WO2023198076A1 - 一种环肽类化合物及其应用 - Google Patents

一种环肽类化合物及其应用 Download PDF

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WO2023198076A1
WO2023198076A1 PCT/CN2023/087687 CN2023087687W WO2023198076A1 WO 2023198076 A1 WO2023198076 A1 WO 2023198076A1 CN 2023087687 W CN2023087687 W CN 2023087687W WO 2023198076 A1 WO2023198076 A1 WO 2023198076A1
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group
ring
compound
substituted
unsubstituted
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PCT/CN2023/087687
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English (en)
French (fr)
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杨财广
张涛
王蓬宇
魏柄妍
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中国科学院上海药物研究所
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Publication of WO2023198076A1 publication Critical patent/WO2023198076A1/zh

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    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4985—Pyrazines or piperazines ortho- or peri-condensed with heterocyclic ring systems
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/4995—Pyrazines or piperazines forming part of bridged ring systems
    • 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
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/519—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with heterocyclic rings
    • 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
    • A61K31/53—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with three nitrogens as the only ring hetero atoms, e.g. chlorazanil, melamine
    • 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
    • A61K31/5377—1,4-Oxazines, e.g. morpholine not condensed and containing further heterocyclic rings, e.g. timolol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/04—Antibacterial agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D471/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00
    • C07D471/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, at least one ring being a six-membered ring with one nitrogen atom, not provided for by groups C07D451/00 - C07D463/00 in which the condensed system contains two hetero rings
    • C07D471/08—Bridged systems
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems

Definitions

  • the present invention relates to the field of medicinal chemistry, specifically to a cyclic peptide compound or a pharmaceutically acceptable salt or stereoisomer thereof, and its use.
  • ClpP protease is an important component of the Clp hydrolysis system. It is an ATP-dependent proteolytic enzyme containing a serine protease catalytic triad domain. It is usually combined with ClpX, a molecular chaperone of the AAA + family, to form a ClpXP protease complex, which can unfold abnormal or misfolded protein substrates in bacteria, and then transfer the substrate molecules to the hydrolysis cavity of ClpP protease for degradation, regulating the bacterial interior. The stability and activity of protein functions, thereby affecting various biological functions of bacteria. Small molecules can effectively inhibit bacterial growth by regulating ClpP function. In addition, studies have shown that activation and inhibition of mitochondrial ClpP function are related to various functions of organisms.
  • Inhibitors of ClpP can occupy the binding pocket between ClpP and the chaperone protein ClpX, thereby inhibiting ClpXP from functioning normally. Studies have shown that small molecule inhibitors of ClpP can affect the formation of bacterial biofilms and change the sensitivity of bacteria to temperature and oxygen content. Inhibition of ClpP function in mitochondria can effectively kill acute myeloid leukemia cells and has no effect on normal cells.
  • ClpP small molecule agonists can trigger the remodeling of the ClpP protease complex structure and rearrange the core structure of ClpP protease, causing the protein to escape the regulation of the normal mechanism and degrade a large number of certain intracellular functional proteins. Studies have proven that small molecule agonists of ClpP can inhibit bacterial growth.
  • the purpose of the present invention is to provide novel small molecule modulators of ClpP.
  • Y is selected from the following group: N or CR 8 ;
  • s 1 or 2;
  • X 1 and X 2 are each independently selected from the following group: O, S, CH 2 or NH;
  • R1 is selected from the group consisting of: H, halogen, unsubstituted C1-C10 alkyl, C1-C4 alkyl substituted by one or more Ra groups (1, 2, 3, 4, 5 or more), C1-C4 haloalkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 alkoxy, C1-C6 alkylthio, -NRbRc, C3-C8 cycloalkyl, C3-C8 heterocyclyl, un Substituted C6-C10 aryl, C5-C8 heteroaryl, or C6-C10 aryl substituted by one or more Ra groups (1, 2, 3, 4, 5 or more); wherein, the The Ra group is selected from the following group: halogen, C1-C4 haloalkyl (preferably fluorinated), C1-C4 alkyl, C1-C4 alkoxy, cyano, amino, or two adjacent Ra groups with their
  • A is selected from unsubstituted C1-C8 alkylene, C1-C4 alkylene substituted by one or more Rd groups, or C1-C8 alkenylene (including monoalkenylene and dialkenylene); And when L is -C(O)-A-, the Rd group is selected from amino or -NHBoc;
  • R 2 is a group selected from the following group:
  • R 7 is selected from the following group: H, CH 3 , F, Cl, Br, I, OH, SH, NH 2 , CN, CF 3 , OCH 3 , SCH 3 , NHCH 3 , N(CH 3 ) 2 , CHF 2. OC 2 H 5 , COOH, CONH 2 ;
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; q is selected from the following group: 0, 1, 2, 3, 4 or 5;
  • R 3 and R 3 ' are each independently selected from the following group: H, halogen, substituted or unsubstituted C1-C4 alkyl, substituted or unsubstituted C2-C6 alkenyl, substituted or unsubstituted C2-C6 alkynyl ; Or R 3 and R 3 ' and their connected carbon atoms together constitute a substituted or unsubstituted saturated, partially unsaturated or aromatic 5-8 membered carbocyclic ring or heterocyclic ring;
  • R 8 is H or C1-C4 alkyl, or R 8 and R 3 together constitute a C1-C4 alkylene group;
  • Ring A and Ring B are each independently a substituted or unsubstituted saturated, partially unsaturated/or aromatic 5-14 membered carbocyclic ring or heterocyclic ring (including single ring, fused ring or spiro ring), and Ring A and Ring B is fused to form a polycyclic fused ring; wherein R 4 is optionally substituted by Re selected from halogen, C1-C4 alkyl, C1-C4 alkoxy;
  • R 5 is selected from H, C1-C3 alkyl
  • substituted means that one or more hydrogen atoms on the group are replaced with a substituent selected from the following group: halogen, oxo, carboxyl, cyano, hydroxyl, phenyl, 5-6 membered heterocycle Base, C1-C6 alkyl, C1-C6 alkoxy, C3-C8 cycloalkyl substitution, amino, N, N-dimethylamino, mercapto, C 1 -C 6 sulfone group.
  • the compound has the structure shown in the following formula II:
  • X 3 is selected from O or S;
  • X 1 , X 2 , R 1 , R 2 , R 3 , R 3 ', R 4 , R 5 , Y, and A are as described above.
  • X 3 is O.
  • Y is N.
  • X 1 and X 2 are each independently O.
  • R 3 and R 3 ' are each independently H or C1-C4 alkyl, preferably H.
  • R 1 is selected from H, halogen, unsubstituted C1-C10 alkyl, C1-C4 haloalkyl, C3-C8 cycloalkyl, C3-C8 heterocyclyl, unsubstituted C6-C10 Aryl, or C6-C10 aryl substituted by one or more Ra groups (1, 2, 3, 4, 5 or more).
  • the Ra group is selected from the following group: halogen, C1-C4 haloalkyl (preferably fluorinated), C1-C4 alkyl, C1-C4 alkoxy, cyano group, amino, or Two adjacent Ra groups and the atoms to which they are connected together form a 5-7 membered carbocyclic ring or heterocyclic ring; and when L is -C(O)NH-CH 2 -, R 1 is not an unsubstituted phenyl group.
  • R 1 is not unsubstituted phenyl.
  • R 1 is selected from H, halogen, C1-C4 haloalkyl, C3-C8 cycloalkyl.
  • the haloalkyl group is preferably a fluoroalkyl group or a chloroalkyl group.
  • A is C1-C8 alkylene, preferably C1-C4 alkylene, more preferably -(CH 2 ) 1-4 -.
  • R 2 is a group selected from the following group:
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10; q is selected from the following group: 0, 1, 2, 3, 4 or 5;
  • R 7 is selected from the following group: H, CH 3 , F, Cl, Br, I, OH, SH, NH 2 , CN, CF 3 , OCH 3 , SCH 3 .
  • R 2 is not
  • n 0 or 1.
  • R 2 is a group selected from the following group:
  • n 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10.
  • R 4 is wherein, Ring A and Ring B are each independently a substituted or unsubstituted aromatic 5-14 membered carbocyclic ring or heterocyclic ring (including a single ring, a fused ring or a spiro ring), and Ring A and Ring B are fused to form a bicyclic ring Fused ring or tricyclic fused ring.
  • the compound of formula I is selected from the following group:
  • the compound further includes a compound selected from the following group:
  • composition comprising the following components:
  • the second therapeutic agent is an antibacterial drug.
  • the second therapeutic agent is selected from the group consisting of ciprofloxacin, rifampicin, linezolid, or a combination thereof.
  • the third aspect of the present invention there is provided a use of the compound described in the first aspect of the present invention, or a pharmaceutically acceptable salt or stereoisomer thereof, for the preparation of prevention and/or treatment of ClpP protease activity Or pharmaceutical compositions for diseases or conditions related to expression levels.
  • the diseases or disorders related to ClpP protease activity or expression level are diseases or disorders caused by infection.
  • the disease or condition associated with ClpP protease activity or expression is cancer.
  • the infection is a bacterial infection.
  • the bacterial infection is a Gram-positive bacterial infection.
  • the bacterium is a Gram-positive bacterium: preferably, the bacterium is selected from the following group: Staphylococcus aureus, methicillin-resistant Staphylococcus aureus, penicillin-resistant Streptococcus pneumoniae, vancomycin-resistant Staphylococcus aureus Staphylococcus aureus, Streptococcus pneumoniae, Enterococcus faecalis, Streptococcus pyogenes.
  • the infection can be caused by one or more bacteria at the same time.
  • the treatment includes using the pharmaceutical composition alone or in combination with commonly used antibacterial drugs.
  • the cancer is selected from the group consisting of glioma, solid tumor, leukemia, or a combination thereof.
  • the solid tumor is selected from the following group: kidney cancer, liver cancer, prostate cancer, melanoma, colon cancer, lung cancer, pancreatic cancer, ovarian cancer, breast cancer, gastric cancer, brain cancer, rectal cancer, Melanoma, ovarian cancer, or combinations thereof.
  • the glioma is selected from the group consisting of glioma, brain glioma, astrocytoma, or a combination thereof.
  • Figure 1 shows the test results of the compound in Biological Test Example 3 in combination with rifampicin and ciprofloxacin.
  • Figure 2 shows the test results of compound A28 combined with rifampicin and ciprofloxacin in biological test example 3.
  • Figure 3 shows the test results of compound B29 combined with rifampicin and ciprofloxacin in biological test example 3.
  • the cyclic peptide compound can be used as a regulator of ClpP protease to treat pathogenic bacterial infections, such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus infections, etc., and is therefore used in the prevention and treatment of bacterial infection diseases.
  • pathogenic bacterial infections such as Staphylococcus aureus, methicillin-resistant Staphylococcus aureus infections, etc.
  • alkyl includes straight or branched chain alkyl groups.
  • C1-C6 alkyl represents a linear or branched chain alkyl group with 1 to 6 carbon atoms, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, etc.
  • alkylene refers to a straight or branched chain alkane with two bonds substituted, for example, C1-C4 alkylene means a straight or branched chain C1-C4 with two bonds substituted.
  • Alkanes such as methylene, ethylene, n-propylene, isopropylene, butylene, etc.
  • alkenyl includes straight or branched chain alkenyl groups.
  • C2-C6 alkenyl refers to a straight-chain or branched alkenyl group with 2-6 carbon atoms, such as vinyl, allyl, 1-propenyl, isopropenyl, 1-butenyl, 2-butenyl alkenyl, or similar groups.
  • alkenylene refers to a straight or branched chain alkene in which two bonds are substituted.
  • the olefins may include monoolefins and dienes, such as conjugated dienes.
  • C2-C6 alkenylene represents a linear or branched C2-C6 olefin with two substituted bonds, such as vinylene, n-propenylene, isopropenylene, butenylene, 1,3-butylene Dialkenyl etc.
  • alkynyl includes straight or branched chain alkynyl groups.
  • C2-C6 alkynyl refers to a straight-chain or branched-chain alkynyl group with 2-6 carbon atoms, such as ethynyl, propynyl, butynyl, or similar groups.
  • C1-C6 alkoxy refers to a straight or branched chain alkoxy group having 1 to 6 carbon atoms; for example, methoxy, ethoxy, propoxy, isopropoxy base, butoxy group, isobutoxy group, tert-butoxy group, etc.
  • C1-C6 alkylthio refers to -S- (straight or branched chain alkyl having 1 to 6 carbon atoms); for example, methylthio, ethylthio, propylthio , isopropylthio, butylthio, isobutylthio, tert-butylthio, etc.
  • C3-C8 cycloalkyl refers to a cycloalkyl group having 3 to 8 carbon atoms. It may be a monocyclic ring, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, or similar groups. Bicyclic forms, such as bridged or spirocyclic forms, are also possible.
  • C3-C8 heterocyclyl refers to a saturated or partially saturated cyclic group having 3 to 8 atoms, of which 1 to 3 atoms are heteroatoms selected from N, S, and O. . It can be a single ring or a double ring, such as a bridged ring or a spiro ring. Specific examples may be oxetane, azetidine, tetrahydro-2H-pyranyl, piperidinyl, tetrahydrofuranyl, morpholinyl, pyrrolidinyl, and the like.
  • C6-C10 aryl refers to an aryl group having 6 to 10 carbon atoms, for example, phenyl or naphthyl and the like.
  • C5-C8 heteroaryl refers to a cyclic aromatic group having 5-8 atoms, of which 1-3 atoms are heteroatoms selected from N, S, and O. It can be a single ring or a fused ring. Specific examples may be pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, pyrrolyl, pyrazolyl, imidazolyl, (1,2,3)-triazolyl and (1,2, 4)-Triazolyl, tetrazolyl, furyl, thienyl, isoxazolyl, thiazolyl, oxazolyl, etc.
  • the groups described in the present invention can be substituted with substituents selected from the following group: halogen, nitrile group, nitro group, hydroxyl group, amino group , C1-C6 alkyl-amino group, C1-C6 alkyl group, C2-C6 alkenyl group, C2-C6 alkynyl group, C1-C6 alkoxy group, halogenated C1-C6 alkyl group, halogenated C2-C6 alkenyl group , Halogenated C2-C6 alkynyl, Halogenated C1-C6 alkoxy, allyl, benzyl, C6-C12 aryl, C1-C6 alkoxy-C1-C6 alkyl, C1-C6 alkoxy -Carbonyl, phenoxycarbonyl, C2-C6 alkynyl-carbonyl, C2-C6 alkenyl-carbony
  • halogen refers to F, Cl, Br, and I.
  • haloalkyl refers to a C1-C3 alkyl group substituted by halogen.
  • the haloalkyl group is trifluoromethyl, difluoromethyl, or trifluoromethoxy.
  • C1-C3 alkyl refers to a straight-chain or branched alkyl group with 1 to 3 carbon atoms, such as methyl, ethyl, n-propyl, and isopropyl.
  • the structural formulas described in the present invention are intended to include all isomeric forms (such as enantiomers, diastereomers and geometric isomers (or conformational isomers)): for example, containing asymmetric The R and S configurations of the center, the (Z) and (E) isomers of the double bond, etc. Therefore, individual stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers or geometric isomers (or conformational isomers) are within the scope of the present invention.
  • enantiomers means isomers of compounds with the same molecular formula that are mirror images of each other due to differences in the arrangement of atoms in space. For example, R and S configurations containing asymmetric centers.
  • the active ingredient structure of the present invention is designed through the recognition mechanism based on the crystal structure of the complex of small molecules and ClpP, that is: the small molecule regulator is composed of a "rigid skeleton" and two parts of “side arms". Under the support of the rigid skeleton, the two "side arms" of the molecule bind to the two hydrophobic pockets of the ClpP protein respectively, regulating the activity of ClpP and affecting various biological functions. This part of the rigid skeleton structure is different from the skeletons of ClpP-related regulators reported in the existing literature. By optimizing the "skeleton" and "side arm” structures, the present invention further obtains small molecules with different regulatory activities for the ClpP protein. Molecular compounds.
  • the compounds involved in the present invention are all designed based on the molecular recognition mechanism of small molecules and ClpP protein, and the sources of their structural design are well documented.
  • compounds of the present invention refer to compounds of formula I and other compounds listed in the present invention that have ClpP-modulating effects, and also include various pharmaceutically acceptable salts or stereoisomers of the above compounds.
  • Preferred compounds of the present invention include compounds (including various R-configuration and/or S-configuration stereoisomers of each compound, and/or E-/Z-cis-trans isomers).
  • the pharmaceutically acceptable salts include salts formed by combining with inorganic acids, organic acids, alkali metal ions, alkaline earth metal ions or organic bases that can provide physiologically acceptable cations, and ammonium salts.
  • the inorganic acid is selected from hydrochloric acid, hydrobromic acid, phosphoric acid or sulfuric acid;
  • the organic acid is selected from methanesulfonic acid, p-toluenesulfonic acid, trifluoroacetic acid, citric acid, maleic acid Tartaric acid, fumaric acid, citric acid or lactic acid;
  • the alkali metal ions are selected from lithium ions, sodium ions and potassium ions;
  • the alkaline earth metal ions are selected from calcium ions and magnesium ions; the alkali metal ions can provide physiologically reliable
  • the organic base that accepts the cation is selected from methylamine, dimethylamine, trimethylamine, piperidine, morpholine or tris(2-hydroxyethyl)amine.
  • the starting materials and intermediates in the preparation process of the compounds of the present invention are easy to obtain, and each step of the reaction can be easily synthesized based on reported literature or by conventional methods in organic synthesis for those skilled in the art.
  • the compounds of formula I may exist in the form of solvates or unsolvates, and different solvates may be obtained by crystallization using different solvents.
  • the present invention provides methods for synthesizing compounds of formula IA, analogs thereof, and pharmaceutically acceptable salts thereof.
  • the synthetic route of this compound is as follows:
  • step 1 The synthesis of the compounds begins with the commercially available starting materials ⁇ -naphthaldehyde and aminoacetal.
  • compound I1 and compound I2 are refluxed and dehydrated at 100°C to form Schiff base I3 (step 1).
  • step 2 Compound I4 is obtained by reductive amination (step 2), which is further condensed with an Fmoc-protected amino acid to obtain compound I5 (step 3).
  • step 4 The free primary amine I6 is obtained by removing Fmoc under ethylamine conditions (step 4).
  • step 4 Then compound I6 is condensed with Fmoc-protected ⁇ -alanine to form compound I7 (step 5).
  • step 6 deprotection obtains the key intermediate compound I9 (step 7), and the intermediate I9 reacts with isocyanate to obtain the compound of formula IA (step 8).
  • compositions and methods of administration are provided.
  • the compound of the present invention has excellent ClpP regulating activity
  • the compound of the present invention and its pharmaceutically acceptable inorganic or organic salts, or stereoisomers, as well as pharmaceutical compositions containing the compound of the present invention as the main active ingredient can be used for prevention and/or treat (stabilize, alleviate, or cure) ClpP-related diseases (e.g., infectious diseases, cancer).
  • ClpP-related diseases e.g., infectious diseases, cancer
  • the pharmaceutical composition of the present invention contains a compound of the present invention and a pharmaceutically acceptable excipient or carrier within a safe and effective amount.
  • the “safe and effective dose” refers to the amount of compound that 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/dose, more preferably, it contains 10-200 mg of the compound of the present invention/dose.
  • the "dose" is a capsule or tablet.
  • “Pharmaceutically acceptable carrier” refers to one or more compatible solid or liquid filler or gel substances that are suitable for human use and must be of sufficient purity and low enough toxicity. "Compatibility” here means that the components of the composition can be blended with the compounds of the present invention and with each other without significantly reducing the efficacy of the compounds.
  • Examples of pharmaceutically acceptable carriers include cellulose and its derivatives (such as sodium carboxymethylcellulose, sodium ethylcellulose, 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, glycerin, mannitol, sorbitol, etc.), emulsifiers (such as Tween ), wetting agents (such as sodium lauryl sulfate), colorants, flavorings, stabilizers, antioxidants, preservatives, pyrogen-free water, etc.
  • cellulose and its derivatives such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • gelatin such as sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.
  • the administration mode of the compounds or pharmaceutical compositions of the present invention is not particularly limited, and representative administration modes include (but are not limited to): oral, parenteral (intravenous, intramuscular or subcutaneous).
  • 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 the following ingredients: (a) fillers or compatibilizers, for example, Starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) Binders, for example, hydroxymethylcellulose, alginate, gelatin, polyvinylpyrrolidone, sucrose and gum arabic; (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) retarder, such as paraffin; (f) Absorption accelerators, such as quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glyceryl mono
  • Solid dosage forms such as tablets, dragees, capsules, pills and granules may be prepared using coatings and shell materials such as enteric casings 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 released in a delayed manner in a certain part of the digestive tract. Examples of embedding components that can be used are polymeric substances and waxy substances. If necessary, the active compounds can 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, isopropyl alcohol, 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.
  • inert diluents conventionally employed in the art, such as water or other solvents, solubilizers and emulsifiers, for example, ethanol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1 , 3-butanediol, dimethylformamide and oils,
  • compositions may 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 may contain, in addition to the active compound, suspending agents, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar or mixtures of these substances and the like.
  • suspending agents for example, 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 contain 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.
  • the compounds of the present invention may be administered alone or in combination with other pharmaceutically acceptable compounds (eg, other therapeutic agents).
  • other pharmaceutically acceptable compounds eg, other therapeutic agents.
  • the pharmaceutical composition When administered in combination, the pharmaceutical composition also includes one or more (2, 3, 4, or more) other pharmaceutically acceptable compounds.
  • One or more (2, 3, 4, or more) of the other pharmaceutically acceptable compounds can be used simultaneously, separately, or sequentially with the compound of the present invention to prevent and/or treat ClpP related diseases.
  • a safe and effective amount of the compound of the present invention is applied to a mammal (such as a human) in need of treatment, and the dosage when administered is a pharmaceutically effective dosage.
  • a mammal such as a human
  • the daily dose is usually 1 to 2000 mg, preferably 20 to 500 mg.
  • the specific dosage should also take into account factors such as the route of administration and the patient's health condition, which are all within the skill of a skilled physician.
  • the compound of the present invention is not modified based on the structure in the existing literature, but is rationally designed through the molecular recognition mechanism based on ClpP protein and small molecule compounds. The molecular structures of this series of compounds are completely different from those currently reported.
  • the compound of the present invention can effectively regulate ClpP, and has both excellent ClpP activating activity and excellent ClpP inhibitory activity;
  • the silica gel for column chromatography was commercial silica gel of 200-300 mesh produced by Sinopharm Chemical Reagent Co., Ltd.; 1 H-NMR, the compounds were separated using Lisui EZ Plus 100D medium pressure preparation instrument.
  • ⁇ -Naphthaldehyde (1.56g, 10.0mmol) and aminoacetal (1.33g, 10.0mmol) were heated to reflux at 100°C for 1 hour, cooled to room temperature, diluted with ethanol (25mL), and sodium borohydride ( 0.76g, 20mmol), stirred at room temperature for 24 hours.
  • ClpP agonists rearrange the core structure of ClpP protease by triggering the remodeling of the ClpP protease complex structure, causing the protein to escape from the regulation of the normal mechanism, thereby non-specifically degrading a large number of certain intracellular functional proteins and inducing bacteria or cancer.
  • Cell death For example, agonists of HsClpP (human mitochondrial ClpP) can stimulate HsClpP to non-specifically degrade normal proteins in cells, leading to cancer cell death and thus treating various tumors such as pancreatic cancer and leukemia.
  • the function of ClpP relies on the interaction with the molecular chaperone protein ClpX.
  • ClpX depolymerizes misfolded proteins in cells and transports them into the hydrolysis cavity of ClpP, degrading proteins and regulating normal processes. Protein homeostasis in cells. Inhibitors of ClpP occupy the binding pocket between ClpP and the chaperone protein ClpX, hindering the interaction between ClpP and ClpX, thereby making it difficult for misfolded proteins in cells to be degraded and cleared, ultimately causing a series of biological phenotypes.
  • inhibitors of SaClpP can inhibit the interaction between SaClpP and SaClpX (Staphylococcus aureus ClpX), causing the accumulation of misfolded proteins in bacteria, inhibiting the production of bacterial virulence factors, and reducing bacterial virulence.
  • the compounds of the invention can simultaneously inhibit and activate ClpP protease.
  • Compound agonist activity test method uses ⁇ -casein (model protein) as the substrate.
  • ClpP can drive ClpX to degrade ⁇ -casein independently of ATP hydrolysis. Therefore, the activity can be further determined by SDS-PAGE.
  • the detection system is 50 ⁇ L.
  • HsClpP protein 10 ⁇ g HsClpP protein, SaClpP or SpClpP (Streptococcus pyogenes ClpP) protein is first dissolved in PD buffer (25mM HEPES-KOH (pH 7.6), 200mM KCl, 5mM MgCl 2 , 1mM EDTA, 10% glycerol and 2mM DTT (dithiothreitol)). Then add the compound, incubate at room temperature for 10 min, add 0.6 mg/mL ⁇ -casein, incubate at 37°C for 2 hours, immediately add 12.5 ⁇ L SDS-PAGE loading buffer, and heat at 100°C for 10 min.
  • PD buffer 25mM HEPES-KOH (pH 7.6), 200mM KCl, 5mM MgCl 2 , 1mM EDTA, 10% glycerol and 2mM DTT (dithiothreitol)
  • GFP-SsrA is a substrate for the interaction of ClpXP (a protein complex composed of ClpP tetradecamer and ClpX hexamer), so the inhibitory activity of small molecules can be verified by SDS-PAGE. If GFP-SsrA is completely degraded, it means that the compound does not have inhibitory activity; otherwise, the compound has inhibitory activity.
  • ClpXP a protein complex composed of ClpP tetradecamer and ClpX hexamer
  • HsClpP 14 HsClpP protein tetradecamer
  • HsClpX 6 HsClpX protein hexamer
  • ATP regeneration system 5mM ATP, 16mM CP (creatine phosphate), 0.032mg/ mL CK (creatine kinase)), 2mM DTT, and 0.78-100 ⁇ M compound.
  • the hydrolysis system is 60 ⁇ L PD buffer, 0.33 ⁇ M SaClpP 14 (SaClpP protein tetradecamer) or SpClpP 14 (SpClpP protein tetradecamer), 0.6mM SaClpX 6 (SaClpX protein hexamer) or SpClpX 6 (SpClpX protein six polymer), 2 ⁇ M GFP-SsrA (substrate for ClpXP interaction), 0.78-100 ⁇ M compound.
  • the function of the ClpP target protein is related to a variety of biological functions. Studies have shown that the functional stimulation and inhibition of bacterial ClpP protein can affect the survival and virulence of bacteria.
  • the bacterial samples provided in the embodiments of the present invention are associated with the ClpP target.
  • Staphylococcus aureus MIC test method Take out the strain from the -80°C refrigerator and culture it overnight on a TSA (tryptone soybean agar) plate for more than 12 hours.
  • NRS-108 gentamicin-resistant
  • NRS-271 linezolid- and ciprofloxacin-resistant
  • Staphylococcus aureus USA300 methicillin-resistant
  • Staphylococcus aureus The single clone of Newman (susceptible bacteria) was in 10 mL TSB (tryptone soybean liquid) medium. Cultivation was carried out overnight at 37°C and 210 rpm to reach the plateau phase. The next day, dilute 1:1000 into fresh TSB medium. Continue culturing at 37°C and 210 rpm until the OD 600 is 0.6, and then dilute the bacterial solution 400 times with fresh TSB culture medium. The compound was diluted with equal-fold gradients in fresh TSB culture medium in advance, with a final concentration of 0.39-50 ⁇ g/ml.
  • the diluted bacterial solution was added to the diluted compound, and then the 96-well plate was placed in a 37°C incubator for static Incubate for 18 hours. Observe the bacterial growth in each well with the naked eye, and the lowest concentration of a compound that does not inhibit bacterial growth is the MIC value of the compound. After the approximate MIC value range of the compound is obtained in the first experiment, the next experiment is continued to optimize under sub-conditions, and finally the best MIC value is obtained.
  • Streptococcus pyogenes MIC test method Take out the strain from the -80°C refrigerator and culture it overnight on a TSA plate for more than 12 hours. Pick a single clone of Streptococcus pyogenes Sp12344 (ATCC Streptococcus pyogenes 12344) obtained from the American Type Culture Collection and place it in 10 mL of THB (Todd Hewitt Broth) medium. Cultivate overnight at 37°C, 210 rpm to plateau phase. The next day, dilute 1:1000 into fresh THB medium. Continue culturing at 37°C and 210 rpm until the OD 600 is 0.6, and then dilute the bacterial solution 400 times with fresh THB culture medium.
  • THB Todd Hewitt Broth
  • the compound was diluted with equal-fold gradients in fresh THB medium in advance, and the final concentration was 0.39-50 ⁇ g/ml.
  • the diluted bacterial solution was added to the diluted compound, and then the 96-well plate was placed in a 37°C incubator for 18 hours. Observe the bacterial growth in each well with the naked eye, and the lowest concentration of a compound that does not inhibit bacterial growth is the MIC value of the compound.
  • Table 2 The results are shown in Table 2 below:

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Abstract

本发明提供了一类ClpP调节剂及其用途。具体地，本发明提供了一类如下式(I)所示的环肽类化合物，及包含其的组合物。所述化合物可以用于制备预防和/或治疗与ClpP蛋白酶活性或表达量相关的疾病或病症的药物组合物。

Description

一种环肽类化合物及其应用 技术领域
本发明涉及药物化学领域，具体地是涉及一种环肽类化合物或其药学上可接受的盐或立体异构体，以及其用途。
 背景技术
ClpP基因首次发现于大肠杆菌中，几乎在所有的物种中都高度保守。ClpP蛋白酶是Clp水解体系重要组成部分，它是一种包含丝氨酸蛋白酶催化三联体结构域的ATP依赖的蛋白水解酶。通常与AAA+家族的分子伴侣ClpX结合形成ClpXP蛋白酶复合物，能够将细菌中异常或者错误折叠的蛋白底物去折叠，随后将底物分子转移至ClpP蛋白酶的水解腔体进行降解，调控细菌内蛋白功能的稳定性和活性，从而影响细菌的多种生物学功能。小分子调控ClpP功能可以有效抑制细菌的生长。此外，研究表明线粒体的ClpP功能激动和抑制与生物体的多种功能相关。
ClpP的抑制剂。ClpP的抑制剂可以占据ClpP与伴侣蛋白ClpX的结合口袋，从而抑制ClpXP发挥正常的生物学功能。研究表明ClpP小分子抑制剂可以影响细菌生物膜的形成，改变细菌对温度和氧含量的敏感性。线粒体中ClpP功能的抑制可以有效杀死急性髓细胞性白血病细胞，并对正常细胞没有影响。
ClpP的激动剂。ClpP小分子激动剂能引发ClpP蛋白酶复合物结构的重塑，重排ClpP蛋白酶的核心结构，使该蛋白脱离正常机制的调控，大量降解胞内某些功能蛋白。研究证明，ClpP的小分子激动剂可以抑制细菌生长。
因此，本领域迫切需要开发新型的具有ClpP抑制或/和ClpP激活的作用的小分子调节剂。
发明内容
本发明的目的就是提供新型的ClpP的小分子调节剂。
在本发明的第一方面，提供了一种如下式I所示的环肽类化合物，或其药学上可以接受的盐或立体异构体：
其中，Y选自下组：N或CR8；
s为1或2；
X1、X2各自独立地选自下组：O、S、CH2或NH；
L选自下组：H、取代或未取代的C1-C10烷基、-C(O)-A-、-C(O)-(CH2)p-C(O)-A-、-C(O)NH-A-、-C(O)NH-(CH2)p-C(O)-A-、-C(O)NH-(CH2)p-C(O)O-A-、-S(O)0-2-A-、-NHC(O)NH-A-、-C(S)-A-、-C(S)NH-A-、-NHC(S)NH-A-、-C(NH)-A-、-C(NH)NH-A-、-NHC(NH)NH-A-；p选自下组：0、1、2、3、4或5；
R1选自下组：H、卤素、未取代的C1-C10烷基、被一个或多个Ra基团(1、2、3、4、5或更多)取代的C1-C4烷基、C1-C4卤代烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6烷硫基、-NRbRc、C3-C8环烷基、C3-C8杂环基、未取代的C6-C10芳基、C5-C8杂芳基、或被一个或多个Ra基团(1、2、3、4、5或更多)取代的C6-C10芳基；其中，所述的Ra基团选自下组：卤素、C1-C4卤代烷基(优选氟代)、C1-C4烷基、C1-C4烷氧基、氰基、氨基，或两个相邻的Ra基团与其相连的原子共同构成C5-C7碳环或杂环；Rb、Rc各自独立地为C1-C4烷基、或Rb、Rc与其相连的原子共同构成C5-C7碳环或杂环；且当L为-C(O)NH-CH2-时，R1不为未取代的苯基；
A选自未取代的C1-C8亚烷基、被一个或多个Rd基团取代的C1-C4亚烷基、或C1-C8亚烯基(包括亚单烯基和亚二烯基)；且当L为-C(O)-A-时，所述的Rd基团选自氨基或-NHBoc；
R2为选自下组的基团：
或正辛基、正丁基；
其中R7选自下组：H、CH3、F、Cl、Br、I、OH、SH、NH2、CN、CF3、OCH3、SCH3、NHCH3、N(CH3)2、CHF2、OC2H5、COOH、CONH2；
n＝0、1、2、3、4、5、6、7、8、9、10；q选自下组：0、1、2、3、4或5；
R3、R3'各自独立地选自下组：H、卤素、取代或未取代的C1-C4烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基；或R3和R3'及其相连的碳原子共同构成取代或未取代的饱和、部分不饱和或芳香5-8元碳环或杂环；
R8为H或C1-C4烷基，或R8与R3共同构成C1-C4亚烷基；
R4为其中，环A和环B各自独立地为取代或未取代的饱和、部分不饱和/或芳香5-14元碳环或杂环(包括单环、稠环或螺环)，且环A和环B稠合从而形成多环稠环；其中R4任选地被选自卤素、C1-C4烷基、C1-C4烷氧基的Re取代；
R5选自H、C1-C3烷基；
当所述的各个CH2和NH单独或作为其他基团的一部分出现时，可以任选地被取代；
除非特别说明，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、氧代、羧基、氰基、羟基、苯基、5-6元杂环基、C1-C6烷基、C1-C6烷氧基、C3-C8环烷基取代、氨基、N，N-二甲基氨基、巯基、C1-C6砜基。
在另一优选例中，所述的化合物具有如下式II所示的结构：
其中，X3选自O或S；
X1、X2、R1、R2、R3、R3’、R4、R5、Y、A如上所述。
在另一优选例中，X3为O。
在另一优选例中，Y为N。
在另一优选例中，X1、X2各自独立地为O。
在另一优选例中，R3、R3’各自独立地为H或C1-C4烷基，优选为H。
在另一优选例中，R1选自H、卤素、未取代的C1-C10烷基、C1-C4卤代烷基、C3-C8环烷基、C3-C8杂环基、未取代的C6-C10芳基、或被一个或多个Ra基团(1、2、3、4、5或更多)取代的C6-C10芳基。
在另一优选例中，所述的Ra基团选自下组：卤素、C1-C4卤代烷基(优选氟代)、C1-C4烷基、C1-C4烷氧基、氰基、氨基，或两个相邻的Ra基团与其相连的原子共同构成5-7元碳环或杂环；且当L为-C(O)NH-CH2-时，R1不为未取代的苯基。
在另一优选例中，当L为-C(O)NH-A-时，R1不为未取代的苯基。
在另一优选例中，R1选自H、卤素、C1-C4卤代烷基、C3-C8环烷基。
在另一优选例中，所述卤代烷基优选为氟代烷基或氯代烷基。
在另一优选例中，A为C1-C8亚烷基，优选C1-C4亚烷基，更优选为-(CH2)1-4-。
在另一优选例中，R2为选自下组的基团：
n＝0、1、2、3、4、5、6、7、8、9、10；q选自下组：0、1、2、3、4或5；
在另一优选例中，R7选自下组：H、CH3、F、Cl、Br、I、OH、SH、NH2、CN、CF3、OCH3、SCH3。
在另一优选例中，当L为-C(O)NH-A-时，R2不为
在另一优选例中，n＝0或1。
在另一优选例中，R2为选自下组的基团：
n＝0、1、2、3、4、5、6、7、8、9、10。
在另一优选例中，R4为其中，环A和环B各自独立地为取代或未取代的芳香5-14元碳环或杂环(包括单环、稠环或螺环)，且环A和环B稠合从而形成二环稠环或三环稠环。
在另一优选例中，所述式I化合物选自下组：






在另一优选例中，所述化合物还包括选自下组的化合物：
在本发明的第二方面，提供了一种药物组合物，包含如下组分：
1)治疗有效量的一种或多种本发明第一方面所述的化合物、或其药学上可接受的盐或立体异构体；
2)药学上可接受的载体或赋形剂；和
3)任选地第二治疗剂，所述的第二治疗剂为抗菌药物。
在另一优选例中，所述第二治疗剂选自下组：环丙沙星、利福平、利奈唑胺，或其组合。
在本发明的第三方面，提供了一种本发明第一方面所述的化合物，或其药学上可接受的盐或立体异构体的用途，用于制备预防和/或治疗与ClpP蛋白酶活性或表达量相关的疾病或病症的药物组合物。
在另一优选例中。所述的与ClpP蛋白酶活性或表达量相关的疾病或病症为感染引起的疾病或病症。
在另一优选例中，所述的与ClpP蛋白酶活性或表达量相关的疾病或病症为癌症。
在另一优选例中，所述的感染是细菌感染。
在另一优选例中，所述细菌感染是革兰氏阳性菌感染。
在另一优选例中，所述细菌为革兰氏阳性菌：优选地，所述细菌选自下组：葡萄球菌、耐甲氧西林葡萄球菌、耐青霉素肺炎链球菌、耐万古霉素金黄色葡萄球菌、肺炎链球菌、粪肠球菌、酿脓链球菌。
在另一优选例中，所述的感染可以由其中一种或多种菌同时产生。
在另一优选例中，所述的治疗包括单独使用所述的药物组合物，或者与常用抗菌药物联用。
在另一优选例中，所述癌症选自下组：胶质瘤、实体瘤、白血病，或其组合。
在另一优选例中，所述实体瘤选自下组：肾癌、肝癌、***癌、黑素瘤、结肠癌、肺癌、胰腺癌、卵巢癌、乳腺癌、胃癌、脑癌、直肠癌、黑素瘤、卵巢癌，或其组合。
在另一优选例中，所述胶质瘤选自下组：神经胶质瘤、脑胶质瘤、星形细胞瘤，或其组合。
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。
 附图说明
[根据细则26改正 02.06.2023]
图1显示了生物测试例3中化合物与利福平和环丙沙星联用的测试结果。
[根据细则26改正 02.06.2023]
图2显示了生物测试例3中化合物A28与利福平和环丙沙星联用的测试结果。
[根据细则26改正 02.06.2023]
图3显示了生物测试例3中化合物B29与利福平和环丙沙星联用的测试结果。
 具体实施方式
本发明人基于小分子与ClpP蛋白的分子识别机制，理性设计、深入研究，发现了一类与现有ClpP调控小分子结构完全不同且具有优异的调节效果的ClpP调节剂。所述的一种环肽类化合物可以作为ClpP蛋白酶的调控剂，用来治疗病原菌感染，如葡萄球菌、耐甲氧西林葡萄球菌的感染等，因此在细菌感染疾病的防治药物中有所运用。在此基础上完成了本发明。
定义
如本文所用，术语“烷基”包括直链或支链的烷基。例如C1-C6烷基表示具有1-6个碳原子的直链或支链的烷基，例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基等。
如本文所用，术语“亚烷基”是指有两个键被取代的直链或支链的烷烃，例如C1-C4亚烷基表示两个键被取代的直链或支链的C1-C4烷烃，例如亚甲基、亚乙基、亚正丙基、亚异丙基、亚丁基等。
如本文所用，术语“烯基”包括直链或支链的烯基。例如C2-C6烯基指具有2-6个碳原子的直链或支链的烯基，例如乙烯基、烯丙基、1-丙烯基、异丙烯基、1-丁烯基、2-丁烯基、或类似基团。
如本文所用，术语“亚烯基”是指有两个键被取代的直链或支链的烯烃。其中所述烯烃可包括单烯烃和二烯烃，例如共轭二烯烃。例如C2-C6亚烯基表示两个键被取代的直链或支链的C2-C6烯烃，例如亚乙烯基、亚正丙烯基、亚异丙烯基、亚丁烯基、亚1,3-丁二烯基等。
如本文所用，术语“炔基”包括直链或支链的炔基。例如C2-C6炔基是指具有2-6个碳原子的直链或支链的炔基，例如乙炔基、丙炔基、丁炔基、或类似基团。
如本文所用，术语“C1-C6烷氧基”是指具有1-6个碳原子的直链或支链的烷氧基；例如，甲氧基、乙氧基、丙氧基、异丙氧基、丁氧基、异丁氧基、叔丁氧基等。
如本文所用，术语“C1-C6烷硫基”是指-S-(具有1-6个碳原子的直链或支链的烷基)；例如，甲硫基、乙硫基、丙硫基、异丙硫基、丁硫基、异丁硫基、叔丁硫基等。
如本文所用，术语“C3-C8环烷基”指具有3-8个碳原子的环烷基。其可以是单环，例如环丙基、环丁基、环戊基、环己基、或类似基团。也可以是双环形式，例如桥环或螺环形式。
如本文所用，术语“C3-C8杂环基”是指具有3-8个原子的且其中1-3个原子为选自N、S和O的杂原子的饱和或部分饱和的环状基团。其可以是单环，也可以是双环形式，例如桥环或螺环形式。具体的实例可以为氧杂环丁烷、氮杂环丁烷、四氢-2H-吡喃基、哌啶基、四氢呋喃基、吗啉基和吡咯烷基等。
如本文所用，术语“C6-C10芳基”是指具有6-10个碳原子的芳基，例如，苯基或萘基等类似基团。
如本文所用，术语“C5-C8杂芳基”指具有5-8个原子的且其中1-3个原子为选自N、S和O的杂原子的环状芳香基团。其可以是单环，也可以是稠环形式。具体的实例可以为吡啶基、哒嗪基、嘧啶基、吡嗪基、三嗪基、吡咯基、吡唑基、咪唑基、(1,2,3)-***基以及(1,2,4)-***基、四唑基、呋喃基、噻吩基、异恶唑基、噻唑基、恶唑基等。
本发明所述的基团除非特别说明是“取代的或未取代的”，否则本发明的基团均可被选自下组的取代基所取代：卤素、腈基、硝基、羟基、氨基、C1-C6烷基-胺基、C1-C6烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、卤代C1-C6烷基、卤代C2-C6烯基、卤代C2-C6炔基、卤代C1-C6烷氧基、烯丙基、苄基、C6-C12芳基、C1-C6烷氧基-C1-C6烷基、C1-C6烷氧基-羰基、苯氧羰基、C2-C6炔基-羰基、C2-C6烯基-羰基、C3-C6环烷基-羰基、C1-C6烷基-磺酰基等。
如本文所用，“卤素”指F、Cl、Br、和I。
如本文所用，术语“卤代烷基”指被卤素取代的C1-C3的烷基。优选地，卤代烷基为三氟甲基、二氟甲基、三氟甲氧基。这里"C1-C3烷基"指具有1～3个碳原子的直链或支链烷基，例如甲基、乙基、正丙基、异丙基。
除非特别说明，本发明所描述的结构式意在包括所有的同分异构形式(如对映异构，非对映异构和几何异构体(或构象异构体))：例如含有不对称中心的R、S构型，双键的(Z)、(E)异构体等。因此，本发明化合物的单个立体化学异构体或其对映异构体、非对映异构体或几何异构体(或构象异构体)的混合物都属于本发明的范围。
如本文所用，术语“对映异构体”表示具有相同分子式的化合物中，由于原子在空间配置不同而引起的同分异构体，对映异构体之间互为镜像。比如，含有不对称中心的R、S构型。
作为ClpP调控剂的化合物
化合物的设计原理：本发明的活性成分结构是通过基于小分子与ClpP的复合物晶体结构的识别机制设计得到，即：小分子调控剂由“刚性骨架”和两部分“边臂”组成，小分子在刚性骨架的支撑作用下，两部分“边臂”分别与ClpP蛋白的两个疏水口袋结合，调节ClpP的活性，影响多种生物学功能。该部分刚性骨架结构与现有文献报道的ClpP相关调节剂的骨架均不相同，并通过对“骨架”和“边臂”结构的优化，本发明进一步获得了对ClpP蛋白具有不同调节活性的小分子化合物。
本发明所涉及的化合物，均是基于小分子与ClpP蛋白的分子识别作用机制进行设计，其结构设计来源有据可循。
如本文所用，“本发明的化合物”指式I化合物和其他本发明中列举的具有调节ClpP效果的化合物，并且还包括及上述化合物的各种药学上可接受的盐或立体异构体。
优选的本发明化合物包括化合物(包括各化合物的各类R构型和/或S构型的立体异构体，和/或E-/Z-的顺反异构体)。
在另一优选例中，所述的药用盐包括与无机酸、有机酸、碱金属离子、碱土金属离子或能提供生理上可接受的阳离子的有机碱结合形成的盐以及铵盐。
在另一优选例中，所述的无机酸选自盐酸、氢溴酸、磷酸或硫酸；所述的有机酸选自甲磺酸、对甲苯磺酸、三氟乙酸、枸杞酸、马来酸酒石酸、富马酸、柠檬酸或乳酸；所述的碱金属离子选自锂离子、钠离子、钾离子；所述的碱土金属离子选自钙离子、镁离子；所述的能提供生理上可接受的阳离子的有机碱选自甲胺、二甲胺、三甲胺、哌啶、吗啉或三(2-羟乙基)胺。
在本发明范围内的所有这些盐都可采用常规方法制备。
本发明化合物的制备过程中的起始原料及中间体容易得到，各步反应可依据已报道的文献或对本领域熟练技术人员来说可以用有机合成中的常规方法很容易合成。通式I所述化合物可以溶剂化物或非溶剂化物的形式存在，利用不同的溶剂进行结晶可能得到不同的溶剂化物。
制备方法
本发明提供了式IA化合物和其类似物、以及其药学上可接受的盐的合成方法。该化合物的合成路线如下：
化合物的合成始于可商购的初始原料α-萘醛和氨基乙缩醛。首先化合物I1和化合物I2在100℃下回流脱水形成席夫碱I3(步骤1)，还原胺化得到化合物I4(步骤2)，进一步与Fmoc保护的氨基酸缩合得到化合物I5(步骤3)，在二乙胺条件下脱除Fmoc得到游离的伯胺I6(步骤4)，接着化合物I6与Fmoc保护的β丙氨酸缩合形成化合物I7(步骤5)，在甲酸条件下关环得到化合物I8(步骤6)，脱保护得到关键中间体化合物I9(步骤7)，中间体I9与异氰酸酯反应，得到式IA化合物(步骤8)。
药物组合物和施用方法
由于本发明化合物具有优异的ClpP的调节活性，因此本发明化合物及其药学上可接受的无机或有机盐，或立体异构体，以及含有本发明化合物为主要活性成分的药物组合物可用于预防和/或治疗(稳定、减轻或治愈)ClpP相关疾病(例如，感染性疾病、癌症)。
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及药学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，更佳地，含有10-200mg本发明化合物/剂。较佳地，所述的“一剂”为一个胶囊或药片。
“药学上可接受的载体”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)、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。
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、肠胃外(静脉内、肌肉内或皮下)。
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。
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。
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。
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。
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。
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。
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(例如其他治疗剂)联合给药。
联合给药时，所述药物组合物还包括与一种或多种(2种，3种，4种，或更多种)其他药学上可接受的化合物。该其他药学上可接受的化合物中的一种或多种(2种，3种，4种，或更多种)可与本发明的化合物同时、分开或顺序地用于预防和/或治疗ClpP相关疾病。
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20～500mg。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。
本发明的主要优点包括：
1)本发明的化合物不是根据现有文献中的结构改造而来，是通过基于ClpP蛋白与小分子化合物的分子识别机制理性设计得到。该系列化合物的分子结构与现有报道的结构完全不同。
2)本发明的化合物可以有效调节ClpP，既具有优异的ClpP激活活性，同时也具有优异的ClpP抑制活性；
3)本发明的化合物通过调节ClpP靶标，达到优异的抑菌效果；
4)本发明的化合物与药物联用时，对常见抗生素药物难以消除的持留菌具有显著的消除效果。
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，否则百分比和份数是重量百分比和重量份数。
未经特别说明实验所用药品均为市售分析纯或化学纯，除特别说明所有试剂使用前均为纯化处理。
柱层析硅胶为国药集团化学试剂有限公司生产200-300目商业化硅胶；1H-NMR，化合物使用利穗EZ Plus 100D中压制备仪分离。
实施例1.化合物A26的制备
1、化合物4的制备(其余中间体的制备采用相同的方法)
将α-萘醛(1.56g,10.0mmol)和氨基乙缩醛(1.33g,10.0mmol)100℃加热回流1小时，冷却至室温，用乙醇(25mL)稀释，分批次加入硼氢化钠(0.76g，20mmol)，室温搅拌24小时。减压蒸馏除去乙醇，用饱和食盐水洗涤，二氯甲烷萃取，有机层旋干，柱层析纯化(石油醚：乙酸乙酯＝100:5,v:v)，得到黄色油状液体化合物4 2.51g，产率92％。
1H NMR(400MHz,CDCl3)δ8.16(d,J＝8.2Hz,1H,ArH),7.89(d,J＝7.8Hz,1H,ArH),7.80(d,J＝8.1Hz,1H,ArH),7.61–7.48(m,3H,ArH),7.45(t,J＝7.3Hz,1H,ArH),4.68(t,J＝4.8Hz,1H,CH),4.29(s,2H,CH2Ar),3.78–3.63(m,2H,OCH2),3.60–3.47(m,2H,OCH2),2.90(d,J＝5.1Hz,2H,CH2N),1.22(t,J＝7.0Hz,6H,CH3).LC-MS[M+H]+274.2.
2、化合物中间体9的制备(其余中间体的制备采用相同的方法)
将Fmoc-L-异亮氨酸(0.35g,1.0mmol)，HOAT(1-羟基-7-氮杂苯并***)(0.14g,1.0mmol)，HATU(2-(7-氮杂苯并三氮唑)-N,N,N',N'-四甲基脲六氟磷酸酯)(0.46g,1.2mmol)和DIPEA(二异丙基乙基胺)(0.26mL,1.5mmol)溶解在DMF(N,N-二甲基甲酰胺)(10mL)中，室温搅拌5分钟，向体系中加入化合物4(0.27g,1.0mmol)，室温搅拌6小时后，水洗，二氯甲烷萃取。减压蒸馏移除二氯乙烷，反应浓缩液中加入DEA(二乙胺)(10mL)和DCM(二氯甲烷)(10mL)，室温搅拌过夜。混合物旋干除去二乙胺和二氯甲烷，正相柱分离(石油醚：乙酸乙酯2:1,v:v)，得到化合物6，LC-MS[M+H]+386.3。
向混合物中加入Fmoc-β-丙氨酸(217.7mg,0.7mmol)，HOAT(95.2mg,0.7mmol)，HATU(319.2mg,0.84mmol)，DIPEA(0.18mL,1.05mmol)和DMF(5mL)，室温搅拌5分钟，向体系中加入化合物6。室温搅拌6小时，反应液用饱和食盐水洗涤，二氯甲烷萃取。减压蒸馏除去二氯甲烷得到粗产品的化合物7。
将化合物7加入甲酸和二氯甲烷的混合溶液(10mL:10mL)20mL中，密闭下室温搅拌6-12小时，LC-MS检测反应完全，减压蒸馏浓缩，向浓缩液中加入二乙胺(10mL)和二氯甲烷(10mL)，室温搅拌过夜，浓缩，柱层析纯化(乙酸乙酯:甲醇＝90:1,v:v)，得到化合物9为白色固体，收率76％(410mg)。1H NMR(400MHz,CDCl3)δ8.13–8.05(m,1H),7.93–7.83(m,2H),7.59–7.50(m,2H),7.45(t,J＝7.5Hz,1H),7.38(d,J＝6.9Hz,1H),5.39(d,J＝14.5Hz,1H),5.30(dd,J＝8.9,6.0Hz,1H),4.81(d,J＝14.5Hz,1H),4.61(dd,J＝9.9,4.1Hz,1H),3.22–3.01(m,3H),3.00–2.91(m,1H),2.46–2.30(m,2H),2.16–2.05(m,1H),1.86(t,J＝9.1Hz,1H),1.60(s,1H),1.50–1.19(m,12H),0.90(t,J＝6.7Hz,3H).
3、化合物A26的制备
化合物9(73mg,0.2mmol)和异氰酸正丁酯(39.7mg,0.4mmol)在二氯甲烷(5mL)溶液中，搅拌过夜，浓缩后经反相C18柱分离得到化合物A26，为白色固体，收率81％(75mg)。1H NMR(400MHz,CDCl3)δ8.12(d,J＝8.9Hz,1H),7.98–7.80(m,2H),7.55(p,J＝6.9Hz,2H),7.49–7.41(m,1H),7.37(d,J＝6.8Hz,1H),6.07(dd,J＝9.6,5.1Hz,1H),5.26(d,J＝6.1Hz,1H),5.13(dd,J＝35.0,14.6Hz,2H),4.56(s,1H),3.56(dd,J＝14.3,4.6Hz,1H),3.36–3.12(m,5H),2.61–2.49(m,1H),2.36(d,J＝16.8Hz,1H),2.24(dd,J＝6.6,3.5Hz,1H),1.45(dd,J＝14.9,7.3Hz,2H),1.32(td,J＝13.9,6.4Hz,4H),1.14(d,J＝6.8Hz,3H),0.94(dt,J＝14.8,7.3Hz,6H).
替换不同的中间体和反应物，得到如下实施例2-25所示的化合物。







生物测试例1小分子激动ClpP蛋白酶功能的酶学评价
背景：ClpP激动剂通过引发ClpP蛋白酶复合物结构的重塑，重排ClpP蛋白酶的核心结构，使该蛋白脱离正常机制的调控，从而非特异性地大量降解胞内某些功能蛋白，诱导细菌或者癌细胞死亡。如HsClpP(人源线粒体ClpP)的激动剂，可以激动HsClpP非特异性的降解细胞中的正常蛋白，导致癌细胞死亡，从而治疗胰腺癌、白血病等多种肿瘤。正常生物体中，ClpP的功能发挥依赖与分子伴侣蛋白ClpX的相互作用，在ATP水解的驱动下ClpX将细胞中错误折叠的蛋白解聚，并传送入ClpP的水解空腔，降解蛋白，调控正常细胞中蛋白质的稳态。ClpP的抑制剂是通过占据ClpP与伴侣蛋白ClpX的结合口袋，阻碍ClpP与ClpX的相互作用，从而使得细胞内错误折叠蛋白难以被降解和清除，最终引起一系列生物学表型。如SaClpP(金黄色葡萄球菌ClpP)的抑制剂，可以抑制SaClpP与SaClpX(金黄色葡萄球菌ClpX)的相互作用，使得细菌中错误折叠的蛋白不断累积，抑制细菌的毒力因子的产生，降低细菌的毒力。本发明的化合物可以同时抑制和激活ClpP蛋白酶。
化合物激动活性测试方法：化合物活性测试采用α-casein(模式蛋白)作为底物，小分子激动剂与ClpP结合时，可以使ClpP不依赖于ATP水解驱动ClpX降解α-casein。因此，可以通过SDS-PAGE的方法进一步测定活性。检测体系为50μL，10μg HsClpP蛋白、SaClpP或SpClpP(酿脓链球菌ClpP)蛋白先溶解在PD缓冲液(25mM HEPES-KOH(pH 7.6),200mM KCl,5mM MgCl2,1mM EDTA,10％甘油和2mM DTT(二硫苏糖醇))中。之后加入化合物，在室温下孵育10min，加入0.6mg/mLα-casein，37℃孵育2小时，立即加入12.5μL SDS-PAGE上样缓冲液，100℃加热10min。SDS-PAGE电泳后考马斯亮蓝染色后用脱色液脱色拍照，以DMSO为阴性对照，ONC201，ADEP 4为阳性对照，根据α-casein的降解情况判断化合物激动ClpP的活性。结果如下表1所示。
化合物抑制活性测试方法：GFP-SsrA是ClpXP(ClpP十四聚体和ClpX六聚体组成的蛋白复合物)相互作用的底物，因此可以通过SDS-PAGE的方法验证小分子的抑制活性。若GFP-SsrA完全被降解，则说明化合物不具备抑制活性，反之则化合物具备抑制活性。
HsClpP小分子抑制剂活性测试方法。水解体系为60μL，反应在含25mM HEPES-KOH(pH 7.6)，5mM MgCl2，5mM KCl，0.03％吐温20，10％甘油和2mM DTT的缓冲液中进行。0.33μM HsClpP14(HsClpP蛋白十四聚体)，0.6μM HsClpX6(HsClpX蛋白六聚体)，0.4mg/mLα-casein，ATP再生***(5mM ATP,16mM CP(磷酸肌酸),0.032mg/mL CK(肌酸激酶))，2mM DTT，以及0.78-100μM化合物。37℃孵育1小时，立即加入15μL上样缓冲液，100℃加热10min，经SDS-PAGE电泳后考马斯亮蓝染色再脱色后拍照存档，结合Graphpad作图，计算IC50值。
SaClpP和SpClpP小分子抑制剂活性测试方法。水解体系为60μL PD缓冲液，0.33μM SaClpP14(SaClpP蛋白十四聚体)或SpClpP14(SpClpP蛋白十四聚体)，0.6mM SaClpX6(SaClpX蛋白六聚体)或SpClpX6(SpClpX蛋白六聚体)，2μM GFP-SsrA(ClpXP相互作用的底物)，0.78-100μM化合物。37℃孵育1小时，立即加入15μL上样缓冲液，100℃加热10min，经SDS-PAGE电泳后考马斯亮蓝染色再脱色后拍照存档，结合Graphpad作图，计算IC50值，以DMSO为阴性对照，ONC201，ADEP 4为阳性对照。
结果如下表1所示：
表1本发明的化合物激动活性和抑制活性的测试结果

结果表明，本发明的化合物对不同物种的ClpP均具有优异的激动活性和抑制活性。其中，A77、A130、A134、A236针对不同来源的ClpP蛋白酶表现出优异的选择性。
生物测试例2最小抑菌浓度实验(MIC)
背景：ClpP靶标蛋白的功能与多种生物学功能相关，研究表明细菌ClpP蛋白的功能激动与抑制可以影响细菌的生存和毒力，本发明实施例中提供的细菌样本与ClpP靶标关联。
金黄色葡萄球菌MIC测试方法：将菌种从-80℃冰箱取出后在TSA(胰酪大豆胨琼脂培养基)平板上划线培养过夜12小时以上。挑取美国金黄色葡萄球菌耐药库中获得的5种临床耐药菌NRS-1(耐氨基糖苷和四环素)、NRS-70(耐红霉素)、NRS-100(耐苯唑西林和四环素)、NRS-108(耐庆大霉素)、NRS-271(耐利奈唑胺和环丙沙星)和美国社区获得性耐药金黄色葡萄球菌USA300(耐甲氧西林)、金黄色葡萄球菌Newman(敏感菌)的单克隆于10mL TSB(胰酪大豆胨液体)培养基中。分别在37℃，210rpm过夜培养至平台期。第二天1:1000倍稀释至新鲜的TSB培养基中。37℃，210rpm继续培养至OD600为0.6，将菌液用新鲜的TSB培养基再稀释400倍。将化合物提前用新鲜的TSB培养基等倍梯度稀释，终浓度为0.39-50μg/ml，将稀释后的菌液加入到稀释后的化合物中，随后将96孔板置于37℃培养箱中静置培养18小时。用肉眼观察每个孔中细菌生长情况，不抑制细菌生长的化合物的最低浓度为该化合物的MIC值。初次实验得到化合物大致MIC值范围后，再次实验时在次条件下不断优化，最终得最佳MIC值。
酿脓链球菌MIC测试方法：将菌种从-80℃冰箱取出后在TSA平板上划线培养过夜12小时以上。挑取美国模式培养物集存库中获得的酿脓链球菌Sp12344(ATCC Streptococcus pyogenes 12344)的单克隆于10mL THB(Todd Hewitt Broth)培养基中。在37℃，210rpm过夜培养至平台期。第二天1:1000倍稀释至新鲜的THB培养基中。37℃，210rpm继续培养至OD600为0.6，将菌液用新鲜的THB培养基再稀释400倍。将化合物提前用新鲜的THB培养基等倍梯度稀释，终浓度为0.39-50μg/ml。将稀释后的菌液加入到稀释后的化合物中，随后将96孔板置于37℃培养箱中静置培养18小时。用肉眼观察每个孔中细菌生长情况，不抑制细菌生长的化合物的最低浓度为该化合物的MIC值。结果如下表2所示：
表2本发明的化合物的最小抑菌浓度测试结果

生物测试例3与临床常用抗生素药物联用实验测试
背景：过夜培养的金黄色葡萄球菌中，大部分细菌受饥饿等压力胁迫被杀死后，有部分细菌变为持留菌，此时细菌内能量较低，由于细菌内ClpPX(杀菌需要一定能量，此时ClpPX起不了主要的杀菌作用。ClpP激动剂与传统抗生素联用后，一些传统抗生素如：利福平，环丙沙星，加入后可以大量杀菌，但是对这些持留菌具有一定耐受性，此时ClpP小分子激动剂可以在不消耗能量的前提下，激动ClpP的生物学功能，从而消除持留菌。
挑取单克隆USA300于BHI(脑心浸液培养基)培养基，37℃，210rpm过夜培养，分装10mL/管，分别加入8μg/mL环丙沙星、0.4μg/mL利福平及10倍MIC化合物(A28、B29)，37℃，210rpm继续培养分别于不同时间点0/24/48/72小时取样100μL,12000rpm离心5分钟，弃上清，菌体用100μL无菌PBS重悬，10倍梯度稀释至无菌PBS中，各取10μL垂直点在MHA(Mueller-Hinton琼脂)平板。待平板上菌液风干后放37℃倒置培养过夜，第二天记录生成菌落数。用Graphpad作图，绘制曲线。
[根据细则26改正 02.06.2023]
结果如图1-3所示，抗生素单用时，基本没有抑菌效果，而本发明化合物在与环丙沙星和利福平联用情况下，均能体现出优异的联用抑菌效果。
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。

Claims (15)

  1. 一种如下式I所示的环肽类化合物，或其药学上可以接受的盐或立体异构体：
    其中，Y选自下组：N或CR8；
    X1、X2各自独立地选自下组：O、S、CH2或NH；
    L选自下组：H、取代或未取代的C1-C10烷基、-C(O)-A-、-C(O)-(CH2)p-C(O)-A-、-C(O)NH-A-、-C(O)NH-(CH2)p-C(O)-A-、-C(O)NH-(CH2)p-C(O)O-A-、-S(O)0-2-A-、-NHC(O)NH-A-、-C(S)-A-、-C(S)NH-A-、-NHC(S)NH-A-、-C(NH)-A-、-C(NH)NH-A-、-NHC(NH)NH-A-；p选自下组：0、1、2、3、4或5；
    R1选自下组：H、卤素、未取代的C1-C10烷基、被一个或多个Ra基团(1、2、3、4、5或更多)取代的C1-C4烷基、C1-C4卤代烷基、C2-C6烯基、C2-C6炔基、C1-C6烷氧基、C1-C6烷硫基、-NRbRc、C3-C8环烷基、C3-C8杂环基、C5-C8杂芳基、未取代的C6-C10芳基、或被一个或多个Ra基团(1、2、3、4、5或更多)取代的C6-C10芳基；其中，所述的Ra基团选自下组：卤素、C1-C4卤代烷基(优选氟代)、C1-C4烷基、C1-C4烷氧基、氰基、氨基，或两个相邻的Ra基团与其相连的原子共同构成C5-C7碳环或杂环；Rb、Rc各自独立地为H、C1-C4烷基、或Rb、Rc与其相连的原子共同构成C5-C7碳环或杂环；且当L为-C(O)NH-CH2-时，R1不为未取代的苯基；
    A选自未取代的C1-C8亚烷基、被一个或多个Rd基团取代的C1-C4亚烷基、或C1-C8亚烯基(包括亚单烯基和亚二烯基)；且当L为-C(O)-A-时，所述的Rd基团选自氨基或-NHBoc；
    R2为选自下组的基团：
    或正辛基、正丁基(式中*代表连接位点)；
    其中R7选自下组：H、CH3、F、Cl、Br、I、OH、SH、NH2、CN、CF3、OCH3、SCH3、NHCH3、N(CH3)2、CHF2、OC2H5、COOH、CONH2；
    n＝0、1、2、3、4、5、6、7、8、9、10；q选自下组：0、1、2、3、4或5；
    R3、R3'各自独立地选自下组：H、卤素、取代或未取代的C1-C4烷基、取代或未取代的C2-C6烯基、取代或未取代的C2-C6炔基；或R3和R3'及其相连的碳原子共同构成取代或未取代的饱和、部分不饱和、或芳香5-8元碳环或杂环；
    R8为H或C1-C4烷基，或R8与R3共同构成C1-C4亚烷基；
    R4为其中，环A和环B各自独立地为取代或未取代的饱和、部分不饱和、或芳香5-14元碳环或杂环(包括单环、稠环或螺环)，且环A和环B稠合从而形成多环稠环；其中R4任选地被选自卤素、C1-C4烷基、C1-C4烷氧基的Re取代；
    R5选自H、C1-C4烷基；
    当所述的各个CH2和NH单独或作为其他基团的一部分出现时，可以任选地被取代；
    除非特别说明，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、氧代、羧基、氰基、羟基、苯基、5-6元杂环基、C1-C6烷基、C1-C6烷氧基、C3-C8环烷基取代、氨基、N，N-二甲基氨基、巯基、C1-C6砜基。
  2. 如权利要求1所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，所述的化合物具有如下式II所示的结构：
    其中，X3选自O或S；
    X1、X2、R1、R2、R3、R3’、R4、R5、Y、A如权利要求1所述。
  3. 如权利要求1或2所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，Y为N，且X1、X2各自独立地为O。
  4. 如权利要求1或2所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，R1选自H、卤素、未取代的C1-C10烷基、C1-C4卤代烷基、C3-C8环烷基、C3-C8杂环基、未取代的C6-C10芳基、或被一个或多个Ra基团(1、2、3、4、5或更多)取代的C6-C10芳基；
    其中，所述的Ra基团选自下组：卤素、C1-C4卤代烷基(优选氟代)、C1-C4烷基、C1-C4烷氧基、氰基、氨基，或两个相邻的Ra基团与其相连的原子共同构成5-7元碳环或杂环；且当L为-C(O)NH-A-时，R1不为未取代的苯基。
  5. 如权利要求1或2所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，R2为选自下组的基团：
    其中R7选自下组：H、CH3、F、Cl、Br、I、OH、SH、NH2、CN、CF3、OCH3、SCH3、NHCH3、N(CH3)2、CHF2、OC2H5、COOH、CONH2；
    n＝0、1、2或3；q选自下组：0、1、2、3、4或5。
  6. 如权利要求1或2所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，R4为其中，环A和环B各自独立地为取代或未取代的芳香5-14元碳环或杂环(包括单环、稠环或螺环)，且环A和环B稠合从而形成二环稠环或三环稠环。
  7. 如权利要求1所述的化合物，或其药学上可以接受的盐或立体异构体，其特征在于，所述式I化合物选自下组：







  8. 一种化合物，或其药学上可以接受的盐或立体异构体，其特征在于，所述化合物选自下组：

  9. 一种药物组合物，其特征在于，包含如下组分：
    1)治疗有效量的一种或多种权利要求1或8所述的化合物、或其药学上可接受的盐或立体异构体；
    2)药学上可接受的载体或赋形剂；和
    3)任选地第二治疗剂，所述的第二治疗剂为抗菌药物。
  10. 如权利要求9所述的药物组合物，其特征在于，所述第二治疗剂选自下组：环丙沙星、利福平、利奈唑胺，或其组合。
  11. 一种权利要求1-8任一所述化合物，或其药学上可接受的盐或立体异构体的用途， 其特征在于，用于制备预防和/或治疗与ClpP蛋白酶活性或表达量相关的疾病或病症的药物组合物。
  12. 如权利要求11所述的用途，其特征在于，所述的与ClpP蛋白酶活性或表达量相关的疾病或病症为感染引起的疾病或病症。
  13. 如权利要求12所述的用途，其特征在于，所述的感染是细菌感染。
  14. 如权利要求11所述的用途，其特征在于，所述的与ClpP蛋白酶活性或表达量相关的疾病或病症为癌症。
  15. 如权利要求11所述的用途，其特征在于，所述的癌症选自下组：胶质瘤、实体瘤、白血病，或其组合。
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