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WO2017193954A1 - 新型2,4,6-三取代均三嗪化合物及其制备方法和应用 - Google Patents

新型2,4,6-三取代均三嗪化合物及其制备方法和应用 Download PDF

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Publication number
WO2017193954A1
WO2017193954A1 PCT/CN2017/083914 CN2017083914W WO2017193954A1 WO 2017193954 A1 WO2017193954 A1 WO 2017193954A1 CN 2017083914 W CN2017083914 W CN 2017083914W WO 2017193954 A1 WO2017193954 A1 WO 2017193954A1
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phenyl
amino
urea
alkyl
group
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English (en)
French (fr)
Inventor
姚其正
吴剑平
陈瑞环
张磊
姚世宁
莫廉
张青青
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LUODA BIOSCIENCES Inc
Nanjing Shiqi Pharmaceutical Co Ltd
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LUODA BIOSCIENCES Inc
Nanjing Shiqi Pharmaceutical Co Ltd
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Priority to DK17795572.1T priority Critical patent/DK3456712T3/da
Priority to KR1020187035570A priority patent/KR102379959B1/ko
Priority to US16/300,162 priority patent/US10611736B2/en
Priority to AU2017262920A priority patent/AU2017262920B2/en
Priority to ES17795572T priority patent/ES2913982T3/es
Priority to RU2018141762A priority patent/RU2737085C2/ru
Priority to CN201780028741.2A priority patent/CN109311825B/zh
Priority to CA3023854A priority patent/CA3023854A1/en
Priority to SG11201809760TA priority patent/SG11201809760TA/en
Priority to JP2019512032A priority patent/JP6987125B2/ja
Application filed by LUODA BIOSCIENCES Inc, Nanjing Shiqi Pharmaceutical Co Ltd filed Critical LUODA BIOSCIENCES Inc
Priority to EP17795572.1A priority patent/EP3456712B1/en
Priority to BR112018072869-1A priority patent/BR112018072869B1/pt
Publication of WO2017193954A1 publication Critical patent/WO2017193954A1/zh
Anticipated expiration legal-status Critical
Priority to US16/802,378 priority patent/US11390591B2/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/26—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with only hetero atoms directly attached to ring carbon atoms
    • C07D251/40—Nitrogen atoms
    • C07D251/48—Two nitrogen atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/53—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
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • 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
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/14—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom
    • C07D251/16—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D251/00—Heterocyclic compounds containing 1,3,5-triazine rings
    • C07D251/02—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings
    • C07D251/12—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • C07D251/14—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom
    • C07D251/16—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom
    • C07D251/18—Heterocyclic compounds containing 1,3,5-triazine rings not condensed with other rings having three double bonds between ring members or between ring members and non-ring members with hydrogen or carbon atoms directly attached to at least one ring carbon atom to only one ring carbon atom with nitrogen atoms directly attached to the two other ring carbon atoms, e.g. guanamines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/04—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings directly linked by a ring-member-to-ring-member bond
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D403/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00
    • C07D403/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings
    • C07D403/12—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, not provided for by group C07D401/00 containing two hetero rings linked by a chain containing hetero atoms as chain links

Definitions

  • the present invention relates to a class of 2,4,6-trisubstituted-1,3,5-triazine (also known as: s-triazine or s-triazine) compounds or pharmaceutically acceptable salts thereof, prodrugs thereof Or a solvate, as well as a process for its preparation and its use.
  • 2,4,6-trisubstituted-1,3,5-triazine also known as: s-triazine or s-triazine
  • pharmaceutically acceptable salts thereof prodrugs thereof Or a solvate
  • Methoosis In addition to the common forms of death such as apoptosis, necrosis and autophagy, a new form of cell death has been discovered in recent years: Methoosis. This sign of death is characterized by the internalization of the vesicles of the macropinosome into the cells, and the formation of huge liquid vesicles shifts the normal cytoplasm, which in turn loses its metabolic capacity and cell membrane. Integrity, no cell body atrophy and nuclear fragmentation associated with apoptosis, and significant differences between necrosis and autophagy (Maltese et al. Methuosis: nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments. Am J Pathol.
  • drugs that cause cell giant bubble death may overcome the tolerance of tumor cells to drugs that cause apoptosis or other mechanisms of action, and become a new means for humans to fight cancer.
  • indole-based chalcones as inducers of methuosis, a novel type of nonapoptotic cell death. J Med Chem. 2012; 55(5): 1940-56; Kitambi et al. Vulnerability of glioblastoma cells to Catastrophic vacuolization and death induced by a small molecule. Cell.
  • Vimentin is a mesenchymal cell, including the major intermediate filament proteins of connective tissue, muscle, vascular endothelium, and blood cells.
  • the highly conserved evolution and dynamic expression at different developmental stages suggest an important role in the physiological function of vimentin.
  • the intermediate filaments together with the microtubules and actin filaments form the cytoskeleton.
  • vimentin also regulates the function of integrin and affects cell adhesion.
  • Attachment movement can combine the dynamic proteins such as microtubule microfilaments to affect the transport of vesicle membrane; can act as a receptor and a protein kinase scaffold to influence signal transduction (Ivaska et al.Novel functions of vimentin in cell adhesion, Migration, and signaling. Exp Cell Res. 2007; 313(10): 2050-62).
  • Vimentin is closely related to lipid metabolism and controls the transport of cholesterol from low-density lipoprotein (LDL) to its esterification site from lysosomes (Sarria et al.A functional role for vimentin intermediate filaments in the Metabolism of lipoprotein-derived cholesterol in human SW-13cells.J Biol Chem. 1992 Sep 25; 267(27): 19455-63). Given the high expression of vimentin in vascular endothelium and smooth muscle cells, the development of novel drugs for the treatment or prevention of cardiovascular diseases such as atherosclerosis with vimentin may be a worthwhile strategy. Vimentin expression is a hallmark of epithelial to mesenchymal transition (EMT).
  • EMT epithelial to mesenchymal transition
  • EMT is a necessary pathway not only in normal physiological processes such as embryonic development and tissue repair, but also in pathological processes such as organ fibrosis and tumor development (Gonzalez et al. Signaling mechanisms of the epithelial-mesenchymal transition. Sci). Signal. 2014; 7 (344): re8). Therefore, with the use of vimentin as a target to intervene in the process of EMT, it is possible to develop new drugs to promote tissue regeneration, resist organ degradation, treat tissue fibrosis, prevent tumor metastasis, etc. (Davis et al. Targeting EMT in cancer: opportunities for Pharmacological intervention. Trends Pharmacol Sci. 2014; 35(9): 479-88).
  • the 2,4,6-trisubstituted s-triazine compound is represented by the following formula (I):
  • R 1 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 12 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino, hydroxymethyl Base or amine methyl group;
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 haloalkyl, or R 4 and R 5 together with the nitrogen atom to which they are attached Forming a 4 to 6 membered saturated or unsaturated heterocyclic ring optionally containing another hetero atom selected from the group consisting of NR 6 , O and S, which may be a hydroxy group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkane a substituent wherein R 6 is hydrogen, hydroxy, C 1 -C 6 alkyl or C 1 -C 6 haloalkyl;
  • R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 12 alkyl, C 1 -C 6 alkoxy, C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino, hydroxymethyl Base, amine methyl or -COR a ;
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted by one or more substituents selected from halogen or NR 9 R 10 and are 3 -(C 2 -C 6 alkynyl)-3H-biaziridine-substituted C 1 -C 6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached, optionally containing an additional a 4- to 6-membered heterocyclic ring of a hetero atom of N, O and S optionally substituted by a C 1 -C 6 alkyl group;
  • R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 together with the nitrogen atom to which they are attached form 4 which optionally contains an additional hetero atom selected from N, O, S To 6-membered heterocyclic ring; and
  • X is NH or O and is attached to the meta or para position of the phenyl group.
  • R 1 is hydrogen, halogen or nitro.
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl, or R 4 and R 5 together with the nitrogen atom to which they are attached form a 4 to 6 membered saturated or unsaturated heterocyclic ring optionally containing another hetero atom selected from the group consisting of NR 6 , O and S, which may be hydroxy, halogen, nitro , amino or C 1 -C 6 alkyl substituted, wherein R 6 is hydrogen, hydroxy or C 1 -C 6 alkyl.
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 4 and R 5 are attached to the nitrogen atom to which they are attached Forming together a 4 to 6 membered saturated heterocyclic ring optionally containing another hetero atom selected from NR 6 , O and S, which may be substituted by a hydroxy group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkyl group Wherein R 6 is hydrogen or C 1 -C 6 alkyl.
  • R 2 is —NR 4 R 5 , and R 4 and R 5 together with the nitrogen atom to which they are attached form 4 to 6 optionally containing additional heteroatoms selected from NR 6 and O.
  • a mono-saturated heterocyclic ring optionally substituted by a substituent selected from the group consisting of a hydroxyl group and a C 1 -C 6 alkyl group, wherein R 6 is hydrogen or a C 1 -C 6 alkyl group.
  • R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 6 alkyl, hydroxymethyl, aminemethyl or -COR a , wherein R a is OH or NR 7 R 8 , R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted by one or more substituents selected from halogen or NR 9 R 10 and are 3-(C 2 - a C 6 alkynyl-3H-biaziridine-substituted C 1 -C 6 alkyl group, or R 7 and R 8 together with the nitrogen atom to which they are attached, optionally containing an additional moiety selected from N, O and S a 4- to 6-membered heterocyclic ring optionally substituted by a C 1 -C 6 alkyl group; R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 and The nitrogen atoms together form a 4 to 6 membere
  • R 3 is halo, C 1 -C 6 alkoxy or -COR a
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from optionally selected from NR 9
  • R 10 is C 1 -C 6 substituted alkyl, and 3- (C 2 -C 6 alkynyl) -3H- bis aziridinyl substituted C 1 -C 6 alkyl, or R 7 and R 8 to which they are
  • the nitrogen atoms attached together form a 4 to 6 membered saturated heterocyclic ring optionally substituted with a C 1 -C 6 alkyl group optionally containing a hetero atom selected from N or O
  • R 9 and R 10 are independently selected from hydrogen And a C 1 -C 6 alkyl group, or R 9 and R 10 together with the nitrogen atom to which they are attached form a 4 to 6 membered saturated heterocyclic ring optionally containing an additional hetero atom selected from N or O.
  • X is NH, attached to the para or meta position of the phenyl group.
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen, C 1 -C 6 alkyl and C 1 -C 6 haloalkyl, or R 4 and R 5 together with the nitrogen atom to which they are attached Forming a 4 to 6 membered saturated or unsaturated heterocyclic ring optionally containing another hetero atom selected from the group consisting of NR 6 , O and S, which may be a hydroxyl group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkane a substituent wherein R 6 is hydrogen, hydroxy or C 1 -C 6 alkyl;
  • R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 6 alkyl, hydroxymethyl, aminemethyl or -COR a , wherein R a is OH or NR 7 R 8 , R 7 and R 8 independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted by one or more substituents selected from halogen or NR 9 R 10 and by 3-(C 2 -C 6 alkynyl)-3H-double An aziridinyl-substituted C 1 -C 6 alkyl group, or R 7 and R 8 together with the nitrogen atom to which they are attached form an optional C 1 optionally containing a hetero atom selected from N, O and S a -C 6 alkyl substituted 4 to 6 membered heterocyclic ring; R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 together with the nitrogen atom to which they are attached form an optional Further, a 4- to 6-membere
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5 , and R 4 and R 5 together with the nitrogen atom to which they are attached form a 4 to 6 membered saturated heterocyclic ring optionally containing another hetero atom selected from NR 6 and O, said heterocyclic ring Optionally substituted with a substituent selected from hydroxy and C 1 -C 6 alkyl, wherein R 6 is hydrogen or C 1 -C 6 alkyl;
  • R 3 is halogen or -COR a
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from C 1 -C 6 alkyl optionally substituted by NR 9 R 10 and are 3-(C 2 a -C 6 alkynyl-3H-biaziridine-substituted C 1 -C 6 alkyl group, or R 7 and R 8 together with the nitrogen atom to which they are attached, optionally containing an additional N or O selected from a 4- to 6-membered saturated heterocyclic ring of a hetero atom optionally substituted by a C 1 -C 6 alkyl group;
  • R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 and The nitrogen atoms together form a 4 to 6 membered saturated heterocyclic ring optionally containing an additional hetero atom selected from N or O;
  • X is NH and is bonded to the para or meta position of the phenyl group.
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen, C 1 -C 6 alkyl, C 1 -C 6 haloalkyl, or R 4 and R 5 together with the nitrogen atom to which they are attached Forming a 4 to 6 membered saturated or unsaturated heterocyclic ring optionally containing another hetero atom selected from NR 6 , O, S, which may be a hydroxyl group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkane a base substitution wherein R 6 is hydrogen, hydroxy, C 1 -C 6 alkyl;
  • R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 6 alkyl, hydroxymethyl, aminemethyl, -CONR 7 R 8 , wherein R 7 and R 8 are independently selected from hydrogen, C 1 - C 6 optionally substituted alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached form a 4 to 6 membered heterocyclic ring optionally containing an additional hetero atom selected from N, O, S; wherein C 1 - C 6 alkyl may be optionally substituted by one or more halogen, C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino;
  • the X group is the meta and para NH or O.
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5 , R 4 , R 5 are independently selected from hydrogen, C 1 -C 6 alkyl, or R 4 and R 5 are taken together with the nitrogen atom to which they are attached, optionally containing an additional a 4- to 6-membered saturated heterocyclic ring of a hetero atom of NR 6 , O, S, which may be substituted by a hydroxyl group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkyl group, and R 6 is hydrogen, C 1 - C 6 alkyl;
  • R 3 is hydrogen, halogen, -CONR 7 R 8 , wherein R 7 , R 8 are independently selected from hydrogen, optionally C 1 -C 6 substituted alkyl, or R 7 and R 8 are taken together with the nitrogen atom to which they are attached a 4- to 6-membered saturated heterocyclic ring optionally containing another hetero atom selected from N, O, S; wherein the C 1 -C 6 alkyl group may be optionally one or more C 1 -C 6 alkylamino groups, C 1 -C 6 alkylamino substituted;
  • the X group is the meta and para NH or O.
  • the above compound (I) comprises:
  • Figure 1 In vitro inhibition of compounds against different types of tumor cells. Eight compounds with different structural features (#38 ⁇ #45, followed by L20, L21, L16, L18, L14, L10, L13 and L11) were selected for 11 different cell-derived tumor cells to determine the same compound in different tumors. Activity in cells, and sensitivity of the same tumor to compounds of different structural characteristics. For each compound, the columns from left to right were for H522, HCT15, HeLa, HepG2, HT1080, KG1, LNCap, MCF7, OVCAR3, SK-mel28, and U87. Data are from three independent experiments and are expressed as mean ⁇ standard deviation.
  • FIG. 1 Foaming of tumor cells. 100 microliters of medium containing 10 3 human colon adenocarcinoma cells (HCT15) or human glioma cells (U87) and solvent DMSO control or 100 nanomolar #40 compound (L16) (RPMI1640, 2 millimoles of glutamine) The amide, 10% fetal bovine serum was inoculated into a 96-well cell culture plate, 5% CO 2 , and cultured at 37 ° C for 96 hours, and the cell morphology was taken under a phase contrast microscope. Significant foaming occurred in both HCT5 cells (upper right) and U87 cells (bottom right) treated with 100 nanomolar #40 compound.
  • HCT15 human colon adenocarcinoma cells
  • U87 human glioma cells
  • solvent DMSO control 100 nanomolar #40 compound (L16) (RPMI1640, 2 millimoles of glutamine)
  • the amide, 10% fetal bovine serum was inoculated into
  • Figure 3 The ability of a test compound to inhibit cell growth has no correlation with the ability to cause cell foaming.
  • #50 Compound (L1) has the strongest ability to cause cell foaming.
  • FIG. 4 Schematic diagram of the probe fishing target protein flow.
  • the probe fishing process includes in situ specific binding of the probe compound to the protein of the living cell, and the photosensitive group of the probe compound is covalently cross-linked with the target protein under UV irradiation, and the probe is formed.
  • the alkynyl group of the compound and the azide group of the isolated compound are specifically linked by a Click-reaction, and the target compound specifically binding to the probe compound can be caught by separating the biotin of the compound and the streptavidin-agarose beads. protein.
  • Probe fishing target protein tool compound was the most potent in causing cell foaming. Based on the analysis of the structure-activity relationship, the probe #79 compound was attached to the photosensitive group at the site shown in the #50 compound diagram, and may not affect the biological activity of the #50 compound. The experiment also demonstrated that the #79 compound caused the same cellular phenotype as the #50 compound. To facilitate solid phase separation and reduce non-specificity, the designed #80 compound contains a long chain that is linked to biotin and an azide group for the click-link reaction.
  • FIG. 6 Isolation and identification of target proteins.
  • the #79 compound was used as a probe to bind to the target protein in situ and photo-covalently linked, and then reacted with the #80 compound to purify the target protein by solid phase separation.
  • C. Matching protein information indicates that the target protein of the compound is vimentin.
  • Figure 7 Results of docking of #50 compound to target protein.
  • A 3KLT of tetramer vimentin derived from PDB database (system number of the protein, the middle globular part is the active region of the protein); B: for docking #50 compound; C: using software Discovery Studio 3.0 and its LibDock docking method, 3KLT and #50 compounds were docked to obtain the top ten #50 compound conformation score results; D: #50 compound scored the first conformation (reddish) and vimentin effect Figure: E: an amino acid that is hydrogen bonded to the #50 compound after deproteinization. Two amino acids: Arg273 form a hydrogen bond with the triazine ring of Tyr276 and #50 compounds.
  • alkyl refers to C 1 -C 12 alkyl, such as C 1 -C 6 alkyl, such as methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl Base, n-pentyl and the like.
  • Heterocycle refers to a 4 to 6 membered heterocyclic ring optionally containing a heteroatom selected from the group consisting of N, O and S.
  • the heterocyclic ring may be a saturated heterocyclic ring or an unsaturated heterocyclic ring.
  • Exemplary heterocycles include, but are not limited to, morpholinyl, pyrrolidinyl, piperazinyl, piperidinyl, azetidinyl, pyrazolyl, and the like.
  • Halogen includes F, Cl, Br and I.
  • Carboxyl means -COOH.
  • the alkynyl group position of the C 2 -C 6 alkynyl group is usually at the 1-position.
  • the "3-(C 2 -C 6 alkynyl)-3H-biaziridine” is "3-(1-butyn-4-yl)-3H-bi-propyl Acridine-3-yl”.
  • NR 7 R 8 and NR 9 R 10 may be a mono C 1 -C 6 alkylamino group or a di C 1 -C 6 alkylamino group, and the C 1 -C 6 alkyl group may be optionally substituted.
  • the C 1 -C 6 alkyl group may be optionally substituted.
  • substituted by one or more halogens a mono C 1 -C 6 alkylamino group or a di C 1 -C 6 alkylamino group, or a 4 to 6 membered saturated heterocyclic ring containing N and optionally an additional N or O.
  • These heterocycles include, but are not limited to, piperidinyl, piperazinyl, pyrrolidinyl, morpholinyl and the like.
  • the heterocyclic ring may also be optionally substituted, for example by C 1 -C 6 alkyl.
  • the number of substituents may be, for example, 1, 2, 3 or 4 unequal.
  • the substituent may be selected from halogen, C 1 -C 6 alkyl, hydroxy, carboxy, amino, mono C 1 -C 6 alkylamino, di C 1 -C 6 alkylamino, nitro , 3-(C 2 -C 6 alkynyl)-3H-biaziridine,heterocyclyl (eg morpholinyl, pyrrolidinyl, piperazinyl, piperidinyl, azetidinyl, pyridyl) Azolyl, etc.) and C 6 -C 14 aryl (such as phenyl) and the like.
  • the relative terms such as “isomer”, “racemate”, “prodrug”, “solvate” as used herein are not significantly different from the general meaning of the terms described in the art. Those of ordinary skill in the art will be aware of the meaning of these terms.
  • the term “isomer” refers to one of two or more compounds having the same molecular composition but different structures and properties.
  • racemate refers to an equimolar mixture of an optically active chiral molecule and its enantiomer.
  • prodrug is also referred to as a prodrug, a prodrug, a prodrug, etc., and refers to a compound that has a pharmacological action after being transformed in vivo.
  • solvate refers to a mixture of a solvent and a compound.
  • this paper targets multiple signal transduction pathways that are clearly associated with tumor cell proliferation and death, and integrates the pharmacophores of targeting molecules acting on these signaling pathways.
  • a novel class of compounds a s-triazine compound formed by the substitution of a diaryl urea, an arylvinyl group and a saturated nitrogen (oxy) heterocycle. The structure of such a compound is as shown in the following formula (I):
  • R 1 , R 2 , R 3 and X are as described above.
  • R 1 is hydrogen, halogen or nitro; more preferably H, F, Cl or nitro.
  • R 1 when R 1 is a non-hydrogen group, it is usually located at the meta or para position of the benzene ring.
  • R 4 and R 5 are independently selected from hydrogen, C 1 -C 6 alkyl, and C 1 -C 6 haloalkyl, or R 4 and R 5 are attached to the nitrogen to which they are attached.
  • the atoms together form a 4 to 6 membered saturated or unsaturated heterocyclic ring optionally containing another hetero atom selected from NR 6 , O and S, which may be hydroxy, halo, nitro, amino or C 1 -C A 6 alkyl substitution wherein R 6 is hydrogen, hydroxy or C 1 -C 6 alkyl.
  • R 4 and R 5 are, independently, selected from hydrogen and C 1 -C 6 alkyl, or R 4 and R 5 are taken together with the nitrogen atom to which they are attached, optionally containing an additional moiety selected from NR 6 a 4 to 6 membered saturated heterocyclic ring of a hetero atom of O and S, which may be substituted by a hydroxy group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkyl group, wherein R 6 is hydrogen or C 1 - C 6 alkyl.
  • R 2 is —NR 4 R 5 , and R 4 and R 5 together with the nitrogen atom to which they are attached form a 4 to 6 membered saturation optionally containing additional heteroatoms selected from NR 6 and O.
  • a heterocyclic ring which is optionally substituted by a substituent selected from a hydroxyl group and a C 1 -C 6 alkyl group, wherein R 6 is hydrogen or a C 1 -C 6 alkyl group.
  • the 4-6 membered saturated heterocyclic ring includes, but is not limited to, morpholinyl, pyrrolidinyl, piperazinyl, piperidinyl, and azetidinyl.
  • R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1 -C 6 alkyl, hydroxymethyl, aminemethyl or -COR a , wherein R a is OH or NR 7 R 8 , R 7 and R 8 are independently selected from hydrogen, C 1 -C 6 alkyl optionally substituted by one or more substituents selected from halogen or NR 9 R 10 and are 3-(C 2 -C 6 Alkynyl-3H-bisaziridine-substituted C 1 -C 6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached, optionally containing additional hetero-selected from N, O and S a 4- to 6-membered heterocyclic ring optionally substituted by C 1 -C 6 alkyl; R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 are attached thereto The nitrogen atoms together form a 4 to 6 membered heterocyclic
  • R 3 is halo, C 1 -C 6 alkoxy or -COR a
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from NR 9 R 10 substituted C 1 -C 6 alkyl and a 3- (C 2 -C 6 alkynyl) -3H- bis aziridinyl substituted C 1 -C 6 alkyl, or R 7 and R 8 to which they are attached
  • the nitrogen atoms together form a 4 to 6 membered saturated heterocyclic ring optionally substituted with a C 1 -C 6 alkyl group optionally containing a hetero atom selected from N or O
  • R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 together with the nitrogen atom to which they are attached form a 4 to 6 membered saturated heterocyclic ring optionally containing an additional hetero atom selected from N or O.
  • a heterocyclic ring formed by R 7 and R 8 together with the nitrogen atom to which they are attached, and a heterocyclic ring formed by R 9 and R 10 together with the nitrogen atom to which they are attached include, but are not limited to, piperidinyl, Piperazinyl, pyrrolidinyl and morpholinyl.
  • R 3 when R 3 is a non-H substituent, it is usually located at the meta or para position of the phenyl group.
  • X is NH, attached to the para or meta position of the phenyl group. In other embodiments, X is O, which is attached to the para position of the phenyl group.
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are independently selected from hydrogen a C 1 -C 6 alkyl group and a C 1 -C 6 haloalkyl group, or R 4 and R 5 together with the nitrogen atom to which they are attached form 4 which optionally contains an additional hetero atom selected from NR 6 , O and S a 6-membered saturated or unsaturated heterocyclic ring which may be substituted by a hydroxy group, a halogen, a nitro group, an amino group or a C 1 -C 6 alkyl group, wherein R 6 is hydrogen, a hydroxy group or a C 1 -C 6 alkyl group; And R 3 is hydrogen, halogen, nitro, amino, hydroxy, C 1
  • R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 to which they are attached Together with the nitrogen atom optionally containing an additional heteroatom selected N, 4 to six-hetero atom O, S, of.
  • R 1 is hydrogen, halogen or nitro
  • R 2 is -NR 4 R 5
  • R 4 and R 5 are taken together with the nitrogen atom to which they are attached, optionally a further 4- to 6-membered saturated heterocyclic ring selected from the group consisting of NR 6 and O hetero atoms, which heterocyclic ring is optionally substituted by a substituent selected from a hydroxyl group and a C 1 -C 6 alkyl group, wherein R 6 is hydrogen or C 1 -C 6 alkyl
  • R 3 is halogen or -COR a
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from C 1 -C 6 alkane optionally substituted by NR 9 R 10
  • the compound of Formula (I) herein has the structure of Formula (I-1) or Formula (I-2) below:
  • R 1 is selected from the group consisting of H, halogen and nitro
  • R 2 is selected from morpholinyl, pyrrolidinyl, piperazinyl and azetidinyl optionally substituted by hydroxy or C 1 -C 6 alkyl;
  • R 3 is halogen or COR a ; wherein R a is OH or NR 7 R 8 , and R 7 and R 8 are independently selected from C 1 -C 6 alkyl optionally substituted by NR 9 R 10 and are 3-(C a 2 -C 6 alkynyl-3H-biaziridine-substituted C 1 -C 6 alkyl group, or R 7 and R 8 together with the nitrogen atom to which they are attached, optionally containing an additional N or O selected from a 4- to 6-membered saturated heterocyclic ring of a hetero atom optionally substituted by a C 1 -C 6 alkyl group; R 9 and R 10 are independently selected from hydrogen and a C 1 -C 6 alkyl group, or R 9 and R 10 and The nitrogen atoms attached together form a 4 to 6 membered saturated heterocyclic ring optionally containing an additional hetero atom selected from N or O.
  • R 3 is halogen
  • the compound of Formula (I) herein has the structure of Formula (I-3):
  • R 1 is H
  • R 2 is a morpholinyl group
  • R a is OH or NR 7 R 8
  • R 7 and R 8 are independently selected from C 1 -C 6 alkyl optionally substituted by NR 9 R 10 and by 3-(C 2 -C 6 alkynyl)-3H-
  • R 9 and R 10 are independently selected from hydrogen and a C 1 -C 6 alkyl group, or R 9 and R 10 together with the nitrogen atom to which they are attached form an optional Further 4 to 6 membered saturated heterocyclic ring selected from hetero atoms of N or O.
  • R 1 is selected from H, halo and nitro;
  • R 2 is morpholinyl;
  • R 3 is halo or COR a ; wherein R a is OH or NR 7 R 8 , R 7 and R 8 are independently selected from C 1 -C 6 alkyl optionally substituted by NR 9 R 10 and C substituted by 3-(C 2 -C 6 alkynyl)-3H-biaziridine 1 -C 6 alkyl, or R 7 and R 8 together with the nitrogen atom to which they are attached form 4, optionally substituted by a C 1 -C 6 alkyl group, optionally containing another heteroatom selected from N or O a 6-membered saturated heterocyclic ring;
  • R 9 and R 10 are independently selected from hydrogen and C 1 -C 6 alkyl, or R 9 and R 10 are taken together with the nitrogen atom to which they are attached, optionally containing an additional N or O selected from A 4- to 6-membered saturated heterocyclic ring of a
  • these compounds, R 1 and R 3 are each independently a phenyl group positioned meta or para position. In certain embodiments, these compounds, R 1 is a non-hydrogen substituent, meta position of the phenyl, R 3 is located at the para position of the phenyl.
  • the saturated heterocyclic ring includes, but is not limited to, piperazinyl, piperidinyl, pyrrolidinyl, and morpholinyl. In certain embodiments, these compounds are particularly useful for causing vesicle accumulation in tumor cells, resulting in massive cell death.
  • 2,4,6-trisubstituted s-triazine compounds herein can be synthesized by the following methods:
  • Synthetic route 1 using m-phenylenediamine or p-phenylenediamine (1) as a starting material, protecting an amino group with (Boc) 2 O (di-tert-butyl dicarbonate) to obtain intermediate 2, respectively, with various types Substituted phenyl isocyanate to give intermediate 3, the Boc (tert-butoxycarbonyl) protecting group is removed by trifluoroacetic acid to give intermediate 4; cyanuric chloride (5) and methylmagnesium bromide (CH 3 MgBr) The intermediate 6 is obtained by the reaction, and the intermediate 7 is reacted with various types of substituted benzaldehyde under reflux of concentrated hydrochloric acid to obtain intermediate 7, which is obtained by chlorophosphonium chloride, which is obtained by using 4 in diisopropylethylamine. Under the catalysis, intermediate 9,9 and heterocyclic amine such as morpholine or methylpiperazine are respectively obtained under the catalysis of diisopropylethy
  • the synthetic route 1 reaction formula is as follows:
  • R 1 , R 2 and R 3 are as described above.
  • reagents in the above synthetic scheme (a) a base, preferably potassium carbonate, (Boc) 2 O, water, tetrahydrofuran, DMF; (b) substituted phenyl isocyanate, dichloromethane; (c) trifluoroacetic acid, Methyl chloride; (d) tetrahydrofuran solution of methyl magnesium bromide; (e) substituted benzaldehyde, concentrated hydrochloric acid; (f) phosphorus oxychloride; (g) diisopropylethylamine, tetrahydrofuran; (h) heterocyclic ring Amine, diisopropylethylamine, tetrahydrofuran.
  • a base preferably potassium carbonate, (Boc) 2 O, water, tetrahydrofuran, DMF
  • substituted phenyl isocyanate dichloromethane
  • trifluoroacetic acid Methyl chloride
  • the structural formula of the target compound of Scheme 1 may include:
  • Synthetic route 2 using m-phenylenediamine or p-phenylenediamine (1) as a starting material, protecting an amino group with (Boc) 2 O to obtain intermediate 2, which reacts with methyl p-aminobenzoate under the action of triphosgene Intermediate 10 is obtained, the Boc protecting group is removed by trifluoroacetic acid to obtain intermediate 11; and cyanuric chloride (5) is reacted with methyl magnesium bromide to obtain intermediate 6, which is separately reacted under concentrated hydrochloric acid.
  • reaction of the type of substituted benzaldehyde to obtain intermediate 7 is obtained by chlorophosphoryl chloride, which is obtained by the reaction of 11 with diisopropylethylamine to obtain intermediate 12, which is further combined with morpholine or methyl piperazine.
  • the heterocyclic amine such as azine is catalyzed by diisopropylethylamine to obtain compound 13, which is hydrolyzed by lithium hydroxide monohydrate, acidified with hydrochloric acid to obtain intermediate 14, and finally in HOBt (1-hydroxybenzenetriazole).
  • Oxazole) EDCl [1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride] is catalyzed by condensation with various amines to obtain the target compound.
  • the synthetic route 2 reaction formula is as follows:
  • R 1 , R 2 and R 3 are as described above.
  • reagents in the above synthetic route (a) a base, preferably potassium carbonate, (Boc) 2 O, water, tetrahydrofuran, DMF; (b) triphosgene, triethylamine, methyl p-aminobenzoate, dichloromethane (c) trifluoroacetic acid, dichloromethane; (d) methylmagnesium bromide in tetrahydrofuran; (e) substituted benzaldehyde, concentrated hydrochloric acid, refluxed for 12 hours; (f) phosphorus oxychloride, refluxed for 16 hours; (g) diisopropylethylamine, tetrahydrofuran; (h) heterocyclic amine, diisopropylethylamine, tetrahydrofuran; (i) lithium hydroxide monohydrate, tetrahydrofuran, water, concentrated hydrochloric acid; (j) HOBt, EDCI, trie
  • Synthetic route 3 cyanuric chloride (5) is reacted with methyl magnesium bromide to obtain intermediate 6, which is reacted with various types of substituted benzaldehyde under reflux of concentrated hydrochloric acid to obtain intermediate 7 through trichlorocoxide.
  • Oxyphosphorus chlorination gives 8, which is obtained by reacting sodium p-nitrophenolate with intermediate 15,15 and a heterocyclic amine such as morpholine or methylpiperazine under the catalysis of diisopropylethylamine to give compound 16.
  • Compound 16 is reduced in an iron powder or ammonium chloride system to give Intermediate 17, which is reacted with various substituted phenyl isocyanates to obtain the target compound.
  • the synthetic route 3 reaction formula is as follows:
  • R 1 is hydrogen; a hydrogen-absorbing group such as a halogen or a nitro group; an alkyl group, an amino group, a hydroxyl group, a methylol group, an amine methyl group, a carbamoyl group, etc.; and R 2 is an amino group; An alkylamino group, an arylamino group, a heteroarylamino group; R 3 is hydrogen; a hydrogen-absorbing group such as a halogen or a nitro group; an alkyl group, an amino group, a hydroxyl group, a methylol group, an amine methyl group or the like.
  • the reagents in the above reaction scheme are: (a) a solution of methyl magnesium bromide in tetrahydrofuran; (b) a substituted benzaldehyde, concentrated hydrochloric acid, refluxing for 12 hours; (c) phosphorus oxychloride, refluxing for 16 hours; (d) Water, tetrahydrofuran, sodium p-nitrophenolate; (e) heterocyclic amine, diisopropylethylamine, tetrahydrofuran; (f) iron powder, ammonium chloride, ethanol, water, reflux for 3 hours; (g) substituted phenyl Isocyanate, dichloromethane.
  • pharmaceutically acceptable salts examples include inorganic and organic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate, citrate, lactate, tartrate, maleate, fumaric acid Salts, mandelates and oxalates; and inorganic and organic base salts formed with bases such as sodium hydroxy, tris(hydroxymethyl)aminomethane (TRIS, tromethamine) and N-methylglucamine.
  • prodrugs include simple esters of carboxylic acid-containing compounds; esters of hydroxyl-containing compounds; imines of amino-containing compounds; carbamates of amino-containing compounds; aldols or ketals of alcohol-containing compounds alcohol.
  • compositions comprising a pharmaceutically acceptable salt, prodrug or solvate of a compound of (I) herein.
  • the pharmaceutical compositions may also contain various pharmaceutically acceptable carriers conventionally used in the preparation of pharmaceutical compositions.
  • pharmaceutically acceptable carrier refers to an inactive ingredient, such as a solid, semi-solid, or liquid filler, diluent, coating material, formulation, or excipient, in combination with the compounds herein.
  • a “pharmaceutical composition” for administration to a subject is constructed.
  • the pharmaceutically acceptable carrier is non-toxic to the subject to be administered at the dosages and concentrations employed, and is compatible with the other ingredients in the formulation.
  • a pharmaceutically acceptable carrier is suitable for the formulation employed.
  • the carrier can be a gel capsule. If the therapeutic agent is administered subcutaneously, it is desirable that the carrier is non-irritating to the skin and does not cause an injection site reaction.
  • the pharmaceutical compositions may be in any suitable form, such as tablets, troches, and capsules.
  • a pharmaceutical composition comprises a therapeutically or prophylactically effective amount of a pharmaceutically acceptable salt, prodrug or solvate of a compound of (I) herein.
  • effective amount refers to an amount of the compound, pharmaceutically acceptable salt, prodrug or solvate thereof that is effective for treatment or prevention in the prevention or treatment of a disease.
  • the "effective amount” may be based on the active ingredient administered, the mode of administration, the disease and its severity, health, age, weight, family history, genetic composition, pathological development stage, pre-medication and concurrent treatment types, etc. And changes in other individual characteristics of the subject to be treated.
  • the compounds disclosed herein have a strong inhibitory effect on a variety of different types of tumor cells.
  • the compounds of formula (I) and compounds of formula (I-1), (I-2) and (I-3) herein are useful for the prevention and treatment of various solid tumors and hematological tumors including, but not limited to, lung cancer (eg, non-small) Cell lung cancer), colon cancer, cervical cancer, liver cancer, fibrosarcoma, erythroleukemia, prostate cancer, breast cancer, pancreatic cancer, ovarian cancer, melanoma and glioma.
  • the present invention also relates to the use of the compounds disclosed herein to interfere with pathophysiological processes involving vimentin function and to treat diseases associated therewith, including, but not limited to, transformation of epithelial cells to mesenchymal cells, differentiation of stem cells, and invasive growth of tumors. , metastasis, drug resistance and recurrence, tissue fibrosis, infectious diseases and cardiovascular diseases (such as atherosclerosis).
  • a compound described herein in the manufacture of a medicament for the prevention or treatment of a mammalian disease associated with vimentin dysregulation, vesicle formation, transport and secretion, and/or cellular vacuolation.
  • mammalian diseases associated with vimentin dysregulation, vesicle formation, transport and secretion, and/or cellular vacuolation include cancer, neurodegenerative diseases, fibrosis of tissues, and atherosclerosis.
  • the mammalian diseases associated with vimentin dysregulation, vesicle formation, transport and secretion, and/or cell vacuolation include colon cancer, pancreatic cancer, ovarian cancer, gastric cancer, breast cancer, thyroid cancer, Liver cancer, kidney cancer, lung cancer, prostate cancer, sarcoma, glioma and many other solid tumors and leukemia, multiple myeloma.
  • the infectious diseases described herein include, but are not limited to, diseases caused by viral infections, such as diseases caused by infections such as hepatitis C virus, SARS virus, HIV, and dengue virus.
  • provided herein is the use of a compound of formula (I), a pharmaceutically acceptable salt, prodrug or solvate thereof, for the manufacture of a medicament for the treatment or prevention of a tumor.
  • compounds of formula (I), pharmaceutically acceptable salts, prodrugs or solvates thereof, for use in treating or preventing a tumor is as described above.
  • provided herein are compounds of formula (I), pharmaceutically acceptable salts, prodrugs or solvates thereof, for use in the manufacture of a medicament for preventing cell vesicle transfer and release to the extracellular Or in the preparation of a drug that promotes cell giant bubble death, or in the preparation of a medicament for treating or preventing a vimentin-mediated disease.
  • compounds of formula (I) herein for preventing the transport and release of cellular vesicles to the extracellular, promoting cellular macrovesicular death, treating or preventing vimentin-mediated diseases.
  • a pharmaceutically acceptable salt, prodrug or solvate thereof a pharmaceutically acceptable salt, prodrug or solvate thereof.
  • the vimentin-mediated diseases include, inter alia, diseases associated with the transition of epithelial cells to mesenchymal cells, differentiation of stem cells, metastasis of tumors, drug resistance and recurrence, fibrosis of tissues, and cardiovascular diseases (such as atherosclerosis). hardening).
  • the tumor includes colon cancer, pancreatic cancer, ovarian cancer, gastric cancer, breast cancer, thyroid cancer, liver cancer, kidney cancer, lung cancer, prostate cancer, sarcoma, glioma and the like, and various different solid tumors and hematological tumors. Such as leukemia and multiple myeloma.
  • a method of treating or preventing a tumor comprising a solid tumor and a blood tumor, the method comprising administering a therapeutically effective amount of a compound of Formula (I) described herein or a medicament thereof combination.
  • This article also provides for preventing cell vesicle transport and release into the extracellular, promoting cell macrovesicular death or treating or preventing vimentin-mediated A method of the disease, the method comprising administering a therapeutically effective amount of a compound of formula (I) as described herein or a pharmaceutical composition thereof. It can be administered by a suitable route to achieve its intended purpose.
  • the dosage of the drug will be determined by the age, health and weight of the patient, the type of concurrent treatment, the frequency of treatment, and the desired therapeutic benefit.
  • the conventional post-treatment method is: after the completion of the reaction, an appropriate amount of water is added to the reaction liquid, the organic phase and the aqueous phase are separated, and the organic phase is combined. If necessary, it is dried with 5% HCl solution and/or saturated NaSO 4 , filtered, and then dried under reduced pressure to give a crude product.
  • intermediate 8 (78 mg, 0.31 mmol) was dissolved in 5 mL of tetrahydrofuran, sodium p-nitrophenolate (50 mg, 0.31 mmol) was dissolved in 5 mL of water, and sodium p-nitrophenolate solution was slowly dropped onto Intermediate 8. The mixture was stirred at room temperature for 3 hours. After the reaction was completed, half of the solvent was spun off, and the residue was poured into 20 mL of water and extracted with 50 mL of ethyl acetate. The organic layer was dried over anhydrous sodium sulfate, evaporated, evaporated, evaporated, evaporated, evaporated, evaporated,
  • Iron powder 100 mg, 1.79 mmol
  • ammonium chloride 40 mg, 0.74 mmol
  • 10 mL of aqueous ethanol ethanol and water volume ratio: 8:2
  • intermediate 16 100 mg, 0.247 mmol
  • the reaction mixture was added to the above reaction solution, and the reaction was further refluxed for 2 hours.
  • the mixture was filtered, and the filtrate was evaporated.
  • the residue was poured into water (30 mL) and extracted with ethyl acetate (30 mL)
  • the organic phase was collected, dried over anhydrous sodium sulfate, and then passed to a column.
  • Example 5 Test of in vitro anti-tumor activity of a compound (MTT experiment)
  • MTT MTT, RPM1640 medium, fetal bovine serum, 96-well plate, CO 2 incubator, American BIO-RAD680 microplate reader, human non-small cell lung cancer cell line (H522) and human glioma cell line (U87) , all obtained from the market.
  • the cells were seeded, and a single cell suspension was prepared by using a culture solution containing 10% fetal bovine serum, and seeded into a 96-well plate at a cell size of 5000 cells per well at a volume of 100 ⁇ L per well, and cultured overnight.
  • the DMSO stock solution of the compound is diluted with the culture solution to 5 concentrations to be tested, and the dilution between the adjacent concentrations is twice.
  • NA indicates that the compound has little inhibitory effect on tumor cell proliferation ( ⁇ 20%) when tested at the highest concentration (31.6 ⁇ M).
  • Example 6 Compound inhibits broad spectrum and selectivity of tumor cells
  • test cell strains were: human non-small cell lung cancer cell line (H522), human colon adenocarcinoma cell line (HCT15), human cervical cancer cell line (Hela), human liver cancer cell line (HepG2), human fibrosarcoma cell line ( HT1080), human erythroleukemia cell line (KG1), human prostate cancer cell line (LNCaP), human breast cancer cell line (MCF7), human ovarian cancer cell line (OVAR3), human melanoma cell line (SK-mel28) and Human glioma cell line (U87).
  • H522 human non-small cell lung cancer cell line
  • HCT15 human cervical cancer cell line
  • HepG2 human liver cancer cell line
  • HT1080 human fibrosarcoma cell line
  • KG1 human erythroleukemia cell line
  • LNCaP human prostate cancer cell line
  • MCF7 human breast cancer cell line
  • OVAR3 human melanoma cell line
  • SK-mel28 Human gli
  • Figure 3 shows the ability of compounds of different structures to cause cell foaming and their ability to inhibit cell proliferation. The specific results are as follows:
  • Compound# Foaming score IC 50 Compound# Foaming score IC 50 38 0 5.12 59 0 2.2 39 0 4.47 60 4 1.32 40 4 1.71 61 2 1.47 41 0 1.21 62 3.5 1.19 42 0 2.48 63 0 1.8 43 3 4.92 64 0 1 44 0 2.04 65 0 4.4 45 2 2.5 66 0 3.66 48 0 2.91 67 0 1.5 49 2 4.43 68 4 3.28 50 5 3.53 69 2.5 0.22 51 0 >10 70 3.5 0.75 52 4.5 4.74 71 3.5 0.82 53 0 1.19 72 1 1.25 54 4 3.63 73 4.5 2.8 55 0 1.21 74 3.5 1.9 56 0 3.94 75 1 2.1 57 0 3.28 76 0 0.9 58 0 1.33 77 3.5 3
  • Example 8 Further, the compounds 79 and 14 described in Example 8 were tested in accordance with the method described in this example, and the foaming scores were 4.5 and 1.5, respectively.
  • the side chain C1 is referred to the article (Li, Z., Hao P., Li L. et al. Design and synthesis of minimalist terminal alkyne-containing diazirine photo-crosslinkers and their incorporation into kinase inhibitors for cell-and tissue-based Prepared by the method in proteome profiling. Angew. Chem. Int. Ed., 2013, 52, 8551-8556).
  • the reagents N1 and biotin are commercially available and purchased.
  • Biotin (94 mg, 0.385 mmol, biotin), HOBt (74.5 mg, 0.551 mmol), DMF (2 mL), EDCl (106 mg, 0.551 mmol), N1 [80 mg, 0.367 mmol, Name: 2-(2-(2-(2-azidoethoxy)ethoxy)ethoxy)ethane-1-amine] and triethylamine (55.7 mg, 0.551 mmol) in a 25 mL reaction flask The reaction was further stirred for 8 hours. After the reaction was completed, the reaction mixture was poured into 10 mL of ice water, dichloromethane (20 mL), and washed successively with water and brine.
  • Cell culture A certain number of human glioma cells U87 were inoculated in a 150 cm 2 culture dish for 48 hours, and the cells reached a 75-90% confluent state. The medium was replaced with a pre-warmed medium containing a 0.5 micromolar probe (#79 compound) or an equivalent volume of DMSO control, and incubation was continued for 3 hours.
  • UV-induced cross-linking Cells were washed twice with pre-cooled phosphate buffered saline (PBS). Place the cell culture dish on ice, irradiate it from the top with UV (power 100 watt UV lamp, wavelength 350 nm, or 315 to 385 nm) for 1 hour, or add enough pre-cooled PBS (10 ml/150 cm) to the cell culture dish. 2 ), the culture dish is placed on the ultraviolet irradiator, and the cells are irradiated for 30 minutes from the bottom of the culture dish (wavelength 315 to 385 nm, and the time is adjusted according to the ultraviolet power).
  • UV power 100 watt UV lamp, wavelength 350 nm, or 315 to 385 nm
  • pre-cooled PBS 10 ml/150 cm
  • Preparation of cell lysate The cells were detached by trypsin treatment, centrifuged, and washed twice with PBS. The cell pellet was resuspended in HEPES lysis buffer (25 mM Hepes, 150 mM sodium chloride, 2 mM magnesium chloride, 0.1% NP-40, protease inhibitor, pH 7.5), transient ultrasonic homogenization (can be omitted), and placed at 4 ° C for rotary mixing. After 30 minutes, high speed centrifugation was carried out for 10 minutes at 4 ° C, and the lysate protein concentration was measured by the Bradford method.
  • HEPES lysis buffer 25 mM Hepes, 150 mM sodium chloride, 2 mM magnesium chloride, 0.1% NP-40, protease inhibitor, pH 7.5
  • transient ultrasonic homogenization can be omitted
  • Pre-clearing Take 1 mL of cell lysate (1 ⁇ 2mg/mL) and add in advance to HEPES buffer (25mM Hepes, 150 mM sodium chloride, 2 mM magnesium chloride, pH 7.5) equilibrated streptavidin-Agarose-streptavidin, spun at 4 °C for 2 hours or overnight, and centrifuged to retain the liquid phase.
  • HEPES buffer 25mM Hepes, 150 mM sodium chloride, 2 mM magnesium chloride, pH 7.5
  • streptavidin-Agarose-streptavidin spun at 4 °C for 2 hours or overnight, and centrifuged to retain the liquid phase.
  • Click Reaction Click 940 ⁇ l of the above cell lysate and add freshly prepared reaction mixture (20 ⁇ L 50 mM CuSO 4 , 10 ⁇ L of 10 mM TBTA, ie Tris[(1-benzyl-1H-1,2,3) -triazol-4-yl)methyl]amine, 20 ⁇ L 50 mM TCEP, tris (2-carboxyethyl) phosphine, and 10 ⁇ L of 1 mM #80 compound), gently mix at room temperature for 2 hours, add 2 volumes of -20 ° C pre-cooled acetone The reaction was stopped, and the protein was precipitated by high-speed centrifugation (13,000 rpm) at 4 ° C for 10 minutes, washed twice with 500 ⁇ l of pre-cooled methanol, and sonicated in 1 mL of 0.1% SDS PBS.
  • reaction mixture 20 ⁇ L 50 mM CuSO 4 , 10 ⁇ L of 10 mM TBTA, ie Tris[
  • Target protein purification The previously re-dissolved protein solution was added to streptavidin-agarose beads (200 ⁇ l of precipitate) equilibrated with PBS, and mixed by spin mixing at 4 ° C for 2 hours or overnight, and the agarose beads were pelleted by centrifugation. Wash once with 0.1% SDS PBS, wash 4 times with PBS, add 1 volume of 2xSDS loading buffer, boil for 15 minutes, centrifuge to remove the solution, and separate the protein by 10% SDS-PAGE. The gel was stained with 20 mL of Coomassie blue solution for 1 hour, and 2 x 500 ml of double distilled water was stained for 2 x 1 hour, and a specific protein band was cut out (Fig. 6, A).
  • Target protein identification The purified protein was identified using MALDI mass spectrometry and sequence analysis (MALDI-MS/MS). The purified protein was identified as vimentin by matching the sequence of the tested peptide sequences (Fig. 6, B, C).
  • the software Discovery Studio 3.0 was used to interface the 3KLT (see A in Figure 7) and the #50 compound (B in Figure 7) from the vimentin tetramer of the PDB database (protein data bank) using the LibDock docking method.
  • the above pink spherical active pocket was constructed with reference to the ligands supplied with 3KLT, and the radius was set to The number of grid points is 1000, the method of generating the constellation is Best, the maximum number of generated small molecule constellations is set to 255, and the remaining parameter values are default values.

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Abstract

本发明提供了一种通式（I）所示的2,4,6-三取代-1,3,5-三嗪化合物或者其药学上可接受的盐、前药或溶剂合物，其制备方法以及这些化合物在制备用于预防或者治疗与蛋白激酶和波形蛋白调节异常、细胞空泡化有关疾病，尤其是用于治疗或者预防癌症生长与转移、组织的纤维化和动脉粥样硬化的药物。

Description

新型2,4,6-三取代均三嗪化合物及其制备方法和应用 技术领域
本发明涉及一类2,4,6-三取代-1,3,5-三嗪(又名：均三嗪或对称三嗪，s-triazine)化合物或者其药学上可接受的盐、前药或溶剂合物，以及其制备方法及其应用。
背景技术
除了凋亡、坏死和自噬等常见的死亡方式外，近年来发现了一种新的细胞死亡方式：巨泡式死亡(Methuosis)。这种死亡方式的标志是由巨胞饮小体(macropinosome)的小泡被內化到细胞中，而形成的巨大液体囊泡使正常的细胞质移位，细胞进而失去了代谢的能力和细胞膜的完整性，没有与凋亡相关的细胞体萎缩和核碎裂，与坏死和自噬也有显著的差别(Maltese et al.Methuosis:nonapoptotic cell death associated with vacuolization of macropinosome and endosome compartments.Am J Pathol.2014；184(6):1630-42)。鉴于巨泡式死亡与细胞凋亡机制的不同，引起细胞巨泡式死亡的药物有可能克服肿瘤细胞对引起细胞凋亡药物或其它作用机制药物的耐受，成为人类对抗癌症的一个新的手段(Robinson et al.Synthesis and evaluation of indole-based chalcones as inducers of methuosis,a novel type of nonapoptotic cell death.J Med Chem.2012；55(5):1940-56；Kitambi et al.Vulnerability of glioblastoma cells to catastrophic vacuolization and death induced by a small molecule.Cell.2014；157(2):313-28；Trabbic et al.Synthesis and biological evaluation of indolyl-pyridinyl-propenones having either methuosis or microtubule disruption activity.J Med Chem.2015；58(5):2489-512)。
波形蛋白是间充质细胞，包括结缔组织、肌肉、血管内皮以及血液细胞的主要的中间丝蛋白。进化上的高度保守以及在不同发育阶段的动态表达都提示了波形蛋白生理功能的重要。中间丝与微管和肌动蛋白微丝一起共同构成了细胞骨架。波形蛋白除了起着增加细胞机械强度进而起到维持细胞的外形、细胞骨架完整性及亚细胞结构空间分布有序性的作用外，还能调节整联蛋白(integrin)的功能而影响细胞的粘附和移动；能联合微管微丝等动力蛋白而影响囊泡膜的转运；能作为受体及蛋白激酶的支架进而影响信号的转导等(Ivaska et al.Novel functions of vimentin in cell adhesion,migration,and signaling.Exp Cell Res.2007；313(10):2050-62)。
波形蛋白与脂质代谢密切相关，控制着从溶酶体传送源于低密度脂蛋白(LDL)的胆固醇到其酯化位点的运输过程(Sarria et al.A functional role for vimentin intermediate filaments in the metabolism of lipoprotein-derived cholesterol in human SW-13cells.J Biol  Chem.1992Sep 25；267(27):19455-63)。鉴于波形蛋白在血管内皮及平滑肌细胞高度表达，以波形蛋白为靶向开发治疗或预防诸如动脉粥样硬化等心血管疾病的新型药物可能是一个值得尝试的策略。波形蛋白的表达是上皮向间充质细胞转型(EMT)的标志。不仅在胚胎发育和组织修复等正常生理过程，而且在诸如器官的纤维化和肿瘤的发生发展等病理过程中，EMT都是必经途径(Gonzalez et al.Signaling mechanisms of the epithelial-mesenchymal transition.Sci Signal.2014；7(344):re8)。因此，以波形蛋白为靶向来干预EMT的过程，有可能开发出新型的药物来促进组织再生，抵御器官退化，治疗组织纤维化，防止肿瘤转移等(Davis et al.Targeting EMT in cancer:opportunities for pharmacological intervention.Trends Pharmacol Sci.2014；35(9):479-88)。
发明内容
本文通过构效关系的研究获得了特异强效的引起细胞巨泡式死亡作用的化合物，在此基础上运用了可靠高效的探针垂钓靶蛋白的方法，分离鉴定并证明了引起巨泡式死亡的化合物的靶蛋白为波形蛋白。
因此，本文提供一类2,4,6-三取代均三嗪化合物及其制备方法，以及这类化合物参与蛋白激酶、波形蛋白调节和引起细胞巨泡式死亡的应用。
根据本文，所述2,4,6-三取代均三嗪化合物如下式(I)所示：
Figure PCTCN2017083914-appb-000001
其中：
R1为氢、卤素、硝基、氨基、羟基、C1-C12烷基、C1-C6烷氧基、C1-C6烷氨基、二C1-C6烷氨基、羟甲基或胺甲基；
R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基、C1-C6烷基或C1-C6卤代烷基；
R3为氢、卤素、硝基、氨基、羟基、C1-C12烷基、C1-C6烷氧基、C1-C6烷氨基、二C1-C6烷氨基、羟甲基、胺甲基或-CORa；
Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；
R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环；和
X为NH或O，与苯基的间位或对位连接。
本文也包括式(I)所示化合物的药学上可接受的盐、前药或溶剂合物。
在一个或多个实施方案中，R1为氢、卤素或硝基。
在一个或多个实施方案中，R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基或C1-C6烷基。
在一个或多个实施方案中，R2为-NR4R5，R4和R5独立地选自氢和C1-C6烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中，R6为氢或C1-C6烷基。
在一个或多个实施方案中，R2为-NR4R5，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基。
在一个或多个实施方案中，R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基或-CORa，其中，Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环。
在一个或多个实施方案中，R3为卤素、C1-C6烷氧基或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。
在一个或多个实施方案中，X为NH，与苯基的对位或间位连接。
在一个或多个实施方案中，所示式(I)中：
R1为氢、卤素或硝基；
R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或 不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基或C1-C6烷基；和
R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基或-CORa，其中，Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环。
在一个或多个实施方案中，所示式(I)中：
R1为氢、卤素或硝基；
R2为-NR4R5，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基；
R3为卤素或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环；和
X为NH，与苯基的对位或间位连接。
在某些实施方案中，优选地：
R1为氢、卤素或硝基；
R2为-NR4R5，R4、R5独立地选自氢、C1-C6烷基、C1-C6卤代烷基、或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O、S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基、C1-C6烷基；
R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基、-CONR7R8，其中R7、R8独立选自氢、C1-C6任选取代烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环；其中C1-C6烷基可被任选一个或多个卤素、C1-C6烷基氨基、二C1-C6烷基氨基取代；
X基团为间位和对位NH或O。
在某些实施方案中，更优选地，其中：
R1为氢、卤素或硝基；
R2为-NR4R5，R4、R5独立地选自氢、C1-C6烷基、或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O、S的杂原子的4至6元饱和杂环，所述杂环可以被 羟基、卤素、硝基、氨基或C1-C6烷基取代，R6为氢、C1-C6烷基；
R3为氢、卤素、-CONR7R8，其中R7、R8独立选自氢、任选C1-C6取代烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元饱和杂环；其中C1-C6烷基可被任选一个或多个C1-C6烷基氨基、二C1-C6烷基氨基取代；
X基团为间位和对位NH或O。
优选的，上述是(I)化合物包括：
(E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L1)；
(E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L2)；
(E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L3)；
(E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L4)；
(E)-1-(4-氯苯基)-3-(4-(4-(3-羟基氮杂环丁烷-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L5)；
(E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L6)；
(E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L7)；
(E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L8)；
(E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L9)；
(E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L10)；
(E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L11)；
(E)-1-(3-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L12)；
(E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L13)；
(E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L14)；
(E)-1-(3-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L15)；
(E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L16)；
(E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L17)；
(E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L18)；
(E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L19)；
(E)-1-(4-氯苯基)-3-(4-(4-二甲氨基-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L20)；
(E)-1-(4-氯苯基)-3-(4-(4-(四氢吡咯-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L21)；
(E)-1-(4-氯苯基)-3-(4-(4-(3-羟基氮杂环丁烷-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L22)；
(E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L23)；
(E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L24)；
(E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L25)；
(E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L26)；
(E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L27)；
(E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L28)；
(E)-N,N-二甲基-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L29)；
(E)-1-(4-((4-甲基哌啶-1-基)甲酰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L30)；
(E)-1-(4-((4-甲基哌嗪-1-基)甲酰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L31)；
(E)-N-(2-(二甲氨基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L32)；
(E)-N-(2-(二乙氨基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基) 脲)苯甲酰胺(L33)；
(E)-N-(3-(二甲氨基)丙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L34)；
(E)-N-(2-(四氢吡咯-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L35)；
(E)-N-(2-(哌啶-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L36)；
(E)-N-(2-(吗啉-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺(L37)；
(E)-1-(4-甲氧基苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲(L38)；
(E)-1-(4-氯苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲(L39)；
(E)-1-(4-氟苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲(L40)；
(E)-N-(2-(3-(1-丁炔-4-基)-3H-双吖丙啶-3-基)乙基)-4-(3-(4-((4-(吗啉-1-基)-6-苯乙烯-1,3,5-三嗪-2-基)氨基)苯基)脲)苯甲酰胺(79)；和
(E)-1-(4-羧基苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(14)。
附图说明
图1.化合物对不同种类肿瘤细胞的体外抑制作用。选取具有不同结构特征的8个化合物(#38～#45，依次为：L20、L21、L16、L18、L14、L10、L13和L11)针对11种不同细胞来源的肿瘤细胞测定同一化合物在不同肿瘤细胞中的活性，和同一肿瘤对不同结构特征化合物的敏感性。对于每一化合物，从左到右的柱依次针对H522、HCT15、Hela、HepG2、HT1080、KG1、LNCap、MCF7、OVCAR3、SK-mel28和U87。数据来自三批独立的实验，表述为均数±标准差。
图2.肿瘤细胞的泡沫化。将含有103人结肠腺癌细胞(HCT15)或人脑胶质瘤细胞(U87)和溶剂DMSO对照或100纳摩尔#40化合物(L16)的100微升培养基(RPMI1640，2毫摩尔谷氨酰胺，10％胎牛血清)接种于96孔细胞培养板，5％CO2，37℃培养96小时后，相差显微镜下拍摄细胞形态。经100纳摩尔#40化合物处理的HCT5细胞(右上)和U87细胞(右下)均出现明显泡沫化。
图3.受试化合物抑制细胞生长的能力与引起细胞泡沫化的能力不具有相关性。测试了37个化合物(L1～L37)对人胶质瘤U87细胞生长的抑制能力和泡沫化的诱导能力。#50化合物(L1)具有最强的引起细胞泡沫化的能力。
图4.探针垂钓靶蛋白流程示意图。探针垂钓过程包括探针化合物与活体细胞的蛋白质原位特异性结合，探针化合物的光敏基团在UV照射下与靶蛋白原位共价交联，探针化 合物的炔基与分离化合物的叠氮基团经Click-反应特异性地链接，借助分离化合物的生物素和链霉抗生物素蛋白-琼脂糖珠得以钓出与探针化合物特异结合的靶蛋白。
图5.探针垂钓靶蛋白工具化合物。所测试的化合物中，#50化合物引起细胞泡沫化的能力最强。根据对构效关系的分析，在#50化合物图中所示位点连接光敏基团得探针#79化合物，可能不影响#50化合物的生物活性。实验也证明#79化合物能引起与#50化合物相同的细胞表型。为了利于固相分离，减少非特异性，设计的#80化合物含一个长链，连接着生物素和用于点击链接反应的叠氮基团。
图6.靶蛋白的分离与鉴定。以#79化合物为探针与靶蛋白在细胞内原位结合并光促共价连接，再与#80化合物反应得以固相分离纯化靶蛋白。A.左：蛋白免疫印迹检测出能与#79化合物特异性结合的蛋白(箭头所示)；右：考马斯蓝染色显示经PAGE凝胶电泳分离的特异性靶蛋白。B.靶蛋白经MALDI质谱肽段测图和序列分析(MALDI-MS/MS)，与数据库已知蛋白序列匹配。黑体下划线部分为匹配序列。分离出的约60KD和约200KD的两个蛋白与同一个已知蛋白序列匹配。C.匹配蛋白信息表明，化合物的靶蛋白为波形蛋白(vimentin)。
图7.#50化合物与靶蛋白对接结果。A：来源于PDB数据库(protein data bank)的四聚体vimentin的3KLT(该蛋白的系统编号，中间球状部分为该蛋白的活性区域)；B：用于对接#50化合物；C：利用软件Discovery Studio 3.0和其中的LibDock对接方法，将3KLT与#50化合物进行对接，得到的前十的#50化合物构象打分结果；D：#50化合物打分第一的构象(紫红色表示)和波形蛋白作用的图；E：去蛋白后与#50化合物成氢键的氨基酸，两个氨基酸：Arg273与Tyr276与#50化合物中的三嗪环形成氢键。
具体实施方式
应理解，在本文范围内，上述各实施方案所限定的各技术特征(例如各基团的各范围)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，从而构成优选的技术方案。
本文中，“烷基”指C1-C12烷基，如C1-C6烷基，如甲基、乙基、正丙基、异丙基、正丁基、异丁基、叔丁基、正戊基等。
“杂环”指任选含有选自N、O和S的杂原子的4至6元杂环。杂环可以是饱和杂环或不饱和杂环。示例性的杂环包括但不限于如吗啉基、吡咯烷基、哌嗪基、哌啶基、氮杂环丁烷基、吡唑基等。
“卤素”包括F、Cl、Br和I。“羧基”指-COOH。
“3-(C2-C6炔基)-3H-双吖丙啶基”中，C2-C6炔基的炔基位置通常位于1位。在某些实施方案中，所述“3-(C2-C6炔基)-3H-双吖丙啶基”为“3-(1-丁炔-4-基)-3H-双吖丙啶-3-基”。
本文中，NR7R8和NR9R10可以是单C1-C6烷基氨基或二C1-C6烷基氨基，所述C1-C6烷基可任选地被取代，例如被一个或多个卤素、单C1-C6烷基氨基或二C1-C6烷基氨基取代，或被含N和任选的额外的N或O的4到6元饱和杂环取代。这些杂环包括但不限于哌啶基、哌嗪基、吡咯烷基、吗啉基等。所述杂环也可被任选地取代，例如被C1-C6烷基取代。
本文中，当基团被取代时，取代基的数量可以是例如1、2、3或4个不等。通常，除非另有说明，取代基可选自卤素、C1-C6烷基、羟基、羧基、氨基、单C1-C6烷基氨基、二C1-C6烷基氨基、硝基、3-(C2-C6炔基)-3H-双吖丙啶基、杂环基(如吗啉基、吡咯烷基、哌嗪基、哌啶基、氮杂环丁烷基、吡唑基等)和C6-C14芳基(如苯基)等。
此外，本文所用的相关术语诸如“异构体”，“外消旋体”，“前体药物”，“溶剂合物”与所属领域中所述术语的一般含义并无明显不同。本领域的普通技术人员应该知道这些术语的含义。例如，术语“异构体”是指分子组成相同、但结构和性质不同的两种或多种化合物之一。术语“外消旋体”是指一种具有旋光性的手性分子与其对映体的等摩尔混合物。术语“前药”也称前体药物、药物前体、前驱药物等，是指经过生物体内转化后才具有药理作用的化合物。术语“溶剂合物”是指溶剂和化合物组成的混合物。
本文针对肿瘤的可塑性和异质性，以与肿瘤细胞的增殖和死亡有明确关联的多个信号转导通路为靶向，整合了作用于这些信号通路的靶向分子的药效基团，提出一类新型化合物，即由二芳基脲、芳乙烯基和饱和氮(氧)杂环取代形成的均三嗪化合物，这类化合物的结构如下式(I)所示：
Figure PCTCN2017083914-appb-000002
式中，R1、R2、R3和X如前文所述。
在某些实施方案中，R1为氢、卤素或硝基；更优选为H、F、Cl或硝基。
本文的各结构式中，当R1为非氢基团式，通常位于苯环的间位或对位。
在某些实施方案中，R2中，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基或C1-C6烷基。在某些实施方案中，R4和R5独立地选自氢和C1-C6烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中，R6为氢或C1-C6烷基。在某些实施方案中，R2为-NR4R5，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基。在某些实施方案中，所述4－6 元饱和杂环包括但不限于吗啉基、吡咯烷基、哌嗪基、哌啶基和氮杂环丁烷基。
在某些实施方案中，R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基或-CORa，其中，Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环。在某些实施方案中，R3为卤素、C1-C6烷氧基或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。在某些实施方案中，R7和R8与它们所连的氮原子一起形成的杂环以及R9和R10与它们所连的氮原子一起形成的杂环包括但不限于哌啶基、哌嗪基、吡咯烷基和吗啉基。
本文的各结构式中，当R3为非H取代基时，其通常位于苯基的间位或对位上。
在某些实施方案中，X为NH，与苯基的对位或间位连接。在其它实施方案中，X为O，与苯基的对位连接。
如前文所述，本文各实施方案中的各取代基或各技术特征可在式(I)的结构范围内任意组合。因此，举例而言，在某些实施方案中，所示式(I)中：R1为氢、卤素或硝基；R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基或C1-C6烷基；和R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基或-CORa，其中，Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环。在其它实施方案中，所示式(I)中：R1为氢、卤素或硝基；R2为-NR4R5，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基；R3为卤素或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选 自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环；和X为NH，与苯基的对位或间位连接。
在某些实施方案中，本文式(I)化合物具有下式(I-1)或下式(I-2)所示的结构：
Figure PCTCN2017083914-appb-000003
式中，
R1选自H、卤素和硝基；
R2选自任选被羟基或C1-C6烷基取代的吗啉基、吡咯烷基、哌嗪基和氮杂环丁烷基；和
R3为卤素或CORa；其中，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。
在上述式(I-2)的某些实施方案中，R3为卤素。
在某些实施方案中，本文式(I)化合物具有下式(I-3)所示的结构：
Figure PCTCN2017083914-appb-000004
式中，
R1为H；
R2为吗啉基；
Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。
在某些式(I-1)的实施方案中，R1选自H、卤素和硝基；R2为吗啉基；R3为卤素或CORa；其中，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被 3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。在某些实施方案中，这些化合物中，R1(为非氢取代基时)和R3各自独立位于苯基的间位或对位。在某些实施方案中，这些化合物中，R1为非氢取代基时，位于苯基的间位，R3位于苯基的对位。在某些实施方案中，这些化合物中，所述饱和杂环包括但不限于哌嗪基、哌啶基、吡咯烷基和吗啉基。在某些实施方案中，这些化合物尤其可用于在肿瘤细胞中引起囊泡积聚，导致细胞巨泡式死亡。
本文的2,4,6-三取代均三嗪化合物可以采用如下方法进行合成：
合成路线1：以间苯二胺或对苯二胺(1)为起始原料，用(Boc)2O(二碳酸二叔丁酯)保护一个氨基得到中间体2，其分别与各种类型的取代苯基异氰酸酯反应得到中间体3，经三氟乙酸脱去Boc(叔丁氧羰基)保护基团得到中间体4；三聚氯氰(5)与甲基溴化镁(CH3MgBr)反应制得中间体6，在浓盐酸回流条件下，分别与各种类型的取代苯甲醛反应制得中间体7，经三氯氧磷氯代得到8，其与4在二异丙基乙胺催化作用下得到中间体9，9与吗啉、或甲基哌嗪等杂环胺分别在二异丙基乙胺催化作用下得到目标化合物。
合成路线1反应式如下：
Figure PCTCN2017083914-appb-000005
其中R1、R2、R3如前文所述。
上述合成路线反应式中的试剂：(a)碱，优选碳酸钾，(Boc)2O,水，四氢呋喃，DMF；(b)取代苯基异氰酸酯，二氯甲烷；(c)三氟乙酸，二氯甲烷；(d)甲基溴化镁的四氢呋喃溶液；(e)取代苯甲醛，浓盐酸；(f)三氯氧磷；(g)二异丙基乙胺，四氢呋喃；(h)杂环胺，二异丙基乙胺，四氢呋喃。
合成路线1的目标化合物的结构式可包括：
Figure PCTCN2017083914-appb-000006
Figure PCTCN2017083914-appb-000007
Figure PCTCN2017083914-appb-000008
Figure PCTCN2017083914-appb-000009
Figure PCTCN2017083914-appb-000010
Figure PCTCN2017083914-appb-000011
合成路线2：以间苯二胺或对苯二胺(1)为起始原料，用(Boc)2O保护一个氨基得到中间体2，其与对氨基苯甲酸甲酯在三光气作用下反应得到中间体10，经三氟乙酸脱去Boc保护基团得到中间体11；三聚氯氰(5)与甲基溴化镁反应制得中间体6，在浓盐酸回流条件下，分别与各种类型的取代苯甲醛反应制得中间体7，经三氯氧磷氯代得到8，其与11在二异丙基乙胺催化作用下得到中间体12，进而与吗啉、或甲基哌嗪等杂环胺分别在二异丙基乙胺催化作用下得到化合物13，其在氢氧化锂一水合物作用下水解，经盐酸酸化得到中间体14，最终在HOBt(1-羟基苯三氮唑)，EDCl[1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐]催化作用下分别与各种胺缩合得到目标化合物。
合成路线2反应式如下：
Figure PCTCN2017083914-appb-000012
其中R1、R2、R3如前文所述。
上述合成路线反应式中的试剂：(a)碱，优选碳酸钾，(Boc)2O,水，四氢呋喃，DMF；(b)三光气，三乙胺，对氨基苯甲酸甲酯，二氯甲烷；(c)三氟乙酸，二氯甲烷；(d)甲基溴化镁的四氢呋喃溶液；(e)取代苯甲醛，浓盐酸，回流12小时；(f)三氯氧磷，回流16小时；(g)二异丙基乙胺，四氢呋喃；(h)杂环胺，二异丙基乙胺，四氢呋喃；(i)氢氧化锂一水合物，四氢呋喃，水，浓盐酸；(j)HOBt，EDCI，三乙胺，取代胺，DMF。
合成路线2的目标化合物的结构式有如下述例所示：
Figure PCTCN2017083914-appb-000013
Figure PCTCN2017083914-appb-000014
Figure PCTCN2017083914-appb-000015
合成路线3：三聚氯氰(5)与甲基溴化镁反应制得中间体6，在浓盐酸回流条件下，分别与各种类型的取代苯甲醛反应制得中间体7，经三氯氧磷氯代得到8，其与对硝基苯酚钠得到中间体15，15与吗啉、或甲基哌嗪等杂环胺分别在二异丙基乙胺催化作用下得到化合物16。化合物16在铁粉、氯化铵体系中还原得到中间体17，其分别与各种取代苯基异氰酸酯反应，得到目标化合物。
合成路线3反应式如下：
Figure PCTCN2017083914-appb-000016
其中R1、R2和R3如前文所述。在某些实施方案中，R1为氢；卤素、硝基等吸电基团；烷基、氨基、羟基、羟甲基、胺甲基、氨甲酰基等给电集团；R2为氨基、烷基氨基、芳基氨基、杂芳基氨基；R3为氢；卤素、硝基等吸电基团；烷基、氨基、羟基、羟甲基、胺甲基等给电集团。
上述合成路线反应式中的试剂：(a)甲基溴化镁的四氢呋喃溶液；(b)取代苯甲醛，浓盐酸，回流12小时；(c)三氯氧磷，回流16小时；(d)水，四氢呋喃，对硝基苯酚钠；(e)杂环胺，二异丙基乙胺，四氢呋喃；(f)铁粉，氯化铵，乙醇，水，回流3小时；(g)取代苯基异氰酸酯，二氯甲烷。
合成路线3的目标化合物的结构式有如下述例所示：
Figure PCTCN2017083914-appb-000017
Figure PCTCN2017083914-appb-000018
所述化合物的具体操作步骤在实施例中将加以详细说明。
本领域技术人员可以对上述步骤进行变动以提高收率，他们可据本领域的基本知识确定合成的路线，如选择反应物，溶剂和温度，可以通过各种常规保护基以避免副反应的发生以提高收率。
本文也包括式(I)化合物的药学上可接受的盐、前药或溶剂合物。药学上可接受的盐的例子包括无机和有机酸盐，例如盐酸盐、氢溴酸盐、磷酸盐、硫酸盐、柠檬酸盐、乳酸盐、酒石酸盐、马来酸盐、富马酸盐、扁桃酸盐和草酸盐；以及与碱例如钠羟基、三(羟基甲基)胺基甲烷(TRIS，胺丁三醇)和N-甲基葡糖胺形成的无机和有机碱盐。前药的例子包括含有羧酸的化合物的简单酯；含有羟基的化合物的酯；含有氨基的化合物的亚胺；含有氨基的化合物的氨基甲酸酯；含有醇的化合物的醛缩醇或酮缩醇。
本文还包括含有本文(I)化合物的药学上可接受的盐、前药或溶剂合物的药物组合物。药物组合物中还可含有常规用于制备药物组合物的各种药学上可接受的载体。本文中，“药学上可接受的载体”指无活性成分，如固体，半固体，或液体填充剂，稀释剂，包衣材料，制剂佐料，或赋形剂，与本文的化合物合用，一起构成用于给予对象的“药物组合物”。药学上可接受的载体在所用的剂量和浓度对于给药对象而言是无毒的，且与制剂中的其他成分相容。药学上可接受的载体对于所采用的制剂而言是适当的。例如，如果治疗剂将口服给药，则载体可以是凝胶胶囊。如果治疗剂将皮下给药，则理想的是，载体对皮肤无刺激，且不引起注射位点反应。药物组合物可以是任何合适的制剂形式，如片剂、锭剂和胶囊。
通常，药物组合物中含有治疗或预防有效量的本文(I)化合物的药学上可接受的盐、前药或溶剂合物。本文中，“有效量”指在疾病的预防或治疗中能有效进行治疗或预防的所述化合物、其药学上可接受的盐、前药或溶剂合物的量。该“有效量”可根据所给予的活性成分、给药方式、疾病及其严重程度、健康、年龄、体重、家族史、基因组成、病理发展阶段、用药前的和同时进行的治疗的类型等，以及待治疗对象的其它个体特征而变化。
本文所公开的化合物对多种不同类型的肿瘤细胞都有较强的抑制作用。因此，本文式(I)化合物以及式(I-1)、(I-2)和(I-3)化合物可用于预防和治疗各种实体瘤和血液肿瘤，包括但不限于肺癌(如非小细胞肺癌)，结肠癌，宫颈癌，肝癌，纤维肉瘤，红白血病，前列腺癌，乳腺癌，胰腺癌，卵巢癌，黑色素瘤和脑胶质瘤等。
本文的一些化合物则可引起肿瘤细胞非凋亡式死亡——巨泡式死亡。这种死亡方式源于该类化合物与细胞中间丝即波形蛋白的特异性结合。因此，本文也涉及使用本文公开的化合物干预涉及波形蛋白功能的病理生理过程以及治疗与之相关的疾病，包括但不限于上皮细胞向间充质细胞的转型，干细胞的分化，肿瘤的浸润性生长、转移、耐药和复发，组织的纤维化，感染性疾病和心血管疾病(如动脉粥样硬化)等。在某些实施方案中，本文提供本文所述化合物在制备预防或治疗与波形蛋白调节异常，囊泡形成、转运和分泌，和/或细胞空泡化有关的哺乳动物疾病药物中的应用。进一步地，所述波形蛋白调节异常、囊泡形成、转运和分泌和/或细胞空泡化有关的哺乳动物疾病包括癌症、神经变性疾病、组织的纤维化和动脉粥样硬化。更进一步地，所述的与波形蛋白调节异常、囊泡形成、转运和分泌和/或细胞空泡化有关的哺乳动物疾病包括结肠癌、胰腺癌、卵巢癌、胃癌、乳腺癌、甲状腺癌、肝癌、肾癌、肺癌、前列腺癌、肉瘤、胶质瘤等多种不同的实体瘤及白血病、多发性骨髓癌等。在某些实施方案中，本文所述的感染性疾病包括但不限于病毒感染导致的疾病，例如丙肝病毒、SARS病毒、HIV和登革热病毒等感染导致的疾病。
因此，在某些实施方案中，本文提供本文式(I)所示的化合物、其药学上可接受的盐、前药或溶剂合物在制备肿瘤治疗或预防药物中的应用。在某些实施方案中，本文提供用于治疗或预防肿瘤的式(I)所示的化合物、其药学上可接受的盐、前药或溶剂合物。所述肿瘤如前文所述。
在某些实施方案中，本文提供本文式(I)所示的化合物、其药学上可接受的盐、前药或溶剂合物在制备阻止细胞囊泡转移和释放至胞外的药物中的应用，或在制备促进细胞巨泡式死亡的药物中的应用，或在制备治疗或预防波形蛋白介导的疾病的药物中的应用。或者，在某些实施方案中，本文提供用于阻止细胞囊泡转运和释放至胞外、促进细胞巨泡式死亡、治疗或预防波形蛋白介导的疾病的本文式(I)所示的化合物、其药学上可接受的盐、前药或溶剂合物。所述波形蛋白介导的疾病尤其包括与上皮细胞向间充质细胞的转型相关的疾病，干细胞的分化，肿瘤的转移、耐药和复发，组织的纤维化和心血管疾病(如动脉粥样硬化)。更进一步地，所述肿瘤包括结肠癌、胰腺癌、卵巢癌、胃癌、乳腺癌、甲状腺癌、肝癌、肾癌、肺癌、前列腺癌、肉瘤、胶质瘤等多种不同的实体瘤及血液肿瘤如白血病和多发性骨髓癌等。
在某些实施方案中，本文还提供一种治疗或预防肿瘤，包括实体瘤和血液肿瘤的方法，所述方法包括给予治疗有效量的本文所述的式(I)所示的化合物或其药物组合物。本文还提供阻止细胞囊泡转运和释放至胞外、促进细胞巨泡式死亡或治疗或预防波形蛋白介导的 疾病的方法，所示方法包括给予治疗有效量的本文所述的式(I)所示的化合物或其药物组合物。可通过合适的途径给药，以达到其预期目的。例如，可以通过肠外、皮下、静脉、肌肉、腹腔内、透皮、口腔、鞘内、颅内、鼻腔或外用途径给药。药的剂量将根据病人的年龄、健康与体重、并行治疗的种类、治疗的频率、以及所需治疗效益来决定。
为了更好的说明本发明的技术内容，下面结合具体实例对本发明作进一步阐述，但不限于此。应理解的是，当提及式(I)化合物时，包括本文所述的式(I-1)、(I-2)和(I-3)所示的化合物。实施例中所用到的方法和试剂，除非另有说明，否则为本领域常规的方法和试剂。
下述化合物合成的实施例中，常规后处理方法是：反应完成后，在反应液中加入适量的水，分离有机相和水相，合并有机相。如有需要，依次使用5％HCl溶液和/或饱和NaSO4干燥，过滤之后减压选干，得到粗产物，再经过柱层析分离纯化之后得到最终产物。
实施例1：L16的合成
1)4-((叔丁氧羰基)氨基)苯胺的制备(2)
对苯二胺(3.24g，30.0mmol)，碳酸钾(1.52g，11.0mmol)溶于10mL DMF和5mL水中。在室温下，缓慢滴加(Boc)2O(2.18g，10.0mmol)的四氢呋喃(30mL)溶液。滴完后，继续室温搅拌反应4小时。反应液倒入100mL冰水，二氯甲烷(200mL)萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到白色固体1.79g，产率86％。
2)1-(4-氯苯基)-3-(4-((叔丁氧羰基)氨基)苯基)脲的制备(3)
室温条件下，向中间体2(625mg,3.0mmol)的二氯甲烷(10mL)溶液中，缓慢滴加对氯苯异氰酸酯(461mg，3.0mmol)的二氯甲烷(5mL)溶液，滴定完全后，室温下搅拌5小时。反应液直接抽滤，滤饼干燥得到白色固体977mg，产率90％。
3)1-(4-氯苯基)-3-(4-氨基苯基)脲的制备(4)
室温条件下，向中间体3(188mg,0.52mmol)的二氯甲烷(10mL)溶液中，缓慢滴加三氟乙酸(47mg，4.16mmol)的二氯甲烷(5mL)溶液，滴定完全后，室温下搅拌3小时。反应液直接旋干，饱和碳酸钠调节pH至弱碱性。乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，得到白色固体129mg，产率95％。1H NMR(300MHz,DMSO):δ8.63(s,1H,NH)；8.16(s,1H,NH)；7.45(d,2H,J＝8.8Hz)；7.29(d,2H,J＝8.8Hz)；7.06(d,2H,J＝8.6Hz)；6.50(d,2H,J＝8.6Hz)；4.80(s,2H,-NH2)。
4)2-甲基-4，6-二氯-1,3,5-三嗪(6)
在氮气保护，零下20度条件下，向三聚氯氰(1.38g，7.5mmol)的8mL四氢呋喃溶液中中，缓慢滴加甲基溴化镁乙醚溶液(3mL，9mmol)，滴完，放置常温搅拌反应2小 时，停止反应。加入20mL饱和的氯化铵淬灭反应。用20mL乙酸乙酯20mL饱和的氯化钠萃取两次，有机相用无水硫酸钠干燥。硅胶柱层析得到白色固体615mg，产率为50％。1H NMR(CDCl3)δ:2.71(s,3H,CH3)。
5)2,4-二羟基-6-(3-硝基苯乙烯基)-1,3,5-三嗪(7)
中间体6(1.60g，9.76mmol)，3-硝基苯甲醛(1.47g，9.76mmol)溶于30mL浓盐酸中，加热至100度，继续搅拌反应12小时。停止反应，降到室温，反应液倒入60mL冰水，抽滤，依次用10mL乙酸乙酯，10mL水石油醚洗涤，滤饼干燥得到白色粗品2.01g，产率79.2％。
6)2,4-二氯-6-(3-硝基苯乙烯基)-1,3,5-三嗪(8)
中间体7(960mg，3.69mmol)，三氯氧磷(10mL)混合液，加热至140度，继续搅拌反应19小时。停止反应，降到室温，将反应夜旋干，剩余物倒入60mL冰水，60mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到白色固体660mg，产率60.2％。
7)1-(4-氯苯基)-3-(4-氯-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(9)
在氮气保护条件下，中间体4(342mg，1.15mmol)、中间体8(300mg，1.15mmol)和二异丙基乙胺(149mg，1.15mmol)溶于50mL四氢呋喃中，室温搅拌反应5小时，反应结束后，旋掉一半溶剂，剩余物倒入60mL水中，120mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入2mL无水乙醇，超声30秒，抽滤，滤饼干燥得到粗品522mg,收率86.9％。
8)1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲(L16)
在氮气保护条件下，中间体9(270mg，0.52mmol)、吗啉(181mg，2.08mmol)和二异丙基乙胺(202mg，1.56mmol)溶于60mL四氢呋喃中，室温搅拌反应4小时，反应结束后，旋掉一半溶剂，剩余物倒入100mL水中，100mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入1mL无水乙醇，超声30秒，抽滤，滤饼干燥得到淡黄色固体206mg,收率69.0％。1H NMR(300MHz,DMSO-d6):δ9.46(s,1H),9.19(s,1H),8.96(s,1H),8.40(s,1H),8.10(d,J＝8.0Hz,2H),7.95(d,J＝15.9Hz,1H),7.62(d,J＝8.0Hz,2H),7.53-7.58(m,1H),7.39(d,J＝8.6Hz,2H),7.31(d,J＝8.6Hz,2H),7.21(d,J＝8.6Hz,2H)6.95(d,J＝15.9Hz,1H),3.73(m,4H),3.59(m,4H)ppm。HRMS calcd for C28H26ClN8O4[M+H]+,573.1760,found 573.1750。
实施例2：L31的合成
1)4-((叔丁氧羰基)氨基)苯胺的制备(2)
对苯二胺(3.24g，30.0mmol)，碳酸钾(1.52g，11.0mmol)溶于10mL DMF和5mL 水中。在室温下，缓慢滴加(Boc)2O(2.18g，10.0mmol)的四氢呋喃(30mL)溶液。滴完后，继续室温搅拌反应4小时。反应液倒入100mL冰水，二氯甲烷(200mL)萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到白色固体1.79g，产率86％。
2)4-(3-(4-((叔丁氧羰基)氨基)苯基)脲)苯甲酸甲酯的制备(10)
冰浴条件下，向三光气(980.1mg,3.3mmol)的二氯甲烷(10mL)溶液中，缓慢滴加对氨基苯甲酸甲酯(500mg，3.3mmol)的二氯甲烷(5mL)溶液，滴定完全后，继续滴加三乙胺(667mg，6.6mmol)的二氯甲烷(5mL)溶液，滴加完成后，将反应液中的溶剂旋干，重新加入二氯甲烷(20mL)，将中间体2(618mg，2.97mmol)加入，室温下搅拌5小时。反应液加入饱和碳酸钠，调节pH至弱碱性，用乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，用体积比乙酸乙酯/石油醚＝1/4洗涤，抽滤，滤饼干燥得到白色固体600mg，产率52.4％。1H NMR(300MHz,DMSO)δ9.20(s,1H),9.01(s,1H),8.64(s,1H),7.88(d,J＝8.7Hz,2H),7.58(d,J＝8.7Hz,2H),7.41–7.30(m,4H),3.82(s,3H),1.47(s,9H)ppm。
3)4-(3-(4-氨基苯基)脲)苯甲酸甲酯的制备(11)
室温条件下，向中间体10(200mg,0.52mmol)的二氯甲烷(10mL)溶液中，缓慢滴加三氟乙酸(474mg，4.16mmol)的二氯甲烷(5mL)溶液，滴定完全后，室温下搅拌3小时。反应液直接旋干，饱和碳酸钠调节pH至弱碱性。乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，得到白色固体141mg，产率95％。1H NMR(300MHz,DMSO):δ8.63(s,1H)；8.16(s,1H)；7.45(d,J＝8.8Hz，2H,)；7.29(d,,J＝8.8Hz，2H)；7.06(d,8.6Hz，2H)；6.50(d,J＝8.6Hz，2H)；4.80(s,2H)，3.82(s,3H)。
4)2-甲基-4,6-二氯-1,3,5-三嗪(6)
在氮气保护，零下20度条件下,向三聚氯氰(1.38g，7.5mmol)的8mL四氢呋喃溶液中中，缓慢滴加甲基溴化镁乙醚溶液(3mL，9mmol)，滴完，放置常温搅拌反应2小时，停止反应。加入20mL饱和的氯化铵淬灭反应。用20mL乙酸乙酯20mL饱和的氯化钠萃取两次，有机相用无水硫酸钠干燥。硅胶柱层析得到白色固体615mg，产率为50％。1H NMR(CDCl3)δ:2.71(s,3H,CH3)。
5)2,4-二羟基-6-苯乙烯基-1,3,5-三嗪(7)
中间体6(1.60g，9.76mmol)，苯甲醛(1.04g，9.76mmol)溶于30mL浓盐酸中，加热至100度，继续搅拌反应12小时。停止反应，降到室温，反应液倒入60mL冰水，抽滤，依次用10mL乙酸乙酯，10mL水石油醚洗涤，滤饼干燥得到白色粗品1.66g，产率79.2％。
6)2,4-二氯-6-苯乙烯基-1,3,5-三嗪(8)
中间体7(794mg，3.69mmol)，三氯氧磷(10mL)混合液，加热至140度，继续搅 拌反应19小时。停止反应，降到室温，将反应夜旋干，剩余物倒入60mL冰水，60mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到白色固体560mg，产率60.2％。
7)1-(4-(甲氧基羰基)苯基)-3-(4-氯-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(12)
在氮气保护条件下，中间体11(441mg，1.75mmol)、中间体8(500mg，1.75mmol)和二异丙基乙胺(226mg，1.75mmol)溶于50mL四氢呋喃中，室温搅拌反应5小时，反应结束后，旋掉一半溶剂，剩余物倒入60mL水中，120mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入2mL无水乙醇，超声30秒，抽滤，滤饼干燥得到粗品762mg,收率86.9％。
8)1-(4-(甲氧基羰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(13)
在氮气保护条件下，中间体12(840mg，1.68mmol)、吗啉(584mg，6.72mmol)和二异丙基乙胺(651mg，5.04mmol)溶于60mL四氢呋喃中，室温搅拌反应4小时，反应结束后，旋掉一半溶剂，剩余物倒入100mL水中，100mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入1mL无水乙醇，超声30秒，抽滤，滤饼干燥得到淡黄色固体639mg,收率69.0％。1H NMR(300MHz,DMSO)δ9.61(s,1H),9.37(s,1H),8.98(s,1H),7.95(d,J＝15.9Hz,1H),7.89(d,J＝8.7Hz,2H),7.75–7.64(m,4H),7.60(d,J＝8.7Hz,2H),7.42(t,J＝7.6Hz,5H),6.89(d,J＝15.9Hz,1H),3.82(s,7H),3.69(s,4H)ppm。
9)4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酸的制备(14)
在室温条件下，中间体13(300mg，0.54mmol)、氢氧化锂一水合物(68.5mg，1.63mmol)溶于四氢呋喃(10mL)、甲醇(5mL)和水(1mL)中，加热至回流，继续搅拌反应12小时，反应结束后，抽滤，滤饼用2M盐酸调节pH至4，抽滤，滤饼干燥，加入1mL无水乙醇，超声30秒，抽滤，滤饼干燥得到淡黄色固体171mg,收率59.0％。1H NMR(300MHz,DMSO)δ12.33(s,1H),9.59(s,1H),9.41(s,1H),9.09(s,1H),7.94(d,J＝15.9Hz,1H),7.88(d,J＝8.3Hz,2H),7.77–7.63(m,4H),7.59(d,J＝8.3Hz,2H),7.51–7.36(m,5H),6.89(d,J＝15.9Hz,1H),3.83(s,4H),3.69(s,4H)ppm。
10)1-(4-((4-甲基哌嗪-1-基)甲酰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L31)
在室温条件下，依次按照下列顺序将中间体14(100mg，0.186mmol)、HOBt(32.7mg，0.242mmol)、DMF(2mL)、EDCI(53.5mg，0.279mmol)、4-甲基哌嗪(24.2mg，0.242mmol)和三乙胺(37.6mg，0.372mmol)加入25mL反应瓶中，继续搅拌反应8小时，反应结束后， 反应液倒入10mL冰水，二氯甲烷(20mL)萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到淡黄色固体87.6mg，产率76％。1H NMR(300MHz,DMSO-d6):δ9.60(s,1H),8.91(s,1H),8.67(s,1H),7.95(d,J＝15.9Hz,1H),7.65-7.72(m,4H),7.52(d,J＝8.5Hz,2H),7.41-7.43(m,5H),7.34(d,J＝8.5Hz,2H),6.89(d,J＝15.9Hz,1H)3.83(m,4H),3.69(m,4H),3.50(m,4H),2.33(m,4H),2.21(s,3H)ppm。13C NMR(75MHz,DMSO-d6)δ:169.98,168.92,164.41,152.41,141.17,138.65,135.20,134.18,134.09,129.45,128.91,128.55,128.18,127.76,127.14,120.57,118.71,117.30,65.96,54.78,53.58,47.60,45.50,43.38.HRMS calcd for C34H38N9O3[M+H]+,620.3092,found 620.3105。
实施例3：L39的合成
1)2-甲基-4,6-二氯-1,3,5-三嗪(6)
在氮气保护，零下20度条件下,向三聚氯氰(1.38g，7.5mmol)的8mL四氢呋喃溶液中中，缓慢滴加甲基溴化镁乙醚溶液(3mL，9mmol)，滴完，放置常温搅拌反应2小时，停止反应。加入20mL饱和的氯化铵淬灭反应。用20mL乙酸乙酯20mL饱和的氯化钠萃取两次，有机相用无水硫酸钠干燥。硅胶柱层析得到白色固体615mg，产率为50％。1H NMR(CDCl3)δ:2.71(s,3H,CH3)。
2)2,4-二羟基-6-(3-硝基苯乙烯基)-1,3,5-三嗪(7)
中间体6(1.60g，9.76mmol)，3-硝基苯甲醛(1.47g，9.76mmol)溶于30mL浓盐酸中，加热至100度，继续搅拌反应12小时。停止反应，降到室温，反应液倒入60mL冰水，抽滤，依次用10mL乙酸乙酯，10mL水石油醚洗涤，滤饼干燥得到白色粗品2.01g，产率79.2％。
3)2,4-二氯-6-(3-硝基苯乙烯基)-1,3,5-三嗪(8)
中间体7(960mg，3.69mmol)，三氯氧磷(10mL)混合液，加热至140度，继续搅拌反应19小时。停止反应，降到室温，将反应夜旋干，剩余物倒入60mL冰水，60mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到白色固体660mg，产率60.2％。
4)2-氯-4-(4-硝基苯氧基)-6-(苯乙烯基)-1,3,5-三嗪(15)
在氮气保护条件下，中间体8(78mg，0.31mmol)溶于5mL四氢呋喃，对硝基苯酚钠(50mg，0.31mmol)溶于5mL水，将对硝基苯酚钠水溶液缓慢滴到中间体8的混合液中，室温搅拌反应3小时，反应结束后，旋掉一半溶剂，剩余物倒入20mL水中，50mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入0.5mL无水乙醇，超声30秒，抽滤，滤饼干燥得到粗品84mg,收率76.9％。
5)2-(吗啉-1-基)-4-(4-硝基苯氧基)-6-(苯乙烯基)-1,3,5-三嗪(16)
在氮气保护条件下，中间体15(84mg，0.24mmol)、吗啉(63mg，0.72mmol)和二异丙基乙胺(93mg，0.72mmol)溶于15mL四氢呋喃中，室温搅拌反应4小时，反应结束后，旋掉一半溶剂，剩余物倒入30mL水中，30mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，加入0.5mL无水乙醇，超声30秒，抽滤，滤饼干燥得到淡黄色固体84mg,收率86.0％。
6)4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪-2-氧)-苯胺(17)
铁粉(100mg，1.79mmol)，氯化铵(40mg，0.74mmol)溶于10mL乙醇水溶液(乙醇和水体积比8:2)中，加热至回流，将中间体16(100mg，0.247mmol)快速加到上述反应液中，继续回流反应2小时，反应结束后，抽滤，将滤液中乙醇旋掉，剩余物倒入30mL水中，30mL乙酸乙酯萃取，依次用水，饱和食盐水洗涤。收集有机相，无水硫酸钠干燥，制砂，过柱，洗脱液为石油醚：乙酸乙酯＝3:1。得到纯品85mg，收率91.5％。1H NMR(300MHz,DMSO)：δ7.95(d,J＝15.9Hz,1H),7.73(d,J＝7.6Hz,2H),7.43–7.41(m,3H),6.92(d,J＝15.9Hz,1H),6.87(d,J＝8.7Hz,2H),6.58(d,J＝8.7Hz,2H),5.04(s,2H),3.87(s,2H),3.66-3.60(m,4H)ppm.
7)(E)-1-(4-氯苯基)-3-(4-((4-(吗啉-1-基)-6-(苯乙烯基)-1,3,5-三嗪基-2-基)氧)苯基)脲(L39)
室温条件下，向中间体17(92.5mg,0.246mmol)的二氯甲烷(5mL)溶液中，缓慢滴加对氯苯异氰酸酯(37.8mg，0.346mmol)的二氯甲烷(3mL)溶液，滴定完全后，室温下搅拌5小时。反应液直接抽滤，滤饼干燥得到白色固体117mg，产率90％。1H NMR(300MHz,DMSO-d6):δ8.86(s,1H),8.80(s,1H),7.96(d,J＝15.9Hz,1H),7.73-7.11(m,2H),7.52-7.45(m,4H),7.42-7.40(m,3H),7.34(d,J＝8.7Hz,2H),7.17(d,J＝8.7Hz,2H),6.93(d,J＝15.9Hz,1H),3.89(s,2H),3.67(s,2H),3.60(s,4H)ppm。
实施例4：化合物L1-L15、L17-L30、L32-L38和L40的制备
采用实施例1相同的方法和相应的材料制备化合物L1-L15和L17-L28；采用实施例2相同的方法和相应的材料制备化合物L29-L30和L32-L37；采用实施例3相同的方法和相应的材料制备化合物L38和L40。所得化合物的相关数据如下所示：
L1(#50)：
1H NMR(300MHz,DMSO-d6)δ:9.57(s,1H),8.88(s,1H),8.59(s,1H),7.93(d,J＝15.9Hz,1H),7.70(d,J＝8.7Hz,2H),7.65(d,J＝8.7Hz,2H),7.49(d,J＝8.7Hz,2H),7.43-7.38(m,5H),7.32(d,J＝8.7Hz,2H),6.88(d,J＝15.9Hz,1H),3.82(m,4H),3.68(m,4H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.99,164.43,163.80,152.46,138.83,138.63,135.22,134.17,134.11,129.44,128.90,128.55,127.76,127.16,125.13,120.58,119.58,118.75,65.96,43.37.HRMS calcd for C28H27ClN7O2[M+H]+,528.1909；found 528.1918.
L2(#52)：
1H NMR(300MHz,DMSO-d6)δ:9.65(s,1H),8.78(s,1H),8.66(s,1H),7.95(d,J＝15.9Hz,1H),7.71(d,J＝6.6Hz,2H),7.62(d,J＝8.5Hz,2H),7.48-7.38(m,7H),7.11(t,J＝8.5Hz,2H),6.90(d,J＝15.9Hz,1H),3.82(m,4H),3.69(m,4H)ppm.HRMS calcd for C28H27FN7O2[M+H]+,512.2205,found 512.2215.
L3(#51)
1H NMR(300MHz,DMSO-d6)δ:9.53(s,1H),8.80(s,1H),8.61(s,1H),7.93(d,J＝15.9Hz,1H),7.71(d,J＝8.7Hz,2H),7.66(d,J＝8.7Hz,2H),7.50(d,J＝8.7Hz,2H),7.44-7.39(m,5H),7.32(d,J＝8.7Hz,2H),6.88(d,J＝15.9Hz,1H),3.83(m,4H),2.39(m,4H),2.23(s,3H)ppm.13C-NMR(75MHz,DMSO-d6)δ:169.99,164.28,163.80,152.47,138.83,138.51,135.24,134.25,134.05,129.40,128.89,128.55,127.75,127.27,125.12,120.52,119.57,118.76,54.34,45.74,42.73.HRMS calcd for C29H30ClN8O[M+H]+,541.2175,found 541.2190.
L4(#53)：
1H NMR(300MHz,DMSO-d6)δ:9.55(s,1H),8.69(s,1H),8.56(s,1H),7.92(d,J＝15.9Hz,1H),7.70(d,J＝6.6Hz,2H),7.64(d,J＝8.5Hz,2H),7.48-7.38(m,7H),7.12(t,J＝8.5Hz,2H),6.88(d,J＝15.9Hz,1H),3.83(m,4H),2.39(m,4H),2.23(s,3H)ppm.HRMS calcd for C29H30FN8O[M+H]+,525.2521,found 525.2534.
L5(#57)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.87(s,1H),8.68(s,1H),8.50(s,1H),8.22(d,J＝8.0Hz,2H),7.98(d,J＝15.9Hz,1H),7.74-7.69(m,3H),7.49(d,J＝8.0Hz,2H),7.39(d,J＝8.7Hz,2H),7.32(d,J＝8.7Hz,2H),7.08(d,J＝15.9Hz,1H),5.78(d,J＝6.6Hz,1H),4.61-4.59(m,1H),4.37-4.32(m,2H),3.90-3.86(m,2H)ppm.13C-NMR(75MHz,DMSO-d6)δ:169.8,165.4,163.5,152.5,138.6,135.2,134.4,133.9,129.4,128.9,128.6,127.8,127.2,125.1,120.3,119.5,118.7,60.9,59.2.HRMS calcd for C27H25ClN7O2[M+H]+,514.1753,found 514.1765.
L6(#54)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.81(s,1H),8.62(s,1H),7.90(d,J＝15.9Hz,1H),7.83(s,1H),7.66(d,J＝9.0Hz,3H),7.50(d,J＝8.8Hz,2H),7.43(dd,J＝13.2,6.6Hz,4H),7.33(d,J＝8.8Hz,2H),6.96(d,J＝15.9Hz,1H),3.82(m,4H),3.69(m,4H)ppm.HRMS calcd for C28H26Cl2N7O2[M+H]+,562.1520,found 562.1505.
L7(#60)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.83(s,1H),8.70(s,1H),7.91(d,J＝15.9Hz,1H)7.81(s,1H),7.63-7.69(m,3H),7.44-7.49(m,4H),7.40(d,J＝8.8Hz,2H),7.12(t,J＝8.8Hz,2H),6.95(d,J＝15.9Hz,1H),3.82(m,4H),3.69(m,4H)ppm.13C NMR(75MHz, DMSO-d6)δ:169.74,164.37,163.83,158.75,155.60,152.72,137.54,137.07,136.23,134.43,133.91,133.72,130.63,129.00,127.36,126.32,120.67,119.72,119.62,118.55,115.33,115.03,65.95,43.35.HRMS calcd for C28H26Cl FN7O2[M+H]+,546.1815,found 546.1821.
L8(#55)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.81(s,1H),8.62(s,1H),7.90(d,J＝15.9Hz,1H),7.83(s,1H),7.66(d,J＝9.0Hz,3H),7.50(d,J＝8.8Hz,2H),7.43(dd,J＝13.2,6.6Hz,4H),7.33(d,J＝8.8Hz,2H),6.96(d,J＝15.9Hz,1H),3.84(m,4H),2.39(m,4H),2.23(s,3H).13C-NMR(75MHz,DMSO-d6)δ:169.75,164.23,163.84,152.47,138.83,137.57,136.96,134.19,134.10,133.71,130.63,128.98,128.55,127.37,127.31,125.13,120.59,119.59,118.78,54.34,45.73,42.74.HRMS calcd for C29H29 Cl2N8O[M+H]+,575.1836,found 575.1840.
L9(#61)：
1H NMR(300MHz,DMSO-d6)δ:9.50(s,1H),8.67(s,1H),8.55(s,1H),7.89(d,J＝15.9Hz,1H)7.82(s,1H),7.63-7.69(m,3H),7.44-7.49(m,4H),7.39(d,J＝8.8Hz,2H),7.12(t,J＝8.8Hz,2H),6.95(d,J＝15.9Hz,1H),3.84(m,4H),2.39(m,4H),2.23(s,3H)ppm.13C-NMR(75MHz,DMSO-d6)δ:169.7,164.2,163.8,158.8,155.6,152.8,137.5,136.9,134.3,133.7,130.6,129.0,127.4,126.3,120.6,119.8,119.7,118.6,115.4,115.1,54.3,45.7,42.7.HRMScalcd for C29H29Cl FN8O[M+H]+,559.2131,found 559.2144.
L10(#43)：
1H NMR(300MHz,DMSO-d6)δ:10.46(s,1H),10.22(s,1H),9.52(s,1H),7.90(d,J＝15.9Hz,1H),7.75(d,J＝8.0Hz,2H),7.53-7.61(m,4H),7.44-7.52(m,4H),7.28(d,J＝8.8Hz,1H),6.89(d,J＝15.9Hz,1H),3.83(m,4H),3.68(m,4H)ppm.HRMS calcd for C28H26Cl2N7O2[M+H]+,562.152,found 562.1517.
L11(#45)：
1H NMR(300MHz,DMSO-d6)δ:9.6(s,1H),8.66(s,1H),8.54(s,1H),7.88(d,J＝15.9Hz,1H),7.75(d,J＝8.4Hz,2H),7.63(d,J＝8.4Hz,1H),7.43-7.49(m,4H),7.38(d,J＝8.8Hz,2H),7.11(t,J＝8.8Hz,1H),6.89(d,J＝15.9Hz,1H),3.82(m,4H),3.68(m,4H)ppm.HRMS calcdfor C28H26Cl FN7O2[M+H]+,546.1815,found 546.1822.
L12(#49)：
1H NMR(300MHz,DMSO-d6)δ:9.60(s,1H),8.86(s,1H),8.62(s,1H),7.91(d,J＝15.9Hz,1H),7.75(d,J＝8.0Hz,2H),7.53-7.58(m,1H),7.39(d,J＝8.6Hz,2H),7.31(d,J＝8.6Hz,2H),7.21(d,J＝8.6Hz,2H),6.95(d,J＝15.9Hz,1H),3.73(m,4H),3.59(m,4H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.8,164.0,152.4,141.8,141.6,137.3,134.2,134.0,133.9,130.3,130.2,129.5,128.9,120.6,118.8,113.7,108.0,107.8,104.8,104.5,65.9,43.3.HRMS calcd for C28H26ClFN7O2[M+H]+,546.1815,found 546.1823.
L13(#44)：
1H NMR(300MHz,DMSO-d6)δ:9.50(s,1H),8.84(s,1H),8.64(s,1H),7.89(d,J＝15.9Hz,1H),7.75(d,J＝8.6Hz,2H),7.64(d,J＝8.6Hz,2H),7.46-7.50(m,4H),7.39(d,J＝8.9Hz,2H),7.32(d,J＝8.9Hz,1H),6.91(d,J＝15.9Hz,1H),3.83(m,4H),2.38(m,4H),2.23(s,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.9,164.5,163.8,152.5,138.9,137.1,134.2,133.8,129.5,128.9,128.6,128.1,125.1,120.5,119.5,118.7,54.3,45.7,42.7.HRMS calcd for C29H29Cl2N8O[M+H]+,575.1836,found 575.1837.
L14(#42)：
1H NMR(300MHz,DMSO-d6)δ:9.51(s,1H),8.88(s,1H),8.66(s,1H),7.89(d,J＝15.9Hz,1H),7.78(d,J＝8.6Hz,2H),7.64(d,J＝8.6Hz,2H),7.51-7.47(m,4H),7.39(d,J＝8.9Hz,2H),7.32(d,J＝8.9Hz,1H),6.91(d,J＝15.9Hz,1H),3.85(m,4H),2.38(m,4H),2.25(s,3H)ppm.HRMS calcd for C29H29Cl FN8O[M+H]+,559.2131,found 559.2134.
L15(#48)：
1H NMR(300MHz,DMSO-d6)δ:9.61(s,1H),9.37(s,1H),8.98(s,1H),7.95(d,J＝15.9Hz,1H),7.89(d,J＝8.8Hz,2H),7.66-7.72(m,4H),7.60(d,J＝8.8Hz,2H),7.39-7.44(m,4H),6.89(d,J＝15.9Hz,1H),3.82(m,4H),3.82(s,3H),3.69(m,4H)ppm.13C-NMR(75MHz,DMSO-d6)δ:164.2,163.8,160.8,152.4,141.7,137.2,134.2,134.0,133.8,130.3,130.2,129.5,118.7,113.7,107.7,104.8,104.4,54.0,45.3,42.3.HRMS calcd for C29H29ClFN8O[M+H]+,559.2131,found 559.2136.
L17(#62)：
1H NMR(300MHz,DMSO-d6)δ:9.58(s,1H),8.84(s,1H),8.72(s,1H),8.52(s,1H),8.22(d,J＝8.0Hz,2H),8.06(d,J＝15.9Hz,1H),7.74(d，J＝8.0Hz，1H),7.63-7.69(m,2H),7.44-7.49(m,2H),7.40(d,J＝8.8Hz,2H),7.13(d,J＝8.8Hz,2H),7.07(d,J＝15.9Hz,1H),3.84(m,4H),3.70(m,4H)ppm.13C NMR(75MHz,DMSO-d6)δ:164.33,163.86,158.69,152.79,148.33,137.14,136.31,134.58,133.76,133.56,130.31,130.13,123.60,122.39,120.71,119.57,119.46,118.39,115.32,115.03,65.96,43.36.HRMS calcd for C28H26FN8O4[M+H]+,557.2056,found 557.2060.
L18(#41)：
1H NMR(300MHz,DMSO-d6)δ:9.53(s,1H),8.84(s,1H),8.66(s,1H),8.50(s,1H),8.20(d,J＝7.6Hz,2H),8.03(d,J＝15.9Hz,1H),7.73-7.64(m,3H),7.50(d,J＝8.5Hz,2H),7.41(d,J＝8.5Hz,2H),7.31(d,J＝8.5Hz,2H),7.05(d,J＝15.9Hz,1H),3.84(m,4H),2.39(m,4H),2.23(s,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.53,164.18,163.86,152.48,148.31,138.85,137.15,136.20,134.16,133.55,130.28,128.53,125.09,123.58,122.37,120.60,119.56,118.74,54.33,45.72,42.72.HRMS calcd for C29H29ClN9O3[M+H]+,586.2076,found 586.2082.
L19(#63)：
1H NMR(300MHz,DMSO-d6)δ:9.52(s,1H),8.67(s,1H),8.55(s,1H),8.51(s,1H),8.22(d,J＝8.0Hz,2H),8.04(d,J＝15.9Hz,1H),7.73(d,J＝8.0Hz,1H),7.64-7.69(m,2H),7.44-7.49(m,2H),7.40(d,J＝8.8Hz,2H),7.13(d,J＝8.8Hz,2H),7.06(d,J＝15.9Hz,1H),3.85(m,4H),2.40(m,4H),2.24(s,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.6,164.2,158.8,152.7,148.3,137.2,136.2,134.3,134.0,133.6,130.3,123.6,122.4,120.6,119.8,119.7,118.7,115.4,115.1,54.4,45.7,42.7.HRMS calcd for C29H29FN9O3[M+H]+,570.2372,found 570.2384.
L20(#38)：
1H NMR(300MHz,DMSO-d6)δ:9.50(s,1H),8.81(s,1H),8.62(s,1H),8.50(s,1H),8.21(dd,J＝ 8.0Hz,J＝1.8Hz,2H),8.03(d,J＝15.9Hz,1H),7.74-7.69(m,3H),7.49(d,J＝8.9Hz,2H),7.40(d,J＝8.9Hz,2H),7.32(d,J＝8.5Hz,2H),7.06(d,J＝15.9Hz,1H),3.23-3.17(m,6H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.19,164.90,163.64,152.48,148.35,138.86,137.19,135.97,134.36,133.98,133.59,130.32,128.55,125.09,123.56,122.29,120.41,119.56,118.74,35.86.HRMS calcd for C28H24ClN8O3[M+H]+,531.1654,found 531.1667.
L21(#39)：
1H NMR(300MHz,DMSO-d6)δ:9.49(s,1H),8.79(s,1H),8.59(s,1H),8.49(s,1H),8.21(dd,J＝8.0Hz,J＝1.8Hz,2H),8.01(d,J＝15.9Hz,1H),7.77-7.69(m,3H),7.49(d,J＝8.9Hz,2H),7.39(d,J＝8.9Hz,2H),7.32(d,J＝8.5Hz,2H),7.06(d,J＝15.9Hz,1H),3.62-3.57(m,4H),1.96-1.94(m,4H)ppm.13C NMR(75MHz,DMSO-d6):162.87,152.47,148.35,138.85,137.19,134.55,133.83,133.61,130.33,128.55,125.09,123.55,122.24,120.21,119.55,118.75,45.99,24.76.HRMS calcd for C28H26ClN8O3[M+H]+,557.1811,found 557.1816.
L22(#58)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.88(s,1H),8.69(s,1H),8.50(s,1H),8.22(d,J＝8.0Hz,2H),7.98(d,J＝15.9Hz,1H),7.68-7.74(m,3H),7.49(d,J＝8.8Hz,2H),7.39(d,J＝8.8Hz,2H),7.32(d,J＝8.8Hz,2H),7.08(d,J＝15.9Hz,1H),5.78(d,J＝6.6Hz,1H),4.58-4.61(m,1H),4.32-4.37(m,2H),3.86-3.90(m,2H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.36,165.26,163.55,152.51,148.31,138.88,137.09,136.14,134.29,134.05,133.65,130.27,128.54,125.06,123.57,122.25,120.42,119.49,118.67,60.93,59.23.HRMScalcd for C27H24ClN8O4[M+H]+,559.1604,found 559.1609.
L23(#64)：
1H NMR(300MHz,DMSO-d6)δ:9.63(s,1H),8.81(s,1H),8.70(s,1H),8.32(s,1H)8.01(d,J＝15.9Hz,1H),7.69-7.71(m,2H),7.49(d,J＝8.8Hz,2H),7.39(m,3H),7.33(d,J＝8.8Hz,2H),7.25-7.16(m,2H),6.93(m,1H),6.89(d,J＝15.9Hz,1H),3.87(m,4H),3.69(m,4H)ppm.13C NMR(75MHz,DMSO-d6)δ:170.09,164.44,163.90,152.30,140.28,139.66,138.92, 138.71,135.25,129.42,128.85,128.55,127.77,127.06,125.28,119.68,113.68,112.19,109.91,66.03,43.41.HRMS calcd for C28H27ClN7O2[M+H]+,528.1909,found 528.1916.
L24(#65)：
1H NMR(300MHz,DMSO-d6)δ:9.58(s,1H),8.90(s,1H),8.76(s,1H),8.34(s,1H)8.00(d,J＝15.9Hz,1H),7.69-7.71(m,2H)7.50(d,J＝8.8Hz,2H)7.38(m,3H)7.32(d,J＝8.8Hz,2H)7.18-7.21(m,2H),6.94(m,1H),6.89(d,J＝15.9Hz,1H),3.87(m,4H),2.39(m,4H),2.23(s,3H)ppm.13C-NMR(75MHz,DMSO-d6)δ:170.1,164.3,163.9,152.3,140.3,139.7,138.8,135.3,129.4,128.8,128.6,128.5,127.8,127.1,125.2,119.6,113.6,112.1,109.9,54.5,45.8,42.8.HRMS calcd for C29H30ClN8O[M+H]+,541.2226,found 541.2236.
L25(#59)：
1H NMR(300MHz,DMSO-d6)δ:9.64(s,1H),8.82(s,1H),8.71(s,1H),8.31(s,1H)7.98(d,J＝15.9Hz,1H),7.80(s,1H),7.68(d,J＝7.0Hz,1H),7.43-7.50(m,4H),7.32(d,J＝8.8Hz,2H),7.16-7.26(m,2H),6.96(d,J＝15.9Hz,1H),6.94(d,J＝7.0Hz,1H),3.88(m,4H),3.69(m,4H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.84,164.38,163.91,152.30,140.20,139.67,138.72,137.55,137.36,133.71,130.57,129.01,128.76,128.61,128.53,127.32,126.37,125.25,119.62,113.72,112.23,109.94,66.02,43.40.HRMS calcd for C28H26Cl2N7O2[M+H]+,562.1520,found 562.1530.
L26(#56)：
1H NMR(300MHz,DMSO-d6)δ:9.62(s,1H),8.83(s,1H),8.72(s,1H),8.52(s,1H)8.34(s,1H)8.22(d,J＝8.0Hz,2H),8.12(d,J＝15.9Hz,1H),7.67(t,J＝7.0Hz,1H),7.50(d,J＝8.0Hz,2H),7.17-7.34(m,4H),7.06(d,J＝15.9Hz,1H),6.94(t,J＝7.0Hz,1H),3.91(m,4H),2.41(m,4H),2.25(s,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.8,164.2,163.9,152.3,140.3,139.7,138.7,137.6,137.2,133.7,130.6,129.0,128.9,128.6,127.3,126.4,125.3,119.6,113.7,112.2,109.9,54.5,45.8,42.8.HRMS calcd for C29H29Cl2N8O[M+H]+,575.1836,found 575.1842.
L27(#66)：
1H NMR(300MHz,DMSO-d6)δ:9.62(s,1H),8.83(s,1H),8.72(s,1H),8.47(s,1H)8.30(s,1H)8.18(d,J＝8.0Hz,2H),8.11(d,J＝15.9Hz,1H),7.65(t,J＝7.0Hz,1H)7.45(d,J＝8.0Hz,2H),7.14-7.32(m,4H),7.05(d,J＝15.9Hz,1H),6.93(t,J＝7.0Hz,1H),3.87(m,4H),3.67(m,4H)ppm.13C-NMR(75MHz,DMSO-d6)δ:169.63,164.37,163.94,152.32,148.28,140.18,139.95,139.70,138.72,137.13,136.59,133.59,130.21,129.96,128.60,128.49,125.24,123.56,122.31,119.59,113.72,112.22,109.91,66.03,43.42,30.37.HRMS calcd for C28H26Cl2N8O4[M+H]+,573.1687,found 573.1760.
L28(#67)：
1H NMR(300MHz,DMSO-d6)δ:9.60(s,1H),8.81(s,1H),8.70(s,1H),8.50(s,1H)8.33(s,1H)8.20(d,J＝8.0Hz,2H),8.12(d,J＝15.9Hz,1H),7.67(t,J＝7.0Hz,1H)7.48(d,J＝8.0Hz,2H)7.17-7.34(m,4H),7.06(d,J＝15.9Hz,1H),6.94(t,J＝7.0Hz,1H),3.89(m,4H),2.39(m,4H),2.23(s,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.6,164.2,163.9,152.3,148.3,140.2,139.7,138.7,137.1,133.6,130.2,130.0,128.6,128.5,123.6,122.4,119.6,113.7,112.1,109.9,54.5,45.8,42.8.HRMS calcd for C29H29ClN9O3[M+H]+,586.2076,found 586.2085.
L29(#70)：
1H NMR(300MHz,DMSO-d6)δ:9.60(s,1H),8.86(s,1H),8.64(s,1H),7.94(d,J＝15.9Hz,1H),7.72-7.65(m,4H),7.51(d,J＝8.5Hz,2H),7.46-7.40(m,5H),7.36(d,J＝8.5Hz,2H),6.89(d,J＝15.9Hz,1H),3.83(m,4H),3.69(m,4H),2.97(s,6H)ppm.13C NMR(75MHz,DMSO-d6)δ:170.0,164.4,163.8,152.4,141.0,138.7,135.2,134.1,129.5,129.2,128.9,128.2,127.8,120.6,118.7,117.2,66.0,43.4.HRMS calcd for C31H33N8O3[M+H]+,565.1670,found 565.2676.
L30(#76)：
1H NMR(300MHz,DMSO-d6)δ:9.56(s,1H),8.83(s,1H),8.62(s,1H),7.94(d,J＝15.9Hz,1H),7.71-7.64(m,4H),7.50(d,J＝8.5Hz,2H),7.44-7.39(m,5H),7.30(d,J＝8.5Hz,2H),6.88(d,J＝15.9Hz,1H),3.82(m,4H),3.69(m,4H),3.33(m,2H),2.89(m,1H),2.73(m,1H),1.63-1.61(m,3H),1.12-1.01(m,2H),0.92(d,J＝6.1Hz,3H)ppm.13C NMR(75MHz,DMSO-d6)δ:168.85,164.44,163.81,152.42,140.92,135.22,134.18,134.11,129.25,128.91,128.08,127.87,127.76,120.59,118.72,117.34,65.96,43.37,30.47,21.57.HRMS calcd for C35H39N8O3[M+H]+,619.3140,found 619.3151.
L32(#73)：
1H NMR(300MHz,DMSO-d6)δ:9.64(s,1H),9.11(s,1H),8.84(s,1H),8.37(t,＝5.4Hz,1H)，7.99(d，J＝15.9Hz，1H)，7.85(d，J＝8.5Hz，2H)，7.77-7.70(m，4H),7.58(d,J＝8.5Hz,2H),7.52-7.45(m,5H),6.93(d,J＝15.9Hz,1H),3.88(m,4H),3.74(m,4H),3.49-3.45(m,2H),2.67(t,J＝6.1Hz,2H),2.41(s,6H)ppm.13C NMR(75MHz,DMSO-d6)δ:165.83,164.45,163.81,152.38,142.63,138.65,134.21,134.08,129.44,128.91,128.13,127.75,127.19,120.59,118.70,116.90,65.96,57.73,44.50,43.37,36.55.HRMS calcd for C33H38N9O3[M+H]+,608.3092,found 608.3104.
L33(#74)：
1H NMR(300MHz,DMSO-d6)δ:9.61(s,1H),9.54(s,1H),9.25(s,1H),8.50(s,1H),7.95(d,J＝15.9Hz,1H),7.82(d,J＝8.5Hz,2H),7.72(d,J＝6.7Hz,2H),7.66(d,J＝8.3Hz,2H),7.55(d,J＝8.5Hz,2H),7.43(d,J＝5.8Hz,5H),6.89(d,J＝15.8Hz,1H),3.82(s,4H),3.70(s, 4H),3.00(dd,J＝14.2,7.0Hz,4H),2.85(s,4H),1.17(t,J＝7.2Hz,6H).HRMS calcd for C35H42N9O3[M+H]+,636.3405,found 636.3417.
L34(#77)：
1H NMR(300MHz,DMSO-d6)δ:9.61(s,1H),9.02(s,1H),8.77(s,1H),8.37(t,J＝5.4Hz,1H),7.94(d,J＝15.9Hz,1H),7.77(d,J＝8.5Hz,2H),7.72-7.64(m,4H),7.52(d,J＝8.5Hz,2H),7.43-7.41(m,5H),6.88(d,J＝15.9Hz,1H),3.82(m,4H),3.69(m,4H),3.29-3.24(m,2H),2.27(t,J＝6.9Hz,2H),2.15(s,6H),1.69-1.62(m,2H)ppm.13C NMR(75MHz,DMSO-d6)δ:165.6,164.4,152.4,142.5,135.2,134.2,134.1,129.5,128.9,128.0,127.8,127.5,120.6,118.7,116.9,65.9,56.9,45.1,43.3,37.6,27.1.HRMS calcd for C34H40N9O8[M+H]+,662.3249,found 662.3255.
L35(#75)：
1H NMR(300MHz,DMSO-d6)δ:9.61(s,1H),9.51(s,1H),9.23(s,1H),8.43(s,1H),7.95(d,J＝15.9Hz,1H),7.82(d,J＝8.5Hz,2H),7.69(dd,J＝17.5,7.5Hz,4H),7.54(d,J＝8.5Hz,2H),7.43(d,J＝4.6Hz,5H),6.89(d,J＝15.7Hz,1H),3.83(s,4H),3.70(s,4H),3.44(d,J＝5.4Hz,2H),2.83(d,J＝45.5Hz,6H),1.76(s,4H).13C NMR(75MHz,DMSO-d6)δ:169.95,165.83,164.41,163.77,152.51,142.80,138.66,135.21,134.26,134.05,129.44,128.91,128.19,127.76,127.15,126.95,124.86,120.59,118.41,116.69,65.96,54.46,53.48,45.44,43.35,37.46,29.27,29.00,22.89.HRMS calcd for C35H40N9O3[M+H]+,634.3249,found 634.3264.
L36(#72)：
1H NMR(300MHz,DMSO-d6)δ:9.66(s,1H),9.50(s,1H),9.22s,1H),8.52(s,1H),7.99(d,J＝15.9Hz,1H),7.87(d,J＝8.5Hz,2H),7.77-7.70(m,4H),7.60(d,J＝8.5Hz,2H),7.49-7.43(m,5H),6.94(d,J＝15.9Hz,1H),3.87(m,4H),3.74(m,4H),3.54-3.52(m,2H),2.80(m,6H),1.66(m,4H),1.49(m,2H)ppm.13C NMR(75MHz,DMSO-d6)δ:169.98,165.91,164.40,163.76,152.47,142.80,138.62,135.19,134.19,134.10,129.43,128.91,128.20,127.75,127.12,126.92,120.58,118.49,116.76,65.96,56.77,53.26,43.35,35.56,24.15,22.77.HRMS calcd for C36H42N9O3[M+H]+,648.3405,found 648.3423.
L37(#71)：
1H NMR(300MHz,DMSO-d6)δ:9.60(s,1H),8.93(s,1H),8.68(s,1H),8.27(t,J＝5.2Hz,1H),7.94d,J＝15.9Hz,1H),7.78(d,J＝8.5Hz,2H),7.65-7.72(m,4H),7.52(d,J＝8.5Hz,2H),7.40-7.46(m,5H),7.34(d,J＝8.5Hz,2H),6.89(d,J＝15.9Hz,1H),3.83(m,4H),3.69(m,4H),3.56-3.59(m,4H),3.35(m,2H),2.43-2.50(m,6H)ppm.13C-NMR(75MHz,DMSO-d6)δ:169.99,165.67,164.41,163.78,152.34,142.49,138.67,135.19,134.23,134.01,129.45,128.91,128.08,127.76,127.40,127.12,120.56,118.77,116.98,66.15,65.96,57.44,53.27,43.35,36.41.HRMS calcd for C35H40N9O4[M+H]+,650.3198,found 650.3212.
L38(#84)：
1H NMR(300MHz,DMSO-d6):δ8.67(s,1H),8.50(s,1H),7.96(d,J＝15.9Hz,1H),7.74-7.72(m,2H),7.49(d,J＝8.9Hz,2H),7.42-7.40(m,2H),7.36(d,J＝8.9Hz,2H),7.14(d,J＝8.9Hz,2H),6.94(d,J＝15.9Hz,1H),6.88(d,J＝8.9Hz,2H),3.72(s,3H),3.67(s,2H),3.60(s,4H)ppm；
L39(#85)：
1H NMR(300MHz,DMSO-d6):δ8.86(s,1H),8.80(s,1H),7.96(d,J＝15.9Hz,1H),7.73-7.11(m,2H),7.52-7.45(m,4H),7.42-7.40(m,3H),7.34(d,J＝8.7Hz,2H),7.17(d,J＝8.7Hz,2H),6.93(d,J＝15.9Hz,1H),3.89(s,2H),3.67(s,2H),3.60(s,4H)ppm；
L40(#86)：
1H NMR(300MHz,DMSO-d6):δ8.76(s,1H),8.74(s,1H),8.70(s,1H),7.96(d,J＝15.9Hz,1H),7.72-7.70(m,2H),7.50-7.45(m,5H),7.42-7.40(m,2H),7.15-7.10(m,4H),6.94(d,J＝15.9Hz,1H),3.89(s,2H),3.67(s,2H),3.60(s,4H)ppm.
实施例5：化合物的体外抗肿瘤细胞活性的测试(MTT实验)
1)实验材料
MTT，RPM1640培养基，胎牛血清，96孔板，CO2恒温培养箱，美国BIO-RAD680型酶标仪，人非小细胞肺癌细胞株(H522)和人脑胶质瘤细胞株(U87)，均从市售途径获得。
2)实验步骤
(1)接种细胞，用含10％胎牛血清的培养液配成单个细胞悬液，以每孔5000个细胞接种到96孔板，每孔体积100μL，培养过夜。
(2)化合物溶液的配制，在无菌台中，将化合物的DMSO储备液以培养液稀释成待测5个浓度，相邻浓度之间为两倍稀释。
(3)将不同浓度的化合物溶液加入已经培养过夜的96孔板中，每孔中加入100μL，每个浓度加三个副孔。周围由于具有边缘效应，易染菌，因此不加细胞，不加化合物，而加100μL的培养液用作空白。另设置100％孔，即加入细胞和不含化合物的培养液100μL，在37度恒温培养箱中孵育48小时。
(4)染色，向96孔板加入10μL MTT溶液(5mg/mL用PBS配制)染色，孵育4小时后，2500rps离心10分钟，然后用排枪将培养液从孔中吸出，注意枪尖朝下不要将细胞吸出，加150μL DMSO，在振荡板震荡5-10分钟，使甲昱充分溶解用酶标仪测定570nm测定每孔的OD值。如下计算抑制率：
Figure PCTCN2017083914-appb-000019
化合物实验结果如下表所示：
Figure PCTCN2017083914-appb-000020
Figure PCTCN2017083914-appb-000021
“NA”表示测试最高浓度(31.6μM)时，化合物对肿瘤细胞增殖的抑制作用几乎没有(<20％)。
“>30”表示测试最高浓度(31.6μM)时，化合物对肿瘤细胞有一定的抑制作用，但抑制率<50％。
实施例6：化合物抑制肿瘤细胞的广谱性和选择性
选取8个化合物(#38～#45，依次为：L20、L21、L16、L18、L14、L10、L13和L11)测试其对11种不同的人肿瘤细胞生长的抑制能力。方法同实施例5。受试细胞株为：人非小细胞肺癌细胞株(H522)，人结肠腺癌细胞株(HCT15)，人宫颈癌细胞株(Hela)，人肝癌细胞株(HepG2)，人纤维肉瘤细胞株(HT1080)，人红白血病细胞株(KG1)，人前列腺癌细胞株(LNCaP)，人乳腺癌细胞株(MCF7)，人卵巢癌细胞株(OVAR3)，人黑色素瘤细胞株(SK-mel28)和人脑胶质瘤细胞株(U87)。实验结果如图1所示。
实施例7：肿瘤细胞的泡沫化和巨泡式死亡
多种肿瘤细胞经某些特定结构的化合物处理后，都观察到细胞质出现大量囊泡积聚，细胞呈现泡沫化形态，部分细胞逐渐变圆，以致最终破裂。这些变化符合细胞巨泡式死亡的特征(图2)。
为了比较不同化合物引起细胞泡沫化的能力，将含有103人胶质瘤U87细胞、测试化合物终浓度分别为0.01，0.0316，0.1，0.316，1.0，3.160微摩尔的100微升培养基(RPMI1640，2毫摩尔谷氨酰胺，10％胎牛血清)接种于96孔培养板，5％CO2、37℃培养96小时后，高倍显微镜下观察细胞形态，对化合物引起细胞泡沫化的能力打分。在10纳摩尔即出现明显泡沫化打分为5，31.6纳摩尔打分为4，100纳摩尔打分为3，316纳摩尔打分为2，1000纳摩尔打分为1，在所有浓度均无泡沫化打分为0。
图3显示了不同结构的化合物引起细胞泡沫化的能力以及其抑制细胞增殖的能力。具体结果如下所示：
化合物# 泡沫化打分 IC50 化合物# 泡沫化打分 IC50
38 0 5.12 59 0 2.2
39 0 4.47 60 4 1.32
40 4 1.71 61 2 1.47
41 0 1.21 62 3.5 1.19
42 0 2.48 63 0 1.8
43 3 4.92 64 0 1
44 0 2.04 65 0 4.4
45 2 2.5 66 0 3.66
48 0 2.91 67 0 1.5
49 2 4.43 68 4 3.28
50 5 3.53 69 2.5 0.22
51 0 >10 70 3.5 0.75
52 4.5 4.74 71 3.5 0.82
53 0 1.19 72 1 1.25
54 4 3.63 73 4.5 2.8
55 0 1.21 74 3.5 1.9
56 0 3.94 75 1 2.1
57 0 3.28 76 0 0.9
58 0 1.33 77 3.5 3
注：化合物#51未显示在图3中；化合物#68为化合物L16(#40)的盐酸盐：
Figure PCTCN2017083914-appb-000022
另外，按照本实施例所述的方法测试实施例8所述的化合物79和14，其泡沫化打分分别为4.5分和1.5分。
实施例8：靶蛋白探针垂钓
引起细胞泡沫化的化合物的靶蛋白的分离与鉴定方法概括于图4，相关步骤简述如下。
探针的合成(图5)：
#79化合物的合成路线与合成：
Figure PCTCN2017083914-appb-000023
式中，侧链C1参照文章(Li,Z.,Hao P.,Li L.et al.Design and synthesis of minimalist terminal alkyne-containing diazirine photo-crosslinkers and their incorporation into kinase inhibitors for cell-and tissue-based proteome profiling.Angew.Chem.Int.Ed.,2013,52,8551-8556)中的方法制得。
采用实施例2的方法和相应的材料制备化合物14((E)-1-(4-羧基苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)，其质谱数据如下：
1H NMR(300MHz,DMSO-d6)δ:12.33(s,1H),9.59(s,1H),9.41(s,1H),9.09(s,1H),7.94(d,J＝15.9Hz,1H),7.88(d,J＝8.3Hz,2H),7.77–7.63(m,4H),7.59(d,J＝8.3Hz,2H),7.51–7.36(m,5H),6.89(d,J＝15.9Hz,1H),3.83(s,4H),3.69(s,4H)ppm。
(E)-N-(2-(3-(1-丁炔-4-基)-3H-双吖丙啶-3-基)乙基)-4-(3-(4-((4-(吗啉-1-基)-6-苯乙烯-1,3,5-三嗪-2-基)氨基)苯基)脲)苯甲酰胺(79)
在室温条件下，依次按照下列顺序将中间体14(100mg，0.186mmol)、HOBt(32.7mg，0.242mmol)、DMF(2mL)、EDCI(53.5mg，0.279mmol)、C1(33.0mg，0.242mmol)和三乙胺(37.6mg，0.372mmol)加入25mL反应瓶中，继续搅拌反应8小时，反应结束后，反应液倒入10mL冰水，二氯甲烷(20mL)萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到淡黄色固体92.8mg，产率76％。1H NMR(300MHz,DMSO-d6):δ9.62(s,1H),8.94(s,1H),8.68(s,1H),8.34(t,J＝5.2Hz,1H),7.96(d,J＝15.9Hz,1H),7.80(d,J＝8.6Hz,2H),7.67-7.74(m,4H),7.55(d,J＝8.6Hz,2H),7.42-7.48(m,5H),6.90(d,J＝15.9Hz,1H),3.84(m,4H),3.71(m,4H),3.13-3.20(m,2H),2.87(d,J＝2.5Hz,1H),2.04(td,J＝7.4Hz,J＝2.5Hz,2H),1.62-1.69(m,4H)ppm。13C NMR(75MHz,DMSO-d6)δ:170.05,165.66,164.41,163.87,152.16,142.45,140.30,139.59,138.91,135.22,129.44,128.87,128.63,128.07,127.77,127.40,127.01,117.02,113.69,112.13,109.78,83.13,71.72,66.05,43.41,34.27,31.99,31.30,27.28,12.67.ES-MSm/Z:655.5[M-H]-,C36H36N10O3(MW＝656.7)。
#80化合物的合成路线与合成：
Figure PCTCN2017083914-appb-000024
式中，试剂N1和biotin为商品，购置。
N-(2-(2-(2-(2叠氮乙氧基)乙氧基)乙氧基)乙烷-1-基)生物素酰胺(80)
在室温条件下，依次按照下列顺序将生物素(94mg，0.385mmol,biotin)、HOBt(74.5mg，0.551mmol)、DMF(2mL)、EDCl(106mg，0.551mmol)、N1[80mg，0.367mmol，名为：2-(2-(2-(2叠氮乙氧基)乙氧基)乙氧基)乙烷-1-胺]和三乙胺(55.7mg，0.551mmol)加入25mL反应瓶中，继续搅拌反应8小时，反应结束后，反应液倒入10mL冰水，二氯甲烷(20mL)萃取，依次用水，饱和食盐水洗涤。有机层用无水硫酸钠干燥，蒸干溶剂，硅胶柱层析得到淡黄色固体123mg，产率72％。1H NMR(300MHz,DMSO-d6)δ:4.31(m,1H,CH-1-biotin),4.14(m,1H,CH-4-biotin),3.55-3.52(m,8H,O(CH2CH2O)-PEG),3.40-3.38(m 4H,CH2NH and CH2N3-PEG),3.10(m,1H,CH-3-biotin),2.83(dd,1H,J＝4.8,12.3Hz,CH-2a-biotin),2.60-2.56(m,1H,CH-2b-biotin),2.07(t,2H,J＝7.2Hz,CH2CO-biotin),1.50-1.30(m,6H,(CH2)3-biotin)。
细胞培养：接种一定数量的人胶质瘤细胞U87于150cm2培养皿培养48小时，细胞达到75～90％融合状态。用预温的含有0.5微摩尔浓度探针(#79化合物)或同等体积DMSO对照的培养液更换培养液，继续培养3小时。
紫外光促交联：细胞以预冷的磷酸缓冲液(PBS)洗2次。将细胞培养皿置于冰上，从上部以紫外(功率100瓦紫外灯，波长350nm,或315～385nm)照射1小时，或在细胞培养皿里加入足够少的预冷的PBS(10ml/150cm2)，置培养皿于紫外辐照器上，细胞自培养皿底部接受辐照30分钟(波长315～385nm，根据紫外功率调整时间)。
细胞裂解液的制备：细胞以胰蛋白酶处理脱壁分散，离心沉淀，以PBS洗2次。细胞块重新悬浮于HEPES裂解缓冲液(25mMHepes，150mM氯化钠，2mM氯化镁，0.1％NP-40，蛋白酶抑制剂，pH7.5)，短暂超声匀浆(可省略)，置于4℃旋转混合30分钟，然后4℃高速离心10分钟，以Bradford法测定裂解液蛋白浓度。
预先清除：取1mL细胞裂解液(1～2mg/mL)，加入预先以HEPES缓冲液(25mM Hepes， 150mM氯化钠，2mM氯化镁，pH7.5)平衡过的链霉抗生物素蛋白-琼脂糖珠(Agarose-streptavidin)，4℃旋转混合2小时或过夜，离心保留液相。
点击链接反应(Click Reaction)：取940微升前述细胞裂解液，加入新鲜配制的反应混合物(20μL 50mM CuSO4，10μL现配的10mM TBTA即Tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine，20μL 50mM TCEP即tris(2-carboxyethyl)phosphine，和10μL 1mM#80化合物)，室温温和混合2小时，加入2倍体积的－20℃预冷的丙酮以终止反应，4℃高速离心(13000rpm)10分钟沉淀蛋白，以500微升预冷的甲醇洗2次，超声溶于1mL 0.1％SDS PBS中。
靶蛋白纯化：前述重新溶解的蛋白溶液，加入经PBS平衡过的链霉抗生物素蛋白-琼脂糖珠(200微升沉淀物)，4℃旋转混合2小时或过夜，离心沉淀琼脂糖珠，以0.1％SDS PBS洗1次，PBS洗4次，加入1倍体积的2xSDS加样缓冲液，煮沸15分钟，离心取溶液，10％SDS-PAGE分离蛋白。以20mL考马斯蓝溶液染胶1小时，2×500ml双蒸水脱染2×1小时，切取特异的蛋白带(图6，A)。
靶蛋白鉴定：使用MALDI质谱肽段测图和序列分析法(MALDI-MS/MS)鉴定所纯化蛋白。以所测肽段序列数据库匹配鉴定出所纯化蛋白为波形蛋白(vimentin)(图6,B、C)。
实施例9：#50化合物与靶蛋白对接结果
选用软件Discovery Studio 3.0，采用其中LibDock对接方法，将来源于PDB数据库(protein data bank)的波形蛋白四聚体的3KLT(见图7中A)与#50化合物(图7中B)进行对接。参考3KLT自带的配体构建上述粉红色球形活性口袋，半径设置为
Figure PCTCN2017083914-appb-000025
格点数为1000，产生构象的方法为Best，生成小分子构象数量最大值设置为255，其余参数值为默认值。得到前十的#50化合物构象打分结果(见图7中C)；由此给出#50化合物打分第一的构象和波形蛋白作用的图(图7中D)，以及去蛋白后与#50化合物成氢键的氨基酸(图7中E)，可见仅有两个氨基酸：Arg273与Tyr276与#50化合物中的三嗪环形成氢键。

Claims (10)

  1. 通式I所示的化合物、其药学上可接受的盐、前药或溶剂合物：
    式中，
    R1为氢、卤素、硝基、氨基、羟基、C1-C12烷基、C1-C6烷氧基、C1-C6烷氨基、二C1-C6烷氨基、羟甲基或胺甲基；
    R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基、C1-C6烷基或C1-C6卤代烷基；
    R3为氢、卤素、硝基、氨基、羟基、C1-C12烷基、C1-C6烷氧基、C1-C6烷氨基、二C1-C6烷氨基、羟甲基、胺甲基或-CORa；
    Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基取代的4至6元杂环；
    R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环；和
    X为NH或O，与苯基的间位或对位连接。
  2. 如权利要求1所述的化合物、其药学上可接受的盐、前药或溶剂合物，其中，
    R1为氢、卤素或硝基；和/或
    R2为-NR4R5，R4和R5独立地选自氢、C1-C6烷基和C1-C6卤代烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和或不饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中R6为氢、羟基或C1-C6烷基；和/或
    R3为氢、卤素、硝基、氨基、羟基、C1-C6烷基、羟甲基、胺甲基或-CORa，其中，Ra为OH或NR7R8，R7和R8独立选自氢、任选被一个或多个选自卤素或NR9R10的取代基取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N、O和S的杂原子的任选被C1-C6烷基 取代的4至6元杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N、O、S的杂原子的4至6元杂环；和/或
    X为NH，与苯基的对位或间位连接。
  3. 如权利要求2所述的化合物、其药学上可接受的盐、前药或溶剂合物，其中，
    R2为-NR4R5，R4和R5独立地选自氢和C1-C6烷基，或者R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6、O和S的杂原子的4至6元饱和杂环，所述杂环可以被羟基、卤素、硝基、氨基或C1-C6烷基取代，其中，R6为氢或C1-C6烷基；优选地，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基；和/或
    R3为卤素、C1-C6烷氧基或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。
  4. 如权利要求1所述的化合物、其药学上可接受的盐、前药或溶剂合物，其中，
    R1为氢、卤素或硝基；
    R2为-NR4R5，R4和R5与它们所连的氮原子一起形成任选含有另外的选自NR6和O的杂原子的4至6元饱和杂环，所述杂环任选被选自羟基和C1-C6烷基的取代基取代，其中，R6为氢或C1-C6烷基；
    R3为卤素或-CORa，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环；和
    X为NH，与苯基的对位或间位连接。
  5. 如权利要求1所述的化合物、其药学上可接受的盐、前药或溶剂合物，其中，所述化合物具有下式(I-1)、式(I-2)或式(I-3)所示的结构：
    Figure PCTCN2017083914-appb-100002
    Figure PCTCN2017083914-appb-100003
    式(I-1)或式(I-2)中，
    R1选自H、卤素和硝基；
    R2选自任选被羟基或C1-C6烷基取代的吗啉基、吡咯烷基、哌嗪基和氮杂环丁烷基；和
    R3为卤素或CORa；其中，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环；
    式(I-3)中，
    R1为H；
    R2为吗啉基；
    Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环。
  6. 如权利要求5所示的化合物、其药学上可接受的盐、前药或溶剂合物，其中，式(I-1)中，
    R1选自H、卤素和硝基；
    R2为吗啉基；和
    R3为卤素或CORa；其中，Ra为OH或NR7R8，R7和R8独立选自任选被NR9R10取代的C1-C6烷基和被3-(C2-C6炔基)-3H-双吖丙啶基取代的C1-C6烷基，或者R7和R8与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的任选被C1-C6烷基取代的4至6元饱和杂环；R9和R10独立选自氢和C1-C6烷基，或者R9和R10与它们所连的氮原子一起形成任选含有另外的选自N或O的杂原子的4至6元饱和杂环；
    优选地，R1为非氢取代基时，与R3各自独立位于苯基的间位或对位；更优选地R1为非氢取代基时，位于苯基的间位，R3位于苯基的对位；所述饱和杂环选自哌嗪基、哌啶基、吡咯烷基和吗啉基。
  7. 如权利要求1所述的化合物、其药学上可接受的盐、前药或溶剂合物，其中，所述化合物选自：
    (E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L1)；
    (E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲(L2)；
    (E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(3-羟基氮杂环丁烷-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(3-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(3-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(4-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-二甲氨基-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(四氢吡咯-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-(4-(3-羟基氮杂环丁烷-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-(3-氯苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(吗啉-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-氯苯基)-3-(3-(4-(4-甲基哌嗪-1-基)-6-(3-硝基苯乙烯基)-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-N,N-二甲基-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-1-(4-((4-甲基哌啶-1-基)甲酰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-1-(4-((4-甲基哌嗪-1-基)甲酰基)苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲；
    (E)-N-(2-(二甲氨基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-N-(2-(二乙氨基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-N-(3-(二甲氨基)丙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-N-(2-(四氢吡咯-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-N-(2-(哌啶-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲)苯甲酰胺；
    (E)-N-(2-(吗啉-1-基)乙基)-4-(3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基) 脲)苯甲酰胺；
    (E)-1-(4-甲氧基苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲；
    (E)-1-(4-氯苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲；
    (E)-1-(4-氟苯基)-3-(4-((4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-基)氧)苯基)脲；
    (E)-N-(2-(3-(1-丁炔-4-基)-3H-双吖丙啶-3-基)乙基)-4-(3-(4-((4-(吗啉-1-基)-6-苯乙烯-1,3,5-三嗪-2-基)氨基)苯基)脲)苯甲酰胺；和
    (E)-1-(4-羧基苯基)-3-(4-(4-(吗啉-1-基)-6-苯乙烯基-1,3,5-三嗪基-2-氨基)苯基)脲。
  8. 一种药物组合物，其中，所述药物组合物含有权利要求1－7中任一项所述的化合物、其药学上可接受的盐、前药或溶剂合物和药学上可接受的载体。
  9. 权利要求1－7中任一项所述的化合物、其药学上可接受的盐、前药或溶剂合物在制备治疗和预防实体瘤和血液肿瘤用的药物中的应用；
    优选地，所述实体瘤选自：结肠癌、胰腺癌、卵巢癌、胃癌、乳腺癌、甲状腺癌、肝癌、肾癌、肺癌、前列腺癌、肉瘤和胶质瘤；所述血液肿瘤选自白血病和多发性骨髓癌。
  10. 权利要求1－7中任一项所述的化合物、其药学上可接受的盐、前药或溶剂合物在制备诊断、治疗和预防与细胞内囊泡形成、转运和释放至胞外相关的疾病的药物中的应用，或在制备促进细胞巨泡式死亡的药物中的应用，或在制备治疗或预防波形蛋白介导的疾病的药物中的应用；
    优选地，所述疾病选自与上皮细胞向间充质细胞转型相关的疾病，干细胞的分化，肿瘤的浸润性生长、转移、耐药和复发，组织的纤维化，感染性疾病和心血管疾病；
    优选地，所述疾病选自：实体瘤如结肠癌、胰腺癌、卵巢癌、胃癌、乳腺癌、甲状腺癌、肝癌、肾癌、肺癌、前列腺癌、肉瘤和胶质瘤；血液肿瘤，如白血病和多发性骨髓癌；和动脉粥样硬化。
PCT/CN2017/083914 2016-05-12 2017-05-11 新型2,4,6-三取代均三嗪化合物及其制备方法和应用 Ceased WO2017193954A1 (zh)

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SG11201809760TA SG11201809760TA (en) 2016-05-12 2017-05-11 Novel 2,4,6-trisubstituted s-triazine compound, preparation method therefor, and use thereof
US16/300,162 US10611736B2 (en) 2016-05-12 2017-05-11 2,4,6-trisubstituted s-triazine compound, preparation method therefor, and use thereof
AU2017262920A AU2017262920B2 (en) 2016-05-12 2017-05-11 Novel 2,4,6-trisubstituted s-triazine compound, preparation method therefor, and use thereof
ES17795572T ES2913982T3 (es) 2016-05-12 2017-05-11 Nuevo compuesto S-triazina 2,4,6-trisustituido, método de preparación y uso del mismo
RU2018141762A RU2737085C2 (ru) 2016-05-12 2017-05-11 Новое соединение 2,4,6-тризамещенного s-триазина, способ его получения и его применение
CN201780028741.2A CN109311825B (zh) 2016-05-12 2017-05-11 2,4,6-三取代均三嗪化合物及其制备方法和应用
CA3023854A CA3023854A1 (en) 2016-05-12 2017-05-11 Novel 2,4,6-trisubstituted s-triazine compounds, preparation method therefor, and use thereof
DK17795572.1T DK3456712T3 (da) 2016-05-12 2017-05-11 Hidtil ukendte 2,4,6-trisubstitueret S-triazinforbindelse, fremgangsmåde til fremstilling deraf og anvendelse deraf
KR1020187035570A KR102379959B1 (ko) 2016-05-12 2017-05-11 신규 2,4,6-트리치환된-s-트리아진 화합물, 그 제조방법 및 용도
JP2019512032A JP6987125B2 (ja) 2016-05-12 2017-05-11 新規２，４，６−三置換ｓ−トリアジン化合物並びにその製造方法および使用
EP17795572.1A EP3456712B1 (en) 2016-05-12 2017-05-11 Novel 2,4,6-trisubstituted s-triazine compound, preparation method therefor, and use thereof
BR112018072869-1A BR112018072869B1 (pt) 2016-05-12 2017-05-11 Compostos de s-triazina 2,4,6-trissubstituídos, seus usos, e composição farmacêutica
US16/802,378 US11390591B2 (en) 2016-05-12 2020-02-26 2,4,6-trisubstituted s-triazine compound, preparation method therefor, and use thereof

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