WO2022166990A1 - Anti-tumor pharmaceutical combination - Google Patents

Anti-tumor pharmaceutical combination Download PDF

Info

Publication number
WO2022166990A1
WO2022166990A1 PCT/CN2022/075561 CN2022075561W WO2022166990A1 WO 2022166990 A1 WO2022166990 A1 WO 2022166990A1 CN 2022075561 W CN2022075561 W CN 2022075561W WO 2022166990 A1 WO2022166990 A1 WO 2022166990A1
Authority
WO
WIPO (PCT)
Prior art keywords
substituted
unsubstituted
group
alkyl
amino
Prior art date
Application number
PCT/CN2022/075561
Other languages
French (fr)
Chinese (zh)
Inventor
杨财广
董泽
徐洪蛟
王传辉
黄悦
Original Assignee
中国科学院上海药物研究所
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海药物研究所 filed Critical 中国科学院上海药物研究所
Publication of WO2022166990A1 publication Critical patent/WO2022166990A1/en

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D231/00—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings
    • C07D231/02—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings
    • C07D231/10—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members
    • C07D231/12—Heterocyclic compounds containing 1,2-diazole or hydrogenated 1,2-diazole rings not condensed with other rings having two or three double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/415—1,2-Diazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4192—1,2,3-Triazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/42—Oxazoles
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/4409—Non condensed pyridines; Hydrogenated derivatives thereof only substituted in position 4, e.g. isoniazid, iproniazid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/535—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with at least one nitrogen and one oxygen as the ring hetero atoms, e.g. 1,2-oxazines
    • A61K31/5375—1,4-Oxazines, e.g. morpholine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D249/00—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms
    • C07D249/02—Heterocyclic compounds containing five-membered rings having three nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D249/04—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles
    • C07D249/06—1,2,3-Triazoles; Hydrogenated 1,2,3-triazoles with aryl radicals directly attached to ring atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D257/00—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms
    • C07D257/02—Heterocyclic compounds containing rings having four nitrogen atoms as the only ring hetero atoms not condensed with other rings
    • C07D257/04—Five-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D261/00—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings
    • C07D261/02—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings
    • C07D261/06—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members
    • C07D261/08—Heterocyclic compounds containing 1,2-oxazole or hydrogenated 1,2-oxazole rings not condensed with other rings having two or more double bonds between ring members or between ring members and non-ring members with only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/30—1,4-Oxazines; Hydrogenated 1,4-oxazines not condensed with other rings

Definitions

  • the present invention relates to the field of pharmaceutical compounds, and specifically, the present invention provides a combination of an FTO inhibitor represented by formula (I) and an immune checkpoint inhibitor for the treatment of solid tumors.
  • tumors can weaken and evade immune T cells.
  • tumors can express aggressive molecules on the cell surface that bind T cells and thereby inhibit the killing activity of T cells. These molecules are called immune checkpoints, such as PD. -1 and CTLA-4. Inhibiting immune checkpoint molecules to regulate the body's immune system is the core of tumor immunotherapy.
  • Tumor immunotherapy stimulates the recognition of the immune system by modulating T cell receptor signaling or using natural biomolecules and monoclonal antibodies to relevant tumor antigens.
  • the immune checkpoint inhibitor anti-PD-1/PD-L1 antibody has shown good efficacy in various advanced solid tumors such as melanoma and renal cancer, and its objective response rate (ORR) is about 10 in different solid tumors. %-40%, with the highest in malignant melanoma (about 36%-53%).
  • ORR objective response rate
  • anti-PD-1/PD-L1 antibody alone has not achieved the expected efficacy in many cancer patients, especially for patients with advanced colorectal cancer. Therefore, it is urgent to find small molecule compounds that can be combined with PD-1 or PD-L1 antibodies and enhance their efficacy.
  • m 6 A modification affects tumor antigen-specific T cell immune responses by regulating lysosomal cathepsin translation efficiency of dendritic cells.
  • m 6 A demethylase FTO its inhibitor can change the abundance of m 6 A in tumor cells, so as to improve the efficiency of tumor antigen-specific T cell immune response.
  • the present invention provides a type of FTO inhibitor, which can achieve the purpose of anti-tumor by being used in combination with an immune checkpoint inhibitor PD-1 or PD-L1 antibody.
  • the object of the present invention is to provide a pharmaceutical combination containing an FTO inhibitor suitable for tumor treatment.
  • the first aspect of the present invention provides a compound represented by the following formula (I), and the use of a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof,
  • M is selected from the group consisting of CR'2 , NH, O or S;
  • X has the structure shown in the following formula: carboxyl group, O-substituted or unsubstituted hydroxamic acid group, substituted or unsubstituted C2 - C12 ester group, substituted or unsubstituted amide group (C(O) NH2 ), substituted or unsubstituted 3-12-membered heterocyclyl;
  • Y is selected from the group consisting of substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 3-12 membered heterocyclyl;
  • the compound of formula (I) is used together with an immune checkpoint inhibitor to prepare a pharmaceutical composition for treating or preventing solid tumors.
  • R a and R b are each independently selected from the group consisting of halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted of C 1 -C 6 alkoxy;
  • R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy.
  • the compound of formula (I) has the structure shown in the following formula (II):
  • a 1 , A 2 , A 3 , A 4 are each independently CR' or N;
  • R 0 is selected from the group consisting of hydrogen, hydroxy, substituted or unsubstituted C 1 -C 10 alkyl
  • R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic , bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
  • n is selected from the group consisting of 0, 1, 2, 3 or 4;
  • R x , R y are each independently selected from the group consisting of halogen, substituted or unsubstituted C 1 -C 4 alkyl;
  • Het is selected from the group consisting of substituted or unsubstituted C6 - C10 aryl, substituted or unsubstituted 4-7 membered saturated heterocyclyl, substituted or unsubstituted 3-12 membered heterocyclyl;
  • the R 0 has the structure shown in the following formula:
  • R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic, bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
  • described Het is selected from the following group: substituted or unsubstituted pyridine, substituted or unsubstituted tetrazolium, substituted or unsubstituted triazole, substituted or unsubstituted pyrimidine, substituted or unsubstituted pyrimidine, substituted or unsubstituted tetrazolium Unsubstituted pyrazoles, substituted or unsubstituted isoxazoles, substituted or unsubstituted morpholines, substituted or unsubstituted thiomorpholines, substituted or unsubstituted piperidines, substituted or unsubstituted piperazines, substituted or unsubstituted or unsubstituted oxetane, substituted or unsubstituted thietane, substituted or unsubstituted azetidine.
  • described Het is selected from the following group:
  • the A 2 and A 3 are each independently CR'.
  • the A 1 , A 2 , A 3 , and A 4 are each independently CR'.
  • the R' has the structure shown in the following formula:
  • R" is H or a substituted or unsubstituted C1-C6 alkyl group.
  • the compound of formula (I) is selected from the following group:
  • the solid tumor is selected from the group consisting of melanoma, lung cancer, colon cancer, renal cancer, pancreatic cancer, lung cancer, and osteosarcoma.
  • Figure 1 shows that after the compounds of the present invention Dac51, Dac258, Dam62, and Dam128 treat tumor cells, the m 6 A abundance changes in a concentration-dependent manner with FTO inhibitors.
  • Figure 2 shows the improvement of tumor growth and body weight change of the compound Dam59 of the present invention in combination with anti-PD-L1 antibody in tumor-bearing mice.
  • Figure 4 shows the improvement of tumor growth and body weight change in tumor-bearing mice when the compound Dam60 of the present invention is combined with anti-PD-1 antibody.
  • substituted refers to the substitution of one or more hydrogen atoms on a group with a substituent selected from the group consisting of C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 1 -C 10 alkoxy group, halogen, hydroxyl, carboxyl group (-COOH), C 1 -C 10 aldehyde group, C 2 -C 10 acyl group, C 2 -C 10 ester group, amino group, phenyl group; the described Phenyl includes unsubstituted phenyl or substituted phenyl with 1-3 substituents selected from: halogen, C 1 -C 10 alkyl, cyano, OH, nitro, C 3 -C 10 cycloalkyl, C 1 -C 10 alkoxy, amino.
  • C 1 -C 6 alkoxy refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy , isobutoxy, sec-butoxy, tert-butoxy, or similar groups.
  • carbonyl C 1 -C 6 alkyl refers to a group in the form of "-COO-straight or branched chain alkyl of 1 to 6 carbon atoms", such as carbonyl-methyl, carbonyl-ethyl, carbonyl- propyl, carbonyl-isopropyl, carbonyl-butyl, carbonyl-isobutyl, carbonyl-sec-butyl, carbonyl-tert-butyl, or the like.
  • C 6 -C 12 aryl refers to aryl groups having 6 to 12 carbon atoms, including monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, or the like.
  • 3-12 membered heterocyclyl refers to a 3- to 12-membered ring system substituted with one or more heteroatoms selected from O, S, N, or P, saturated or unsaturated (including aromatic) ring systems group, such as pyridyl, thienyl, piperidinyl, or similar groups, preferably a 4-9 membered heterocyclic group.
  • halogen refers to F, Cl, Br and I.
  • the terms “comprising”, “comprising” or “including” mean that the various ingredients can be used together in the mixture or composition of the present invention.
  • the terms “consisting essentially of” and “consisting of” are encompassed by the term “comprising”.
  • the term "pharmaceutically acceptable” ingredients refers to substances that are suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergy), ie, have a reasonable benefit/risk ratio.
  • the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect.
  • the precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to prespecify the exact effective amount. However, for a given situation, routine experimentation can be used to determine the effective amount, as is the judgment of the clinician.
  • substituted refers to the replacement of one or more hydrogen atoms on a group with a substituent selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted Substituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.
  • each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.
  • the term "compounds of the present invention” refers to compounds of formula I.
  • the term also includes the various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I).
  • prodrug refers to a pharmaceutically acceptable metabolic precursor of a compound of the present invention, which can be converted to a compound of the biologically active compound of the present invention under physiological conditions or by solvolysis.
  • a prodrug may be inactive when administered to an individual in need thereof, but be converted in vivo to an active compound of the present invention.
  • Prodrugs are typically rapidly transformed in vivo to yield the parent compounds of the invention, eg, by hydrolysis in blood.
  • Prodrug compounds generally provide the advantages of solubility, histocompatibility or sustained release in mammalian organisms.
  • the FTO inhibitor compound is used in combination with the immune checkpoint inhibitor, thereby achieving a synergistic effect.
  • the FTO inhibitor is a compound represented by the following formula (I), and a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof:
  • a 1 , A 2 , A 3 , A 4 are each independently CR' or N;
  • M is selected from the group consisting of CR'2 , NH, O or S;
  • X has the structure shown in the following formula: carboxyl group, hydroxamic acid group, substituted or unsubstituted C 2 -C 12 ester group, substituted or unsubstituted amide group (C(O)NH 2 ), substituted or unsubstituted 3 -12-membered heterocyclyl;
  • Y is selected from the group consisting of substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 3-12 membered heterocyclyl;
  • R a , R b , R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted Substituted C 1 -C 6 alkoxy;
  • substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen atom, carbonyl ( O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group, substituted or unsubstituted C 1 -C 10 alkane base, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C 2 -C 10 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, or substituted or unsubstituted five-membered or Six-membered heteroaryl, 3-12-membered heterocyclic, 3-12-membered cycloalkyl, preferably C 1 -C 6 alkyl, hal
  • Preferred compounds have structures as shown in the Examples herein.
  • the pharmaceutical combination of the present invention has excellent inhibitory activity on tumor cell proliferation
  • Pharmaceutical combinations of inhibitors can be used to treat, prevent and alleviate diseases caused by tumor cell proliferation.
  • the compounds of the present invention can be used to treat the following diseases: melanoma, lung cancer, colon cancer and other cancers.
  • the pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount.
  • the "safe and effective amount” refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects.
  • the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-200 mg of the compound of the present invention per dose.
  • the "one dose” is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gel substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility” as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.
  • Examples of pharmaceutically acceptable carrier moieties 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, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethyl cellulose, sodium ethyl cellulose, cellulose acetate
  • the mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
  • Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules.
  • the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostea
  • 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 well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
  • Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures.
  • liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylform
  • compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
  • Suspensions in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
  • 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.
  • 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 if necessary.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
  • a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is
  • the administration dose is usually 1 to 2000 mg, preferably 5 to 500 mg.
  • the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
  • the present invention provides the use of a compound of formula (I) and an immune checkpoint inhibitor in combination to treat tumors.
  • the method can effectively inhibit the proliferation of tumor cells at a very low dosage, and compared with the two used alone , resulting in a clear synergistic effect.
  • the first step 30g (120mmol, 1.2eq) of o-iodobenzoic acid, 24g (100mmol, 1.0eq) of 2,6-dichloro-4-bromoaniline, 24g (100mmol, 1.0eq) of triethylamine (150mmol, 1.5eq) and anhydrous copper acetate 9g (5.0mmol, 0.5eq) was dissolved in 500mL of DMF, heated to 120°C under argon protection and reacted for 24 hours, after the reaction was completed.
  • Step 2 Dissolve 3.6 g of 2-(4-bromo-2,6-dichlorophenyl)amino)benzoic acid in 200 mL of absolute ethanol, add 20 mL of concentrated sulfuric acid to it under cooling in an ice-water bath, and heat to 100 The reaction was refluxed at °C for 12 hours. After the reaction was completed, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL ⁇ 3, and the organic phases were combined and washed with saturated brine for 10 mL ⁇ 3.
  • the third step 2-((4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), 3,5-dimethylpyrazole-4- Pinacol borate 2.13g (9.6mmol, 1.2eq), Pd(dppf)Cl 2 584mg (0.8mmol, 0.1eq), Potassium carbonate 1.68g (12.0mmol, 1.5eq) dissolved in 1,4-dioxane In 160 mL of a mixed solvent of ring and water, the mixture was heated to 100°C and reacted for 24 hours.
  • Step 4 Dissolve 1.10g (2.7mmol, 1.0eq) of ethyl 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoate in tetrahydrofuran
  • a mixed solvent of 13.5 mL and 27 mL of absolute ethanol under cooling in an ice-water bath, a solution of 540 mg (13.5 mmol, 5.0 eq) of sodium hydroxide in 7 mL of water was slowly added dropwise. The reaction was heated to 45 °C overnight.
  • the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. . After suction filtration, the solid was washed with water to obtain 930 mg of the target product, 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid as a white solid.
  • the first and second reactions were prepared according to the synthetic procedure of Dac51.
  • the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 930 mg of the target product, 2-((2,6-dichloro-4-(3-methylpyrazole)phenyl)amino)benzoic acid as a white solid.
  • the fifth step in a 10mL round-bottom flask, add 2-((2,6-dichloro-4-(pyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was added at 45 °C reaction. TLC detection, the raw material consumption was completed.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), 114 mg (0.3 mmol, 1.5 eq) of HATU, dissolved in 4.0 mL of anhydrous DMF at room temperature, stirred for 5 min, and 77 mg (0.6 mmol, 3.0 eq) of DIEA, 18 mg (0.24 mmol) of 3-aminopropanol were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenedi Amine 28mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), 114 mg (0.3 mmol, 1.5 eq) of HATU, dissolved in 4.0 mL of anhydrous DMF at room temperature, stirred for 5 min, and 77 mg (0.6 mmol, 3.0 eq) of DIEA, 18 mg (0.24 mmol) of 3-aminopropanol were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours.
  • the first step 29.8g (120mmol, 1.2eq) of o-iodobenzoic acid, 22.8g (100mmol, 1.0eq) of 2,6-diethyl-4-bromoaniline, 22.8g (100mmol, 1.0eq) of triethylamine (150mmol, 1.5eq) and no 9.1 g (5.0 mmol, 0.5 eq) of water copper acetate was dissolved in 360 mL of DMF, heated to 120° C. for 24 h under argon protection, and the reaction was completed.
  • Step 2 Dissolve 10.4 g of 2-(4-bromo-2,6-diethylphenyl)amino)benzoic acid in 300 mL of absolute ethanol, add 30 mL of concentrated sulfuric acid to it under cooling in an ice-water bath, and heat to The reaction was refluxed at 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL ⁇ 3, and the organic phases were combined and washed with saturated brine for 10 mL ⁇ 3.
  • the fourth step dissolve 650 mg (1.7 mmol, 1.0 eq) of ethyl 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoate in A solution of 199 mg (8.3 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise to a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg (0.24mmol) were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenedi Amine 28mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second reactions were prepared according to the synthetic procedure for Dam128.
  • the third step 2-(4-bromo-2,6-diethylphenyl)amino)ethyl benzoate 1.13g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2eq) ), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 160mL (4:1/v:v) , heated to 100 °C for 24 hours.
  • Step 4 Dissolve 775 mg (2.1 mmol, 1.0 eq) of ethyl 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol 249 mg (10.5 mmol, 5.0 eq) of a solution of lithium hydroxide in 2 mL of water was slowly added dropwise thereto under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and methylamine hydrochloride 16mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenediamine 28mg (0.24mmol, 1.2eq) were successively added to the above system ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-dimethylaminoethylenediamine 21mg (0.24mmol, 1.2eq) were added to the above system successively ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and 2-aminoethanol 15mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system was The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system was heated at 45°C React for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step 30.7g (124mmol, 1.2eq) of o-iodobenzoic acid, 22.7g (103mmol, 1.0eq) of 2-chloro-6-methyl-4-bromoaniline, 15.7g (155mmol, 1.5eq) of triethylamine ) and 9.3 g (51.5 mmol, 0.5 eq) of anhydrous copper acetate were dissolved in 372 mL of DMF, heated to 120° C. under argon protection and reacted for 24 h, after the reaction was completed.
  • the second step dissolve 32 g of 2-(4-bromo-2-chloro-6-methylphenyl) amino) benzoic acid in 300 mL of absolute ethanol, add 50 mL of concentrated sulfuric acid to it, and heat to The reaction was refluxed at 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL ⁇ 3, and the organic phases were combined and washed with saturated brine for 20 mL ⁇ 3.
  • the third step 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), 3,5-dimethylisoxazole- 4-boronic acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixture of dioxane and water In 37 mL of solvent (4:1/v:v), the mixture was heated to 100°C and reacted for 24 hours.
  • the fourth step 911mg (2.37mmol, 1.0eq) of ethyl 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoate It was dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 16 mL of absolute ethanol, and a solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to it under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • Step 5 In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second steps are prepared according to the synthetic operation of Dam106.
  • the third step 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 1.1g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2g) eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 15mL (4:1/v:v) , heated to 100 °C for 24 hours.
  • Step 4 Dissolve 734 mg (2.0 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoate in a mixture of 8 mL of tetrahydrofuran and 16 mL of anhydrous ethanol In the solvent, under ice-water bath cooling, a solution of 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac406.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were added to the above system in turn, and the resulting mixed system was The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second reactions were prepared according to the synthetic procedure of Dac406.
  • Step 5 In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-di Ethylaminoethylenediamine 18mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • Step 5 In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol were added to the above system successively 18mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenediamine 24mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second reactions were prepared according to the synthetic procedure of Dac406.
  • Step 5 In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 14mg (0.24mg) were successively added to the above system mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second reactions were prepared according to the synthetic procedure of Dac406.
  • the first step o-iodobenzoic acid 30g (120mmol, 1.2eq), 2,6-diisopropyl-4-bromoaniline 25.6g (100mmol, 1.0eq), triethylamine (150mmol, 1.5eq) and no 9 g (5.0 mmol, 0.5 eq) of water copper acetate was dissolved in 500 mL of DMF, heated to 120° C. under argon protection and reacted for 24 h, after the reaction was completed.
  • the third step 2-(4-bromo-2,6-diisopropylphenyl)amino)ethyl benzoate 1.2g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2g) eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 15mL (4:1/v:v) , heated to 100 °C for 24 hours.
  • Step 4 Dissolve 748 mg (2.0 mmol, 1.0 eq) of ethyl 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise thereto under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoic acid 75mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step 29.8g (120mmol, 1.2eq) of o-iodobenzoic acid, 20.4g (100mmol, 1.0eq) of 2-fluoro-6-methyl-4-bromoaniline, 15.2g (150mmol, 1.5eq) of triethylamine ) and 9.1 g (50 mmol, 0.5 eq) of anhydrous copper acetate were dissolved in 360 mL of DMF, heated to 120 °C under argon protection and reacted for 24 h, after the reaction was completed.
  • the first step 20.2g (100mmol, 1.0eq) of 2-chloro-5-nitrobenzoic acid, 26.5g (110mmol, 1.1eq) of 2,6-dichloro-4-bromoaniline, 28.8g of sodium tert-butoxide (300mmol, 3.0eq) was dissolved in 500mL of DMF, heated to 80°C under argon protection and reacted for 24 hours, after the reaction was completed. Cooled to room temperature, adjusted pH with 2M dilute hydrochloric acid, a yellow solid was precipitated, suction filtered, and washed with water to obtain 28.0 g of the target product, a yellow solid.
  • the fourth step 2-((2,6-dichloro-4-(3,5-dimethylisoxazole) phenyl) amino)-5-nitrobenzoic acid ethyl ester 1.3g (2.9mmol, 1.0eq), zinc powder 943mg (14.5mmol, 5.0eq), ammonium formate 1.8g (29.0mmol, 10.0eq) was dissolved in DMF 58mL, heated to 45°C, reacted overnight, water was added to the system, EA extraction, silica gel column After chromatographic separation, 1.22 g of ethyl 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)-5-aminobenzoate was obtained as a yellow target product.
  • Step 5 Dissolve 79mg (0.55mmol, 1.1eq) of monoethyl fumarate, 228mg (0.6mmol, 1.2eq) of HATU in DMF (4mL), stir for 5min, and add 194mg (1.5mg) of DIEA to the above system in turn. mmol, 3.0eq), 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)-5-aminobenzoic acid ethyl ester 210mg (0.5mmol, 1.0 eq), the resulting mixed system was reacted at 45 °C. TLC detection, the raw material consumption was completed. An ice-water mixture was added to the system, and a yellow solid was precipitated, which was filtered off with suction and washed with water to obtain the target product, Dac258 (180 mg) as a yellow solid.
  • the fourth step Compound Dac258 55mg (0.1mmol, 1.0eq) was dissolved in a mixed solvent of tetrahydrofuran (2mL) and absolute ethanol (4mL), and lithium hydroxide 24mg (1.0mmol, 10.0eq) was slowly added dropwise thereto. solution in water (1 mL). The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, water was added, and 2M dilute hydrochloric acid was added to adjust the pH to 3. The obtained suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain the target product Dac58 (46 mg).
  • the third step 2-(4-bromo-2,6-diisopropylphenyl)amino)ethyl benzoate 1.2g (3.0mmol, 1.0eq), morpholine 313mg (3.6mmol, 1.2eq), BINAP 224mg (0.36mmol, 0.12eq) catalyst Pd(OAc) 2 68mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) was dissolved in 15mL of toluene, heated to 110 °C and reacted for 24 hours.
  • the third step 1.2g (3.0mmol, 1.0eq) of ethyl 2-(4-bromo-2-fluoro-6-methylphenyl)amino)benzoate, 313mg (3.6mmol, 1.2eq) of morpholine, BINAP 224mg (0.36mmol, 0.12eq) catalyst Pd(OAc) 2 68mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) was dissolved in 15mL of toluene, heated to 110 °C and reacted for 24 hours.
  • Step 4 Dissolve 720 mg (2.0 mmol, 1.0 eq) of ethyl 2-(2-fluoro-6-methyl-4-morpholine)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol, Under ice-water bath cooling, a solution of 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dam266.
  • the first step and the second step are prepared according to the synthetic method of Dac51.
  • the third step 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), triazole boronic acid 1.1g (8.8mmol, 1.2eq), Catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) was dissolved in a mixed solvent of dioxane and water 37mL (4:1/v:v), Heated to 100°C and reacted for 24 hours.
  • the fourth step 927 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(5-methyltriazole))phenyl)amino)benzoate was dissolved in tetrahydrofuran A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 8 mL and 16 mL of anhydrous ethanol under cooling in an ice-water bath. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(5-methyltriazole))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0 eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step and the second step are prepared according to the synthetic method of Dac51.
  • the third step 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), 3,5-dimethyltriazole-4 - Boric acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/v:v), heated to 100°C and reacted for 24 hours.
  • the fourth step 893mg (2.37mmol, 1.0eq) of ethyl 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoate It was dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 16 mL of absolute ethanol, and a solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to it under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
  • the third step 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), 3,5-dimethyltriazole- 4-boronic acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixture of dioxane and water In 37 mL of solvent (4:1/v:v), the mixture was heated to 100°C and reacted for 24 hours.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg ( 0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step and the second step are prepared according to the synthetic method of Dac51.
  • the third step 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), tetrazolium-5-boronic acid 1.2g (8.8g) mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/ In v:v), the reaction was heated to 100° C. for 24 hours.
  • the fourth step 830 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoate was dissolved in 8 mL of tetrahydrofuran and anhydrous A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 16 mL of ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 70mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting The mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 70mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were sequentially added to the above system, The resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylamino-1,4- Butanediamine 35mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol 18mg (0.24mmol, 1.2eq) were added to the above system in turn ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylethylenediamine 24mg ( 0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first and second steps are prepared according to the synthetic method of Dac406.
  • the third step 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), tetrazolium-5-boronic acid 1.2g (8.8g) mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/ In v:v), the reaction was heated to 100° C. for 24 hours.
  • the fourth step 848 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoate was dissolved in 8 mL of tetrahydrofuran and anhydrous A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 16 mL of ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting The mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • the first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac118.
  • the fifth step in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were sequentially added to the above system, The resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed.
  • FTO enzyme activity inhibition reaction system is as follows: 50mM Tris ⁇ HCl, pH 7.5, 0.3 ⁇ M FTO, 1 ⁇ M 39nt-m 6 A modified double-stranded DNA, 300 ⁇ M 2OG, 280 ⁇ M (NH 4 ) 2 Fe(SO 4 ) 2 , 2mM L -Ascorbic Acid and compounds of different concentrations, incubated at room temperature for 2 hours, slowly heated at 65°C for inactivation, and annealed to double-stranded DNA by adding 1 ⁇ M 39nt antisense DNA. Take 8ul of the reaction solution and digest the double-stranded substrate with the methylation-sensitive enzyme DpnII.
  • the digested samples were detected by 15% non-denaturing polyacrylamide electrophoresis, and photographed under the gel imaging system after Gel-Red staining.
  • the obtained bands were read for the inhibition rate in grayscale, and the IC 50 for inhibiting the demethylation activity of FTO was evaluated for the compounds with better inhibition rate. The results are shown in the following table.
  • Solid tumor cell lines such as the melanoma cell line B16-OVA, the lung cancer cell line LLC, and the colon cancer cell line MC38 were cultured separately, seeded at a density of 1000 cells per well in a 96-well plate, and placed in a 37°C CO 2 incubator. Incubate the cells until they adhere to the wall, add different compounds and continue to culture for 72h, add 10uL of MTT solution to each well, continue to incubate for 4h, detect the absorbance value at 490nm, calculate the inhibition rate with the DMSO group as the control, and focus on the cells with better inhibition rate. Compounds were evaluated for cell proliferation inhibitory activity IC50 , and the results are shown in the table below.
  • Tumor cells were seeded in a 6cm culture dish at a certain density, the compound was added the next day (the concentration was set according to the IC 50 value of MTT), and after 48-72 hours of treatment, the cell culture medium was removed, and 1 mL of TRIzol reagent was added to it, and the cells were collected. Lyse, pipette into RNase free tube, and incubate on ice for 5 min to completely separate nucleoprotein complexes; add 200 ⁇ L of chloroform to lyse, vortex for 15 s, incubate; centrifuge for layers.
  • mice 5-week-old C57BL/6 strain mice with a body weight of 18-20 g were selected.
  • tumor cells MC38 were cultured.
  • the candidate compounds Dam59 and Dam159 were respectively dissolved in DMSO to prepare a 20 mM stock solution and diluted with PBS.
  • the content of DMSO in the finally obtained dosing solution was 20%, which was ready for use.
  • the candidate compounds Dam59 (2 mg/kg) and Dam159 (0.5 mg/kg) were injected by intraperitoneal administration from the 10th, 11th, and 12th days, respectively.
  • On days 13 and 16 100 mg/kg of antibody PD-L1 and antibody negative control IgG were injected intraperitoneally. Tumor volume and mouse body weight were measured every two days. The results are shown in Fig. 2 and Fig. 3 , when used in combination with the antibody PD-L1, the compounds of the present invention exhibited obvious synergistic effects.
  • mice 5-week-old C57BL/6 strain mice with a body weight of 18-20 g were selected.
  • tumor cells MC38 were cultured.
  • the candidate compound Dam60 was dissolved in DMSO, and a 20 mM stock solution was prepared and diluted with PBS. The content of DMSO in the finally obtained dosing solution was 20%, which was ready for use.
  • the candidate compound Dam60 (1 mg/kg) was injected by intraperitoneal administration from day 7, 8, and 9, respectively. On days 10 and 12, 100 mg/kg of antibody PD-1 and antibody negative control IgG were injected intraperitoneally. Tumor volume and mouse body weight were measured every two days. The results are shown in FIG. 4 , when the compound is used in combination with the antibody PD-1, the compound of the present invention exhibits an obvious synergistic effect.

Abstract

Provided in the present invention are a 2-(substituted phenoxy)aromatic formic acid FTO inhibitor, a preparation method therefor and the use thereof, and specifically disclosed are a compound represented by formula (I), and the use of a pharmaceutically acceptable salt, a hydrate, a solvate, or a prodrug thereof. The compound can be used in combination with an immune checkpoint inhibitor for treating solid tumors, such as melanoma, lung cancer, colon cancer, renal cancer, pancreatic cancer, lung cancer and osteosarcoma.

Description

用于抗肿瘤的药物组合Drug Combinations for Antitumor 技术领域technical field
本发明涉及药物化合物领域，具体地，本发明提供了一种如式(I)所示的FTO抑制剂与免疫检查点抑制剂联合用药用于治疗实体瘤的用途。The present invention relates to the field of pharmaceutical compounds, and specifically, the present invention provides a combination of an FTO inhibitor represented by formula (I) and an immune checkpoint inhibitor for the treatment of solid tumors.
背景介绍Background introduction
癌症发生的原因之一是肿瘤可以削弱和逃避免疫T细胞，例如肿瘤可以在细胞表面表达攻击性分子，绑定T细胞从而抑制T细胞的杀伤活性，这些分子被称为免疫检查点，例如PD-1和CTLA-4。抑制免疫检查点分子从而调节机体免疫***是肿瘤免疫疗法的核心。One of the reasons why cancer occurs is that tumors can weaken and evade immune T cells. For example, tumors can express aggressive molecules on the cell surface that bind T cells and thereby inhibit the killing activity of T cells. These molecules are called immune checkpoints, such as PD. -1 and CTLA-4. Inhibiting immune checkpoint molecules to regulate the body's immune system is the core of tumor immunotherapy.
近年，肿瘤免疫疗法取得了突破性进展。肿瘤免疫治疗通过调节T细胞受体信号或使用天然的生物分子和相关肿瘤抗原的单克隆抗体刺激免疫***的识别。例如，免疫检查点抑制剂抗PD-1/PD-L1抗体在黑色素瘤、肾癌等多种晚期实体瘤中显示了较好的疗效，其客观有效率(ORR)在不同实体瘤中约10％-40％，其中在恶性黑色素瘤中最高(约36％-53％)。但是，许多癌症患者单用抗PD-1/PD-L1抗体并未获得预期疗效，尤其针对晚期结直肠癌患者。因此找到能与PD-1或PD-L1抗体联用并增强其疗效的小分子化合物迫在眉睫。In recent years, breakthroughs have been made in tumor immunotherapy. Tumor immunotherapy stimulates the recognition of the immune system by modulating T cell receptor signaling or using natural biomolecules and monoclonal antibodies to relevant tumor antigens. For example, the immune checkpoint inhibitor anti-PD-1/PD-L1 antibody has shown good efficacy in various advanced solid tumors such as melanoma and renal cancer, and its objective response rate (ORR) is about 10 in different solid tumors. %-40%, with the highest in malignant melanoma (about 36%-53%). However, anti-PD-1/PD-L1 antibody alone has not achieved the expected efficacy in many cancer patients, especially for patients with advanced colorectal cancer. Therefore, it is urgent to find small molecule compounds that can be combined with PD-1 or PD-L1 antibodies and enhance their efficacy.
靶向m 6A修饰的小分子抑制剂为这些困扰提供了新思路。m 6A修饰通过调控树突状细胞的溶酶体组织蛋白酶翻译效率，影响肿瘤抗原特异性的T细胞免疫应答。作为m 6A最为重要的去甲基化酶FTO，其抑制剂能够改变肿瘤细胞内m 6A的丰度，从而达到提高肿瘤抗原特异性的T细胞免疫应答效率的目的。 Small-molecule inhibitors targeting m 6 A modification provide new ideas for these problems. m 6 A modification affects tumor antigen-specific T cell immune responses by regulating lysosomal cathepsin translation efficiency of dendritic cells. As the most important m 6 A demethylase FTO, its inhibitor can change the abundance of m 6 A in tumor cells, so as to improve the efficiency of tumor antigen-specific T cell immune response.
本发明提供了一类FTO抑制剂，通过与免疫检查点抑制剂PD-1或PD-L1抗体联用达到抗肿瘤的目的。The present invention provides a type of FTO inhibitor, which can achieve the purpose of anti-tumor by being used in combination with an immune checkpoint inhibitor PD-1 or PD-L1 antibody.
发明内容SUMMARY OF THE INVENTION
本发明的目的是提供一种适用于肿瘤治疗的含有FTO抑制剂的药物组合。The object of the present invention is to provide a pharmaceutical combination containing an FTO inhibitor suitable for tumor treatment.
本发明的第一方面，提供了一种如下式(I)所示的化合物，及其药学上可接受的盐，水合物，溶剂合物或前药的用途，The first aspect of the present invention provides a compound represented by the following formula (I), and the use of a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof,
Figure PCTCN2022075561-appb-000001
Figure PCTCN2022075561-appb-000001
其中，in,
A 1、A 2、A 3、A 4各自独立地为CR'或N； A 1 , A 2 , A 3 , A 4 are each independently CR' or N;
M选自下组：CR' 2、NH、O或S； M is selected from the group consisting of CR'2 , NH, O or S;
R'选自下组：H、卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基、C 1-C 6烷氧基羰基、取代或未 取代的C 1-C 6酰氨基、取代或未取代的C 2-C 12酯基、或取代或未取代的C 1-C 10烷基、或取代或未取代的C3-C10烯酰胺基； R' is selected from the group consisting of H, halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, substituted or unsubstituted C 1 -C 6 amido, substituted or unsubstituted C 2 -C 12 ester group, or substituted or unsubstituted C 1 - C10 alkyl, or substituted or unsubstituted C3-C10 enamido;
X具有如下式所示的结构：羧基、O-取代或未取代的羟肟酸基、取代或未取代的C 2-C 12酯基、取代或未取代的酰胺基(C(O)NH 2)、取代或未取代3-12元杂环基； X has the structure shown in the following formula: carboxyl group, O-substituted or unsubstituted hydroxamic acid group, substituted or unsubstituted C2 - C12 ester group, substituted or unsubstituted amide group (C(O) NH2 ), substituted or unsubstituted 3-12-membered heterocyclyl;
Y选自下组：取代或未取代的C 6-C 12的芳基、取代或未取代3-12元杂环基； Y is selected from the group consisting of substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 3-12 membered heterocyclyl;
R a、R b、R c、R d各自独立地选自下组：H、卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基； R a , R b , R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted Substituted C 1 -C 6 alkoxy;
所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 1-C 6烷氧基羰基、C 1-C 6酰氨基、C 2-C 12酯基、取代或未取代的C 1-C 10烷基，取代或未取代的C 2-C 10烯基，取代或未取代的C 2-C 10炔基，取代或未取代的C 6-C 10芳基、或取代或未取代的五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或C 1-C 6烷氨基；其中，所述的取代或未取代的C 1-C 10烷基，取代或未取代的C 6-C 10芳基或五元或六元杂芳基的取代基选自下组：卤素原子、羰基(＝O)、羟基、羧基、C 1-C 6烷氧基羰基、氨基、C 1-C 6酰氨基、硝基、氰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 6-C 10芳基或五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为卤素原子、C 1-C 6烷氧基羰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或苯基、5-6元杂环基； Said substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group, substituted or unsubstituted C 1 -C 10 alkane base, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C 2 -C 10 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, or substituted or unsubstituted five-membered or Six-membered heteroaryl, 3-12-membered heterocyclic, 3-12-membered cycloalkyl, preferably C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy Or C 1 -C 6 alkylamino; wherein, the substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 6 -C 10 aryl or the substitution of five- or six-membered heteroaryl The group is selected from the group consisting of halogen atom, carbonyl group (=O), hydroxyl group, carboxyl group, C 1 -C 6 alkoxycarbonyl group, amino group, C 1 -C 6 amido group, nitro group, cyano group, C 1 -C 6 group Alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 6 -C 10 aryl or five- or six-membered heteroaryl, 3-12 membered heterocyclic group, 3-12 membered cycloalkyl group, preferably halogen atom, C 1 -C 6 alkoxycarbonyl group, C 1 -C 6 alkyl group, halogenated C 1 -C 6 alkyl group, C 1 -C 6 alkyl group 6 alkoxy or phenyl, 5-6 membered heterocyclic group;
其中，所述的式(I)化合物与免疫检查点抑制剂共同用于制备治疗或预防实体瘤的药物组合物。Wherein, the compound of formula (I) is used together with an immune checkpoint inhibitor to prepare a pharmaceutical composition for treating or preventing solid tumors.
在另一优选例中，R a、R b各自独立地选自下组：卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基； In another preferred embodiment, R a and R b are each independently selected from the group consisting of halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted of C 1 -C 6 alkoxy;
R c、R d各自独立地选自下组：H、卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基。 R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy.
在另一优选例中，所述的式(I)化合物具有如下式(II)所示的结构：In another preferred example, the compound of formula (I) has the structure shown in the following formula (II):
Figure PCTCN2022075561-appb-000002
Figure PCTCN2022075561-appb-000002
其中，in,
A 1、A 2、A 3、A 4各自独立地为CR’或N； A 1 , A 2 , A 3 , A 4 are each independently CR' or N;
M选自下组：NH或S；M is selected from the group: NH or S;
R’选自下组：H、卤素、羟基、氨基、硝基、氰基、取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基、取代或未取代的C 1-C 6酰氨； R' is selected from the group consisting of H, halogen, hydroxy, amino, nitro, cyano, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or Unsubstituted C 1 -C 6 alkylamino, substituted or unsubstituted C 1 -C 6 amido;
R 0选自下组：氢、羟基、取代或非取代的C 1-C 10烷基、
Figure PCTCN2022075561-appb-000003
其中，R a，R b各自独立地选自下组：氢、取代或未取代的C 1-C 6烷基，取代或未取代的C 3-C 10环 烷基(包括单环、多环、桥环结构)、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基； R 0 is selected from the group consisting of hydrogen, hydroxy, substituted or unsubstituted C 1 -C 10 alkyl,
Figure PCTCN2022075561-appb-000003
Wherein, R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic , bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
m选自下组：0、1、2、3或4；m is selected from the group consisting of 0, 1, 2, 3 or 4;
R x、R y各自独立地选自下组：卤素、取代或非取代的C 1-C 4烷基； R x , R y are each independently selected from the group consisting of halogen, substituted or unsubstituted C 1 -C 4 alkyl;
Het选自下组：取代或未取代的C 6-C 10的芳基、取代或未取代的4-7元饱和杂环基、取代或未取代3-12元杂环基； Het is selected from the group consisting of substituted or unsubstituted C6 - C10 aryl, substituted or unsubstituted 4-7 membered saturated heterocyclyl, substituted or unsubstituted 3-12 membered heterocyclyl;
所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、氧原子(＝O)、羧基、羟基、氨基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 1-C 6烷氧基羰基、C 1-C 6酰氨基、C 2-C 12酯基；或未取代或被一个或多个卤素或C 1-C 6烷基取代的选自下组的基团：C 1-C 10烷基，C 2-C 10烯基，C 2-C 10炔基，C 6-C 10芳基、5-6元杂芳基、3-12元杂环基、3-12元环烷基。 Said substitution refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of halogen, oxygen atom (=O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group; or unsubstituted or by one or more halogen or C 1 -C 6 alkyl substituted group selected from the group consisting of C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 6 -C 10 aryl, 5 -6-membered heteroaryl, 3-12-membered heterocyclyl, 3-12-membered cycloalkyl.
在另一优选例中，所述的R 0具有如下式所示的结构： In another preferred example, the R 0 has the structure shown in the following formula:
Figure PCTCN2022075561-appb-000004
Figure PCTCN2022075561-appb-000004
其中，in,
R a，R b各自独立地选自下组：氢、取代或未取代的C 1-C 6烷基，取代或未取代的C 3-C 10环烷基(包括单环、多环、桥环结构)、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基； R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic, bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
m选自以下组：1、2或3。m is selected from the group: 1, 2 or 3.
在另一优选例中，所述的Het选自下组：取代或未取代的吡啶、取代或未取代的四氮唑、取代或未取代的三氮唑、取代或未取代的嘧啶、取代或未取代的吡唑、取代或未取代的异恶唑、取代或未取代的吗啉、取代或未取代的硫代吗啉、取代或未取代的哌啶、取代或未取代的哌嗪、取代或未取代的氧杂环丁烷、取代或未取代的硫杂环丁烷、取代或未取代的氮杂环丁烷。In another preferred embodiment, described Het is selected from the following group: substituted or unsubstituted pyridine, substituted or unsubstituted tetrazolium, substituted or unsubstituted triazole, substituted or unsubstituted pyrimidine, substituted or unsubstituted pyrimidine, substituted or unsubstituted tetrazolium Unsubstituted pyrazoles, substituted or unsubstituted isoxazoles, substituted or unsubstituted morpholines, substituted or unsubstituted thiomorpholines, substituted or unsubstituted piperidines, substituted or unsubstituted piperazines, substituted or unsubstituted or unsubstituted oxetane, substituted or unsubstituted thietane, substituted or unsubstituted azetidine.
在另一优选例中，所述的Het选自下组：In another preferred embodiment, described Het is selected from the following group:
Figure PCTCN2022075561-appb-000005
Figure PCTCN2022075561-appb-000005
其中，各个R 1、R 2、R 3或R 4各自独立地选自下组：氢、卤素、羟基、氨基、氧原子(＝O)、羧基(COOH)、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 6-C 10芳基、取代或未取代的5-6元杂芳基、取代或未取代的C 1-C 6烷氨基、取代或未取代的C 1-C 6烷氧基羰基、取代或未取代的C 1-C 6酰氨基；其中，所述的取代基选自下组：F、Cl、C 1-C 6烷基。 wherein each R 1 , R 2 , R 3 or R 4 is each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, oxygen atom (=O), carboxyl (COOH), substituted or unsubstituted C 1 - C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted C 1 -C 6 alkylamino group, substituted or unsubstituted C 1 -C 6 alkoxycarbonyl group, substituted or unsubstituted C 1 -C 6 amido group; wherein, the substituent is selected from the following group: F, Cl, C 1 -C 6 alkyl.
在另一优选例中，各个R 1，R 2，R 3，R 4各自独立地选自下组：H、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基。 In another preferred embodiment, each R 1 , R 2 , R 3 , R 4 is independently selected from the group consisting of H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkyl C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino.
在另一优选例中，所述的A 2、A 3各自独立地为CR'。 In another preferred embodiment, the A 2 and A 3 are each independently CR'.
在另一优选例中，所述的A 1、A 2、A 3、A 4各自独立地为CR'。 In another preferred embodiment, the A 1 , A 2 , A 3 , and A 4 are each independently CR'.
在另一优选例中，所述的R'具有如下式所示的结构：In another preferred example, the R' has the structure shown in the following formula:
Figure PCTCN2022075561-appb-000006
其中，所述的R”为H或取代或未取代的C1-C6烷基。
Figure PCTCN2022075561-appb-000006
Wherein, the R" is H or a substituted or unsubstituted C1-C6 alkyl group.
在另一优选例中，所述的式(I)化合物选自下组：In another preference, the compound of formula (I) is selected from the following group:
Figure PCTCN2022075561-appb-000007
Figure PCTCN2022075561-appb-000007
在另一优选例中，所述的免疫检查点抑制剂选自下组：抗PD-L1抗体、抗PD-1抗体，或其组合。In another preferred embodiment, the immune checkpoint inhibitor is selected from the group consisting of anti-PD-L1 antibody, anti-PD-1 antibody, or a combination thereof.
在另一优选例中，所述的实体瘤选自下组：黑色素瘤、肺癌、结肠癌、肾癌、胰腺癌、肺癌、骨肉瘤。In another preferred embodiment, the solid tumor is selected from the group consisting of melanoma, lung cancer, colon cancer, renal cancer, pancreatic cancer, lung cancer, and osteosarcoma.
本发明的第二方面，提供了一种化合物，或其药学上可接受的盐，水合物，溶剂合物或前药，所述的化合物选自下组：The second aspect of the present invention provides a compound, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, the compound is selected from the group consisting of:
Figure PCTCN2022075561-appb-000008
Figure PCTCN2022075561-appb-000008
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.
附图说明Description of drawings
图1显示了本发明化合物Dac51、Dac258、Dam62、Dam128处理肿瘤细胞后，m 6A丰度呈FTO抑制剂浓度依赖性变化。 Figure 1 shows that after the compounds of the present invention Dac51, Dac258, Dam62, and Dam128 treat tumor cells, the m 6 A abundance changes in a concentration-dependent manner with FTO inhibitors.
图2显示了本发明化合物Dam59与抗PD-L1抗体联用对荷瘤小鼠的肿瘤生长改善以及体重变化情况。Figure 2 shows the improvement of tumor growth and body weight change of the compound Dam59 of the present invention in combination with anti-PD-L1 antibody in tumor-bearing mice.
图3显示了本发明化合物Dam159与抗PD-L1抗体联用对荷瘤小鼠的肿瘤生长改善以及体重变化情况。Figure 3 shows the improvement of tumor growth and body weight change of the compound Dam159 of the present invention in combination with anti-PD-L1 antibody in tumor-bearing mice.
图4显示了本发明化合物Dam60与抗PD-1抗体联用对荷瘤小鼠的肿瘤生长改善以及体重变化情况。Figure 4 shows the improvement of tumor growth and body weight change in tumor-bearing mice when the compound Dam60 of the present invention is combined with anti-PD-1 antibody.
具体实施方式Detailed ways
本发明人经过长期而深入的研究，发现如式(I)所示的一类化合物与免疫检查点抑制剂的药物组合能够协同作用于实体瘤并有效抑制肿瘤生长。基于上述发现，发明人完成了本发明。After long-term and in-depth research, the inventors found that the drug combination of a class of compounds represented by formula (I) and an immune checkpoint inhibitor can synergistically act on solid tumors and effectively inhibit tumor growth. Based on the above findings, the inventors have completed the present invention.
术语the term
除非特别说明，在本文中，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：C 1～C 10烷基、C 3～C 10环烷基、C 1～C 10烷氧基、卤素、羟基、羧基(-COOH)、C 1～C 10醛基、C 2～C 10酰基、C 2～C 10酯基、氨基、苯基；所述的苯基包括未取代的苯基或具有1-3个取代基的取代苯基，所述取代基选自：卤素、C 1-C 10烷基、氰基、OH、硝基、C 3～C 10环烷基、C 1～C 10烷氧基、氨基。 Unless otherwise specified, as used herein, the term "substituted" refers to the substitution of one or more hydrogen atoms on a group with a substituent selected from the group consisting of C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 1 -C 10 alkoxy group, halogen, hydroxyl, carboxyl group (-COOH), C 1 -C 10 aldehyde group, C 2 -C 10 acyl group, C 2 -C 10 ester group, amino group, phenyl group; the described Phenyl includes unsubstituted phenyl or substituted phenyl with 1-3 substituents selected from: halogen, C 1 -C 10 alkyl, cyano, OH, nitro, C 3 -C 10 cycloalkyl, C 1 -C 10 alkoxy, amino.
术语“C 1～C 6烷基”指具有1～6个碳原子的直链或支链烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、仲丁基、叔丁基、或类似基团。 The term "C 1 -C 6 alkyl" refers to straight or branched chain alkyl groups having 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl group, tert-butyl group, or similar groups.
术语“C 1～C 6烷氧基”指具有1-6个碳原子的直链或支链烷氧基，例如甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、仲丁氧基、叔丁氧基、或类似基团。 The term "C 1 -C 6 alkoxy" refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms, such as methoxy, ethoxy, propoxy, isopropoxy, butoxy , isobutoxy, sec-butoxy, tert-butoxy, or similar groups.
术语“羰基C 1～C 6烷基”指形如“-COO-1～6个碳原子的直链或支链烷基”的基团，例如羰基-甲基、羰基-乙基、羰基-丙基、羰基-异丙基、羰基-丁基、羰基-异丁基、羰基-仲丁基、羰基-叔丁基、或类似基团。 The term "carbonyl C 1 -C 6 alkyl" refers to a group in the form of "-COO-straight or branched chain alkyl of 1 to 6 carbon atoms", such as carbonyl-methyl, carbonyl-ethyl, carbonyl- propyl, carbonyl-isopropyl, carbonyl-butyl, carbonyl-isobutyl, carbonyl-sec-butyl, carbonyl-tert-butyl, or the like.
术语“C 6～C 12芳基”指具有6～12个碳原子的芳基，包括单环或二环芳基，例如苯基、萘基，或类似基团。 The term "C 6 -C 12 aryl" refers to aryl groups having 6 to 12 carbon atoms, including monocyclic or bicyclic aryl groups, such as phenyl, naphthyl, or the like.
术语“3-12元杂环基”指具有3-12元的环系上具有一个或多个选自O、S、N或P的杂原子的饱和或非饱和(包括芳香性)环系取代基，如吡啶基、噻吩基、哌啶基，或类似基团，优选为4-9元的杂环基。The term "3-12 membered heterocyclyl" refers to a 3- to 12-membered ring system substituted with one or more heteroatoms selected from O, S, N, or P, saturated or unsaturated (including aromatic) ring systems group, such as pyridyl, thienyl, piperidinyl, or similar groups, preferably a 4-9 membered heterocyclic group.
术语“卤素”指F、Cl、Br和I。The term "halogen" refers to F, Cl, Br and I.
本发明中，术语“含有”、“包含”或“包括”表示各种成分可一起应用于本发明的混合物或组合物中。因此，术语“主要由...组成”和“由...组成”包含在术语“含有”中。In the present invention, the terms "comprising", "comprising" or "including" mean that the various ingredients can be used together in the mixture or composition of the present invention. Thus, the terms "consisting essentially of" and "consisting of" are encompassed by the term "comprising".
本发明中，术语“药学上可接受的”成分是指适用于人和/或动物而无过度不良副反应(如毒性、刺激和***反应)，即有合理的效益/风险比的物质。In the present invention, the term "pharmaceutically acceptable" ingredients refers to substances that are suitable for use in humans and/or animals without excessive adverse side effects (such as toxicity, irritation and allergy), ie, have a reasonable benefit/risk ratio.
本发明中，术语“有效量”指治疗剂治疗、缓解或预防目标疾病或状况的量，或是表现出可检测的治疗或预防效果的量。对于某一对象的精确有效量取决于该对象的体型和健康状况、病症的性质和程度、以及选择给予的治疗剂和/或治疗剂的组合。因此，预先指定准确的有效量是没用的。然而，对于某给定的状况而言，可以用常规实验来确定该有效量，临床医师是能够判断出来的。In the present invention, the term "effective amount" refers to an amount of a therapeutic agent that treats, alleviates or prevents a target disease or condition, or an amount that exhibits a detectable therapeutic or prophylactic effect. The precise effective amount for a subject depends on the size and health of the subject, the nature and extent of the disorder, and the therapeutic agent and/or combination of therapeutic agents selected for administration. Therefore, it is useless to prespecify the exact effective amount. However, for a given situation, routine experimentation can be used to determine the effective amount, as is the judgment of the clinician.
在本文中，除特别说明之处，术语“取代”指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、未取代或卤代的C1-C6烷基、未取代或卤代的C2-C6酰基、未取代或卤代的C1-C6烷基-羟基。As used herein, unless otherwise specified, the term "substituted" refers to the replacement of one or more hydrogen atoms on a group with a substituent selected from the group consisting of halogen, unsubstituted or halogenated C1-C6 alkyl, unsubstituted Substituted or halogenated C2-C6 acyl, unsubstituted or halogenated C1-C6 alkyl-hydroxy.
除非特别说明，本发明中，所有出现的化合物均意在包括所有可能的光学异构体，如单一手性的化合物，或各种不同手性化合物的混合物(即外消旋体)。本发明的所有化合物之中，各手性碳原子可以任选地为R构型或S构型，或R构型和S构型的混合物。Unless otherwise specified, in the present invention, all occurrences of compounds are intended to include all possible optical isomers, such as single chiral compounds, or mixtures of various chiral compounds (ie, racemates). In all compounds of the present invention, each chiral carbon atom can optionally be in the R configuration or the S configuration, or a mixture of the R and S configurations.
如本文所用，术语“本发明化合物”指式I所示的化合物。该术语还包括及式(I)化合物的各种晶型形式、药学上可接受的盐、水合物或溶剂合物。As used herein, the term "compounds of the present invention" refers to compounds of formula I. The term also includes the various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of formula (I).
在本申请中，术语“前药”是指本发明的化合物的药学上可接受的代谢前体，其可 在生理学条件下或通过溶剂分解而被转化成本发明的生物活性化合物的化合物。当被给予有需要的个体时，前药可以不具有活性，但在体内被转化成本发明的活性化合物。前药通常在体内迅速转化，而产生本发明的母体化合物，例如通过在血液中水解来实现。前药化合物通常在哺乳动物生物体内提供溶解度、组织相容性或缓释的优点。In this application, the term "prodrug" refers to a pharmaceutically acceptable metabolic precursor of a compound of the present invention, which can be converted to a compound of the biologically active compound of the present invention under physiological conditions or by solvolysis. A prodrug may be inactive when administered to an individual in need thereof, but be converted in vivo to an active compound of the present invention. Prodrugs are typically rapidly transformed in vivo to yield the parent compounds of the invention, eg, by hydrolysis in blood. Prodrug compounds generally provide the advantages of solubility, histocompatibility or sustained release in mammalian organisms.
作为FTO抑制剂的式I化合物Compounds of formula I as FTO inhibitors
本发明中，采用FTO抑制剂化合物与免疫检查点抑制剂进行联用，从而起到协同效果。所述的FTO抑制剂为如下式(I)所示的化合物，及其药学上可接受的盐，水合物，溶剂合物或前药：In the present invention, the FTO inhibitor compound is used in combination with the immune checkpoint inhibitor, thereby achieving a synergistic effect. The FTO inhibitor is a compound represented by the following formula (I), and a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof:
Figure PCTCN2022075561-appb-000009
Figure PCTCN2022075561-appb-000009
其中，in,
A 1、A 2、A 3、A 4各自独立地为CR'或N； A 1 , A 2 , A 3 , A 4 are each independently CR' or N;
M选自下组：CR' 2、NH、O或S； M is selected from the group consisting of CR'2 , NH, O or S;
R'选自下组：H、卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基、C 1-C 6烷氧基羰基、取代或未取代的C 1-C 6酰氨基、取代或未取代的C 2-C 12酯基、或取代或未取代的C 1-C 10烷基、或取代或未取代的C3-C10烯酰胺基； R' is selected from the group consisting of H, halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, substituted or unsubstituted C 1 -C 6 amido, substituted or unsubstituted C 2 -C 12 ester group, or substituted or unsubstituted C 1 - C10 alkyl, or substituted or unsubstituted C3-C10 enamido;
X具有如下式所示的结构：羧基、羟肟酸基、取代或未取代的C 2-C 12酯基、取代或未取代的酰胺基(C(O)NH 2)、取代或未取代3-12元杂环基； X has the structure shown in the following formula: carboxyl group, hydroxamic acid group, substituted or unsubstituted C 2 -C 12 ester group, substituted or unsubstituted amide group (C(O)NH 2 ), substituted or unsubstituted 3 -12-membered heterocyclyl;
Y选自下组：取代或未取代的C 6-C 12的芳基、取代或未取代3-12元杂环基； Y is selected from the group consisting of substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 3-12 membered heterocyclyl;
R a、R b、R c、R d各自独立地选自下组：H、卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基； R a , R b , R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted Substituted C 1 -C 6 alkoxy;
所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 1-C 6烷氧基羰基、C 1-C 6酰氨基、C 2-C 12酯基、取代或未取代的C 1-C 10烷基，取代或未取代的C 2-C 10烯基，取代或未取代的C 2-C 10炔基，取代或未取代的C 6-C 10芳基、或取代或未取代的五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或C 1-C 6烷氨基；其中，所述的取代或未取代的C 1-C 10烷基，取代或未取代的C 6-C 10芳基或五元或六元杂芳基的取代基选自下组：卤素原子、羰基(＝O)、羟基、羧基、C 1-C 6烷氧基羰基、氨基、C 1-C 6酰氨基、硝基、氰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 6-C 10芳基或五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为卤素原子、C 1-C 6烷氧基羰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或苯基、5-6元杂环基。 Said substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group, substituted or unsubstituted C 1 -C 10 alkane base, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C 2 -C 10 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, or substituted or unsubstituted five-membered or Six-membered heteroaryl, 3-12-membered heterocyclic, 3-12-membered cycloalkyl, preferably C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy Or C 1 -C 6 alkylamino; wherein, the substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 6 -C 10 aryl or the substitution of five- or six-membered heteroaryl The group is selected from the group consisting of halogen atom, carbonyl group (=O), hydroxyl group, carboxyl group, C 1 -C 6 alkoxycarbonyl group, amino group, C 1 -C 6 amido group, nitro group, cyano group, C 1 -C 6 group Alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 6 -C 10 aryl or five- or six-membered heteroaryl, 3-12 membered heterocyclic group, 3-12 membered cycloalkyl group, preferably halogen atom, C 1 -C 6 alkoxycarbonyl group, C 1 -C 6 alkyl group, halogenated C 1 -C 6 alkyl group, C 1 -C 6 alkyl group 6 -alkoxy or phenyl, 5-6 membered heterocyclic group.
优选的化合物具有如本申请实施例中所示的结构。Preferred compounds have structures as shown in the Examples herein.
药物组合物和施用方法Pharmaceutical compositions and methods of administration
由于本发明的药物组合具有优异的对肿瘤细胞增殖的抑制活性，因此式(I)化合物及其各种晶型，药学上可接受的无机或有机盐，水合物或溶剂合物与免疫检查点抑制剂的药物组合可用于治疗、预防以及缓解由于肿瘤细胞增殖而引发的疾病。根据现有技术，本发明化合物可用于治疗以下疾病：黑色素瘤、肺癌、结肠癌等癌症。Since the pharmaceutical combination of the present invention has excellent inhibitory activity on tumor cell proliferation, the compound of formula (I) and its various crystal forms, pharmaceutically acceptable inorganic or organic salts, hydrates or solvates and immune checkpoints Pharmaceutical combinations of inhibitors can be used to treat, prevent and alleviate diseases caused by tumor cell proliferation. According to the prior art, the compounds of the present invention can be used to treat the following diseases: melanoma, lung cancer, colon cancer and other cancers.
本发明的药物组合物包含安全有效量范围内的本发明化合物或其药理上可接受的盐及药理上可以接受的赋形剂或载体。其中“安全有效量”指的是：化合物的量足以明显改善病情，而不至于产生严重的副作用。通常，药物组合物含有1-2000mg本发明化合物/剂，更佳的，含有5-200mg本发明化合物/剂。较佳的，所述的“一剂”为一个胶囊或药片。The pharmaceutical composition of the present invention comprises the compound of the present invention or a pharmacologically acceptable salt thereof and a pharmacologically acceptable excipient or carrier within a safe and effective amount. The "safe and effective amount" refers to: the amount of the compound is sufficient to significantly improve the condition without causing serious side effects. Typically, the pharmaceutical composition contains 1-2000 mg of the compound of the present invention per dose, more preferably 5-200 mg of the compound of the present invention per dose. Preferably, the "one dose" is a capsule or tablet.
“药学上可以接受的载体”指的是：一种或多种相容性固体或液体填料或凝胶物质，它们适合于人使用，而且必须有足够的纯度和足够低的毒性。“相容性”在此指的是组合物中各组份能和本发明的化合物以及它们之间相互掺和，而不明显降低化合物的药效。药学上可以接受的载体部分例子有纤维素及其衍生物(如羧甲基纤维素钠、乙基纤维素钠、纤维素乙酸酯等)、明胶、滑石、固体润滑剂(如硬脂酸、硬脂酸镁)、硫酸钙、植物油(如豆油、芝麻油、花生油、橄榄油等)、多元醇(如丙二醇、甘油、甘露醇、山梨醇等)、乳化剂(如吐温
Figure PCTCN2022075561-appb-000010
)、润湿剂(如十二烷基硫酸钠)、着色剂、调味剂、稳定剂、抗氧化剂、防腐剂、无热原水等。 "Pharmaceutically acceptable carrier" refers to one or more compatible solid or liquid filler or gel substances which are suitable for human use and which must be of sufficient purity and sufficiently low toxicity. "Compatibility" as used herein means that the components of the composition can be admixed with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds. Examples of pharmaceutically acceptable carrier moieties 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)
Figure PCTCN2022075561-appb-000010
), wetting agents (such as sodium lauryl sulfate), colorants, flavors, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
本发明化合物或药物组合物的施用方式没有特别限制，代表性的施用方式包括(但并不限于)：口服、瘤内、直肠、肠胃外(静脉内、肌肉内或皮下)、和局部给药。The mode of administration of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative modes of administration include (but are not limited to): oral, intratumoral, rectal, parenteral (intravenous, intramuscular or subcutaneous), and topical administration .
用于口服给药的固体剂型包括胶囊剂、片剂、丸剂、散剂和颗粒剂。在这些固体剂型中，活性化合物与至少一种常规惰性赋形剂(或载体)混合，如柠檬酸钠或磷酸二钙，或与下述成分混合：(a)填料或增容剂，例如，淀粉、乳糖、蔗糖、葡萄糖、甘露醇和硅酸；(b)粘合剂，例如，羟甲基纤维素、藻酸盐、明胶、聚乙烯基吡咯烷酮、蔗糖和***胶；(c)保湿剂，例如，甘油；(d)崩解剂，例如，琼脂、碳酸钙、马铃薯淀粉或木薯淀粉、藻酸、某些复合硅酸盐、和碳酸钠；(e)缓溶剂，例如石蜡；(f)吸收加速剂，例如，季胺化合物；(g)润湿剂，例如鲸蜡醇和单硬脂酸甘油酯；(h)吸附剂，例如，高岭土；和(i)润滑剂，例如，滑石、硬脂酸钙、硬脂酸镁、固体聚乙二醇、十二烷基硫酸钠，或其混合物。胶囊剂、片剂和丸剂中，剂型也可包含缓冲剂。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 (a) fillers or compatibilizers, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders such as, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, For example, glycerol; (d) disintegrants, such as agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) Absorption accelerators such as quaternary amine compounds; (g) wetting agents such as cetyl alcohol and glyceryl monostearate; (h) adsorbents such as kaolin; and (i) lubricants such as talc, hard Calcium fatty acid, magnesium stearate, solid polyethylene glycol, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage form may also contain buffering agents.
固体剂型如片剂、糖丸、胶囊剂、丸剂和颗粒剂可采用包衣和壳材制备，如肠衣和其它本领域公知的材料。它们可包含不透明剂，并且，这种组合物中活性化合物或化合物的释放可以延迟的方式在消化道内的某一部分中释放。可采用的包埋组分的实例是聚合物质和蜡类物质。必要时，活性化合物也可与上述赋形剂中的一种或多种形成微胶囊形式。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 well known in the art. They may contain opacifying agents, and the release of the active compound or compounds in such compositions may be in a certain part of the digestive tract in a delayed manner. Examples of embedding components that can be employed are polymeric substances and waxes. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.
用于口服给药的液体剂型包括药学上可接受的乳液、溶液、悬浮液、糖浆或酊剂。除了活性化合物外，液体剂型可包含本领域中常规采用的惰性稀释剂，如水或其它溶剂，增溶剂和乳化剂，例知，乙醇、异丙醇、碳酸乙酯、乙酸乙酯、丙二醇、1,3-丁二醇、二甲基甲酰胺以及油，特别是棉籽油、花生油、玉米胚油、橄榄油、蓖麻油和芝麻油或这些物质的混合物等。Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compound, liquid dosage forms may contain inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropanol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils, especially cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil or mixtures of these substances, and the like.
除了这些惰性稀释剂外，组合物也可包含助剂，如润湿剂、乳化剂和悬浮剂、甜味剂、矫味剂和香料。Besides these inert diluents, the compositions can also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening, flavoring and perfuming agents.
除了活性化合物外，悬浮液可包含悬浮剂，例如，乙氧基化异十八烷醇、聚氧乙 烯山梨醇和脱水山梨醇酯、微晶纤维素、甲醇铝和琼脂或这些物质的混合物等。Suspensions, in addition to the active compounds, may contain suspending agents such as ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.
用于肠胃外注射的组合物可包含生理上可接受的无菌含水或无水溶液、分散液、悬浮液或乳液，和用于重新溶解成无菌的可注射溶液或分散液的无菌粉末。适宜的含水和非水载体、稀释剂、溶剂或赋形剂包括水、乙醇、多元醇及其适宜的混合物。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.
用于局部给药的本发明化合物的剂型包括软膏剂、散剂、贴剂、喷射剂和吸入剂。活性成分在无菌条件下与生理上可接受的载体及任何防腐剂、缓冲剂，或必要时可能需要的推进剂一起混合。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 if necessary.
本发明化合物可以单独给药，或者与其他药学上可接受的化合物联合给药。The compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds.
使用药物组合物时，是将安全有效量的本发明化合物适用于需要治疗的哺乳动物(如人)，其中施用时剂量为药学上认为的有效给药剂量，对于60kg体重的人而言，日给药剂量通常为1～2000mg，优选5～500mg。当然，具体剂量还应考虑给药途径、病人健康状况等因素，这些都是熟练医师技能范围之内的。When using the pharmaceutical composition, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) in need of treatment, and the dose is the effective dose considered pharmaceutically, for a 60kg body weight, the daily dose is The administration dose is usually 1 to 2000 mg, preferably 5 to 500 mg. Of course, the specific dosage should also take into account the route of administration, the patient's health and other factors, which are all within the skill of the skilled physician.
本发明的主要优点包括：The main advantages of the present invention include:
本发明提供了一类式(I)化合物和免疫检查点抑制剂联用***的用途，所述的方法可以以非常低的用药剂量下有效抑制肿瘤细胞的增殖，且相对于二者单用，产生了明显的协同作用。The present invention provides the use of a compound of formula (I) and an immune checkpoint inhibitor in combination to treat tumors. The method can effectively inhibit the proliferation of tumor cells at a very low dosage, and compared with the two used alone , resulting in a clear synergistic effect.
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. In the following examples, the experimental methods without specific conditions are usually in accordance with conventional conditions, or in accordance with the conditions suggested by the manufacturer. Percentages and parts are by weight unless otherwise indicated.
化学实施例FTO抑制剂的合成及表征CHEMICAL EXAMPLES Synthesis and Characterization of FTO Inhibitors
实施例1化合物Dac51的合成Example 1 Synthesis of compound Dac51
Figure PCTCN2022075561-appb-000011
Figure PCTCN2022075561-appb-000011
第一步：将邻碘苯甲酸30g(120mmol,1.2eq)、2,6-二氯-4-溴苯胺24g(100mmol,1.0eq)、三乙胺(150mmol,1.5eq)和无水乙酸铜9g(5.0mmol,0.5eq)溶于DMF 500mL中，氩气保护下加热至120℃反应24小时，反应结束后。降温到室温，加入等体积的水，乙酸乙酯萃取300mL×3，用水洗涤，有机相旋干，通过硅胶柱色谱分离(石油醚:乙酸乙酯＝20:1到1:1)，得到目标产物黄色固体2-(4-溴-2,6-二氯苯基)氨基)苯甲酸9.8g。The first step: 30g (120mmol, 1.2eq) of o-iodobenzoic acid, 24g (100mmol, 1.0eq) of 2,6-dichloro-4-bromoaniline, 24g (100mmol, 1.0eq) of triethylamine (150mmol, 1.5eq) and anhydrous copper acetate 9g (5.0mmol, 0.5eq) was dissolved in 500mL of DMF, heated to 120°C under argon protection and reacted for 24 hours, after the reaction was completed. Cool to room temperature, add an equal volume of water, extract with 300 mL×3 of ethyl acetate, wash with water, spin dry the organic phase, and separate by silica gel column chromatography (petroleum ether:ethyl acetate=20:1 to 1:1) to obtain the target The product was 9.8 g of 2-(4-bromo-2,6-dichlorophenyl)amino)benzoic acid as a yellow solid.
第二步：将2-(4-溴-2,6-二氯苯基)氨基)苯甲酸3.6g溶解于无水乙醇200mL中，冰水浴冷却下，向其中加入浓硫酸20mL，加热至100℃回流反应12小时。反应结束后， 反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水100mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝99:1)，得到目标产物白色固体2-((4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.1g。Step 2: Dissolve 3.6 g of 2-(4-bromo-2,6-dichlorophenyl)amino)benzoic acid in 200 mL of absolute ethanol, add 20 mL of concentrated sulfuric acid to it under cooling in an ice-water bath, and heat to 100 The reaction was refluxed at °C for 12 hours. After the reaction was completed, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL×3, and the organic phases were combined and washed with saturated brine for 10 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, the filtrate was concentrated, and the filtrate was separated by silica gel column chromatography (petroleum ether:ethyl acetate=99:1) to obtain the target product as a white solid 2-((4-bromo-2,6-dichloromethane). Phenyl)amino)ethyl benzoate 3.1 g.
第三步：将2-((4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),3,5-二甲基吡唑-4-硼酸频哪醇酯2.13g(9.6mmol,1.2eq),Pd(dppf)Cl 2 584mg(0.8mmol,0.1eq),碳酸钾1.68g(12.0mmol,1.5eq)溶于1,4-二氧六环和水的混合溶剂160mL中，加热至100℃反应24小时。冷却至室温，浓缩至干，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得到目标产物白色固体2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸乙酯1.10g。 The third step: 2-((4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), 3,5-dimethylpyrazole-4- Pinacol borate 2.13g (9.6mmol, 1.2eq), Pd(dppf)Cl 2 584mg (0.8mmol, 0.1eq), Potassium carbonate 1.68g (12.0mmol, 1.5eq) dissolved in 1,4-dioxane In 160 mL of a mixed solvent of ring and water, the mixture was heated to 100°C and reacted for 24 hours. Cooled to room temperature, concentrated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain the target product as a white solid 2-((2,6-dichloro-4-(3,5-dichloromethane). Methylpyrazole)phenyl)amino)ethyl benzoate 1.10 g.
第四步：将2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸乙酯1.10g(2.7mmol,1.0eq)溶于四氢呋喃13.5mL和无水乙醇27mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化钠540mg(13.5mmol,5.0eq)的水7mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，用2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得目标产物白色固体2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸930mg。Step 4: Dissolve 1.10g (2.7mmol, 1.0eq) of ethyl 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoate in tetrahydrofuran In a mixed solvent of 13.5 mL and 27 mL of absolute ethanol, under cooling in an ice-water bath, a solution of 540 mg (13.5 mmol, 5.0 eq) of sodium hydroxide in 7 mL of water was slowly added dropwise. The reaction was heated to 45 °C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. . After suction filtration, the solid was washed with water to obtain 930 mg of the target product, 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得目标产物白色固体Dac51(22mg)。 1H NMR(500MHz,DMSO)δ11.31(s,1H),9.43(s,1H),7.50(d,J＝7.7Hz,1H),7.45(s,2H),7.24(dd,J＝11.4,4.2Hz,1H),6.76(t,J＝7.5Hz,1H),6.30(d,J＝8.3Hz,1H),2.23(s,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn ), the resulting mixed system was reacted at 45 °C. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain the target product Dac51 (22 mg) as a white solid. 1 H NMR (500MHz, DMSO) δ 11.31 (s, 1H), 9.43 (s, 1H), 7.50 (d, J=7.7Hz, 1H), 7.45 (s, 2H), 7.24 (dd, J=11.4 ,4.2Hz,1H),6.76(t,J=7.5Hz,1H),6.30(d,J=8.3Hz,1H),2.23(s,6H).
实施例2化合物Dac50的合成Example 2 Synthesis of compound Dac50
Figure PCTCN2022075561-appb-000012
Figure PCTCN2022075561-appb-000012
第一步和第二步反应按照Dac51的合成操作制备。The first and second reactions were prepared according to the synthetic procedure of Dac51.
第三步：将2-(4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),3-甲基吡唑-4-硼酸频哪醇酯2.13g(9.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 584mg(0.8mmol,0.1eq),碳酸钾1.68g(12.0mmol,1.5eq)溶于二氧六环和水的混合溶剂160mL中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2,6-二氯-4-(3-甲基吡唑)苯基)氨基)苯甲酸乙酯1.10g。 The third step: 3.0g (8.0mmol, 1.0eq) of ethyl 2-(4-bromo-2,6-dichlorophenyl)amino)benzoate, 3-methylpyrazole-4-boronic acid pinacol Ester 2.13g (9.6mmol, 1.2eq), catalyst Pd(dppf)Cl 2 584mg (0.8mmol, 0.1eq), potassium carbonate 1.68g (12.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 160mL , heated to 100 °C for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2,6-dichloro-4-(3- Methylpyrazole)phenyl)amino)ethyl benzoate 1.10 g.
第四步：将2-((2,6-二氯-4-(3-甲基吡唑)苯基)氨基)苯甲酸乙酯1.10g(2.7mmol,1.0eq)溶于四氢呋喃13.5mL和无水乙醇27mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化钠540mg(13.5mmol,5.0eq)的水7mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得目标产物白色固体2-((2,6-二氯-4-(3-甲基吡唑)苯基)氨基)苯甲酸930mg。Step 4: Dissolve 1.10 g (2.7 mmol, 1.0 eq) of ethyl 2-((2,6-dichloro-4-(3-methylpyrazole)phenyl)amino)benzoate in 13.5 mL of tetrahydrofuran and A solution of 540 mg (13.5 mmol, 5.0 eq) of sodium hydroxide in 7 mL of water was slowly added dropwise to a mixed solvent of 27 mL of anhydrous ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 930 mg of the target product, 2-((2,6-dichloro-4-(3-methylpyrazole)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3-甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dac50(17mg)。 1H NMR(500MHz,DMSO)δ11.31(s,1H),9.43(s,1H),7.50(d,J＝7.7Hz,1H),7.45(s,2H),7.24(dd,J＝11.4,4.2Hz,1H),6.76(t,J＝7.5Hz,1H),6.30(d,J＝8.3Hz,1H),2.23(s,3H). Step 5: In a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3-methylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were successively added to the above system, the resulting The mixed system was reacted at 45°C. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain Dac50 (17 mg) as a white solid. 1 H NMR (500MHz, DMSO) δ 11.31 (s, 1H), 9.43 (s, 1H), 7.50 (d, J=7.7Hz, 1H), 7.45 (s, 2H), 7.24 (dd, J=11.4 ,4.2Hz,1H),6.76(t,J=7.5Hz,1H),6.30(d,J=8.3Hz,1H),2.23(s,3H).
实施例3化合物Dac52的合成Example 3 Synthesis of compound Dac52
Figure PCTCN2022075561-appb-000013
Figure PCTCN2022075561-appb-000013
第一步和第二步反应按照Dac51的合成操作制备。The first and second reactions were prepared according to the synthetic procedure of Dac51.
第三步：将2-(4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),吡唑-4-硼酸频哪醇酯2.13g(9.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 584mg(0.8mmol,0.1eq),碳酸钾1.68g(12.0mmol,1.5eq)溶于二氧六环和水的混合溶剂160mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚：乙酸乙酯20:1)，得白色固体2-((2,6-二氯-4-(吡唑)苯基)氨基)苯甲酸乙酯1.10g。 The third step: 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), pyrazole-4-boronic acid pinacol ester 2.13g ( 9.6mmol, 1.2eq), catalyst Pd(dppf)Cl 2 584mg (0.8mmol, 0.1eq), potassium carbonate 1.68g (12.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 160mL (4:1 /v:v), heated to 100°C and reacted for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate 20:1) to obtain 2-((2,6-dichloro-4-(pyrazole) as a white solid) Phenyl) amino) ethyl benzoate 1.10 g.
第四步：将2-((2,6-二氯-4-(吡唑)苯基)氨基)苯甲酸乙酯1.10g(2.7mmol,1.0eq)溶于四氢呋喃13.5mL和无水乙醇27mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化钠540mg(13.5mmol,5.0eq)的水7mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得白色固体2-((2,6-二氯-4-(吡唑)苯基)氨基)苯甲酸930mg。Step 4: Dissolve 1.10g (2.7mmol, 1.0eq) of ethyl 2-((2,6-dichloro-4-(pyrazole)phenyl)amino)benzoate in 13.5mL of tetrahydrofuran and 27mL of absolute ethanol In the mixed solvent of , under cooling in an ice-water bath, a solution of 540 mg (13.5 mmol, 5.0 eq) of sodium hydroxide in 7 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 930 mg of 2-((2,6-dichloro-4-(pyrazole)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24 mmol,1.2eq),所得混合体系在45℃反应。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dac52(18mg)。 1H NMR(500MHz,DMSO)δ11.31(s,1H),9.43(s,1H),7.50(d,J＝7.7Hz,1H),7.45(s,2H),7.24(dd,J＝11.4,4.2Hz,1H),6.76(t,J＝7.5Hz,1H),6.30(d,J＝8.3Hz,1H),2.23(s,6H). The fifth step: in a 10mL round-bottom flask, add 2-((2,6-dichloro-4-(pyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was added at 45 °C reaction. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain Dac52 (18 mg) as a white solid. 1 H NMR (500MHz, DMSO) δ 11.31 (s, 1H), 9.43 (s, 1H), 7.50 (d, J=7.7Hz, 1H), 7.45 (s, 2H), 7.24 (dd, J=11.4 ,4.2Hz,1H),6.76(t,J=7.5Hz,1H),6.30(d,J=8.3Hz,1H),2.23(s,6H).
实施例4化合物Dam62的合成Example 4 Synthesis of compound Dam62
Figure PCTCN2022075561-appb-000014
Figure PCTCN2022075561-appb-000014
第一步、第二步、第三步、第四步反应按照Dac51的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF4.0mL，搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam62(46mg)。 1H NMR(400MHz,DMSO)δ12.47(s,1H),10.13(s,1H),8.12(s,1H),7.74(d,J＝7.8Hz,1H),7.51(s,1H),7.48(s,2H),7.28(t,J＝7.7Hz,1H),6.78(t,J＝7.5Hz,1H),6.31(d,J＝8.2Hz,1H),2.29(s,3H),2.23(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain white solid Dam62 (46 mg). 1 H NMR (400MHz, DMSO)δ12.47(s,1H), 10.13(s,1H), 8.12(s,1H), 7.74(d, J=7.8Hz,1H), 7.51(s,1H), 7.48(s, 2H), 7.28(t, J=7.7Hz, 1H), 6.78(t, J=7.5Hz, 1H), 6.31(d, J=8.2Hz, 1H), 2.29(s, 3H), 2.23(s,3H).
实施例5化合物Dam253的合成Example 5 Synthesis of compound Dam253
Figure PCTCN2022075561-appb-000015
Figure PCTCN2022075561-appb-000015
第一步、第二步反应按照Dac51的合成操作制备。The first and second steps are prepared according to the synthetic operation of Dac51.
第三步：将2-(4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),2,5-二甲基异恶唑-4-硼酸1.36g(9.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 584mg(0.8mmol,0.1eq),碳酸钾1.68g(12.0mmol,1.5eq)溶于二氧六环和水的混合溶剂160mL中，加热至100 ℃反应24小时。冷却至室温，浓缩至干，硅胶柱色谱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸乙酯1.98g。 The third step: 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), 2,5-dimethylisoxazole-4- Boric acid 1.36g (9.6mmol, 1.2eq), catalyst Pd(dppf)Cl 2 584mg (0.8mmol, 0.1eq), potassium carbonate 1.68g (12.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 160mL , heated to 100 °C and reacted for 24 hours. Cooled to room temperature, concentrated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain white solid 2-((2,6-dichloro-4-(3,5-dimethylisobutylene) 1.98 g of ethyl oxazole)phenyl)amino)benzoate.
第四步：将2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸乙酯1.98g(4.4mmol,1.0eq)溶于四氢呋喃36mL和无水乙醇72mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化钠880mg(22mmol,5.0eq)的水9mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得白色固体2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸1.6g。The fourth step: dissolve ethyl 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)benzoate 1.98g (4.4mmol, 1.0eq) in In a mixed solvent of 36 mL of tetrahydrofuran and 72 mL of anhydrous ethanol, under ice-water bath cooling, a solution of 880 mg (22 mmol, 5.0 eq) of sodium hydroxide in 9 mL of water was slowly added dropwise. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 1.6 g of 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF 4.0mL，搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq)，N,N-二乙基丁二胺35mg(0.24mmol,1.2eq)，所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam253(66mg)。 1H NMR(400MHz,DMSO)δ9.86(s,1H),8.62(s,1H),7.66(d,J＝7.6Hz,1H),7.63(s,2H),7.27(t,J＝7.8Hz,1H),6.83(t,J＝7.4Hz,1H),6.36(d,J＝8.2Hz,1H),3.30(d,J＝6.3Hz,3H),2.46(s,6H),2.42(d,J＝8.4Hz,2H),2.28(s,3H),1.55(dd,J＝13.8,6.8Hz,2H),1.46(d,J＝6.8Hz,2H),0.94(t,J＝7.1Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0 eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylbutane were sequentially added to the above system 35 mg of diamine (0.24 mmol, 1.2 eq), the resulting mixed system was reacted at 45° C. for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain white solid Dam253 (66 mg). 1 H NMR(400MHz, DMSO)δ9.86(s,1H),8.62(s,1H),7.66(d,J=7.6Hz,1H),7.63(s,2H),7.27(t,J=7.8 Hz, 1H), 6.83(t, J＝7.4Hz, 1H), 6.36(d, J＝8.2Hz, 1H), 3.30(d, J＝6.3Hz, 3H), 2.46(s, 6H), 2.42( d, J＝8.4Hz, 2H), 2.28(s, 3H), 1.55(dd, J＝13.8, 6.8Hz, 2H), 1.46(d, J＝6.8Hz, 2H), 0.94(t, J＝7.1 Hz, 6H).
实施例6化合物Dam59的合成Example 6 Synthesis of compound Dam59
Figure PCTCN2022075561-appb-000016
Figure PCTCN2022075561-appb-000016
第一步、第二步、第三步、第四步反应按照Dac51的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF4.0mL，搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq)，3-氨基丙醇18mg(0.24mmol,1.2eq)，所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam59(48mg)。 1H NMR(400MHz,MeOD)δ7.67–7.54(m,1H),7.46–7.34(m,2H),7.25(t,J＝7.8Hz,1H),6.83(t,J＝7.5Hz,1H),6.41(d,J＝8.3Hz,1H),3.71(t,J＝6.2Hz,2H),3.51(t,J＝6.9Hz,2H),2.31(s,6H),1.98–1.81(m,2H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), 114 mg (0.3 mmol, 1.5 eq) of HATU, dissolved in 4.0 mL of anhydrous DMF at room temperature, stirred for 5 min, and 77 mg (0.6 mmol, 3.0 eq) of DIEA, 18 mg (0.24 mmol) of 3-aminopropanol were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain white solid Dam59 (48 mg). 1 H NMR(400MHz,MeOD)δ7.67-7.54(m,1H),7.46-7.34(m,2H),7.25(t,J=7.8Hz,1H),6.83(t,J=7.5Hz,1H) ), 6.41(d, J＝8.3Hz, 1H), 3.71(t, J＝6.2Hz, 2H), 3.51(t, J＝6.9Hz, 2H), 2.31(s, 6H), 1.98–1.81(m , 2H).
实施例7化合物Dam60的合成Example 7 Synthesis of compound Dam60
Figure PCTCN2022075561-appb-000017
Figure PCTCN2022075561-appb-000017
第一步、第二步、第三步、第四步反应按照Dac51的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF4.0mL，搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq)，N,N-二乙氨基乙二胺28mg(0.24mmol,1.2eq)，所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam60(48mg)。 1H NMR(500MHz,MeOD)δ8.13(dd,J＝7.5,1.4Hz,1H),8.01(s,1H),7.77(s,2H),7.63(dd,J＝7.5,1.4Hz,1H),7.58(td,J＝7.5,1.4Hz,1H),7.08–6.98(m,2H),3.66(t,J＝7.3Hz,1H),3.52(t,J＝7.3Hz,1H),3.02–2.92(m,2H),2.66(t,J＝7.3Hz,2H),2.43(q,J＝6.4Hz,2H),2.07(s,3H),1.95(s,3H),1.03(t,J＝6.4Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenedi Amine 28mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain white solid Dam60 (48 mg). 1 H NMR (500MHz, MeOD) δ 8.13 (dd, J=7.5, 1.4Hz, 1H), 8.01 (s, 1H), 7.77 (s, 2H), 7.63 (dd, J=7.5, 1.4Hz, 1H ),7.58(td,J＝7.5,1.4Hz,1H),7.08–6.98(m,2H),3.66(t,J＝7.3Hz,1H),3.52(t,J＝7.3Hz,1H),3.02 –2.92(m, 2H), 2.66(t, J=7.3Hz, 2H), 2.43(q, J=6.4Hz, 2H), 2.07(s, 3H), 1.95(s, 3H), 1.03(t, J=6.4Hz, 6H).
实施例8化合物Dam61的合成Example 8 Synthesis of compound Dam61
Figure PCTCN2022075561-appb-000018
Figure PCTCN2022075561-appb-000018
第一步、第二步、第三步、第四步反应按照Dac51的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac51.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF4.0mL，搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq)，3-氨基丙醇18mg(0.24mmol,1.2eq)，所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam61(46mg)。 1H NMR(500MHz,MeOD)δ8.38(s,1H),8.03(dd,J＝7.4,1.5Hz,1H),7.79(d,J＝7.1Hz,3H),7.61(dd,J＝7.5,1.4Hz,1H),7.59–7.53(m,1H),6.95(td,J＝7.5,1.4Hz,1H),3.46(dd,J＝16.6,7.8Hz,2H),2.43(s,6H),1.69(ddd,J＝7.8,4.6,3.1Hz,2H),1.35(s,1H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), 114 mg (0.3 mmol, 1.5 eq) of HATU, dissolved in 4.0 mL of anhydrous DMF at room temperature, stirred for 5 min, and 77 mg (0.6 mmol, 3.0 eq) of DIEA, 18 mg (0.24 mmol) of 3-aminopropanol were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse phase, and the separated solution was lyophilized to obtain white solid Dam61 (46 mg). 1 H NMR (500MHz, MeOD) δ 8.38 (s, 1H), 8.03 (dd, J=7.4, 1.5Hz, 1H), 7.79 (d, J=7.1Hz, 3H), 7.61 (dd, J=7.5 ,1.4Hz,1H),7.59–7.53(m,1H),6.95(td,J=7.5,1.4Hz,1H),3.46(dd,J=16.6,7.8Hz,2H),2.43(s,6H) ,1.69(ddd,J=7.8,4.6,3.1Hz,2H),1.35(s,1H).
实施例9化合物Dam128的合成Example 9 Synthesis of compound Dam128
Figure PCTCN2022075561-appb-000019
Figure PCTCN2022075561-appb-000019
第一步：将邻碘苯甲酸29.8g(120mmol,1.2eq),2,6-二乙基-4-溴苯胺22.8g(100mmol,1.0eq),三乙胺(150mmol,1.5eq)和无水乙酸铜9.1g(5.0mmol,0.5eq)溶于DMF360mL中，氩气保护下加热至120℃反应24h，反应结束后。降温到室温，加入等体积的水，母液用DCM萃取300mL×3，用水洗涤DMF，有机相旋干，硅胶柱色谱分离(石油醚:乙酸乙酯＝20:1到1:1)，得到目标产物黄色固体13.6g。The first step: 29.8g (120mmol, 1.2eq) of o-iodobenzoic acid, 22.8g (100mmol, 1.0eq) of 2,6-diethyl-4-bromoaniline, 22.8g (100mmol, 1.0eq) of triethylamine (150mmol, 1.5eq) and no 9.1 g (5.0 mmol, 0.5 eq) of water copper acetate was dissolved in 360 mL of DMF, heated to 120° C. for 24 h under argon protection, and the reaction was completed. Cool to room temperature, add an equal volume of water, extract the mother liquor with DCM 300 mL×3, wash DMF with water, spin dry the organic phase, and separate by silica gel column chromatography (petroleum ether:ethyl acetate=20:1 to 1:1) to obtain the target The product was a yellow solid, 13.6 g.
第二步：将2-(4-溴-2,6-二乙基苯基)氨基)苯甲酸10.4g溶解于无水乙醇300mL中，冰水浴冷却下，向其中加入浓硫酸30mL，加热至100℃回流反应12小时。反应结束后，反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水100mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶柱色谱分离，得白色固体2-(4-溴-2,6-二乙基苯基)氨基)苯甲酸乙酯10.2g。Step 2: Dissolve 10.4 g of 2-(4-bromo-2,6-diethylphenyl)amino)benzoic acid in 300 mL of absolute ethanol, add 30 mL of concentrated sulfuric acid to it under cooling in an ice-water bath, and heat to The reaction was refluxed at 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL×3, and the organic phases were combined and washed with saturated brine for 10 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain 10.2 g of ethyl 2-(4-bromo-2,6-diethylphenyl)amino)benzoate as a white solid.
第三步：将2-(4-溴-2,6-二乙基苯基)氨基)苯甲酸乙酯1.13g(3.0mmol,1.0eq),3,5-二甲基异恶唑-4-硼酸508mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于二氧六环和水的混合溶剂160mL中，加热至100℃反应24小时。冷却至室温，浓缩至干，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸乙酯650mg。 The third step: 2-(4-bromo-2,6-diethylphenyl)amino)ethyl benzoate 1.13g (3.0mmol, 1.0eq), 3,5-dimethylisoxazole-4 - Boric acid 508mg (3.6mmol, 1.2eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in 160mL of a mixed solvent of dioxane and water , heated to 100 °C for 24 hours. Cooled to room temperature, concentrated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain white solid 2-((2,6-diethyl-4-pyridine)phenyl)amino) Ethyl benzoate 650mg.
第四步：将2-((2,6-二乙基-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸乙酯650mg(1.7mmol,1.0eq)溶于四氢呋喃4mL和无水乙醇8mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂199mg(8.3mmol,5.0eq)的水2mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得白色固体2-((2,6-二乙基-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸620mg。The fourth step: dissolve 650 mg (1.7 mmol, 1.0 eq) of ethyl 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoate in A solution of 199 mg (8.3 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise to a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 620 mg of 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸73mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam128(52mg)。 1H NMR(400MHz,DMSO)δ9.84(s,1H),8.05(s,1H),7.72(dd,J＝7.9,1.3Hz,1H),7.39(s,1H),7.24–7.12(m,3H),6.61(dd,J＝11.0,4.0Hz,1H),6.12(d,J＝8.3Hz,1H),2.57–2.51(m,3H),2.45(s,3H),2.28(s,3H),1.09(t,J＝7.5Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg (0.24mmol) were sequentially added to the above system , 1.2eq), the resulting mixed system was reacted at 45 ° C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was prepared and separated by reverse-phase direct alkaline system, and the separated solution was lyophilized to obtain white solid Dam128 (52 mg). 1 H NMR (400MHz, DMSO) δ 9.84(s, 1H), 8.05(s, 1H), 7.72(dd, J=7.9, 1.3Hz, 1H), 7.39(s, 1H), 7.24-7.12(m ,3H),6.61(dd,J＝11.0,4.0Hz,1H),6.12(d,J＝8.3Hz,1H),2.57–2.51(m,3H),2.45(s,3H),2.28(s, 3H), 1.09(t, J=7.5Hz, 6H).
实施例10化合物Dam65的合成Example 10 Synthesis of compound Dam65
Figure PCTCN2022075561-appb-000020
Figure PCTCN2022075561-appb-000020
第一步、第二步、第三步、第四步反应按照Dam128的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二氯-4-(3,5-二甲基吡唑)苯基)氨基)苯甲酸71mg(0.2mmol,1.0eq)，HATU 114mg(0.3mmol,1.5eq)，室温下溶解于无水DMF4.0mL，搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq)，N,N-二乙氨基乙二胺28mg(0.24mmol,1.2eq)，所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接反相制备分离，分离液冻干得白色固体Dam65(33mg)。 1H NMR(500MHz,MEOD)δ8.19(s,1H),8.03(s,1H),7.83–7.73(m,2H),7.66(s,1H),7.63(d,J＝28.6Hz,2H),7.05(s,1H),6.67(s,1H),3.66(s,1H),3.60(s,1H),2.89–2.76(m,2H),2.61–2.50(m,4H),2.42(s,3H),2.40(s,3H),1.10–0.96(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-dichloro-4-(3,5-dimethylpyrazole)phenyl)amino)benzoic acid 71mg (0.2mmol, 1.0eq ), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenedi Amine 28mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly separated by reverse phase preparation, and the separated solution was lyophilized to obtain white solid Dam65 (33 mg). 1 H NMR (500MHz, MEOD) δ 8.19(s, 1H), 8.03(s, 1H), 7.83-7.73(m, 2H), 7.66(s, 1H), 7.63(d, J=28.6Hz, 2H ), 7.05(s, 1H), 6.67(s, 1H), 3.66(s, 1H), 3.60(s, 1H), 2.89–2.76(m, 2H), 2.61–2.50(m, 4H), 2.42( s,3H),2.40(s,3H),1.10–0.96(m,6H).
实施例11化合物Dam159的合成Example 11 Synthesis of compound Dam159
Figure PCTCN2022075561-appb-000021
Figure PCTCN2022075561-appb-000021
第一步和第二步反应按照Dam128的合成操作制备。The first and second reactions were prepared according to the synthetic procedure for Dam128.
第三步：将2-(4-溴-2,6-二乙基苯基)氨基)苯甲酸乙酯1.13g(3.0mmol,1.0eq),吡啶-4-硼酸443mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于二氧六环和水的混合溶剂160mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲 酸乙酯775mg。 The third step: 2-(4-bromo-2,6-diethylphenyl)amino)ethyl benzoate 1.13g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2eq) ), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 160mL (4:1/v:v) , heated to 100 °C for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain 2-((2,6-diethyl-4-pyridine) as a white solid Phenyl)amino)ethyl benzoate 775 mg.
第四步：将2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸乙酯775mg(2.1mmol,1.0eq)溶于四氢呋喃4mL和无水乙醇8mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂249mg(10.5mmol,5.0eq)的水2mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得白色固体2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸693mg。Step 4: Dissolve 775 mg (2.1 mmol, 1.0 eq) of ethyl 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol 249 mg (10.5 mmol, 5.0 eq) of a solution of lithium hydroxide in 2 mL of water was slowly added dropwise thereto under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 693 mg of 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam159(52mg)。 1H NMR(600MHz,DMSO)δ9.87(s,1H),8.63(d,J＝5.7Hz,2H),8.05(s,1H),7.75(d,J＝6.0Hz,2H),7.72(d,J＝7.9Hz,1H),7.62(s,2H),7.39(s,1H),7.17(t,J＝7.7Hz,1H),6.63(t,J＝7.5Hz,1H),6.12(d,J＝8.3Hz,1H),2.55(q,J＝7.5Hz,4H),1.12(t,J＝7.5Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated in an alkaline system by reverse phase, and the separated solution was lyophilized to obtain white solid Dam159 (52 mg). 1 H NMR(600MHz, DMSO)δ9.87(s,1H),8.63(d,J=5.7Hz,2H),8.05(s,1H),7.75(d,J=6.0Hz,2H),7.72( d, J＝7.9Hz, 1H), 7.62(s, 2H), 7.39(s, 1H), 7.17(t, J＝7.7Hz, 1H), 6.63(t, J＝7.5Hz, 1H), 6.12( d, J=8.3Hz, 1H), 2.55 (q, J=7.5Hz, 4H), 1.12 (t, J=7.5Hz, 6H).
实施例12化合物Dam281的合成Example 12 Synthesis of compound Dam281
Figure PCTCN2022075561-appb-000022
Figure PCTCN2022075561-appb-000022
第一步、第二步、第三步、第四步反应按照Dam159的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),甲胺盐酸盐16mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam281(58mg)。 1H NMR(400MHz,DMSO)δ9.65(s,1H),8.64(dd,J＝4.6,1.5Hz,2H),8.51(d,J＝4.5Hz,1H),7.76(dd,J＝4.6,1.6Hz,2H),7.65(dd,J＝7.9,1.3Hz,1H),7.63(s,2H),7.16(dd,J＝11.3,4.2Hz,1H),6.66(dd,J＝11.0,4.0Hz,1H),6.13(d,J＝8.3Hz,1H),2.82(d,J＝4.5Hz,3H),2.56(q,J＝7.5Hz,4H),1.13(t,J＝7.5Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and methylamine hydrochloride 16mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam281 (58 mg). 1 H NMR (400MHz, DMSO) δ 9.65 (s, 1H), 8.64 (dd, J=4.6, 1.5Hz, 2H), 8.51 (d, J=4.5Hz, 1H), 7.76 (dd, J=4.6 ,1.6Hz,2H),7.65(dd,J＝7.9,1.3Hz,1H),7.63(s,2H),7.16(dd,J＝11.3,4.2Hz,1H),6.66(dd,J＝11.0, 4.0Hz, 1H), 6.13 (d, J=8.3Hz, 1H), 2.82 (d, J=4.5Hz, 3H), 2.56 (q, J=7.5Hz, 4H), 1.13 (t, J=7.5Hz) ,6H).
实施例13化合物Dam156的合成Example 13 Synthesis of compound Dam156
Figure PCTCN2022075561-appb-000023
Figure PCTCN2022075561-appb-000023
第一步、第二步、第三步、第四步反应按照Dam159的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基乙二胺28mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam156(58mg)。 1H NMR(400MHz,DMSO)δ9.49(s,1H),8.63(d,J＝5.6Hz,2H),8.44(t,J＝5.7Hz,1H),7.76(dd,J＝4.6,1.5Hz,2H),7.67–7.59(m,3H),7.22–7.13(m,1H),6.72–6.62(m,1H),6.16–6.09(m,1H),3.35(d,J＝7.2Hz,4H),2.57(ddd,J＝11.9,9.7,5.8Hz,8H),1.12(t,J＝7.5Hz,6H),0.98(t,J＝7.1Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenediamine 28mg (0.24mmol, 1.2eq) were successively added to the above system ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam156 (58 mg). 1 H NMR (400MHz, DMSO) δ 9.49 (s, 1H), 8.63 (d, J=5.6Hz, 2H), 8.44 (t, J=5.7Hz, 1H), 7.76 (dd, J=4.6, 1.5 Hz, 2H), 7.67–7.59 (m, 3H), 7.22–7.13 (m, 1H), 6.72–6.62 (m, 1H), 6.16–6.09 (m, 1H), 3.35 (d, J=7.2Hz, 4H), 2.57(ddd, J＝11.9, 9.7, 5.8Hz, 8H), 1.12(t, J＝7.5Hz, 6H), 0.98(t, J＝7.1Hz, 6H).
实施例14化合物Dam254的合成Example 14 Synthesis of compound Dam254
Figure PCTCN2022075561-appb-000024
Figure PCTCN2022075561-appb-000024
第一步、第二步、第三步、第四步反应按照Dam159的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二甲氨基乙二胺21mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam254(58mg)。 1H NMR(400MHz,DMSO)δ9.47(s,1H),8.64(s,2H),8.44(s,1H),7.76(s,2H),7.62(s,3H),7.17(s,1H),6.67(s,1H),6.12(d,J＝7.8Hz,1H),2.55(d,J＝7.2Hz,5H),2.20(s,6H),1.12(s,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-dimethylaminoethylenediamine 21mg (0.24mmol, 1.2eq) were added to the above system successively ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam254 (58 mg). 1 H NMR (400MHz, DMSO) δ9.47(s,1H), 8.64(s,2H), 8.44(s,1H), 7.76(s,2H), 7.62(s,3H), 7.17(s,1H) ), 6.67(s, 1H), 6.12(d, J=7.8Hz, 1H), 2.55(d, J=7.2Hz, 5H), 2.20(s, 6H), 1.12(s, 6H).
实施例15化合物Dam158的合成Example 15 Synthesis of compound Dam158
Figure PCTCN2022075561-appb-000025
Figure PCTCN2022075561-appb-000025
第一步、第二步、第三步、第四步反应按照Dam159的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),2-氨基乙醇15mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam158(47mg)。 1H NMR(400MHz,DMSO)δ9.57(s,1H),8.63(dd,J＝4.6,1.5Hz,2H),8.50(t,J＝5.5Hz,1H),7.76(dd,J＝4.6,1.6Hz,2H),7.69(dd,J＝7.9,1.3Hz,1H),7.62(s,2H),7.21–7.15(m,1H),6.66(dd,J＝11.0,4.0Hz,1H),6.18–6.10(m,1H),4.80(t,J＝5.6Hz,1H),3.56(q,J＝6.1Hz,2H),3.36(d,J＝3.2Hz,3H),2.55(q,J＝7.5Hz,4H),1.12(t,J＝7.5Hz,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and 2-aminoethanol 15mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system was The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation was separated, and the separated liquid was lyophilized to obtain Dam158 (47 mg) as a white solid. 1 H NMR (400MHz, DMSO) δ 9.57 (s, 1H), 8.63 (dd, J=4.6, 1.5Hz, 2H), 8.50 (t, J=5.5Hz, 1H), 7.76 (dd, J=4.6 ,1.6Hz,2H),7.69(dd,J＝7.9,1.3Hz,1H),7.62(s,2H),7.21-7.15(m,1H),6.66(dd,J＝11.0,4.0Hz,1H) ,6.18–6.10(m,1H),4.80(t,J＝5.6Hz,1H),3.56(q,J＝6.1Hz,2H),3.36(d,J＝3.2Hz,3H),2.55(q, J=7.5Hz, 4H), 1.12(t, J=7.5Hz, 6H).
实施例16化合物Dam160的合成Example 16 Synthesis of compound Dam160
Figure PCTCN2022075561-appb-000026
Figure PCTCN2022075561-appb-000026
第一步、第二步、第三步、第四步反应按照Dam128的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸73mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam160(52mg)。 1H NMR(500MHz,MeOD)δ8.00(s,1H),7.96(s,1H),7.68–7.65(m,2H),7.63(s,1H),7.53(s,1H),6.95(s,1H),6.82(s,1H),2.73–2.58(m,4H),2.43(s,6H),1.34–1.14(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated from the alkaline system in reverse phase, and the separated solution was lyophilized to obtain white solid Dam160 (52 mg). 1 H NMR(500MHz,MeOD)δ8.00(s,1H),7.96(s,1H),7.68-7.65(m,2H),7.63(s,1H),7.53(s,1H),6.95(s ,1H),6.82(s,1H),2.73–2.58(m,4H),2.43(s,6H),1.34–1.14(m,6H).
实施例17化合物Dam161的合成Example 17 Synthesis of compound Dam161
Figure PCTCN2022075561-appb-000027
Figure PCTCN2022075561-appb-000027
第一步、第二步、第三步、第四步反应按照Dam159的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam159.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-吡啶)苯基)氨基)苯甲酸69mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam161(58mg)。 1H NMR(500MHz,MeOD)δ8.75–8.62(m,2H),8.08(s,1H),8.02(s,1H),7.91–7.81(m,2H),7.74–7.67(m,2H),7.66(s,1H),7.56(s,1H),6.97(s,1H),6.85(s,1H),2.67–2.53(m,4H),1.34–1.14(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-pyridine)phenyl)amino)benzoic acid 69mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system was heated at 45°C React for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated in an alkaline system by reverse phase, and the separated solution was lyophilized to obtain white solid Dam161 (58 mg). 1 H NMR(500MHz,MeOD)δ8.75-8.62(m,2H),8.08(s,1H),8.02(s,1H),7.91-7.81(m,2H),7.74-7.67(m,2H) ,7.66(s,1H),7.56(s,1H),6.97(s,1H),6.85(s,1H),2.67–2.53(m,4H),1.34–1.14(m,6H).
实施例18化合物Dam162的合成Example 18 Synthesis of compound Dam162
Figure PCTCN2022075561-appb-000028
Figure PCTCN2022075561-appb-000028
第一步、第二步、第三步、第四步反应按照Dam128的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dam128.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二乙基-4-(3,5-二甲基异恶唑)苯基)氨基)苯甲酸73mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min，向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消 耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam162(52mg)。 1H NMR(500MHz,MeOD)δ8.02(s,1H),7.99(s,1H),7.77(s,1H),7.67–7.64(m,2H),7.61(s,1H),7.53(s,1H),6.94(s,1H),3.73(s,1H),3.57(s,1H),2.87(s,2H),2.77–2.60(m,8H),2.43(s,6H),1.31–1.12(m,6H),1.14–0.94(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diethyl-4-(3,5-dimethylisoxazole)phenyl)amino)benzoic acid 73mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was prepared and separated by reverse-phase direct alkaline system, and the separated solution was lyophilized to obtain white solid Dam162 (52 mg). 1 H NMR(500MHz,MeOD)δ8.02(s,1H),7.99(s,1H),7.77(s,1H),7.67-7.64(m,2H),7.61(s,1H),7.53(s ,1H),6.94(s,1H),3.73(s,1H),3.57(s,1H),2.87(s,2H),2.77–2.60(m,8H),2.43(s,6H),1.31– 1.12(m,6H),1.14–0.94(m,6H).
实施例19化合物Dac406的合成Example 19 Synthesis of compound Dac406
Figure PCTCN2022075561-appb-000029
Figure PCTCN2022075561-appb-000029
第一步：将邻碘苯甲酸30.7g(124mmol,1.2eq),2-氯-6-甲基-4-溴苯胺22.7g(103mmol,1.0eq),三乙胺15.7g(155mmol,1.5eq)和无水乙酸铜9.3g(51.5mmol,0.5eq)溶于DMF 372mL中，氩气保护下加热至120℃反应24h，反应结束后。降温到室温，加入等体积的水，母液用DCM萃取300mL×3，依次用水洗、饱和食盐水洗，有机相旋干，硅胶柱色谱分离(石油醚:乙酸乙酯＝20:1到1:1)，得到黄色固体32g。The first step: 30.7g (124mmol, 1.2eq) of o-iodobenzoic acid, 22.7g (103mmol, 1.0eq) of 2-chloro-6-methyl-4-bromoaniline, 15.7g (155mmol, 1.5eq) of triethylamine ) and 9.3 g (51.5 mmol, 0.5 eq) of anhydrous copper acetate were dissolved in 372 mL of DMF, heated to 120° C. under argon protection and reacted for 24 h, after the reaction was completed. Cool to room temperature, add an equal volume of water, extract the mother liquor with DCM for 300 mL×3, wash with water and saturated brine successively, spin dry the organic phase, and separate by silica gel column chromatography (petroleum ether:ethyl acetate=20:1 to 1:1 ) to obtain 32 g of a yellow solid.
第二步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸32g溶解于无水乙醇300mL中，冰水浴冷却下，向其中加入浓硫酸50mL，加热至100℃回流反应12小时。反应结束后，反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水100mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相50mL×3，合并有机相并用饱和食盐水洗涤20mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离，得白色固体2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯18g。The second step: dissolve 32 g of 2-(4-bromo-2-chloro-6-methylphenyl) amino) benzoic acid in 300 mL of absolute ethanol, add 50 mL of concentrated sulfuric acid to it, and heat to The reaction was refluxed at 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL×3, and the organic phases were combined and washed with saturated brine for 20 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain 18 g of ethyl 2-(4-bromo-2-chloro-6-methylphenyl)amino)benzoate as a white solid.
第三步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯2.7g(7.3mmol,1.0eq),3,5-二甲基异恶唑-4-硼酸1.2g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸乙酯1.86g。 The third step: 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), 3,5-dimethylisoxazole- 4-boronic acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixture of dioxane and water In 37 mL of solvent (4:1/v:v), the mixture was heated to 100°C and reacted for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( 3,5-Dimethylisoxazole))phenyl)amino)ethyl benzoate 1.86g.
第四步：将2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸乙酯911mg(2.37mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸819mg。The fourth step: 911mg (2.37mmol, 1.0eq) of ethyl 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoate It was dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 16 mL of absolute ethanol, and a solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to it under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 819 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水 DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam406(47mg)。 1H NMR(400MHz,DMSO)δ11.31(s,1H),9.31(s,1H),9.16(s,1H),7.52(d,J＝7.6Hz,1H),7.47(s,1H),7.34(s,1H),7.24(t,J＝7.6Hz,1H),6.74(t,J＝7.3Hz,1H),6.23(d,J＝8.2Hz,1H),2.45(s,3H),2.27(s,3H),2.21(s,3H). Step 5: In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam406 (47 mg). 1 H NMR (400MHz, DMSO) δ 11.31(s, 1H), 9.31(s, 1H), 9.16(s, 1H), 7.52(d, J=7.6Hz, 1H), 7.47(s, 1H), 7.34(s, 1H), 7.24(t, J=7.6Hz, 1H), 6.74(t, J=7.3Hz, 1H), 6.23(d, J=8.2Hz, 1H), 2.45(s, 3H), 2.27(s, 3H), 2.21(s, 3H).
实施例20化合物Dam418的合成Example 20 Synthesis of compound Dam418
Figure PCTCN2022075561-appb-000030
Figure PCTCN2022075561-appb-000030
第一步、第二步反应按照Dam106的合成操作制备。The first and second steps are prepared according to the synthetic operation of Dam106.
第三步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯1.1g(3.0mmol,1.0eq),吡啶-4-硼酸443mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于二氧六环和水的混合溶剂15mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，浓缩至干，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸乙酯916mg。 The third step: 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 1.1g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2g) eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 15mL (4:1/v:v) , heated to 100 °C for 24 hours. Cooled to room temperature, concentrated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain white solid 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino ) ethyl benzoate 916 mg.
第四步：将2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸乙酯734mg(2.0mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂240mg(10mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸725mg。Step 4: Dissolve 734 mg (2.0 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoate in a mixture of 8 mL of tetrahydrofuran and 16 mL of anhydrous ethanol In the solvent, under ice-water bath cooling, a solution of 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 725 mg of 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸68mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam418(47mg)。 1H NMR(400MHz,DMSO)δ11.36(s,1H),9.44(s,1H),8.87(d,J＝5.9Hz,2H),8.22(d,J＝6.2Hz,2H),8.09(s,1H),7.96(s,1H),7.53(d,J＝7.7Hz,1H),7.26(t,J＝7.7Hz,1H),6.80(t,J＝7.4Hz,1H),6.28(d,J＝8.2Hz,1H),2.26(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam418 (47 mg). 1 H NMR (400MHz, DMSO) δ 11.36(s, 1H), 9.44(s, 1H), 8.87(d, J=5.9Hz, 2H), 8.22(d, J=6.2Hz, 2H), 8.09( s, 1H), 7.96(s, 1H), 7.53(d, J=7.7Hz, 1H), 7.26(t, J=7.7Hz, 1H), 6.80(t, J=7.4Hz, 1H), 6.28( d, J=8.2Hz, 1H), 2.26(s, 3H).
实施例21化合物Dam226的合成Example 21 Synthesis of compound Dam226
Figure PCTCN2022075561-appb-000031
Figure PCTCN2022075561-appb-000031
第一步、第二步、第三步、第四步反应按照Dac406的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸68mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam226(47mg)。 1H NMR(400MHz,DMSO)δ9.99(s,1H),8.65(dd,J＝4.6,1.5Hz,2H),8.11(s,1H),7.90(d,J＝2.0Hz,1H),7.83–7.77(m,3H),7.74(d,J＝7.9Hz,1H),7.49(s,1H),7.24(t,J＝7.7Hz,1H),6.74(t,J＝7.5Hz,1H),6.21(d,J＝8.0Hz,1H),2.25(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were added to the above system in turn, and the resulting mixed system was The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation was separated, and the separated liquid was lyophilized to obtain white solid Dam226 (47 mg). 1 H NMR(400MHz, DMSO)δ9.99(s,1H),8.65(dd,J=4.6,1.5Hz,2H),8.11(s,1H),7.90(d,J=2.0Hz,1H), 7.83–7.77(m, 3H), 7.74(d, J=7.9Hz, 1H), 7.49(s, 1H), 7.24(t, J=7.7Hz, 1H), 6.74(t, J=7.5Hz, 1H) ), 6.21(d, J=8.0Hz, 1H), 2.25(s, 3H).
实施例22化合物Dam242的合成Example 22 Synthesis of compound Dam242
Figure PCTCN2022075561-appb-000032
Figure PCTCN2022075561-appb-000032
第一步和第二步反应按照Dac406的合成操作制备。The first and second reactions were prepared according to the synthetic procedure of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基乙二胺18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam242(47mg)。 1H NMR(400MHz,DMSO)δ9.59(s,1H),8.48(t,J＝5.5Hz,1H),7.63(d,J＝7.8Hz,1H),7.45(d,J＝1.5Hz,1H),7.33(s,1H),7.23(t,J＝7.7Hz,1H),6.76(t,J＝7.5Hz,1H),6.22(d,J＝8.3Hz,1H),3.36–3.30(m,3H),2.59(t,J＝7.0Hz,2H),2.54(d,J＝7.1Hz,3H),2.44(s,3H),2.27(s,3H),2.21(s,3H),0.98(t,J＝7.1Hz,6H). Step 5: In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-di Ethylaminoethylenediamine 18mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam242 (47 mg). 1 H NMR(400MHz, DMSO)δ9.59(s,1H),8.48(t,J=5.5Hz,1H),7.63(d,J=7.8Hz,1H),7.45(d,J=1.5Hz, 1H), 7.33(s, 1H), 7.23(t, J＝7.7Hz, 1H), 6.76(t, J＝7.5Hz, 1H), 6.22(d, J＝8.3Hz, 1H), 3.36–3.30( m, 3H), 2.59(t, J=7.0Hz, 2H), 2.54(d, J=7.1Hz, 3H), 2.44(s, 3H), 2.27(s, 3H), 2.21(s, 3H), 0.98(t,J=7.1Hz,6H).
实施例23化合物Dam210的合成Example 23 Synthesis of compound Dam210
Figure PCTCN2022075561-appb-000033
Figure PCTCN2022075561-appb-000033
第一步、第二步、第三步、第四步反应按照Dac406的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam210(47mg)。 1H NMR(400MHz,DMSO)δ9.65(s,1H),8.55(t,J＝5.5Hz,1H),7.76–7.60(m,1H),7.46(d,J＝1.8Hz,1H),7.33(d,J＝1.5Hz,1H),7.24(t,J＝7.8Hz,1H),6.76(t,J＝7.5Hz,1H),6.23(d,J＝8.2Hz,1H),4.53(t,J＝5.1Hz,1H),3.50(dd,J＝11.3,6.1Hz,2H),3.33(d,J＝5.8Hz,2H),2.45(s,3H),2.27(s,3H),2.21(s,3H),1.72(p,J＝6.6Hz,2H). Step 5: In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol were added to the above system successively 18mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam210 (47 mg). 1 H NMR(400MHz, DMSO)δ9.65(s,1H),8.55(t,J=5.5Hz,1H),7.76-7.60(m,1H),7.46(d,J=1.8Hz,1H), 7.33(d,J＝1.5Hz,1H),7.24(t,J＝7.8Hz,1H),6.76(t,J＝7.5Hz,1H),6.23(d,J＝8.2Hz,1H),4.53( t, J＝5.1Hz, 1H), 3.50(dd, J＝11.3, 6.1Hz, 2H), 3.33(d, J＝5.8Hz, 2H), 2.45(s, 3H), 2.27(s, 3H), 2.21(s, 3H), 1.72(p, J=6.6Hz, 2H).
实施例24化合物Dam230的合成Example 24 Synthesis of compound Dam230
Figure PCTCN2022075561-appb-000034
Figure PCTCN2022075561-appb-000034
第一步、第二步、第三步、第四步反应按照Dac406的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸68mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam230(41mg)。 1H NMR(500MHz,MeOD)δ8.73–8.65(m,2H),8.17(s,1H),7.89(s,1H),7.86–7.84(m,1H),7.83(s,1H),7.80(s,1H),7.72(dd,J＝7.5,1.4Hz,1H),7.62(s,1H),7.61(s,1H),7.44(s,1H),7.05(s,1H),3.52(s,2H), 3.43(d,J＝20.9Hz,2H),2.12(s,3H),1.52(s,2H),1.32(s,1H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol 18mg (0.24mmol, 1.2eq) were added to the above system successively, the resulting mixture was mixed The system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam230 (41 mg). 1 H NMR(500MHz,MeOD)δ8.73-8.65(m,2H),8.17(s,1H),7.89(s,1H),7.86-7.84(m,1H),7.83(s,1H),7.80 (s,1H),7.72(dd,J=7.5,1.4Hz,1H),7.62(s,1H),7.61(s,1H),7.44(s,1H),7.05(s,1H),3.52( s, 2H), 3.43(d, J=20.9Hz, 2H), 2.12(s, 3H), 1.52(s, 2H), 1.32(s, 1H).
实施例25化合物Dam231的合成Example 25 Synthesis of compound Dam231
Figure PCTCN2022075561-appb-000035
Figure PCTCN2022075561-appb-000035
第一步、第二步、第三步、第四步反应按照Dac406的合成操作制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic operation of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸68mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基乙二胺24mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam231(41mg)。 1H NMR(500MHz,MeOD)δ8.75–8.63(m,2H),8.47(s,1H),8.04(s,1H),7.90–7.84(m,3H),7.75(s,1H),7.68(s,1H),7.56(s,1H),7.29(s,1H),6.97(s,1H),3.64(s,2H),2.94–2.83(m,2H),2.69(s,2H),2.46–2.32(m,2H),2.23(s,3H),1.11–0.96(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 68mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenediamine 24mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam231 (41 mg). 1 H NMR(500MHz,MeOD)δ8.75-8.63(m,2H),8.47(s,1H),8.04(s,1H),7.90-7.84(m,3H),7.75(s,1H),7.68 (s, 1H), 7.56(s, 1H), 7.29(s, 1H), 6.97(s, 1H), 3.64(s, 2H), 2.94–2.83(m, 2H), 2.69(s, 2H), 2.46–2.32 (m, 2H), 2.23 (s, 3H), 1.11–0.96 (m, 6H).
实施例26化合物Dac232的合成Example 26 Synthesis of compound Dac232
Figure PCTCN2022075561-appb-000036
Figure PCTCN2022075561-appb-000036
第一步和第二步反应按照Dac406的合成操作制备。The first and second reactions were prepared according to the synthetic procedure of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam242(21mg)。 1H NMR(500MHz,MeOD)δ8.32(s,1H),8.03(dd,J＝7.5,1.4Hz,1H),7.81(d,J＝1.4Hz,1H),7.74(dd,J＝ 7.5,1.4Hz,1H),7.63(d,J＝1.4Hz,1H),7.54(td,J＝7.5,1.5Hz,1H),7.08(s,1H),6.95(td,J＝7.5,1.5Hz,1H),2.22(s,3H),2.07(s,3H),1.99(s,3H). Step 5: In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 14mg (0.24mg) were successively added to the above system mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam242 (21 mg). 1 H NMR (500MHz, MeOD) δ 8.32 (s, 1H), 8.03 (dd, J=7.5, 1.4Hz, 1H), 7.81 (d, J=1.4Hz, 1H), 7.74 (dd, J=7.5 ,1.4Hz,1H),7.63(d,J=1.4Hz,1H),7.54(td,J=7.5,1.5Hz,1H),7.08(s,1H),6.95(td,J=7.5,1.5Hz) ,1H),2.22(s,3H),2.07(s,3H),1.99(s,3H).
实施例27化合物Dam233的合成Example 27 Synthesis of compound Dam233
Figure PCTCN2022075561-appb-000037
Figure PCTCN2022075561-appb-000037
第一步和第二步反应按照Dac406的合成操作制备。The first and second reactions were prepared according to the synthetic procedure of Dac406.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam233(25mg)。 1H NMR(500MHz,MeOD)δ8.00(dd,J＝7.5,1.6Hz,1H),7.94(s,1H),7.81(d,J＝1.4Hz,1H),7.70(dd,J＝7.5,1.4Hz,1H),7.65(d,J＝1.6Hz,1H),7.53(td,J＝7.4,1.5Hz,1H),7.03(s,1H),6.92(td,J＝7.5,1.4Hz,1H),3.64(t,J＝4.6Hz,2H),3.49(t,J＝4.9Hz,1H),3.42(t,J＝4.9Hz,1H),2.28(s,3H),2.08(s,3H),1.99(s,3H),1.73(p,J＝4.8Hz,2H),1.37(s,1H). Step 5: In a 10mL round-bottomed flask, add 72mg (0.2 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethylisoxazole))phenyl)amino)benzoic acid mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol were added to the above system successively 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam233 (25 mg). 1 H NMR (500MHz, MeOD) δ 8.00 (dd, J=7.5, 1.6Hz, 1H), 7.94 (s, 1H), 7.81 (d, J=1.4Hz, 1H), 7.70 (dd, J=7.5 ,1.4Hz,1H),7.65(d,J=1.6Hz,1H),7.53(td,J=7.4,1.5Hz,1H),7.03(s,1H),6.92(td,J=7.5,1.4Hz) ,1H),3.64(t,J＝4.6Hz,2H),3.49(t,J＝4.9Hz,1H),3.42(t,J＝4.9Hz,1H),2.28(s,3H),2.08(s ,3H),1.99(s,3H),1.73(p,J=4.8Hz,2H),1.37(s,1H).
实施例28化合物Dam264的合成Example 28 Synthesis of compound Dam264
Figure PCTCN2022075561-appb-000038
Figure PCTCN2022075561-appb-000038
第一步：将邻碘苯甲酸30g(120mmol,1.2eq)、2,6-二异丙基-4-溴苯胺25.6g(100mmol,1.0eq)、三乙胺(150mmol,1.5eq)和无水乙酸铜9g(5.0mmol,0.5eq)溶于DMF500mL中，氩气保护下加热至120℃反应24h，反应结束后。降温到室温，加入等体积的水，母液用DCM萃取300mL×3，用水洗涤DMF，有机相旋干，过柱比例由PE:EA＝20:1过渡到PE:EA＝1:1得到黄色固体14.6g。The first step: o-iodobenzoic acid 30g (120mmol, 1.2eq), 2,6-diisopropyl-4-bromoaniline 25.6g (100mmol, 1.0eq), triethylamine (150mmol, 1.5eq) and no 9 g (5.0 mmol, 0.5 eq) of water copper acetate was dissolved in 500 mL of DMF, heated to 120° C. under argon protection and reacted for 24 h, after the reaction was completed. Cool to room temperature, add an equal volume of water, extract the mother liquor with DCM for 300 mL × 3, wash DMF with water, spin dry the organic phase, and transition from PE:EA=20:1 to PE:EA=1:1 to obtain a yellow solid 14.6g.
第二步：将2-(4-溴-2,6-二异丙基苯基)氨基)苯甲酸3.8g溶解于无水乙醇200mL中，冰水浴冷却下，向其中加入浓硫酸20mL，加热至100℃回流反应12小时。反应结束后，反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水100mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离，得白色固体2-(4-溴-2,6-二异丙基苯基)氨基)苯甲酸乙酯3.0g。Step 2: Dissolve 3.8 g of 2-(4-bromo-2,6-diisopropylphenyl)amino)benzoic acid in 200 mL of absolute ethanol, add 20 mL of concentrated sulfuric acid to it under cooling in an ice-water bath, and heat The reaction was refluxed to 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL×3, and the organic phases were combined and washed with saturated brine for 10 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain 3.0 g of ethyl 2-(4-bromo-2,6-diisopropylphenyl)amino)benzoate as a white solid.
第三步：将2-(4-溴-2,6-二异丙基苯基)氨基)苯甲酸乙酯1.2g(3.0mmol,1.0eq),吡啶-4-硼酸443mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于二氧六环和水的混合溶剂15mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-(2,6-二异丙基-4-吡啶苯基)氨基)苯甲酸乙酯913mg。 The third step: 2-(4-bromo-2,6-diisopropylphenyl)amino)ethyl benzoate 1.2g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2g) eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 15mL (4:1/v:v) , heated to 100 °C for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain 2-(2,6-diisopropyl-4-pyridinebenzene as a white solid) base) amino) ethyl benzoate 913 mg.
第四步：将2-((2,6-二异丙基-4-吡啶苯基)氨基)苯甲酸乙酯748mg(2.0mmol,1.0eq)溶于四氢呋喃4mL和无水乙醇8mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂240mg(10mmol,5.0eq)的水2mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2,6-二异丙基-4-吡啶苯基)氨基)苯甲酸693mg。Step 4: Dissolve 748 mg (2.0 mmol, 1.0 eq) of ethyl 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise thereto under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 693 mg of 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二异丙基-4-吡啶苯基)氨基)苯甲酸75mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL，搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam264(58mg)。 1H NMR(400MHz,DMSO)δ9.84(s,1H),8.65(d,J＝6.1Hz,2H),8.06(s,1H),7.82–7.76(m,2H),7.72(d,J＝7.9Hz,1H),7.63(s,2H),7.38(s,1H),7.17(t,J＝7.8Hz,1H),6.62(t,J＝7.4Hz,1H),6.12(d,J＝8.4Hz,1H),3.19–3.00(m,2H),1.16(t,J＝6.6Hz,12H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoic acid 75mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam264 (58 mg). 1 H NMR (400MHz, DMSO) δ 9.84(s, 1H), 8.65(d, J=6.1Hz, 2H), 8.06(s, 1H), 7.82-7.76(m, 2H), 7.72(d, J =7.9Hz,1H),7.63(s,2H),7.38(s,1H),7.17(t,J=7.8Hz,1H),6.62(t,J=7.4Hz,1H),6.12(d,J = 8.4Hz, 1H), 3.19–3.00 (m, 2H), 1.16 (t, J = 6.6Hz, 12H).
实施例29化合物Dam185的合成Example 29 Synthesis of compound Dam185
Figure PCTCN2022075561-appb-000039
Figure PCTCN2022075561-appb-000039
第一步：将邻碘苯甲酸29.8g(120mmol,1.2eq),2-氟-6-甲基-4-溴苯胺20.4g(100mmol,1.0eq),三乙胺15.2g(150mmol,1.5eq)和无水乙酸铜9.1g(50mmol,0.5eq)溶于 DMF 360mL中，氩气保护下加热至120℃反应24h，反应结束后。降温到室温，加入等体积的水，母液用DCM萃取300mL×3，用水洗涤DMF，有机相旋干，过柱比例由PE:EA＝20:1过渡到PE:EA＝1:1得到黄色固体33g。The first step: 29.8g (120mmol, 1.2eq) of o-iodobenzoic acid, 20.4g (100mmol, 1.0eq) of 2-fluoro-6-methyl-4-bromoaniline, 15.2g (150mmol, 1.5eq) of triethylamine ) and 9.1 g (50 mmol, 0.5 eq) of anhydrous copper acetate were dissolved in 360 mL of DMF, heated to 120 °C under argon protection and reacted for 24 h, after the reaction was completed. Cool to room temperature, add an equal volume of water, extract the mother liquor with DCM for 300 mL × 3, wash DMF with water, spin dry the organic phase, and transition from PE:EA=20:1 to PE:EA=1:1 to obtain a yellow solid 33g.
第二步：将2-(4-溴-2-氟-6-甲基苯基)氨基)苯甲酸33g溶解于无水乙醇300mL中，冰水浴冷却下，向其中加入浓硫酸50mL，加热至100℃回流反应12小时。反应结束后，反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水100mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相50mL×3，合并有机相并用饱和食盐水洗涤20mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离，得白色固体2-(4-溴-2-氟-6-甲基苯基)氨基)苯甲酸乙酯13.6g。The second step: 33 g of 2-(4-bromo-2-fluoro-6-methylphenyl) amino) benzoic acid was dissolved in 300 mL of absolute ethanol, and 50 mL of concentrated sulfuric acid was added to it under cooling in an ice-water bath, and heated to The reaction was refluxed at 100°C for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 100 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 50 mL×3, and the organic phases were combined and washed with saturated brine for 20 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography to obtain 13.6 g of ethyl 2-(4-bromo-2-fluoro-6-methylphenyl)amino)benzoate as a white solid.
第三步：将2-(4-溴-2-氟-6-甲基苯基)氨基)苯甲酸乙酯1.1g(3.0mmol,1.0eq),吡啶-4-硼酸443mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于二氧六环和水的混合溶剂15mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚：乙酸乙酯20:1)，得白色固体2-((2-氟-6-甲基-4-吡啶)苯基)氨基)苯甲酸乙酯801mg。 The third step: 2-(4-bromo-2-fluoro-6-methylphenyl)amino)ethyl benzoate 1.1g (3.0mmol, 1.0eq), pyridine-4-boronic acid 443mg (3.6mmol, 1.2 eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in a mixed solvent of dioxane and water 15mL (4:1/v:v) , heated to 100 °C for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate 20:1) to obtain 2-((2-fluoro-6-methyl-4-pyridine) as a white solid Phenyl)amino)ethyl benzoate 801 mg.
第四步：将2-((2-氟-6-甲基-4-吡啶)苯基)氨基)苯甲酸乙酯741mg(2.12mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂255mg(10.6mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-吡啶)苯基)氨基)苯甲酸723mg。Step 4: Dissolve 741 mg (2.12 mmol, 1.0 eq) of ethyl 2-((2-fluoro-6-methyl-4-pyridine)phenyl)amino)benzoate in a mixture of 8 mL of tetrahydrofuran and 16 mL of anhydrous ethanol In the solvent, under cooling in an ice-water bath, a solution of 255 mg (10.6 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 723 mg of 2-((2-chloro-6-methyl-4-pyridine)phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氟-6-甲基-4-吡啶)苯基)氨基)苯甲酸64mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF(4.0mL),搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam185(26mg)。 1H NMR(400MHz,DMSO)δ9.61(s,1H),8.64(d,J＝5.6Hz,2H),8.60(t,J＝5.4Hz,1H),7.78(d,J＝6.1Hz,2H),7.71–7.63(m,3H),7.27(t,J＝7.7Hz,1H),6.79(t,J＝7.6Hz,1H),6.42(dd,J＝8.3,3.8Hz,1H),4.54(t,J＝5.1Hz,1H),3.50(dd,J＝11.6,6.1Hz,2H),3.33(d,J＝6.8Hz,2H),2.30(s,3H),1.81–1.61(m,2H). The fifth step: in a 10mL round bottom flask, add 2-((2-fluoro-6-methyl-4-pyridine)phenyl)amino)benzoic acid 64mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol) , 1.5eq), dissolved in anhydrous DMF (4.0mL) at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol 18mg (0.24mmol, 1.2eq) were sequentially added to the above system, The resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam185 (26 mg). 1 H NMR(400MHz, DMSO)δ9.61(s,1H),8.64(d,J=5.6Hz,2H),8.60(t,J=5.4Hz,1H),7.78(d,J=6.1Hz, 2H), 7.71–7.63 (m, 3H), 7.27 (t, J=7.7Hz, 1H), 6.79 (t, J=7.6Hz, 1H), 6.42 (dd, J=8.3, 3.8Hz, 1H), 4.54(t, J＝5.1Hz, 1H), 3.50(dd, J＝11.6, 6.1Hz, 2H), 3.33(d, J＝6.8Hz, 2H), 2.30(s, 3H), 1.81–1.61(m , 2H).
实施例30化合物Dac258和Dac58的合成Example 30 Synthesis of Compounds Dac258 and Dac58
Figure PCTCN2022075561-appb-000040
Figure PCTCN2022075561-appb-000040
第一步：将2-氯-5-硝基苯甲酸20.2g(100mmol,1.0eq)、2,6-二氯-4-溴苯胺26.5g(110mmol,1.1eq)、叔丁醇钠28.8g(300mmol,3.0eq)溶于DMF 500mL中，氩气保护下加热至80℃反应24小时，反应结束后。降温到室温，2M稀盐酸调节pH，有黄色固体析出，抽滤，水洗，得到目标产物黄色固体28.0g。The first step: 20.2g (100mmol, 1.0eq) of 2-chloro-5-nitrobenzoic acid, 26.5g (110mmol, 1.1eq) of 2,6-dichloro-4-bromoaniline, 28.8g of sodium tert-butoxide (300mmol, 3.0eq) was dissolved in 500mL of DMF, heated to 80°C under argon protection and reacted for 24 hours, after the reaction was completed. Cooled to room temperature, adjusted pH with 2M dilute hydrochloric acid, a yellow solid was precipitated, suction filtered, and washed with water to obtain 28.0 g of the target product, a yellow solid.
第二步：将2-(4-溴-2,6-二氯苯基)氨基)-5-硝基苯甲酸28.0g溶解于无水乙醇500mL中，冰水浴冷却下，向其中加入浓硫酸50mL，加热至100℃回流反应12小时。反应结束后，反应体系冷却至室温，旋蒸浓缩除去乙醇，向体系中加水50mL，饱和碳酸钠中和不产生气泡为止。乙酸乙酯萃取有机相20mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝99:1)，得到目标产物白色固体21.0g。Step 2: Dissolve 28.0 g of 2-(4-bromo-2,6-dichlorophenyl)amino)-5-nitrobenzoic acid in 500 mL of absolute ethanol, add concentrated sulfuric acid to it under cooling in an ice-water bath 50 mL, heated to 100 °C and refluxed for 12 hours. After the reaction, the reaction system was cooled to room temperature, concentrated by rotary evaporation to remove ethanol, 50 mL of water was added to the system, and neutralized with saturated sodium carbonate until no bubbles were generated. The organic phase was extracted with ethyl acetate 20 mL×3, and the organic phases were combined and washed with saturated brine for 10 mL×3. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel chromatography (petroleum ether:ethyl acetate=99:1) to obtain 21.0 g of the target product as a white solid.
第三步：将2-(4-溴-2,6-二氯苯基)氨基)-5-硝基苯甲酸乙酯1.3g(3.0mmol,1.0eq),3,5-二甲基异恶唑-4-硼酸508mg(3.6mmol,1.2eq),催化剂Pd(dppf)Cl 2 220mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于1,4-二氧六环和水的混合溶剂30mL中，加热至100℃反应24小时。冷却至室温，浓缩至干，硅胶层析柱分离(石油醚:乙酸乙酯＝10:1)，得到目标产物白色固体2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)-5-硝基苯甲酸乙酯1.3g。 The third step: 2-(4-bromo-2,6-dichlorophenyl)amino)-5-nitrobenzoic acid ethyl ester 1.3g (3.0mmol, 1.0eq), 3,5-dimethyliso Oxazole-4-boronic acid 508mg (3.6mmol, 1.2eq), catalyst Pd(dppf)Cl 2 220mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) dissolved in 1,4-dioxane In 30 mL of a mixed solvent of ring and water, the mixture was heated to 100°C and reacted for 24 hours. Cooled to room temperature, concentrated to dryness, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=10:1) to obtain the target product as a white solid 2-((2,6-dichloro-4-(3,5-dichloromethane). Methylisoxazole)phenyl)amino)-5-nitrobenzoic acid ethyl ester 1.3g.
第四步：将2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)-5-硝基苯甲酸乙酯1.3g(2.9mmol,1.0eq)，锌粉943mg(14.5mmol,5.0eq)，甲酸铵1.8g(29.0mmol,10.0eq)溶于DMF 58mL中，加热至45℃，反应过夜，向体系中加水，EA萃取，硅胶柱色谱分离，得黄色目标产物2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)-5-氨基苯甲酸乙酯1.22g。The fourth step: 2-((2,6-dichloro-4-(3,5-dimethylisoxazole) phenyl) amino)-5-nitrobenzoic acid ethyl ester 1.3g (2.9mmol, 1.0eq), zinc powder 943mg (14.5mmol, 5.0eq), ammonium formate 1.8g (29.0mmol, 10.0eq) was dissolved in DMF 58mL, heated to 45°C, reacted overnight, water was added to the system, EA extraction, silica gel column After chromatographic separation, 1.22 g of ethyl 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)-5-aminobenzoate was obtained as a yellow target product.
第五步：将富马酸单乙酯79mg(0.55mmol,1.1eq),HATU 228mg(0.6mmol,1.2eq)溶解于DMF(4mL)中，搅拌5min，向上述体系中依次加入DIEA 194mg(1.5mmol,3.0eq),2-((2,6-二氯-4-(3,5-二甲基异恶唑)苯基)氨基)-5-氨基苯甲酸乙酯210mg(0.5mmol, 1.0eq),所得混合体系在45℃反应。TLC检测，原料消耗完毕。向体系中加入冰水混合物，即有黄色固体析出，抽滤，水洗，得目标产物黄色固体Dac258(180mg)。Step 5: Dissolve 79mg (0.55mmol, 1.1eq) of monoethyl fumarate, 228mg (0.6mmol, 1.2eq) of HATU in DMF (4mL), stir for 5min, and add 194mg (1.5mg) of DIEA to the above system in turn. mmol, 3.0eq), 2-((2,6-dichloro-4-(3,5-dimethylisoxazole)phenyl)amino)-5-aminobenzoic acid ethyl ester 210mg (0.5mmol, 1.0 eq), the resulting mixed system was reacted at 45 °C. TLC detection, the raw material consumption was completed. An ice-water mixture was added to the system, and a yellow solid was precipitated, which was filtered off with suction and washed with water to obtain the target product, Dac258 (180 mg) as a yellow solid.
第四步：将化合物Dac258 55mg(0.1mmol,1.0eq)溶于四氢呋喃(2mL)和无水乙醇(4mL)的混合溶剂中，向其中缓缓滴加氢氧化锂24mg(1.0mmol,10.0eq)的水(1mL)溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加水，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水冲洗，得目标产物Dac58(46mg)。The fourth step: Compound Dac258 55mg (0.1mmol, 1.0eq) was dissolved in a mixed solvent of tetrahydrofuran (2mL) and absolute ethanol (4mL), and lithium hydroxide 24mg (1.0mmol, 10.0eq) was slowly added dropwise thereto. solution in water (1 mL). The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, water was added, and 2M dilute hydrochloric acid was added to adjust the pH to 3. The obtained suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain the target product Dac58 (46 mg).
实施例31化合物Dam265的合成Example 31 Synthesis of compound Dam265
Figure PCTCN2022075561-appb-000041
Figure PCTCN2022075561-appb-000041
第一步、第二步按照Dam264的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dam264.
第三步：将2-(4-溴-2,6-二异丙基苯基)氨基)苯甲酸乙酯1.2g(3.0mmol,1.0eq),吗啉313mg(3.6mmol,1.2eq),BINAP 224mg(0.36mmol,0.12eq)催化剂Pd(OAc) 2 68mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于甲苯15mL中，加热至110℃反应24小时。冷却至室温，旋蒸，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-(2,6-二异丙基-4-吗啉)氨基)苯甲酸乙酯723mg。 The third step: 2-(4-bromo-2,6-diisopropylphenyl)amino)ethyl benzoate 1.2g (3.0mmol, 1.0eq), morpholine 313mg (3.6mmol, 1.2eq), BINAP 224mg (0.36mmol, 0.12eq) catalyst Pd(OAc) 2 68mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) was dissolved in 15mL of toluene, heated to 110 ℃ and reacted for 24 hours. Cooled to room temperature, rotary evaporated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain ethyl 2-(2,6-diisopropyl-4-morpholino)amino)benzoate as a white solid Ester 723mg.
第四步：将2-((2,6-二异丙基-4-吡啶苯基)氨基)苯甲酸乙酯820mg(2.0mmol,1.0eq)溶于四氢呋喃4mL和无水乙醇8mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂240mg(10mmol,5.0eq)的水2mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2,6-二异丙基-4-吗啉)氨基)苯甲酸648mg。Step 4: Dissolve 820 mg (2.0 mmol, 1.0 eq) of ethyl 2-((2,6-diisopropyl-4-pyridylphenyl)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise thereto under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. After suction filtration, the solid was washed with water to obtain 648 mg of 2-((2,6-diisopropyl-4-morpholino)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2,6-二异丙基-4-吗啉)氨基)苯甲酸77mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL，搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam265(48mg)。 1H NMR(400MHz,DMSO)δ9.84(s,1H),8.65(d,J＝6.1Hz,2H),8.06(s,1H),7.82–7.76(m,2H),7.72(d,J＝7.9Hz,1H),7.63(s,2H),7.38(s,1H),7.17(t,J＝7.8Hz,1H),6.62(t,J＝7.4Hz,1H),6.12(d,J＝8.4Hz,1H),3.19–3.00(m,2H),1.16(t,J＝6.6Hz,12H). The fifth step: in a 10mL round bottom flask, add 2-((2,6-diisopropyl-4-morpholino)amino)benzoic acid 77mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5 eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were added to the above system successively, and the resulting mixed system was heated at 45°C React for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam265 (48 mg). 1 H NMR (400MHz, DMSO) δ 9.84(s, 1H), 8.65(d, J=6.1Hz, 2H), 8.06(s, 1H), 7.82-7.76(m, 2H), 7.72(d, J =7.9Hz,1H),7.63(s,2H),7.38(s,1H),7.17(t,J=7.8Hz,1H),6.62(t,J=7.4Hz,1H),6.12(d,J = 8.4Hz, 1H), 3.19–3.00 (m, 2H), 1.16 (t, J = 6.6Hz, 12H).
实施例32化合物Dam266的合成Example 32 Synthesis of compound Dam266
Figure PCTCN2022075561-appb-000042
Figure PCTCN2022075561-appb-000042
第一步、第二步按照Dam185的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dam185.
第三步：将2-(4-溴-2-氟-6-甲基苯基)氨基)苯甲酸乙酯1.2g(3.0mmol,1.0eq),吗啉313mg(3.6mmol,1.2eq),BINAP 224mg(0.36mmol,0.12eq)催化剂Pd(OAc) 2 68mg(0.3mmol,0.1eq),碳酸钾828mg(6.0mmol,1.5eq)溶于甲苯15mL中，加热至110℃反应24小时。冷却至室温，浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-(2-氟-6-甲基-4-吗啉)氨基)苯甲酸乙酯848mg。 The third step: 1.2g (3.0mmol, 1.0eq) of ethyl 2-(4-bromo-2-fluoro-6-methylphenyl)amino)benzoate, 313mg (3.6mmol, 1.2eq) of morpholine, BINAP 224mg (0.36mmol, 0.12eq) catalyst Pd(OAc) 2 68mg (0.3mmol, 0.1eq), potassium carbonate 828mg (6.0mmol, 1.5eq) was dissolved in 15mL of toluene, heated to 110 ℃ and reacted for 24 hours. Cooled to room temperature, concentrated, and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain ethyl 2-(2-fluoro-6-methyl-4-morpholine)amino)benzoate as a white solid 848mg.
第四步：将2-(2-氟-6-甲基-4-吗啉)氨基)苯甲酸乙酯720mg(2.0mmol,1.0eq)溶于四氢呋喃4mL和无水乙醇8mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂240mg(10mmol,5.0eq)的水2mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水20mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氟-6-甲基-4-吗啉)氨基)苯甲酸691mg。Step 4: Dissolve 720 mg (2.0 mmol, 1.0 eq) of ethyl 2-(2-fluoro-6-methyl-4-morpholine)amino)benzoate in a mixed solvent of 4 mL of tetrahydrofuran and 8 mL of anhydrous ethanol, Under ice-water bath cooling, a solution of 240 mg (10 mmol, 5.0 eq) of lithium hydroxide in 2 mL of water was slowly added dropwise thereto. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 20 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 691 mg of 2-((2-fluoro-6-methyl-4-morpholino)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氟-6-甲基-4-吗啉)氨基)苯甲酸66mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL，搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam266(66mg)。 1H NMR(500MHz,MeOD)δ8.39(dd,J＝15.0,3.1Hz,1H),7.90–7.53(m,2H),7.16–6.85(m,2H),6.69(dd,J＝16.0,3.0Hz,1H),6.53(d,J＝3.1Hz,1H),6.24(s,1H),3.73(t,J＝9.3Hz,4H),3.54–3.36(m,4H),3.27(t,J＝9.3Hz,4H),2.13(s,3H),1.62(tt,J＝15.2,10.1Hz,2H),1.30(s,1H). The fifth step: in a 10mL round-bottom flask, add 2-((2-fluoro-6-methyl-4-morpholino)amino)benzoic acid 66mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5 eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol 18mg (0.24mmol, 1.2eq) were successively added to the above system, and the resulting mixed system was The reaction was carried out at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam266 (66 mg). 1 H NMR (500MHz, MeOD) δ 8.39 (dd, J=15.0, 3.1 Hz, 1H), 7.90-7.53 (m, 2H), 7.16-6.85 (m, 2H), 6.69 (dd, J=16.0, 3.0Hz, 1H), 6.53(d, J=3.1Hz, 1H), 6.24(s, 1H), 3.73(t, J=9.3Hz, 4H), 3.54–3.36(m, 4H), 3.27(t, J=9.3Hz, 4H), 2.13(s, 3H), 1.62(tt, J=15.2, 10.1Hz, 2H), 1.30(s, 1H).
实施例33化合物Dam267的合成Example 33 Synthesis of compound Dam267
Figure PCTCN2022075561-appb-000043
Figure PCTCN2022075561-appb-000043
第一步、第二步、第三步、第四步按照Dam266的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dam266.
第五步：在10mL圆底烧瓶中，加入2-((2-氟-6-甲基-4-吗啉)氨基)苯甲酸66mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL，搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基乙二胺28mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反应液直接碱性体系反相制备分离，分离液冻干得白色固体Dam267(58mg)。 1H NMR(500MHz,MeOD)δ8.37(dd,J＝15.0,3.0Hz,1H),7.88–7.54(m,2H),6.97(td,J＝15.0,3.0Hz,1H),6.74–6.64(m,2H),6.52(d,J＝2.9Hz,1H),4.90(s,1H),3.72(t,J＝9.2Hz,4H),3.59(t,J＝10.0Hz,2H),3.26(t,J＝9.3Hz,4H),2.47(dt,J＝25.2,11.4Hz,6H),2.13(s,3H),0.93(t,J＝12.6Hz,6H). The fifth step: in a 10mL round-bottom flask, add 2-((2-fluoro-6-methyl-4-morpholino)amino)benzoic acid 66mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5 eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylaminoethylenediamine 28mg (0.24mmol, 1.2eq) were successively added to the above system , the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. The reaction solution was directly prepared and separated by reverse-phase alkaline system, and the separated solution was lyophilized to obtain white solid Dam267 (58 mg). 1 H NMR (500MHz, MeOD) δ 8.37 (dd, J=15.0, 3.0Hz, 1H), 7.88-7.54 (m, 2H), 6.97 (td, J=15.0, 3.0Hz, 1H), 6.74-6.64 (m, 2H), 6.52(d, J=2.9Hz, 1H), 4.90(s, 1H), 3.72(t, J=9.2Hz, 4H), 3.59(t, J=10.0Hz, 2H), 3.26 (t, J=9.3Hz, 4H), 2.47 (dt, J=25.2, 11.4Hz, 6H), 2.13 (s, 3H), 0.93 (t, J=12.6Hz, 6H).
实施例34化合物Dac103的合成Example 34 Synthesis of compound Dac103
Figure PCTCN2022075561-appb-000044
Figure PCTCN2022075561-appb-000044
第一步、第二步按照Dac51的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dac51.
第三步：2-(4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),三氮唑硼酸1.1g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(5-甲基三氮唑))苯基)氨基)苯甲酸乙酯1.13g。 The third step: 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), triazole boronic acid 1.1g (8.8mmol, 1.2eq), Catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) was dissolved in a mixed solvent of dioxane and water 37mL (4:1/v:v), Heated to 100°C and reacted for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( 5-Methyltriazole))phenyl)amino)ethyl benzoate 1.13g.
第四步：将2-((2-氯-6-甲基-4-(5-甲基三氮唑))苯基)氨基)苯甲酸乙酯927mg(2.37 mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(5-甲基三氮唑))苯基)氨基)苯甲酸819mg。The fourth step: 927 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(5-methyltriazole))phenyl)amino)benzoate was dissolved in tetrahydrofuran A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 8 mL and 16 mL of anhydrous ethanol under cooling in an ice-water bath. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 819 mg of 2-((2-chloro-6-methyl-4-(5-methyltriazole))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(5-甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam103(47mg)。 1H NMR(500MHz,MeOD)δ8.38(dd,J＝15.0,3.0Hz,1H),7.80(s,2H),7.76–7.61(m,2H),7.31(s,1H),6.98(td,J＝15.0,3.0Hz,1H),6.68(s,1H),4.56(s,1H),2.38(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(5-methyltriazole))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0 eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24mmol, 1.2 eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, the separation liquid was lyophilized to obtain white solid Dam103 (47 mg). 1 H NMR(500MHz,MeOD)δ8.38(dd,J=15.0,3.0Hz,1H),7.80(s,2H),7.76-7.61(m,2H),7.31(s,1H),6.98(td , J=15.0, 3.0Hz, 1H), 6.68(s, 1H), 4.56(s, 1H), 2.38(s, 3H).
实施例35化合物Dac105的合成Example 35 Synthesis of compound Dac105
Figure PCTCN2022075561-appb-000045
Figure PCTCN2022075561-appb-000045
第一步、第二步按照Dac51的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dac51.
第三步：将2-(4-溴-2,6-二氯苯基)氨基)苯甲酸乙酯3.0g(8.0mmol,1.0eq),,3,5-二甲基三氮唑-4-硼酸1.2g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸乙酯1.33g。 The third step: 2-(4-bromo-2,6-dichlorophenyl)amino)ethyl benzoate 3.0g (8.0mmol, 1.0eq), 3,5-dimethyltriazole-4 - Boric acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/v:v), heated to 100°C and reacted for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( 3,5-Dimethyltriazole))phenyl)amino)ethyl benzoate 1.33g.
第四步：将2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸乙酯893mg(2.37mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸763mg。The fourth step: 893mg (2.37mmol, 1.0eq) of ethyl 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoate It was dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 16 mL of absolute ethanol, and a solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to it under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 763 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消 耗完毕。反相制备分离，分离液冻干得白色固体Dam105(47mg)。 1H NMR(400MHz,DMSO)δ11.31(s,1H),9.31(s,1H),9.16(s,1H),7.52(d,J＝7.6Hz,1H),7.47(s,1H),7.34(s,1H),7.24(t,J＝7.6Hz,1H),6.74(t,J＝7.3Hz,1H),6.23(d,J＝8.2Hz,1H),2.45(s,3H),2.27(s,3H),2.21(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam105 (47 mg). 1 H NMR (400MHz, DMSO) δ 11.31(s, 1H), 9.31(s, 1H), 9.16(s, 1H), 7.52(d, J=7.6Hz, 1H), 7.47(s, 1H), 7.34(s, 1H), 7.24(t, J=7.6Hz, 1H), 6.74(t, J=7.3Hz, 1H), 6.23(d, J=8.2Hz, 1H), 2.45(s, 3H), 2.27(s, 3H), 2.21(s, 3H).
实施例36化合物Dam268的合成Example 36 Synthesis of compound Dam268
Figure PCTCN2022075561-appb-000046
Figure PCTCN2022075561-appb-000046
第一步、第二步、第三步、第四步按照Dac105的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam268(33mg)。 1H NMR(500MHz,MeOD)δ8.39(dd,J＝15.0,3.1Hz,1H),7.81(s,2H),7.77–7.61(m,2H),6.99(td,J＝15.0,2.9Hz,1H),6.54(s,2H),4.75(s,1H),4.30(s,3H),2.38(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam268 (33 mg). 1 H NMR (500MHz, MeOD) δ 8.39 (dd, J=15.0, 3.1Hz, 1H), 7.81 (s, 2H), 7.77-7.61 (m, 2H), 6.99 (td, J=15.0, 2.9Hz) ,1H),6.54(s,2H),4.75(s,1H),4.30(s,3H),2.38(s,3H).
实施例37化合物Dam269的合成Example 37 Synthesis of compound Dam269
Figure PCTCN2022075561-appb-000047
Figure PCTCN2022075561-appb-000047
第一步、第二步、第三步、第四步按照Dac105的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基-1,4-丁二胺35mg(0.24mmol,1.2eq),17mg(0.24mmol,1.2eq),所得混合体系在 45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam269(67mg)。 1H NMR(400MHz,DMSO)δ11.31(s,1H),9.31(s,1H),9.16(s,1H),7.52(d,J＝7.6Hz,1H),7.47(s,1H),7.34(s,1H),7.24(t,J＝7.6Hz,1H),6.74(t,J＝7.3Hz,1H),6.23(d,J＝8.2Hz,1H),2.45(s,3H),2.27(s,3H),2.21(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-di Ethylamino-1,4-butanediamine 35mg (0.24mmol, 1.2eq), 17mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam269 (67 mg). 1 H NMR (400MHz, DMSO) δ 11.31(s, 1H), 9.31(s, 1H), 9.16(s, 1H), 7.52(d, J=7.6Hz, 1H), 7.47(s, 1H), 7.34(s, 1H), 7.24(t, J=7.6Hz, 1H), 6.74(t, J=7.3Hz, 1H), 6.23(d, J=8.2Hz, 1H), 2.45(s, 3H), 2.27(s, 3H), 2.21(s, 3H).
实施例38化合物Dam270的合成Example 38 Synthesis of compound Dam270
Figure PCTCN2022075561-appb-000048
Figure PCTCN2022075561-appb-000048
第一步、第二步、第三步、第四步按照Dac105的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam270(53mg)。 1H NMR(400MHz,DMSO)δ11.31(s,1H),9.31(s,1H),9.16(s,1H),7.52(d,J＝7.6Hz,1H),7.47(s,1H),7.34(s,1H),7.24(t,J＝7.6Hz,1H),6.74(t,J＝7.3Hz,1H),6.23(d,J＝8.2Hz,1H),2.45(s,3H),2.27(s,3H),2.21(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol were added to the above system successively 18mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, the separation liquid was lyophilized to obtain white solid Dam270 (53 mg). 1 H NMR (400MHz, DMSO) δ 11.31(s, 1H), 9.31(s, 1H), 9.16(s, 1H), 7.52(d, J=7.6Hz, 1H), 7.47(s, 1H), 7.34(s, 1H), 7.24(t, J=7.6Hz, 1H), 6.74(t, J=7.3Hz, 1H), 6.23(d, J=8.2Hz, 1H), 2.45(s, 3H), 2.27(s, 3H), 2.21(s, 3H).
实施例39化合物Dam271的合成Example 39 Synthesis of compound Dam271
Figure PCTCN2022075561-appb-000049
Figure PCTCN2022075561-appb-000049
第一步、第二步、第三步、第四步按照Dac105的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac105.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基乙二胺28mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测， 原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam406(51mg)。 1H NMR(500MHz,MeOD)δ8.43(s,1H),8.05(s,1H),7.85–7.80(m,2H),7.71(s,1H),7.55(s,1H),7.52(s,1H),6.94(s,1H),4.24(s,3H),3.63(s,2H),2.90–2.73(m,2H),2.71(s,2H),2.61–2.49(m,2H),2.39(s,3H),1.10–0.89(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-di Ethylaminoethylenediamine 28mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detected that the raw materials were consumed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam406 (51 mg). 1 H NMR(500MHz,MeOD)δ8.43(s,1H),8.05(s,1H),7.85-7.80(m,2H),7.71(s,1H),7.55(s,1H),7.52(s ,1H),6.94(s,1H),4.24(s,3H),3.63(s,2H),2.90–2.73(m,2H),2.71(s,2H),2.61–2.49(m,2H), 2.39(s, 3H), 1.10–0.89(m, 6H).
实施例40化合物Dac109的合成Example 40 Synthesis of compound Dac109
Figure PCTCN2022075561-appb-000050
Figure PCTCN2022075561-appb-000050
第一步、第二步按照Dac51的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dac51.
第三步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯2.7g(7.3mmol,1.0eq),3,5-二甲基三氮唑-4-硼酸1.2g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸乙酯1.11g。 The third step: 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), 3,5-dimethyltriazole- 4-boronic acid 1.2g (8.8mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixture of dioxane and water In 37 mL of solvent (4:1/v:v), the mixture was heated to 100°C and reacted for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( 3,5-Dimethylisoxazole))phenyl)amino)ethyl benzoate 1.11 g.
第四步：将2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸乙酯912mg(2.37mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸715mg。The fourth step: 912mg (2.37mmol, 1.0eq) of ethyl 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoate It was dissolved in a mixed solvent of 8 mL of tetrahydrofuran and 16 mL of absolute ethanol, and a solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to it under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 715 mg of 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dac109(24mg)。 1H NMR(500MHz,MeOD)δ8.38(s,1H),8.03(s,1H),7.81(s,1H),7.61(d,J＝17.6Hz,1H),7.61(d,J＝17.6Hz,2H),7.54(s,1H),7.04(s,1H),6.94(s,1H),4.25(s,3H),2.38(s,3H),2.22(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), hydroxylamine hydrochloride 17mg (0.24 mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain Dac109 (24 mg) as a white solid. 1 H NMR (500MHz, MeOD) δ 8.38(s, 1H), 8.03(s, 1H), 7.81(s, 1H), 7.61(d, J=17.6Hz, 1H), 7.61(d, J=17.6 Hz, 2H), 7.54(s, 1H), 7.04(s, 1H), 6.94(s, 1H), 4.25(s, 3H), 2.38(s, 3H), 2.22(s, 3H).
实施例41化合物Dam273的合成Example 41 Synthesis of compound Dam273
Figure PCTCN2022075561-appb-000051
Figure PCTCN2022075561-appb-000051
第一步、第二步、第三步、第四步按照Dac109的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac109.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(3,5-二甲基三氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam273(31mg)。 1H NMR(500MHz,MeOD)δ8.38(s,1H),8.03(s,1H),7.81(s,1H),7.62(s,1H),7.58(s,1H),7.54(s,1H),7.34(s,2H),6.94(s,1H),4.25(s,3H),2.37(s,3H),2.22(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(3,5-dimethyltriazole))phenyl)amino)benzoic acid 72mg (0.2 mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq), dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), ammonium chloride 14mg ( 0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam273 (31 mg). 1 H NMR(500MHz,MeOD)δ8.38(s,1H),8.03(s,1H),7.81(s,1H),7.62(s,1H),7.58(s,1H),7.54(s,1H) ),7.34(s,2H),6.94(s,1H),4.25(s,3H),2.37(s,3H),2.22(s,3H).
实施例42化合物Dac111的合成Example 42 Synthesis of Compound Dac111
Figure PCTCN2022075561-appb-000052
Figure PCTCN2022075561-appb-000052
第一步、第二步按照Dac51的合成方法制备。The first step and the second step are prepared according to the synthetic method of Dac51.
第三步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯2.7g(7.3mmol,1.0eq),四氮唑-5-硼酸1.2g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸乙酯973g。 The third step: 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), tetrazolium-5-boronic acid 1.2g (8.8g) mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/ In v:v), the reaction was heated to 100° C. for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( Tetrazolium))phenyl)amino)ethyl benzoate 973g.
第四步：将2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸乙酯830mg(2.37mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀 盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸710mg。The fourth step: 830 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoate was dissolved in 8 mL of tetrahydrofuran and anhydrous A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 16 mL of ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 710 mg of 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸70mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dac111(21mg)。 1H NMR(500MHz,MeOD)δ8.50–8.30(m,1H),8.10–7.97(m,1H),7.88–7.78(m,2H),7.69–7.59(m,1H),7.59–7.44(m,1H),7.10–7.01(m,1H),6.99–6.88(m,1H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 70mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting The mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation was separated, and the separated liquid was lyophilized to obtain Dac111 (21 mg) as a white solid. 1 H NMR(500MHz,MeOD)δ8.50-8.30(m,1H),8.10-7.97(m,1H),7.88-7.78(m,2H),7.69-7.59(m,1H),7.59-7.44( m, 1H), 7.10–7.01 (m, 1H), 6.99–6.88 (m, 1H).
实施例43化合物Dam274的合成Example 43 Synthesis of compound Dam274
Figure PCTCN2022075561-appb-000053
Figure PCTCN2022075561-appb-000053
第一步、第二步、第三步、第四步按照Dac111的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸70mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam274(21mg)。 1H NMR(500MHz,MeOD)δ9.36–9.26(m,1H),8.54–8.33(m,1H),8.12–8.00(m,1H),7.93–7.81(m,2H),7.60(s,0H),7.55–7.48(m,1H),7.46–7.19(m,2H),7.03–6.78(m,1H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 70mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were sequentially added to the above system, The resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain Dam274 (21 mg) as a white solid. 1 H NMR(500MHz,MeOD)δ9.36-9.26(m,1H),8.54-8.33(m,1H),8.12-8.00(m,1H),7.93-7.81(m,2H),7.60(s, 0H), 7.55–7.48 (m, 1H), 7.46–7.19 (m, 2H), 7.03–6.78 (m, 1H).
实施例44化合物Dam275的合成Example 44 Synthesis of compound Dam275
Figure PCTCN2022075561-appb-000054
Figure PCTCN2022075561-appb-000054
第一步、第二步、第三步、第四步按照Dac111的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙氨基-1,4-丁二胺35mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam275(35mg)。 1H NMR(500MHz,MeOD)δ9.51–9.16(m,1H),8.27–8.13(m,1H),7.79(s,0H),7.78–7.69(m,2H),7.64–7.46(m,2H),7.09–6.81(m,1H),3.40(q,J＝7.5Hz,1H),3.31(q,J＝7.5Hz,1H),3.03(q,J＝6.4Hz,2H),2.96(q,J＝7.5Hz,2H),2.85(q,J＝6.3Hz,2H),1.81(dt,J＝15.5,7.8Hz,2H),1.70–1.52(m,2H),1.37–1.14(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylamino-1,4- Butanediamine 35mg (0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation were performed, and the separated liquid was lyophilized to obtain Dam275 (35 mg) as a white solid. 1 H NMR(500MHz,MeOD)δ9.51-9.16(m,1H),8.27-8.13(m,1H),7.79(s,0H),7.78-7.69(m,2H),7.64-7.46(m, 2H),7.09–6.81(m,1H),3.40(q,J=7.5Hz,1H),3.31(q,J=7.5Hz,1H),3.03(q,J=6.4Hz,2H),2.96( q, J=7.5Hz, 2H), 2.85 (q, J=6.3Hz, 2H), 1.81 (dt, J=15.5, 7.8Hz, 2H), 1.70–1.52 (m, 2H), 1.37–1.14 (m ,6H).
实施例45化合物Dam276的合成Example 45 Synthesis of compound Dam276
Figure PCTCN2022075561-appb-000055
Figure PCTCN2022075561-appb-000055
第一步、第二步、第三步、第四步按照Dac111的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),3-氨基丙醇18mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam276(29mg)。 1H NMR(500MHz,MeOD)δ9.38(s,1H),8.38(s,1H),8.03(dd,J＝7.5,1.4Hz,1H),7.79(s,0H),7.78–7.69(m,2H),7.60(s,1H),7.54(s,1H),6.96(s,1H),3.58(s,2H),3.48(s,1H),3.42(s,1H),1.68(s,2H),1.34(s,1H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), 3-aminopropanol 18mg (0.24mmol, 1.2eq) were added to the above system in turn ), the resulting mixed system was reacted at 45 °C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam276 (29 mg). 1 H NMR(500MHz,MeOD)δ9.38(s,1H),8.38(s,1H),8.03(dd,J=7.5,1.4Hz,1H),7.79(s,0H),7.78-7.69(m ,2H),7.60(s,1H),7.54(s,1H),6.96(s,1H),3.58(s,2H),3.48(s,1H),3.42(s,1H),1.68(s, 2H), 1.34(s, 1H).
实施例46化合物Dam277的合成Example 46 Synthesis of compound Dam277
Figure PCTCN2022075561-appb-000056
Figure PCTCN2022075561-appb-000056
第一步、第二步、第三步、第四步按照Dac111的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac111.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),N,N-二乙基乙二胺24mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam406(29mg)。 1H NMR(500MHz,Chloroform)δ9.38(s,1H),8.38(s,1H),8.09(s,1H),8.03(s,1H),7.82–7.71(m,2H),7.60(s,1H),7.54(s,1H),6.96(s,1H),3.72(s,1H),3.57(s,1H),3.02–2.87(m,2H),2.61(s,2H),2.52–2.37(m,2H),1.11–0.98(m,6H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in anhydrous DMF 4.0mL at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq), N,N-diethylethylenediamine 24mg ( 0.24mmol, 1.2eq), the resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain white solid Dam406 (29 mg). 1 H NMR (500MHz, Chloroform) δ 9.38(s, 1H), 8.38(s, 1H), 8.09(s, 1H), 8.03(s, 1H), 7.82–7.71(m, 2H), 7.60(s) ,1H),7.54(s,1H),6.96(s,1H),3.72(s,1H),3.57(s,1H),3.02–2.87(m,2H),2.61(s,2H),2.52– 2.37(m,2H),1.11–0.98(m,6H).
实施例47化合物Dac118的合成Example 47 Synthesis of compound Dac118
Figure PCTCN2022075561-appb-000057
Figure PCTCN2022075561-appb-000057
第一步、第二步按照Dac406的合成方法制备。The first and second steps are prepared according to the synthetic method of Dac406.
第三步：将2-(4-溴-2-氯-6-甲基苯基)氨基)苯甲酸乙酯2.7g(7.3mmol,1.0eq),四氮唑-5-硼酸1.2g(8.8mmol,1.2eq),催化剂Pd(dppf)Cl 2 534mg(0.73mmol,0.1eq),碳酸钾2.1g(14.6mmol,2.0eq)溶于二氧六环和水的混合溶剂37mL(4:1/v:v)中，加热至100℃反应24小时。冷却至室温，旋蒸除去1/2溶剂，用乙酸乙酯萃取50mL×3，合并有机相并用饱和食盐水洗涤10mL×3。无水硫酸钠干燥有机相，过滤，滤液浓缩，硅胶层析柱分离(石油醚:乙酸乙酯＝20:1)，得白色固体2-((2-氯-6-甲基-4-(3,5-二甲基异恶唑))苯基)氨基)苯甲酸乙酯967mg。 The third step: 2-(4-bromo-2-chloro-6-methylphenyl)amino)ethyl benzoate 2.7g (7.3mmol, 1.0eq), tetrazolium-5-boronic acid 1.2g (8.8g) mmol, 1.2eq), catalyst Pd(dppf)Cl 2 534mg (0.73mmol, 0.1eq), potassium carbonate 2.1g (14.6mmol, 2.0eq) dissolved in a mixed solvent of dioxane and water 37mL (4:1/ In v:v), the reaction was heated to 100° C. for 24 hours. Cool to room temperature, remove 1/2 of the solvent by rotary evaporation, extract 50 mL×3 with ethyl acetate, combine the organic phases and wash 10 mL×3 with saturated brine. The organic phase was dried over anhydrous sodium sulfate, filtered, and the filtrate was concentrated and separated by silica gel column chromatography (petroleum ether:ethyl acetate=20:1) to obtain a white solid 2-((2-chloro-6-methyl-4-( 3,5-Dimethylisoxazole))phenyl)amino)ethyl benzoate 967 mg.
第四步：将2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸乙酯848mg(2.37mmol,1.0eq)溶于四氢呋喃8mL和无水乙醇16mL的混合溶剂中，冰水浴冷却下，向其中缓缓滴加氢氧化锂284mg(11.9mmol,5.0eq)的水4mL溶液。加热至45℃反应过夜，反应结束后，体系冷却至室温，浓缩除去有机溶剂，加入水10mL，置于冰水浴中，2M稀盐酸调节pH至3，所得悬浊液继续在室温下搅拌30min。抽滤，固体用水洗，得白色固体2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸723mg。The fourth step: 848 mg (2.37 mmol, 1.0 eq) of ethyl 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoate was dissolved in 8 mL of tetrahydrofuran and anhydrous A solution of 284 mg (11.9 mmol, 5.0 eq) of lithium hydroxide in 4 mL of water was slowly added dropwise to a mixed solvent of 16 mL of ethanol under ice-water bath cooling. The reaction was heated to 45°C overnight. After the reaction was completed, the system was cooled to room temperature, concentrated to remove the organic solvent, added with 10 mL of water, placed in an ice-water bath, adjusted to pH 3 with 2M dilute hydrochloric acid, and the resulting suspension was continuously stirred at room temperature for 30 min. Suction filtration, and the solid was washed with water to obtain 723 mg of 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid as a white solid.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),盐酸羟胺17mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam118(19mg)。 1H NMR(500MHz,MeOD)δ9.32(s,1H),8.38(s,1H),8.04(s,1H),7.87(s,1H),7.60(d,J＝3.9Hz,2H),7.54(s,1H),7.03(s,1H),6.94(s,1H),2.21(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and hydroxylamine hydrochloride 17mg (0.24mmol, 1.2eq) were added to the above system in turn, the resulting The mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation and separation, and the separation liquid was lyophilized to obtain Dam118 (19 mg) as a white solid. 1 H NMR(500MHz,MeOD)δ9.32(s,1H),8.38(s,1H),8.04(s,1H),7.87(s,1H),7.60(d,J=3.9Hz,2H), 7.54(s, 1H), 7.03(s, 1H), 6.94(s, 1H), 2.21(s, 3H).
实施例48化合物Dam279的合成Example 48 Synthesis of compound Dam279
Figure PCTCN2022075561-appb-000058
Figure PCTCN2022075561-appb-000058
第一步、第二步、第三步、第四步按照Dac118的合成方法制备。The first step, the second step, the third step and the fourth step are prepared according to the synthetic method of Dac118.
第五步：在10mL圆底烧瓶中，加入2-((2-氯-6-甲基-4-(四氮唑))苯基)氨基)苯甲酸72mg(0.2mmol,1.0eq),HATU 114mg(0.3mmol,1.5eq),室温下溶解于无水DMF 4.0mL,搅拌5min,向上述体系中依次加入DIEA 77mg(0.6mmol,3.0eq),氯化铵14mg(0.24mmol,1.2eq),所得混合体系在45℃反应2小时。TLC检测，原料消耗完毕。反相制备分离，分离液冻干得白色固体Dam279(29mg)。 1H NMR(500MHz,MeOD)δ9.37(s,1H),8.24(s,1H),7.95(s,1H),7.72(s,1H),7.63(s,1H),7.56(s,1H),7.43(s,1H),7.34(s,2H),6.95(s,1H),2.15(s,3H). The fifth step: in a 10mL round bottom flask, add 2-((2-chloro-6-methyl-4-(tetrazolium))phenyl)amino)benzoic acid 72mg (0.2mmol, 1.0eq), HATU 114mg (0.3mmol, 1.5eq) was dissolved in 4.0mL of anhydrous DMF at room temperature, stirred for 5min, DIEA 77mg (0.6mmol, 3.0eq) and ammonium chloride 14mg (0.24mmol, 1.2eq) were sequentially added to the above system, The resulting mixed system was reacted at 45°C for 2 hours. TLC detection, the raw material consumption was completed. Reverse-phase preparation was separated, and the separated liquid was lyophilized to obtain Dam279 (29 mg) as a white solid. 1 H NMR(500MHz,MeOD)δ9.37(s,1H),8.24(s,1H),7.95(s,1H),7.72(s,1H),7.63(s,1H),7.56(s,1H) ), 7.43(s, 1H), 7.34(s, 2H), 6.95(s, 1H), 2.15(s, 3H).
生物实施例1：FTO抑制活性测定Biological Example 1: FTO Inhibitory Activity Assay
通过镍柱亲和层析纯化得到高纯度的FTO蛋白。Purified by nickel column affinity chromatography to obtain high-purity FTO protein.
FTO酶活抑制反应体系如下：50mM Tris·HCl,pH 7.5,0.3μM FTO,1μM 39nt-m 6A修饰的双链DNA,300μM 2OG,280μM(NH 4) 2Fe(SO 4) 2,2mM L-Ascorbic Acid及不同浓度化合物，室温孵育2h后，65℃缓慢加热失活，加1μM 39nt的反义链DNA退火成双链。取8ul反应液，用甲基化敏感的酶DpnII对双链底物进行酶切，酶切后的样品经15％非变性聚丙烯酰胺电泳检测，Gel-Red染色后凝胶成像***下拍照，对获得的条带进行灰度的读取抑制率，并针对抑制率较好的化合物评估抑制FTO去甲基活性的IC 50，结果如下表。 FTO enzyme activity inhibition reaction system is as follows: 50mM Tris·HCl, pH 7.5, 0.3μM FTO, 1μM 39nt-m 6 A modified double-stranded DNA, 300μM 2OG, 280μM (NH 4 ) 2 Fe(SO 4 ) 2 , 2mM L -Ascorbic Acid and compounds of different concentrations, incubated at room temperature for 2 hours, slowly heated at 65°C for inactivation, and annealed to double-stranded DNA by adding 1 μM 39nt antisense DNA. Take 8ul of the reaction solution and digest the double-stranded substrate with the methylation-sensitive enzyme DpnII. The digested samples were detected by 15% non-denaturing polyacrylamide electrophoresis, and photographed under the gel imaging system after Gel-Red staining. The obtained bands were read for the inhibition rate in grayscale, and the IC 50 for inhibiting the demethylation activity of FTO was evaluated for the compounds with better inhibition rate. The results are shown in the following table.
表1.化合物对FTO酶学活性抑制IC 50 Table 1. Compound IC50 for inhibition of FTO enzymatic activity
Figure PCTCN2022075561-appb-000059
Figure PCTCN2022075561-appb-000059
Figure PCTCN2022075561-appb-000060
Figure PCTCN2022075561-appb-000060
结果显示，上述化合物具有一定的活性，其IC 50小于10μM，且大多数小于1μM。 The results show that the above compounds have certain activities, and their IC 50 is less than 10 μM, and most of them are less than 1 μM.
生物实施例2：肿瘤细胞增殖抑制活性评估Biological Example 2: Evaluation of Tumor Cell Proliferation Inhibitory Activity
分别培养黑色素瘤细胞系B16-OVA、肺癌细胞系LLC、结肠癌细胞系MC38等实体瘤细胞系，以1000个每孔的密度种细胞于96孔板中，置于37℃ CO 2培养箱，培养细胞至贴壁，加入不同化合物继续培养72h，向每个孔中加入MTT溶液10uL，继续孵育4h，检测490nm处的吸光度值，以DMSO组为对照计算抑制率，并针对抑制率较好的化合物评估细胞增殖抑制活性IC 50，结果如下表。 Solid tumor cell lines such as the melanoma cell line B16-OVA, the lung cancer cell line LLC, and the colon cancer cell line MC38 were cultured separately, seeded at a density of 1000 cells per well in a 96-well plate, and placed in a 37°C CO 2 incubator. Incubate the cells until they adhere to the wall, add different compounds and continue to culture for 72h, add 10uL of MTT solution to each well, continue to incubate for 4h, detect the absorbance value at 490nm, calculate the inhibition rate with the DMSO group as the control, and focus on the cells with better inhibition rate. Compounds were evaluated for cell proliferation inhibitory activity IC50 , and the results are shown in the table below.
表2.FTO抑制物对肿瘤细胞的增殖抑制活性IC 50 Table 2. The proliferation inhibitory activity IC50 of FTO inhibitors on tumor cells
Figure PCTCN2022075561-appb-000061
Figure PCTCN2022075561-appb-000061
生物实施例3：斑点印记杂交检测肿瘤细胞内m 6A丰度改变 Biological Example 3: Detection of m 6 A abundance changes in tumor cells by dot blot hybridization
肿瘤细胞以一定密度种板于6cm培养皿中，次日加入化合物(浓度设置依据MTT的IC 50值)，处理48-72hr后，移除细胞培养基，向其中加入1mL的TRIzol试剂，收集细胞裂解，吸至RNase free管中，冰上孵育5min，使核蛋白复合物完全分离；加入200μL氯仿裂解，涡旋15s，孵育；离心分层。将含有RNA水相的上层转移到一个新的1.5mL RNase free管中，移出溶液；加入500μL预冷的异丙醇，颠倒混匀，孵育10min；4℃离心10min，移除上清，加入1mL DEPC·H 2O-乙醇溶液，重悬沉淀，4℃离心5min；吸走上清，冰上干燥RNA沉淀10min，加入20-50μL DEPC·H 2O溶解沉淀，涡旋溶解，离心，重复3次，测定浓度。测完浓度之后，95℃变性3min，立即冰上放置5min，短暂离心，轻弹管底，再次离心，放冰上；将RNA浓度稀释为浓度梯度,点样；暖光干燥20-30min，12000W光交联3min，用PBST配制的5％脱脂牛奶室温封闭1h,m 6A一抗封闭过夜，用PBST洗膜，孵育二抗，用PBST洗膜三遍，发色。结果如图1所示，FTO抑制剂能够浓度依赖性的改变肿瘤细胞内m 6A的丰度。 Tumor cells were seeded in a 6cm culture dish at a certain density, the compound was added the next day (the concentration was set according to the IC 50 value of MTT), and after 48-72 hours of treatment, the cell culture medium was removed, and 1 mL of TRIzol reagent was added to it, and the cells were collected. Lyse, pipette into RNase free tube, and incubate on ice for 5 min to completely separate nucleoprotein complexes; add 200 μL of chloroform to lyse, vortex for 15 s, incubate; centrifuge for layers. Transfer the upper layer containing the RNA aqueous phase to a new 1.5 mL RNase free tube, remove the solution; add 500 μL of pre-cooled isopropanol, invert and mix, incubate for 10 min; centrifuge at 4°C for 10 min, remove the supernatant, and add 1 mL DEPC·H 2 O-ethanol solution, resuspend the pellet, centrifuge at 4°C for 5 min; aspirate the supernatant, dry the RNA pellet on ice for 10 min, add 20-50 μL DEPC·H 2 O to dissolve the pellet, vortex to dissolve, centrifuge, repeat 3 Next, measure the concentration. After measuring the concentration, denature at 95°C for 3 minutes, immediately place on ice for 5 minutes, centrifuge briefly, flick the bottom of the tube, centrifuge again, and put on ice; dilute the RNA concentration to a concentration gradient, and spot; warm light drying for 20-30 minutes, 12000W Photocrosslinking for 3 min, blocking with 5% skim milk prepared with PBST for 1 h at room temperature, blocking with m 6 A primary antibody overnight, washing the membrane with PBST, incubating with the secondary antibody, washing the membrane three times with PBST, and developing color. The results are shown in Fig. 1, FTO inhibitor can change the abundance of m 6 A in tumor cells in a concentration-dependent manner.
生物实施例4：动物药效学评价Biological Example 4: Animal Pharmacodynamic Evaluation
选用5周龄、体重18-20g的C57BL/6品系小黑鼠。同时培养肿瘤细胞MC38。小鼠前右腋下注射2.5×10 5细胞(100uL,细胞密度2.5×10 6细胞/mL)。种瘤6天后，开始量肿瘤长和宽，并计算肿瘤体积(Volume＝0.5×Length×Width 2)，第9天选取肿瘤体积在60mm 3-100mm 3范围的荷瘤小鼠，每组4只。 5-week-old C57BL/6 strain mice with a body weight of 18-20 g were selected. At the same time, tumor cells MC38 were cultured. The mice were injected with 2.5×10 5 cells (100uL, cell density 2.5×10 6 cells/mL) in the anterior right armpit. 6 days after the tumor was implanted, the length and width of the tumor were measured, and the tumor volume was calculated (Volume=0.5×Length×Width 2 ). On the 9th day, tumor-bearing mice with a tumor volume ranging from 60 mm 3 to 100 mm 3 were selected, with 4 mice in each group. .
候选化合物Dam59和Dam159分别用DMSO溶解，配置20mM的母液，并用PBS稀释，最终获得的给药溶液DMSO的含量为20％，备用。The candidate compounds Dam59 and Dam159 were respectively dissolved in DMSO to prepare a 20 mM stock solution and diluted with PBS. The content of DMSO in the finally obtained dosing solution was 20%, which was ready for use.
从第10、11、12天分别采用腹腔给药方式注射候选化合物Dam59(2mg/kg)以及Dam159(0.5mg/kg)。第13、16天采用腹腔给药方式注射剂量为100mg/kg的抗体PD-L1以及抗体阴性对照IgG。隔两天量取肿瘤的体积和小鼠体重。结果如图2和图3所示，当与抗体PD-L1联用情况下，本发明的化合物体现出明显的协同效果。The candidate compounds Dam59 (2 mg/kg) and Dam159 (0.5 mg/kg) were injected by intraperitoneal administration from the 10th, 11th, and 12th days, respectively. On days 13 and 16, 100 mg/kg of antibody PD-L1 and antibody negative control IgG were injected intraperitoneally. Tumor volume and mouse body weight were measured every two days. The results are shown in Fig. 2 and Fig. 3 , when used in combination with the antibody PD-L1, the compounds of the present invention exhibited obvious synergistic effects.
生物实施例5：动物药效学评价Biological Example 5: Animal Pharmacodynamic Evaluation
选用5周龄、体重18-20g的C57BL/6品系小黑鼠。同时培养肿瘤细胞MC38。小鼠前右腋下注射2.5×10 5细胞(100uL,细胞密度2.5×10 6细胞/mL)。种瘤6天后，开始量肿瘤长和宽，并计算肿瘤体积(Volume＝0.5×Length×Width 2)，第9天选取肿瘤体积在60mm 3-100mm 3范围的荷瘤小鼠，每组4只。 5-week-old C57BL/6 strain mice with a body weight of 18-20 g were selected. At the same time, tumor cells MC38 were cultured. The mice were injected with 2.5×10 5 cells (100uL, cell density 2.5×10 6 cells/mL) in the anterior right armpit. 6 days after the tumor was implanted, the length and width of the tumor were measured, and the tumor volume was calculated (Volume=0.5×Length×Width 2 ). On the 9th day, tumor-bearing mice with a tumor volume ranging from 60 mm 3 to 100 mm 3 were selected, with 4 mice in each group. .
候选化合物Dam60用DMSO溶解，配置20mM的母液，并用PBS稀释，最终获得的给药溶液DMSO的含量为20％，备用。The candidate compound Dam60 was dissolved in DMSO, and a 20 mM stock solution was prepared and diluted with PBS. The content of DMSO in the finally obtained dosing solution was 20%, which was ready for use.
从第7、8、9天分别采用腹腔给药方式注射候选化合物Dam60(1mg/kg)。第10、12天采用腹腔给药方式注射剂量为100mg/kg的抗体PD-1以及抗体阴性对照IgG。隔两天量取肿瘤的体积和小鼠体重。结果如图4所示，当化合物与抗体PD-1联用情况下，本发明的化合物体现出明显的协同效果。The candidate compound Dam60 (1 mg/kg) was injected by intraperitoneal administration from day 7, 8, and 9, respectively. On days 10 and 12, 100 mg/kg of antibody PD-1 and antibody negative control IgG were injected intraperitoneally. Tumor volume and mouse body weight were measured every two days. The results are shown in FIG. 4 , when the compound is used in combination with the antibody PD-1, the compound of the present invention exhibits an obvious synergistic effect.
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (14)

  1. 一种如下式(I)所示的化合物，及其药学上可接受的盐，水合物，溶剂合物或前药的用途，A compound represented by the following formula (I), and the use of a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof,
    Figure PCTCN2022075561-appb-100001
    Figure PCTCN2022075561-appb-100001
    其中，in,
    A 1、A 2、A 3、A 4各自独立地为CR'或N； A 1 , A 2 , A 3 , A 4 are each independently CR' or N;
    M选自下组：CR' 2、NH、O或S； M is selected from the group consisting of CR'2 , NH, O or S;
    R'选自下组：H、卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基、C 1-C 6烷氧基羰基、取代或未取代的C 1-C 6酰氨基、取代或未取代的C 2-C 12酯基、或取代或未取代的C 1-C 10烷基、或取代或未取代的C3-C10烯酰胺基； R' is selected from the group consisting of H, halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, substituted or unsubstituted C 1 -C 6 amido, substituted or unsubstituted C 2 -C 12 ester group, or substituted or unsubstituted C 1 - C10 alkyl, or substituted or unsubstituted C3-C10 enamido;
    X具有如下式所示的结构：羧基、O-取代或未取代的羟肟酸基、取代或未取代的C 2-C 12酯基、取代或未取代的酰胺基(C(O)NH 2)、取代或未取代3-12元杂环基； X has the structure shown in the following formula: carboxyl group, O-substituted or unsubstituted hydroxamic acid group, substituted or unsubstituted C2 - C12 ester group, substituted or unsubstituted amide group (C(O) NH2 ), substituted or unsubstituted 3-12-membered heterocyclyl;
    Y选自下组：取代或未取代的C 6-C 12的芳基、取代或未取代3-12元杂环基； Y is selected from the group consisting of substituted or unsubstituted C 6 -C 12 aryl, substituted or unsubstituted 3-12 membered heterocyclyl;
    R a、R b各自独立地选自下组：卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基； R a , R b are each independently selected from the group consisting of halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkane Oxygen;
    R c、R d各自独立地选自下组：H、卤素、-OH、CN、NO 2、NH 2、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基； R c , R d are each independently selected from the group consisting of H, halogen, -OH, CN, NO 2 , NH 2 , substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy;
    所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素原子、羰基(＝O)、羧基、羟基、氨基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 1-C 6烷氧基羰基、C 1-C 6酰氨基、C 2-C 12酯基、取代或未取代的C 1-C 10烷基，取代或未取代的C 2-C 10烯基，取代或未取代的C 2-C 10炔基，取代或未取代的C 6-C 10芳基、或取代或未取代的五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或C 1-C 6烷氨基；其中，所述的取代或未取代的C 1-C 10烷基，取代或未取代的C 6-C 10芳基或五元或六元杂芳基的取代基选自下组：卤素原子、羰基(＝O)、羟基、羧基、C 1-C 6烷氧基羰基、氨基、C 1-C 6酰氨基、硝基、氰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 6-C 10芳基或五元或六元杂芳基、3-12元杂环基、3-12元环烷基，优选为卤素原子、C 1-C 6烷氧基羰基、C 1-C 6烷基、卤代C 1-C 6烷基、C 1-C 6烷氧基或苯基、5-6元杂环基； Said substitution means that one or more hydrogen atoms on the group are replaced by a substituent selected from the group consisting of halogen atom, carbonyl (=O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group, substituted or unsubstituted C 1 -C 10 alkane base, substituted or unsubstituted C 2 -C 10 alkenyl, substituted or unsubstituted C 2 -C 10 alkynyl, substituted or unsubstituted C 6 -C 10 aryl, or substituted or unsubstituted five-membered or Six-membered heteroaryl, 3-12-membered heterocyclic, 3-12-membered cycloalkyl, preferably C 1 -C 6 alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy Or C 1 -C 6 alkylamino; wherein, the substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 6 -C 10 aryl or the substitution of five- or six-membered heteroaryl The group is selected from the group consisting of halogen atom, carbonyl group (=O), hydroxyl group, carboxyl group, C 1 -C 6 alkoxycarbonyl group, amino group, C 1 -C 6 amido group, nitro group, cyano group, C 1 -C 6 group Alkyl, halogenated C 1 -C 6 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 6 -C 10 aryl or five- or six-membered heteroaryl, 3-12 membered heterocyclic group, 3-12 membered cycloalkyl group, preferably halogen atom, C 1 -C 6 alkoxycarbonyl group, C 1 -C 6 alkyl group, halogenated C 1 -C 6 alkyl group, C 1 -C 6 alkyl group 6 alkoxy or phenyl, 5-6 membered heterocyclic group;
    其中，所述的式(I)化合物与免疫检查点抑制剂共同用于制备治疗或预防实体瘤的药物组合物。Wherein, the compound of formula (I) is used together with an immune checkpoint inhibitor to prepare a pharmaceutical composition for treating or preventing solid tumors.
  2. 如权利要求1所述的化合物，及其药学上可接受的盐，水合物，溶剂合物或前药的用途，其中，所述的式(I)化合物具有如下式(II)所示的结构：The compound of claim 1, and the use of pharmaceutically acceptable salts, hydrates, solvates or prodrugs thereof, wherein the compound of formula (I) has the structure shown in the following formula (II) :
    Figure PCTCN2022075561-appb-100002
    Figure PCTCN2022075561-appb-100002
    其中，in,
    A 1、A 2、A 3、A 4各自独立地为CR’或N； A 1 , A 2 , A 3 , A 4 are each independently CR' or N;
    M选自下组：NH或S；M is selected from the group: NH or S;
    R’选自下组：H、卤素、羟基、氨基、硝基、氰基、取代或未取代的C 1-C 10烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基、取代或未取代的C 1-C 6酰氨； R' is selected from the group consisting of H, halogen, hydroxy, amino, nitro, cyano, substituted or unsubstituted C 1 -C 10 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or Unsubstituted C 1 -C 6 alkylamino, substituted or unsubstituted C 1 -C 6 amido;
    R 0选自下组：氢、羟基、取代或非取代的C 1-C 10烷基、
    Figure PCTCN2022075561-appb-100003
    其中，R a，R b各自独立地选自下组：氢、取代或未取代的C 1-C 6烷基，取代或未取代的C 3-C 10环烷基(包括单环、多环、桥环结构)、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基；     R 0 is selected from the group consisting of hydrogen, hydroxy, substituted or unsubstituted C 1 -C 10 alkyl,
    Figure PCTCN2022075561-appb-100003
    Wherein, R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic , bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
    m选自下组：0、1、2、3或4；m is selected from the group consisting of 0, 1, 2, 3 or 4;
    R x、R y各自独立地选自下组：卤素、取代或非取代的C 1-C 4烷基； R x , R y are each independently selected from the group consisting of halogen, substituted or unsubstituted C 1 -C 4 alkyl;
    Het选自下组：取代或未取代的C 6-C 10的芳基、取代或未取代的4-7元饱和杂环基、取代或未取代3-12元杂环基； Het is selected from the group consisting of substituted or unsubstituted C6 - C10 aryl, substituted or unsubstituted 4-7 membered saturated heterocyclyl, substituted or unsubstituted 3-12 membered heterocyclyl;
    所述的取代指基团上的一个或多个氢原子被选自下组的取代基取代：卤素、氧原子(＝O)、羧基、羟基、氨基、硝基、氰基、C 1-C 6烷氧基、C 1-C 6烷氨基、C 1-C 6烷氧基羰基、C 1-C 6酰氨基、C 2-C 12酯基；或未取代或被一个或多个卤素或C 1-C 6烷基取代的选自下组的基团：C 1-C 10烷基，C 2-C 10烯基，C 2-C 10炔基，C 6-C 10芳基、5-6元杂芳基、3-12元杂环基、3-12元环烷基。 Said substitution refers to the substitution of one or more hydrogen atoms on a group by a substituent selected from the group consisting of halogen, oxygen atom (=O), carboxyl, hydroxyl, amino, nitro, cyano, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, C 1 -C 6 alkoxycarbonyl, C 1 -C 6 amido, C 2 -C 12 ester group; or unsubstituted or by one or more halogen or C 1 -C 6 alkyl substituted group selected from the group consisting of C 1 -C 10 alkyl, C 2 -C 10 alkenyl, C 2 -C 10 alkynyl, C 6 -C 10 aryl, 5 -6-membered heteroaryl, 3-12-membered heterocyclyl, 3-12-membered cycloalkyl.
  3. 如权利要求1所述的用途，其特征在于，所述的R 0具有如下式所示的结构： The use according to claim 1, wherein the R 0 has the structure shown in the following formula:
    Figure PCTCN2022075561-appb-100004
    Figure PCTCN2022075561-appb-100004
    其中，in,
    R a，R b各自独立地选自下组：氢、取代或未取代的C 1-C 6烷基，取代或未取代的C 3-C 10环烷基(包括单环、多环、桥环结构)、取代或未取代的C 2-C 6烯基、取代或未取代的C 2-C 6炔基； R a , R b are each independently selected from the group consisting of hydrogen, substituted or unsubstituted C 1 -C 6 alkyl, substituted or unsubstituted C 3 -C 10 cycloalkyl (including monocyclic, polycyclic, bridged ring structure), substituted or unsubstituted C 2 -C 6 alkenyl, substituted or unsubstituted C 2 -C 6 alkynyl;
    m选自以下组：1、2或3。m is selected from the group: 1, 2 or 3.
  4. 如权利要求1所述的用途，其特征在于，所述的Het选自下组：取代或未取代的吡啶、取代或未取代的四氮唑、取代或未取代的三氮唑、取代或未取代的嘧啶、取代或未取代的吡唑、取代或未取代的异恶唑、取代或未取代的吗啉、取代或未取代的硫代吗啉、取代或未取代的哌啶、取代或未取代的哌嗪、取代或未取代的氧杂环丁烷、取代或未取代的硫杂环丁烷、取代或未取代的氮杂环丁烷。The use according to claim 1, wherein the Het is selected from the group consisting of substituted or unsubstituted pyridine, substituted or unsubstituted tetrazolium, substituted or unsubstituted triazole, substituted or unsubstituted pyridine Substituted pyrimidine, substituted or unsubstituted pyrazole, substituted or unsubstituted isoxazole, substituted or unsubstituted morpholine, substituted or unsubstituted thiomorpholine, substituted or unsubstituted piperidine, substituted or unsubstituted Substituted piperazine, substituted or unsubstituted oxetane, substituted or unsubstituted thietane, substituted or unsubstituted azetidine.
  5. 如权利要求1所述的用途，其特征在于，所述的Het选自下组：The purposes of claim 1, wherein the Het is selected from the group consisting of:
    Figure PCTCN2022075561-appb-100005
    Figure PCTCN2022075561-appb-100005
    其中，各个R 1、R 2、R 3或R 4各自独立地选自下组：氢、卤素、羟基、氨基、氧原子(＝O)、羧基(COOH)、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 6-C 10芳基、取代或未取代的5-6元杂芳基、取代或未取代的C 1-C 6烷氨基、取代或未取代的C 1-C 6烷氧基羰基、取代或未取代的C 1-C 6酰氨基；其中，所述的取代基选自下组：F、Cl、C 1-C 6烷基。 wherein each R 1 , R 2 , R 3 or R 4 is each independently selected from the group consisting of hydrogen, halogen, hydroxyl, amino, oxygen atom (=O), carboxyl (COOH), substituted or unsubstituted C 1 - C 6 alkyl, substituted or unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 6 -C 10 aryl, substituted or unsubstituted 5-6 membered heteroaryl, substituted or unsubstituted C 1 -C 6 alkylamino group, substituted or unsubstituted C 1 -C 6 alkoxycarbonyl group, substituted or unsubstituted C 1 -C 6 amido group; wherein, the substituent is selected from the following group: F, Cl, C 1 -C 6 alkyl.
  6. 如权利要求5所述的用途，其特征在于，各个R 1，R 2，R 3，R 4各自独立地选自下组：H、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基。 The use of claim 5, wherein each R 1 , R 2 , R 3 , R 4 is independently selected from the group consisting of H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or Unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino.
  7. 如权利要求6所述的用途，其特征在于，各个R 1，R 2，R 3，R 4各自独立地选自下组：H、取代或未取代的C 1-C 6烷基、取代或未取代的C 1-C 6烷氧基、取代或未取代的C 1-C 6烷氨基。 The use of claim 6, wherein each R 1 , R 2 , R 3 , R 4 is independently selected from the group consisting of H, substituted or unsubstituted C 1 -C 6 alkyl, substituted or Unsubstituted C 1 -C 6 alkoxy, substituted or unsubstituted C 1 -C 6 alkylamino.
  8. 如权利要求1所述的用途，其特征在于，所述的A 2、A 3各自独立地为CR'。 The use according to claim 1, wherein the A 2 and A 3 are each independently CR'.
  9. 如权利要求1所述的用途，其特征在于，所述的A 1、A 2、A 3、A 4各自独立地为CR'。 The use according to claim 1, wherein said A 1 , A 2 , A 3 and A 4 are each independently CR'.
  10. 如权利要求1所述的用途，其特征在于，所述的R'为H或
    Figure PCTCN2022075561-appb-100006
    其中，所述的R”为H或取代或未取代的C1-C6烷基。     The use according to claim 1, wherein the R' is H or
    Figure PCTCN2022075561-appb-100006
    Wherein, the R" is H or a substituted or unsubstituted C1-C6 alkyl group.
  11. 如权利要求1所述的用途，其特征在于，所述的式(I)化合物选自下组：The use according to claim 1, wherein the compound of formula (I) is selected from the group consisting of:
    Figure PCTCN2022075561-appb-100007
    Figure PCTCN2022075561-appb-100007
    Figure PCTCN2022075561-appb-100008
    Figure PCTCN2022075561-appb-100008
  12. 如权利要求1所述的用途，其特征在于，所述的免疫检查点抑制剂选自下组：抗PD-L1抗体、抗PD-1抗体，或其组合。The use of claim 1, wherein the immune checkpoint inhibitor is selected from the group consisting of an anti-PD-L1 antibody, an anti-PD-1 antibody, or a combination thereof.
  13. 如权利要求1所述的用途，其特征在于，所述的实体瘤选自下组：黑色素瘤、肺癌、结肠癌、肾癌、胰腺癌、肺癌、骨肉瘤。The use of claim 1, wherein the solid tumor is selected from the group consisting of melanoma, lung cancer, colon cancer, kidney cancer, pancreatic cancer, lung cancer, and osteosarcoma.
  14. 一种化合物，或其药学上可接受的盐，水合物，溶剂合物或前药，其特征在于，所述的化合物选自下组：A compound, or a pharmaceutically acceptable salt, hydrate, solvate or prodrug thereof, characterized in that the compound is selected from the group consisting of:
    Figure PCTCN2022075561-appb-100009
    Figure PCTCN2022075561-appb-100009
PCT/CN2022/075561 2021-02-08 2022-02-08 Anti-tumor pharmaceutical combination WO2022166990A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202110184609.9A CN114907267A (en) 2021-02-08 2021-02-08 Pharmaceutical composition for resisting tumor
CN202110184609.9 2021-02-08

Publications (1)

Publication Number Publication Date
WO2022166990A1 true WO2022166990A1 (en) 2022-08-11

Family

ID=82742006

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2022/075561 WO2022166990A1 (en) 2021-02-08 2022-02-08 Anti-tumor pharmaceutical combination

Country Status (2)

Country Link
CN (1) CN114907267A (en)
WO (1) WO2022166990A1 (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000041505A2 (en) * 1999-01-13 2000-07-20 Warner-Lambert Company Anthranilic acid derivatives
CN102348689A (en) * 2009-03-13 2012-02-08 奥米罗有限公司 Addition salts of amines containing hydroxyl and/or carboxylic groups with amino nicotinic acid derivatives as DHODH inhibitors
CN104069092A (en) * 2014-07-14 2014-10-01 中国科学院上海药物研究所 Use of 2-(substituted phenylamino) benzoic acid and ester compound thereof in preparation of FTO (Fat Mass and Obesity-Associated Protein) inhibitor
WO2018157843A1 (en) * 2017-03-02 2018-09-07 中国科学院上海药物研究所 2-(substituted benzene matrix) aromatic formate fto inhibitor, preparation method therefor, and applications thereof
WO2019204257A1 (en) * 2018-04-16 2019-10-24 Arrys Therapeutics, Inc. Ep4 inhibitors and use thereof

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3835323A1 (en) * 2014-10-29 2021-06-16 Five Prime Therapeutics, Inc. Combination therapy for cancer
RU2765785C2 (en) * 2017-05-24 2022-02-02 Аббиско Тхерапеутицс Цо., Лтд. N-(azaaryl)cyclolactam-1-carboxamide derivative, its preparation method and its application
CA3075638A1 (en) * 2017-09-25 2019-03-28 Chemocentryx, Inc. Combination therapy using a chemokine receptor 2 (ccr2) antagonist and a pd-1/pd-l1 inhibitor
AU2018101488A4 (en) * 2018-10-05 2018-11-08 Macau University Of Science And Technology Method and kit for treating cancer through combination therapy

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000041505A2 (en) * 1999-01-13 2000-07-20 Warner-Lambert Company Anthranilic acid derivatives
CN102348689A (en) * 2009-03-13 2012-02-08 奥米罗有限公司 Addition salts of amines containing hydroxyl and/or carboxylic groups with amino nicotinic acid derivatives as DHODH inhibitors
CN104069092A (en) * 2014-07-14 2014-10-01 中国科学院上海药物研究所 Use of 2-(substituted phenylamino) benzoic acid and ester compound thereof in preparation of FTO (Fat Mass and Obesity-Associated Protein) inhibitor
WO2018157843A1 (en) * 2017-03-02 2018-09-07 中国科学院上海药物研究所 2-(substituted benzene matrix) aromatic formate fto inhibitor, preparation method therefor, and applications thereof
WO2019204257A1 (en) * 2018-04-16 2019-10-24 Arrys Therapeutics, Inc. Ep4 inhibitors and use thereof

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
HUANG YUE, ET AL: "Small-Molecule Targeting of Oncogenic FTO Demethylase in Acute Myeloid Leukemia", CANCER CELL, CELL PRESS, US, vol. 35, no. 4, 1 April 2019 (2019-04-01), US , pages 677 - 691.e10, XP055957252, ISSN: 1535-6108, DOI: 10.1016/j.ccell.2019.03.006 *
WWW, ET AL: "Chemical Science A strategy based on nucleotide specificity leads to a subfamily-selective and cell-active inhibitor of N 6 -methyladenosine demethylase FTO", CHEMICAL SCIENCE, 1 January 2015 (2015-01-01), pages 112 - 122, XP055957255, Retrieved from the Internet <URL:https://pubs.rsc.org/en/content/articlepdf/2015/sc/c4sc02554g> [retrieved on 20220902] *

Also Published As

Publication number Publication date
CN114907267A (en) 2022-08-16

Similar Documents

Publication Publication Date Title
CN111138301A (en) Biphenyl compound, intermediate thereof, preparation method, pharmaceutical composition and application
JP4405602B2 (en) Histone deacetylase inhibitor
WO2021129824A1 (en) New-type k-ras g12c inhibitor
WO2020259432A1 (en) Kras-g12c inhibitor
CN108524482B (en) Use of 2- (substituted phenylamino) benzoic acid FTO inhibitors for treating leukemia
TW202026271A (en) Small molecule pd-1/pd-l1 inhibitor, pharmaceutical composition thereof with pd-l1 antibody and application thereof
JP2005521698A (en) New tricyclic compounds
CN111051300B (en) Novel heteroaryl amide derivatives as selective inhibitors of histone deacetylase 1 and/or 2 (HDAC 1-2)
JP2022126780A (en) Diaryl macrocyclic compound, pharmaceutical composition, and use thereof
WO2012031563A1 (en) Heterocyclic amino berbamine derivatives, preparation method and use thereof
JP2022527892A (en) Pharmaceutical composition with small molecule PD-1 / PD-L1 inhibitor, PD-L1 antibody and its use
CN111303121A (en) 4-phenoxypyridine compound containing quinoxalinone and application thereof
CN114516832A (en) Tubulin inhibitor and preparation method and application thereof
KR102238012B1 (en) Carboline derivatives, bromodomain inhibitors
CN110240587B (en) Aryl difluoro benzyl ether compounds, preparation method and application
CN107056789B (en) With substitution pyrazine and imidazole derivative, preparation and its application in medicine
CN113924288B (en) Quinolinyl-containing compound, pharmaceutical composition and application thereof
CN115304603A (en) Preparation and application of quinazoline inhibitor
WO2021143729A1 (en) Compound, composition and functional molecule with multi-target inhibiting effect and use thereof
WO2020173417A1 (en) Acryloyl-containing nuclear transport regulator and uses thereof
WO2022166990A1 (en) Anti-tumor pharmaceutical combination
JP2024516194A (en) Compounds as PD1/PD-L1 inhibitors and methods thereof
JP2002322162A (en) Thiazolidine derivative
WO2022122044A1 (en) Heterocyclic compound serving as gls1 inhibitor
CN107793360B (en) Indoleamine 2, 3-dioxygenase inhibitor and application thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 22749269

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 22749269

Country of ref document: EP

Kind code of ref document: A1