sulfate (1 g), and acetic acid (10 mL) were added to a 50 mL three-necked flask. Under argon, the mixture was stirred at 105°C for 24 h. The reaction was complete. Filtered, the filter cake was washed with EA, and concentrated. The crude product was separated by column chromatography (MeOH / DCM = 1 / 40-1 / 20) to give B8-a (131 mg, 67%) as a light brown solid. LC-MS: 496.2 [M+H] + .

[0169] Step 2: Synthesis of (9S)-1-amino-9-ethyl-5-fluoro-9-hydroxy-4-methyl-1,9,12,15-tetrahydro-13H-pyrano[3′,4′:6,7]indolizine[1,2-b]thiopyrano[4,3,2-de]quinoline-10,13(2H)-dione

[0170]

[0171] B8-a (131 mg, 0.26 mmol, 1 eq) and 1,4-dioxane (5 mL) were added to a 50 mL three-necked flask. After the system dissolved, concentrated hydrochloric acid (37%, 5 mL) was added and stirred at 80°C under argon for 24 h. After cooling to room temperature, the mixture was concentrated and the crude product was slurried with ACN / EA (1 / 1) to obtain a brown solid compound 1 (hydrochloride) (107 mg, yield 84.6%). LC-MS: 454.2 [M+H] + .

[0172] Similar to the synthesis of compound 1, the compounds listed in the following table can be obtained:

[0173] Table 3. List of compounds 2-31

[0174]

[0175]

[0176]

[0177] Example 2 Chiral separation of compound 1

[0178] Compound 1 is a pair of diastereoisomer mixtures. Two diastereoisomers 1-1 and 1-2 of compound 1 can be obtained by salt formation recrystallization or pre-HPLC separation and purification.

[0179]

[0180] Chromatographic conditions: Shimadzu LC-20AP preparative liquid chromatograph; chromatographic column: Waters SunFire PrepC18 OBD (50×150 mm, 5 μm); mobile phase: acetonitrile-0.5‰ trifluoroacetic acid aqueous solution = 66:34; flow rate: 48.0 mL / min; detection wavelength: 254 nm; injection volume: 3000 μL.

[0181] Experimental steps: Take an appropriate amount of 1 and dilute to volume with 50% acetonitrile aqueous solution to prepare a test solution with a concentration of 25 mg / mL. Take the above test solution and inject it into the preparative liquid chromatograph, detect according to the chromatographic conditions of the present invention and record the data.

[0182] Results: 1-1 (33 mg) and 1-2 (31 mg) were obtained by preparative separation. The retention times of the two components were 5.419 min and 7.614 min, respectively, and the purities were 99.38% and 99.21%, respectively.

[0183] Similar chiral separation methods to compound 1 can yield the compounds listed in the following table:

[0184] Table 4. Pre-HPLC separation of compounds

[0185]

[0186]

[0187]

[0188] Example 3 Synthesis of Compound 32-1 and Compound 32-2

[0189]

[0190] In a 50 mL three-necked flask, component 1-2 (98 mg, 0.2 mmol, 1 eq), 2-hydroxyacetic acid (18.4 mg, 0.24 mmol, 1.2 eq) and anhydrous DCM (5 mL) were added, and HATU (84 mg, 0.3 mmol, 1.5 eq) and DIPEA (90.3 mg, 0.7 mmol, 3.5 eq) were added in sequence under ice bath. After maintaining the temperature for 0.5 h, water (5 mL) was added to dilute the system, the liquids were separated, and DCM (5 mL*2) was extracted. The organic phase was washed with saturated brine, dried, and concentrated by column chromatography (MeOH / DCM=1 / 40) to give compound 32-2 (75 mg, yield 73%).

[0191] 1 H NMR (400MHz, DMSO-d6) δ: 8.21 (dd, J=13.3, 8.5Hz, 1H), 7.78 (d, J=10.6Hz, 1H), 7.31(d, J=1.2Hz, 1H), 6.53(s, 1H), 5.89-5.79(m, 1H), 5.56-5.49(m, 1H), 5.43( s, 2H), 5.38 (s, 1H), 5.31 (d, J = 4.8Hz, 1H), 3.90 (d, J = 4.7Hz, 2H), 3.50-3.35 (m , 3H), 2.44 (s, 3H), 1.91-1.80 (m, 2H), 0.87 (t, J=6.5Hz, 3H), LC-MS: 512.2[M+H] + .

[0192] Similar to the synthesis of compound 32-2, the diastereomer 32-1 of compound 32-2 can be obtained by using another component 1-1 with a shorter retention time.

[0193] Similar to the synthesis of compound 32-1 and compound 32-2, using different intermediates, the compounds listed in the following table can be obtained:

[0194] Table 5. List of compounds 33-96

[0195]

[0196]

[0197]

[0198]

[0199]

[0200]

[0201]

[0202] Example 4 Preparation of Antibody-Drug Conjugate (ADC-1)

[0203] Step 1: Preparation of compound LD-1

[0204]

[0205] To a 50 mL single-necked flask, L-1 (76 mg, 0.12 mmol, 1.0 eq), the longer-retention component compound 1-2 (55 mg, 0.12 mmol, 1.0 eq), NMI (50.6 mg, 0.62 mmol, 5.0 eq), and DMF (2 mL) were added, stirred, and cooled to 0°C. TCFH (41.5 mg, 0.15 mmol, 1.2 eq) was added to the reaction solution, and stirred for 30 min. LC-MS analysis was performed. After completion of the reaction, the reaction solution was purified by reverse-phase C18 column chromatography (MeCN / water = 0-60%). The target fraction was lyophilized to obtain LD-1 (80 mg, 61.5% yield) as a yellow solid.

[0206] 1 H-NMR (400MHz, DMSO-d6) δ: 8.56 (t, J=6.4Hz, 1H), 8.30 (dd, J=14.0, 8.3Hz, 2H), 8.11 (d, J=7.9Hz, 1H), 8.06 (t, J=5.7Hz, 1H), 7.99 (t, J=5.7Hz, 1H), 7.76 (d, J=10.7Hz, 1H), 7.31 (d, J=2.8Hz, 1H), 7.27-7.12 (m, 5H), 6.98 (s, 2H), 6.54 (d, J=1.8Hz, 1H), 5.84-5.83 (m, 1H), 5.49-5.31 (m, 3H), 5.27-5.2 1(m, 1H), 4.66-4.53(m, 2H), 4.48-4.41(m, 1H), 3.98(s, 2H), 3.75-3.54(m , 6H), 3.42(s, 2H), 3.36-3.35(m, 2H), 3.04-2.98(m, 1H), 2.80-2.74(m, 1H ), 2.42 (s, 3H), 2.08 (t, J=7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J=1 3.2, 5.9Hz, 4H), 1.25-1.12(m, 2H), 0.88-0.84(m, 3H), LC-MS: 1052.1[M+H] +, 1050.1[MH] - .

[0207] Step 2 ADC-1 preparation

[0208] At 37°C, a solution of trastuzumab in PBS buffer (pH = 6.5, 0.05% PBS buffer; 2.5 mL, 9.96 mg / mL, 0.168 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL.

[0209] Compound LD-1 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to the above 2.0 mL solution. The mixture was shaken in a water bath at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL) and stored frozen at 4°C. UV-HPLC calculated average: n = 7.2.

[0210] Example 5 Preparation of Antibody-Drug Conjugate (ADC-2)

[0211]

[0212] Step 1: Preparation of compound LD-2

[0213]

[0214] To a 50 mL single-necked flask, L-1 (76 mg, 0.12 mmol, 1.0 eq), the shorter-retention-time component 1-1 (55 mg, 0.12 mmol, 1.0 eq), NMI (50.6 mg, 0.62 mmol, 5.0 eq), and DMF (2 mL) were added, stirred, and cooled to 0°C. TCFH (41.5 mg, 0.15 mmol, 1.2 eq) was added to the reaction solution, and stirred for 30 min. LC-MS analysis was performed. After completion of the reaction, the reaction solution was purified by reverse-phase C18 column chromatography (MeCN / water = 0-60%). The target fraction was lyophilized to obtain LD-2 (80 mg, 61.5% yield) as a yellow solid.

[0215] 1 H-NMR (400 MHz, DMSO-d6) 1H-NMR (400MHz, DMSO-d6) δ: 8.56 (t, J=6.4Hz, 1H), 8.30 (dd, J=14.0, 8.3Hz, 2H), 8.11 (d, J=7.9Hz, 1H), 8.06 (t, J=5.7Hz, 1H), 7.99 (t, J=5.7Hz, 1H), 7.76 (d, J=10.7Hz, 1H), 7.31 (d, J=2.8Hz, 1H), 7.30-7.16 (m, 5H), 6.98 (s, 2H), 6.54 (d, J=1.8Hz, 1H), 5.90-5.86 (m, 1H), 5.46-5.30 (m, 3H), 5.26-5.2 1(m, 1H), 4.68-4.53(m, 2H), 4.46-4.41(m, 1H), 3.98(s, 2H), 3.75-3.54(m , 6H), 3.42(s, 2H), 3.36-3.35(m, 2H), 3.04-2.98(m, 1H), 2.80-2.74(m, 1H ), 2.42 (s, 3H), 2.08 (t, J=7.4, 2H), 1.93-1.79 (m, 2H), 1.50-1.41 (dd, J=1 3.2, 5.9Hz, 4H), 1.25-1.12(m, 2H), 0.88-0.84(m, 3H), LC-MS: 1052.1[M+H] + , 1050.1[MH] - .

[0216] Step 2 ADC-2 preparation

[0217] At 37°C, a solution of trastuzumab in PBS buffer (pH = 6.5, 0.05% PBS buffer; 2.5 mL, 9.96 mg / mL, 0.168 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath and diluted to 5.0 mg / mL.

[0218] Compound LD-2 (2.02 nmol) was dissolved in DMSO (0.10 mL) and added to the above 2.0 mL solution. The mixture was shaken in a water bath at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL), which was stored frozen at 4°C. UV-HPLC calculated average: n = 7.2.

[0219] Example 6 Preparation of Antibody-Drug Conjugates (ADC-3 and ADC-4)

[0220]

[0221] Step 1: Preparation of compounds LD-3 and LD-4

[0222]

[0223] L-2 (80 mg, 0.12 mmol, 1.0 eq) and the component compound 1-1 with a shorter retention time (55 mg, 0.12 mmol, 1.0 eq), NMI (50.6 mg, 0.62 mmol, 5.0 eq) and DMF (2 mL) were added to a 50 mL single-necked bottle, stirred evenly and cooled to 0°C. TCFH (41.5 mg, 0.15 mmol, 1.2 eq) was added to the reaction solution and stirred for 30 min. LC-MS detection was performed. After the reaction was completed, the reaction solution was purified by reverse phase C18 column chromatography to obtain two fractions. The one with a shorter retention time was LD-3 and the one with a longer retention time was LD-4. The target fractions were lyophilized to obtain yellow solids LD-3 (20 mg) and LD-4 (25 mg).

[0224] LC-MS: 1092.4 [M+H] + .

[0225] Chromatographic conditions: Thermo Fisher Scientific semi-preparative liquid chromatograph U3000; chromatographic column: Welch Ultimate XB-Phenyl; mobile phase: 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution = 50:50; flow rate: 30.0 mL / min; detection wavelength: 370 nm; injection volume: 100 μL.

[0226] Experimental Procedure: Dissolve an appropriate amount of LD-3 and LD-4 mixture in DMF to prepare a 10 mg / mL test solution. Inject this solution into a preparative liquid chromatograph and analyze according to the chromatographic conditions of this invention. Record the data and repeat the injection multiple times.

[0227] Results: LD-3 and LD-4 were obtained by preparative separation. The retention times of the two components were 5.29 min and 5.87 min, respectively. The purities were 99.38% and 99.21%.

[0228] Step 2 Preparation of ADC-3 and ADC-4

[0229] At 37°C, a solution of trastuzumab in PBS buffer (pH = 6.5, 0.05% PBS buffer; 2.5 mL, 9.96 mg / mL, 0.168 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / mL, and two equal portions were prepared in parallel.

[0230] 2.0 nmol of compound LD-3 was dissolved in DMSO (0.10 mL) and added to the above 2.0 mL solution. The mixture was shaken in a water bath at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1 mL) and stored frozen at 4°C. UV-HPLC calculated average: n = 7.3.

[0231] ADC-4 was prepared by the same method using compound LD-4, with n=7.3.

[0232] Example 7 Preparation of Antibody-Drug Conjugates (ADC-5 and ADC-6)

[0233]

[0234] Step 1: Preparation of compounds LD-5 and LD-6

[0235]

[0236] To a 50 mL single-necked vial, L-2 (80 mg, 0.12 mmol, 1.0 eq), compound 1-2 (55 mg, 0.12 mmol, 1.0 eq), NMI (50.6 mg, 0.62 mmol, 5.0 eq), and DMF (2 mL) were added, stirred, and cooled to 0°C. TCFH (41.5 mg, 0.15 mmol, 1.2 eq) was added to the reaction solution, and stirred for 30 min. LC-MS analysis was performed. After completion of the reaction, the reaction solution was purified by reverse-phase C18 column chromatography to yield two fractions: LD-5 with the earlier retention time and LD-6 with the later retention time. The target fractions were lyophilized to yield yellow solids LD-5 (20 mg) and LD-6 (23 mg).

[0237] LC-MS: 1092.4 [M+H] + .

[0238] Chromatographic conditions: Thermo Fisher Scientific semi-preparative liquid chromatograph U3000; chromatographic column: Welch Ultimate XB-Phenyl; mobile phase: 0.1% formic acid acetonitrile-0.1% formic acid aqueous solution = 50:50; flow rate: 30.0 mL / min; detection wavelength: 370 nm; injection volume: 100 μL.

[0239] Experimental Procedure: Dissolve an appropriate amount of LD-5 and LD-6 mixture in DMF to prepare a 10 mg / mL test solution. Inject this solution into a preparative liquid chromatograph and analyze according to the chromatographic conditions of this invention. Record the data and repeat the injection multiple times.

[0240] Results: LD-5 and LD-6 were obtained by preparative separation. The retention times of the two components were 6.04 min and 6.48 min, respectively. The purities were 98.58% and 99.13%, respectively.

[0241] Step 2 Preparation of ADC-5 and ADC-6

[0242] At 37°C, a solution of trastuzumab in PBS buffer (pH = 6.5, 0.05% PBS buffer; 2.5 mL, 9.96 mg / mL, 0.168 nmol) was added with a prepared aqueous solution of tris(2-carboxyethyl)phosphine (10 mM, 0.082 mL). The solution was placed in a water bath shaker and shaken at 37°C for 3 hours to stop the reaction. The reaction solution was cooled to 25°C in a water bath, diluted to 5.0 mg / mL, and two equal portions were prepared in parallel.

[0243] 2.0 nmol of compound LD-5 was dissolved in DMSO (0.10 mL) and added to the above 2.0 mL solution. The mixture was shaken in a water bath at 25°C for 3 hours, after which the reaction was stopped. The reaction solution was desalted and purified using a Sephadex G25 gel column (elution phase: 0.05 M PBS buffer solution, pH 6.5, containing 0.001 M EDTA) to obtain ADC in PBS buffer (5.0 mg / mL, 1.1 mL) and stored frozen at 4°C. UV-HPLC calculated average: n = 7.3.

[0244] ADC-6 was prepared by the same method using compound LD-6, with n=7.3.

[0245] Example 8 Other ADCs

[0246] Other compounds similar to LD-1, LD-2, LD-3, LD-4, LD-5, or LD-6 (the camptothecin derivatives in this application are small molecule toxins) can be prepared using the same method. LD-1, LD-2, LD-3, LD-4, LD-5, or LD-6 and similar compounds can be further combined with the antibody trastuzumab or other similar antibodies to prepare antibody-drug conjugates containing the camptothecin derivatives in this application as small molecule toxins.

[0247] Example 9 Determination of antiproliferative activity of SK-BR-3 cells

[0248] The activity of the antibody-drug conjugate of the present invention can be determined by measuring the in vitro anti-SK-BR-3 cell proliferation activity of a small molecule toxin camptothecin derivative.

[0249] SK-BR-3 cells were seeded in 384-well plates (Fisher 142762) with 3000 cells per well. A gradient of dilutions of the compound was added on the second day. 72 hours after addition of the compound, the ATP content in the cells was measured by CellTiter-Lumi (Biyuntian C0068XL). Cell growth was evaluated and the relative IC of the compound for inhibiting cell growth was calculated. 50 The screening results are shown in Table 6.

[0250] Table 6. Antiproliferative activity of the compounds of the present invention on SK-BR-3 cells

[0251] serial number <![CDATA[IC 50 (nM)]]> serial number <![CDATA[IC 50 (nM)]]> 1 2.98 1-1 5.81 1-2 1.73 3-2 2.85 4-2 2.74 7-2 2.68 14-2 4.13 20 5.83 32 4.30 34 5.84 35 6.53 39 4.78 41 6.47 44 6.29 48 6.73 49 7.32 Exatecan 5.35 Deruxtecan 13.61 Topotecan 58.30

[0252] Compared to Exatecan, Deruxtecan, and the marketed camptothecin drug topotecan, the compounds of the present invention exhibit strong in vitro anti-SK-BR-3 cell proliferation activity, particularly when X in the general formula (1) is S or O. For example, compound 1-2 exhibits a two-fold higher activity than Exatecan, and compound 32 exhibits three times the activity of Deruxtecan. In particular, the compounds of the general formula (1) of the present invention, because they contain conveniently attached groups such as OH or NH2 in their side chains, exhibit strong cellular activity and are therefore suitable as small molecule toxins for ADCs.

[0253] Example 10 In vitro antitumor activity of the antibody-drug conjugate of the present invention

[0254] SK-BR-3 cells, which highly express HER2, were selected as the cell line for in vitro activity testing in this experiment to evaluate the dose-effect of the antibody-drug conjugate on cell killing. The initial seeding density of each cell was selected: 1500-2000 cells / well, and the cytotoxic activity was determined after 12 hours. The final concentration of the antibody-drug conjugate after addition was set to 10nM as the starting concentration, and a series of concentrations (3-10 times dilution) were designed. The killing effect was observed for 144 hours, and the CellTiter-Glo@Luminescent Cell Viability Assay chemiluminescence staining was performed, and the IC was calculated after reading the fluorescence data. 50 .

[0255] Judging from the activity test results, all ADCs showed certain anti-tumor activity, and the activity of some ADCs exceeded that of DS-8201a.

[0256] Table 7 Anti-proliferative activity of the antibody-drug conjugates of the present invention on SK-BR-3 cells

[0257] sample <![CDATA[IC 50 (nM)]]> ADC-1 0.26 ADC-2 0.13 ADC-3 0.08 ADC-4 0.06 ADC-5 0.02 ADC-6 0.10 Exatecan 0.73 DS-8201a 0.09

[0258] Judging from the activity test results, all ADCs showed certain anti-tumor activity, and the activity of some ADCs exceeded that of DS-8201a.

[0259] Example 11 In vivo anti-tumor activity of the antibody-drug conjugate of the present invention

[0260] Human gastric cancer cells (NCI-N87) dissolved in 100 μL PBS were subcutaneously injected into the right side of the neck of 6-8 week old female Balb / c nude mice. 3 The 32 nude mice were randomly divided into 4 groups according to tumor size, with 8 animals in each group. The mice were injected into the tail vein: 01 was a blank control group, 02 was DS-8201a (4.5 mg / kg), 03 was ADC-1 (4.5 mg / kg), and 04 was ADC-2 (4.5 mg / kg). The body weight and tumor volume of the experimental animals were measured twice a week, and the survival status of the animals was observed during the experiment. The specific results of the changes in tumor volume in each group are shown in Figure 2. Figure 1 .

[0261] from Figure 1 It can be seen that both ADC samples of the present invention exhibited in vivo anti-tumor activity comparable to that of DS-8201a.

[0262] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A camptothecin derivative compound represented by general formula (1) or any of its optical isomers or pharmaceutically acceptable salts thereof: In the general formula (1): m is an integer of 0, 1 or 2; X is selected from -O- and -S-; R 1 and R 2 independently selected from H, halogen, OH, C1-6 alkyl, C1-6 alkoxy, C1-6 haloalkyl, NH2, NO2 or CN; R 3 Selected from C1-6 alkyl, C1-3 alkoxy substituted C1-3 alkyl or C1-6 haloalkyl; R 5 Selected from H; R 6 and R 7 Independently selected from H, C1-6 alkyl, C1-6 haloalkyl or C3-6 cycloalkyl; R 8 Selected from OH.

2. The camptothecin derivative compound represented by formula (1) according to claim 1, or its optical isomers or pharmaceutically acceptable salts thereof, wherein in formula (1), R 1 and R 2 independently selected from H, halogen, OH, Me, Et, OMe, OEt, CF3, NH2, NO2 or CN.

3. The camptothecin derivative compound represented by formula (1) according to claim 1, or its optical isomers or pharmaceutically acceptable salts thereof, wherein in formula (1), R 3 Selected from Me, Et, 4. The camptothecin derivative compound represented by formula (1) according to claim 1, or its optical isomers or pharmaceutically acceptable salts thereof, wherein in formula (1), R 6 and R 7 Independently selected from H, Me, Et, CHF2, CF3, CH2CF3, 5. The camptothecin derivative compound represented by formula (1) according to claim 1, or each optical isomer thereof, or a pharmaceutically acceptable salt thereof, wherein in formula (1), Selected from 6. The camptothecin derivative compound represented by formula (1) according to claim 1 or its optical isomers or pharmaceutically acceptable salts thereof, wherein the compound has one of the following structures:

7. The camptothecin derivative compound represented by formula (1) according to claim 1 or its optical isomers or pharmaceutically acceptable salts thereof, wherein the compound has one of the following structures:

8. A pharmaceutical composition, characterized in that It contains a pharmaceutically acceptable excipient or carrier, and a camptothecin derivative compound represented by general formula (1) as described in any one of claims 1 to 7, or its optical isomers or pharmaceutically acceptable salts thereof as an active ingredient.

9. Use of a camptothecin derivative compound represented by general formula (1) or any of its optical isomers or pharmaceutically acceptable salts according to any one of claims 1 to 7, or a pharmaceutical composition according to claim 8, in the preparation of a drug for treating breast cancer.

10. An antibody-drug conjugate, characterized in that The antibody-drug conjugate has one of the following structures: What it does not do n is 7.2; Wherein Ab represents trastuzumab; n is 7.2-7.

3.

11. An antibody-drug conjugate, characterized in that The antibody-drug conjugate has one of the following structures: n is 7.2; n is 7.2; n is 7.3; n is 7.3; The antibody-drug conjugate is not n is 7.2.

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