HK40095722B - Isochroman compound - Google Patents

Description

heterochromatic compounds

This application claims the following priority:

CN202010991814.1, application date: September 18, 2020.

Technical Field

This invention relates to novel heterochromatic compounds, particularly to compounds of formula (I) or pharmaceutically acceptable salts thereof, and the use of compounds of formula (I) or pharmaceutically acceptable salts thereof in therapeutic fields.

Background Technology

Malignant tumors are a serious disease that severely threatens human life and health. Current treatment methods mainly include surgery, chemotherapy, and targeted therapy. Chemotherapy is a systemic treatment that uses chemical drugs to kill tumor cells and inhibit their growth. Due to the heterogeneity of malignant tumors, chemotherapy remains an important method for treating cancer. However, it is precisely this systemic treatment that leads to significant side effects from chemotherapy. There is a huge unmet clinical need to develop targeted chemotherapy drugs.

Aldehyde reductase (AKR1C3) is a member of the aldehyde reductase family and is mainly involved in hormone synthesis and toxin clearance. AKR1C3 can be overexpressed by factors such as smoking, alcohol, and infection with hepatitis B or hepatitis C. AKR1C3 is overexpressed in a variety of refractory cancers, such as liver cancer, lung cancer, gastric cancer, esophageal cancer, colorectal cancer, prostate cancer, and acute lymphoblastic leukemia, especially liver cancer, where the overexpression rate is over 60%.

Currently, AKR1C3 inhibitor drugs are being developed in clinical practice, but significant progress has not been made. OBI-3424, a compound targeting the AKR1C3 enzyme, has been reported by OBI-3424, a selective prodrug that releases a potent DNA alkylating agent in tumor cells that highly express the AKR1C3 enzyme, selectively killing these cells and giving the chemotherapy drug a significant targeting effect.

Research on this target is currently in its early stages. Only OBI-3424 has entered Phase I clinical trials, primarily for hepatocellular carcinoma (HCC) and castration-induced prostate cancer (CRPC). Its efficacy and safety are still being validated. Therefore, this area requires further exploration and research.

Summary of the Invention

This invention provides compounds of formula (I) or pharmaceutically acceptable salts thereof.

Where T is N or CH;

_R1 and _R2 are each independently H, F, Cl, Br, I or _C1-3 alkyl, wherein the _C1-3 alkyl is optionally substituted by 1, 2 or 3 _Ra ; each _Ra is independently F, Cl, Br, I, -CN, -OH or _-NH2 ;

_R3 and _R4 are each independently H, F, Cl, Br, I, CN, _C1-3 alkyl, _C1-3 alkoxy, wherein the _C1-3 alkyl is optionally substituted by 1, 2 or 3 _Re ;

_Rb and _Rc are each independently H, _-CH3 , _-CH2CH3 , -( _CH2 ) _{2CH3 , or} -CH( _CH3 ) ₂ ;

R _d is -CH ₃ , -CH ₂ CH ₃ , -(CH ₂ ) ₂ CH ₃ or -CH(CH ₃ ) ₂ ;

Each _Re is independently F, Cl, Br, I, -CN, -OH or -NH ₂ .

In some embodiments of the present invention, the above-mentioned compound has the structure shown in formula (I-1) or formula (I-2):

Wherein, _R1 , _R2 , _R3 and _R4 are as defined in this invention.

In some embodiments of the present invention, the above-mentioned compounds have the structures shown in formula (I-3) or (I-4):

The carbon atoms marked with "*" are chiral carbon atoms, existing in a single enantiomer (R) or (S) form or rich in one enantiomer form; _R1 , _R2 , _R3 and _R4 are as defined in this invention.

In some embodiments of the present invention, _R1 and _R2 are each independently H, F, Cl, Br, I or _-CH3 , wherein _-CH3 is optionally replaced by 1, 2 or 3 _Ra , and _Ra and other variables are as defined in the present invention.

In some embodiments of the present invention, _R1 is H, F, Cl, Br, I or _-CH3 , and other variables are as defined in the present invention.

In some embodiments of the present invention, _R2 is H, and other variables are as defined in the present invention.

In some embodiments of the present invention, _R1 is H, F, Cl, Br, I or _-CH3 ; _R2 is H, and other variables are as defined in the present invention.

In some embodiments of the present invention, _R3 and _R4 are each independently H, F, Cl, Br, I, CN, _-CH3 , _-OCH3 , wherein _-CH3 is optionally replaced by 1, 2 or 3 _Re , and _Re and other variables are as defined in the present invention.

In some embodiments of the present invention, _R3 and _R4 are each independently H, F, _-CH3 , _-CHF2 , _-OCH3 , and other variables as defined in the present invention.

In some embodiments of the present invention, _R3 and _R4 are each independently H, F, _-CH3 , _-CHF2 , and _-OCH3 , respectively, and other variables are as defined in the present invention.

In some embodiments of the present invention, _R3 and _R4 are each independently H.

Some solutions in this invention are derived from arbitrary combinations of the above variables.

The present invention also provides compounds of the following formula or pharmaceutically acceptable salts thereof.

The present invention also provides compounds of the following formula or pharmaceutically acceptable salts thereof.

The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments targeting the AKR1C3 enzyme.

The present invention also provides the use of the above-mentioned compounds or pharmaceutically acceptable salts thereof in the preparation of medicaments for treating liver cancer.

Technical effect

This invention provides a novel compound targeting AKR1C3. The compound exhibits excellent anti-proliferative activity against tumor cells highly expressing AKR1C3, while showing weak activity against tumor cells with low AKR1C3 expression, demonstrating excellent selectivity. In both subcutaneous and orthotopic models of liver cancer, the compound of this invention exhibits significant antitumor efficacy.

definition

Unless otherwise stated, the following terms and phrases used herein are intended to have the following meanings. A particular term or phrase should not be considered uncertain or unclear unless specifically defined, but should be understood in its ordinary sense. When a trade name appears herein, it is intended to refer to the corresponding product or its active ingredient. The term "pharmaceutically acceptable" as used herein refers to compounds, materials, compositions, and/or dosage forms that, within the bounds of reliable medical judgment, are suitable for use in contact with human and animal tissues without undue toxicity, irritation, allergic reactions, or other problems or complications, in proportion to a reasonable benefit/risk ratio.

The term "pharmaceutically acceptable salt" refers to a salt of the compounds of this invention, prepared by reacting a compound having specific substituents discovered in this invention with a relatively non-toxic acid or base. When the compounds of this invention contain relatively acidic functional groups, base addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of base in a pure solution or a suitable inert solvent. Pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amine, or magnesium salts or similar salts. When the compounds of this invention contain relatively basic functional groups, acid addition salts can be obtained by contacting a neutral form of such compound with a sufficient amount of acid in a pure solution or a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include inorganic acid salts, such as hydrochloric acid, hydrobromic acid, nitric acid, carbonic acid, bicarbonate, phosphoric acid, monohydrogen phosphate, dihydrogen phosphate, sulfuric acid, hydrogen sulfate, hydroiodic acid, phosphorous acid, etc.; and organic acid salts, such as acetic acid, propionic acid, isobutyric acid, maleic acid, malonic acid, benzoic acid, succinic acid, octanoic acid, fumaric acid, lactic acid, mandelic acid, phthalic acid, benzenesulfonic acid, p-toluenesulfonic acid, citric acid, tartaric acid, and methanesulfonic acid; as well as salts of amino acids (such as arginine) and salts of organic acids such as glucuronic acid. Certain specific compounds of the present invention contain both basic and acidic functional groups, and thus can be converted into either a base or an acid addition salt.

The pharmaceutically acceptable salts of the present invention can be synthesized from parent compounds containing acid radicals or bases by conventional chemical methods. Generally, such salts are prepared by reacting these compounds in free acid or base form with a stoichiometric amount of a suitable base or acid in water or an organic solvent or a mixture thereof.

In addition to the salt form, the compounds provided by this invention also exist in prodrug form. The prodrugs of the compounds described herein readily undergo chemical changes under physiological conditions to be converted into the compounds of this invention. Furthermore, the prodrugs can be converted into the compounds of this invention in the in vivo environment via chemical or biochemical methods.

Some compounds of this invention may exist in non-solventized or solvated forms, including hydrated forms. Generally, solvated and non-solventized forms are equivalent and both are included within the scope of this invention.

The compounds of this invention can exist in specific geometric or stereoisomeric forms. This invention contemplates all such compounds, including cis and trans isomers, (-)- and (+)- enantiomers, (R)- and (S)- enantiomers, diastereomers, (D)- isomers, (L)- isomers, and racemic mixtures thereof, as well as other mixtures, such as mixtures enriched with enantiomers or diastereomers, all of which are within the scope of this invention. Additional asymmetric carbon atoms may be present in substituents such as alkyl groups. All such isomers and mixtures thereof are included within the scope of this invention.

Unless otherwise stated, the terms "enantiomer" or "optical isomer" refer to stereoisomers that are mirror images of each other.

Unless otherwise stated, the terms "cis-trans isomers" or "geometric isomers" arise because the single bonds of double bonds or cyclic carbon atoms cannot rotate freely.

Unless otherwise stated, the term "diastereomer" refers to a stereoisomer of a molecule having two or more chiral centers and being in a non-mirror relationship with each other.

Unless otherwise stated, "(D)" or "(+)" indicates right-handed rotation, "(L)" or "(-)" indicates left-handed rotation, and "(DL)" or "(±)" indicates racemic rotation.

Unless otherwise specified, wedge-shaped solid and wedge-shaped dashed lines represent the absolute configuration of a solid center, straight solid and straight dashed lines represent the relative configuration of a solid center, and wavy lines represent wedge-shaped solid or wedge-shaped dashed lines, or wavy lines represent straight solid and straight dashed lines.

The compounds of this invention can exist in specific forms. Unless otherwise stated, the terms "tautomer" or "tautomer form" refer to isomers of different functional groups in dynamic equilibrium at room temperature, capable of rapidly interconverting into each other. If tautomerization is possible (e.g., in solution), chemical equilibrium of the tautomer can be achieved. For example, proton tautomers (also called prototropic tautomers) include interconversions via proton migration, such as keto-enol isomerization and imine-enamine isomerization. Valence tautomers include interconversions involving the rearrangement of some bonding electrons. A specific example of keto-enol tautomerization is the interconversion between the two tautomers, pentane-2,4-dione and 4-hydroxypent-3-en-2-one.

Unless otherwise stated, the terms "rich in one isomer," "isomer enrichment," "rich in one enantiomer," or "enantiomer enrichment" mean that the content of one isomer or enantiomer is less than 100%, and the content of the isomer or enantiomer is greater than or equal to 60%, or greater than or equal to 70%, or greater than or equal to 80%, or greater than or equal to 90%, or greater than or equal to 95%, or greater than or equal to 96%, or greater than or equal to 97%, or greater than or equal to 98%, or greater than or equal to 99%, or greater than or equal to 99.5%, or greater than or equal to 99.6%, or greater than or equal to 99.7%, or greater than or equal to 99.8%, or greater than or equal to 99.9%.

Unless otherwise stated, the terms "isomer excess" or "enantiomer excess" refer to the difference between the relative percentages of two isomers or two enantiomers. For example, if one isomer or enantiomer is 90% and the other isomer or enantiomer is 10%, then the isomer or enantiomer excess (ee value) is 80%.

Optically active (R)- and (S)- isomers, as well as D- and L- isomers, can be prepared by chiral synthesis, chiral reagents, or other conventional techniques. To obtain an enantiomer of a compound of the present invention, it can be prepared by asymmetric synthesis or derivatization with a chiral auxiliary, wherein the resulting diastereomeric mixture is separated and the auxiliary group is cleaved to provide a pure desired enantiomer. Alternatively, when the molecule contains a basic functional group (such as an amino group) or an acidic functional group (such as a carboxyl group), a salt of the diastereomeric isomer is formed with a suitable optically active acid or base, followed by diastereomeric resolution by conventional methods known in the art, and then the pure enantiomer is recovered. Furthermore, the separation of enantiomers and diastereomeric isomers is typically accomplished by using chromatography with a chiral stationary phase, optionally combined with chemical derivatization (e.g., from amines to carbamates). The compounds of the present invention may contain atomic isotopes in non-natural proportions on one or more atoms constituting the compound. For example, compounds can be labeled with radioactive isotopes, such as tritium ( ^3H ), iodine-125 ( ^125I ), or C-14 ( ^14C ). As another example, deuterium can be used to replace hydrogen to form deuterated drugs. The bond between deuterium and carbon is stronger than that between ordinary hydrogen and carbon. Compared to undeuterated drugs, deuterated drugs have advantages such as reduced toxicity, increased drug stability, enhanced efficacy, and prolonged biological half-life. All isotopic variations in the compounds of this invention, regardless of radioactivity, are included within the scope of this invention.

The terms “optional” or “optionally” refer to events or conditions that may occur but are not required to occur as described below, and the description includes both cases where said events or conditions occur and cases where said events or conditions do not occur.

The term "substituted" means that any one or more hydrogen atoms on a particular atom are replaced by a substituent, which can include deuterium and hydrogen variants, provided that the valence state of the particular atom is normal and the substituted compound is stable. When the substituent is oxygen (i.e., =O), it means that two hydrogen atoms are replaced. Oxygen substitution does not occur on aromatic groups. The term "optionally substituted" means that it may or may not be substituted, unless otherwise specified, and the type and number of substituents can be arbitrary on a chemically feasible basis.

When any variable (e.g., R) appears more than once in the composition or structure of a compound, its definition is independent in each case. Thus, for example, if a group is substituted by 0-2 Rs, the group can optionally be substituted by at most two Rs, and the Rs in each case have independent options. Furthermore, combinations of substituents and/or their variants are only permitted if such combinations produce a stable compound.

When the number of a linking group is 0, such as -(CRR) _0- , it indicates that the linking group is a single bond.

When one of the variables is selected as a single bond, it means that the two groups it connects to are directly connected. For example, when L in A-L-Z represents a single bond, it means that the structure is actually A-Z.

When a substituent is vacant, it means that the substituent does not exist. For example, in A-X, if X is vacant, it means that the structure is actually A. When the listed substituents do not specify which atom they are attached to the substituted group through, such substituents can be bonded to any of their atoms. For example, a pyridinium group as a substituent can be attached to the substituted group through any carbon atom on the pyridine ring.

When the connecting group listed does not specify its connection direction, the connection direction is arbitrary. For example, if the connecting group L is -M-W-, then -M-W- can connect ring A and ring B in the same direction as the reading order from left to right, or in the opposite direction to the reading order from left to right. The combination of the connecting group, substituent and/or its variants is only permitted if such a combination produces a stable compound.

Unless otherwise specified, when a group has one or more connectable sites, any one or more sites of that group can be linked to other groups by chemical bonds. The chemical bonds connecting these sites to other groups can be represented by solid lines, dashed lines, or wavy lines. For example, a solid line in -OCH ₃ indicates a connection to another group through the oxygen atom in that group; a dashed line in indicates a connection to another group through the two ends of the nitrogen atom in that group; and a wavy line in indicates a connection to another group through the carbon atoms at positions 1 and 2 of the phenyl group.

Unless otherwise specified, the number of atoms in a ring is usually defined as the elemental number of the ring. For example, a “5-7 elemental ring” refers to a “ring” with 5-7 atoms arranged around it.

Unless otherwise specified, the term " _C1-3 alkyl" is used to denote a straight-chain or branched saturated hydrocarbon group consisting of one to three carbon atoms. The _C1-3 alkyl group includes _C1-2 and _C2-3 alkyl groups, etc.; it can be monovalent (e.g., methyl), divalent (e.g., methylene), or polyvalent (e.g., methine). Examples of _C1-3 alkyl groups include, but are not limited to, methyl (Me), ethyl (Et), propyl (including n-propyl and isopropyl), etc.

Unless otherwise specified, the term " _C1-3 alkoxy" refers to alkyl groups comprising one to three carbon atoms that are attached to the remainder of a molecule by an oxygen atom. _C1-3 alkoxy groups include _C1-2 , _C2-3 , _C3 , and _C2 alkoxy groups, etc. Examples of _C1-3 alkoxy groups include, but are not limited to, methoxy, ethoxy, propoxy (including n-propoxy and isopropoxy), etc.

Unless otherwise specified, C _{_n-n+m} or C<sub> _n -C_{_n+m} includes any specific case of n to n+m carbons, such as C _{_1-12} including C_₁, C _{₂ ,} C _₃ , C₄, C_₅, C₆, C₇ , C ₈, C _₉ , C _₁₀, C _₁₁, and C _₁₂, and also includes any range from n to n+m, such as C _{_1-12} including C_{_1-3}, C _{_1-6}, C_1-9, C_3-6, C_3-9, C_3-12 , C _{_6-9}, C _{_6-12} , and C₁. _9-12 , etc.; similarly, n-membered to n+m-membered indicates that the number of atoms on the ring is n to n+m. For example, 3-12-membered rings include 3-membered rings, 4-membered rings, 5-membered rings, 6-membered rings, 7-membered rings, 8-membered rings, 9-membered rings, 10-membered rings, 11-membered rings, and 12-membered rings. It also includes any range from n to n+m. For example, 3-12-membered rings include 3-6-membered rings, 3-9-membered rings, 5-6-membered rings, 5-7-membered rings, 6-7-membered rings, 6-8-membered rings, and 6-10-membered rings, etc.

The term "leaving group" refers to a functional group or atom that can be replaced by another functional group or atom through a substitution reaction (such as a nucleophilic substitution reaction). For example, representative leaving groups include trifluoromethanesulfonates; chlorine, bromine, and iodine; sulfonate groups, such as methanesulfonates, toluenesulfonates, p-bromobenzenesulfonates, p-toluenesulfonates, etc.; acyloxy groups, such as acetoxy groups, trifluoroacetoxy groups, etc.

The term "protecting group" includes, but is not limited to, "amino protecting group," "hydroxy protecting group," or "thiol protecting group." The term "amino protecting group" refers to a protecting group suitable for preventing side reactions at the nitrogen position of an amino group. Representative amino protecting groups include, but are not limited to: formyl; acyl, such as alkanoyl (e.g., acetyl, trichloroacetyl, or trifluoroacetyl); alkoxycarbonyl, such as tert-butoxycarbonyl (Boc); arylmethoxycarbonyl, such as benzyloxycarbonyl (Cbz) and 9-fluorenemethoxycarbonyl (Fmoc); arylmethyl, such as benzyl (Bn), triphenylmethyl (Tr), 1,1-di-(4'-methoxyphenyl)methyl; silyl, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc. The term "hydroxyl protecting group" refers to a protecting group suitable for preventing hydroxyl side reactions. Representative hydroxyl protecting groups include, but are not limited to: alkyl groups, such as methyl, ethyl, and tert-butyl; acyl groups, such as alkanolyl groups (e.g., acetyl); arylmethyl groups, such as benzyl (Bn), p-methoxybenzyl (PMB), 9-fluorenylmethyl (Fm), and diphenylmethyl (diphenylmethyl, DPM); silyl groups, such as trimethylsilyl (TMS) and tert-butyldimethylsilyl (TBS), etc.

The compounds of the present invention can be prepared by a variety of synthetic methods known to those skilled in the art, including the specific embodiments listed below, embodiments formed by combining them with other chemical synthetic methods, and equivalent substitutions known to those skilled in the art. Preferred embodiments include, but are not limited to, the embodiments of the present invention.

This invention uses the following abbreviations:

Pd/C Pd/C catalyst, palladium content 10 wt%. DCM dichloromethane THF Tetrahydrofuran EtOAc Ethyl acetate TBME tert-butyl methyl ether Boc tert-Butoxycarbonyl is an amine protecting group. Cbz benzyloxycarbonyl is an amine protecting group. DMF N,N-Dimethylformamide TFA Trifluoroacetic acid PE petroleum ether DMSO Dimethyl sulfoxide EtOH ethanol MeOH methanol AcOH Acetic acid DIPEA Diisopropylethylamine <![CDATA[SiO₂]]> 100-200 mesh silica gel powder, used for column chromatography. psi pounds per square inch (lbs) is a unit of pressure. p-HPLC Preparative high-performance liquid chromatography (HPLC) for the purification of compounds.

The compounds of this invention are named according to conventional naming principles in the art or using software, and commercially available compounds are named according to the supplier's catalog.

The solvents used in this invention are commercially available and require no further purification. Reactions are generally carried out under inert nitrogen atmosphere in an anhydrous solvent. Proton NMR data are recorded on a Bruker Avance III 400 (400 MHz) spectrometer, with chemical shifts expressed in ppm at the high field of tetramethylsilane. LC/MS or Shimadzu MS includes a DAD: SPD-M20A (LC) and a Shimadzu Micromass 2020 detector. The mass spectrometer is equipped with an electrospray ionization (ESI) source operating in either positive or negative mode.

The structures of the compounds of this invention can be confirmed using conventional methods well known to those skilled in the art. If this invention relates to the absolute configuration of a compound, the absolute configuration can be confirmed using conventional techniques in the art. For example, single-crystal X-ray diffraction (SXRD) can be used. The cultured single crystal is analyzed using a Bruker D8 venture diffractometer to collect diffraction intensity data. The light source is CuKα radiation, and the scanning mode is scanning. After collecting the relevant data, the crystal structure can be further analyzed using the direct method (Shelxs 97) to confirm the absolute configuration.

High-performance liquid chromatography (HPLC) analysis was performed using a Shimadzu LC20AB system equipped with a Shimadzu SIL-20A autosampler and a Shimadzu DAD:SPD-M20A detector, using an Ultimate C18 column (3μm packing material, 2.1×300mm). The 0-60AB_6 min method was employed: a linear gradient was used, starting with 100% A (A being an aqueous solution of 0.0675% TFA) and ending with 60% B (B being a MeCN solution of 0.0625% TFA), for a total run time of 4.2 min, followed by 1 min of 60% B. The column was reequilibrated for 0.8 min to reach 100:0, for a total run time of 6 min. The 10-80AB_6-minute method: A linear gradient was applied, starting with 90% A (A being an aqueous solution of 0.0675% TFA) and ending with 80% B (B being an acetonitrile solution of 0.0625% TFA), for a total run time of 4.2 minutes. Elution was then continued with 80% B for 1 minute. The column was reequilibrated for 0.8 minutes to achieve a 90:10 ratio, for a total run time of 6 minutes. The column temperature was 50°C, and the flow rate was 0.8 mL/min. A diode array detector was used to scan at wavelengths of 200-400 nm.

Thin-layer chromatography (TLC) was performed on a Sanpont-group silica gel GF254. Spots were typically detected by UV light irradiation, but other methods were used in some cases. In these cases, the TLC plate was developed with iodine (prepared by thoroughly mixing approximately 1 g of iodine with 10 g of silica gel), vanillin (prepared by dissolving approximately 1 g of vanillin in 100 mL _of 10% _H₂SO₄ ), ninhydrin (purchased from Aldrich), or a special colorimetric reagent (prepared by thoroughly mixing ₂₅ g of ( _NH₄ ) _{₆Mo₇O₂₄} · _4H₂O , 5 g of ( _NH₄ )₂Ce(IV)( _NO₃ )₆, 450 mL of _H₂O , and 50 _mL of concentrated _H₂SO₄ ) to examine the compounds. A similar method to that disclosed in Still, WC; Kahn, M.; and Mitra, M., Journal of Organic Chemistry, 1978, 43, 2923-2925, was used for rapid column chromatography on 40-63 μm (230-400 mesh) silica gel in Silicycle. Common solvents for rapid column chromatography or thin-layer chromatography are mixtures of dichloromethane/methanol, EtOAc/methanol, and petroleum ether/EtOAc.

Preparative chromatography was performed on a Gilson-281 Prep LC 322 system using a Gilson UV/VIS-156 detector. The columns used were Agella Venusil ASB Prep C18 (5 μm packing, 150 × 21.2 mm), Phenomenex Gemini C18 (5 μm packing, 150 × 30 mm), Boston Symmetrix C18 (5 μm packing, 150 × 30 mm), or Phenomenex Synergi C18 (4 μm packing, 150 × 30 mm). The compounds were eluted with a low gradient of acetonitrile/water (containing 10 mM ammonium bicarbonate) at a flow rate of approximately 25 mL/min, with a total run time of 8–15 minutes.

Attached Figure Description

Figure 1 shows the tumor volume growth curves for each group during the drug administration period.

Figure 2 shows the relative weight gain curves of animals in each group during the drug administration period.

Figure 3 shows the tumor growth signal-time curve.

Figure 4 is a schematic diagram of the tumor weight at the experimental endpoint.

Figure 5 shows the animal's weight-time curve.

Figure 6 shows the animal's weight change-time curve.

Detailed Implementation

The present invention will be described in detail below with reference to embodiments, but this does not imply any adverse limitation on the invention. The present invention has been described in detail, and specific embodiments thereof have been disclosed. It will be apparent to those skilled in the art that various changes and modifications can be made to the specific embodiments of the present invention without departing from the spirit and scope thereof.

Example 1

Step A: Compound 1-1 (5 g, 44.21 mmol) was dissolved in DMF (50 mL), and potassium carbonate (18.33 g, 132.64 mmol) and compound 1-2 (9 g, 48.63 mmol) were added. The reaction mixture was stirred at 50°C for 12 hours. The reaction mixture was concentrated under reduced pressure, and the residue was added with water (50 mL). The pH was adjusted to 1 with dilute hydrochloric acid (1 mol/L). The mixture was filtered, and the filter cake was dried under vacuum to obtain compound 1-3. ¹ H NMR (DMSO-d ₆ ,400MHz)δ13.88-13.37(m,1H),8.55(d,J＝2.0Hz,1H),8.21-8.16(m,2H),7.97( ddd,J＝1.6,8.1,9.9Hz,1H),7.50(dd,J＝5.0,7.8Hz,1H),7.29(d,J＝8.8Hz,1H).

Step B: Compounds 1-3 (6 g, 21.57 mmol) were dissolved in dioxane (150 mL), and dichlorobis(4-methylisopropylphenyl)ruthenium(II) (1.32 g, 2.16 mmol), guanidine carbonate (1.94 g, 10.78 mmol), compounds 1-4 (3.91 g, 29.55 mmol), and AcOH (1.30 g, 21.57 mmol) were added. The reaction mixture was stirred at 105°C for 16 hours under nitrogen protection. The reaction mixture was concentrated under reduced pressure. The crude product was purified by column chromatography ( _SiO₂ , PE:EtOAc = 1:0-5:1) to obtain compounds 1-5. ¹ H NMR (DMSO-d ₆ ,400MHz)δ8.64(s,1H),8.14(td,J＝1.5,4.8Hz,1H),7.92(ddd,J＝1.7,8.1,10.0Hz,1H) ,7.80(s,1H),7.60(s,1H),7.52-7.42(m,4H),7.39-7.32(m,2H),5.42(d,J=1.2Hz,2H).

Step C: Compounds 1-5 (2.5 g, 6.37 mmol) were added to toluene (50 mL), cooled to -60°C, and diisobutylaluminum hydride (1 mol/L, 12.11 mL) was added. The reaction mixture was stirred at -60°C for 2 hours. The reaction mixture was quenched at -60°C with water (1 mL), followed by the addition of sodium tartrate aqueous solution (4.5 g dissolved in 100 mL). The mixture was stirred for 1.5 hours, then extracted with EtOAc (100 mL × 2). The combined organic phases were washed with brine (50 mL × 1), dried over anhydrous sodium sulfate, and concentrated under reduced pressure to obtain crude compounds 1-6.

Step D: Compounds 1-6 (2.4 g, 1.06 mmol) and triethylsilane (2.12 g, 18.26 mmol) were added to DCM (50 mL), and TFA (2.08 g, 18.26 mmol) was added at 0°C. The mixture was slowly heated to 25°C and stirred for 2 hours. The reaction mixture was added to DCM (100 mL), washed with sodium bicarbonate (50 mL × 1), and the combined organic phases were washed with brine (20 mL). The organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography ( _SiO₂ , PE/DCM/EtOAc = 20:1:1-5:1:1) to give compounds 1-7. ¹ H NMR (DMSO-d ₆ ,400MHz) δ8.07(s,1H),8.04(s,1H),7.99(br d,J＝4.9Hz,1H),7.57(ddd,J＝1.4,8.2,10.1Hz,1H),7.52(s,1H),7.47-7.40(m,2H),7.38-7.30(m,4H),4.84(s,2H),4.75(s,2H).

Step E: Dissolve compounds 1-7 (400 mg, 1.06 mmol) in DCM (50 mL), cool to -70°C, and purge with ozone for approximately 15 minutes. Excess ozone is then removed with a nitrogen stream. A mixture of sodium borohydride (131.09 mg, 3.47 mmol) and methanol (5 mL) is added at -20°C, and the reaction mixture is stirred at 0-20°C for 1 hour. The reaction mixture is quenched by adding water (10 mL) at 20°C and concentrated under reduced pressure. The crude product is purified by column chromatography ( _SiO₂ , PE:EtOAc = 100:0-1:1) to obtain compounds 1-8. ¹ H NMR (DMSO-d ₆ ,400MHz)δ8.10(td,J＝1.5,4.8Hz,1H),7.94(s,1H),7.77(ddd,J＝1.5,8.1,10.1Hz,1H),7.44(ddd,J＝0.6,4.8,7.9Hz,1H),7.25(s ,1H),5.76(d,J＝6.2Hz,1H),4.83-4.67(m,2H),4.55(q,J＝6.1Hz,1H),3.94(dd,J＝4.9,11.4Hz,1H),3.58(dd,J＝6.8,11.4Hz,1H).

Step F: Dissolve compounds 1-8 (190 mg, 620.42 μmol) in THF (8 mL). Add hexamethyldisilamide lithium (1 mol/L, 930.63 μL) at -60°C. Stir the mixture at -60°C for 15 minutes under nitrogen protection. Add phosphorus oxychloride (190.26 mg, 1.24 mmol) at -60°C. Stir the mixture at -60°C for 15 minutes under nitrogen protection. Then add 2-bromoethylamine hydrobromide (1.02 g, 4.96 mmol) and diisopropylethylamine (641.46 mg, 4.96 mmol). Stir the reaction mixture at 0°C for 1 hour under nitrogen protection. The reaction mixture was extracted with water (10 mL) and EtOAc (50 mL × 3). The combined organic phases were washed with brine (10 mL × 1), dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure. The crude product was purified by column chromatography ( _SiO₂ , PE:EtOAc = 100:0-0:1) to give compounds 1-9.

Step G: Compounds 1-9 (360 mg, 601.85 μmol) were dissolved in THF (18 mL), and silver oxide (4.18 g, 18.06 mmol) was added. The mixture was stirred at 63°C for 12 hours. The reaction solution was filtered, and the filtrate was concentrated under reduced pressure. The crude product was subjected to p-HPLC (column: Welch Ultimate XB-SiOH (size: 250 mm × 50 mm, particle size: 10 μm); mobile phase: [n-hexane-isopropanol]; elution gradient: isopropanol 20%-60%, 15 min) to obtain compound 1. ^¹H NMR ( _CDCl³⁺ , 400MHz) δ 8.00 (td, J = 1.6, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J = 1.7, 7.8, 9.6 Hz, 1H), 7.20–7.16 (m, 2H), 5.42–5.29 (m, 1H), 4.87–4.59 (m, 2H), 4.00 (d, J = 4.4 Hz, 2H), 2.13–1.96 (m, 8H). Step H:

Compound 1 was chirally resolved (separation column: DAICL CHIRALPAK AD (size: 250 mm × 30 mm, particle size: 10 μm); mobile phase: [neutral-isopropanol]; elution gradient: isopropanol 35%-35%) to give compound 1A (retention time = 1.671 min) and compound 1B (retention time = 1.870 min).

Compound 1A: ^¹H NMR ( _CDCl₃ , 400MHz) δ 8.00 (td, J = 1.6, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J = 1.7, 7.9, 9.6 Hz, 1H), 7.21–7.15 (m, 2H), 5.35 (dt, J = 8.76, 4.25 Hz, 1H), 4.87–4.57 (m, 2H), 4.00 (d, J = 4.4 Hz, 2H), 2.14–1.96 (m, 8H). ee value (enantiomer excess): 100%.

Compound 1B: ^¹H NMR ( _CDCl₃ , 400MHz) δ 8.00 (td, J = 1.5, 4.8 Hz, 1H), 7.66 (s, 1H), 7.48 (ddd, J = 1.7, 7.9, 9.6 Hz, 1H), 7.19–7.16 (m, 2H), 5.35 (dt, J = 8.63, 4.32 Hz, 1H), 4.84–4.63 (m, 2H), 4.00 (d, J = 4.4 Hz, 2H), 2.13–1.95 (m, 8H). ee value (enantiomer excess): 98%.

Example 2

The synthesis of compound 2 follows the same steps A to G as that of compound 1. In the synthetic route for compound 2, starting material 1-1 is simply replaced with 2-fluorophenol.

Compound 2: ^¹H NMR (400MHz, _CDCl₃ ) δ 7.62 (s, 1H), 7.18–7.10 (m, 4H), 7.08 (s, 1H), 5.31 (td, J = 4.2, 8.7 Hz, 1H), 4.82–4.73 (m, 1H), 4.68–4.58 (m, 1H), 3.99 (d, J = 4.4 Hz, 2H), 2.08–1.92 (m, 8H).

Bioactivity

Experimental Example 1: Antiproliferative Activity of the Compounds of the Present Invention on the NCI-H460 Cell Line

Experimental materials:

RPMI-1640 medium and penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (a chemiluminescent cell viability assay) reagent was purchased from Promega. The NCI-H460 cell line was purchased from Nanjing Kebai Biotechnology Co., Ltd. A Nivo multilabel analyzer (PerkinElmer) was used.

Experimental methods:

NCI-H460 cells (lung cancer) were seeded in 96-well plates, with 80 μL of cell suspension per well, containing 4000 NCI-H460 cells. The cell culture plates were incubated overnight in a CO2 incubator. The test compound was diluted 5-fold to the 9th concentration using a multi-channel pipette, i.e., from 2 mM to 5.2 nM, in duplicate. 78 μL of culture medium was added to the intermediate plate, and then 2 μL of serially diluted compound was transferred to each well of the intermediate plate according to the corresponding positions. After mixing, 20 μL of the compound was transferred to each well of the cell culture plate. The concentration range of the compound transferred to the cell culture plate was 10 μM to 0.026 nM. The cell culture plates were incubated in a CO2 incubator for 2 hours, after which the drug-containing culture medium was removed, the cell culture plate was rinsed once with fresh culture medium, and 100 μL of drug-free fresh culture medium was added to each well for further incubation for 70 hours. A separate cell culture plate was prepared, and the signal value on the day of drug addition was recorded as the maximum value (Max value in the equation below) for data analysis. Add 25 μL of chemiluminescent cell viability assay reagent to each well of the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Read the values using a multi-label analyzer.

Data Analysis:

The original data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)*100%. The _IC50 value can then be obtained by curve fitting using four parameters (obtained in the "log(inhibitor) vs. response--Variable slope" mode of GraphPad Prism). Table 1 shows the inhibitory activity of the compounds of this invention on the proliferation of NCI-H460 cells.

Table 1. Data on the antiproliferative activity of the compounds of this invention against the NCI-H460 cell line.

Compound numbering <![CDATA[IC₅₀(nM)]]> Compound 1 3.8 Compound 1A 10.1 Compound 1B 16.6 Compound 2 21.6

Conclusion: The compounds of this invention exhibit excellent anti-proliferative activity against NCI-H460 cells that highly express AKR1C3.

Experimental Example 2: Antiproliferative Activity of the Compounds of the Present Invention on the HepG2 Cell Line

Experimental materials:

DMEM culture medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (a chemiluminescence immunoassay reagent for cell viability) reagent was purchased from Promega. HepG2 cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multilabel analyzer (PerkinElmer) was used.

Experimental methods:

HepG2 cells (liver cancer) were seeded in white 384-well plates, with 25 μM cell suspension per well, containing 1000 HepG2 cells. The cell culture plates were incubated overnight in a CO2 incubator. The test compound was diluted 3-fold to the 9th concentration using a multi-channel pipette, i.e., from 200 μM to 30 nM, in duplicate. 99 μM of culture medium was added to the intermediate plate, and then 1 μM of serially diluted compound was transferred to each well of the intermediate plate according to the corresponding positions. After mixing, 25 μM was transferred to each well of the cell culture plate. The concentration range of the compound transferred to the cell culture plate was 1 μM to 0.15 nM. The cell culture plates were incubated in a CO2 incubator for 5 days. A separate cell culture plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the equation below) for data analysis. 20 μM of chemiluminescent cell viability assay reagent was added to each well of this cell culture plate, and incubated at room temperature for 10 minutes to stabilize the luminescence signal. The readings were taken using a multi-label analyzer. Add 20 μM of chemiluminescent cell viability assay reagent to each well of the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Read the values using a multi-label analyzer.

Data Analysis:

The original data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)*100%. The _IC50 value can then be obtained by curve fitting using four parameters (obtained in the "log(inhibitor) vs. response--Variable slope" mode of GraphPad Prism). Table 2 shows the inhibitory activity of the compounds of this invention on the proliferation of HepG2 cells.

Table 2. Data on the antiproliferative activity of the compounds of this invention against the HepG2 cell line.

Compound numbering <![CDATA[IC₅₀(nM)]]> Compound 1A 9.3 Compound 1B 7.4

Conclusion: The compounds of this invention exhibit excellent anti-proliferative activity against HepG2 cells that highly express AKR1C3.

Experimental Example 3: Antiproliferative Activity of the Compounds of the Present Invention on Hep3B Cell Line

Experimental materials:

EMEM medium, penicillin/streptomycin antibiotics were purchased from Vicente, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (a chemiluminescent cell viability assay) reagent was purchased from Promega. Hep3B cell line was purchased from the Cell Bank of the Chinese Academy of Sciences. Nivo multilabel analyzer (PerkinElmer) was used.

Experimental methods:

Hep3B cells (liver cancer) were seeded in white 96-well plates, with 80 μL of cell suspension per well, containing 3000 Hep3B cells. The cell culture plates were incubated overnight in a CO2 incubator. The test compound was diluted 5-fold to the 9th concentration using a multi-channel pipette, i.e., from 2 mM to 5.12 nM, in duplicate. 78 μL of culture medium was added to the intermediate plate, and then 2 μL of serially diluted compound was transferred to each well of the intermediate plate according to the corresponding positions. After mixing, 20 μL of the compound was transferred to each well of the cell culture plate. The concentration range of the compound transferred to the cell culture plate was 10 μM to 0.0256 nM. The cell culture plates were incubated in a CO2 incubator for 3 days. A separate cell culture plate was prepared, and the signal value was read on the day of drug addition as the maximum value (Max value in the equation below) for data analysis. 25 μL of chemiluminescent cell viability assay reagent was added to each well of this cell culture plate, and the plate was incubated at room temperature for 10 minutes to stabilize the luminescence signal. The readings were taken using a multi-label analyzer. Add 25 μL of chemiluminescent cell viability assay reagent to each well of the cell plate and incubate at room temperature for 10 minutes to stabilize the luminescence signal. Read the values using a multi-label analyzer.

Data Analysis:

The original data were converted into inhibition rate using the equation (Sample-Min)/(Max-Min)*100%. The _IC50 value can then be obtained by curve fitting using four parameters (obtained in the "log(inhibitor) vs. response--Variable slope" mode in GraphPad Prism). Table 3 shows the inhibitory activity of the compounds of this invention on Hep3B cell proliferation.

Table 3. Data on the antiproliferative activity of the compounds of this invention against Hep3B cell lines.

Compound numbering <![CDATA[IC₅₀(nM)]]> Compound 1A >10,000 Compound 1B >10,000

Conclusion: The compounds of this invention have no anti-proliferative activity against Hep3B cells with low AKR1C3 expression, exhibiting high selectivity.

Experimental Example 4: In vivo pharmacodynamic study of the compound of the present invention on a nude mouse xenograft model of human hepatocellular carcinoma Hep G2.

Experimental objective:

This study investigated the inhibitory effect on tumor growth in a nude mouse xenograft model of human hepatocellular carcinoma Hep G2.

Experimental materials:

Female NU/NU nude mice (number: 90; age: 6-8 weeks), human hepatocellular carcinoma cells HepG2, MEM culture medium, fetal bovine serum (FBS), trypsin, penicillin-streptomycin antibiotics, PBS, matrix gel, etc.

Experimental methods and procedures:

1. Cell Culture

Routine cell culture was performed at 5% _CO2 , 37°C, and in MEM medium containing 10% fetal bovine serum; cells were passaged by digestion with 0.25% trypsin; and passaged 2 to 3 times per week at a passage ratio of 1:3 to 1:6, depending on cell growth.

2. Animal model preparation

Hep G2 cells in logarithmic growth phase were collected, counted, and resuspended in 50% serum-free MEM medium and 50% matrix gel, adjusting the cell concentration to 2.5 × ^10⁷ cells/mL. The cells were dispersed evenly by pipetting and transferred to 50 mL centrifuge tubes, which were then placed in an icebox. 200 μL (5 × 10⁶ cells/mouse) of the cell suspension was injected subcutaneously into the right axilla of nude mice using a 1 mL syringe to establish a Hep G2 nude mouse xenograft model. Animal condition and tumor growth were observed regularly after inoculation. Tumor diameter was measured using electronic calipers, and the data were directly entered into an Excel spreadsheet to calculate tumor volume. Once the tumor volume reached 100–300 ^mm³ , 48 healthy animals with similar tumor volumes were selected and randomly divided into 8 groups (n = 6) based on tumor volume, while maintaining a consistent average body weight within each group. The day of grouping was designated as the first day of the experiment (D1). After the experiment began, the tumor diameter was measured twice a week, the tumor volume was calculated, and the animal's weight was weighed and recorded.

The formula for calculating tumor volume (TV) is as follows: TV ( ^mm³ ) = l × ^w² / 2

Where l represents the long diameter of the tumor (mm); w represents the short diameter of the tumor (mm).

3. Animal grouping and administration:

Animal grouping and administration regimens are shown in Table 4. Administration began on the day of grouping and ended after 3 weeks (or when the tumor volume in the solvent control group reached 2000 ^mm³ or higher, whichever came first). The administration volume was 10 mL· ^kg⁻¹ . Group 1 served as the solvent control group, receiving intravenous injection of DMSO & 30% HP-β-CD (10:90, v:v) once a week for 3 consecutive weeks. Groups 2 and 3 received intravenous injections of compounds 1A and 1B, respectively, at a dose of 1 mg· ^kg⁻¹ .

Table 4. Dosing regimen for the Hep G2 nude mouse xenograft model drug efficacy experiment

4. Experimental Indicators:

The formula for calculating the tumor growth inhibition rate (GI) is: TGI = 100% × [1 - (TV _t(T) - TV _initial(T) ) / (TV _t(C) - TV _initial(C) )]

Where TV _{_t(T)} represents the tumor volume measured in the treatment group at each administration; TV _{_initial(T)} represents the tumor volume in the treatment group at the time of administration; TV_{_t(C)} represents the tumor volume measured in the solvent control group at each administration; and TV _{_initial(C)} represents the tumor volume in the solvent control group at the time of administration. The formula for calculating relative animal body weight is: Relative animal body weight = BW _{_t} / BW_{_initial * 100}

Wherein, BW _t represents the animal's body weight measured each time during the administration period; BW _initial represents the animal's body weight when the administration was administered in groups.

5. Inhibitory effect of the compound on the growth of subcutaneously transplanted HepG2 hepatocellular carcinoma tumors in nude mice:

This experiment evaluated the efficacy of compounds 1A and 1B in a HepG2 hepatocellular carcinoma xenograft model. After 21 days of administration, compound 1B at a dose of 1 mg/kg significantly inhibited tumor growth, with p < 0.05 compared to the solvent control group. Tumor volume and inhibition rate are shown in Table 5, and tumor growth curves are detailed in Figure 1.

Table 5. Tumor volume and tumor growth inhibition rate in each group of animals during drug administration.

6. Weight changes:

In this model, the body weight of all treated animals did not fluctuate significantly, and the overall average decrease in animal body weight did not exceed 10% (see Figure 2). Conclusion: The compounds of this invention exhibit good antitumor efficacy.

Experimental Example 5: In vivo pharmacodynamic study of the compound of the present invention against a human Hep G2 orthotopic xenograft tumor model.

Experimental objective:

This study used the HepG2 orthotopic xenograft tumor nude mouse model to evaluate the antitumor effects of the compounds.

Experimental materials:

Female Balb/C nude mice, 6-8 weeks old, weighing 18-22 grams, were used in the following environments: fetal bovine serum (PBS), EMEM culture medium (catalog number 30-2003), phosphate buffer, penicillin-dextrose antibody (catalog number 15240-062), Matrigel, and trypsin.

Experimental methods and procedures

1. Cell Culture Preparation: HepG2-luc cells were cultured in vitro in a monolayer using EMEM medium supplemented with 10% heat-inactivated fetal bovine serum at 37°C in a 5% _CO2 incubator. Cells were passaged twice a week using trypsin-EDTA digestion. When cell saturation reached 80%-90%, cells were digested with trypsin-EDTA, counted, and resuspended in PBS and Matrigel (PBS:Matrigel = 1:1) at a density of 166.67 × ^10⁶ cells/mL.

2. Tumor Cell Inoculation and Grouping: Animals were anesthetized via intramuscular injection of 60 mg/kg of 50mg/kg of methylphenidate + 1.5 mg/kg of toluidine. Once the animals were deeply anesthetized, they were properly secured, and the abdominal skin was cleaned with 75% alcohol swabs. A surgical incision of approximately 10 mm was made, and 0.03 mL of HepG2-luc cells (PBS: Matrigel = 1:1) was in situ inoculated into the left lobe of the liver of each mouse. The muscle wound was then sutured with absorbable catgut, and the skin wound was sutured with a stapler. The surgically operated animals were kept warm on a heat-insulating blanket until they regained consciousness. To reduce pain, 2 mg/kg of meloxicam (subcutaneous injection, once daily) was administered for 3 consecutive days post-surgery. Fifteen animals were randomly selected to monitor signal growth. When the signal began to rise, they were randomly grouped according to the biofluorescence signal value and treatment was initiated. Detailed treatment protocols are shown in Table 6.

Table 6. Grouping and Dosing Regimens of Experimental Animals

3. Experimental Indicators

The experimental endpoints were whether tumor growth could be delayed or whether the tumor could be cured. Animal bioluminescence signals and body weight were measured weekly after tumor inoculation until the end of the observation period. Bioluminescence signal values were used to calculate T/C (where T is the treatment group and C is the average bioluminescence intensity of the blank control group at the set time). The tumor inhibition rate (TGI) was calculated using the formula: TGI(%) = [1 - ( _Ti - _T0 ) / ( _Vi - _V0 )] × 100, where _Ti is the average bioluminescence intensity of the treatment group at the set time; _T0 is the average bioluminescence intensity at the start of administration; _Vi is the average bioluminescence intensity of the blank control group at the set time; and _V0 is the average bioluminescence intensity at the start of administration.

4. Inhibitory effect of the compound on the growth of subcutaneously transplanted HepG2 hepatocellular carcinoma tumors in nude mice

This experiment evaluated the efficacy of compound 1B in a HepG2 hepatocellular carcinoma orthotopic xenograft model. After 21 days of administration, compound 1B at a dose of 1 mg/kg significantly inhibited tumor growth, with p < 0.05 compared to the solvent control group. Increasing the dose of compound 1B to 3 mg/kg significantly enhanced the tumor-inhibiting effect.

Experimental results: see Figure 3 and Tables 7, 8, and 9. The tumor weight at the experimental endpoint is shown in Figure 4 and Table 10.

Table 7. Antitumor effects of the compounds of this invention on the HepG2 orthotopic xenograft tumor model.

Note: a. Mean ± SEM, n = 6.

Table 8. p-values for comparing relative tumor signal growth values (RBL) among groups in the HepG2 xenograft model.

Group Group 1 Group 2 Group 3 Group 4 1 N/A 0.038 0.029 0.024 2 0.029 0.921 N/A 0.682 3 0.024 0.384 0.682 N/A

Note: p-values were obtained from one-way ANOVA analysis of relative tumor volume (RBL). There were significant differences in F-values among the groups (p<0.001), and the Games-Howell test was used.

Table 9. Bioluminescent signal values of tumor tissue at different time points in each group.

Note: a. Mean ± SEM, n = 6

Table 10 Tumor weight in each group

Note: a. Mean ± SEM, n = 6.

b. Tumor growth inhibition is calculated by T/C _weight = TW _treatment / TW _solvent .

c.p values were obtained by analyzing tumor weight using one-way ANOVA and solvent therapy groups. The F values showed a significant difference (p<0.001), and the Games-Howell method was used for analysis.

5. Weight changes

In this model, the body weight of all treated animals did not fluctuate significantly, and the overall average decrease in body weight did not exceed 5% (see Figures 5 and 6). Conclusion: The compounds of this invention have a significant inhibitory effect on tumor growth, and the body weight of the treated animals did not decrease significantly, demonstrating good safety.

Claims (10)

1. The compound represented by formula (I) or a pharmaceutically acceptable salt thereof, Where T is N or CH; _R1 and _R2 are each independently H, F, Cl, Br, I or _C1-3 alkyl; _R3 and _R4 are each independently H, F, Cl, Br, I, _C1-3 alkyl or _C1-3 alkoxy. 2. The compound according to claim 1, or a pharmaceutically acceptable salt thereof, wherein the compound has the structure shown in formula (I-1) or formula (I-2): _R1 , _R2 , _R3 and _R4 are as defined in claim 1. 3. The compound of claim 2 or a pharmaceutically acceptable salt thereof, wherein the compound has the structure shown in formula (I-3) or (I-4): Wherein, the carbon atom marked with "*" is a chiral carbon atom, existing in a single enantiomer form (R) or (S) or rich in one enantiomer form; _R1 , _R2 , _R3 and _R4 are as defined in claim 2. 4. The compound according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof, wherein _R1 and _R2 are each independently H, F, Cl, Br, I or _-CH3 . 5. The compound of claim 4 or a pharmaceutically acceptable salt thereof, wherein _R1 is H, F, Cl, Br, I or _-CH3 ; and _R2 is H. 6. The compound or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 3 or 5, wherein _R3 and _R4 are each independently H, F, Cl, Br, I, _-CH3 or _-OCH3 . 7. The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein _R3 and _R4 are each independently H, F, _-CH3 or _-OCH3 . 8. The compound of claim 7 or a pharmaceutically acceptable salt thereof, wherein _R3 and _R4 are each independently H. 9. A compound of the following formula or a pharmaceutically acceptable salt thereof, 10. A compound of the following formula or a pharmaceutically acceptable salt thereof,