CN114685520B - Tri-fused ring compound and pharmaceutical composition and application thereof - Google Patents

Tri-fused ring compound and pharmaceutical composition and application thereof Download PDF

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CN114685520B
CN114685520B CN202111602260.2A CN202111602260A CN114685520B CN 114685520 B CN114685520 B CN 114685520B CN 202111602260 A CN202111602260 A CN 202111602260A CN 114685520 B CN114685520 B CN 114685520B
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CN114685520A (en
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方华祥
杨秀眉
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Wuhan Yuxiang Medical Technology Co ltd
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    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D491/00—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00
    • C07D491/12—Heterocyclic compounds containing in the condensed ring system both one or more rings having oxygen atoms as the only ring hetero atoms and one or more rings having nitrogen atoms as the only ring hetero atoms, not provided for by groups C07D451/00 - C07D459/00, C07D463/00, C07D477/00 or C07D489/00 in which the condensed system contains three hetero rings
    • C07D491/14—Ortho-condensed systems
    • C07D491/147—Ortho-condensed systems the condensed system containing one ring with oxygen as ring hetero atom and two rings with nitrogen as ring hetero atom
    • 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

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Abstract

The invention relates to a tricyclic compound, a pharmaceutical composition and application thereof. The compound is a compound shown in a formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a tautomer thereof, or a hydrate thereof, or a solvate thereof, or a metabolite thereof, or a prodrug thereof, wherein R 1～R3, L, X, Y and Z groups are defined as in the specification. The compound and the pharmaceutical composition containing the same have good PLK1 enzyme inhibition activity, so the compound and the pharmaceutical composition can be used as PLK1 inhibitors and can be used for preparing medicines for treating and/or preventing diseases such as cancers over-expressed by PLK 1.

Description

Tri-fused ring compound and pharmaceutical composition and application thereof
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a tricyclic compound, a pharmaceutical composition containing the compound and application of the compound in the field of medicines.
Background
Proteins expressing the normal KRAS gene (murine sarcoma virus oncogene) play an important role in normal tissue signal transduction. Mutation of the KRAS gene due to single amino acid substitutions, particularly single nucleotide substitutions, is responsible for activating the mutation, which is an essential step in many cancer developments. The muteins produced are involved in a variety of malignancies, including lung adenocarcinoma, mucous adenocarcinoma, pancreatic ductal carcinoma, and colorectal carcinoma. Similar to other members of the Ras family, KRAS proteins are gtpases and are involved in many signal transduction pathways.
KRAS acts as a molecular on/off switch that, once turned on, recruits and activates proteins necessary for growth factor and other receptor signaling, such as c-Raf and PI-3 kinase. Normal KRAS binds GTP in the active state and has an inherent enzymatic activity, i.e. cleaves the terminal phosphate of a nucleotide, converting it into GDP. After converting GTP to GDP, KRAS is turned off. The conversion is generally slow but can be significantly accelerated by auxiliary proteins of the GTPase Activating Protein (GAP) class, such as RasGAP. In turn, KRAS may bind to proteins of the guanine nucleotide exchange factor (GEF) class, such as SOS1, which forces release of the bound nucleotides. Subsequently, KRAS binds GTP present in the cytoplasm and GEF is released from ras-GTP. In mutant KRAS, its gtpase activity is directly removed, such that KRAS is constitutively active. Mutant KRAS are generally characterized by: mutation of codons 12, 13, 61 or a mixture thereof.
PLK1 is a serine/threonine kinase consisting of 603 amino acids and having a molecular weight of 66kDa, and is an important regulator of the cell cycle. In particular, PLK1 is important for mitosis and is involved in the formation and changes of the mitotic spindle and the activation of CDK/cyclin complexes during the M phase of the cell cycle.
The viability of cancer cells harboring mutant KRAS is known to be dependent on Polo-like kinase 1 (PLK 1), and silencing PLK1 has been shown to result in cell death containing mutant KRAS. Thus, compounds that inhibit PLK1 are useful in the treatment of cancers caused by KRAS mutations.
Disclosure of Invention
The invention aims to provide a tricyclic compound used as a PLK1 inhibitor with a novel structure, which has good inhibition activity on tumor cells, good patentability and wide drug development prospect.
In a first aspect, the present invention provides a compound of formula I, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite, or prodrug thereof, wherein
Wherein,
L is- (CH 2)m-(A)p-(CH2)n -, wherein A is selected from oxygen, CR 10 or NR 10, m, p and n are independently any integer from 0 to 3;
X, Y and Z are each independently selected from CR 4 or N;
r 1 is selected from hydrogen, C 1-C6 alkyl, C 1-C6 heteroalkyl, C 2-C6 alkenyl, C 2-C6 alkynyl, C 3-C8 cycloalkyl, C 3-C8 heterocycloalkyl, C 3-C8 cycloalkoxy, or C 3-C8 heterocycloalkoxy, and wherein each of said alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkoxy, and heterocycloalkoxy is optionally substituted with at least 1R 5;
R 2 is selected from cyano 、P(＝O)(CH3)2、N＝S(＝O)(CH3)2、S(＝O)(＝NH)CH3、SO2NR6R7、NR6-COR7、NR6-CO-NR7、CO-OR6 or CO-NR 6R7;
R 3 is selected from C 6-C10 aryl, 5 to 6 membered monocyclic heteroaryl, or 9 to 10 membered bicyclic heteroaryl, and wherein each of said aryl, monocyclic heteroaryl, and bicyclic heteroaryl is optionally substituted with up to 5R 8;
R 4 and R 5 are each independently selected from hydrogen, halogen, cyano, hydroxy, amino, C 1-C6 alkyl, C 3-C8 cycloalkyl, C 3-C8 heterocycloalkyl, C 1-C3 alkoxy, or C 1-C6 haloalkyl, each optionally substituted with at least 1R 9.
R 6 and R 7 are each independently selected from hydrogen, C 1-C8 alkyl, C 3-C6 cycloalkyl or C 3-C8 heterocycloalkyl, or R 6 and R 7 together with the nitrogen atom to which they are attached form C 3-C8 cycloalkyl or C 3-C8 heterocycloalkyl, and wherein each of said alkyl and cycloalkyl is optionally substituted with at least 1R 9;
R 8 is independently selected from hydrogen, halogen, cyano, hydroxy, amino, C 1-C6 alkyl, C 1-C6 heteroalkyl, C 2-C6 alkenyl, C 2-C6 alkynyl, C 3-C8 cycloalkyl, C 3-C8 heterocycloalkyl, C 3-C8 cycloalkoxy, or C 3-C8 heterocycloalkoxy, and wherein the alkyl, heteroalkyl, alkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkoxy, and heterocycloalkoxy are each optionally substituted with at least 1R 9;
Each R 9 is independently selected from hydrogen, halogen, cyano, hydroxy, amino, carbamoyl, C 1-C6 alkyl, C 1-C6 heteroalkyl, C 3-C8 cycloalkyl, 3 to 14 membered heterocycloalkyl, C 1-C3 alkoxy, C 1-C3 haloalkoxy, C 6-C10 aryl, 5 to 6 membered monocyclic heteroaryl, or 9 to 10 membered bicyclic heteroaryl, and wherein each of the alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, alkoxy, haloalkoxy, aryl, monocyclic heteroaryl bicyclic heteroaryl is optionally substituted with at least 1R 10;
The heteroatoms or heteroatom groups contained in the heteroalkyl, heterocycloalkyl, heterocycloalkoxy, heteroaryl groups in R 1 to R 9 are each independently selected from -C(＝O)N(R10)-、-N(R10)-、-NH-、-N＝、-O-、-S-、-C(＝O)O-、-C(＝O)-、-C(＝S)-、-S(＝O)-、-S(＝O)2- and-N (R 10)C(＝O)N(R10) -, and the number of heteroatoms or heteroatom groups are each independently selected from 1, 2 and 3;
each R 10 is independently selected from hydrogen, chloro, fluoro, cyano, hydroxy, amino, isopropyl, cyclopropyl, methyl, difluoromethyl, trifluoromethyl, methoxy, trifluoromethoxy, ethoxy, 2-difluoroethoxy, 2-trifluoroethoxy and phenyl.
Preferably, it is a compound as shown in any one of formulas I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8 or I-9,
Wherein o is independently any integer from 0 to 5.
More preferably, it is a compound as shown in any one of the formulas I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1,
More preferably, the present invention provides specific compounds as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1 having the structural formula:
In a second aspect, the invention provides a pharmaceutical composition comprising a compound as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1, or one or more pharmaceutically acceptable salts, hydrates, solvates, stereoisomers, tautomers, metabolites or prodrugs thereof.
Preferably, the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
In a third aspect, the invention provides the use of a compound as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for the prevention and/or treatment of a disease caused by overexpression of PLK 1.
In a fourth aspect, the invention provides the use of a compound as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition comprising the same, for the preparation of a PLK1 inhibitor drug.
In a fifth aspect, the invention provides the use of a compound as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof, or a pharmaceutical composition comprising the same, in the manufacture of a medicament for the treatment and/or prophylaxis of cancer.
Preferably, the cancer is any one or more of hematological tumor, pancreatic cancer, colorectal cancer and lung cancer.
In a sixth aspect, the present invention provides a method for preventing and/or treating a disease or disorder caused by overexpression of PLK1, comprising administering to a subject in need thereof a prophylactically and/or therapeutically effective amount of a compound as shown in formula I, I-1, I-2, I-3, I-4, I-5, I-6, I-7, I-8, I-9, I-1-1, I-2-1, I-3-1, I-4-1, I-5-1, I-6-1, I-7-1, I-8-1 or I-9-1, or a pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof or a pharmaceutical composition comprising the same.
The invention provides novel PLK1 inhibitor compounds which not only have good PLK1 enzyme inhibition activity, but also can obviously reduce ERK phosphorylation in KRAS mutant cancer cell lines and have excellent anti-tumor activity on KRAS mutant cancer cells.
Compared with the prior art, the invention has the following beneficial effects:
The invention provides a series of tricyclic compounds with novel structures, and related enzyme and cell activity tests prove that the compounds have excellent cell proliferation inhibition activity, and in vitro experiments show that the IC 50 value of cell proliferation reaches nM level, so that the compounds can be well applied to various tumors. In particular, the compounds of the invention can significantly reduce ERK phosphorylation in KRAS mutant cancer cell lines, and have excellent antitumor activity on KRAS mutant cancer cells. Is suitable for preparing PLK1 inhibitor for preventing and/or treating diseases or symptoms related to PLK1 activation, such as cancers (including but not limited to blood tumor, pancreatic cancer, colorectal cancer and lung cancer).
Detailed Description
General terms and definitions
Unless stated to the contrary, the terms used in the present invention have the following meanings.
"Alkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms, which may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-dimethylpropyl, 1, 2-dimethylpropyl, 2-dimethylpropyl, 1-ethylpropyl, 2-methylbutyl, 3-methylbutyl, n-hexyl, 1-ethyl-2-methylpropyl, 1, 2-trimethylpropyl, 1-dimethylbutyl, 1, 2-dimethylbutyl, 2-dimethylbutyl, 1, 3-dimethylbutyl, 2-ethylbutyl, and various branched isomers thereof, and the like. Non-limiting examples also include, but are not limited to, methylene, ethylene, propylene, butylene, and various branched isomers thereof. In addition, in the present invention, "alkyl" may be optionally substituted or unsubstituted.
"Alkoxy" refers to an "-O-alkyl" group, where "alkyl" is defined above.
"Alkenyl" refers to unsaturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms and at least 1 carbon-carbon double bond, and may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkenyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, vinyl (-ch=ch 2), propen-1-yl (-ch=ch-CH 3), propen-2-yl (-C (CH 3)＝CH2), buten-1-yl (-ch=ch-CH 2-CH3), buten-2-yl (-C (C 2H5)＝CH2), 1-methylpropen-1-yl (-C (CH 3)＝CH-CH3) and various branched isomers thereof, etc. non-limiting examples also include, but are not limited to, 1-vinylidene (=c=ch 2), 1, 2-vinylidene (-ch=ch-), 1-propenylene (=c=ch-CH 3), 1, 2-propenylene (-ch=c (CH 3) -), 1, 3-propenylene (-ch=ch-CH 2 -) and various branched isomers thereof.
"Alkynyl" refers to unsaturated aliphatic hydrocarbon groups, including straight and branched chain groups of 1 to 20 carbon atoms and at least 1 carbon-carbon triple bond, and may be, for example, straight and branched chain groups of 1 to 18 carbon atoms, 1 to 12 carbon atoms, 1 to 8 carbon atoms, 1 to 6 carbon atoms, or 1 to 4 carbon atoms. In the present invention, "alkynyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, ethynylPropynyl groupButynyl groupPentynyl groupAnd various branched isomers thereof. Non-limiting examples also include, but are not limited to, ethynylenePropynyl groupButynyl groupAnd various branched isomers thereof. In addition, in the present invention, "alkynyl" may be optionally substituted or unsubstituted.
"Heteroalkyl" refers to saturated aliphatic hydrocarbon groups, including straight and branched chain groups of 2 to 20 atoms, such as may be straight and branched chain groups of 2 to 18 atoms, 2 to 12 atoms, 2 to 8 atoms, 2 to 6 atoms, or 2 to 4 atoms, where one or more atoms are heteroatoms selected from nitrogen, oxygen, or S (O) m (where m is 0, 1, or 2) and the remainder are carbon. In the present invention, "heteroalkyl" may be a monovalent, divalent or trivalent group. Non-limiting examples include, but are not limited to, methoxymethyl (2-oxapropyl), methylthiomethyl (2-thiapropyl), methylaminomethyl (2-aza-propyl), and various branched isomers thereof, and the like. In addition, in the present invention, "heteroalkyl" may be optionally substituted or unsubstituted.
"Cycloalkyl" refers to a saturated or partially unsaturated, mono-or polycyclic aliphatic hydrocarbon group comprising 3 to 12 ring atoms, which may be, for example, 3 to 12, 3 to 10, or 3 to 6 ring atoms (i.e., 3 to 6 membered rings). Non-limiting examples of monocyclic cycloalkyl groups include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl, cyclohexadienyl, cycloheptyl, cycloheptatrienyl, cyclooctyl, and the like. In the present invention, "cycloalkyl" may be optionally substituted or unsubstituted.
"Heterocycloalkyl" means a saturated or partially unsaturated, mono-or polycyclic aliphatic hydrocarbon group comprising 3 to 20 ring atoms, which may be, for example, 3 to 16, 3 to 12, 3 to 10 or 3 to 6 ring atoms, wherein one or more of the ring atoms is a heteroatom selected from nitrogen, oxygen or S (O) m (where m is 0, 1 or 2) and the remaining ring atoms are carbon. Preferably the heterocycloalkyl group comprises 3 to 12 ring atoms, of which 1 to 4 ring atoms are heteroatoms, more preferably 3 to 10 ring atoms, most preferably 5 or 6 ring atoms, of which 1 to 4, preferably 1 to 3, more preferably 1 to 2 are heteroatoms. Non-limiting examples of monocyclic heterocycloalkyl groups include, but are not limited to, pyrrolidinyl, piperidinyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. Non-limiting examples of polycyclic heterocycloalkyl groups include, but are not limited to, a fused, spiro, or bridged heterocycloalkyl group.
"Halogen" means fluorine, chlorine, bromine and iodine, preferably fluorine, chlorine and bromine.
"Haloalkyl" or "haloalkoxy" refers to an alkyl or alkoxy group substituted with one or more halogen atoms, which may be the same or different, examples of preferred alkyl or alkoxy groups include, but are not limited to: trifluoromethyl, trifluoroethyl, trifluoromethoxy.
"Cyano" refers to the "-CN" group.
"Hydroxy" refers to an "-OH" group.
"Amino" refers to the "-NH 2" group.
"Carbamoyl" refers to the "- (c=o) -NH 2" group.
"Aryl" refers to monocyclic, bicyclic, and tricyclic carbocyclic ring systems containing 6 to 14 ring atoms, wherein at least one ring system is aromatic, wherein each ring system contains rings of 3 to 7 atoms and has one or more points of attachment to the remainder of the molecule. Examples include, but are not limited to: phenyl, naphthyl, anthracene, and the like. Preferably, the aryl group is a carbocyclic ring system of 6 to 10 or 6 to 7 ring atoms.
"Heteroaryl" refers to monocyclic, bicyclic, and tricyclic ring systems containing 5 to 14 ring atoms, wherein at least one ring system is aromatic and at least one ring system contains one or more heteroatoms selected from nitrogen, oxygen, and sulfur, wherein each ring system contains a ring of 5 to 7 atoms and has one or more points of attachment to the remainder of the molecule. The term "heteroaryl" may be used interchangeably with the term "heteroaromatic ring" or "heteroaromatic compound". Examples include, but are not limited to: furyl, imidazolyl, 2-pyridyl, 3-pyridyl, thiazolyl, purinyl, and quinolinyl. Preferably, the heteroaryl group is a ring system of 5 to 10 ring atoms.
"Optional" or "optionally" means that the subsequently described event or circumstance may but need not occur, and that the description includes instances where the event or circumstance occurs or does not. For example, "a heterocyclic group optionally substituted with an alkyl group" means that an alkyl group may be, but is not necessarily, present, and the description includes cases where the heterocyclic group is substituted with an alkyl group and cases where the heterocyclic group is not substituted with an alkyl group.
"Substituted" means that one or more hydrogen atoms, preferably up to 5, more preferably 1 to 3 hydrogen atoms in the group are independently substituted with a corresponding number of substituents.
By "pharmaceutically acceptable salts" is meant salts prepared from the compounds of the present invention with relatively non-toxic acids or bases. When the compounds of the present invention contain relatively acidic functional groups (e.g., carboxyl or sulfonic groups), the base addition salts may be obtained by contacting the free form thereof with a sufficient amount of a base in pure solution or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable base addition salts include, but are not limited to, sodium, potassium, ammonium, calcium, magnesium, organic amine salts, or the like. When the compounds of the present invention contain relatively basic functional groups (e.g., amino or guanidino), the acid addition salts may be obtained by contacting the free form with a sufficient amount of an acid in a pure solution or in a suitable inert solvent. Non-limiting examples of pharmaceutically acceptable acid addition salts include, but are not limited to, inorganic acid salts (e.g., hydrochloride, hydrobromide, hydroiodide, nitrate, carbonate, bicarbonate, phosphate, monohydrogen phosphate, dihydrogen phosphate, phosphite, sulfate, bisulfate, etc.), organic acid salts (e.g., acetate, propionate, isobutyrate, malonate, succinate, suberate, maleate, fumarate, citrate, tartrate, lactate, mandelate, benzoate, phthalate, methanesulfonate, benzenesulfonate, p-toluenesulfonate, glucuronate, etc.), and amino acid salts (e.g., arginate, etc.). Specific forms of pharmaceutically acceptable salts can also be found in Berge et al, "Pharmaceutical Salts", journal of Pharmaceutical Science,1977, 66:1-19). Certain specific compounds of the invention contain basic and acidic functionalities that can be converted to either base or acid addition salts. Preferably, the salt is contacted with a base or acid in a conventional manner to isolate the parent compound, thereby regenerating the neutral form of the compound. The parent form of a compound differs from its various salt forms in certain physical properties, such as solubility in polar solvents. According to an embodiment of the present invention, the pharmaceutically acceptable salt of the compound of formula I is preferably an acid addition salt, preferably a hydrochloride, hydrobromide, phosphate or sulfate salt, more preferably a hydrochloride salt.
"Pharmaceutical composition" refers to a pharmaceutically acceptable composition comprising one or more compounds of formula I or a pharmaceutically acceptable form thereof (e.g., salts, hydrates, solvates, stereoisomers, tautomers, metabolites, prodrugs, etc.), as well as other components (e.g., pharmaceutically acceptable excipients).
In the present invention, "pharmaceutically acceptable excipients" refers to auxiliary materials widely used in the field of pharmaceutical production. The main purpose of the use of auxiliary substances is to provide a pharmaceutical composition which is safe to use, stable in nature and/or has specific functionalities, and to provide a method so that the active ingredient can be dissolved at a desired rate after administration of the drug to a subject, or so that the active ingredient is effectively absorbed in the subject to whom it is administered. Pharmaceutically acceptable excipients may be inert fillers or may be functional ingredients that provide some function to the pharmaceutical composition (e.g., to stabilize the overall pH of the composition or to prevent degradation of the active ingredients in the composition). Non-limiting examples of pharmaceutically acceptable excipients include, but are not limited to, binders, suspending agents, emulsifiers, diluents (or fillers), granulating agents, binders, disintegrants, lubricants, anti-adherent agents, glidants, wetting agents, gelling agents, absorption delaying agents, dissolution inhibitors, reinforcing agents, adsorbents, buffers, chelating agents, preservatives, coloring agents, flavoring agents, sweetening agents, and the like.
The pharmaceutical compositions of the present invention may be prepared using any method known to those skilled in the art. For example, conventional mixing, dissolving, granulating, emulsifying, milling, encapsulating, entrapping and/or lyophilizing processes.
In the present invention, the purpose of the pharmaceutical composition is to promote the administration to a living body, facilitate the absorption of an active ingredient, and further exert biological activity. The pharmaceutical compositions of the present invention may be administered by any form including injection (intra-arterial, intravenous, intramuscular, intraperitoneal, subcutaneous), mucosal, oral (oral solid, oral liquid), rectal, inhalation, implantation, topical (e.g. ocular) administration, and the like. Non-limiting examples of oral solid formulations include, but are not limited to, powders, capsules, lozenges, granules, tablets, and the like. Non-limiting examples of liquid formulations for oral or mucosal administration include, but are not limited to, suspensions, tinctures, elixirs, solutions, and the like. Non-limiting examples of topical formulations include, but are not limited to, emulsions, gels, ointments, creams, patches, pastes, foams, lotions, drops or serum formulations. Non-limiting examples of parenteral formulations include, but are not limited to, solutions for injection, dry powders for injection, suspensions for injection, emulsions for injection, and the like. The pharmaceutical compositions of the invention may also be formulated in controlled-or delayed-release dosage forms (e.g. liposomes or microspheres).
Preferably, the compounds of the present invention or pharmaceutical compositions comprising the same are administered orally or intravenously to an individual in need thereof. Depending on the specific circumstances of the subject, other routes of administration may also be employed or even preferred. For example, transdermal administration would be a very important mode of administration for patients with amnesia or irritability to oral medications. In the present invention, the route of administration can be varied or adjusted in any suitable manner to meet the nature of the drug, the convenience of the patient and medical personnel, and other related factors.
The compound or pharmaceutically acceptable salt, hydrate, solvate, stereoisomer, tautomer, metabolite or prodrug thereof or the pharmaceutical composition containing the compound has excellent PLK1 enzyme inhibition activity and cell proliferation inhibition activity, can be used as a PLK inhibitor, is used for preventing and/or treating diseases or symptoms caused by PLK over-expression, and has good clinical application and medical application. Preferably, non-limiting examples of diseases or conditions caused by PLK1 overexpression are cancers, including but not limited to hematological tumors, pancreatic cancers, colorectal cancers and lung cancers.
The following examples are provided to further illustrate the invention and are not intended to limit the scope of the invention. Various changes and modifications to the specific embodiments of the invention will be apparent to those skilled in the art without departing from the spirit and scope of the invention.
The preparation of the compounds of the present invention may be accomplished by synthetic methods well known to those skilled in the art, including but not limited to the specific embodiments listed below, embodiments formed in combination with other chemical synthetic methods, and equivalent alternatives well known to those skilled in the art, preferred embodiments including but not limited to the examples of the present invention. The known starting materials used in the present invention may be synthesized by methods known in the art or purchased through conventional commercial means (e.g., from Shaohuan chemical technology, beijing coupling technology, etc.). Unless otherwise indicated, the reactions were carried out under argon or nitrogen atmosphere. The hydrogenation reaction is usually vacuumized, filled with hydrogen and repeatedly operated for 3 times. The reaction temperature is room temperature and the temperature range is 20-30 ℃. Monitoring of the progress of the reaction may be accomplished by synthetic methods well known to those skilled in the art, including but not limited to Thin Layer Chromatography (TLC). Thin layer chromatography silica gel plates using Qingdao ocean GF254 silica gel plates, the developer system includes but is not limited to A: methylene chloride and methanol systems; b: petroleum ether and ethyl acetate system, and the volume ratio of the solvent can be adjusted according to the polarity of the compound.
The isolation and purification of the compounds of the present invention may be accomplished by synthetic methods well known to those skilled in the art, including, but not limited to, column Chromatography (CC), high Performance Liquid Chromatography (HPLC), ultra-high performance liquid chromatography (UPLC), and the like. Column chromatography typically uses Qingdao ocean 200-300 mesh silica gel as a carrier, and eluent systems include, but are not limited to, A: methylene chloride and methanol systems; b: the volume ratio of the petroleum ether to the ethyl acetate can be adjusted according to the polarity of the compound, and a small amount of acidic or alkaline tailing-preventing agent can be added for adjustment. HPLC spectra were determined using an Agilent1200DAD HPLC chromatograph (column: sunfireC18, 150X4.6 mm,5 μm) or a Waters 2695-2996HPLC chromatograph (column: giminiC18, 150X4.6 mm,5 μm).
Structural identification of the compounds of the present invention may be accomplished by methods well known to those skilled in the art, including but not limited to Nuclear Magnetic Resonance (NMR), mass Spectrometry (MS), and the like. NMR spectra were determined using Bruker AVANCE-400 or VarianOxford-300 nuclear magnetic instruments using deuterated dimethyl sulfoxide (DMSO-d 6), deuterated chloroform (CDC 1 3) or deuterated methanol (CD 3 OD), internal standard Tetramethylsilane (TMS), chemical shifts in 10 -6 (ppm). MS spectra were determined using AGILENTSQD (ESI) mass spectrometers (model 6110) or Shimadzu SQD (ESI) mass spectrometers (model 2020).
Preparation of intermediates
Preparation of intermediate INT-1
The preparation method comprises the following steps:
The first step: synthesis of Compound INT-1B
Compound INT-1A (10.0 g,39.2 mmol) was dissolved in dioxane (100.0 mL), and acetic anhydride (4.8 g,47.0 mmol) and triethylamine (7.92 g,78.4 mmol) were added at room temperature to react at 60℃for 18 hours. TLC showed that after the reaction was completed, the reaction mixture was dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10:1 to 1:2 (volume ratio)) to give compound INT-1B (10.4 g, pale yellow solid, yield 89%).
MS(ESI):m/z298.0[M+1]+。
And a second step of: synthesis of Compound INT-1C
Compound INT-1B (2.0 g,6.71 mmol) was dissolved in dioxane (30.0 mL), N-methylpiperazine (671mg,6.71mmol),Cs2CO3(4.4g,13.4mmol),Pd2(dba)3(0.3g,0.34mmol),4,5- bis (diphenylphosphine) -9, 9-dimethylxanthene (0.39 g,0.67 mmol) was added at room temperature, and the reaction mixture was reacted at 95℃for 18 hours under N 2. TLC showed that after the reaction was completed, the reaction mixture was dried by spin-drying and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10:1 to 1:2 (volume ratio)) to give compound INT-1C (2.6 g, pale yellow solid, yield 87%).
MS(ESI):m/z318.1[M+1]+。
And a third step of: synthesis of Compound INT-1D
Compound INT-1C (0.89 g,2.8 mmol) was added to ethanol (10 mL), and then concentrated hydrochloric acid (2.5 mL) was added under ice-bath, and the reaction mixture was reacted at 80℃for 18h. TLC showed that after the reaction was completed, ethanol was turned off, the reaction solution was diluted with water (50 mL), ph=9 was adjusted with aqueous ammonia, extracted with ethyl acetate (15 ml×2), the organic phases were combined, washed with saturated aqueous sodium chloride solution (10 ml×2), the organic phase was dried over anhydrous sodium sulfate, and the organic phase was dried by spin-drying to give compound INT-1D (0.72 g, pale yellow solid, yield 93%).
MS(ESI):m/z276.1[M+1]+。
Fourth step: synthesis of Compound INT-1
Compound INT-1D (0.61 g,2.22 mmol) was added to acetonitrile (10 mL) at room temperature followed by INT-1E (0.35 g,2.89 mmol). The reaction mixture was reacted at 80℃for 18 hours. TLC showed that after the reaction was completed, the reaction solution was spin-dried to give compound INT-1 (0.64 g, brown solid, yield 90%).
MS(ESI):m/z318.2[M+1]+。
Preparation of intermediate INT-2
The preparation method comprises the following steps:
The first step: synthesis of Compound INT-2B
Compound INT-1B (2 g,6.71 mmol) was dissolved in dioxane (30 mL), INT-2A(1.52g,6.71mmol),Cs2CO3(4.4g,13.4mmol),Pd2(dba)3(0.3g,0.34mmol),4,5- bis (diphenylphosphine) -9, 9-dimethylxantheneXanths (0.39 g,0.67 mmol) was added at room temperature, and the reaction mixture was reacted at 95℃for 18 hours under N 2. TLC showed that after the reaction was completed, the reaction mixture was dried by spin-drying and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10:1 to 1:2 (volume ratio)) to give compound INT-2B (2.6 g, pale yellow solid, yield 87%).
MS(ESI):m/z444.2[M+1]+。
And a second step of: synthesis of Compound INT-2C
Compound INT-2B (3.2 g,7.22 mmol) was dissolved in ethyl acetate (15 mL), followed by dropwise addition of 8% strength by mass ethyl acetate hydrochloride (30 mL) and reaction at room temperature for 18h. TLC showed that after completion of the reaction, the reaction solution was dried by spin-drying, the residue was slurried with EtOAc, and the filter cake was filtered to give compound INT-2C (3.0 g, pale yellow solid, purity: 50%, yield 55%).
MS(ESI):m/z344.2[M+1]+。
And a third step of: synthesis of Compound INT-2D
Compound INT-2C (3.0 g,50% purity, 4.37 mmol) was added to dichloromethane (30 mL) and water (10 mL), and 37% aqueous formaldehyde (2.1 g,26.2 mmol) and acetic acid (0.2 mL) were added dropwise over ice and reacted at room temperature for 1h. Sodium cyanoborohydride (0.82 g,13.1 mmol) was then added in portions under ice bath, and the reaction was allowed to proceed at room temperature for 12h. TLC showed that after the reaction was completed, aqueous ammonia was adjusted to ph=9, and the reaction solution was diluted with water (150 mL), extracted with dichloromethane (50 ml×2), extracted, the organic phases were combined, washed with saturated aqueous sodium chloride solution (30 ml×2), the organic phase was dried over anhydrous sodium sulfate, the resulting organic solution was filtered, and the residue after spin-drying was purified by basic alumina column chromatography (eluent: petroleum ether/ethyl acetate=10:1 to 1:2 (volume ratio)) to give compound INT-2D (1.0 g, tan oil, yield 64%).
MS(ESI):m/z358.1[M+1]+。
Fourth step: synthesis of Compound INT-2E
Compound INT-2D (1 g,2.8 mmol) was added to ethanol (10 mL), then concentrated hydrochloric acid (2.5 mL) was added under ice-bath, and the reaction mixture was reacted at 80℃for 18h. TLC showed that after the reaction was completed, ethanol was turned off, the reaction mixture was diluted with water (50 mL), ph=9 was adjusted with aqueous ammonia, extracted with ethyl acetate (15 ml×2), the organic phases were combined, washed with saturated aqueous sodium chloride solution (10 ml×2), the organic phase was dried over anhydrous sodium sulfate, and the resulting organic solution was filtered and dried by spinning to give compound INT-2E (0.8 g, pale yellow solid, yield 91%).
MS(ESI):m/z316.1[M+1]+。
Fifth step: synthesis of Compound INT-2
Compound INT-2F (0.7 g,2.22 mmol) was added to acetonitrile (10 mL) at room temperature followed by INT-1E (0.35 g,2.89 mmol). The reaction mixture was reacted at 80℃for 18 hours. TLC showed that after the reaction was completed, the reaction solution was spin-dried to give compound INT-2 (0.8 g, brown solid, yield 91%).
MS(ESI):m/z358.2[M+1]+。
Preparation and functional verification of target compounds
Example 1: preparation of Compound 1
The structural formula of the compound 1 is as follows:
The synthetic route of compound 1 is:
The specific preparation method of the compound 1 comprises the following steps:
the first step: synthesis of Compound 1C
Compound 1A (3 g,30 mmol) was dissolved in DCM (25 mL), tetrahydropyrrole pyrrolidine (1.1 g,15 mmol) was added under ice-bath and reacted at room temperature for 2h, then 1B (3.6 g,30 mmol) was added under ice-bath and the reaction was reacted at 40℃for 16 h. TLC showed that after the reaction was completed, the reaction mixture was dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=10:1 (volume ratio)), to give compound 1C (3.5 g, yellow solid, yield 58%).
MS(ESI):m/z203[M+1]+。
And a second step of: synthesis of Compound 1D
Compound 1C (6.6 g,32.6 mmol) was dissolved in THF (60 mL), liHMDS (49 mL,1M,48.9 mmol) was added dropwise at-60℃under nitrogen, reaction was performed at-60℃for 0.5 and then diethyl oxalate (7.2 g,48.9 mmol) was added dropwise, and the mixture was allowed to react at room temperature for 12h. TLC showed that after the reaction was completed, quenched by pouring ice water, extracted with ethyl acetate, washed with brine, the organic phase was dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=5:1 (volume ratio)) to give compound 1D (3.1 g, yellow solid, yield 31%).
MS(ESI):m/z303[M+1]+。
And a third step of: synthesis of Compound 1E
Compound 1D (2.9 g,9.6 mmol) was added to methanol (25 mL) and acetic acid (5 mL), and 1D-1 (876 mg,9.6 mmol) was added dropwise under ice-bath and reacted at room temperature for 16h. TLC showed that after the reaction was completed, a large amount of solid was precipitated, and the reaction solution was filtered to obtain compound 1E (2.3 g, yellow solid, purity 75%, yield 53%).
MS(ESI):m/z343[M+1]+。
Fourth step: synthesis of Compound 1F
Compound 1E (2.2 g,6.4 mmol) and RuCl3.H2O were added to acetonitrile (30 mL) and water (6 mL), followed by addition of sodium periodate (2.2 g,9.6 mmol) in an ice bath, and the reaction was allowed to react at room temperature for 16h. TLC showed that after the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, washed with brine, the organic phase was dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=1:1 (volume ratio)) to give compound 1F (1.2 g, pale yellow solid, yield 73%).
MS(ESI):m/z255[M+1]+。
Fifth step: synthesis of Compound 1G
Compound 1F (1 g,3.9 mmol) was added to DMF (6 mL) at room temperature, followed by N, N-dimethylformamide dimethyl acetal DMF-DMA (6 mL). The reaction was carried out at 120℃for 2h. TLC showed that after the reaction was completed, compound 1G (1.1G, brown oil, 90% yield) was obtained by spin-drying.
MS(ESI):m/z310[M+1]+。
Sixth step: synthesis of Compound 1H
Compound 1G (1.1G, 3.6 mmol) was added to DMF (10 mL) at room temperature, and compound INT-1 (1.25G, 3.6 mmol) and potassium carbonate (0.98G, 7.1 mmol) were added at room temperature. Then heated to 110℃for reaction 32h. TLC showed that after the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, washed with brine, the organic phase was dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=5:1 (volume ratio)), to give compound 1H (0.35 g, yellow solid, yield 17%)
MS(ESI):m/z564[M+1]+。
Seventh step: synthesis of Compound 1
Compound 1H (350 mg,0.06 mmol) was added to a jar containing NMP (5 mL) at room temperature, followed by 7.0M NH 3 in methanol (5 mL). Then heated to 95℃and reacted for 48 hours. TLC showed that after the reaction was completed, the solid precipitated after cooling to room temperature, filtration and washing with ethyl acetate gave compound 1 (200 mg, yellow solid, purity 70%, yield 42%).
MS(ESI):m/z535[M+1]+。
1H-NMR(400MHz,DMSO):δppm 8.81(s,1H),8.20(s,1H),7.69(brs,1H),7.49(brs,1H)7.26(d,J＝4Hz,1H),7.21-7.19(m,1H),6.76-6.73(m,1H),5.46(s,2H),4.64-4.59(m,2H),3.72-3.65(m,2H),3.13(t,J＝4Hz,4H),2.44(t,J＝4Hz,4H),2.21(s,3H).
Example 2: preparation of Compound 2
The structural formula of the compound 2 is as follows:
the synthetic route for compound 2 is:
The specific preparation method of the compound 2 comprises the following steps:
the first step: synthesis of Compound 2B
Compound 2A (8.0 g,34.3 mmol) was dissolved in DCM (100.0 mL), and tetrahydropyrrole (1.22 g,17.2 mmol) was added dropwise under ice-bath, and stirred at room temperature for 2 hours. Then benzaldehyde is added dropwise in ice bath, and the mixture is stirred for 36 hours after being warmed to room temperature. TLC showed that after the reaction was completed, the reaction solution was dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3/1 (volume ratio)) to give compound 2B (6.6 g, yellow oil, yield: 60%).
MS(ESI):m/z 322[M+1]+。
1H NMR(400MHz,CDCl3)δppm 7.45-7.25(m,11H),5.22(s,2H),4.75(s,2H),3.78-3.76(m,2H),2.75-2.52(m,2H).
And a second step of: synthesis of Compound 2C
Compound 2B (6 g,18.7 mmol) was dissolved in THF (80 mL), a 1.0M solution of LiHMDS in THF (28 mL,28 mmol) was added dropwise at-60℃and stirred at-60℃for 1 hour, followed by a solution of diethyl oxalate (4.1 g,28 mmol) in THF (6 mL). The reaction was slowly warmed to room temperature and stirred for 12 hours. TLC showed that after the reaction was completed, ethyl acetate and water were added, extraction was performed, the organic phase was washed with brine, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3/1 (volume ratio)) to give compound 2C (5 g, yellow solid, yield: 64%).
MS(ESI):m/z 422[M+1]+。
And a third step of: synthesis of Compound 2D
Compound 2C (5 g,11.86 mmol) was dissolved in methanol (30 mL), THF (30 mL) and acetic acid (6 mL), and compound 1D-1 (0.9 g,11.86 mmol) was added dropwise under ice-bath and stirred at room temperature for 16 hours. TLC showed that after the reaction was completed, most of the methanol was removed by rotation, ethyl acetate and water were added, extraction was performed, the organic phase was washed with saturated sodium bicarbonate and brine, and the residue was purified by beating (petroleum ether/ethyl acetate=1/1, 40 ml) to give compound 2D (4.0 g, white solid, yield: 73%).
MS(ESI):m/z 462[M+1]+。
Fourth step: synthesis of Compound 2E
Compound 2D (4.0 g,8.67 mmol) was dissolved in acetonitrile (80 mL) and water (20 mL), ruthenium trichloride hydrate (0.19 g,0.87 mmol) and sodium periodate (2.4 g,10.4 mmol) were added while ice-cooling, and stirred for 4h at room temperature. After TLC showed that acetonitrile was removed by spin, ethyl acetate and water were added, the organic phase was extracted, washed with brine, spin-dried, and the residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=1/1 (volume ratio)) to give compound 2E (2 g, pale yellow oil, yield: 60%).
MS(ESI):m/z 388[M+1]+。
1H NMR(400MHz,CDCl3)δppm 7.41-7.25(m,5H),5.24(s,2H),5.00(s,2H),4.75-4.71(m,2H),4.48-4.45(m,2H),4.32(s,2H),4.10-4.05(m,2H),1.45-1.40(m,3H).
Fifth step: synthesis of Compound 2F
Compound 2E (1.0 g,2.58 mmol) was dissolved in DMF (12 mL) and then DMF-DMA (6 mL) was added and stirred at 110℃for 2h. After completion of TLC, the reaction solution was dried by spin to give compound 2F (1.1 g, brown yellow oil, yield: 96%), and the crude product was used directly in the next step.
MS(ESI):m/z 443[M+1]+。
Sixth step: synthesis of Compound 2G
Compound 2F (1.1 g,2.49 mmol) was dissolved in DMF (15 mL) and then INT-1 (0.29 g,2.49 mmol) and potassium carbonate (1.03 g,7.46 mmol) were added and stirred at 100deg.C for 3 days. After TLC showed that ethyl acetate and water were added, extraction was performed, the organic phase was washed with brine, dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=10/1 (volume ratio)) to give compound 2G (0.55G, brown oil, purity: 60%, yield: 19%).
MS(ESI):m/z 697[M+1]+。
Seventh step: synthesis of Compound 2H
Compound 2G (0.55G, 60% purity, 0.47 mmol) was dissolved in NMP (8 mL) and then 30% ammonia in methanol (8 mL) was added and stirred at 100deg.C for 3 days. After TLC showed that it was completed, it was dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=10/1 (volume ratio)) to give compound 2H (0.4 g, brown oil, purity: 50%, yield: 63%).
MS(ESI):m/z 668[M+1]+。
Eighth step: synthesis of Compound 2
Compound 2H (100 mg,0.07 mmol) was dissolved in methanol (4 mL), then 10% wet palladium on carbon (10 mg), hydrogen balloon was substituted for the gas, and stirred at room temperature for 16 hours. TLC showed that after the reaction was complete, filtration, spin-drying and purification of the residue by HPLC (Waters Sunfire OBD 100X30mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile in 1min,52% -52% acetonitrile to 10min,95% acetonitrile to 14min,10% acetonitrile to 16min end) gave compound 2 (15 mg, yellow solid, yield: 38%).
MS(ESI):m/z 534[M+1]+。
1H NMR(400MHz,DMSO)δppm 9.67(s,1H),9.33(s,1H),7.98(s,1H),7.68(s,1H),7.30-7.21(m,2H),6.86(dd,J＝9.2,2.9Hz,1H),4.90-4.82(m,2H),4.66(s,1H),3.75(d,J＝4.8Hz,2H),3.20-3.10(m,4H),2.46-2.40(m,4H),2.20(s,3H).
Example 3: preparation of Compound 3
The structural formula of the compound 3 is as follows:
the synthetic route for compound 3 is:
The specific preparation method of the compound 3 comprises the following steps:
The first step: synthesis of Compound 3B
The hydrochloride salt of compound INT-2 (0.4 g,1 mmol) was added to DMF (6 mL) at room temperature, and 3A (0.35 g,1.1mmol, synthesized by the procedure and method described on pages 42-44 of WO 200874788) and potassium carbonate (0.28 g,2 mmol) were added at room temperature. Then heated to 100 ℃ and reacted for 48 hours. TLC showed that after the reaction was completed, the reaction solution was diluted with water, extracted with ethyl acetate, washed with brine, the organic phase was dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=5:1 (volume ratio)), to give compound 3B (0.3 g, brown oil, yield 29%)
MS(ESI):m/z602.2[M+1]+。
And a second step of: synthesis of Compound 3
Compound 3B (300 mg,60% purity, 0.3 mmol) was added to a jar containing NMP (5 mL) at room temperature, followed by 7M NH3 in methanol (5 mL). Then heated to 95℃and reacted for 3 days. TLC showed that after the reaction was completed, methanol, ethyl acetate and water were diluted, extracted, dried, and the residue was purified by HPLC (Waters Sunfire OBD X30mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile running 1min,52% -52% acetonitrile running 10min,95% acetonitrile running 14min,10% acetonitrile running 16min ended) to give compound 3 (20 mg, pale yellow solid, purity 90%, yield 12%).
MS(ESI):m/z573.2[M+1]+。
1H-NMR(400MHz,DMSO-d6):δppm8.89(s,1H),8.24(s,1H),7.49(brs,1H),7.29(brs,1H),7.23(d,J＝4Hz,1H),7.19(d,J＝8Hz,1H),6.78(dd,J＝8Hz,4Hz,1H),4.64-4.61(m,2H),3.64-3.61(m,2H),3.08(t,J＝4Hz,4H),2.97(t,J＝8Hz,2H),2.93(s,4H),2.79(t,J＝8Hz,2H),2.22(s,3H),1.74(t,J＝4Hz,4H).
Example 4: preparation of Compound 4
The structural formula of the compound 4 is as follows:
The synthetic route for compound 4 is:
The specific preparation method of the compound 4 comprises the following steps:
the first step: synthesis of Compound 4B
Compound 4A (10.0 g,71.3 mmol) was dissolved in DMF (100 mL), DMF-DMA (30 mL) was added dropwise, and the mixture was warmed to 120℃and stirred for 2h. TLC showed that after the reaction was completed, the reaction solution was cooled to room temperature and dried by spin-drying to give compound 4B (10.0 g, brown oil, yield: 72%) which was used in the next step without further purification.
MS(ESI):m/z 196.1[M+1]+。
And a second step of: synthesis of Compound 4C
Compound 4B (10.0 g,51.2 mmol) was dissolved in methanol (100 mL) and acetic acid (10 mL), and compound 1D-1 (3.9 g,51.2 mmol) was added dropwise and stirred at room temperature for 12h. TLC showed that after the reaction was completed, methanol was swirled off, water (200 mL) was added, and extracted with ethyl acetate (60 mL. Times.2), the organic phases were combined, washed successively with saturated sodium hydrogencarbonate (60 mL. Times.1) and saturated aqueous sodium chloride (60 mL. Times.1), the organic phase was dried over anhydrous sodium sulfate, and the obtained residue was purified by column chromatography on silica gel (eluent: petroleum ether/ethyl acetate=2/1 (volume ratio)) to give compound 4C (2.2 g, pale yellow solid, yield: 24%).
MS(ESI):m/z 181[M+1]+。
And a third step of: synthesis of Compound 4D
Compound 4C (1.8 g,9.99 mmol) was dissolved in acetonitrile (30 mL), NBS (2.13 g,11.99 mmol) was added in portions under ice bath, and the mixture was stirred at 50℃for 4 hours. TLC showed that after the reaction was completed, most of acetonitrile was swirled off, water (100 mL) was added, and extracted with ethyl acetate (30 ml×2), and after the organic phases were combined, washed with saturated sodium bicarbonate (30 ml×1) and saturated aqueous sodium chloride (30 ml×1) in this order, the organic phase was dried over anhydrous sodium sulfate, and the residue was purified by silica gel column chromatography (eluent: petroleum ether/ethyl acetate=3/1 (volume ratio)) to give compound 4D (2.0 g, pale yellow solid, yield: 77%).
MS(ESI):m/z 259.0[M+1]+,261.0[M+3]+。
1H NMR(400MHz,CDCl3)δppm 4.71-4.65(m,2H),4.08-3.95(m,2H),2.69(t,J＝6.1Hz,2H),2.63-2.56(m,2H),2.53(s,1H),2.22-2.11(m,2H).
Fourth step: synthesis of Compound 4E
Compound 4D (2.0 g,7.72 mmol) was dissolved in DMF (25 mL) and then DMF-DMA (10 mL) was added and stirred at 120℃for 1h. After completion of TLC, the reaction solution was dried by spin to give compound 4E (2.4 g, brown yellow oil, yield: 99%), and the crude product was used in the next step.
MS(ESI):m/z 314.0[M+1]+,316.0[M+3]+。
Fifth step: synthesis of Compound 4F
Compound 4E (2.4 g,7.64 mmol) was dissolved in DMF (30 mL) and then added to compound INT-1 (2.42 g,7.64 mmol) and DIPEA (1.97 g,15.28 mmol) for 16h at 100deg.C. After TLC showed that water (100 mL) was added and extracted with ethyl acetate (30 ml×2), the organic phases were combined, washed with saturated aqueous sodium chloride (30 ml×1), dried over anhydrous sodium sulfate, and the organic phase was dried by spin-drying, and the residue was purified by silica gel column chromatography (eluent: ethyl acetate/methanol=10/1 (volume ratio)) to give compound 4F (1.0 g, pale yellow solid, yield: 23%).
MS(ESI):m/z 568.1[M+1]+,570.1[M+3]+。
Sixth step: synthesis of Compound 4
Compound 4F (100 mg,0.18 mmol) was dissolved in dioxane (2.0 mL), then compound 4F-1 (25 mg,0.26 mmol), cesium carbonate (115 mg,0.35 mmol), pd 2(dba)3 (16 mg,0.02 mmol), xantphos (20 mg,0.04 mmol) was added and the reaction stirred under nitrogen at 110℃for 16 hours. TLC showed that after the reaction was completed, ethyl acetate and water were added, the organic phase was washed with brine, dried by spin, and the residue was purified by HPLC (Waters Sunfire OBD X30mm,5 μm, mobile phase A: 0.1% TFA in water, mobile phase B: acetonitrile, gradient: 10% acetonitrile to 1min,52% -52% acetonitrile to 10min,95% acetonitrile to 14min,10% acetonitrile to 16min end), to give compound 4 (6.6 mg, white solid, yield: 6%).
MS(ESI):m/z 581[M+1]+。
1H NMR(400MHz,DMSO)δppm 8.74(s,1H),8.26(s,1H),7.27(d,J＝2.4Hz,1H),7.18(d,J＝7.9Hz,1H),6.75(dd,J＝9.1,2.9Hz,1H),4.61-4.35(m,3H),3.60-3.56(m,2H),3.28-3.24(m,5H),3.15-3.09(m,4H),2.72(t,J＝7.3Hz,2H),2.45-2.40(m,4H),2.20(s,3H).
Example 5: preparation of Compound 5
The structural formula of the compound 5 is as follows:
synthesis of Compound 5 was synthesized by the step of synthesizing Compound 4 in reference example 4, in which zinc cyanide was used in place of Compound 4F-1 in the sixth step.
MS(ESI):515.2[M+1]+。
1H NMR(400MHz,DMSO)δ(ppm)9.44(s,1H),9.16(s,1H),8.40(s,1H),7.34-7.16(m,2H),6.95-6.77(m,1H),4.78-4.72(m,2H),3.98-3.95(m,2H),3.35-3.29(m,8H),2.86-2.62(m,7H).
Example 6: preparation of Compound 6
The structural formula of the compound 6 is as follows:
Synthesis of Compound 6 reference the step of synthesizing Compound 4 in example 4, wherein in the sixth step, compound 4F-1 is replaced with phosphorus oxide dimethyl, compound 6 is synthesized.
MS(ESI):566.2[M+1]+。
1H NMR(400MHz,DMSO)δ(ppm)8.90(s,1H),8.33(s,1H),7.25-7.15(m,2H),6.78(dd,J＝9.1,2.7Hz,1H),4.66-4.54(m,3H),3.63-3.53(m,2H),3.15-3.09(m,4H),2.95-2.75(m,4H),2.42(s,4H),2.20(s,3H),1.68(s,3H),1.63(s,3H).
Example 7: preparation of Compound 7
The structural formula of the compound 7 is as follows:
Synthesis of Compound 7 reference example 4 the step of synthesizing Compound 4 in which Compound 4F-1 was replaced with acetamide in the sixth step, compound 7 was synthesized.
MS(ESI):547.2[M+1]+。
Experimental example 1: anti-proliferative Activity test of the inventive Compounds against HT29 cells
Experimental materials:
McCoy's 5A medium, penicillin/streptomycin antibiotics were purchased from vitamin, and fetal bovine serum was purchased from Biosera. CellTiter-Glo (cell viability chemiluminescent detection reagent) reagent was purchased from Promega. HT29 cell line was purchased from the marsupenario life technologies Co. Nivo Multilabel Analyzer (Perkinelmer).
The experimental method comprises the following steps:
HT29 cells were seeded in white 96-well plates, 80 μl of cell suspension per well, containing 3000 HT29 cells. Cell plates were placed in a carbon dioxide incubator overnight for culture.
The test compounds were diluted 3-fold to the 9 th concentration, i.e. from 0.6mM to 91.45nM, using a row gun and a double multiplex assay was set up. 78. Mu.L of medium was added to the intermediate plate, and 2. Mu.L of the gradient diluted compound per well was transferred to the intermediate plate at the corresponding position, and 20. Mu.L of the gradient diluted compound per well was transferred to the cell plate after mixing. The concentration of compound transferred into the cell plate ranged from 3. Mu.M to 0.457nM. The cell plates were placed in a carbon dioxide incubator for 3 days. A cell plate was also prepared and the signal value read on the day of dosing as the maximum value (Max value in the following equation) was used in the data analysis. To this plate, 25. Mu.L of cell viability chemiluminescent detection reagent was added per well and incubated at room temperature for 10 minutes to stabilize the luminescent signal. Multiple marker analyzer readings were used.
To the cell plate, 25. Mu.L of a cell viability chemiluminescent detection reagent per well was added, and the luminescent signal was stabilized by incubation at room temperature for 10 minutes. Multiple marker analyzer readings were used.
Data analysis:
The raw data is converted to inhibition rate using equation (Sample-Min)/(Max-Min) ×100%, and the IC50 values can be obtained by curve fitting four parameters (obtained in "log (inhibitor) vs. response-Variable slope" mode in GRAPHPAD PRISM). Table 1 provides the inhibitory activity of the compounds of the invention on HT29 cell proliferation.
TABLE 1 anti-cell proliferation Activity data (IC 50) for the compounds of the invention
From the experimental results in table 1, it can be seen that the compounds of the present invention have good activity in inhibiting HT29 cell proliferation. Compound 1 had an activity of less than 100nM. Showing extremely important antitumor potential.
Experimental example 2: PLK1 enzyme Activity test experiment
This assay was used to examine the efficacy of compounds to inhibit PLK1 enzymatic activity, with lower IC 50 values indicating high efficacy of compounds as PLK1 inhibitors in the following assay settings.
Experimental materials:
PLK1 Active was purchased from CARNA;
Casein Protein is available from SIGNALCHEM;
ADP-Glo KINASE ASSAY was purchased from Promega;
KINASE ASSAY buffer III was purchased from SIGNALCHEM;
Nivo Multilabel Analyzer (Perkinelmer).
The experimental method comprises the following steps:
the enzyme, substrate, ATP and inhibitor were diluted using a kinase buffer in the kit.
The test compound was diluted to 1mM with 100% DMSO as the first concentration, and then 5-fold diluted to the 8 th concentration with a row gun,
I.e., from 1mM to 0.013. Mu.M. Compound working solutions containing 5% dmso were prepared by diluting each concentration point of the compound 20-fold with 1X kinase buffer, and 1 μl of each concentration gradient working solution of the compound was added to the microplates to set up double wells. To the microplate was added 2. Mu.l PLK1 enzyme (15 ng), 2. Mu.l of a mixture of substrate and ATP (20. Mu.M ATP, 0.2. Mu.g/. Mu.l Casein protein) at a final concentration gradient of 10. Mu.M diluted to 0.13nM and the reaction was allowed to react at 25℃for 60 minutes. After the reaction was completed, 5. Mu.l of ADP-Glo reagent was added to each well, the reaction was continued at 25℃for 40 minutes, 10. Mu.l of the kinase detection reagent was added to each well after the completion of the reaction, and after 30 minutes of the reaction at 25℃the chemiluminescence was read by using a PERKINELMER NIVO-multiple-tag analyzer, and the integration time was 0.5 seconds.
Max wells positive control wells read no enzyme blank wells
Min well: negative control wells read as wells containing 1% dmso solvent
Data analysis:
The raw data is converted to inhibition rate using the equation (Sample-Min)/(Max-Min) ×100%, and the value of IC 50 can be obtained by curve fitting four parameters (log (inhibitor) vs. response-Variable slope mode in GRAPHPAD PRISM).
Wherein the inhibition effect of the PLK1 enzyme inhibitor NMS-1286937 prepared by the invention on PLK1 enzyme activity is shown in table 2.
TABLE 2 IC 50 data for PLK1 enzyme inhibition by the compounds of the invention
Numbering of compounds IC50(nM)
NMS-1286937 4.60
Compound 1 17.20
As shown in Table 2, the compound of the invention, specifically compound 1, has a better inhibition effect on PLK1 enzyme, the activity reaches nM level, and compared with clinical compound NMS-1286937, the compound has only three to four times of gap, is an extremely excellent lead compound, has great potential to further optimize the structure, obtains better compound structure and generates good clinical application prospect.
While embodiments of the present invention have been illustrated and described above, it will be appreciated that the above described embodiments are illustrative and should not be construed as limiting the invention. Variations, modifications, substitutions, and alterations are also possible in the above described embodiments without departing from the principles and spirit of the invention, and such variations, modifications, substitutions, and alterations are to be within the scope of this disclosure.

Claims (4)

1. A tri-fused ring compound characterized in that the compound is a compound shown as a formula 4, a formula 6 or a formula 7 or a pharmaceutically acceptable salt thereof or a stereoisomer thereof,
。
2. A pharmaceutical composition comprising an effective amount of a compound as claimed in claim 1, or a pharmaceutically acceptable salt thereof, or one or more stereoisomers thereof.
3. The pharmaceutical composition according to claim 2, wherein the pharmaceutical composition further comprises at least one pharmaceutically acceptable excipient.
4. Use of a compound according to claim 1, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a pharmaceutical composition according to claim 2, for the manufacture of a medicament for the treatment and/or prophylaxis of cancer; the cancer is any one or more of hematological tumor, pancreatic cancer, colorectal cancer and lung cancer.
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