CN112679470B - (E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof - Google Patents
(E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof Download PDFInfo
- Publication number
- CN112679470B CN112679470B CN202110011243.5A CN202110011243A CN112679470B CN 112679470 B CN112679470 B CN 112679470B CN 202110011243 A CN202110011243 A CN 202110011243A CN 112679470 B CN112679470 B CN 112679470B
- Authority
- CN
- China
- Prior art keywords
- substituted
- ddo
- phenyl
- compound
- group
- 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.)
- Active
Links
Images
Abstract
The invention discloses (E) -1-phenyl 4-alkenyl-1H-pyrazole compounds and application thereof, belonging to the field of pharmaceutical chemistry. The compounds can be covalently combined with a specific sulfydryl on Hsp90 protein, and are used for treating various diseases mediated by Hsp90, such as cancer or other proliferative diseases.
Description
Technical Field
The invention belongs to the field of pharmaceutical chemistry, and particularly relates to a series of (E) -1-phenyl 4-alkenyl-1H-pyrazole Hsp90 inhibitors and application thereof.
Background
The heat shock protein Hsp90 is a highly conserved molecular chaperone, in normal cells of a body, the expression of the Hsp90 can accurately regulate and control the steady state of cell functions such as gene expression, cell proliferation, cell cycle and the like, but in tumor cells, the abnormal regulation and control of the Hsp90 can cause imbalance of the intracellular steady state and cause error folding and over expression of client proteins, thereby promoting the generation and development of tumors. Therefore, the development of Hsp90 inhibitors is an effective way to suppress tumor cell growth.
Post-transcriptional modification of Hsp90 plays an important role in regulating the conformational changes of Hsp90, the binding of co-chaperones and the assembly of client proteins. Research shows that various kinases or endogenous molecules can modify amino acid residues (such as cysteine, serine and tyrosine) of Hsp90 in a manner of phosphorylation, acetylation, nitration, oxidation and ubiquitination, so that multiple downstream signal pathways are regulated to promote tumor occurrence and development. The literature reports that exogenous molecules such as natural products containing Michael acceptors or NO donors can be covalently bound to Hsp90 to form Hsp 90-small molecule covalent complexes, and the covalent modification can improve the energy barrier of the conformational transition, regulate the binding of chaperone proteins and client proteins to Hsp90, thereby selectively inhibiting the function of Hsp90 and playing an anti-tumor role. However, the design of small molecule inhibitors based on the Hsp90 covalent modification principle has not been studied and reported.
Of the Hsp90 inhibitors that have been developed, more than ten small molecules have entered clinical research, and their mechanism of action is mainly competitive binding to the ATP pocket at the N-terminus of Hsp90, thereby inhibiting the atpase activity of Hsp 90. The inhibitor shows strong toxic and side effects or limited treatment effects in clinical tests, so that a novel Hsp90 inhibitor is developed based on protein post-transcriptional modification, the selectivity of the inhibitor is improved by partially regulating and controlling the function of Hsp90, an idea is provided for the development of the Hsp90 inhibitor, and the inhibitor has potential clinical application prospects.
Disclosure of Invention
The invention aims to provide a (E) -1-phenyl 4-alkenyl-1H-pyrazole compound which can be used as an Hsp90 inhibitor and used for researching and developing medicaments for Hsp90 related tumors.
In order to achieve the purpose, the invention adopts the following technical scheme:
a compound having the general formula (I) or a pharmaceutically acceptable salt thereof,
R 1 selected from substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted C 3 -C 10 Cycloalkyl, or substituted or unsubstituted 3 to 11 membered heterocycloalkyl; the above-mentioned substituted aryl, substituted heteroaryl, substituted C 3 -C 10 The number of substituents in cycloalkyl, substituted 3-to 11-membered heterocycloalkyl is 1 to 3, and the above substituents are each independently selected from C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, nitrile, C 1 -C 6 Alkoxycarbonyl, nitro, carboxyl or C 1 -C 6 A haloalkyl group;
R 2 selected from H, C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, amino, nitro, nitrile or C 1 -C 6 A haloalkyl group;
R 3 selected from-C (= O) NR a R b 、-C(＝O)OR c OR-S (= O) OR d ，
Wherein: r a 、R b Each independently selected from H, C unsubstituted or substituted by 3-to 8-membered heterocycloalkyl 1 -C 6 Alkyl, and satisfy R a And R b Not H at the same time;
or, R a 、R b Together with the N atom to which they are attached form a 5-to 8-membered heterocycloalkyl optionally substituted by R e R f N-C (= O) -substitution, wherein R e And R f Each independently is H or C 1 -C 6 An alkyl group;
R c selected from C unsubstituted or substituted by 3-to 8-membered heterocycloalkyl 1 -C 6 Alkyl radical, C 4 -C 10 Cycloalkyl, or 3 to 8 membered heterocycloalkyl;
R d is a substituted or unsubstituted aryl group in which the number of substituents is 1 to 3, and each of the above substituents is independently C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy radicalNitrile group, C 1 -C 6 Alkoxycarbonyl, nitro, carboxyl or haloalkyl.
Preferably, R 1 Selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted C 5 -C 7 Cycloalkyl, or substituted or unsubstituted 3 to 11 membered heterocycloalkyl; substituted phenyl, substituted pyridyl, substituted furyl, substituted thienyl, substituted C 5 -C 7 The substituents in the cycloalkyl group and the substituted heterocycloalkyl group are 1 or 2, and each of the above substituents is independently selected from C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, nitrile, C 1 -C 6 Alkoxycarbonyl, nitro, carboxyl, or C 1 -C 6 A haloalkyl group.
Preferably, R 2 Selected from H, C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, or C 1 -C 6 A haloalkyl group.
Preferably, R a 、R b Each independently selected from H, unsubstituted C 1 -C 6 Alkyl, or is substituted by
Substituted C 1 -C 6 Alkyl, and satisfy R a And R b Not H at the same time;
or, R a 、R b Together with the N atom to which they are attached
The
The
The device is
Each independently of the others is optionally substituted by H 2 N-C (= O) -substitution.
Preferably, R c Selected from unsubstituted C 1 -C 6 Alkyl, cyclopentyl, cyclohexyl, or are substituted by
Substituted C 1 -C 6 An alkyl group.
Preferably, R d Selected from unsubstituted phenyl, or by C 1 -C 6 Alkyl or halogen substituted phenyl.
More specifically, the present invention also provides the following specific compounds useful as Hsp90 inhibitors:
in another aspect of the invention, there is provided a process for the preparation of the compound, as follows:
(1) When R is 2 When the compound is H, the preparation method comprises the following steps:
the method comprises the following steps that a raw material I-1 generates an intermediate I-2 under the action of phenylhydrazine and acetic acid, the intermediate I-2 reflows in the presence of phosphorus oxychloride and DMF to generate Vilsmeier-Haack reaction, and then the pH =7 is adjusted by 2M NaOH solution to generate an intermediate I-3; then, the intermediate 1-3 and malonic acid are subjected to reflux reaction under the catalysis of pyridine to obtainAn intermediate I-4; intermediates I-4 and R b R a NH reacts overnight at room temperature under the catalysis of a Kate condensing agent BOP and DIPEA to obtain a corresponding amide target product; or intermediates I-4 and R c OH is refluxed in the presence of oxalyl chloride to obtain a corresponding target product of esters; or intermediates I-4 and R d SO 3 Na is refluxed in the presence of iodobenzene diacetic acid to obtain a corresponding sulfone target product;
(2) When R is 2 When other substituent groups are adopted, the preparation method comprises the following steps:
reacting phenylboronic acid compounds II-1 and 3-bromoimidazole in dioxane under the action of copper acetate and pyridine to generate an intermediate II-2, then reacting II-2 and 3-methoxyphenylboronic acid in dimethyl ether under the catalysis of 1,1' -bis (diphenylphosphino) ferrocene palladium dichloride and sodium carbonate to obtain an intermediate II-3, refluxing the intermediate II-3 in the presence of phosphorus oxychloride and DMF to generate Vilsmeier-Haack reaction, then adjusting the pH =7 by 2M NaOH solution to generate an intermediate II-4, and reacting the intermediate II-4 and malonic acid in a reflux manner under the catalysis of pyridine to obtain an intermediate II-5; and reacting the intermediate II-5 and 2-piperidine formamide at room temperature overnight under catalysis of a Kate condensing agent BOP and DIPEA to obtain a target product II-6.
The compound of the general formula I or the pharmaceutically acceptable salt thereof can be used for preparing medicines for treating or preventing diseases mediated by Hsp 90.
Further, the disease mediated by Hsp90 is breast cancer, colon cancer, gastric cancer or liver cancer.
A pharmaceutical composition comprising a compound of formula I or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier or excipient.
Has the advantages that:
the reactivity of a cysteine sulfydryl at 598 position of Hsp90 is determined by a computational chemistry means in the previous research, then a lead compound DDO-6600 is obtained by covalent virtual screening and experimental verification based on a structure and a Michael addition mechanism, a secondary mass spectrum LC-MS/MS experiment proves that the lead compound DDO-6600 can covalently modify 598 position of Hsp90, alpha, beta-unsaturated ketone in the structure is used as a Michael addition receptor to form a covalent bond with cysteine residue at 598 position, and the intermediate antitumor activity is shown in vitro.
The structural formula of compound DDO-6600 is as follows:
the compound of the invention as an Hsp90 inhibitor can be clinically applied in oral administration, intravenous injection and other administration modes. Can be clinically used for single-drug therapy, and can also be combined with other clinically used therapeutic means such as chemotherapy drugs, radiotherapy and the like for treating the cancers. The clinical dosage of the compound of the invention is 0.01 mg-1000 mg/day, and the dosage can deviate from the range according to the severity of the disease condition or different dosage forms.
The invention discloses (E) -1-phenyl 4-alkenyl-1H-pyrazole Hsp90 covalent inhibitors and medical application thereof, wherein the inhibitors can cause Hsp90 protein dysfunction through a covalent binding mechanism, and regulate and control the degradation of client protein at the downstream of Hsp90, thereby inhibiting the growth of tumor cells. The invention provides a thought for the design of a novel Hsp90 inhibitor, and has potential medicinal value and development prospect; meanwhile, the micromolecule regulating agent disclosed by the invention can be used for designing a chemical probe, and provides a basis for further researching protein posttranscriptional modification.
Drawings
FIG. 1A is an LC-MS/MS secondary mass spectrum of cysteine residue of compound DDO-6600 covalently modified Hsp90, in which "CCIVTSTYGWTANMER" represents the amino acid fragment identified by DDO-6600 by secondary mass spectrum; "y", "b" and "a" represent ion fragments formed by different fragmentation patterns, respectively; "" indicates a cysteine residue forming a covalent bond with DDO-6600; b is LC-MS/MS secondary mass spectrum obtained by mutating cysteine at position 598 of Hsp90 into alanine and incubating with compound DDO-6600, wherein the figure is 'LVTSPCAIVTSTY'Representing the corresponding amino acid fragments identified by the secondary mass spectrum; "a" and "b" represent ion fragments formed by different fragmentation patterns, respectively, "-NH3" indicates the removal of a molecule of ammonia, "-H 2 O' represents the removal of one molecule of water.
FIG. 2 is a graph showing the effect of compound DDO-6600 on the thermal stability of Hsp 90C-terminal protein;
FIG. 3 is a graph of the effect of compound DDO-6600 on a client protein downstream of Hsp90 in HCT116 cells;
FIG. 4 is a graph of the effect of compound DDO-6647 on the thermal stability of Hsp 90C-terminal protein;
FIG. 5 is a graph of the effect of compound DDO-6647 on the client protein downstream of Hsp90 in HCT116 cells.
Detailed Description
The invention is described in further detail below with reference to the figures and the examples, but the invention should not be construed as being limited thereto. Modifications or substitutions to methods, procedures, or conditions of the invention may be made without departing from the spirit and scope of the invention. The experimental methods and reagents of the formulations not specified in the examples are in accordance with the conventional conditions in the art.
Definition of terms
The term "alkyl" as used herein refers to monovalent saturated groups remaining after removal of one hydrogen atom from a saturated alkane, including straight and branched chain monovalent saturated groups of 1 to 6 carbon atoms. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, n-pentyl, isopentyl, n-hexyl, isohexyl and the like.
The term "cycloalkyl" as used herein denotes a monovalent non-aromatic cyclic hydrocarbon group of 3 to 8 carbons. Examples of cycloalkyl groups are cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and the like.
The term "heterocycloalkyl" as used herein denotes a non-aromatic radical having 3 to 8 atoms in the cycloalkyl radical, containing 1 to 2 heteroatoms in the heterocycle, and selected from nitrogen, oxygen or sulfur atoms. The heterocycloalkyl radical may be unsubstituted or substituted by C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, nitrile, C 1 -C 6 Alkoxycarbonyl, nitro, carboxyl, or C 1 -C 6 Haloalkyl substitution.
The term "C" as used herein 1 -C 6 Haloalkyl "refers to a C containing at least one, e.g., one, two or more, halogen atoms including a halogen atom selected from fluorine (-F), chlorine (-Cl), and bromine (-Br) 1 -C 6 An alkyl group.
As used herein, the term "alkoxy" refers to the substituent-OR, where R is C 1 -C 6 An alkyl group. Examples of alkoxy groups are methoxy, ethoxy, propoxy, isopropoxy, n-butoxy, isobutoxy, tert-butoxy, n-pentoxy, isopentoxy, n-hexoxy, isohexoxy and the like.
The term "alkoxycarbonyl" as used herein denotes a carbonyl-substituted alkoxy group as defined herein. Examples of alkoxycarbonyl are methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl, isopropoxycarbonyl, n-butoxycarbonyl, isobutoxycarbonyl, tert-butoxycarbonyl, n-pentyloxycarbonyl, isopentyloxycarbonyl, n-hexyloxycarbonyl, isohexyloxycarbonyl.
The term "aryl" as used herein denotes an unsaturated aromatic group having 6 carbon atoms, which aryl group may be unsubstituted or substituted with one or two substituents independently selected from the group consisting of: (1) a C1-C6 alkyl group; (2) halogen; (3) a C1-C6 alkoxy group; (4) an amino group; (5) a nitro group; (6) a nitrile group; (7) trifluoromethyl.
The term "heteroaryl" as used herein denotes a 5-or 6-membered ring containing at least one, e.g. one, two or three heteroatoms selected from nitrogen, oxygen and sulfur. The 5-membered ring has 2 double bonds, and the 6-membered ring has three double bonds. Examples of heteroaryl groups may be thienyl, furyl and pyridyl.
The term "halogen" as used herein denotes a group selected from fluorine (-F), chlorine (-Cl) and bromine (-Br).
The term "amino" as used herein means-NH 2 A group.
The term "nitro" as used herein means-NO 2 A group.
The term "nitrile group" as used herein denotes the group — CN.
The term "carboxy" as used herein denotes a-C (= O) OH group.
Example 1
(E) Preparation of (E) -1- (3- (1,3-diphenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6601)
(1) Preparation of (E) -3- (1,3-diphenyl-1H-pyrazol-4-yl) acrylic acid
Acetophenone (5 g, 42mmol) was dissolved in 30ml of dichloromethane, phenylhydrazine (5.4 g, 50mmol) and 1ml of glacial acetic acid were added, stirred at room temperature for 1 hour, concentrated and recrystallized from methanol to give 6.8g of yellow powder. The resulting yellow powder was dissolved in 20ml DMF and POCl was slowly added dropwise under ice bath 3 (6 g, 40mmol), the solution was transferred to 70 ℃ and reacted for 6 hours. And (3) concentrating the reaction solution, adding 10 times of water by volume, adjusting the pH to be =7 by using 2M sodium hydroxide solution, separating out a large amount of solid, carrying out suction filtration and water washing for three times, and recrystallizing a filter cake by using methanol to obtain 2.4g of light yellow solid. The pale yellow solid was dissolved in 20ml of pyridine, malonic acid (2g, 20mmol) was added thereto, and the mixture was refluxed for 6 hours, and then the reaction mixture was poured into 200ml of water to precipitate 1.8g of a pale yellow solid in 62% yield.
(2) Preparation of the title Compound
The (E) -3- (1,3-diphenyl-1H-pyrazol-4-yl) acrylic acid (10.12mmol, 0.500g, 1eq) was dissolved in 5ml of DMF, a Kate condensing agent (2 eq), 2-piperidinecarboxamide (2 eq) and DIPEA (3 eq) were added, 100ml of water was added after completion of the reaction, EA was extracted 3 times, the organic phases were combined, washed with saturated brine, dried over sodium sulfate, and column chromatography (PE: EA = 2:1) was performed to obtain 0.34g of a white solid with a yield of 56.4%. m.p.210-211 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.28(s,1H),7.82(dd,J＝11.7,3.4Hz,3H),7.73(d,J＝7.1Hz,2H),7.48(ddd,J＝11.6,10.2,6.0Hz,5H),7.38(t,J＝7.4Hz,1H),6.76(d,J＝15.3Hz,1H),5.55–5.31(m,1H),3.92(d,J＝14.0Hz,1H),3.18(t,J＝12.6Hz,1H),2.33(d,J＝13.2Hz,1H),1.80(dd,J＝26.3,12.2Hz,4H),1.87–1.54(m,6H).HRMS(ESI):calad for C 24 H 24 N 4 O 2 [M+H] + 401.1972,found 401.19691.HPLC:(80:20methanol:water with 1‰TFA):t R ＝10.055min,96.956％.
example 2
(E) Preparation of (E) -1- (3- (1-phenyl-3-m-tolyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6602)
This was obtained from (E) -3- (1-phenyl-3- (m-tolyl) -1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide by the method of example 1, to give 0.27g of a white solid in 52% yield. m.p.209-211 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.23(s,1H),7.86–7.69(m,3H),7.57–7.43(m,4H),7.36(q,J＝7.6Hz,2H),7.25(d,J＝7.5Hz,1H),6.72(d,J＝15.4Hz,1H),6.32(s,1H),5.51–5.23(m,1H),3.88(d,J＝13.3Hz,1H),3.14(t,J＝12.8Hz,1H),2.44(s,3H),2.30(d,J＝13.4Hz,1H),1.67(ddd,J＝29.9,26.0,12.2Hz,6H).HRMS(ESI):calad for C 25 H 26 N 4 O 2 [M+H] + 415.21285,found 415.21258.HPLC:(80:20methanol:water with 1‰TFA):t R ＝9.088min,96.883％.
example 3
(E) Preparation of (E) -1- (3- (1-phenyl-3-p-tolyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6603)
This was obtained from (E) -3- (1-phenyl-3- (p-tolyl) -1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide by the method of example 1, to give 0.22g of a white solid in 66.4% yield. m.p.210-211 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.22(s,1H),7.77(dd,J＝11.6,3.6Hz,3H),7.57(d,J＝7.8Hz,2H),7.48(t,J＝7.8Hz,2H),7.31(dd,J＝18.2,8.1Hz,3H),6.71(d,J＝15.3Hz,1H),6.31(s,1H),5.33(d,J＝24.3Hz,1H),3.89(d,J＝13.6Hz,1H),3.13(t,J＝12.9Hz,1H),2.41(s,3H),2.29(d,J＝13.0Hz,1H),1.89–1.38(m,6H).HRMS(ESI):calad for C 25 H 26 N 4 O 2 [M+H] + 415.21285,found 415.21258.HPLC:(80:20methanol:water with 1‰TFA):t R ＝9.054min,96.476％.
example 4
(E) Preparation of (E) -1- (3- (3- (3-fluorophenyl) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6604)
This was prepared from (E) -3- (3- (3-fluorophenyl) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide by the method of example 1, to give 0.21g of a white solid in 58% yield. m.p.198-200 deg.c; 1 H NMR(300MHz,CDCl 3 )δ8.24(s,1H),7.80–7.73(m,3H),7.53–7.40(m,5H),7.36(t,J＝7.4Hz,1H),7.12(ddd,J＝9.3,6.8,2.8Hz,1H),6.75(d,J＝15.4Hz,1H),5.46–5.19(m,1H),3.92(d,J＝13.9Hz,1H),3.17(t,J＝12.6Hz,1H),2.30(d,J＝12.0Hz,1H),1.61(dd,J＝41.3,27.1Hz,6H).HRMS(ESI):calad for C 24 H 23 FN 4 O 2 [M+Na] + 441.16973,found 441.16982.HPLC:(80:20methanol:water with 1‰TFA):t R ＝10.418min,96.551％.
example 5
(E) Preparation of (E) -1- (3- (3- (3-methoxybenzene) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6608)
This was prepared from (E) -3- (3- (3-methoxybenzene) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide according to the method of example 1, to give 0.34g of a pale yellow solid in 56.4% yield. m.p.211-212 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.23(s,1H),7.78(d,J＝8.2Hz,3H),7.49(t,J＝7.7Hz,2H),7.43–7.30(m,2H),7.26–7.21(m,2H),6.98(dd,J＝8.1,1.9Hz,1H),6.73(d,J＝14.9Hz,1H),6.29(s,1H),5.30(s,1H),3.88(s,1H),3.87(s,3H),3.14(t,1H),2.30(d,J＝12.4Hz,1H),1.65(dd,J＝47.4,13.2Hz,6H).HRMS(ESI):calad for C 25 H 26 N 4 O 3 [M+H] + 431.20777,found 431.20759.HPLC:(80:20methanol:water with 1‰TFA):t R ＝9.74 5min,96.014％.
example 6
(E) Preparation of (E) -1- (3- (3- (3-cyanophenyl) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6609)
This was obtained from (E) -3- (3- (3-cyanophenyl) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide according to the method of example 1, to give 0.32g of a pale yellow solid in 66% yield. m.p.210-212 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.27(s,1H),8.04(s,1H),7.92(d,J＝7.8Hz,1H),7.82–7.67(m,4H),7.56(dt,J＝25.1,8.0Hz,3H),7.38(t,J＝7.4Hz,1H),6.78(d,J＝15.3Hz,1H),6.26(s,1H),5.31(s,1H),3.94(d,J＝14.7Hz,1H),3.21(t,J＝13.3Hz,1H),2.30(d,J＝13.4Hz,1H),1.67(d,J＝53.2Hz,6H).HRMS(ESI):calad for C 25 H 23 N 5 O 2 [M+Na] + 448.1744,found 448.17424.HPLC:(80:20methanol:water with 1‰TFA):t R ＝9.247min,96.358％.
example 7
(E) Preparation of (E) -1- (3- (3- (3,4-dimethylphenyl) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6600)
This was prepared from (E) -3- (3- (3,4-dimethylphenyl) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide as in example 1 to give 0.24g of a pale yellow solid in 52% yield. m.p.211-212 deg.C； 1 H NMR(300MHz,CDCl 3 )δ8.21(s,1H),7.81–7.70(m,3H),7.47(t,J＝7.7Hz,3H),7.34(dt,J＝14.8,7.5Hz,2H),7.29–7.19(m,1H),6.70(d,J＝15.3Hz,1H),6.32(s,1H),5.35(d,J＝37.5Hz,1H),3.87(d,J＝13.3Hz,1H),3.13(t,J＝12.7Hz,1H),2.32(d,J＝3.8Hz,6H),1.71(d,J＝12.0Hz,4H),1.52(m,6H).HRMS(ESI):calad for C 26 H 28 N 4 O 2 [M+H] + 429.2285,found 429.22805.HPLC:(80:20methanol:water with 1‰TFA):t R ＝10.055min,96.956％.
Example 8
(E) Preparation of (E) -1- (3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6613)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide according to the method of example 1 to give 0.15g of a white solid in 46% yield. m.p.200-201 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.99(s,1H),8.66(s,1H),8.23(s,1H),8.00(d,J＝7.7Hz,1H),7.78(d,J＝7.8Hz,2H),7.65(d,J＝15.4Hz,1H),7.50(t,J＝7.4Hz,2H),7.43–7.31(m,2H),6.73(d,J＝15.4Hz,1H),3.57(d,J＝44.0Hz,4H),1.63(d,J＝20.0Hz,4H).HRMS(ESI):calad for C 23 H 23 N 5 O 2 [M+H] + 402.19485,found 402.19827.HPLC:(80:20methanol:water with 1‰TFA):t R ＝11.428min,96.951％.
example 9
(E) Preparation of (E) -1- (3- (3- (furan-2-yl) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6616)
This was prepared from (E) -3- (3- (furan-2-yl) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide as in example 1 to give a brown solid0.17g in bulk, 59% yield. m.p.200-202 deg.c; 1 H NMR(300MHz,CDCl 3 )δ8.14(s,1H),7.97(d,J＝15.3Hz,1H),7.68(d,J＝7.7Hz,2H),7.56–7.22(m,5H),6.80(s,1H),6.74(d,J＝15.5Hz,1H),3.94(d,J＝13.0Hz,1H),3.13(t,J＝12.6Hz,1H),2.36–2.08(m,1H),1.59(d,J＝54.5Hz,7H).HRMS(ESI):calad for C 22 H 22 N 4 O 3 [M+H] + 391.17647,found 391.17599.HPLC:(80:20methanol:water with 1‰TFA):t R ＝8.854min,96.753％.
example 10
(E) Preparation of (E) -1- (3- (3- (thiophen-2-yl) -1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6617)
This was prepared from (E) -3- (3- (thiophen-2-yl) -1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide by the method of example 1 to give 0.17g of a brown solid in 59% yield. m.p.198-200 deg.c; 1 H NMR(300MHz,CDCl 3 )δ8.22(s,1H),7.90(d,J＝15.3Hz,1H),7.76(d,J＝7.9Hz,2H),7.41(ddt,J＝22.8,14.6,7.4Hz,5H),7.18–7.10(m,1H),6.79(d,J＝15.3Hz,1H),6.36(s,1H),5.56–5.26(m,1H),3.98(d,J＝13.2Hz,1H),3.19(t,J＝12.7Hz,1H),2.31(d,J＝13.7Hz,1H),1.62(dd,J＝37.7,24.4Hz,6H).HRMS(ESI):calad for C 23 H 28 N 4 O 2 [M+H] + 407.15362,found 407.15348.HPLC:(80:20methanol:water with 1‰TFA):t R ＝8.928min,97.254％.
example 11
(E) Preparation of (E) -1- (3- (3-cyclohexyl-1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6619)
This product was prepared from (E) -3- (3-cyclohexyl-1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide as in example 1Obtained, 0.17g of a brown solid was obtained, yield 59%. m.p.198-199 deg.C; 1 H NMR(300MHz,CDCl 3 )δ8.10(s,1H),7.70(t,J＝11.5Hz,3H),7.44(t,J＝7.8Hz,2H),7.33–7.24(m,2H),6.68(d,J＝15.3Hz,1H),4.01(d,J＝13.3Hz,1H),3.20(t,J＝12.9Hz,1H),2.86(t,J＝11.7Hz,1H),2.32(d,J＝13.7Hz,1H),1.99–1.36(m,16H).HRMS(ESI):calad for C 24 H 30 N 4 O 2 [M+H] + 407.24415,found 407.24414.HPLC:(80:20methanol:water with 1‰TFA):t R ＝11.241min,96.964％.
example 12
(E) Preparation of (E) -1- (3- (3-cyclopentyl-1-phenyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6618)
This was prepared from (E) -3- (3-cyclopentyl-1-phenyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide according to the method of example 1, to give 0.17g of a brown solid in 59% yield. m.p.200-201 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.12(s,1H),7.78–7.65(m,3H),7.44(t,J＝7.8Hz,2H),7.29(d,J＝7.4Hz,2H),6.70(d,J＝15.3Hz,1H),6.33(s,1H),5.34(s,1H),4.02(d,J＝14.2Hz,1H),3.19(t,J＝13.0Hz,1H),2.32(d,J＝13.3Hz,1H),2.15–1.45(m,14H).HRMS(ESI):calad for C 23 H 28 N 4 O 2 [M+H] + 393.221,found 393.22051.HPLC:(80:20methanol:water with 1‰TFA):t R ＝11.975min,97.274％.
example 13
(E) Preparation of (E) -1- (3- (3- (3-methoxyphenyl) -1-m-tolyl-1H-pyrazol-4-yl) acryloyl) piperidine-2-carboxamide (DDO-6627)
(1) Preparation of (E) -3- (3- (3-methoxyphenyl) -1-m-tolyl-1H-pyrazol-4-yl) acrylic acid
M-methylbenzeneboronic acid (10g, 73mmol) was dissolved in 30ml of dioxane, 3-bromo-1H-pyrazole (5.3g, 36mmol) and copper acetate (10g, 54mmol) were added, and after addition of 5ml of pyridine, reflux reaction was performed overnight, after concentration, 50ml of water was added and extraction was performed three times with ethyl acetate, organic phases were combined, dried over anhydrous sodium sulfate and subjected to sand making, column chromatography (PE: EA = 20) gave a white solid in a yield of 45%. The resulting white solid and M-methoxyphenylboronic acid (7.2g, 48mmol) were dissolved in 10ml of DME, and the reaction mixture was poured into 200ml of water after overnight reflux reaction under catalysis of 1,1' -bisdiphenylphosphinoferrocene palladium dichloride (0.6 g, 0.05mmol) and 2M sodium carbonate solution, extracted three times with ethyl acetate, concentrated, dried over anhydrous sodium sulfate, and subjected to column chromatography (PE: EA = 5:1) to give 1.8g of a pale yellow solid with a yield of 45%. The light yellow solid obtained above is dissolved in 10ml DMF, POCl is slowly dropped under ice bath condition 3 (6 g, 40mmol), the solution was stirred at room temperature for 1h and transferred to an oil bath and reacted at 70 ℃ for 6h. After the reaction was completed, the reaction solution was concentrated and 10 times the volume of water was added, pH =7 was adjusted with 2M sodium hydroxide solution, a large amount of solid was precipitated, filtered with suction through a buchner funnel, and washed with water three times to obtain 2.2g of a pale yellow solid. The pale yellow solid was dissolved in 20ml of pyridine, malonic acid (2g, 20mmol) was added thereto, and after refluxing under heating for 6 hours, the reaction mixture was poured into 200ml of water to precipitate 1.8g of a pale yellow solid, whereby 1.2g of intermediate (E) -3- (3- (3-methoxyphenyl) -1-m-tolyl-1H-pyrazol-4-yl) acrylic acid was obtained in 68% yield.
(2) Preparation of the title Compound
This was prepared from (E) -3- (3- (3-methoxyphenyl) -1-m-tolyl-1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide by the method of example 1, to give 0.17mg of a white solid in a yield of 61%. m.p.210-211 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.20(s,1H),7.78(d,J＝15.4Hz,1H),7.66(d,J＝8.2Hz,2H),7.39(t,J＝8.1Hz,1H),7.30(s,1H),7.26(d,J＝6.7Hz,3H),6.98(dd,J＝8.0,2.2Hz,1H),6.72(d,J＝15.4Hz,1H),6.33(s,1H),3.89(d,J＝5.7Hz,4H),2.93(d,J＝21.8Hz,1H),2.41(s,3H),1.81–1.42(m,6H).HRMS(ESI):calad for C 26 H 28 N 4 O 3 [M+H] + 445.2238,found 445.22346.HPLC:(80:20methanol:water with 1‰TFA):t R ＝13.287min,96.835％.
example 14
(E) Preparation of ethyl (E) -3- (4- (3- (2-carbamoylpiperidin-1-yl) -3-oxoprop-1-en-1-yl) -1-phenyl-1H-pyrazol-3-yl) benzoate (DDO-6612)
This was prepared from 3- (1-phenyl-3- (piperidin-3-yl) -1H-pyrazol-4-yl) acrylic acid and 2-piperidinecarboxamide according to the method of example 1, to give 0.17g of a brown solid in 59% yield. m.p.198-199 deg.C; 1 H NMR(300MHz,CDCl 3 )δ8.38(s,1H),8.24(s,1H),8.10(d,J＝7.8Hz,1H),7.85(d,J＝7.6Hz,1H),7.82–7.73(m,3H),7.70(s,1H),7.61–7.43(m,3H),7.35(t,J＝7.4Hz,1H),6.72(d,J＝15.3Hz,1H),6.30(s,1H),5.32(d,J＝15.3Hz,2H),4.41(q,J＝7.1Hz,2H),3.86(d,J＝13.5Hz,1H),3.14(t,J＝11.4Hz,1H),2.29(d,J＝11.1Hz,1H),1.71(d,J＝12.2Hz,5H),1.44(dt,J＝11.6,8.1Hz,6H).HRMS(ESI):calad for C 27 H 28 N 4 O 4 [M+Na] + 495.20028,found 495.20018.HPLC:(80:20methanol:water with 1‰TFA):t R ＝12.892min,97.682％.
example 15
(E) Preparation of (E) -3- (1-phenyl-3- (piperidin-3-yl) -1H-pyrazol-4-yl) -1- (piperidin-1-yl) prop-2-en-1-one (DDO-6635)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and piperidine according to the procedure in example 1 to give 0.18g of a pale yellow solid in 49% yield. m.p.188-189 ℃; 1 H NMR(300MHz,CDCl 3 )δ9.00(s,1H),8.67(d,J＝4.0Hz,1H),8.23(s,1H),8.02(dt,J＝7.8,1.8Hz,1H),7.75(dd,J＝22.2,11.5Hz,3H),7.51(t,J＝7.9Hz,2H),7.39(dt,J＝14.8,6.2Hz,2H),6.67(d,J＝15.4Hz,1H),3.63(t,J＝6.2Hz,2H),3.54–3.47(m,2H),1.79(d,J＝5.5Hz,2H),1.71(s,1H),1.60(s,1H),1.59(s,2H).HRMS(ESI):calad for C 22 H 22 N 4 O[M+H] + 359.18664,found 359.18649.HPLC:(80:20methanol:water with 1‰TFA):t R ＝7.691min,95.982％.
example 16
(E) Preparation of (E) -3- (1-phenyl-3- (piperidin-3-yl) -1H-pyrazol-4-yl) -1- (pyrrolidin-1-yl) prop-2-en-1-one (DDO-6634)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and cyclopentylamine according to the method of example 1, to give 0.14g of a pale yellow solid in 44% yield. m.p.187-189 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.97(s,1H),8.66(s,1H),8.19(s,1H),8.02(d,J＝7.8Hz,1H),7.75(d,J＝7.8Hz,2H),7.61(d,J＝15.5Hz,1H),7.43(ddd,J＝29.2,15.1,7.4Hz,4H),6.20(d,J＝15.5Hz,1H),5.61(s,1H),4.32(dd,J＝13.9,6.9Hz,1H),2.04(dd,J＝12.2,5.8Hz,2H),1.74–1.56(m,4H),1.42(dd,J＝15.6,9.5Hz,2H).HRMS(ESI):calad for C 21 H 20 N 4 O[M+H] + 345.16792,found 345.16788.HPLC:(80:20methanol:water with 1‰TFA):t R ＝7.258min,96.158％.
example 17
(E) Preparation of (E) -1- (aza-1-yl) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) prop-2-en-1-one (DDO-6636)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and cycloheximide according to the method of example 1, to give 0.15g of a pale yellow solid in 46% yield. m.p.190-191 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.99(s,1H),8.66(s,1H),8.22(s,1H),8.01(d,J＝7.5Hz,1H),7.74(dd,J＝22.4,11.6Hz,3H),7.41(ddd,J＝23.8,14.0,7.5Hz,4H),6.70(d,J＝15.4Hz,1H),3.62(t,J＝6.0Hz,2H),3.49(t,J＝5.7Hz,2H),1.73(d,J＝21.5Hz,4H),1.58(s,4H).HRMS(ESI):calad for C 23 H 24 N 4 O[M+H] + 373.20191,found 373.20187.HPLC:(80:20methanol:water with 1‰TFA):t R ＝12.374min,95.975％.
example 18
(E) Preparation of (E) -N- (2-morpholinoethyl) -3- (3-phenyl-1- (pyridin-3-yl) -1H-pyrazol-5-yl) acrylamide (DD 0-6638)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and N- (2-aminoethyl) morpholine by the method of example 1 to give 0.16g of a pale yellow solid in 51% yield. m.p.195-196 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.97(s,1H),8.67(d,J＝4.6Hz,1H),8.24(s,1H),8.00(dt,J＝7.9,1.9Hz,1H),7.73(t,J＝17.2Hz,2H),7.63(d,J＝15.6Hz,1H),7.50(t,J＝7.8Hz,2H),7.39(dt,J＝15.1,6.1Hz,2H),6.29(d,J＝15.5Hz,1H),3.73(d,J＝3.8Hz,4H),3.49(s,2H),2.65–2.41(m,6H).HRMS(ESI):calad for C 23 H 25 N 5 O 2 [M+H] + 404.2081,found 404.20795.HPLC:(80:20methanol:water with 1‰TFA):t R ＝14.285min,96.237％.
example 19
(E) Preparation of (E) -N- (pent-3-yl) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylamide (DDO-6639)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and 3-aminopentane according to the method of example 1 to give 0.16g of a pale yellow solid in 50% yield. m.p.190-192 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.99(s,1H),8.67(s,1H),8.21(s,1H),8.02(d,J＝7.8Hz,1H),7.75(d,J＝7.8Hz,2H),7.63(d,J＝15.5Hz,1H),7.54–7.33(m,4H),6.25(d,J＝15.5Hz,1H),5.30(d,J＝8.5Hz,1H),3.92(d,J＝7.9Hz,1H),1.65–1.54(m,2H),1.41(dd,J＝14.1,7.2Hz,2H),0.93(t,J＝7.4Hz,6H).HRMS(ESI):calad for C 21 H 22 N 4 O[M+H] + 361.1824,found 361.18251.HPLC:(80:20methanol:water with 1‰TFA):t R ＝12.982min,96.057％.
example 20
Preparation of isopropyl (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylate (DDO-6642)
This was prepared by dissolving (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid (200 mg) in 3ml of anhydrous dichloromethane, slowly adding oxalyl chloride (173mg, 1.4 mmol) dropwise in an ice bath and stirring at room temperature for 30min, then slowly adding isopropanol 3ml dropwise, refluxing for 4H, concentrating, adding 30ml of water and extracting with ethyl acetate three times, combining the organic layers, drying over anhydrous sodium sulfate, and performing column chromatography (PE: EA = 1:3) to obtain 0.24g of a pale yellow solid with a yield of 68%. m.p.194-195 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.96(s,1H),8.69(d,J＝3.8Hz,1H),8.26(s,1H),8.01(dt,J＝7.8,1.8Hz,1H),7.77(d,J＝7.9Hz,2H),7.66(d,J＝16.0Hz,1H),7.55–7.34(m,4H),6.28(d,J＝15.9Hz,1H),5.12(dt,J＝12.5,6.3Hz,1H),1.29(t,J＝6.8Hz,6H).HRMS(ESI):calad for C 20 H 19 N 3 O 2 [M+H] + 334.155,found 334.15477.HPLC:(80:20methanol:water with 1‰TFA):t R ＝13.417min,96.951％.
example 21
(E) Preparation of cyclohexyl (DDO-6645) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylate (DDO-6645)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and cyclohexanol by the method of example 20 to give pale yellowYellow solid 0.21g, yield 55%. m.p.190-192 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.98(s,1H),8.69(s,1H),8.28(s,1H),8.03(d,J＝7.8Hz,1H),7.78(d,J＝7.7Hz,2H),7.67(d,J＝16.0Hz,1H),7.56–7.34(m,4H),6.31(d,J＝15.9Hz,1H),4.89(dd,J＝8.7,3.9Hz,1H),1.90(s,2H),1.75(d,J＝4.0Hz,2H),1.46–1.25(m,6H).HRMS(ESI):calad for C 23 H 23 N 3 O 2 [M+H] + 374.1863,found 374.18694.HPLC:(80:20methanol:water with 1‰TFA):t R ＝14.287min,98.958％.
example 22
Preparation of 2- (piperidin-1-yl) ethyl (E) -3- (3-phenyl-1- (pyridin-3-yl) -1H-pyrazol-5-yl) acrylate (DDO-6646)
This was prepared from (E) -3- (1-phenyl-3- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and N-hydroxyethylpiperidine according to the method of example 20 to give 0.16g of a pale yellow solid in 52% yield. m.p.190-192 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.93(d,J＝1.6Hz,1H),8.68(dd,J＝4.8,1.5Hz,1H),8.30(s,1H),8.00(dt,J＝7.8,1.9Hz,1H),7.73(dd,J＝24.1,11.9Hz,3H),7.58–7.29(m,4H),6.32(d,J＝16.0Hz,1H),4.44(t,J＝5.7Hz,2H),2.83(dd,J＝23.3,17.6Hz,2H),2.68(s,4H),1.73(dd,J＝11.0,5.5Hz,4H),1.49(t,J＝11.2Hz,2H).HRMS(ESI):calad for C 24 H 26 N 4 O 2 [M+H] + 403.21285,found 403.21286.HPLC:(80:20methanol:water with 1‰TFA):t R ＝15.972min,96.332％.
example 23
(E) Preparation of (E) -3- (1-phenyl-4- (2- (phenylsulfonyl) ethenyl) -1H-pyrazol-3-yl) piperidine (DDO-6647)
The product is prepared from (E) -3- (1-phenyl- (pyridine-3-yl) -1H-pyrazol-4-yl) acrylic acid(200mg, 0.68mmol) and sodium benzenesulfonate (490mg, 2.7mmol) were dissolved in 5ml of DMF solvent, iodobenzene diacetic acid (450mg, 1.4 mmol) was added and heated to 100 ℃ to react for 4 hours, the reaction solution was concentrated to 1ml and 30ml of water was added, ethyl acetate was extracted three times and the organic layer was combined, washed with saturated brine, dried over sodium sulfate, the organic layer was spin-dried, made into sand, and subjected to column chromatography (PE: EA = 1:2) to obtain 200mg of a white solid with a yield of 76%. m.p.220-221 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.93(s,1H),8.72(d,J＝4.3Hz,1H),8.23(s,1H),8.01(d,J＝7.8Hz,1H),7.98–7.89(m,2H),7.80–7.67(m,2H),7.68–7.45(m,7H),7.38(t,J＝7.4Hz,1H),6.72(d,J＝15.3Hz,1H).HRMS(ESI):calad for C 22 H 17 N 3 O 2 S[M+H] + 388.10281,found 388.10277.HPLC:(80:20methanol:water with 1‰TFA):t R ＝8.263min,96.586％.
example 24
(E) Preparation of (E) -3- (1-phenyl-4- (2- (p-toluenesulfonyl) vinyl) -1H-pyrazol-3-yl) piperidine (DDO-6648)
This was prepared from (E) -3- (1-phenyl- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and sodium p-toluenesulfonate according to the method of example 23 to give 0.22mg of a pale yellow solid in a yield of 66%. m.p.218-220 ℃; 1 H NMR(300MHz,DMSO)δ9.25(s,1H),8.80(d,J＝42.6Hz,2H),8.08(d,J＝7.9Hz,1H),7.84(dd,J＝19.2,8.1Hz,4H),7.69–7.52(m,3H),7.44(td,J＝14.8,6.7Hz,4H),7.30(d,J＝15.1Hz,1H),2.41(s,3H).HRMS(ESI):calad for C 23 H 19 N 3 O 2 S[M+H] + 402.1288,found 402.12871.HPLC:(80:20methanol:water with 1‰TFA):t R ＝9.575min,96.259％.
example 25
Preparation of 3- (1-phenyl-4- (2- (p-chlorobenzenesulfonyl) ethenyl) -1H-pyrazol-3-yl) piperidine (DDO-6649)
This was prepared from 3- (1-phenyl- (pyridin-3-yl) -1H-pyrazol-4-yl) acrylic acid and sodium p-chlorobenzenesulfonate as in example 23 to give 0.27mg of a pale yellow solid in 79% yield. m.p.224-225 ℃; 1 H NMR(300MHz,CDCl 3 )δ8.90(s,1H),8.71(s,1H),8.24(s,1H),7.94(dd,J＝12.7,5.6Hz,3H),7.80–7.69(m,2H),7.68–7.54(m,3H),7.53–7.33(m,4H),6.73(d,J＝15.3Hz,1H).HRMS(ESI):calad for C 22 H 18 N 3 O 2 S[M+H] + 422.07355,found 422.07303.HPLC:(80:20methanol:water with 1‰TFA):t R ＝8.147min,97.224％.
example 26
LC-MS/MS identification of Hsp90 protein binding to compounds
Hsp90 protein and DDO-6600 (according to 1:6 molar ratio in 37 ℃ environment after incubation for 4h, 200. Mu.g of incubated protein was dissolved in 2M urea, 50mM ammonium bicarbonate solution (pH 8.0), 2mM DTT was added and incubated in 56 ℃ water bath for 30min, and 10mM IAA was incubated away from light for 30min after incubation at 37 ℃ the sample was degraded with sequencing trypsin (Sigma) for 16h, and then concentrated by ultrafiltration centrifugation. The sample was separated using a NanoAcquity UPLC (Waters, ford, USA Mill) system equipped with a Waters Acquity M-Class UPLC HST 3 chromatography column (75. Mu.m. Times.150mm, 1.8 μ M). The mobile phase A: ultrapure water containing 0.1 FA; B: acetonitrile containing 0.1 FA, flow rate: 5% mobile phase B (5 min), mobile phase B (40% mobile phase B; 10% mobile phase MS, TOF-10% mobile phase assay using TOF-MS, TOF-5% mobile phase assay, TOF-5% fragment.
The test result shows that the experiment identifies that the 597-612 peptide segment in the Hsp90 amino acid sequence is modified by DDO-6600 (b) 3 + M/z =804.34 Da), which shows that DDO-6600 covalently modifies the site 597 or 598 of the cysteine residue of Hsp90 protein (as in a of fig. 1), and when the mutation of the cysteine residue 598 to alanine is detected on Hsp90 protein, the peptide segment 592-604 in Hsp90 amino acid sequence is identified by secondary mass spectrometryThe molecular weight change of the modified DDO-6600 (shown as B in figure 1) shows that DDO-6600 interacts with Hsp90 protein through a covalent binding mechanism and is an Hsp90 inhibitor with a brand new action mechanism.
Example 27
Protein thermostability assay for Hsp90 binding Activity (Thermal shift assay)
The fluorescent dye is gathered in a hydrophobic pocket of the protein, the gradient temperature rise causes the gradual reduction of the stability of the protein, the three-dimensional structure is loose and the fluorescent dye is released, thereby obtaining a protein melting curve which is approximately in normal distribution, and the temperature T corresponding to the peak value m . The protein bound to the ligand has increased thermal stability, resulting in a change in the rate at which it releases the fluorescent dye, which shifts the melting curve of the protein by an offset Δ T m Can reflect whether the test compound binds to the protein. The assay was performed using 96-well plates (Life technologies, CF98PV 02) with a final volume of 20. Mu.L per well. mu.L of Hsp 90C-terminal protein (final concentration 2. Mu.M), 1. Mu.L of compound dilution and 16. Mu.L of assay buffer were added to each well, and after incubation for 1h at room temperature, 2. Mu.L of 8 Xfluorescent dye (SYPRO Orange dye, thermo Fisher Scientific) were added, four replicates per compound, and negative controls (1. Mu.L of His-Hsp90C protein and 19. Mu.L of assay buffer) and blank controls (2. Mu.L of 8 Xfluorescent dye and 18. Mu.L of assay buffer) were set for each experiment. After sealing, a gradient heating program was performed using a PCR instrument (StepOnePlus, applied Biosystems) at a temperature of 2 ℃ per minute from 37 ℃ to 99 ℃. Data analysis was performed after the end of the procedure using Protein Thermal Shift software v1.3 (Thermo Fisher Scientific). Delta T m1 Represents the shift in the melting curve of the Hsp 90C-terminal protein from that of the blank when bound to the compound non-covalently, when Δ T m1 <At 2 ℃, the compound is considered not to be bound to the protein; when the compound is covalently bound to the Hsp 90C-terminal protein, the presence of the protein-small molecule adduct causes a biphasic shift in the melting curve, resulting in a Δ T m2 . Protein melting curves for DDO-6600 and DDO-6647 are shown in FIG. 2 and FIG. 4, respectively, and the results of representative compound testing are shown in Table 1.
Table 1 Thermal shift assay of representative compounds
a And (3) testing the protein melting curve of the compound after incubation for 4h with the His-Hsp 90C-terminal protein.
The test result shows that the protein melting curve delta T of the test compounds, namely DDO-6600, DDO-6613, DDO-6635, DDO-6636, DDO-6647, DDO-6648 and DDO-6649 is incubated with His-Hsp90C protein for 4 hours m2 Indicates that the protein is covalently bound to the protein within 4 h; DDO-6602, DDO-6604, DDO-6608, DDO-6612, DDO-6634, DDO-6638, DDO-6639, DDO-6642, DDO-6645 and DDO-6646 are mainly combined by non-covalent acting on Hsp90C terminal protein.
Example 28
Antiproliferative activity assay of tumor cells (MTT assay)
The compounds of the invention were tested for their antiproliferative activity on the human colon cancer cell line HCT 116. HCT116 was first seeded into 96-well plates (approximately 4000 cells per well), incubated for 24h at 37 deg.C, 5% CO2, compounds diluted in medium at various concentrations were added to the cells, incubated for 72h, 20. Mu.L MTT solution (5 mg/ml, 0.22. Mu.M filter sterilized after PBS formulation) was added to each well, after further incubation for 4h at 37 deg.C, medium was removed, 150. Mu.L DMSO was added to each well, absorbance was read at 490nM using Molecular devices SpectraMax i3X, and the results were analyzed using GraphPad Prism 6.0, and IC was calculated 50 . Representative compound test results are shown in table 2.
Table 2 MTT test results for representative compounds
| Compound numbering | MTT experiment IC50 (μ M) | Compound numbering | MTT experiment IC50 (μ M) |
| DDO-6600 | 7.83±1.24 | DDO-6627 | 14.21±1.87 |
| DDO-6601 | 16.55±3.35 | DDO-6634 | 10.11±0.42 |
| DDO-6602 | 12.12±1.87 | DDO-6635 | 8.11±0.85 |
| DDO-6603 | 14.93±1.32 | DDO-6636 | 7.17±0.16 |
| DDO-6604 | 13.20±1.17 | DDO-6638 | 10.18±0.25 |
| DDO-6608 | 12.34±1.42 | DDO-6639 | 10.25±1.31 |
| DDO-6609 | 15.65±1.86 | DDO-6642 | 12.94±1.49 |
| DDO-6612 | 13.12±1.74 | DDO-6645 | 12.03±1.52 |
| DDO-6613 | 7.12±1.22 | DDO-6646 | 12.20±1.37 |
| DDO-6616 | 22.79±2.54 | DDO-6647 | 4.98±0.34 |
| DDO-6617 | 18.46±2.50 | DDO-6648 | 6.77±0.96 |
| DDO-6618 | 19.21±2.45 | DDO-6649 | 7.28±0.43 |
| DDO-6619 | 18.02±1.58 |
The test results show that the antiproliferative activity of DDO-6613, DDO-6635 and DDO-6636 is equivalent to that of DDO-6600, and the antiproliferative activity of DDO-6647, DDO-6648 and DDO-6649 is improved.
Example 29
Effect of the Compounds DDO-6600 and DDO-6647 on Hsp90 and its downstream client proteins (Western Blotting)
When the HCT116 cells in the culture dish are fused to reach 80%, the cells are treated by the compounds with different concentrations and incubated for 24 hours at 37 ℃, the cells are collected by centrifugation, the RIPA lysate is cracked on ice for 45min, then the supernatant is collected by centrifugation, the BCA protein quantification method is adopted, the detection is carried out after the sample preparation of the 1 Xprotein loading buffer solution, and the beta-actin is used as an internal reference.
The experimental results show that the compounds DDO-6600 and DDO-6647 can effectively reduce the level of Hsp90 client protein, and the inhibition effect is concentration-dependent. While compound treatment did not cause the up-regulation of Hsp70, indicating that the compound did not cause a heat shock response in the cells, as shown in fig. 3 and 5.
Claims (6)
1. A compound of formula I or a pharmaceutically acceptable salt thereof,
R 1 selected from substituted or unsubstituted phenyl, substituted or unsubstituted pyridyl, substituted or unsubstituted furyl, substituted or unsubstituted thienyl, substituted or unsubstituted C 5 -C 7 Cycloalkyl, or substituted or unsubstituted 3 to 11 membered heterocycloalkyl; the above-mentioned substituted phenyl group, substituted pyridyl group, substituted furyl group, substituted thienyl group, substituted C 5 -C 7 The substituents in the cycloalkyl group, the substituted 3-to 11-membered heterocycloalkyl group are 1 or 2, and the above substituents are each independently selected from C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, nitrile, C 1 -C 6 Alkoxycarbonyl radicalsRadical, nitro, carboxyl, or C 1 -C 6 A haloalkyl group;
R 2 selected from H, C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, amino, nitro, nitrile or C 1 -C 6 A haloalkyl group.
2. A compound of formula I or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: r 2 Selected from H, C 1 -C 6 Alkyl, halogen, C 1 -C 6 Alkoxy, or C 1 -C 6 A haloalkyl group.
3. The compound of formula I, or a pharmaceutically acceptable salt thereof, according to claim 1, characterized in that:
the compound of formula I is selected from the following compounds:
4. use of a compound of general formula I according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof for the manufacture of a medicament for the treatment or prevention of a disease mediated by Hsp 90.
5. Use according to claim 4, characterized in that: the disease mediated by Hsp90 is breast cancer, colon cancer, stomach cancer or liver cancer.
6. A pharmaceutical composition for treating or preventing a disease mediated by Hsp90, wherein: comprising a compound of general formula I, or a pharmaceutically acceptable salt thereof, as claimed in any one of claims 1-3, and a pharmaceutically acceptable carrier or excipient.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110011243.5A CN112679470B (en) | 2021-01-06 | 2021-01-06 | (E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110011243.5A CN112679470B (en) | 2021-01-06 | 2021-01-06 | (E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112679470A CN112679470A (en) | 2021-04-20 |
| CN112679470B true CN112679470B (en) | 2023-01-13 |
Family
ID=75457422
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110011243.5A Active CN112679470B (en) | 2021-01-06 | 2021-01-06 | (E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112679470B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| GB0229618D0 (en) * | 2002-12-19 | 2003-01-22 | Cancer Rec Tech Ltd | Pyrazole compounds |
| EP1682143A2 (en) * | 2003-10-28 | 2006-07-26 | Pharmacia Corporation | Combinations comprising an hsp90 inhibitor and a phophodiesterase inhibitor for treating or preventing neoplasia |
-
2021
- 2021-01-06 CN CN202110011243.5A patent/CN112679470B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN112679470A (en) | 2021-04-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106916101B (en) | NAMPT/HDAC double-target inhibitor and preparation method thereof | |
| JP6783663B2 (en) | New glutaminase inhibitor | |
| ES2671354T3 (en) | DNA-PK inhibitors | |
| CN104080455A (en) | Certain chemical entities, compositions, and methods | |
| HUE027149T2 (en) | Pyrrolidinones as metap2 inhibitors | |
| US11325908B2 (en) | CaMKII inhibitors and uses thereof | |
| JP2021523168A (en) | Cancer treatments that target cancer stem cells | |
| AU2014225889B2 (en) | CaMKII inhibitors and uses thereof | |
| CN112300082B (en) | Phenyl piperazine quinazoline compound or pharmaceutically acceptable salt thereof, preparation method and application | |
| CN110003204B (en) | BET protein inhibitor, preparation method and application thereof | |
| WO2016023330A1 (en) | Quinazoline derivative | |
| CN103102352B (en) | Tyrosine kinase inhibitor indolinone derivative | |
| CN103833756A (en) | Pyridazinone compound as well as preparation method and application thereof | |
| CN112521336B (en) | Indazole and pyrrolopyridine compounds and application thereof | |
| CN112679470B (en) | (E) -1-phenyl-4-alkenyl-1H-pyrazole compound and medical application thereof | |
| CN114437113B (en) | Thiazolopyridine cyclotriazole compound, and preparation method and application thereof | |
| CN110551102B (en) | ALK covalent inhibitors and uses thereof | |
| CN108329232B (en) | Hydrazide derivative and application thereof | |
| CN102617478B (en) | Synthesis of benzimidazole, oxazole and thiazole derivatives and application thereof | |
| MX2015002310A (en) | Novel phenyl-pyridine/pyrazine amides for the treatment of cancer. | |
| CN111247143B (en) | Pyridoquinazoline derivatives useful as inhibitors of protein kinases | |
| CN114573567B (en) | Indazole cyclotriazole compound and preparation method and application thereof | |
| CN114573562B (en) | Niacin-containing triazole compound and preparation method and application thereof | |
| CN108383837A (en) | A kind of amino substituted-tetrahydro pyrido-pyrimidines or its available salt and the preparation method and application thereof | |
| CN108239081A (en) | A kind of compound for inhibiting ROCK and its application |
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 |