WO2018045969A1 - 活化腺苷单磷酸活化蛋白激酶之化合物 - Google Patents

活化腺苷单磷酸活化蛋白激酶之化合物 Download PDF

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WO2018045969A1
WO2018045969A1 PCT/CN2017/100735 CN2017100735W WO2018045969A1 WO 2018045969 A1 WO2018045969 A1 WO 2018045969A1 CN 2017100735 W CN2017100735 W CN 2017100735W WO 2018045969 A1 WO2018045969 A1 WO 2018045969A1
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compound
substituted
group
phenyl
protein kinase
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PCT/CN2017/100735
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French (fr)
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萧崇玮
苏溶真
林彦儒
黄瑞雯
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法玛科技顾问股份有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D233/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings
    • C07D233/96—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, not condensed with other rings having three double bonds between ring members or between ring members and non-ring members
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/06—Antihyperlipidemics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/32—One oxygen, sulfur or nitrogen atom
    • C07D239/42—One nitrogen atom
    • 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
    • C07D401/14—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 three or more hetero rings
    • 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
    • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
    • C07D409/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings
    • C07D409/04—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing two hetero rings directly linked by a ring-member-to-ring-member bond

Definitions

  • the present invention relates to a novel compound and its use, in particular to a compound which is an adenosine monophosphate-activated protein kinase (AMPK) activator and can be applied to the treatment of adenosine Monophosphate-activated protein kinase-mediated cancer or a disease associated with fat metabolism or syndrome.
  • AMPK adenosine monophosphate-activated protein kinase
  • ATP-activated protein kinase is a protein complex that contains three subunits of ⁇ , ⁇ , and ⁇ (AMPK ⁇ , AMPK ⁇ , and AMPK ⁇ ) and is a eukaryote.
  • An important regulator of metabolic related reactions in cells when adenosine monophosphate-activated protein kinase is activated by the regulation of upstream factors, further regulates downstream signaling molecules, such as: HIF-1, TSC1, TSC2, Raptor, ULK1 SREBP-1, SirT1, P300, HNF4 ⁇ , Torc2, HDAC4, MBS85, MYPT1, Tau, CLIP170, ATGL, HSL, ACC, HMG-CoA, PFKFB3, GS, and TCB1D1, etc., thereby regulating metabolic-related message transmission pathways.
  • downstream signaling molecules such as: HIF-1, TSC1, TSC2, Raptor, ULK1 SREBP-1, SirT1, P300, HNF4 ⁇ , Torc2, HDAC4, MBS85, MYPT1, Tau, CLIP170, ATGL, HSL, ACC, HMG-CoA, PFKFB3, GS, and TCB1D1, etc.
  • the metabolic response regulated by adenosine monophosphate-activated protein kinase directly affects cell growth and cell survival, and the phenomenon that adenosine monophosphate-activated protein kinase is disabled or exhibits a low activation state is common in cancer, obesity, diabetes, and aging.
  • adenosine monophosphate-activated protein kinase In disease, when adenosine monophosphate-activated protein kinase is phosphorylated, it is converted to an activated state and has protein kinase activity, that is, activated adenosine monophosphate-activated protein kinase can further phosphorylate downstream molecules to initiate or inhibit The message transmission pathway; in some studies, it was pointed out that phosphorylation-activated adenosine monophosphate-activated protein kinase can further inhibit the growth and survival of cancer cells and fat cells, and the same phenomenon can be observed in C. elegans. Thus, activation of adenosine monophosphate-activated protein kinases plays a key role in organisms, making it a potential target for drug development and disease treatment.
  • Adenosine monophosphate-activated protein kinase activator (agonist/activator) can be subdivided into indirect (indirect) adenosine monophosphate-activated protein kinase activator and direct adenosine monophosphate-activated protein kinase activator due to direct adenosine monophosphate-activated protein kinase activator for adenosine monophosphate-activated protein kinase complex
  • Each subunit in (complex) has binding specificity, that is, it directly affects its activity by directly binding to adenosine monophosphate-activated protein kinase, rather than indirectly affecting adenosine monophosphate activation through other molecules.
  • the activity of protein kinase is so high that the direct adenosine monophosphate-activated protein kinase activator is higher than the indirect adenosine monophosphate-activated protein kinase activator; the direct adenosine monophosphate-activated protein has been developed.
  • the kinase activator enters the clinical trial phase, and the compound A-769662 and the compound 911 are used as the index compounds, and most of the existing compounds are the subunits (AMPK ⁇ 2) or sense in the adenosine monophosphate-activated protein kinase complex.
  • the ATP concentration subunit (AMPK ⁇ ) is specific and has less specificity for the active subunit (AMPK ⁇ ).
  • the present invention is based on the above objects, and in one aspect, proposes a novel compound which is of the formula I, (Formula I),
  • R 1 is an unsubstituted or substituted aromatic group
  • R 3 is a substituted phenyl amide group or a phenyl urea group
  • Ar is an unsubstituted Or a substituted phenylene group.
  • the unsubstituted or substituted phenyl group is or
  • R 2 is a hydrogen, a halide or an alkyl group.
  • R 1 is an unsubstituted or substituted phenyl or substituted
  • R 1 is an unsubstituted pyrrolic group, a substituted pyrrolyl group or a substituted
  • R 1 is an unsubstituted thiophene group, a substituted thienyl group or a substituted
  • R 1 is an unsubstituted naphthalenic group, a substituted naphthyl group or a substituted
  • R 1 is a disubstituted phenyl group, once substituted or
  • the substituted anilino group of R 3 is substituted
  • the substituted phenylurea of R 3 is substituted.
  • the halide of R 2 is fluorine or chlorine.
  • the alkyl group of R 2 is a methyl or ethane group.
  • the present invention is based on the above compounds, and provides a use of the compound for the preparation of a pharmaceutical composition, and the pharmaceutical composition is for use with an adenosine monophosphate-activated protein kinase (AMP-activated protein) ELISA; AMPK) related cancer, lipid metabolism disease or lipid metabolism syndrome.
  • AMP-activated protein adenosine monophosphate-activated protein kinase
  • the present invention is also directed to the use of the compound described above for the treatment of a disease in an effective amount of the compound or a pharmaceutically acceptable salt thereof, and the disease is associated with adenosine monophosphate-activated protein kinase (AMP-activated protein kinase; AMPK) related cancer, lipid metabolism disease or lipid metabolism syndrome.
  • AMP-activated protein kinase AMP-activated protein kinase
  • the cancer is liver cancer or breast cancer.
  • the compound has binding specificity for the alpha subunit of adenosine monophosphate-activated protein kinase and phosphorylates an alpha subunit of adenosine monophosphate-activated protein kinase.
  • the compound is a cell death that induces adipocytes.
  • 1A-1B are diagrams showing the activation of AMPK by the compounds of the present invention.
  • 2A-2C are graphs showing that a compound of the invention can be phosphorylated to activate AMPK.
  • Figure 2D is a graph showing that the compounds of the present invention can further modulate downstream signaling molecules upon phosphorylation of activated AMPK.
  • Figure 3 is a graph showing that the compound of the present invention inhibits the growth of liver cancer cells.
  • Figure 4 is a graph showing that the compounds of the present invention inhibit the growth of triple negative breast cancer cells.
  • Figures 5A-5B are graphs showing that the compounds of the invention induce cell death in adipocytes.
  • Figure 6 is a graph showing that feeding a compound of the present invention to a high fat diet obese mice can reduce their body weight.
  • Ar refers to a group of aromatic rings, a compound containing hydrocarbons in its structure, and a general term for a large class of compounds containing one or more different types of atomic planar rings covalently bonded in two different types. .
  • Aromatic ring refers to a cyclic planar molecule with a resonant bond ring that is more stable than other geometric or linked structures of the same atomic group. Since the most common aromatic compound is benzene, "aromatic” can also It is called a benzene derivative.
  • Alino refers to a group having a basic structure of C 6 H 6 N - .
  • Phenylurea means a group having a basic structure of C 6 H 5 NHCONH.
  • Phenylene refers to a group whose basic structure is a di-substituted benzene ring.
  • halide means fluorine, chlorine, bromine and iodine.
  • Alkyl means a chain-like organic functional group containing only two atoms of carbon and hydrogen.
  • Substituted means that a plurality of substitutions are made by a named substituent; if a plurality of substituent moieties are disclosed or claimed, the substituted compound may independently be derived from one or more of the disclosed or claimed substituent moieties. Single or Multiple substitutions; in addition, independently substituted means that the (two or more) substituents may be the same or different.
  • “Pharmaceutically acceptable” in this specification means a compound, material, composition, salt and/or dosage form that is safe and suitable for administration to humans or animals, using reasonable medical judgment and in compliance with all applicable government regulations.
  • Effective dose means a dose of a compound of the invention which means (1) treating or preventing a particular disease, condition or disorder; (2) reducing, ameliorating or eliminating one or more symptoms of a particular disease, condition or condition. Or (3) prevent or delay the onset of one or more symptoms of a particular disease, condition or condition described herein.
  • Treatment means reversing, alleviating, inhibiting the progression of, or delaying the onset of, or preventing the onset or prevention of one or more symptoms of the condition or condition to which the term applies; unless otherwise stated, as used, “treatment” refers to a therapeutic act such as “treating” as defined above; “treatment” also includes adjuvant treatment and new adjuvant treatment for an individual; for the avoidance of doubt, in this specification Reference to “treatment” includes reference to curative, palliative and prophylactic treatment, and administration of an agent for such treatment.
  • the compound in the pharmaceutical composition and method of treatment of the present invention, can be administered in any suitable manner to provide a mammalian (especially human) effective amount of the compound of the present invention; for example, oral, rectal, or topical. , parenteral, ocular, transpulmonary, nasal, and the like; dosage forms include troches, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like, but not To the extent that it is preferred, the compounds of the invention are administered orally.
  • the compound in the pharmaceutical composition and method of treatment of the present invention, can be administered in any suitable manner to provide a mammalian (especially human) effective amount of the compound of the present invention; for example, oral, rectal, or topical. , parenteral, ocular, transpulmonary, nasal, and the like; dosage forms include troches, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like, preferably The compound of the present invention is orally administered.
  • a mammalian especially human
  • dosage forms include troches, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like, preferably The compound of the present invention is orally administered.
  • Suitable individuals of the invention include mammalian subjects including, but not limited to, large, cat, cow, goat, horse, sheep, pig, rodent, rabbit, primate, and the like. And covered A mammal born; in a preferred embodiment, the human is a suitable individual, and the human individual can be of any gender and at any stage of development.
  • the compound disclosed by the present invention is an activator of AMP-activated protein kinase (AMPK), which binds to adenosine monophosphate-activated protein kinase alpha subunit (AMPK ⁇ ) and is activated by phosphorylation.
  • AMPK ⁇ which in turn regulates the downstream signaling pathway mediated by AMPK ⁇ , therefore, the compounds of the present invention and pharmaceutically acceptable salts are effective for treating diseases mediated by adenosine monophosphate-activated protein kinases, such as cancer and fatty metabolic diseases. , fat metabolism syndrome, diabetes and aging related diseases.
  • reaction formula I for synthesizing the compound of the present invention:
  • the compound A and the compound B are subjected to a coupling reaction in an environment of N,N-diisopropylethylamine (DIEA) to produce an intermediate product C
  • DIEA N,N-diisopropylethylamine
  • this step DIEA is first added to Compound B dissolved in DMSO at 4 ° C, and then Compound A is added to obtain a mixed solution, which is then stirred at 4 ° C for 5 minutes, and then reacted at room temperature. After 3 hours, ice water was added to terminate the reaction; then, extraction was carried out with 100 ml of ethyl acetate to obtain an organic layer, and the organic layer was washed with brine and dried over anhydrous magnesium sulfate.
  • R 1 is an unsubstituted or substituted aromatic ring group, for example, an unsubstituted or substituted phenyl group or a substituted one.
  • R 3 may be a substituted anilino group, for example, once substituted R 3 may also be a substituted phenyl urea, for example: once substituted
  • Ar may be an unsubstituted or substituted phenyl group, for example: Wherein, and R 2 may be monohydrogen, monohalide or monoalkyl.
  • the halide may be fluorine or chlorine
  • the alkyl group may be methyl or ethyl.
  • SCT-1011 is synthesized by the following Chemical Formula II:
  • the intermediate product 4-(2-chloropyrimidin-4-yl)benzonitrile is synthesized; 2,4-dichloropyrimidine, 4-cyano group dissolved in a dimethoxyethane solution.
  • the final product compound SCT-1001 is synthesized; first, 0.61 mmol of 3-amino-N-(4) dissolved in 1.0 equivalent of 4-(2-chloropyrimidin-4-yl)benzonitrile -chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide is mixed with isopropyl alcohol (IPA) and added with a catalytic amount of HCl.
  • IPA isopropyl alcohol
  • N-(4- Chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)-4-methylbenzamide N-(4- Chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)-4-methylbe Nzamide
  • N-(4- Chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)-4-methylbe Nzamide is the compound SCT-1001.
  • reaction formula III for synthesizing the compound of the present invention:
  • the compound I and the compound B are subjected to a coupling reaction in the presence of THF to produce an intermediate product J.
  • triphosgene and THF are first added to the THF solution containing the compound I at 4 ° C.
  • the compound B is further added to obtain a mixed solution, which is further stirred at 4 ° C for 5 minutes, and then reacted at room temperature for 3 hours, and then added with ice water to terminate the reaction; 100 ml of 3 times of ethyl acetate was extracted to obtain an organic layer, and the organic layer was washed with brine, dried over anhydrous magnesium sulfate (MgSO 4 ), filtered, and filtered, and then filtered and purified by chromatography.
  • Intermediate product J is obtained; then intermediate product J is subjected to hydration reaction under hydrogen and Palladium/charcoal to obtain compound K.
  • R 1 is an unsubstituted or substituted aromatic ring group, for example, an unsubstituted or substituted phenyl group or a substituted one.
  • R 3 may be a substituted anilino group, for example, once substituted R 3 may also be a substituted phenyl urea, for example: once substituted
  • Ar may be an unsubstituted or substituted phenyl group, for example: Wherein, and R 2 may be monohydrogen, monohalide or monoalkyl.
  • the halide may be fluorine or chlorine, and the alkyl group may be methyl or ethyl.
  • SCT-1015 is synthesized by the following Chemical Formula IV:
  • the final product compound SCT-1015 is synthesized; first, 0.61 mmol in 1.0 equivalent of t-butyl 2-(2-chloropyrimidin-4-yl)-1H-pyrrole-1-carboxylate 1-(3-aminophenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea is mixed with isopropyl alcohol (IPA) and added with a catalytic amount of HCl. The mixture is then concentrated and chromatographed.
  • IPA isopropyl alcohol
  • the liver cancer cell line Huh-7 is obtained from Health Science Research Resources Bank (HSRRB, Osaka, Japan; JCRB0403); the liver cancer cell line PLC/PRF/5 (PLC5), Sk-Hep-1 and HCC1806 are Taken from the American Type Culture Collection (ATCC, Manassas, VA); all cell lines were scaled up and frozen immediately after harvesting, so the frozen cells were thawed every 3 months to allow experiments in the same batch of cells.
  • the extract and the anti-AMPK ⁇ 1 antibody were co-cultured in an immunoprecipitation buffer solution (G-Biosciences) overnight, followed by protein magnetic beads (Protein A/ G Magnetic Bead; PureProteome TM ) was added to each sample and cultured at 4 ° C for 4 hours.
  • protein magnetic beads Protein A/ G Magnetic Bead; PureProteome TM
  • different doses of the compound were combined with 12.5 ng of recombinant protein human AMPK ( ⁇ 1). , ⁇ 1, ⁇ 1) co-culture, and then the analysis was performed according to the manual of the AMPK SAMS peptide assay (Cyclex) for detecting AMPK activity.
  • AMPK activity was tested after addition of a protein extract of 20-20,000 nanomolar (nM) of compound SCT-1015 to AMPK ⁇ 1, which was confirmed to be active compared to the group in which no compound was added.
  • nM nanomolar
  • Fig. 1B another 20-2,000 nanomolar compound SCT-1015 was co-cultured with the recombinant protein AMPK ⁇ 1/ ⁇ 1/ ⁇ 1, and the recombinant protein AMPK ⁇ 1/ ⁇ 1/ ⁇ 1 was compared to the group without compound addition.
  • the activity was also significantly increased; the SCT-1015 compound was shown to increase the activity of AMPK in liver cancer cells by Figures 1A and 1B.
  • the commercial kit AMPK [pT172] Phospho-ELISA Kit (KHO0651) sold by ThermoFisher Science was used, and the liver cancer cell line PLC5 was co-cultured with a compound derivative of 10 micromolar ( ⁇ M) for 24 hours.
  • the analysis was performed according to the user manual disclosed therein, and the absorbance was detected at a wavelength of 450 nm (nm).
  • the results are shown in Table 3.
  • the first column is the compound number
  • the third column is the test result of the compound activating AMPK
  • the control group is the solvent DMSO added only the compound derivative
  • the value ⁇ standard deviation shows; from the results, it is known that the compound derivative can phosphorylate and activate AMPK at the position of T172, and the activation effect (1.8) of the compound SCT1015 is optimal.
  • liver cancer cell line PCL5 and the triple negative breast cancer cell line (TNBC) MDA-MB-231 and MDA-MB-453 co-cultured with the compound SCT-1015 were separately collected, and after rinsing, the cells were lysed with RIPA buffer to obtain a protein extract.
  • TNBC triple negative breast cancer cell line
  • the protein concentration was measured by Bio-Rad Protein Assay dye reagent (Bio-Rad), followed by 2 times SDS-loading buffer (100 mM Tris HCl, pH 6.8, 200 mM ⁇ -mercaptoethanol, 4% SDS, 0.02% bromophenol blue, and 20% glycerol) adjust the protein sample volume, then electrophorese the protein sample with 10% SDS polyacrylamide gels and transfer it to the PVDF membrane, then add the grade 1 antibody to the membrane to detect the target protein, then add with horseradish
  • the secondary antibody of peroxidase (horseradish peroxidase) is combined with a grade 1 antibody, followed by the addition of a reaction signal (enhanced SuperSignal West Pico Cheminluminescent Substrate; Pierce) to obtain a specific protein signal, protein content
  • the ⁇ -actin was used as the internal control group for analysis; the results are shown in Figures 2A-2C, in the liver cancer cell line PCL5 and the
  • the addition of compound SCT-1015 showed a significant increase in the expression of phosphorylated AMPK ⁇ (p-AMPK ⁇ ) compared to the unadded group, as shown in Figure 2D, in three liver cancers.
  • phosphorylation-activated AMPK further regulates the downstream signaling molecules HIF1 and PDHK1; from the above results, the compound SCT-1015 of the present application can indeed Phosphorylation activates AMPK ⁇ and further regulates downstream signaling pathways, thereby affecting the relevant biochemical reactions within the cell.
  • the analysis was performed using the commercialized kit Prestoblue assay sold by Thermo Fisher Scientific. Three liver cancer cell lines PLC/PRF/5, SK-hep1 and Huh-7 were co-cultured with compound SCT-1015 for 48 hours and 72 respectively. The analysis was performed according to the manual of the commercial kit. The results are shown in Figure 3. Compared with the control group DMSO, the three liver cancer cell lines were supplemented with 5-20 micromolar ( ⁇ M) compound SCT-1015. After the culture, cell proliferation was indeed inhibited; thus, it was found that the compound of the present invention can inhibit the growth and division of liver cancer cells by activating AMPK.
  • ⁇ M micromolar
  • the experiment established an in situ triple-negative breast cancer mouse model (HCC1806/luc2-bearing orthotopic mice) with a triple-negative breast cancer cell HCC1806 (HCC1806/luc2) carrying the luciferase (luc2) gene, and divided the experimental mice into two groups.
  • the two groups were (1) control group (vehicle): oral solvent; (2) experimental group: (SCT-1015): oral compound SCT-1015 (20 mg/kg), and the above samples were fed daily.
  • the mice were continued for 14 days, and on the 0th day and the 14th day, the reaction substrate of luciferase was injected into the re-cavity of the mouse, and the HCC1806/luc2 was detected by In vivo imaging system (IVIS). A cold light signal from the cell to observe the growth of the cell.
  • IVIS In vivo imaging system
  • the results are shown in Figure 4.
  • the first line of the image taken on day 0, the second line is the image taken on day 14; compared to the mouse image taken on day 0 of the experimental group (SCT-1015), fed
  • the mice of compound SCT-1015 did not significantly increase the number of HCC1806/luc2 cells, and the mice fed compound SCT-1015, HCC1806/, compared with the mouse images of the control group on day 14
  • the number of luc2 cells was significantly reduced; from the results of the experiments, it was found that the compound of the present invention can activate AMPK to inhibit the growth of triple-negative breast cancer cells.
  • the experiment was based on the standard process of adipocyte differentiation, and human human-adipocytes (Gibco StemPro adipogenesis differentiation reagent) was cultured for 14 days to induce differentiation into mature adipocytes, and then, Mature adipocytes were treated with different concentrations of compound SCT-1015 (100 ⁇ M, 25 ⁇ M, 12.5 ⁇ M, 5 ⁇ M, and 2.5 ⁇ M) or only solvent (control group Mock) or untreated (untreated) and cultured 48 After an hour, the fat cells were stained with the dye adipoRed (Lonza inc.), and the fluorescence signal of the fat cells was measured at an excitation light wavelength of 485 nm and a color light wavelength of 572 nm to calculate the number of cells; in addition, the cells were treated at 2.5 ⁇ M. The compound SCT-1015 was also observed by taking a cell image to observe the cell type.
  • the number of cells of the mature adipocytes after treatment of the compound SCT-1015 was significantly decreased, and the amount of cell decrease gradually increased as the concentration of the compound SCT-1015 increased;
  • the cells under the treatment of 2.5 ⁇ M of the compound SCT-1015 the mature adipocytes (marked *) showed a shrinking and dead form, whereas the undifferentiated cells (marked **)
  • the healthy cell type is still maintained; from the above experimental results, it is known that the compound of the present invention can induce cell death of fat cells and is not toxic to undifferentiated cells.
  • DIO high-fat obesity
  • Vehicle mixed solvent
  • mice treated with the compound SCT-1015 had an average weight loss of 5% compared with the control group of mice, and were similar to the reduced body weight of the mice treated with the standard orlistat.
  • the compound of the present invention can reduce the body weight of the high fat diet obese mice by affecting the activity of AMPK and thereby inducing apoptosis of the adipocytes.
  • the compounds disclosed in the present invention have a novel chemical structure and act as an activator of adenosine monophosphate-activated protein kinase; it is confirmed by the foregoing experimental results that the compound of the present invention can bind to adenosine monophosphate Activated protein kinase alpha subunit (AMPK ⁇ ), which phosphorylates and activates AMPK ⁇ , further regulates downstream signaling molecules, and inhibits the growth and division of liver cancer cells and breast cancer cells. In addition, it can also induce cell death of fat cells.
  • AMPK ⁇ adenosine monophosphate Activated protein kinase alpha subunit
  • the compound provided by the invention is more useful for preparing a pharmaceutical composition, and the pharmaceutical composition is used for
  • the adenosine monophosphate-activated protein kinase-mediated cancer or fat metabolism-related diseases or syndromes on the other hand, the compounds provided by the present invention can also be applied to the treatment of diseases, and the disease is caused by adenosine monophosphate Activated protein kinase-mediated cancer or fat metabolism-related diseases or syndromes; therefore, the present invention provides a novel compound with medical potential that has better activated AMPK efficacy and better therapeutic specificity than conventional techniques.

Abstract

本发明揭示一种如结构式I之化合物，其作为腺苷单磷酸活化蛋白激酶之活化剂，而得以磷酸化活化腺苷单磷酸活化蛋白激酶，进而调控下游讯息传递路径，而可抑制肝癌细胞及乳癌细胞之生长***，亦可诱发脂肪细胞死亡；由此，本发明所揭示之化合物可应用于制备与腺苷单磷酸活化蛋白激酶相关之癌症及脂肪代谢疾病或症候群之医药组合物上，亦可用于治疗与腺苷单磷酸活化蛋白激酶相关之癌症及脂肪代谢疾病或症候群。

Description

活化腺苷单磷酸活化蛋白激酶之化合物 【技术领域】
本发明是关于一种新颖之化合物及其应用，尤其指一种作为腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase；AMPK)活化剂(agonist)之化合物，并能应用于治疗由腺苷单磷酸活化蛋白激酶介导之癌症、脂肪代谢相关疾病或症候群。
【背景技术】
腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase；AMPK)是一蛋白复合体(complex)，其是包含α、β及γ三个次单元(AMPKα、AMPKβ及AMPKγ)，且为真核生物细胞中代谢相关反应之重要调控者，当腺苷单磷酸活化蛋白激酶受到上游因子的调控而被活化时，是进一步调控下游讯息传递分子，例如：HIF-1、TSC1、TSC2、Raptor、ULK1、SREBP-1、SirT1、P300、HNF4α、Torc2、HDAC4、MBS85、MYPT1、Tau、CLIP170、ATGL、HSL、ACC、HMG-CoA、PFKFB3、GS及TCB1D1等，进而调控与代谢相关之讯息传递路径。
腺苷单磷酸活化蛋白激酶所调控之代谢反应是直接影响细胞生长及细胞存活，且腺苷单磷酸活化蛋白激酶的失能或呈现低活化态的现象常见于癌症、肥胖症、糖尿病及老化相关疾病中；当腺苷单磷酸活化蛋白激酶发生磷酸化修饰时即转变为活化态，并具有蛋白激酶的活性，亦即活化之腺苷单磷酸活化蛋白激酶可进一步磷酸化下游分子以启动或抑制讯息传递路径；在一些研究中指出，磷酸化活化之腺苷单磷酸活化蛋白激酶可进一步抑制癌细胞及脂肪细胞之生长及存活，在线虫模式(C.elegans)中亦可观察到相同的现象；由此可知，腺苷单磷酸活化蛋白激酶之活化在生物体细胞中扮演关键的角色，使其成为药物开发以及疾病治疗之潜在标的。
腺苷单磷酸活化蛋白激酶的活化剂(agonist/activator)又可细分为间接型 (indirect)腺苷单磷酸活化蛋白激酶活化剂及直接型(direct)腺苷单磷酸活化蛋白激酶活化剂，由于直接型腺苷单磷酸活化蛋白激酶活化剂对于腺苷单磷酸活化蛋白激酶复合体(complex)中各个次单元分别具有结合专一性(binding specificity)，亦即其是通过直接结合于腺苷单磷酸活化蛋白激酶进而影响其活性，而非通过其他分子间接影响腺苷单磷酸活化蛋白激酶之活性，故直接型腺苷单磷酸活化蛋白激酶活化剂之临床应用性较间接型腺苷单磷酸活化蛋白激酶活化剂为高；目前已有已开发之直接型腺苷单磷酸活化蛋白激酶活化剂进入临床试验阶段，且以化合物A-769662及化合物911为指标性化合物，而绝大部分现有的化合物是对腺苷单磷酸活化蛋白激酶复合体中调控次单元(AMPKβ2)或感测ATP浓度次单元(AMPKγ)具有专一性，较少对活性次单元(AMPKα)具有专一性之化合物，故本申请即以此为方向着手开发新颖之化合物。
【发明内容】
本发明基于上述之目的，于一方面，是提出一种新颖之化合物，其是如结构式I所式，
Figure PCTCN2017100735-appb-000001
(式I)，
其中，R1是一未经取代或一经取代之芳香环基(aromatic group)，R3是一经取代之苯胺基(phenyl amide group)或苯基脲(phenyl urea group)且Ar是一未经取代或一经取代之伸苯基(phenylene group)。
于本发明之一实施例中，该未经取代或该经取代之伸苯基是
Figure PCTCN2017100735-appb-000002
或
Figure PCTCN2017100735-appb-000003
且R2是一氢、一卤化物(halide)或一烷基(alkyl group)。
于本发明之一实施例中，R1是一未经取代或一经取代之苯基或一经取代之
Figure PCTCN2017100735-appb-000004
于本发明之一实施例中，R1是一未经取代之吡咯基(pyrrolic group)、一经取代之吡咯基或一经取代之
Figure PCTCN2017100735-appb-000005
于本发明之一实施例中，R1是一未经取代之噻吩基(thiophene group)、一经取代之噻吩基或一经取代之
Figure PCTCN2017100735-appb-000006
于本发明之一实施例中，R1是一未经取代之萘基(naphthalenic group)、一经取代之萘基或一经取代之
Figure PCTCN2017100735-appb-000007
于本发明之一实施例中，R1是一双取代苯基、一经取代之
Figure PCTCN2017100735-appb-000008
或
Figure PCTCN2017100735-appb-000009
于本发明之一实施例中，R3之该经取代之苯胺基是一经取代之
Figure PCTCN2017100735-appb-000010
于本发明之一实施例中，R3之该经取代之苯基脲是一经取代之
Figure PCTCN2017100735-appb-000011
Figure PCTCN2017100735-appb-000012
于本发明之一实施例中，R2之该卤化物是氟或氯。
于本发明之一实施例中，R2之该烷基是甲烷基或乙烷基。
于另一方面，本发明基于上述之化合物，提出一种将该化合物用于制备医药组成物之用途，且所述之医药组成物是用于与腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase；AMPK)相关之癌症、脂质代谢疾病或脂质代谢症候群。
在另一方面，本发明亦针对上述之化合物，提出一种以有效剂量之该化合物或其医药上可接受之盐治疗疾病之用途，且所述之疾病是与腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase；AMPK)相关之癌症、脂质代谢疾病或脂质代谢症候群。
于本发明之一实施例中，该癌症是肝癌或乳癌。
于本发明之一实施例中，该化合物是对腺苷单磷酸活化蛋白激酶之α次单元具有结合特异性，并磷酸化活化腺苷单磷酸活化蛋白激酶之α次单元。
于本发明之一实施例中，该化合物是诱发脂肪细胞之细胞死亡。
【附图说明】
图1A-1B是显示本发明之化合物可活化AMPK。
图2A-2C是显示本发明之化合物可磷酸化活化AMPK。
图2D是显示本发明之化合物在磷酸化活化AMPK后可进一步调控下游讯息传递分子。
图3是显示本发明之化合物可抑制肝癌细胞之生長。
图4是显示本发明之化合物可抑制三阴性乳癌细胞之生长。
图5A-5B是显示本发明之化合物可诱发脂肪细胞之细胞死亡。
图6是显示喂养本发明之化合物于高脂饮食肥胖小鼠可减少其体重。
【具体实施方式】
名词定义
「Ar」是指含有芳香环之基团，其结构中含有碳氢的有机化合物，分子结构中含有一个或多个两种不同类型共价键联结成的原子平面环的一大类化合物的总称。
「芳香环」是指具有共振键环的环状平面分子，其相较于相同原子组的其它几何或连接结构更为稳定，由于最常见的芳香族化合物是苯，因此「芳香族」亦可称之为苯衍生物。
「苯胺基」是指基础结构为C6H6N-之基团。
「苯基脲」是指基础结构为C6H5NHCONH之基团。
「伸苯基」是指基础结构为双取代苯环(di-substituted benzene ring)之基团。
「卤化物」是指氟、氯、溴以及碘。
「烷基」是指仅含有碳、氢两种原子的链状有机官能团。
「经取代」是指由所命名之取代基实施多种程度之取代；如揭示或主张多个取代基部分，则经取代化合物可独立地由一或多个所揭示或所主张取代基部分来单一地或 复数个地取代；此外，独立地经取代意指(两个或更多个)取代基可相同或不同。
「医药上可接受之」在本说明书中是指使用合理的医学判断且遵循所有可应用之政府法规，能安全且适于投与人类或动物之化合物、材料、组合物、盐及/或剂型。
「有效剂量」是指本发明化合物的一种剂量，其意谓(1)治疗或预防特定疾病、病状或病症；(2)减轻、改善或消除特定疾病、病状或病症之一或多种症状；或(3预防或延迟本文所述之特定疾病、病状或病症之一或多种症状发作。
「治疗」是指逆转、缓解、抑制该术语所适用之病症或病状或该病症或病状之一或多种症状的进展、延迟其发作或预防该病症或病状；除非另外说明，否则如本说明书所用，「治疗(treatment)」是指治疗行为，如「治疗(treating)」在上文中刚刚定义；「治疗」亦包括对个体之佐剂治疗及新佐剂治疗；为避免疑问，本说明书中提及「治疗」包括提及治愈性、姑息性及预防性治疗，及给予用于此类治疗的药剂。
本发明之医药组成物及治疗方法中，化合物给予方式及剂量范围，可采用任何适宜给予途径以提供哺乳动物(尤其人类)有效剂量之本发明化合物；例如，可采用经口、经直肠、局部、非经肠、经眼、经肺、经鼻及诸如此类；剂型包括锭剂、***锭(troches)、分散剂、悬浮液、溶液、胶囊、乳霜、软膏、气溶胶及诸如此类但不以此为限，较佳者，系经口给予本发明化合物。
本发明之医药组成物及治疗方法中，化合物给予方式及剂量范围，可采用任何适宜给予途径以提供哺乳动物(尤其人类)有效剂量之本发明化合物；例如，可采用经口、经直肠、局部、非经肠、经眼、经肺、经鼻及诸如此类；剂型包括锭剂、***锭(troches)、分散剂、悬浮液、溶液、胶囊、乳霜、软膏、气溶胶及诸如此类，较佳者，是经口给予本发明化合物。
本发明之适合个体包括哺乳动物个体；所述之哺乳动物包括(但不限于)大、猫、牛、山羊、马、绵羊、猪、啮齿动物、兔类动物、灵长类动物及其类似动物，且涵盖未 出生之哺乳动物；于一较佳实施例中，人类为一适合个体，人类个体可为任一性别且处于任何发育阶段。
功效
本发明所揭示之化合物是腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase;AMPK)之活化剂，其是结合至腺苷单磷酸活化蛋白激酶α次单元(AMPKα)，并通过磷酸化活化AMPKα，进而调控AMPKα所介导之下游讯息传递路径，故，本发明之化合物及医药上可接受之盐可有效治疗由腺苷单磷酸活化蛋白激酶所介导之疾病，诸如癌症、脂肪代谢疾病、脂肪代谢症候群、糖尿病及老化相关疾病。
本发明之其他特色及优点将于下列实施范例中被进一步举例与说明，而该实施范例仅作为辅助说明，并非用于限制本发明之范围。
本发明化合物之合成方法
以下化学反应式图示及实施例是用于合成本发明如结构式I所示之化合物之流程图，而此些化学反应式及实施例仅用以阐释本发明之技术精神，不应以此些化学反应式及实施例来限制本发明。
合成本发明之化合物之第一反应通式(反应式I)：
Figure PCTCN2017100735-appb-000013
反应式I
于上述反应式I之第一步骤中，化合物A与化合物B在N,N-二异丙基乙基胺(N,N-Diisopropylethylamine；DIEA)的环境下进行耦合反应以产生中间产物C，于此步骤中，DIEA首先于4℃下加入溶于DMSO之化合物B中，接着，再将化合物A加入以获得一混合溶液，该混合溶液接续在4℃下搅拌5分钟，而后，于室温下反应3小时，再加入冰水以终止反应；接着，以100毫升之3倍乙酸乙酯(ethyl acetate)进行萃取以获得有机层，再将有机层以盐水(brine)清洗后以无水硫酸镁(MgSO4)除水干燥后过滤，过滤后之溶液经浓缩后通过层析管柱纯化以获得中间产物C；接着将中间产物C在氢及钯/碳(Palladium/charcoal)的环境下进行水合反应以获得化合物D，待此反应结束后，取得Palladium/charcoal之过滤物并通过层析管柱纯化以获得化合物D；另一方面，化合物E与经取代之硼酸(boronic acid)F在(1,1′-双(二苯基膦)二茂铁)二氯化钯(1,1′-Bis(diphenylphosphino)ferrocene)palladium(II)dichloride；Pd(dppf)Cl2)环境下进行耦合反应以获得化合物G，于此反应中，溶于二甲醚(methoxymethane；DME)溶液之化合物E经干燥后与boronic acid F及Pd(dppf)2Cl2共同混合后于氮气环境下加热至90℃，待混合溶液之温度降至室温后，将其过滤并浓缩在通过层析管柱进行纯化以获得化合物G；于最终步骤中，化合物G及化合物D在异丙醇(isopropanol alcohol；IPA)溶液中加入催化剂盐酸(HCl)进行反应，此混合物于100℃下加热8小时候，以100毫升之3倍乙酸乙酯进行萃取，再将所获得之有机层以盐水清洗后，以无水硫酸镁(MgSO4)除水干燥后过滤，过滤后之溶液经浓缩后通过层析管柱纯化以获得化合物H。
于上述之反应式I中，R1是一未经取代或一经取代之芳香环基，例如：一未经取代或一经取代之苯基或一经取代之
Figure PCTCN2017100735-appb-000014
Figure PCTCN2017100735-appb-000015
一未经取代之吡咯基(pyrrolic group)、一经取代之吡咯基、一经取代之一未经取代之噻吩基(thiophene group)、一经取代之噻吩基、一经取代 之
Figure PCTCN2017100735-appb-000017
一未经取代之萘基(naphthalenic group)、一经取代之萘基、一经取代之
Figure PCTCN2017100735-appb-000018
一双取代苯基或一经取代之
Figure PCTCN2017100735-appb-000019
此外，于上述之反应式I中，R3可为一经取代之苯胺基，例如：一经取代之
Figure PCTCN2017100735-appb-000020
R3亦可为一经取代之苯基脲，例如：一经取代之
Figure PCTCN2017100735-appb-000021
Figure PCTCN2017100735-appb-000022
又，于上述之反应式I中，Ar可为一未经取代或一经取代之伸苯基，例如：
Figure PCTCN2017100735-appb-000023
其中，且R2可为一氢、一卤化物或一烷基，于较佳实施例中，该卤化物可为氟或氯，该烷基可为甲基或乙基。
通过上述之反应式I可合成如表1所列之本发明衍生之化合物。
表1.本发明之化合物衍生物
Figure PCTCN2017100735-appb-000024
实施例1、合成化合物SCT-1011
本发明所揭示之化合物之一实施例SCT-1011是由下列之化学式II所合成：
Figure PCTCN2017100735-appb-000025
反应式II
在合成化合物SCT1001之流程中，4-甲基-3-硝基苯甲酰氯(4-methyl-3-nitrobenzoylchloride)首先与5-氯-3-(三氟甲基)苯胺(5-chloro-3-(trifluoromethyl)aniline)进行耦合反应，接着在钯/碳(Pd/C)的催化下进行中间产物之还原反应使得氮基还原为胺基，另一方面，2,4-二氯嘧啶(2,4-dichloropyrimidine)是与4-氰基苯硼酸((4-cyanophenyl)boronic acid)进行耦合反应以获得4-(2-氯嘧啶-4-基)苯甲腈(4-(2-chloropyrimidin-4-yl)benzonitrile)，接着4-(2-chloropyrimidin-4-yl)benzonitrile是耦合于前述之胺基而获得化合物SCT1001。
于反应式II之第一步骤中，是合成中间产物4-(2-chloropyrimidin-4-yl)benzonitrile；溶于二甲氧基乙烷(dimethoxyethane)溶液之2,4-dichloropyrimidine,4-氰基硼酸((4-cyanophenyl)boronic acid)及催化剂量之(1,1′-双(二苯基膦)二茂铁)二氯化钯(1,1′-Bis(diphenylphosphino)ferrocene)palladium(II)dichloride,Pd(dppf)Cl2)是与3个当量之三乙胺(triethylamine)混合，并将混合物以90℃加热3小时，将混合物过滤取得过滤物后经浓缩及纯化而获得中间产物4-(2-chloropyrimidin-4-yl)benzonitrile。
4-(2-氯嘧啶-4-基)苯甲腈(4-(2-chloropyrimidin-4-yl)benzonitrile)之分析结 果：
1H NMR(400MHz,CDCl3):δ8.73(d,J＝5.2Hz,1H),8.20(d,J＝8.4Hz,2H),7.81(d,J＝8.4Hz,2H),7.62(d,J＝5.2Hz,1H)ppm.
于反应式II之第一步骤中，是合成3-氨基-N-(4-氯-3-(三氟甲基)苯基)-4-甲基苯甲酰胺(3-amino-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide)；于此一步骤中，首先，1当量之4-氯-3-(三氟甲基)苯胺(4-chloro-3-(trifluoromethyl)aniline)及1.2当量之triethylamine是共同溶于3毫升之二氯甲烷(dichloromethane)中，并于冰浴中缓慢加入溶于3毫升dichloromethane之4-甲基-3-硝基苯甲酰氯(4-Methyl-3-nitrobenzoyl chloride)(0.28毫摩尔)，再于室温下搅拌3小时，接着，于氢气及钯(palladium)环境下，粗中间产物之氮基还原为胺基，并于过滤后以层析管柱(己烷/乙酸乙酯＝9:1)(hexane/ethyl acetate＝9:1)纯化以获得3-氨基-N-(4-氯-3-(三氟甲基)苯基)-4-甲基苯甲酰胺(3-amino-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide)。
3-氨基-N-(4-氯-3-(三氟甲基)苯基)-4-甲基苯甲酰胺(3-amino-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide)之分析结果：1H NMR(400MHz,MeOH):δ8.24(s,1H),7.95(d,J＝8.8Hz,1H),7.57(d,J 8.8Hz,1H),7.25(s,1H),7.16(q,J＝8.0Hz,2H),2.22(s,3H)ppm.HRMS calculated for C 15 H 12 ClF 3 N 2 O(M-H)－:328.72.Found:327.22.
于反应式II之最终步骤中，是合成最终产物化合物SCT-1001；首先，溶于1.0当量之4-(2-chloropyrimidin-4-yl)benzonitrile之0.61毫摩尔之3-amino-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide是混合于isopropyl alcohol(IPA)，并加入催化剂量之HCl，接着，混合物是经浓缩并以层析管柱纯化以获得N-(4-氯-3-(三氟甲基)苯基)-3-((4-(4-氰基苯基)嘧啶-2-基)氨基)-4-甲基苯甲酰胺(N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)-4-methylbe nzamide)，即为化合物SCT-1001。
合成本发明之化合物之第二反应通式(反应式III)：
Figure PCTCN2017100735-appb-000026
反应式III
于上述反应式III之第一步骤中，化合物I与化合物B在THF的环境下进行耦合反应以产生中间产物J，于此步骤中，triphosgene及THF首先于4℃下加入包含化合物I之THF溶液及TEA之中，接着，再将化合物B加入以获得一混合溶液，该混合溶液接续在4℃下搅拌5分钟，而后，于室温下反应3小时，再加入冰水以终止反应；接着，以100毫升之3倍ethyl acetate进行萃取以获得有机层，再将有机层以brine清洗后以无水硫酸镁(MgSO4)除水干燥后过滤，过滤后之溶液经浓缩后通过层析管柱纯化以获得中间产物J；接着将中间产物J在氢及Palladium/charcoal的环境下进行水合反应以获得化合物K，待此反应结束后，取得Palladium/charcoal之过滤物并通过层析管柱纯化以获得化合物D；另一方面，化合物E与经取代之boronic acid F在Pd(dppf)2Cl2环境下进行耦合反应以获得化合物G，于此反应中，溶于DME溶液之化合物E经干燥后与boronic acid F及Pd(dppf)2Cl2共同混合后于氮气环境下加热至90℃，待混合溶液之温度降至室温后，将其过滤并浓缩在通过层析管柱进行纯化以获得化合物G；于最终步骤中，化合物G及化合物K在IPA溶液中加入催化剂HCl进行反应，此混合物于100℃下加热8小时后，以100毫升之3倍ethyl acetate进行萃取，再将所获得之有机层以brine清洗后，以无水硫酸镁(MgSO4)除水干燥后过滤，过滤后之溶液经浓缩后通过层析管柱纯化以获得化合物H。
于上述之反应式III中，R1是一未经取代或一经取代之芳香环基，例如：一未经取代或一经取代之苯基或一经取代之
Figure PCTCN2017100735-appb-000027
Figure PCTCN2017100735-appb-000028
一未经取代之吡咯基(pyrrolic group)、一经取代之吡咯基、一经取代之
Figure PCTCN2017100735-appb-000029
一未经取代之噻吩基(thiophene group)、一经取代之噻吩基、一经取代之
Figure PCTCN2017100735-appb-000030
一未经取代之萘基(naphthalenic group)、一经取代之萘基、一经取代之
Figure PCTCN2017100735-appb-000031
一双取代苯基或一经取代之
Figure PCTCN2017100735-appb-000032
此外，于上述之反应式III中，R3可为一经取代之苯胺基，例如：一经取代之
Figure PCTCN2017100735-appb-000033
R3亦可为一经取代之苯基脲，例如：一经取代之
Figure PCTCN2017100735-appb-000034
Figure PCTCN2017100735-appb-000035
又，于上述之反应式III中，Ar可为一未经取代或一经取代之伸苯基，例如：
Figure PCTCN2017100735-appb-000036
其中，且R2可为一氢、一卤化物或一烷基，于较佳实施例中，该卤化物可为氟或氯，该烷基可为甲基或乙基。
通过上述之反应式III可合成如表1所列之本发明衍生之化合物。
表2.本发明之化合物衍生物
Figure PCTCN2017100735-appb-000037
Figure PCTCN2017100735-appb-000038
实施例2、合成化合物SCT-1015
本发明所揭示之化合物之一实施例SCT-1015是由下列之化学式IV所合成：
Figure PCTCN2017100735-appb-000039
反应式IV
在合成化合物SCT1015之流程中，首先，0.148克、0.61毫摩尔之4-氯-4-3-(三氟甲基)苯胺(chloro-3-(trifluoromethyl)aniline)及2.2毫摩尔之三乙胺(triethylamine)是与3毫升干燥之四氢呋喃(THF)混合，再将之于冰浴中缓慢加入0.30毫摩尔三光气(triphosgene)之干燥之THF溶液中，接着于室温下搅拌30分钟后进行浓缩，再以1当量之3-硝基苯胺(3-nitroaniline)于65℃下反应30分钟，而后再次进行浓缩；接着于氢气的环境下借由催化剂钯/碳(Pd/C)将粗中间产物之转化为胺基中间产物，并于过滤后以层析管柱(hexane/ethyl acetate＝1:1)纯化以获得1-(3-氨基苯基)-3-(4-氯-3-(三氟甲基)苯基)脲(1-(3-aminophenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea)。
1-(3-氨基苯基)-3-(4-氯-3-(三氟甲基)苯基)脲(1-(3-aminophenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea)之分析结果：1H NMR(400MHz,MeOD-d4):δ7.98(d,J＝2.4Hz,1H),7.60(dd,J＝8.8,2.4Hz,1H),7.49(d,J＝8.8Hz,1H),7.02(t,J＝8.0Hz,1H),6.91(s,1H),6.70(dd,J＝8.0,1.2Hz,1H),6.44(dd,J＝8.0, 1.2Hz,1H)ppm。
于反应式IV之第二步骤中，是合成中间产物叔丁基-2-(2-氯嘧啶-4-基)-1H-吡咯-1-甲酸叔丁酯(t-butyl-2-(2-chloropyrimidin-4-yl)-1H-pyrrole-1-carboxylate)；2,4-二氯嘧啶(2,4-dichloropyrimidine)之二甲氧基乙烷(dimethoxyethane)溶液、1-(叔丁氧基羰基)-1H-吡咯-2-基-吡咯)硼酸(1-(tert-butoxycarbonyl)-1H-pyrrol-2-yl-pyrrol)boronic acid)及催化剂量之1′-Bis(diphenylphosphino)ferrocene)palladium(II)dichloride,Pd(dppf)Cl 2)是与3个当量之triethylamine共同混合，并将混合物于90℃下加热3小时，并于过滤后以层析管柱(hexane/ethyl acetate＝9:1)纯化以获得t-butyl 2-(2-chloropyrimidin-4-yl)-1H-pyrrole-1-carboxylate。
叔丁基-2-(2-氯嘧啶-4-基)-1H-吡咯-1-甲酸叔丁酯(Tert-butyl 2-(2-chloropyrimidin-4-yl)-1H-pyrrole-1-carboxylate)之分析结果：1H NMR(400MHz,CDCl3):δ8.47(d,J＝5.2Hz,1H),7.36(dd,J＝3.6,2.0Hz,1H),7.28(d,J＝5.2Hz,1H),6.64(dd,J＝3.6,1.6Hz,1H),6.20(t,J＝3.6Hz,1H),1.40(s,9H)ppm。
于反应式IV之最终步骤中，是合成最终产物化合物SCT-1015；首先，溶于1.0当量t-butyl 2-(2-chloropyrimidin-4-yl)-1H-pyrrole-1-carboxylate之0.61毫摩尔之1-(3-aminophenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea是混合于isopropyl alcohol(IPA)，并加入催化剂量之HCl，接着，混合物是经浓缩并以层析管柱(hexane/ethyl acetate＝2:1)纯化以获得1-(3-((4-(1H-吡咯-2-基)嘧啶-2-基)氨基)苯基)-3-(4-氯-3-(三氟甲基)苯基)脲(1-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-chloro-3-(trifle oromethyl)phenyl)urea)，即为化合物SCT-1015。
1-(3-((4-(1H-吡咯-2-基)嘧啶-2-基)氨基)苯基)-3-(4-氯-3-(三氟甲基)苯基)脲(1-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-chloro-3-(trifle oromethyl)phenyl)urea)之分析结果：1H NMR(400MHz,DMSO-d6):δ11.75(s,1H),10.55(s,1H),9.91(s,1H),9.61(s,1H),8.45(s, 1H),8.35(d,J＝6.4Hz,1H),8.08(s,1H),7.64(s,2H),7.38(s,1H),7.34(d,J＝6.4Hz,1H),7.28(t,J＝8.0Hz,1H),7.22(s,1H),7.12(d,J＝8.0Hz,1H),7.03(d,J＝7.6Hz,1H),6.36(s,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.5,153.1,152.3,148.3,139.2,138.8,137.8,131.6,128.8,127.9,126.8,126.3(q),122.5,122.3(q),121.9,116.7,116.1(q),113.7,113.1,111.4,109.8,104.7ppm.HRMS calculated forC22H16ClF3N6O(M-H)-:471.0942.Found:471.0957.
本发明之化合物及其相关衍生物之图谱结果
实施例1、
N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)-4-methy lbenzamide(化合物SCT-1001)
Figure PCTCN2017100735-appb-000040
分析结果
1H NMR(400MHz,CDCl3):δ8.79(s,1H),8.53(d,J＝5.2Hz,1H),8.23(s,1H),8.17(d,J＝8.4Hz,2H),7.90～7.87(m,2H),7.74(d,J＝8.4Hz,2H),7.47(t,J＝8.0Hz,2H),7.30(d,J＝8.0Hz,1H),7.19(d,J＝5.2Hz,1H),7.12(s,1H),2.40(s,3H)ppm.HRMS calculated for C26H17ClF3N5O(M-H)-:506.0990.Found:506.1010.
实施例2、
N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(3-cyanophenyl)pyrimidin-2-yl)amino)-4-methylbenzamide(化合物SCT-1002)
Figure PCTCN2017100735-appb-000041
分析结果
1H NMR(400MHz,CDCl3):δ8.88(s,1H),8.56(d,J＝5.2Hz,1H),8.41(s,1H),8.32(d,J＝8.0Hz,1H),8.00(s,1H),7.95(dd,J＝8.4,2.8Hz,1H),7.87(s,1H),7.76(d,J＝8.0Hz,1H),7.59(t,J＝8.0Hz,1H),7.51～7.48(m,2H),7.30(d,J＝7.6Hz,1H),7.20(d,J＝5.2Hz,1H),7.09(s,1H),2.44(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ164.9,160.9,160.4,159.4,138.2,137.6,137.2,135.9,133.7,131.4,131.3,130.8,130.0,129.9,129.6,126.0(q),124.4,123.6,122.9,120.9,118.5(q),117.9,111.6,107.4,17.7ppm.HRMS calculated for C26H17ClF3N5O(M-H)-506.0990.Found:506.1008.
实施例3、
3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)-N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methylbenzamide(化合物SCT-1003)
Figure PCTCN2017100735-appb-000042
分析结果
1H NMR(400MHz,MeOD-d4):δ8.43(d,J＝2.0Hz,1H),8.26(d,J＝2.4Hz,1H),8.19(d,J＝5.6Hz,1H),7.96(dd,J＝8.8,2.4Hz,1H),7.63(dd,J＝8.0,2.0Hz,1H),7.58(d,J＝8.8Hz,1H),7.40(d,J＝8.0Hz,1H),7.02(d,J＝5.6Hz,1H),6.99(dd,J＝2.8,1.6Hz,1H),6.95(dd,J＝3.6,1.6Hz,1H),6.25(dd,J＝3.6,2.8Hz,1H),2.41(s,3H)ppm.HRMS calculated for C23H17ClF3N5O(M-H)-:470.0990.Found:470.0997.
实施例4、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((4-phenylpyrimidin-2-yl)amino)benzami de(化合物SCT-1004)
Figure PCTCN2017100735-appb-000043
分析结果
1H NMR(400MHz,MeOD-d4):δ8.46(d,J＝6.4Hz,1H),8.36(s,1H),8.30(s,1H),8.29(d,J＝7.2Hz,2H),8.08(dd,J＝8.8,2.8Hz,1H),7.99(dd,J＝8.0,1.6Hz,1H),7.75(d,J＝6.4Hz,1H),7.71(t,J＝6.4Hz,1H),7.66～7.59(m,4H),2.48(s,3H)ppm.13C NMR(100MHz,MeOD-d4):δ172.4,167.2,156.0,150.0,140.2,139.7,135.8,134.8,133.1,132.7,130.5,129.7,129.0(q),127.9,127.1,127.0,126.2,124.3(q),120.5(q),108.7,18.4ppm.HRMS calculated for C25H18ClF3N4O(M-H)-:481.1038.Found:481.1043.
实施例5、
N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(2-methoxynaphthalen-1-yl)pyrimidin-2-yl)amino)-4-methylbenzamide(化合物SCT-1005)
Figure PCTCN2017100735-appb-000044
分析结果
1H NMR(400MHz,MeOD-d4):δ8.49(d,J＝5.2Hz,1H),8.30(s,1H),8.13(s,1H),7.95(d,J＝9.2Hz,1H),7.78(d,J＝8.0Hz,2H),7.56～7.52(m,3H),7.44(d,J＝9.2Hz,1H),7.37～7.32(m,2H),7.24(t,J＝7.6Hz,1H),6.91(d,J＝5.2Hz,1H),3.87(s,3H),2.38(s,3H)ppm.HRMS calculated  for C30H22ClF3N4O2(M-H)-:561.1300.Found:561.1311.
实施例6、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((6-phenylpyrimidin-4-yl)amino)benzamide(化合物SCT-1006)
Figure PCTCN2017100735-appb-000045
分析结果
1H NMR(400MHz,MeOD-d4):δ8.76(s,1H),8.27(d,J＝2.4Hz,1H),8.05(s,1H),7.97(dd,J＝8.8,2.4Hz,1H),7.94(dd,J＝8.0,2.0Hz,1H),7.85(d,J＝7.6Hz,2H),7.73～7.56(m,5H),7.11(s,1H),2.42(s,3H)ppm.13C NMR(100MHz,MeOD-d4):δ166.2,163.6,154.5,152.4,139.0,138.0,134.9,133.1,132.6,131.6,131.3,130.1,129.5,127.9(q),127.1,126.7,126.1,125.9,124.8,124.2,121.5,119.3(q),16.8ppm.HRMS calculated for C25H18ClF3N4O(M-H)-:481.1038.Found:481.1043.
实施例7、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((4-(thiophen-2-yl)pyrimidin-2-yl)amino)benzamide(化合物SCT-1007)
Figure PCTCN2017100735-appb-000046
分析结果
1H NMR(400MHz,MeOD-d4):δ10.7(s,1H),9.86(s,1H),8.45(d,J＝5.6 Hz,1H),8.37(d,J＝2.4Hz,1H),8.24(s,1H),8.15(dd,J＝8.8,2.4Hz,1H),8.12(d,J＝4.0Hz,1H),7.86(d,J＝4.0Hz,1H),7.82(d,J＝8.0Hz,1H),7.69(d,J＝8.0Hz,1H),7.48～7.45(m,2H),7.25(dd,J＝4.8,4.0Hz,1H),2.35(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ165.0,161.2,156.8,153.3,140.9,138.3,136.8,135.9,132.8,131.8,131.5,130.3,130.2,128.7,126.1(q),124.5,124.4,124.3,123.7,120.9,118.5(q),105.6,17.7ppm.HRMScalculated for C23H16ClF3N4OS(M-H)-:487.0602.Found:487.0609.59.
实施例8、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((4-(3-(methylthio)phenyl)pyrimidin-2-yl)amino)benzamide(化合物SCT-1008)
Figure PCTCN2017100735-appb-000047
分析结果
1H NMR(400MHz,DMSO-d6):δ10.71(s,1H),9.70(s,1H),8.56(d,J＝5.6Hz,1H),8.40(s,1H),8.34(s,1H),8.15(d,J＝8.8Hz,1H),7.97(s,1H),7.89(d,J＝6.0Hz,1H),7.81(d,J＝8.0Hz,1H),7.69(d,J＝8.8Hz,1H),7.55(d,J＝5.6Hz,1H),7.46-7.41(m,3H),2.42(s,3H),2.36(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ165.0,164.5,158.0,155.6,138.9,138.3,136.5,136.4,135.9,131.6,131.5,130.1,129.0,128.3,126.1(q),124.5,124.0,123.8,123.7,123.6,123.3,121.0,118.5(q),107.3,17.7,13.9ppm.HRMS calculated for C26H20ClF3N4OS(M-H)-:527.0915.Found:527.0922.
实施例9、
N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(4-fluoro-3-methylphenyl)pyrimidin-2-yl)amino) -4-methylbenzamide(化合物SCT-1009)
Figure PCTCN2017100735-appb-000048
分析结果
1H NMR(400MHz,DMSO-d6):δ10.69(s,1H),9.52(s,1H),8.53(d,J＝5.6Hz,1H),8.48(s,1H),8.42(d,J＝2.4Hz,1H),8.18(d,J＝6.4Hz,1H),8.12(d,J＝8.8Hz,1H),8.03～8.00(m,1H),7.78(d,J＝8.0Hz,1H),7.69(d,J＝8.8Hz,1H),7.50(d,J＝5.6Hz,1H),7.43(d,J＝8.0Hz,1H),7.24(t,J＝8.8Hz,1H),2.37(s,3H),2.26(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ166.1,164.9,164.6,162.1,159.0,156.7,139.3,137.7,136.5,132.4,131.5(d),130.9,127.6(d),126.9(q),125.6,125.4,125.3,124.6,124.4,124.1,121.9,119.3(q),116.0(d),108.0,18.6,14.5(d)ppm.HRMScalculated for C26H19ClF4N4O(M-H)-513.1100.Found:513.1106.
实施例10、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)benzamide(化合物SCT-1010)
Figure PCTCN2017100735-appb-000049
分析结果
1H NMR(400MHz,MeOD-d4):δ8.45(d,J＝6.4Hz,1H),8.40(d,J＝8.4Hz,2H),8.33(s,1H), 8.27(s,1H),8.02(d,J＝9.2Hz,1H),7.97(d,J＝8.0Hz,1H),7.73(d,J＝6.4Hz,1H),7.61(d,J＝8.4Hz,2H),7.49(d,J＝8.4Hz,2H),2.47(s,3H)ppm.13C NMR(100MHz,MeOD-d4):δ171.2,167.4,156.1,154.1,150.1,140.3,139.5,135.6,134.8,134.5,133.1,132.8,131.9,129.0(m),128.0,127.2,127.0,126.1,125.6,123.0,122.9,122.3,120.6(m),120.4,108.7,18.2ppm.HRMS calculated for C26H17ClF6N4O2(M-H)-:565.0860.Found:565.0864.
实施例11、
N-(4-chloro-3-(trifluoromethyl)phenyl)-4-methyl-3-((4-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)benzamide(化合物SCT-1011)
Figure PCTCN2017100735-appb-000050
分析结果
1H NMR(400MHz,MeOD-d4):δ8.45～8.41(m,3H),8.27(d,J＝8.4Hz,2H),7.97(d,J＝9.2Hz,1H),7.92(d,J＝7.6Hz,1H),7.87(d,J＝8.4Hz,2H),7.70(d,J＝6.4Hz,1H),7.59(t,J＝7.6Hz,2H),2.46(s,3H)ppm.HRMS calculated for C26H17ClF6N4O(M-H)-:549.0911.Found:549.0916.
实施例12、
N-(4-chloro-3-(trifluoromethyl)phenyl)-3-((4-(3,5-dichlorophenyl)pyrimidin-2-yl)amino)-4-methylbenzamide(化合物SCT-1012)
Figure PCTCN2017100735-appb-000051
分析结果
1H NMR(400MHz,DMSO-d6):δ10.64(s,1H),9.33(s,1H),8.56(d,J＝5.2Hz,1H),8.41(s,1H),8.39(s,1H),8.16(s,2H),8.11(d,J＝8.8Hz,1H),7.76～7.74(m,2H),7.69(d,J＝8.8Hz,1H),7.55(d,J＝5.2Hz,1H),7.41(d,J＝8.0Hz,1H),2.35(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ165.0,160.6,159.7,158.7,139.4,138.4,137.2,135.7,134.3,131.4,131.3,129.9,129.8,126.0(q),125.2,124.5,123.6,123.1,122.3,121.0,118.5(q),107.7,17.7ppm.HRMS calculated for C25H16Cl3F3N4O(M-H)-:549.0258.Found:549.0270.
实施例13、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(4-cyanophenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1013)
Figure PCTCN2017100735-appb-000052
分析结果
1H NMR(400MHz,DMSO-d6):δ9.76(s,1H),9.57(s,1H),9.08(s,1H),8.62(d,J＝5.2Hz,1H),8.44(d,J＝8.0Hz,2H),8.21(s,1H),8.18(s,1H),7.96(d,J＝8.0Hz,2H),7.60(s,2H),7.51(d,J＝5.2Hz,1H),7.32(d,J＝8.0Hz,1H),7.20(t,J＝8.0Hz,1H),7.04(d,J＝6.8Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ161.2,159.6,159.1,151.9,140.3,139.1,139.0,132.3,131.5,128.3,127.4,126.2(q),123.7,122.3,121.6,121.0,118.0,115.9(q),112.9,112.6,111.5,108.6,108.0ppm.HRMS calculated for C25H16ClF3N6O(M-H)-:507.0942.Found:507.0955.
实施例14、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(3-cyanophenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1014)
Figure PCTCN2017100735-appb-000053
分析结果
1H NMR(400MHz,MeOD-d4):δ8.66(s,1H),8.59(d,J＝8.8Hz,1H),8.47(d,J＝6.4Hz,1H),8.19(s,1H),8.04(d,J＝2.4Hz,1H),7.96(d,J＝8.0Hz,1H),7.75(t,J＝8.0Hz,1H),7.64(d,J＝6.0Hz,1H),7.61(dd,J＝8.8,2.4Hz,1H),7.49(d,J＝8.4Hz,1H),7.39(t,J＝8.0Hz,1H),7.22(d,J＝8.0Hz,1H),7.15(d,J＝8.0Hz,1H)ppm.HRMS calculated for C25H16ClF3N6O(M-H)-:507.0942.Found:507.0954.
实施例15、
1-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea(化合物SCT-1015)
Figure PCTCN2017100735-appb-000054
分析结果
1H NMR(400MHz,DMSO-d6):δ11.75(s,1H),10.55(s,1H),9.91(s,1H),9.61(s,1H),8.45(s,1H),8.35(d,J＝6.4Hz,1H),8.08(s,1H),7.64(s,2H),7.38(s,1H),7.34(d,J＝6.4Hz,1H),7.28(t,J＝8.0Hz,1H),7.22(s,1H),7.12(d,J＝8.0Hz,1H),7.03(d,J＝7.6Hz,1H),6.36(s,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.5,153.1,152.3,148.3,139.2,138.8,137.8,131.6,128.8,127.9,126.8,126.3(q),122.5,122.3(q),121.9,116.7,116.1(q),113.7,113.1,111.4,109.8, 104.7ppm.HRMS calculated for C22H16ClF3N6O(M-H)-:471.0942.Found:471.0957.
实施例16、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-phenylpyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1016)
Figure PCTCN2017100735-appb-000055
分析结果
1H NMR(400MHz,DMSO-d6):δ10.00(s,1H),9.85(s,1H),9.31(s,1H),8.56(d,J＝5.6Hz,1H),8.28～8.26(m,2H),8.17(s,1H),8.14(s,1H),7.62(s,2H),7.54～7.53(m,3H),7.49(d,J＝5.6Hz,1H),7.37(d,J＝8.0Hz,1H),7.24(t,J＝8.0Hz,1H),7.08(d,J＝8.0Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ164.5,158.1,156.4,152.0,139.7,139.2,139.1,135.6,131.6,130.9,128.5,128.4,127.0,126.3(q),122.4(q),122.2,121.5,115.8(q),113.3,112.0,109.2,107.3ppm.HRMS calculated for C24H17ClF3N5O(M-H)-:482.0990.Found:482.1002.
实施例17、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(thiophen-2-yl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1017)
Figure PCTCN2017100735-appb-000056
分析结果
1H NMR(400MHz,MeOD-d4):δ8.23(d,J＝6.4Hz,1H),8.18(d,J＝4.0Hz,1H),8.03(d,J＝2.4 Hz,1H),7.94(d,J＝4.0Hz,1H),7.91(s,1H),7.63(dd,J＝8.4,2.4Hz,1H),7.51(d,J＝7.6Hz,1H),7.50(d,J＝6.8Hz,1H),7.43(t,J＝8.4Hz,1H),7.31～7.24(m,3H)ppm.13C NMR(100MHz,DMSO-d6):δ160.3,156.9,154.7,152.0,141.2,139.1,139.1,132.1,131.5,129.7,128.5,128.5,126.2(q),122.2,121.6,121.0(q),115.8(q),113.7,112.7,109.8,105.8ppm.HRMS calculated for C22H15ClF3N5OS(M-H)-:488.0554.Found:488.0568.
实施例18、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(3-(methylthio)phenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1018)
Figure PCTCN2017100735-appb-000057
分析结果
1H NMR(400MHz,MeOD-d4):δ8.37(d,J＝6.8Hz,1H),8.18(s,1H),8.05～8.04(m,3H),7.67(d,J＝6.4Hz,1H),7.62(dd,J＝8.8,2.4Hz,1H),7.55～7.48(m,3H),7.44(t,J＝8.0Hz,1H),7.24(t,J＝8.0Hz,2H),2.53(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ163.5,158.5,157.1,152.0,139.8,139.1,138.7,136.4,131.5,129.0,128.3,128.0,126.4(q),123.6,123.3,122.3(q),122.2,121.5,115.9(q),113.3,112.0,109.3,107.5,14.0ppm.HRMS calculated for C25H19ClF3N5OS(M-H)-:528.0867.Found:528.0878.
实施例19、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(4-fluoro-3-methylphenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1019)
Figure PCTCN2017100735-appb-000058
分析结果
1H NMR(400MHz,DMSO-d6):δ9.90(s,1H),9.75(s,1H),9.25(s,1H),8.54(d,J＝5.6Hz,1H),8.19～8.13(m,4H),7.60(s,1H),7.59(s,1H),7.45(d,J＝5.6Hz,1H),7.32～7.20(m,3H),7.04(d,J＝8.0Hz,1H),2.27(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ163.4,163.3,161.0,158.3,156.8,152.0,139.8,139.1,131.8(d),131.5,130.4(d),128.3,126.8(d),126.2(q),124.4(d),122.3(q),122.2,121.6,115.9(q),115.0(d),113.2,111.9,109.2,107.1,13.7(d)ppm.HRMS calculated for C25H18ClF4N5O(M-H)-:514.1052.Found:514.1062.
实施例20、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(4-(trifluoromethoxy)phenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1020)
Figure PCTCN2017100735-appb-000059
分析结果
1H NMR(400MHz,DMSO-d6):δ9.83(s,1H),9.72(s,1H),9.20(s,1H),8.58(d,J＝5.6Hz,1H),8.41(d,J＝9.2Hz,2H),8.30(s,1H),8.19(s,1H),7.60(s,1H),7.59(s,1H),7.46(d,J＝9.2Hz,2H),7.46(s,1H),7.29(d,J＝8.4Hz,1H),7.21(t,J＝8.0Hz,1H),7.01(d,J＝8.0Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ162.1,159.1,158.2,152.0,149.7,140.2,139.1,139.0,135.0,131.5,129.0,128.3,126.3(q),122.3,121.6,121.0(q),120.8,120.6,115.8(q),113.0,111.6,108.8,107.4ppm.HRMS calculated for C25H16ClF6N5O2(M-H)-:566.0813.Found:566.0826.
实施例21、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(4-(trifluoromethyl)phenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1021)
Figure PCTCN2017100735-appb-000060
分析结果
1H NMR(400MHz,DMSO-d6):δ9.77(s,1H),9.22(s,1H),8.88(s,1H),8.62(d,J＝4.8Hz,1H),8.47(d,J＝8.0Hz,2H),8.31(s,1H),8.18(s,1H),7.82(d,J＝8.0Hz,2H),7.60(s,2H),7.50(d,J＝4.8Hz,1H),7.32(d,J＝8.0Hz,1H),7.20(t,J＝8.0Hz,1H),6.99(d,J＝8.0Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ161.5,159.7,159.1,151.9,140.4,140.0,138.9(d),131.40,130.2(q),128.2,127.4,126.2(q),125.1(d),124.8,124.2(q),122.5,121.7,121.5(q),116.1(q),112.9,111.6,108.8,107.9ppm.HRMS calculated for C25H16ClF6N5O(M-H)-:550.0864.Found:550.0874.
实施例22、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((6-phenylpyrimidin-4-yl)amino)phenyl)urea(化合物SCT-1022)
Figure PCTCN2017100735-appb-000061
分析结果
1H NMR(400MHz,DMSO-d6):δ11.30(s,1H),9.93(s,1H),9.59(s,1H),8.91(s,1H),8.12(s,1H),7.92(d,J＝7.2Hz,2H),7.87(s,1H),7.67～7.58(m,5H),7.41(d,J＝8.0Hz,1H),7.36(s,1H),7.33(t,J＝8.0Hz,1H),7.23(d,J＝8.0Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ161.1,153.4,153.2,152.1,139.5,138.9,137.2,131.7,131.5,130.5,129.0,128.9,126.7,126.1(q),123.7,122.2,121.7,121.0(q),115.9(q),115.3,114.8,111.2,102.9,102.6ppm.HRMS calculated for C24H17ClF3N5O(M-H)-:482.0990.Found:482.1001.
实施例23、
1-(4-chloro-3-(trifluoromethyl)phenyl)-3-(3-((4-(3,5-dichlorophenyl)pyrimidin-2-yl)amino)phenyl)urea(化合物SCT-1023)
Figure PCTCN2017100735-appb-000062
分析结果
1H NMR(400MHz,MeOD-d4):δ8.44(d,J＝6.4Hz,1H),8.22(d,J＝2.0Hz,2H),8.13(s,1H),8.02(d,J＝2.8Hz,1H),7.68(t,J＝2.0Hz,1H),7.61(dd,J＝8.8,2.8Hz,1H),7.58(d,J＝6.4Hz,1H),7.49(d,J＝8.8Hz,1H),7.38(t,J＝8.0Hz,1H),7.22(d,J＝8.0Hz,1H),7.15(d,J＝8.0Hz,1H) ppm.HRMS calculated for C24H15Cl3F3N5O(M-H)-:550.0211.Found:550.0228.
实施例24、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(3-(trifluoromethyl)phenyl)urea(化合物SCT-1029)
Figure PCTCN2017100735-appb-000063
分析结果
1H NMR(400MHz,DMSO-d6):δ11.74(s,1H),10.47(s,1H),9.72(s,1H),9.53(s,1H),8.56(s,1H),8.36(d,J＝6.4Hz,1H),8.02(s,1H),7.60(d,J＝8.0Hz,1H),7.55(t,J＝8.0Hz,1H),7.38(s,1H),7.34(s,1H),7.32(s,1H),7.28(t,J＝8.0Hz,1H),7.20(s,1H),7.10(d,J＝8.0Hz,1H),7.00(d,J＝8.0Hz,1H),6.36(m,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.2,153.6,152.5,149.1,140.0,139.3,138.0,129.5,128.9(q),128.7,128.0,126.3,123.7(q),121.5,117.8(d),116.3,113.6(q),113.5(d),112.9,111.3,109.6,104.8ppm.HRMS calculated for C22H17F3N6O(M+H)-:439.1489.Found:439.1479.
实施例25、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-(trifluoromethoxy)phenyl)urea(化合物SCT-1030)
Figure PCTCN2017100735-appb-000064
分析结果
1H NMR(400MHz,DMSO-d6):δ11.76(s,1H),10.55(s,1H),9.54(s,1H),9.50(s,1H),8.58(s,1H),8.36(d,J＝6.4Hz,1H),7.58(d,J＝9.2Hz,2H),7.39～7.23(m,6H),7.07(d,J＝8.0Hz,1H),7.01(d,J＝8.0Hz,1H),6.38(m,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.5,153.2,152.6,148.3,142.3,139.5,138.4,137.8,128.7,128.0,126.9,121.3,120.0(q),119.2,116.8,113.4,112.9,111.6,109.7,104.8ppm.HRMS calculated for C22H17F3N6O2(M+H)-:455.1438.Found:455.1429.
实施例26、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(3-chlorophenyl)urea(化合物SCT-1031)
Figure PCTCN2017100735-appb-000065
分析结果
1H NMR(400MHz,MeOD-d4):δ8.92(s,1H),8.12(d,J＝6.8Hz,1H),7.81(s,1H),7.41(d,J＝4.0Hz,1H),7.39～7.31(m,4H),7.27(d,J＝8.0Hz,1H),7.09(d,J＝8.0Hz,1H),6.96(d,J＝8.0Hz,1H),6.92(d,J＝8.0Hz,1H),6.48(t,J＝4.0Hz,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.2,153.0,152.2,148.2,140.4,139.2,137.7,132.5,129.7,128.5,127.8,126.4,120.9,116.9,116.5,116.1,113.2,112.6,112.3,109.4,104.6ppm.HRMS calculated for C21H17ClN6O(M+H)-:405.1225.Found:405.1217.
实施例27、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(3,5-dichlorophenyl)Urea(化合物SCT-1032)
Figure PCTCN2017100735-appb-000066
分析结果
1H NMR(400MHz,DMSO-d6):δ11.67(s,1H),10.34(s,1H),9.77(s,1H),9.50(s,1H),8.62(s,1H),8.34(d,J＝6.4Hz,1H),7.56(d,J＝6.4Hz,2H),7.36(s,1H),7.30(d,J＝6.4Hz,1H),7.26(s,1H),7.25(d,J＝8.0Hz,1H),7.17(s,1H),7.07(d,J＝8.0Hz,1H),6.94(d,J＝8.0Hz,1H),6.38(s,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.0,154.1,152.2,149.7,141.7,139.0,138.2,133.7,128.7,128.1,125.9,120.6,115.9,115.8,113.6,112.8,111.2,109.6,104.8ppm.HRMS calculated for C21H16Cl2N6O(M+H)-:439.0835.Found:439.0827.
实施例28、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(3-ethylphenyl)urea(化合物SCT-1033)
Figure PCTCN2017100735-appb-000067
分析结果
1H NMR(400MHz,DMSO-d6):δ11.82(s,1H),10.54(s,1H),9.45(s,1H),9.24(s,1H),8.70(s,1H),8.36(d,J＝6.4Hz,1H),7.40(s,1H),7.38～7.20(m,6H),7.04(d,J＝8.0Hz,1H),6.94(d,J＝8.0Hz,1H),6.86(d,J＝7.6Hz,1H),6.39～6.37(m,1H),2.58(q,J＝7.6Hz,2H),1.17(t,J＝7.6Hz,3H) ppm.13C NMR(100MHz,DMSO-d6):δ159.3,153.4,152.6,148.7,143.9,139.7,139.0,137.9,128.6,128.2,128.0,126.5,121.1,117.4,116.5,115.5,113.0,112.6,111.4,109.4,104.7,27.8,15.1ppm.HRMS calculated for C23H22N6O(M+H)-:399.1928.Found:399.1919.
实施例29、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(2-fluoro-5-methylphenyl)urea(化合物SCT-1034)
Figure PCTCN2017100735-appb-000068
分析结果
1H NMR(400MHz,DMSO):δ11.74(s,1H),10.35(s,1H),9.57(s,1H),8.75(s,1H),8.71(s,1H),8.37(d,J＝5.6Hz,1H),7.94(d,J＝7.6Hz,1H),7.31～7.24(m,3H),7.15～7.07(m,3H),6.91(d,J＝7.6Hz,1H),6.85(s,1H),6.36(s,1H),2.29(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ158.7,154.5,152.1,151.4,150.4,149.0,139.3,138.5,132.8(d),128.5,128.1,126.2(d),125.4,122.7(d),121.1,115.3,114.2(d),113.0,112.1,111.0,109.1,104.8,20.1ppm.HRMS calculated for C22H19FN6O(M+H)-:403.1677.Found:403.1669.
实施例30、
1-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)-3-(3,5-bis(trifluoromethyl)phenyl)urea(化合物SCT-1035)
Figure PCTCN2017100735-appb-000069
分析结果
1H NMR(400MHz,DMSO-d6):δ11.75(s,1H),10.57(s,1H),10.26(s,1H),9.72(s,1H),8.46(s,1H),8.37(d,J＝6.4Hz,1H),8.12(s,2H),7.64(s,1H),7.42(s,1H),7.35(d,J＝6.4Hz,1H),7.31(t,J＝8.0Hz,1H),7.23(s,1H),7.15(d,J＝8.0Hz,1H),7.06(d,J＝8.0Hz,1H),6.37～6.35(m,1H)ppm.13C NMR(100MHz,DMSO-d6):δ159.5,153.0,152.2,148.2,141.3,139.0,137.7,130.3(q),128.8,127.9,126.8,122.8(q),117.2,116.8,114.0,113.9,113.3,111.3,109.9,104.7ppm.
实施例31、
1-(4-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)-3-(4-chloro-3-(trifluoromethyl)phenyl)urea(化合物SCT-1036)
Figure PCTCN2017100735-appb-000070
分析结果
1H NMR(400MHz,DMSO-d6):δ11.42(s,1H),9.27(s,1H),9.08(s,1H),8.66(s,1H),8.32(d,J＝5.2Hz,1H),8.11(s,1H),7.74(d,J＝8.8Hz,2H),7.64～7.58(m,2H),7.37(d,J＝8.8Hz,2H),7.06～7.04(m,2H),6.95(s,1H),6.22(s,1H)ppm.HRMS calculated for C22H16ClF3N6O(M+H)-:473.1099.Found:473.1092.
实施例32、Methyl
3-(3-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)ureido)benzo-ate(化合物SCT-1037)
Figure PCTCN2017100735-appb-000071
分析结果
1H NMR(400MHz,DMSO-d6):δ11.72(s,1H),10.49(s,1H),9.60(s,1H),9.51(s,1H),8.65(s,1H),8.35(d,J＝6.4Hz,1H),8.24(s,1H),7.63(d,J＝8.0Hz,1H),7.59(d,J＝7.6Hz,1H),7.47(t,J＝7.6Hz,1H),7.38(s,1H),7.33(d,J＝6.4Hz,1H),7.30(s,1H),7.27(d,J＝8.0Hz,1H),7.06(d,J＝8.0Hz,1H),6.96(d,J＝8.0Hz,1H),6.40～6.38(m,1H),3.84(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ165.7,159.2,153.6,152.5,149.0,140.0,139.4,138.0,130.0,128.8,128.7,127.9,126.6,122.4,122.3,118.0,116.4,113.3,112.7,111.4,109.5,104.8,51.7ppm.HRMS calculated for C23H20N6O3(M+H)-:429.1670.Found:429.1663.
实施例33、Methyl
5-(3-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)ureido)-2-methylbenzoate(化合物SCT-1039)
Figure PCTCN2017100735-appb-000072
分析结果
1H NMR(400MHz,DMSO-d6):δ11.67(s,1H),10.25(s,1H),9.32(s,1H),9.30(s,1H),8.70(s,1H),8.34(d,J＝5.6Hz,1H),8.06(s,1H),7.49(d,J＝8.4Hz,1H),7.29～7.23(m,4H),7.19(s,1H),7.04(d,J＝8.0Hz,1H),6.90(d,J＝8.0Hz,1H),6.36(s,1H),3.81(s,3H),2.45(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ166.8,158.7,154.6,152.5,150.6,139.4,138.5,137.0,131.9,131.5,129.1,128.5,128.1,125.6,121.6,119.1,115.3,113.0,112.3,111.1,109.3,104.8,51.4,19.9ppm.HRMS calculated for C24H22N6O3(M+H)-:443.1826.Found:443.1822.
实施例34、
Methyl5-(3-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)ureido)-2-chlorobenzoate(化合物SCT-1041)
Figure PCTCN2017100735-appb-000073
分析结果
1H NMR(400MHz,MeOD-d4):δ8.95(s,1H),8.33(s,1H),8.15(d,J＝6.8Hz,1H),7.75(d,J＝7.6Hz,1H),7.64(d,J＝8.4Hz,1H),7.50(t,J＝7.6Hz,1H),7.44～7.41(m,2H),7.38(t,J＝8.0Hz,1H),7.34(d,J＝7.6Hz,1H),6.98～6.92(m,2H),6.49～6.47(m,1H),3.93(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ165.7,159.2,153.5,152.5,149.0,139.5,139.4,138.0,129.7,128.8,128.7,127.9,126.6,122.4,122.3,118.0,116.4,113.3,112.7,111.4,109.5,104.8,51.7ppm.HRMS calculated for C23H19ClN6O3(M+H)-:463.1280.Found:463.1280.
实施例35、
3-(3-(3-((4-(1H-pyrrol-2-yl)pyrimidin-2-yl)amino)phenyl)ureido)benzoic acid(化合物SCT-1038)
Figure PCTCN2017100735-appb-000074
分析结果
1H NMR(400MHz,DMSO-d6):δ11.36(s,1H),9.44(s,1H),9.03(s,1H),8.85(s,2H),8.33(d,J＝5.2Hz,1H),8.19(s,1H),7.63～7.59(m,2H),7.44(t,J＝8.0Hz,1H),7.16(t,J＝8.0Hz,1H), 7.10～7.06(m,3H),7.02(s,1H),6.76(d,J＝8.0Hz,1H),6.22～6.21(m,1H)ppm.HRMS calculated for C22H18N6O3(M+H)-:415.1513.Found:415.1507.
实施例36、
5-(3-(3-((4-(1H-pyrrol-3-yl)pyrimidin-2-yl)amino)phenyl)ureido)-2-methylbenzoic acid(化合物SCT-1040)
Figure PCTCN2017100735-appb-000075
分析结果
1H NMR(400MHz,DMSO-d6):δ12.89(s,1H),11.39(s,1H),9.48(s,1H),8.86(s,2H),8.76(s,1H),8.33(d,J＝5.2Hz,1H),8.01(s,1H),7.51(dd,J＝8.4,2.4Hz,1H),7.25(d,J＝8.4Hz,1H),7.16(t,J＝8.0Hz,1H),7.10～7.02(m,4H),6.74(d,J＝8.0Hz,1H),6.24～6.22(m,1H),2.47(s,3H)ppm.13C NMR(100MHz,DMSO-d6):δ168.2,159.3,157.4,156.3,152.4,140.8,139.1,136.8,131.9,131.4,130.3,128.8,128.1,122.2,121.4,119.6,112.1,111.0,110.4,109.7,108.0,105.1,20.1ppm.
实施例37
Figure PCTCN2017100735-appb-000076
实施例38
Figure PCTCN2017100735-appb-000077
实施例39
Figure PCTCN2017100735-appb-000078
实施例40
Figure PCTCN2017100735-appb-000079
实施例41
Figure PCTCN2017100735-appb-000080
实施例42
Figure PCTCN2017100735-appb-000081
实施例43
Figure PCTCN2017100735-appb-000082
实施例44
Figure PCTCN2017100735-appb-000083
实施例45
Figure PCTCN2017100735-appb-000084
实施例46
Figure PCTCN2017100735-appb-000085
实施例47
Figure PCTCN2017100735-appb-000086
实施例48
Figure PCTCN2017100735-appb-000087
实施例49
Figure PCTCN2017100735-appb-000088
实施例50
Figure PCTCN2017100735-appb-000089
实施例51
Figure PCTCN2017100735-appb-000090
实施例52
Figure PCTCN2017100735-appb-000091
实施例53
Figure PCTCN2017100735-appb-000092
实施例54
Figure PCTCN2017100735-appb-000093
实施例55
Figure PCTCN2017100735-appb-000094
实施例56
Figure PCTCN2017100735-appb-000095
实施例57
Figure PCTCN2017100735-appb-000096
实施例58
Figure PCTCN2017100735-appb-000097
实施例59
Figure PCTCN2017100735-appb-000098
实施例60
Figure PCTCN2017100735-appb-000099
实施例61
Figure PCTCN2017100735-appb-000100
实施例62
Figure PCTCN2017100735-appb-000101
实施例63
Figure PCTCN2017100735-appb-000102
实施例64
Figure PCTCN2017100735-appb-000103
实施例65
Figure PCTCN2017100735-appb-000104
实施例66
Figure PCTCN2017100735-appb-000105
实施例67
Figure PCTCN2017100735-appb-000106
实施例68
Figure PCTCN2017100735-appb-000107
实施例69
Figure PCTCN2017100735-appb-000108
实施例70
Figure PCTCN2017100735-appb-000109
实施例71
Figure PCTCN2017100735-appb-000110
实施例72
Figure PCTCN2017100735-appb-000111
生物活性测试
为确认本发明之化合物之药理特性及对生物之影响性，故分别分析其对腺苷单磷酸活化蛋白激酶活性之影响，以及其对癌症细胞存活率之影响，以下，将依序说明本发明之化合物之测 试结果数据。
细胞株
肝癌细胞株Huh-7是取自健康科学研究资源银行(Health Science Research Resources Bank；HSRRB,Osaka,Japan；JCRB0403)；肝癌细胞株PLC/PRF/5(PLC5)、Sk-Hep-1及HCC1806是取自美国典型培养物保存中心(ATCC,Manassas,VA)；所有细胞株自取得后立即放大培养并冻存，因此每3个月即可以解冻冻存之细胞而得以以同一批细胞进行实验。
AMPK激酶活性测试(AMPK kinase enzyme assay)
自肝癌细胞株PLC5取得蛋白质萃取物后，将萃取物及抗AMPKα1之抗体(anti-AMPKa1antibody)共同培养于免疫共沉淀缓冲溶液(G-Biosciences)中隔夜培养，接着，蛋白磁珠(Protein A/G Magnetic Bead；PureProteomeTM)分别加入各样品中并于4℃下旋转培养4小时；于重组蛋白AMPK之分析中，是将不同剂量之化合物与12.5奈克(ng)之重组蛋白人类AMPK(α1,β1,γ1)共同培养，而后，是依据用以侦测AMPK活性之分析法AMPK SAMS peptide assay(Cyclex)之使用手册执行分析。
结果如图1A，在加入20-20,000奈体积摩尔浓度(nM)之化合物SCT-1015至AMPKα1免疫共沉淀之蛋白萃取物后测试AMPK活性，相较于未加入化合物之组别，AMPK之活性确实明显增加；又如图1B，另将20-2,000奈体积摩尔浓度之化合物SCT-1015与重组蛋白AMPKα1/β1/γ1共同培养，相较于未加入化合物之组别，重组蛋白AMPKα1/β1/γ1之活性亦明显增加；由图1A及1B显示SCT-1015化合物可增加肝癌细胞中AMPK之活性。
以酵素连结免疫分析法(ELISA assay)侦测AMPK磷酸化活性
于此分析中是使用ThermoFisher Science所贩卖之商业化套组AMPK[pT172]Phospho-ELISA Kit(KHO0651)，肝癌细胞株PLC5是与10微体积摩尔浓度(μM)之化合物衍生物共同培养24小时，并依据其所揭示之使用手册执行分析，并以波长450纳米(nm)侦测吸光值。
结果如表3所示，第一栏为化合物编号，第三栏为化合物活化AMPK之测试结果，控制组是仅添加化合物衍生物之溶剂DMSO，数值则以3组样品(N＝3)之平均值±标准偏差显示；由结果可以得知，化合物衍生物确实能够于T172的位置磷酸化并活化AMPK，其中，又以化合物SCT1015之活化效果(1.8)为最佳。
表3.AMPK活性测试结果
Figure PCTCN2017100735-appb-000112
以免疫墨点分析法(Immuno-blot assay)侦测AMPK之磷酸化
分别收取与化合物SCT-1015共同培养之肝癌细胞株PCL5及三阴性乳癌细胞株(TNBC)MDA-MB-231及MDA-MB-453，于清洗后以RIPA缓冲液裂解细胞以获得蛋白萃取物，接着以Bio-Rad Protein Assay dye reagent(Bio-Rad)量测蛋白浓度，再以2倍SDS-loading buffer(100mM Tris HCl,pH 6.8,200mM β-mercaptoethanol,4％SDS,0.02％bromophenol blue,and 20％glycerol)调整蛋白样本体积，而后将蛋白样本以10％SDS polyacrylamide gels进行电泳分离后转渍到PVDF膜上，接着加入1级抗体至膜上以侦测目标蛋白，再加入带有辣根过氧化物酶(horseradish peroxidase)之二级抗体以结合1级抗体，接着加入辣根过氧化物酶之反应基质(enhanced SuperSignal West Pico Cheminluminescent Substrate；Pierce)进行反应以获得特定蛋白之讯号，蛋白含量是以β-actin作为内控制组以进行分析；结果如图2A-2C所示，于肝癌细胞株PCL5及三阴性乳癌细胞株(TNBC)MDA-MB-231及MDA-MB-453中，添加化合物SCT-1015相较于未添加之组别，其磷酸化AMPKα(p-AMPKα)之蛋白表现量均显着增加，又如图2D所示，在三株肝癌细胞株PLC/PRF/5、SK-hep1及Huh-7中，经磷酸化活化之AMPK是进一步调控下游讯息传递分子HIF1及PDHK1；由上述结果可以得知，本申请之化合物SCT-1015确实能够磷酸化活化AMPKα，并得以进一步调控下游讯息传递路径，进而影响细胞内相关的生化反应。
肝癌細胞株之细胞存活率测试(cytotoxicity effect)
此分析是以Thermo Fisher Scientific所贩卖之商业化套组Prestoblue assay进行分析，三株肝癌细胞株PLC/PRF/5、SK-hep1及Huh-7是分别与化合物SCT-1015共同培养48小时及72小时，依据商业化套组之使用手册执行分析；结果如图3所示，相较于控制组DMSO，三株肝癌细胞株在分别添加5-20微体积摩尔浓度(μM)之化合物SCT-1015培养之后，确实抑制细胞增生；由此可知，本发明之化合物确实能够通过活化AMPK而抑制肝癌细胞之生长***。
三阴性乳癌细胞之细胞存活率测试
实验是以带有荧光素酶(luc2)基因之三阴性乳癌细胞HCC1806(HCC1806/luc2)建立原位性三阴性乳癌小鼠模式(HCC1806/luc2-bearing orthotopic mice)，并将实验小鼠分为两个组别，分别为(1)控制组(vehicle)：口服溶剂；(2)实验组：(SCT-1015)：口服化合物SCT-1015(20毫克/公斤)，分别将前述之样品每天喂食小鼠并持续14天，且于第0天及第14天时，分别注射荧光素酶之反应基质至小鼠复腔中，再以活体影像***(In vivo imaging system；IVIS)侦测HCC1806/luc2细胞所发出的冷光讯号，以观察细胞生长情形。
结果如图4所示，第一行为第0天拍摄之影像，第二行则为第14天拍摄之影像；相较于实验组(SCT-1015)于第0天拍摄之小鼠影像，喂食化合物SCT-1015之小鼠，其带有的HCC1806/luc2细胞数量并未显著增加，且相较于控制组于第14天拍摄之小鼠影像，喂食化合物SCT-1015之小鼠，其HCC1806/luc2细胞数量显著减少；由此实验结果可以得知，本发明之化合物可以透过活化AMPK以抑制三阴性乳癌细胞之生长。
脂肪细胞之细胞生长情形测试
实验是依据脂肪细胞分化标准流程，将人类脂肪前驱细胞(humanpre-adipocytes)处理分化试剂(Gibco StemPro adipogenesis differentiation reagent)并培养14天，以诱导其分化为成熟脂肪细胞(mature adipocytes)，接着，将成熟脂肪细胞分别处理不同浓度之化合物SCT-1015(100μM、25μM、12.5μM、5μM及2.5μM)或仅处理溶剂(控制组Mock)或未处理任何样品(未处理组non-treated)并培养48小时，接着再以染剂adipoRed(Lonza inc.)进行脂肪细胞呈色，并以激发光波长485nm及散色光波长572nm量测脂肪细胞之荧光讯号以计算细胞数量；此外，在细胞处理2.5μM之化合物SCT-1015，亦透过拍摄细胞影像以观察细胞型态。
结果如图5A所示，相较于控制组(Mock)，成熟脂肪细胞在处理化合物SCT-1015后细胞数量显著降低，且细胞减少的数量随着化合物SCT-1015浓度增加而渐增； 又如图5B所示，细胞在处理2.5μM之化合物SCT-1015之下，成熟脂肪细胞(标记*处)呈现皱缩且死亡的型态，反之，未分化之细胞(标记**处)则仍维持健康的细胞型态；由上述实验结果可以得知，本发明之化合物能够诱发脂肪细胞之细胞死亡，并且对未分化之细胞不具毒杀性。
以本发明之化合物处理高脂饮食肥胖症小鼠模式(diet-induced obesity(DIO)mouse models)
雄性实验小鼠C57BL/6是购自国家实验研究院国家实验动物中心(National Laboratory Animal Center；NLAC,Taipei,Taiwan)，并连续4周喂养小鼠高脂肪饮食以诱发肥胖症(diet-induced obesity；DIO)，而后取得所建立之高脂饮食肥胖症小鼠进行后续实验；高脂饮食肥胖症小鼠系分为三个组别，一组5只小鼠，分别为(1)控制组(vehicle)：混合溶剂(N-甲基吡咯酮：聚氧乙烯蓖麻油：生理食盐水＝1：2：7(NMP:Cre:Saline＝1:2:7)；(2)标准品(orlistat)：溶于混合溶剂(NMP:Cre:Saline＝1:2:7)之orlistat(50毫克/公斤)及(3)实验组(SCT-1015)：溶于混合溶剂(NMP:Cre:Saline＝1:2:7)之化合物SCT-1015(50毫克/公斤)，分别将前述之样品以口服处理小鼠，每天处理一次并持续32天，记录小鼠之体重及饮食状况。
结果如图6所示，相较于控制组之小鼠，处理化合物SCT-1015之小鼠其体重平均减少5％，且与处理标准品orlistat之小鼠所减少的体重相仿，由此可知，本发明之化合物可透过影响AMPK之活性，进而诱导脂肪细胞之细胞凋亡，而得以减少高脂饮食肥胖症小鼠之体重。
综合上述，本发明所揭示之化合物，是具有新颖之化学结构，并可作为腺苷单磷酸活化蛋白激酶之活化剂；通过前述之实验结果证实，本发明之化合物能够通过结合至腺苷单磷酸活化蛋白激酶α次单元(AMPKα)，进而磷酸化活化AMPKα，而能够进一步调控下游讯息传递分子，并且得以抑制肝癌细胞及乳癌细胞之生长***，此外，亦可诱发脂肪细胞之细胞死亡，故本发明所提供之化合物更可用于制备医药组成物，而该医药组成物是用于 由腺苷单磷酸活化蛋白激酶所介导之癌症或脂肪代谢相关疾病或症候群，另一方面，亦可将本发明所提供之化合物应用于疾病治疗上，而该疾病则是由腺苷单磷酸活化蛋白激酶所介导之癌症或脂肪代谢相关疾病或症候群；故，本发明提供一具医疗潜力之新颖化合物，其相较习知技术具有较佳的活化AMPK效力及较佳之治疗专一性。

Claims (19)

  1. 一种如结构式I所示之化合物，
    Figure PCTCN2017100735-appb-100001
    或其医药上可接受之盐；其中，
    R1是一未经取代或一经取代之芳香环基(aromatic group)，R3是一经取代之苯胺基(phenyl amide group)或苯基脲(phenyl urea group)且Ar是一未经取代或一经取代之伸苯基(phenylene group)。
  2. 如权利要求1所述的化合物，其中该未经取代或该经取代之伸苯基是
    Figure PCTCN2017100735-appb-100002
    Figure PCTCN2017100735-appb-100003
    且R2是一氢、一卤化物(halide)或一烷基(alkyl group)。
  3. 如权利要求1所述的化合物，其中，R1是一未经取代或一经取代之苯基或一经取代之
    Figure PCTCN2017100735-appb-100004
  4. 如权利要求1所述的化合物，其中，R1是一未经取代之吡咯基(pyrrolic group)、一经取代之吡咯基或一经取代之
    Figure PCTCN2017100735-appb-100005
  5. 如权利要求1所述的化合物，其中，R1是一未经取代之噻吩基(thiophene group)、一经取代之噻吩基或一经取代之
    Figure PCTCN2017100735-appb-100006
  6. 如权利要求1所述的化合物，其中，R1是一未经取代之萘基(naphthalenic group)、一经取 代之萘基或一经取代之
    Figure PCTCN2017100735-appb-100007
  7. 如权利要求1所述的化合物，其中，R1是一双取代苯基、一经取代之
    Figure PCTCN2017100735-appb-100008
    Figure PCTCN2017100735-appb-100009
  8. 如权利要求1所述的化合物，其中，R3之该经取代之苯胺基是一经取代之
    Figure PCTCN2017100735-appb-100010
  9. 如权利要求1所述的化合物，其中，R3之该经取代之苯基脲是一经取代之
    Figure PCTCN2017100735-appb-100011
  10. 如权利要求2所述的化合物，其中，R2之该卤化物是氟或氯。
  11. 如权利要求2所述的化合物，其中，R2之该烷基是甲烷基或乙烷基。
  12. 一种如权利要求1的化合物或其医药上可接受之盐之用途，其是用以制备用于与腺苷单磷酸活化蛋白激酶(AMP-activated protein kinase；AMPK)相关癌症、脂质代谢疾病或脂质代谢症候群之医药组成物。
  13. 如权利要求12所述的用途，其中该癌症是肝癌或乳癌。
  14. 如权利要求12所述的用途，其中该化合物是结合于腺苷单磷酸活化蛋白激酶之α次单元。
  15. 如权利要求12所述的用途，其中该化合物是诱发脂肪细胞之细胞凋亡。
  16. 一种治疗或预防与腺苷单磷酸活化蛋白激酶相关癌症、脂质代谢疾病或脂质代谢症候群之方法，其是给予有效剂量之权利要求1的之化合物，或其医药上可接受之盐。
  17. 如权利要求16所述的方法，其中该癌症是肝癌或乳癌。
  18. 如权利要求16所述的方法，其中该化合物是对腺苷单磷酸活化蛋白激酶之α次单元具有结合特异性，并磷酸化活化腺苷单磷酸活化蛋白激酶之α次单元。
  19. 如权利要求16所述的方法，其中该化合物是诱发脂肪细胞之细胞死亡。
PCT/CN2017/100735 2016-09-07 2017-09-06 活化腺苷单磷酸活化蛋白激酶之化合物 WO2018045969A1 (zh)

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