CN114702439A - Naphthylurea-piperazine compounds and preparation method and application thereof - Google Patents

Naphthylurea-piperazine compounds and preparation method and application thereof Download PDF

Info

Publication number
CN114702439A
CN114702439A CN202210405822.2A CN202210405822A CN114702439A CN 114702439 A CN114702439 A CN 114702439A CN 202210405822 A CN202210405822 A CN 202210405822A CN 114702439 A CN114702439 A CN 114702439A
Authority
CN
China
Prior art keywords
acid
compound
piperazine
cells
cell
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210405822.2A
Other languages
Chinese (zh)
Other versions
CN114702439B (en
Inventor
徐学军
杨玉坡
段超群
杨争艳
徐红运
裴梦富
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Henan Radiomedical Science And Technology Co ltd
Original Assignee
Henan Radiomedical Science And Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Henan Radiomedical Science And Technology Co ltd filed Critical Henan Radiomedical Science And Technology Co ltd
Priority to PCT/CN2022/092953 priority Critical patent/WO2023109004A1/en
Publication of CN114702439A publication Critical patent/CN114702439A/en
Application granted granted Critical
Publication of CN114702439B publication Critical patent/CN114702439B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/36—Radicals substituted by singly-bound nitrogen atoms
    • C07D213/40—Acylated substituent nitrogen atom
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • 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
    • C07D239/26—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 only hydrogen atoms, hydrocarbon or substituted hydrocarbon radicals, directly attached to ring carbon atoms
    • 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
    • C07D239/34—One oxygen 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
    • C07D401/02—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
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
    • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
    • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Abstract

The invention discloses a naphthyl urea-piperazine compound and a preparation method and application thereof, wherein the compound is prepared by reacting a naphthyl urea-piperazine compound with a low dose, can obviously inhibit the activation of cell signal conduction JAKs/STATs signals, and the results of experiments such as cell proliferation experiments, immunoblotting experiments, cell cycle experiments, protein membrane transport experiments, cell invasion and migration experiments and the like show, the compound can specifically inhibit JAK2 signal activation and expression of downstream STAT3, cyclinD1, cyclinB1, MMP9 and other target genes, induces cell cycle G1 phase retardation and apoptosis, the compound can obviously inhibit the proliferation of various tumor cell strains such as breast cancer, liver cancer, lung cancer, colon cancer, leukemia, lymphoma, multiple myeloma, retinoblastoma and the like, and shows that the compound has the prospect of being developed into a targeted anticancer medicament for JAKs/STATs cell regulation and control signal transduction pathway related targets.

Description

Naphthylurea-piperazine compounds, and preparation method and application thereof
Technical Field
The invention belongs to the field of tumor targeted therapy, and particularly relates to a naphthylurea-piperazine compound and a preparation method and application thereof.
Background
There have been numerous studies demonstrating that abnormal activation of STATs (signal transducers and activators of transducers) signals is associated with a number of diseases, including cancer and immune related diseases. Overexpression and constitutive activation of STAT3 are common in a variety of solid tumors and hematologic cancers. STAT3 is one of the members of the STATs family, a JAK2(Janus kinase2) substrate protein, which has been shown to be closely related to the development, progression and malignant transformation of cancer. Under normal conditions, STAT3 exists in the cytosol as an inactive monomer and there is a strict negative feedback regulatory mechanism. When the negative feedback regulation mechanism of JAK2 or STAT3 is abnormal or the genes are mutated, the phosphorylation level of STAT3 is continuously increased and endogenous excitement is caused, a homodimer or a heterodimer is formed with an SH2 structural domain of another STAT3 protein and enters a cell nucleus, and the protein is combined to a specific gene promoter sequence through a DNA binding domain to start the transcription and protein expression of downstream genes, wherein the expression of a series of anti-apoptosis factors such as BCL-2 and BCL-XL of a BCL-2 protein family related to mitochondrial apoptosis and Cyclin-D1 related to cell cycle regulation are included.
As a plurality of proliferation promoting, invasion and anti-apoptosis genes such as Cyclin-D1, BCL-XL, MMP9, VEGF, c-Myc and the like are target genes of STAT3 signals, in an animal tumor model continuously activated by STAT3 or tumor cells cultured in vitro, the STAT3 protein can be inhibited to effectively inhibit the growth of the tumor cells or induce the apoptosis of the tumor cells, and reduce the metastasis of the tumor cells. STAT3 has become a hot target for tumor therapy. Although three JAK inhibitors are currently marketed in immune diseases abroad, the research on the tumor treatment by a plurality of JAKs inhibitors is in the later clinical stage, some targeted inhibitors against STAT3 are also in the clinical research stage, and the demand of the JAKs/STAT3 inhibitor in the tumor market is far from being met.
In order to develop STAT3 targeted antitumor drugs, a class of naphthylurea-piperazine compounds with a brand new structural formula is synthesized recently. Through some biological technical analyses, the compounds can obviously inhibit the activation of STAT3 signals, inhibit the cell proliferation of breast cancer and liver cancer cell lines, induce cells to generate G1/S or G2/M phase retardation, promote tumor cell apoptosis and show extremely strong tumor inhibition activity.
The invention aims to provide the antitumor effect and the potential pharmacological mechanism of a novel naphthyl urea-piperazine compound and a derivative thereof, and the potential application of the compound in clinical treatment of breast cancer, liver cancer, lung cancer, colon cancer and leukemia.
Disclosure of Invention
The invention aims to provide a naphthylurea-piperazine compound and a preparation method and application thereof.
Based on the purpose, the invention adopts the following technical scheme:
a structural formula of the naphthyl urea-piperazine compound is shown as a general formula I:
Figure BDA0003601812520000021
wherein R is1Is selected from
Figure BDA0003601812520000022
Figure BDA0003601812520000023
Figure BDA0003601812520000024
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51Each independently selected from H, F, Cl, Br, -CN, -CH3、-CF3、-OCH3、-OCF3；
R2Is selected from
Figure BDA0003601812520000031
Figure BDA0003601812520000032
R3Is selected from
Figure BDA0003601812520000033
Figure BDA0003601812520000034
H、CH3、CF3；
m represents CH2The number of the substituents, m is 1, 2, 3, 4.. 10;
k, z represent CH2The number of the substituents, k and z, is 0, 1, 2, 3, 4, 5 or 6.
The above-mentioned naphthylurea-piperazine compound is specifically a compound with the following structure:
Figure BDA0003601812520000035
Figure BDA0003601812520000041
and (2) a biologically acceptable salt of the aforementioned naphthylurea-piperazine compound with at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.
The preparation method of the naphthylurea-piperazine compound comprises the following steps:
(1) will be provided with
Figure BDA0003601812520000042
Dissolving in 1, 4-dioxane, adding potassium tert-butoxide at room temperature, and adding
Figure BDA0003601812520000043
Stirring and reacting at 80-85 ℃ until the reaction is complete, and carrying out post-treatment to obtain the product
Figure BDA0003601812520000044
(2) Will be provided with
Figure BDA0003601812520000045
Dissolving the mixture in a mixed solution of tetrahydrofuran, ethanol and a saturated ammonium chloride aqueous solution, adding iron powder at 45-55 ℃, stirring and reacting at 45-55 ℃ until the mixture is complete, and performing post-treatment to obtain the iron-containing water-soluble organic silicon dioxide
Figure BDA0003601812520000046
(3) Dissolving triphosgene in dichloromethane, cooling to 0 +/-5 ℃ with ice bath, and reacting
Figure BDA0003601812520000047
Dissolving N, N-diisopropylethylamine in dichloromethane, dripping into dichloromethane solution of triphosgene, reacting at 0 + -5 deg.C for 1.5-2.5 h, adding the rest N, N-diisopropylethylamine and R1NH2Adding toluene, reacting at 110-120 deg.c and column chromatography to obtain
Figure BDA0003601812520000051
Wherein, the
Figure BDA0003601812520000052
The preparation process of (A) is as follows:
(a) will be provided with
Figure BDA0003601812520000053
Dissolving triphenylphosphine in tetrahydrofuran, adding diisopropyl azodicarboxylate under the protective atmosphere of-5 ℃, stirring at room temperature for reaction till completion, and performing post-treatment to obtain
Figure BDA0003601812520000054
(b) The compound
Figure BDA0003601812520000055
Dissolving in tetrahydrofuran at-5 deg.CAdding sodium borohydride in batches, stirring at room temperature until the reaction is complete, and carrying out aftertreatment to obtain the sodium borohydride
Figure BDA0003601812520000056
Further, in the step (1)
Figure BDA0003601812520000057
The molar ratio of the potassium tert-butoxide to the potassium tert-butoxide is 1:1.2: 1.2;
in the step (2)
Figure BDA0003601812520000058
The mol ratio of the iron powder to the tetrahydrofuran to the ethanol to the saturated ammonium chloride aqueous solution is 1:2.4: 2.4;
in the step (3), triphosgene and R1NH2、
Figure BDA0003601812520000061
And N, N-diisopropylethylamine at a molar ratio of 1:2.3:3:11, and N, N-diisopropylethylamine at a molar ratio of 4: 7.
Further, in the step (a),
Figure BDA0003601812520000062
the molar ratio of triphenylphosphine to diisopropyl azodicarboxylate is 1:1:1.2: 1.2;
in the step (b), the step (c),
Figure BDA0003601812520000063
and sodium borohydride in a molar ratio of 1: 4.
The naphthyl urea-piperazine compound and the biologically acceptable salt thereof are used for preparing the antitumor drug, wherein the antitumor drug is a drug for treating tumors related to STAT3 signal transduction.
Preferably, the anti-tumor drug is a drug for treating breast cancer, liver cancer, lung cancer, colon cancer or leukemia.
Another purpose of the invention is to provide a kind of small molecule compound with targeting antitumor activity.
The tumor can be particularly a STAT3 high-expression or constitutive activation tumor, and comprises but is not limited to liver cancer, breast cancer, lung cancer, colon cancer, leukemia and the like.
Specifically, the invention synthesizes a new-structure naphthylurea-piperazine compound ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, ID211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1, ID211012B-1 and the like. The proliferation inhibition effect of the compound on tumor cells is detected by an MTT method, the influence of the compound on the cell cycle and apoptosis of the tumor cells is detected by flow cytometry, and the inhibition effect of the compound on JAK2/STAT3 signals is clarified by methods such as immunoblotting and the like.
The results show that the compounds ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 and the like can effectively inhibit the proliferation of breast cancer cells and liver cancer cells, and induce the G1/S or G2/M blocking stage and apoptosis of the cancer cells.
In conclusion, the invention provides a novel naphthyl urea-piperazine compound and the application and potential molecular mechanism of the derivative thereof in tumor treatment.
Drawings
FIG. 1 shows the results of half inhibition ratios (IC50 values) of tumor cells such as ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 against breast cancer cell MB-MDA-468, liver cancer cell HepG2, lung cancer cell PC9, Afatinib-resistant lung cancer cell PC9-AR, colon cancer cell HT29 and leukemia cell Jurkat;
FIG. 2 is the effect of ID210916B-1 on the induction of TNBC cell cycle arrest;
FIG. 3 is the inhibitory effect of ID210916B-1 on TNBC cell scratch repair;
FIG. 4 is the inhibitory effect of ID210916B-1 on TNBC cell migration;
FIG. 5 is a graph showing the inhibitory effect of ID210916B-1 on p-STAT3 nuclear entry in TNBC cells;
FIG. 6 is a graph showing the inhibitory effect of ID210916B-1 on STAT3 transcriptional activity in TNBC cells;
FIG. 7 shows the inhibition of the phosphorylation of STAT3 and the expression of Cyclin D1 in TNBC cells by ID210916B-1, with MDA-MB-468 cells on the left and 4T1 cells on the right.
Detailed Description
In order to make the technical purpose, technical solutions and advantages of the present invention clearer, the technical solutions of the present invention are further described below with reference to the accompanying drawings and specific embodiments.
In the process of the present invention for the synthesis of compounds of formula I, the various starting materials for the reaction are either prepared by methods known in the literature or are commercially available, as known to the person skilled in the art. The intermediates, starting materials, reagents, reaction conditions, etc. used in the above reaction schemes may be appropriately modified according to the knowledge of those skilled in the art.
In the present invention, unless otherwise specified, wherein: (i) the temperature is expressed in degrees centigrade (DEG C), and the operation is carried out in a room temperature environment; more specifically, the room temperature is 20-30 ℃; (ii) drying the organic solvent by a common drying method, evaporating the solvent by using a rotary evaporator for reduced pressure evaporation, and keeping the bath temperature not higher than 50 ℃; the developing agent and the eluent are in volume ratio; (iii) the reaction process was followed by Thin Layer Chromatography (TLC); (iv) the final product has satisfactory proton NMR1H-NMR)。
EXAMPLE 1 Synthesis of Compounds
Figure BDA0003601812520000081
ID210916B-1：
Figure BDA0003601812520000082
ID210917B-1：
Figure BDA0003601812520000083
ID210918B-1：
Figure BDA0003601812520000084
ID210919B-1：
Figure BDA0003601812520000085
ID211130C-1：
Figure BDA0003601812520000086
ID211203B-1：
Figure BDA0003601812520000087
IY211214C-1：
Figure BDA0003601812520000088
IY211228B-1：
Figure BDA0003601812520000089
IY220302B-1：
Figure BDA0003601812520000091
IY220209A-1：
Figure BDA0003601812520000092
IY220313A-1：
Figure BDA0003601812520000093
IY220219A-1：
Figure BDA0003601812520000094
ID210928B-1：
Figure BDA0003601812520000095
ID210929B-1：
Figure BDA0003601812520000096
IY220319A-1：
Figure BDA0003601812520000097
ID211008B-1：
Figure BDA0003601812520000098
ID211009B-1：
Figure BDA0003601812520000099
R3＝H；
ID211010B-1：
Figure BDA00036018125200000910
R3＝CH3；
ID211116B-1：
Figure BDA00036018125200000911
ID211012B-1：
Figure BDA00036018125200000912
The specific synthesis method takes compound ID210916B-1 as an example, and the structural formula is as follows:
Figure BDA0003601812520000101
the name of compound ID210916B-1 is tert-butyl 4- (2- (4- (((4- (3- (pyridine-4-yl) ureido) naphthalen-1-yl) oxy) methyl) phenoxy) ethyl) piperazine-1-carboxylate,
the synthetic route is as follows:
Figure BDA0003601812520000102
step 1.tert-butyl 4- (2- (4-formalphenoxy) ethyl) piperazine-1-carboxylate (int 1)
Weighing 520.00g (equivalent to 4.26mol) of p-hydroxybenzaldehyde (compound 1), placing the p-hydroxybenzaldehyde (compound 1) in a 50L double-layer glass reaction kettle, dissolving the p-hydroxybenzaldehyde (compound 1) by using 20L of tetrahydrofuran, and stirring the mixture to completely dissolve the p-hydroxybenzaldehyde;
weighing 980.65g (equivalent to 4.26mol) of tert-butyl-4- (2-hydroxyethyl) piperazine-1-carboxylic ester (compound 1a) and 1.34kg (equivalent to 5.11mol) of triphenylphosphine, adding the weighed materials into the mixture I, stirring the materials to completely dissolve the materials, and then stirring and cooling the materials to 0 ℃;
weighing 1.03kg (equivalent to 5.11mol) of diisopropyl azodicarboxylate (DIAD), and then slowly dripping into the solution II by using a constant-pressure dropping funnel to keep the temperature of the system at 0 +/-4 ℃;
fourthly, after the dripping is finished, keeping the temperature at 0 ℃ and stirring for 0.50 hour, and then heating the system to 20 ℃ for reacting for 16 hours;
performing rotary distillation on the reaction solution by using a rotary evaporator until no fraction is obtained;
sixthly, diluting the obtained liquid with 10L of ethyl acetate, pouring the diluted liquid into a 50L double-layer glass reaction kettle, adding 10L of water, adjusting the pH value of the solution to 2-4 with 5.9L of diluted hydrochloric acid (1M), separating out a water phase and an organic phase, extracting the water phase twice with ethyl acetate, wherein the dosage is 8L each time, combining the three organic phases, discarding the water phase, and performing subsequent treatment on the water phase;
seventhly, adding the water phase into a 50L double-layer glass reaction kettle, adjusting the pH value of the water phase to 8 by using sodium bicarbonate, extracting twice by using ethyl acetate, using 8L of the water phase each time, combining organic phases, discarding the water phase (no product residue is monitored by TLC), drying the organic phases by using anhydrous sodium sulfate, removing the sodium sulfate by suction filtration, performing rotary evaporation on the filtrate by using a rotary evaporator until no fraction flows out, and obtaining 717.67g of a viscous brownish red liquid which is int 1;
1H NMR(CDCl3,300MHz)δ:9.93(s,1H),7.87(d,J＝9.0Hz,2H),7.04(d,J＝9.0Hz,2H),4.23(t,J＝6.0Hz,2H),3.50(t,J＝6.0Hz,4H),2.89(t,J＝6.0Hz,2H),2.57(t,J＝6.0Hz,4H),,1.50(s,9H)
step 2.tert-butyl 4- (2- (4- (hydroxymethy) phenoxy) ethyl) piperazine-1-carboxylate (int 2)
Weighing int 1707.34 g (equivalent to 2.12mol), placing the int 1707.34 g in a 50L double-layer glass reaction kettle, dissolving the int 1707.34 g in 4.8L tetrahydrofuran, and stirring to completely dissolve the int 1707.34 g;
weighing 320.08g (equivalent to 8.46mol) of sodium borohydride, preparing the sodium borohydride into a turbid liquid (preventing the sodium borohydride from absorbing moisture in the feeding process) by using 1.2L of methanol, and slowly adding the turbid liquid into the solution I; the color of the solution changes from brown red to light yellow;
placing the mixture at room temperature for reaction;
fourthly, the reaction time is 14 hours, then TLC monitoring is carried out, the result shows that the raw materials are reacted completely and new products are generated, then the reaction is stopped, and the subsequent treatment is started;
performing rotary evaporation on the reaction liquid by using a rotary evaporator, removing most of solvent, stopping the rotary evaporation after 1.20L of liquid is remained, adding 5L of dichloromethane into the remained liquid for dilution (the purpose of using dichloromethane is to completely extract products into an organic phase during extraction and improve the extraction efficiency), then adding 5L of water for extraction, stirring for 15 minutes, standing for layering, separating out a water phase and an organic phase, extracting the water phase by using 5L of dichloromethane, stirring for 15 minutes, standing for layering, separating out the water phase and the organic phase, monitoring by TLC (thin layer chromatography) to remove product residues, combining the two organic phases, and drying by using anhydrous sodium sulfate;
sixthly, removing sodium sulfate by suction filtration, performing rotary evaporation on the filtrate by using a rotary evaporator until no fraction is evaporated to obtain 666.74g of light yellow viscous liquid, and solidifying the light yellow viscous liquid after standing to obtain int 2.
1H NMR(CDCl3,300MHz)δ:7.33(d,J＝9.0Hz,2H),6.92(d,J＝9.0Hz,2H),4.66(s,2H),4.14(t,J＝6.0Hz,2H),3.49(t,J＝6.0Hz,4H),2.85(t,J＝6.0Hz,2H),2.54(t,J＝6.0Hz,4H),,1.50(s,9H)
Step 3 tert-butyl 4- (2- (4- (((4-nitrilophen-1-yl) oxy) methyl) phenoxy
)ethyl)piperazine-1-carboxylate(int 3)
Weighing int 2512.14 g (equivalent to 1.52mol), placing the int 2512.14 g in a 50L double-layer glass reaction kettle, dissolving the int 2512.14 g by using 8L 1, 4-dioxane, and stirring to completely dissolve the int 2512.14 g;
weighing 170.82g (equivalent to 1.52mol) of potassium tert-butoxide, slowly adding the potassium tert-butoxide into the solution I, and stirring the mixture for 15 minutes at room temperature;
③ 242.50g of 1-fluoro-4-nitronaphthalene (compound 2, equivalent to 1.27mol) is weighed and slowly added into the solution II, then the temperature of the reaction system is raised to 80 ℃, and the reaction is carried out with heat preservation;
fourthly, after the reaction is carried out for 2 hours, monitoring by TLC, and detecting results show that new products are generated after the raw materials are reacted, stopping the reaction and starting subsequent treatment;
fifthly, carrying out suction filtration on the reaction liquid to remove insoluble substances, assisting the filtration with diatomite, but still not carrying out suction filtration, wherein the suction filtration rate is slow, and after the suction filtration is finished, placing the filtrate for concentration and sample mixing in the tomorrow;
sixthly, performing rotary evaporation on the filtrate obtained by suction filtration yesterday by using a rotary evaporator, adding silica gel for sample mixing, performing column loading after rotary drying, and purifying a product by using column chromatography;
seventhly, purifying the product by column chromatography, and eluting the glass column by using eluent of a methanol/dichloromethane (1/100 (V/V) system to finally obtain 323.64g of a sticky brown liquid which is int 3. However, both nuclear magnetic and TLC assays contained impurity sites.
Step 4 tert-butyl 4- (2- (4- (((4-aminophthalalen-1-yl) oxy) methyl) phenoxy) ethyl) piperazine-1-carboxylate (int 4)
Weighing int 3320.66 g (equivalent to 631.73mmol), placing the int 3320.66 g into a 5L double-neck round-bottom flask, dissolving the int 3320.66 g in tetrahydrofuran (500mL), and then continuously adding 95% ethanol (1.2L) to completely dissolve the int 3320.66 g, so that the solution becomes dark brown;
secondly, adding a saturated ammonium chloride solution (1.2L) into the solution I, separating out solids in the solution at the moment, changing the color of the solution into brown yellow, raising the temperature of the solution to 50 ℃, and dissolving the solids;
③ 282.23g (equivalent to 5.05mol) of iron powder is weighed and slowly added into the solution II, and the reaction is kept at 50 ℃ for 2 hours;
fourthly, after the reaction is carried out for 2 hours, monitoring by TLC, and starting subsequent treatment when the result shows that the reaction of the raw materials is finished;
adding 200.00g of diatomite into the reaction liquid for filtration assistance, then removing iron powder and diatomite in the reaction liquid by suction filtration, soaking and washing a filter cake by dichloromethane, and pouring the filtrate into a 50L double-layer glass reaction kettle;
sixthly, adding 8.00L of water and 10L of dichloromethane into a 50L reaction kettle, stirring for 25 minutes, standing for layering, separating out a water phase and an organic phase, and detecting by using a water phase TLC (thin layer chromatography) to ensure that no product is left and is discarded; the organic phase was dried over anhydrous sodium sulfate;
seventhly, removing sodium sulfate by suction filtration, then carrying out rotary evaporation on the filtrate by using a rotary evaporator, weighing 340.12g of crude product after distillation till no fraction is obtained, placing the crude product in a refrigerator for storage at low temperature, and carrying out column chromatography purification after sample mixing in tomorrow;
dissolving a crude product obtained yesterday by using dichloromethane (200mL), adding about 400.00g of silica gel with the mass of 1.2 times of that of the crude product, mixing, performing rotary evaporation by using a rotary evaporator, removing a solvent, rotating to be powder, mixing, and packing (firstly, about 1.80kg of silica gel with the mass of 5 times of that of the crude product is added into a glass column, then pouring the mixed silica gel into a top packing column), and preparing for column chromatography purification;
ninthly, eluting the silica gel column by pure dichloromethane, wetting the silica gel column, removing impurities with polarity smaller than that of the product, and then using methanol: eluting with a dichloromethane-100 (v/v) eluent, collecting a part of the eluted eluent containing both products and impurities for treatment, eluting continuously to obtain a pure product, and spin-drying the eluent by using a rotary evaporator to obtain 119.37g of brown viscous liquid, namely int 4.
Step 5 tert-butyl 4- (2- (4- (((4- (3- (pyridine-4-ylmethyl) ureido) naphthalen-1-yl) oxy) methyl) phenoxy) ethyl) piperazine-1-carboxylate (ID210916B-1)
Weighing 33.15g of triphosgene (equivalent to 111.72mmol) and placing the triphosgene in a 1L single-neck round-bottom flask, dissolving the triphosgene in dichloromethane (300mL), stirring the mixture to completely dissolve the triphosgene, and then cooling the temperature in the system to 0 ℃ by using an ice-water bath
Weighing int 4160.07 g (equivalent to 335.15mmol) and N, N-diisopropylethylamine 57.76g (equivalent to 448.87mmol), mixing, adding dichloromethane (350mL) for dissolving, slowly adding the mixture into the solution I by using a constant-pressure first funnel, and then keeping the temperature at 0 ℃ for reaction for 2 hours; (white smoke is generated in the process of dripping and disappears after dripping)
③ after 2 hours of reaction, 99.63g (corresponding to 770.85mmol) of N, N-diisopropylethylamine was added dropwise to the reaction mixture;
fourthly, after the dropwise addition is finished, weighing 27.79g (equivalent to 256.95mmol) of 4-picolylamine, slowly adding the weighed 4-picolylamine into the reaction solution, then supplementing 600mL of methylbenzene into the reaction system, then heating to 110 ℃, keeping the temperature for reaction overnight, and monitoring by TLC (thin layer chromatography) in tomorrow;
fifthly, the reaction time is 18 hours, then TLC is used for monitoring, the result shows that the raw materials are reacted completely and new products are generated, and then the reaction solution is subjected to subsequent treatment;
sixthly, adding about 450g of silica gel with the mass of 1.2 times of the crude product (about 300g of the total feeding amount) into the reaction solution for sample mixing, then spin-drying the solvent in the solution by using a rotary evaporator, and loading a column after the solvent is spun into powder (1.50 kg of silica gel with the mass of 5 times of the crude product is firstly added into a glass column, and then the mixed silica gel sample is poured above the silica gel) for purification of the product by column chromatography;
and seventhly, purifying the product by using column chromatography, eluting the product by using an eluent of a MeOH/DCM (1/20) ═ V/V) system, carrying out rotary evaporation on the obtained eluent containing a pure product, concentrating the eluent until the residual 12.5L of the eluent is obtained, transferring the eluent into a 50L double-layer glass reaction kettle, adding 8L of water into the eluent for washing, stirring the eluent for 15 minutes, standing and layering the eluent to separate out an organic phase and a water phase, discarding the water phase, transferring the organic phase into the 50L double-layer glass reaction kettle again, washing the organic phase and the water phase once by using 8L of water, stirring the mixture for 15 minutes, standing and layering the organic phase and the water phase, discarding the water phase, drying the organic phase by using anhydrous sodium sulfate, carrying out suction filtration to remove the sodium sulfate, carrying out rotary evaporation on the filtrate by using a rotary evaporator, and evaporating the filtrate to obtain 101.94g of light brown (slightly whitish) solid, namely ID 210916B-1.
1H NMR(CDCl3,300MHz)δ:8.52(d,J＝6.0Hz,2H),8.44(s,1H),8.19(d,J＝6.0Hz,1H),8.01(d,J＝6.0Hz,1H),7.62-7.45(m,5H),7.32(d,J＝6.0Hz,2H),7.06-6.92(m,4H),5.20(s,2H),4.36(d,J＝6.0Hz,2H),4.10(t,J＝6.0Hz,2H),3.31(t,J＝6.0Hz,4H),2.72(t,J＝6.0Hz,2H),2.44(t,J＝6.0Hz,4H),1.40(s,9H)
Other compounds were synthesized by referring to example 1, except that in step 1, the starting material 1 was changed to the corresponding aldehyde, or 1a was changed to piperazine having the corresponding substituent, or in step 5, the starting material 4-pyridinemethylamine was changed to the corresponding amine.
Example 2: proliferation inhibitory effects of ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 on cells such as breast cancer and liver cancer
Collecting tumor cells in logarithmic growth phase, respectively, and adjusting cell suspension concentration to 5 × 104Per mL, add 96 well cell culture plates, 100ul per well volume. Control with DMSO as solvent, WP1066 (Chinese name: (2E) -3- (6-bromo-2-pyridyl) -2-cyano-N- [ (1S) -1-phenylethyl)]-2-acrylamide, CAS:857064-38-1, having the structure
Figure BDA0003601812520000141
) As a positive control, the novel naphthylurea-piperazine compounds ID210916B-1, ID210917B-1, ID210918B-1 and the like according to the present invention were diluted with DMSO and added to the culture wells so that the final concentrations of the compounds in the system were 0.1, 0.3, 1, 3, 10, 30, 100 and 300 (. mu.mol/L), respectively. Continuing culturing for 48h, adding 10 μ L MTT solvent (5mg/ml) into each well, incubating at 37 deg.C for 4h, removing culture supernatant, adding 150 μ L DMSO into each well, shaking and decolorizing for 10min, reading with enzyme-labeling instrument, and determining in the adsorption phaseThe OD at 490nm was recorded, and the cell growth curve was plotted with the dose of the compound as abscissa and the absorbance as ordinate. The statistical results of the half inhibition rate (IC50 value) of the ID210916B-1, ID210917B-1, ID210918B-1, ID210919B-1, D211130C-1, ID211203B-1, IY211214C-1, IY211228B-1, IY220302B-1, IY220209A-1, IY220313A-1, IY220219A-1, ID210928B-1, ID210929B-1, IY220319A-1, ID211008B-1, ID211009B-1, ID211010B-1, ID211116B-1 and ID211012B-1 on the tumor cells are shown in FIG. 1.
The results of fig. 1 show that: compared with a positive control medicament WP1066, the compounds ID210916B-1, IY211214C-1, ID211130C-1 and the like have good proliferation inhibition effects on tumor cells such as breast cancer and liver cancer, particularly have stronger tumor inhibition activity on breast cancer and liver cancer cells, and the application focuses on further research on the anti-tumor effects of the 3 compounds on the breast cancer and the liver cancer.
Example 3: effect of ID210916B-1 on inducing TNBC cell cycle arrest
MDA-MB-468 or 4T1 cells are taken from logarithmic growth phase, digested, centrifuged and made into single cell suspension. After counting, the cells were plated in 1 12-well plate, and both cells were seeded 2X 10 cells per well5Cells were plated in 3 wells for parallel control. After plating for 16h, cells were treated with compound. DMSO was used as a solvent for the compound, and the final concentrations of compound ID210916B-1 in 4T1 cell suspension were 0, 2.5, 5, and 10. mu.M, respectively, and compound ID210916B-1 in MDA-MB-468 cell suspension were 0, 0.025, 0.05, and 0.1. mu.M, respectively. Adding medicine for 48 hr, digesting each empty cell with pancreatin, resuspending, counting, adjusting cell concentration of each well to 5 × 105And (4) respectively. After digestion was completed, the supernatant was centrifuged and discarded, cells were washed twice with PBS (2000rpm, 5min centrifugation), and the supernatant was discarded, and 980. mu.l of 70% cold ethanol and 20. mu.l of 5% BSA (addition of a small amount of BSA reduces cell loss during the procedure) were added to each tube and fixed overnight at 4 ℃. The fixative was discarded and washed 3 times with PBS to remove residual fixative (1000rpm, 3min centrifugation). After the cell washing was completed, the subsequent operations were performed according to the requirements of the instructions of the DNA content detection kit (product of Beijing Solebao Co.). 100. mu.l of RNase A for 3 each sampleAfter 30min incubation at 7 ℃, 500. mu.l of the prepared PI (propidium iodide) working solution was added to each sample and incubated for 30min at room temperature in the absence of light. Finally, the cell cycle was determined by flow cytometry. And analyzing the experimental result by adopting ModFit software, and further analyzing by Graphpad prism 6.0 to obtain the respective cell cycle ratio of the two cells.
As shown in FIG. 2, the ratio of the cells in the G2 phase of the MDA-MB-468 cells in the group treated with the compound ID210916B-1 at higher concentrations (0.05 and 0.1. mu.M) was increased as compared with the solvent (DMSO) control group, and was 19.67%, 25.63% and 36.04%, respectively. The S phase cell rate also increased significantly (25.67%: 41.66%) and the G1 phase rate decreased correspondingly (54.65%: 22.3%) with the 0.1 μ M dose treatment. Thus, compound ID210916B-1 induced MDA-MB-468 cells to block S/G2 phase. Among 4T1 cells, the rate of G1 cells in the higher concentration (5 and 10 μ M) compound ID210916B-1 treated group was significantly increased and dose-dependent. Accordingly, the ratio of G2 phase cells was significantly reduced. The compound ID210916B-1 is shown to be capable of inducing 4T1 cells to generate G1 phase block and inhibiting cell cycle progress.
Example 4: inhibition of TNBC cell scratch repair by ID210916B-1
MDA-MB-468 or 4T1 cells in logarithmic growth phase were seeded in 6-well plates at 5X 10/well5And (4) cells. After about 24h, the cells spread evenly as a monolayer across the bottom of the wells. The vertical uniform scratch was made with a 200 μ L tip. After washing 2 times with PBS, different doses of ID210916B-1(MDA-MB-468 cell treatment concentrations of 0, 0.015, 0.03 and 0.06. mu.M, 4T1 cell treatment concentrations of 0, 0.25, 0.5 and 1. mu.M) were added to DMEM medium containing 0.5% fetal bovine serum and photographed under a microscope at 0, 24 and 36h (MDA-MB-468 cells) and 0, 24 and 48h (4T1 cells), respectively.
The results in FIG. 3 show that the control group without ID210916B-1 treatment showed close to 50% of the area of the closure at 24 hours and close to complete closure at 36 hours in MDA-MB-468 cells and 4T1 cells. Whereas the healing rate of the cells was significantly delayed after treatment with 0.015 μ M and 0.03 μ M of ID 210916B-1. Scratch repair of cells was almost completely inhibited with 0.06. mu.M ID 210916B-1. A similar phenomenon was observed in 4T1 cells, indicating that the compound can inhibit the lateral migration ability of TNBC cells.
Example 5: inhibition of TNBC cell migration by ID210916B-1
MDA-MB-468 or 4T1 cells in logarithmic growth phase are taken, subjected to conventional digestion and centrifugation, washed with PBS for 1 time, centrifuged at 500g for 5min, and then re-suspended and counted by using a basic culture medium. The upper chamber of the Transwell (8 μm pore sizes, Corning, US) was baited with 30 μ L of pre-cooled Matrigel and the lower chamber with 600 μ L of medium containing 20% serum. The upper chamber is seeded with 1X 10 seeds per well5And (4) cells. DMSO is used as a solvent control hole, cells are respectively treated by ID210916B-1 with different concentrations (MDA-MB-468 cell treatment concentrations are 0, 0.015 and 0.03 mu M, and 4T1 cell treatment concentrations are 0, 2 and 4 mu M), and the cells are placed in an incubator for continuous culture. After 48h, the cells were stained with crystal violet.
And (3) crystal violet dyeing: 1) discard the culture medium, wash the chamber 3 times with 1 × PBS; 2) discard PBS, fix cell for 15min with 75% ethanol; 3) after fixation, the ethanol was discarded, and the chamber was washed 3 times with 1 × PBS; 4) discarding PBS, and dyeing the small chamber with 0.1% crystal violet for 30 min; 5) the water flow is flushed from the small chamber until the water flow is clear and purple can not be seen; 6) cells that failed to migrate from the upper chamber were carefully wiped off by dipping with a cotton swab, and the upper chamber was then returned to the 24-well plate. The cells were photographed under a microscope to record migration and invasion.
From the results of Transwell (FIG. 4), it was observed that the control wells had a large number of purple-red particles, indicating that both MDA-MB-468 and 4T1 cells without ID210916B-1 had strong ability to migrate longitudinally through the bottom membrane of the upper chamber of the insert. While positive staining cells were significantly reduced in each well after ID210916B-1 treatment, indicating that the longitudinal migration ability of TNBC cells was inhibited.
Example 6: inhibition of p-STAT3 nuclear entry in TNBC cells by ID210916B-1
Subculture was carried out according to the conventional cell culture method of example 2, followed by the following operations: (1) MDA-MB-468 and 4T1 cells were plated in 35mm dishes at a cell count of 1X 105Culturing in 37 deg.C incubator overnight; (2) MDA-MB-468 or 4T1 cells were treated with ID210916B-1, and MDA-MB-468 was treated with ID210916B-1 to a final concentration of 0.04 μ M and ID210916B-1 treatment 4T1 at a final concentration of 6 μ M, and incubation was continued for 48 hours with 1 μ l DMSO/ml culture medium as solvent Control (Control); (3) using a suction head to discard the culture medium; (4) adding 1ml of immune fixative into each well, and fixing at room temperature for 15min (note: adding on the periphery of the dish, shaking quickly, not impacting cells, not drying the plate); (5) discarding the fixative, washing with 1 × PBS for 3 times, each for 5min, and shaking with shaker at slowest speed; (6) permeabilizing with precooled methanol at-20 deg.C for 10min or at 4 deg.C for 15 min; (7) removing methanol, washing with 1 × PBS for 3 times, each for 5 min; (8) adding sealing liquid into the container at a concentration of 150-; (9) diluting the primary antibody with an antibody diluent 1:100, removing the blocking solution, adding 150 mu l of the primary antibody per hole, and incubating overnight in a wet box; (10) discarding the primary antibody, washing with 1 × PBS for 3 times, each for 5 min; (11) adding a secondary antibody prepared by using an antibody diluent, and reacting for 30min at room temperature in a dark place; (12) discarding the secondary antibody, washing with 1 × PBS for 3 times, each for 5 min; (13) staining nuclei with DAPI, adding a small amount of DAPI, covering the sample, and incubating at room temperature for 5 min; (14) removing DAPI, washing with 1 × PBS for 3 times, each for 5 min; (15) 1ml of 1 XPBS was added to each dish and observed in the dark or fixed.
The results in FIG. 5 show that p-STAT3 expression in MDA-MB-468 cells, particularly in the nucleus, was significantly reduced after 24h of treatment with 0.04. mu.M ID210916B-1 compared to cells in control wells. p-STAT3 expression was significantly reduced in both 4T1 cytoplasm and nucleus 24h after treatment with 6. mu.M ID 210916B-1. ID210916B-1 was shown to interfere with nuclear entry of p-STAT3 in TNBC cells.
Example 7: inhibition of STAT3 transcriptional activity in TNBC cells by ID210916B-1
Subculture was carried out according to the conventional cell culture method of example 2, followed by the following operations: (1) taking MDA-MB-468 cells as an example: MDA-MB-468 cells were seeded in 12-well plates at a cell count of 1.5X 10 per well5Putting the mixture into an incubator at 37 ℃ for incubation for 24 hours; (2) taking out the 12-hole plate, discarding the culture solution, washing the cells once by using 1mL of PBS buffer solution, and washing once again by using 500 mu L of serum-free culture solution for later use; (3) STAT 3-luciferase reporter plasmid (purchased from Shanghai Yangyang Biotech, Inc., TSB10127-1) and pRL-TK Renilla plasmid (purchased from Promega, US) were diluted with serum-free medium along with liposomes(Lipofectamine TM 2000, CAT. NO. 11668-027). 250 μ L of serum-free medium was diluted to 4 μ g of plasmid per well and gently pipetted and mixed well. Diluting 10 μ g liposome with 250 μ L serum-free culture medium per well, gently blowing, mixing, and standing at room temperature for 5 min. The diluted plasmid is added into the diluted liposome, gently blown and kept stand at room temperature for 20 min. The mixture was slowly added dropwise to the cells, gently mixed and incubated at 37 ℃ for 4 h. 1mL of fresh medium containing 10% FBS was replaced per well and the incubator was incubated at 37 ℃ for a further 60 h. (4) MDA-MB-468 cells (0, 0.015, 0.03, 0.06 and 0.12. mu.M) and 4T1 cells (0, 3, 6 and 12. mu.M) were pretreated with a gradient dose of compound ID210916B-1 for 2h each. Cells were then stimulated with IL-6(50ng/ml) for 1 h. The supernatant was discarded and the cells were washed once with PBS. The cells were lysed with 200. mu.L of 5 XPLB lysate for 20min at room temperature. The cell lysate was transferred to a 1.5ml Ep tube. According to the following steps: 50, adding a dual-luciferase reporter gene detection kit (Promega, US, E1910) detection buffer solution, and respectively detecting the activities of the firefly luciferase and the sea cucumber luciferase in a multifunctional microplate reader (Varioskan Flash, Thermo-Fisher, US). (5) Relative fluorescence intensity (RF ═ firefly fluorescence value/renilla fluorescence value) was calculated and compared with that of the blank control group.
As shown in FIG. 6, in MDA-MB-468 cells, the ratio of activity without IL-6 treatment to luciferase activity was about 10, indicating that the cells had a strong background STAT3 transcriptional activity. After the cells are stimulated for 1 hour by 50ng/ml of IL-6, the relative luciferase activity is improved by about 5 times, and the sensitivity and the specificity of the response of detection systems such as the cells and a report carrier to STAT3 signals are shown. The luciferase activity ratio was decreased after pretreatment with ID210916B-1 at 0, 0.015, 0.03, 0.06 and 0.12. mu.M. STAT3 transcriptional activity was reduced by approximately 50% under 0.03. mu.M ID 210916B-1. While IL-6-induced STAT3 transcriptional activation was suppressed to background levels at a concentration of 0.12. mu.M ID 210916B-1. Similar results were obtained in 4T1 cells. ID210916B-1 was shown to dose-dependently inhibit the gene transcriptional regulatory function of STAT3 in TNBC cells.
Example 8: inhibition of phosphorylation of STAT3 and expression of Cyclin D1 in TNBC cells by ID210916B-1
A, cellsTaking MDA-MB-468 or 4T1 cells in logarithmic phase, digesting with pancreatin, and preparing into culture medium with L-15 or DMEM containing 10% fetal calf serum and density of 4 × 105Single cell suspension per mL, seeded into 6-well plates at 2mL cell suspension per well. b.37 ℃ and 5% CO2Incubator incubation (MDA-MB-468 cell culture requires no 5% CO2) After the cells adhered to the wall, different concentrations of ID210916B-1 were added. c. After further 48h of culture, the cells were lysed with RIPA lysate and the protein was collected.
II, collecting and cracking cells: a. discard the upper medium and wash the cells twice with pre-cooled PBS. 100. mu.L of pre-cooled RIPA cell lysate (protease inhibitor, PMSF and lysate are added in advance and mixed at a ratio of 1: 100) is added into each well.
b. Lysing on ice for 3min, scraping the cells with a cell scraper, and collecting into 1.5mL EP tubes; the cells were lysed on ice for 30min and vortexed every 6 min. c.4 ℃ and centrifugation at 12000g for 10 min. d. The cell supernatant was transferred to a new EP tube. The cell supernatant is divided into two parts: adding 5 mu L of the mixture into a 1.5mL EP tube for BCA protein content determination, adding 45 mu L of 1 XPBS, and mixing uniformly for later use; the residual cell supernatant was quantitatively sampled to 80. mu.L, added to 5 XSDS Loading Buffer 20. mu.L, mixed well and boiled in boiling water for 10min, centrifuged and then applied to the sample or stored in a-20 ℃ refrigerator.
e. Protein concentration determination step: (1) preparing a BCA working solution: and calculating the total required amount of the mixed working solution A and B according to the number of the standard substances and the samples to be detected. And (3) according to the volume ratio of the BCA firming agent A to the B of 50: 1, preparing the working solution, and carrying out vortex oscillation and uniform mixing for later use.
(2)1 × PBS diluted protein standard:
7 tube number 1×PBS(μl) Amount of BSA standard BSA standard (μ g/ml)
A 0 100 2000
B 200 200 1000
C 200 200 (take from B tube) 500
D 200 200 (taken from the C tube) 250
E 200 200 (taken from D tube) 125
F 400 100 (taken from E tube) 25
G 200 0 0 (blank)
(3) The protein standard and the sample supernatant diluted with PBS (10-fold dilution) were added to a new 96-well plate in 25 μ L each. Then respectively adding 200 mu L of BCA working solution prepared in advance and fully mixing. The reaction solution is subjected to air-blowing to generate bubbles, a 96-well plate cover is tightly covered, and the reaction solution is reacted in a 37 ℃ incubator for 30 min. (4) And taking out the 96-well plate, recovering to room temperature for 3-5 min, measuring the absorbance value of A562 on an enzyme-labeling instrument, copying the obtained value and storing in an Excel table. A standard curve was made and the protein content of 1. mu.L of each sample was calculated for protein loading.
Third, SDS-PAGE: (1) the gel plate was fixed and a 10% SDS-PAGE separation gel was prepared.
The separation gel was formulated as follows: 10mL
Figure BDA0003601812520000181
Figure BDA0003601812520000191
(2) And respectively adding the mixed separation gel into 2 rubber plates, adding the rubber plates to a position 1.0cm away from the top, filling the rubber plates with absolute ethyl alcohol, and standing for 30-45 min. (3) After the separation and gelation are finished, the residual absolute ethyl alcohol is poured out and is completely absorbed by filter paper. (4) 5mL of 5% concentrated gum was prepared according to the following table
Deionized water 2.77mL
30％(m/v)Acrylamide 830μL
0.5M Tris-HCl (pH 6.8) buffer 1.26mL
10％(m/v)SDS 50μL
10％(m/v)APS 50μL
TEMED 5μL
Total 5mL
(5) Slowly adding the prepared concentrated glue into a rubber plate to avoid generating bubbles, inserting a comb, and standing for 30-45 min.
(6) Taking out protein sample, heating in water bath at 100 deg.C for 5min, centrifuging at 10000rpm for 5 min. (7) Fixing the gel plate in an electrophoresis tank, adding SDS-PAGE electrophoresis buffer solution, pulling out a comb, and sequentially adding the processed protein samples into the sample tank, wherein each hole of the protein samples is 50 mu g. (8)80V electrophoresis for 40 min. (9) Changing the voltage to 120V for electrophoresis for about 1.5h until bromophenol blue comes out of the colloid;
fourthly, Western-blot: (1) and (3) putting the SDS-PAGE gel after electrophoresis into a TBST buffer solution for rinsing once, and putting the albumin gel into a membrane transfer buffer solution for soaking. (2) Soaking a layer of cotton pad in a membrane transfer buffer solution, clamping the cotton pad on a membrane transfer instrument by using tweezers, putting the cotton pad, the filter paper, the albumin glue, the PVDF membrane, the filter paper, the cotton pad and the white board in sequence, clamping, and putting the membrane transfer instrument. If there are air bubbles between each layer, it is driven out by the light rolling of the glass solid tube. (3) The transfer apparatus was turned on and constant current transfer was carried out at 300mA for 80 min. (4) The membrane was placed in TBST buffer and rinsed 3 times for 8min each. (5) Blocking with 20mL of 5% BSA-TBST blocking solution at room temperature for 2 h. (6) Primary antibody was added and incubated overnight at 60rpm at 4 ℃. (7) The membranes were washed three times each for 10min with TBST at room temperature on a shaker at 60 rpm. (8) Secondary antibody was added and incubated for 1h at room temperature. (9) The membranes were washed three times for 10min each with TBST at room temperature on a shaker at 60 rpm. (10) Taking 1ml of each of the chemiluminescence substrate reagent solution A and the chemiluminescence substrate reagent solution B, and developing for 2min at room temperature. (11) The liquid on the membrane was blotted with filter paper and the plate was exposed on the machine.
Fifthly, reagent preparation:
(1) 10% SDS: 1g of high purity (electrophoresis grade) SDS was weighed into a 10mL centrifuge tube, and about 8mL of deionized water was added thereto, heated to dissolve, and the volume was adjusted to 10mL, and the mixture was stored at room temperature.
(2) Ammonium persulfate (Ammonium persulfate, AP) 10%: ammonium persulfate, 1g, was weighed, dissolved with stirring after adding about 10mL of deionized water, and stored at 4 ℃.
(3)5 × electrophoresis buffer: weighing 15.1g of Tris, 94g of Glycine and 5.0g of SDS into a beaker, adding 1L of double distilled water for dissolving, storing at room temperature, and diluting by 5 times when in use.
(4) Membrane transfer buffer: weighing 5.8g of Tris, 11.6g of glycine and 0.75g of SDS into a beaker, adding 700mL of double distilled water, dissolving, and then fixing the volume to 800mL, and finally adding 200mL of methanol.
(5)1.5mol/L Tris-HCl, 100ml: dissolving 18.15gtris in 80ml of water, adjusting the solution to 8.8 by using 4N HCl, and fixing the volume to 100 ml.
(6)0.5mol/L Tris-HCl, 1000 ml: 60.5 g tris base was weighed, water was added to 850ml, concentrated hydrochloric acid was added and stirred until all the base was dissolved, the pH was adjusted to 6.8, and water was added to 1L.
(7) TBS buffer: weighing NaCl 8.8g in 800mL distilled water, dissolving, adding 10mL 1mol/L Tris HCl (pH7.5), diluting to 1L, and storing at room temperature.
(8) TBST buffer: 20% Tween 20500. mu.L was added to 1L of TBS buffer so that the final concentration of Tween20 was 0.1%, and the buffer was prepared as it was.
(9) Blocking solution, antibody dilution: 5% skimmed milk powder or BSA is added into TBST buffer solution, and the mixture is prepared in situ.
To further clarify the inhibitory effect of ID210916B-1 on phosphorylation of STAT3 in TNBC cells. We treated MDA-MB-468 and 4T1 cells with different concentrations of ID210916B-1 for 48h each. Western blot results showed that 0.06. mu.M and 4. mu.M ID210916B-1 were effective in down-regulating the expression levels of p-STAT3(Y705), the total protein of STAT3 (T-STAT3), and the downstream target protein Cyclin D1 of STAT3, which are key markers of STAT3 activation, in two cells, respectively (FIG. 7).
The data fully show that the compound ID210916B-1 can effectively inhibit the growth and migration of TNBC cells, can inhibit the phosphorylation and activation of STAT3, and is a potential therapeutic drug for STAT3 targeted inhibitors and TNBC.
The results show that the piperazine compound represented by ID210916B-1 can obviously inhibit proliferation and metastasis of breast cancer and liver cancer cells, induce the cycle arrest of tumor cells, has obvious inhibition effect on STAT3 and related proteins thereof, and shows good anticancer effect. According to the general way of drug development (firstly carrying out conventional antitumor in vitro screening and then carrying out targeted research), the compound can be applied to cancer treatment drugs related to abnormal cell proliferation, and can be used for preparing antitumor drugs by being mixed with human body acceptable salt or medicinal carriers.
Finally, it should be noted that: the above embodiments are merely illustrative and not restrictive of the technical solutions of the present invention, and any equivalent substitutions and modifications or partial substitutions made without departing from the spirit and scope of the present invention should be covered by the claims of the present invention.

Claims (9)

1. A naphthyl urea-piperazine compound is characterized in that the structural formula is shown as the general formula I:
Figure FDA0003601812510000011
wherein R is1Is selected from
Figure FDA0003601812510000012
Figure FDA0003601812510000013
Figure FDA0003601812510000014
R1、R2、R3、R4、R5、R6、R7、R8、R9、R10、R11、R12、R13、R14、R15、R16、R17、R18、R19、R20、R21、R22、R23、R24、R25、R26、R27、R28、R29、R30、R31、R32、R33、R34、R35、R36、R37、R38、R39、R40、R41、R42、R43、R44、R45、R46、R47、R48、R49、R50、R51Each independently selected from H, F, Cl, Br, -CN, -CH3、-CF3、-OCH3、-OCF3；
R2Is selected from
Figure FDA0003601812510000015
Figure FDA0003601812510000021
R3Is selected from
Figure FDA0003601812510000022
Figure FDA0003601812510000023
H、CH3、CF3；
m represents CH2The number of the substituents, m is 1, 2, 3, 4.. 10;
k, z representCH2The number of the substituents, k and z, is 0, 1, 2, 3, 4, 5 or 6.
2. The naphthylurea-piperazine based compound of claim 1, which is specifically a compound of the following structure:
Figure FDA0003601812510000024
3. the naphthyl urea-piperazine compound of claim 1 or 2, which forms a biologically acceptable salt with at least one of acetic acid, dihydrofolic acid, benzoic acid, citric acid, sorbic acid, propionic acid, oxalic acid, fumaric acid, maleic acid, hydrochloric acid, malic acid, phosphoric acid, sulfurous acid, sulfuric acid, vanillic acid, tartaric acid, ascorbic acid, boric acid, lactic acid, and ethylenediaminetetraacetic acid.
4. The method for producing a naphthylurea-piperazine compound according to claim 1 or 2, comprising the steps of:
(1) will be provided with
Figure FDA0003601812510000031
Dissolving in 1, 4-dioxane, adding potassium tert-butoxide at room temperature, and adding
Figure FDA0003601812510000032
Stirring and reacting at 80-85 ℃ until the reaction is complete, and carrying out post-treatment to obtain the product
Figure FDA0003601812510000033
(2) Will be provided with
Figure FDA0003601812510000034
Dissolving the mixture in a mixed solution of tetrahydrofuran, ethanol and a saturated ammonium chloride aqueous solution, adding iron powder at 45-55 ℃, stirring and reacting at 45-55 ℃ until the mixture is complete, and performing post-treatment to obtain the iron-containing water-soluble organic silicon dioxide
Figure FDA0003601812510000035
(3) Dissolving triphosgene in dichloromethane, cooling to 0 +/-5 ℃ with ice bath, and reacting
Figure FDA0003601812510000036
And N, N-diisopropylethylamine is dissolved in dichloromethane, the solution is dripped into the dichloromethane solution of triphosgene, the reaction is carried out for 1.5h to 2.5h under the temperature of 0 plus or minus 5 ℃, and the rest N, N-diisopropylethylamine and R are added again1NH2Adding toluene, reacting at 110-120 deg.c and column chromatography to obtain
Figure FDA0003601812510000037
5. The method for producing a naphthylurea-piperazine-based compound according to claim 4, wherein the naphthylurea-piperazine-based compound is produced by the method
Figure FDA0003601812510000041
The preparation process is as follows:
(a) will be provided with
Figure FDA0003601812510000042
Dissolving triphenylphosphine in tetrahydrofuran, adding diisopropyl azodicarboxylate at-5 deg.C, stirring at room temperature for reaction, and post-treating to obtain
Figure FDA0003601812510000043
(b) The compound
Figure FDA0003601812510000044
Dissolving in tetrahydrofuran, adding sodium borohydride in batches at-5 ℃, stirring at room temperature until the reaction is complete, and carrying out post-treatment to obtain the sodium borohydride
Figure FDA0003601812510000045
6. The method for producing a naphthylurea-piperazine-based compound according to claim 4, wherein in step (1), the compound is produced by
Figure FDA0003601812510000046
The molar ratio of the potassium tert-butoxide to the potassium tert-butoxide is 1:1.2: 1.2;
in the step (2)
Figure FDA0003601812510000047
The mol ratio of the iron powder to the tetrahydrofuran to the ethanol to the saturated ammonium chloride aqueous solution is 1:2.4: 2.4;
in step (3), triphosgene, R1NH2、
Figure FDA0003601812510000048
And N, N-diisopropylethylamine at a molar ratio of 1:2.3:3:11, and N, N-diisopropylethylamine at a molar ratio of 4: 7.
7. The process for producing a naphthylurea-piperazine-based compound according to claim 5, wherein in step (a),
Figure FDA0003601812510000049
the molar ratio of triphenylphosphine to diisopropyl azodicarboxylate is 1:1:1.2: 1.2;
in the step (b), the step (c),
Figure FDA0003601812510000051
and sodium borohydride in a molar ratio of 1: 4.
8. Use of the naphthylurea-piperazine based compound and the biologically acceptable salt thereof according to any one of claims 1 to 3, in the preparation of an antitumor drug, wherein the antitumor drug is a drug for treating diseases associated with STAT3 cell signaling abnormality.
9. The use according to claim 8, wherein the antitumor drug is a drug for treating breast cancer, liver cancer, lung cancer, colon cancer or leukemia.
CN202210405822.2A 2021-12-13 2022-04-18 Naphthyl urea-piperazine compound and preparation method and application thereof Active CN114702439B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/CN2022/092953 WO2023109004A1 (en) 2021-12-13 2022-05-16 Naphthylurea-piperazine compound, preparation method therefor and use thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111520886 2021-12-13
CN2021115208869 2021-12-13

Publications (2)

Publication Number Publication Date
CN114702439A true CN114702439A (en) 2022-07-05
CN114702439B CN114702439B (en) 2023-11-10

Family

ID=82174173

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210405822.2A Active CN114702439B (en) 2021-12-13 2022-04-18 Naphthyl urea-piperazine compound and preparation method and application thereof

Country Status (2)

Country Link
CN (1) CN114702439B (en)
WO (1) WO2023109004A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214106A1 (en) * 2021-04-08 2022-10-13 河南省锐达医药科技有限公司 Naphthyl urea compound having anti-cancer effect, preparation method therefor, and use thereof
CN116102514A (en) * 2023-01-17 2023-05-12 河南省锐达医药科技有限公司 Naphthalene amide compound and preparation method and application thereof

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043384A1 (en) * 1999-01-19 2000-07-27 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
CN110526881A (en) * 2019-08-27 2019-12-03 河南省锐达医药科技有限公司 A kind of naphthylamine compounds and its acceptable salt of biology, preparation method and application
CN111559991A (en) * 2020-06-01 2020-08-21 河南省锐达医药科技有限公司 Preparation method and application of naphthylamine compound and salt thereof
CN113444035A (en) * 2021-04-08 2021-09-28 河南省锐达医药科技有限公司 Naphthyl urea compounds with brand new chemical structure and preparation method and application thereof

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000043384A1 (en) * 1999-01-19 2000-07-27 Boehringer Ingelheim Pharmaceuticals, Inc. Aromatic heterocyclic compounds as antiinflammatory agents
CN110526881A (en) * 2019-08-27 2019-12-03 河南省锐达医药科技有限公司 A kind of naphthylamine compounds and its acceptable salt of biology, preparation method and application
CN111559991A (en) * 2020-06-01 2020-08-21 河南省锐达医药科技有限公司 Preparation method and application of naphthylamine compound and salt thereof
CN113444035A (en) * 2021-04-08 2021-09-28 河南省锐达医药科技有限公司 Naphthyl urea compounds with brand new chemical structure and preparation method and application thereof

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022214106A1 (en) * 2021-04-08 2022-10-13 河南省锐达医药科技有限公司 Naphthyl urea compound having anti-cancer effect, preparation method therefor, and use thereof
CN116102514A (en) * 2023-01-17 2023-05-12 河南省锐达医药科技有限公司 Naphthalene amide compound and preparation method and application thereof
CN116102514B (en) * 2023-01-17 2024-03-15 河南省锐达医药科技有限公司 Naphthalene amide compound and preparation method and application thereof

Also Published As

Publication number Publication date
WO2023109004A1 (en) 2023-06-22
CN114702439B (en) 2023-11-10

Similar Documents

Publication Publication Date Title
CN114702439A (en) Naphthylurea-piperazine compounds and preparation method and application thereof
UA124271C2 (en) PROCESS FOR THE PREPARATION OF PYRAZOLO[1,5-a]PYRIMIDINES AND SALTS THEREOF
CN110028475B (en) Novel CDK9 inhibitor based on benzopyran structure, preparation method and application thereof
CN113444035B (en) Naphthyl urea compounds with anticancer effect and preparation method and application thereof
CN110526881B (en) Naphthylamine compound and biologically acceptable salt thereof, and preparation method and application thereof
WO2022166994A1 (en) Naphthyl urea compound, preparation method therefor and use thereof
EP4361141A1 (en) Protein degradation agent
CN113387892B (en) Imidazole heterocyclic derivative containing nitrogen mustard and preparation method and application thereof
Zhou et al. Synthesis and evaluation of novel 12-aryl berberine analogues with hypoxia-inducible factor-1 inhibitory activity
Sun et al. Design and synthesis of β-carboline derivatives with nitrogen mustard moieties against breast cancer
CN109369620B (en) Pyridine compound, preparation method thereof and application thereof in resisting gastric cancer
CN110885332B (en) PDE delta protein degradation targeting chimera and preparation method and application thereof
CN113582864B (en) PRMTI type methyltransferase inhibiting active compound and preparation and application thereof
CN114133390B (en) Harmine derivative as well as preparation method and application thereof
Zhu et al. Design, synthesis and antitumor activity evaluation of Chrysamide B derivatives
CN110272464B (en) Compound, preparation method thereof and application thereof in preparing antitumor drugs
CN111484495B (en) Preparation method and application of derivative containing dihydropteridine diketone framework
CN114539129B (en) Allylamine bifunctional compound and application thereof
CN115475253B (en) Lapatinib-cancer cell stem inhibitor conjugate, preparation method, pharmaceutical composition and application
CN114380800B (en) Pyridine-pyrimidine amine-benzimidazole derivative, and preparation method and application thereof
CN117263851A (en) Pyridine-naphthyl urea-piperazine derivative, and preparation method and application thereof
CN115304605B (en) Oxetane derivatives with antitumor activity, and preparation method and application thereof
CN116178248A (en) Naphthyl urea-piperidine compounds, and preparation method and application thereof
CN115611867B (en) (1, 1-Trichloro-2) carbamate derivative and preparation method and application thereof
CN109251189B (en) 3-piperazinyl chalcone derivatives, pharmaceutical compositions thereof and application thereof

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant