Compounds as protein phosphatase 5 agonists and preparation methods and uses thereof

Abstract

The present invention belongs to the field of pharmaceutical chemistry, and specifically relates to a class of compounds shown in formula I or formula II as protein phosphatase 5 agonists, preparation methods thereof, and pharmaceutical compositions containing these compounds and applications of these compounds or compositions in drug preparation. The present invention provides a class of 2-imine-2H-chromene-3-thioamide protein phosphatase (PP5) agonists and derivatives thereof, by discovering active small molecules with novel structures and good physicochemical properties, and the small molecules have shown good activity in various experiments, directly targeting PP5 to produce agonistic effects, and provide a new solution for the treatment of related diseases caused by PP5 abnormalities, and is expected to be developed into specific drugs for the treatment of Alzheimer's disease, obesity caused by glucocorticoid treatment, and cancer caused by protease dephosphorylation dysfunction, and has clinical application value.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a class of 2-imine-2H-chromene-3-thioamide protein phosphatase 5 (PP5) agonists and their derivatives, and their use in preparing drugs for related diseases caused by PP5 abnormalities. Background Art

[0002] Protein phosphatase 5 (PP5) is a serine / threonine protein phosphatase that mainly regulates the dephosphorylation process of proteins. Under normal physiological conditions, PP5 is usually in an autoinhibitory state. In this state, the TPR domain and catalytic domain at the N-terminus of the PP5 protein interact with each other, so that the catalytic site of the phosphatase activity of PP5 is completely covered, thereby preventing the substrate from entering the catalytic active site. In addition, the αJ helix at the C-terminus interacts with the TPR domain to further stabilize the autoinhibitory state of PP5. At present, the reported PP5 activators can be divided into TPR-dependent activators and non-TPR-dependent activators. TPR-dependent activators include long-chain fatty acids with more than 16 carbons, suramin, and the MEEVD motif at the C-terminus of Hsp90 (heat shock protein 90). This type of activator allosterically activates PP5 by binding to the TPR domain of PP5. Currently, only one compound, P5SA-2, has been reported as a non-TPR-dependent activator, which activates PP5 by binding to the non-TPR allosteric activation site of PP5.

[0003] PP5 activation plays a vital role in cells. For example, a downward trend in PP5 activation levels was found in cells from patients with Alzheimer's disease and in visceral cells with excessive fat accumulation due to glucocorticoid treatment. The reason may be that the reduction of endogenous activating substances in these cells leads to a decrease in PP5 activity, which in turn causes the substrate protein of PP5 to be overphosphorylated, leading to its normal function being disordered and accelerating the occurrence and development of the disease. Therefore, PP5 agonists can regulate the dephosphorylation of overphosphorylated proteins to achieve the purpose of treating a series of diseases caused by dysfunctional dephosphorylation.

[0004] At present, the research on PP5-mediated diseases mainly focuses on the development of its inhibitors, because PP5 overexpression has been found to varying degrees in many different cancer cells. However, the research on PP5 agonism is equally important. Because the activity of PP5 must be maintained at a normal level, overexpression or overinhibition will lead to different diseases. In addition, there is no particularly in-depth research on PP5 activation. The currently discovered PP5 activators have the following problems: (1) TPR-dependent activators are non-specific PP5 activators, which are prone to off-target effects and thus lead to a series of toxic side effects; (2) The activators reported so far all have the problem of poor drugability, such as the poor membrane permeability of the MEEVD sequence and the poor water solubility of long-chain fatty acids; (3) The non-TPR-dependent PP5 small molecule agonist activity is weak and it is difficult to efficiently activate the activity of PP5, while the TPR-dependent small molecules will affect the co-chaperone function of PP5 and cannot play their due function. In addition, low PP5 activity can lead to a series of signal transduction problems, thereby causing a variety of diseases. Therefore, PP5 agonists with high selectivity and strong agonistic effects can help further understand the role of PP5 in cell signal transduction, so as to solve many diseases caused by low PP5 activity, and have great clinical research prospects. Summary of the invention

[0005] The main technical problem solved by the present invention is to provide a class of 2-imine-2H-chromene-3-thioamide PP5 agonists and their derivatives, which can be used for preparing treatments for related diseases caused by PP5 abnormalities.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0007] In the first aspect of the present invention, a compound represented by formula I or formula II is provided,

[0008]

[0009] in:

[0010] R in Formula I 1-4 Independently selected from hydrogen atom, halogen, alkyl, alkoxy, nitro, amino, hydroxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; wherein the substituted or unsubstituted heteroaryl is selected from:

[0011] The substituted or unsubstituted aryl group in Formula I is

[0012] In Formula I, R 8-12are independently selected from hydrogen, halogen, alkyl, alkoxy, nitro, amino, hydroxy, cyano, aryl, substituted or unsubstituted heteroaryl; the substituted or unsubstituted heteroaryl is selected from:

[0013] In formula I, R5 is selected from hydrogen atom, alkyl, alkoxy, amide, sulfonamide;

[0014] In formula I, R6 is selected from hydrogen atom, alkyl, alkoxy, aldehyde, amide, sulfonamide;

[0015] In formula I, X1 is a carbon atom or a nitrogen atom; X2 is an oxygen atom or a sulfur atom; X3 is selected from a carbon atom, an oxygen atom, and a sulfur atom;

[0016] R in Formula II 1-4 are independently selected from hydrogen atom, halogen, alkyl, alkoxy, nitro, amino, hydroxyl, cyano, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; the substituted or unsubstituted heteroaryl is selected from:

[0017] The substituted or unsubstituted aryl group in Formula II is

[0018] In formula II, R 8-12 are independently selected from hydrogen, halogen, alkyl, alkoxy, nitro, amino, hydroxy, cyano, aryl, substituted or unsubstituted heteroaryl; the substituted or unsubstituted heteroaryl is selected from:

[0019] In formula II, R6 is selected from hydrogen atom, alkyl, alkoxy, aldehyde, amide, sulfonamide;

[0020] In formula II, R7 is selected from hydrogen atom, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl; wherein the substituted or unsubstituted heteroaryl is selected from: The substituted or unsubstituted aryl group is Where R 8-12 Independently selected from hydrogen atom, halogen, alkyl, alkoxy, nitro, amino, hydroxyl, cyano, aryl, substituted or unsubstituted heteroaryl; wherein the substituted or unsubstituted heteroaryl is selected from:

[0021] In formula II, X1 is a carbon atom or a nitrogen atom; X2 is an oxygen atom or a sulfur atom; and X3 is selected from a carbon atom, an oxygen atom, and a sulfur atom.

[0022] In certain preferred embodiments, the compound is any one of the following compounds or a pharmaceutically acceptable salt thereof:

[0023]

[0024]

[0025] In another aspect, the present invention provides use of the above-mentioned compound as a protein phosphatase 5 agonist or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating / preventing PP5-related diseases.

[0026] In yet another aspect, the present invention provides a pharmaceutical composition comprising a compound of the present invention, or a pharmaceutically acceptable salt thereof, or optionally a pharmaceutically acceptable carrier, excipient, diluent, adjuvant, vehicle, or a combination thereof.

[0027] In another aspect, the present invention provides a method for preparing the compound as shown in Formula I, wherein the reaction formula of the preparation method is as follows:

[0028]

[0029] Among them, X1, X2, X3, R 1-4 , R5, R6 as described in claim 1;

[0030] The specific steps include:

[0031] After raw materials I-1 and I-2 are dissolved in isopropanol, piperidine is used as a catalyst to undergo Knoevenagel condensation reaction and nucleophilic addition reaction to generate different target products I-3; I-3 undergoes nucleophilic substitution reaction with alkyl / alkoxyamine hydrochloride using acetic acid as solvent and catalyst, or undergoes Suzuki coupling with different types of commercially available borate compounds, or undergoes Buchwald-Hartwing coupling with commercially available substituted or unsubstituted aniline compounds to generate target compound I-4; I-4 undergoes amide condensation reaction with different alkyl acid solutions to generate compounds of formula I;

[0032] The present invention also provides a method for preparing the compound of formula II, the reaction formula of which is as follows:

[0033]

[0034] Among them, X1, X2, X3, R 1-4 , R6, R7 as described in claim 1;

[0035] The specific steps include:

[0036] The compound II-1 is prepared by the synthesis method of the formula I. II-1 and commercially available formaldehyde compounds undergo Knoevenagel condensation reaction and nucleophilic substitution reaction in the presence of piperidine as a catalyst and tetrahydrofuran as a solvent to generate the compound of formula II.

[0037] Another aspect of the present invention provides the use of the compound of the present invention, or a pharmaceutically acceptable salt thereof, in the preparation of a medicament for treating any one or more of Alzheimer's disease, obesity, and cancer caused by dysfunctional dephosphorylation of certain transcription factors or proteases.

[0038] definition

[0039] Definitions of specific functional groups and chemical terms are described in more detail below.

[0040] As described herein, the compounds of the present invention may be optionally substituted with one or more substituents, such as the general formula compounds of the present invention, or as the specific examples, subclasses, and classes of compounds included in the embodiments. It should be understood that the term "substituted and unsubstituted" can be used interchangeably with the term "optionally substituted". In general, unless otherwise indicated, an optionally substituted group can have a substituent substituted at each substitutable position of the group. When more than one position in the given structural formula can be substituted by one or more substituents selected from a specific group, the substituents can be substituted at each position in the same or different manner.

[0041] The term "alkyl" used in the present invention includes saturated straight or branched monovalent hydrocarbon groups of 1 to 6 carbon atoms, some examples of which are methyl and ethyl.

[0042] The term "alkoxy" as used herein refers to an -OR substituent, wherein R comprises C 1-6 Some examples are methoxy and ethoxy.

[0043] The term "aryl" as used in the present invention is an unsaturated aromatic group having 6 carbon atoms. The aryl group may be unsubstituted or substituted by one or two substituents independently selected from the following groups: (1) C1-C6 alkyl; (2) halogen; (3) C1-C6 alkoxy; (4) amino; (5) nitro; (6) cyano; (7) trifluoromethyl.

[0044] The term "heteroaryl" as used herein includes a 5- or 6-membered ring containing a heteroatom selected from nitrogen, oxygen and sulfur; the 5-membered ring has two double bonds and the 6-membered ring has three double bonds; some examples of said heteroaryl are pyridyl, thienyl, furanyl, pyrimidinyl, imidazolyl, quinolinyl and morpholine rings.

[0045] The term "halogen" is a group selected from fluorine (-F), chlorine (-Cl) and bromine (-Br). The term "amino" as used herein refers to a -NH2 group. The term "nitro" as used herein refers to a -NO2 group. The term "cyano" as used herein refers to a -CN group. The term "aldehyde" as used herein refers to a -CHO group. The term "amide" as used herein refers to a -NH2CO group. The term "sulfonamide" as used herein refers to a -NH2SO2 group.

[0046] Unless otherwise indicated, the structural formulas described in the present invention include all isomeric forms (such as enantiomers, diastereomers, and geometric isomers (or conformational isomers)); for example, R, S configurations containing asymmetric centers, (Z), (E) isomers of double bonds, and (Z), (E) conformational isomers. Therefore, single stereochemical isomers of the compounds of the present invention or mixtures of their enantiomers, diastereomers, or geometric isomers (or conformational isomers) are all within the scope of the present invention.

[0047] The term "pharmaceutically acceptable" as used herein refers to any substance that does not interfere with the effectiveness of the biological activity of the active ingredient and is non-toxic to the host to which it is administered.

[0048] Unless otherwise indicated, all tautomeric forms of the compounds of the present invention are included within the scope of the present invention. In addition, unless otherwise indicated, the structural formulas of the compounds described in the present invention include enriched isotopes of one or more different atoms.

[0049] The beneficial effects of the present invention are as follows: the present invention provides a class of compounds as PP5 agonists, experiments show that they directly produce agonistic effects on PP5, and active small molecules with novel structures and good physical and chemical properties are discovered. The small molecules have shown good activity in various experiments and are expected to be developed into specific drugs for the treatment of Alzheimer's disease, obesity caused by glucocorticoid treatment, and cancer caused by protease dephosphorylation dysfunction. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 EC of compounds DDO3728, DDO3733, DDO3750 and DDO3752 50 and E max Test data;

[0051] Figure 2 The ITC binding affinity activity of compound DDO3728 with PP5 truncated protein and PP5 full-length protein was evaluated;

[0052] Figure 3 ASK1 and p-ASK1 in HCT116 cells after administration of positive compound P5SA-2 at different concentrations for 24 hoursT838 , CDC37, p-CDC37 S13 Content;

[0053] Figure 4 ASK1 and p-ASK1 in HCT116 cells after administration of compound DDO3728 at different concentrations for 24 hours T838 , CDC37, p-CDC37 S13 Content;

[0054] Figure 5 ASK1 and p-ASK1 in HCT116 cells after administration of compound DDO3733 at different concentrations for 24 hours T838 , CDC37, p-CDC37 S13 Content;

[0055] Figure 6 ASK1 and p-ASK1 in HCT116 cells after administration of compound DDO3733 at 50 μM for different time periods T838 , CDC37, p-CDC37 S13 The content. DETAILED DESCRIPTION

[0056] The following examples are provided so as to provide those skilled in the art with a complete disclosure and description of how to perform, prepare and evaluate the methods and compounds claimed herein and are intended to merely illustrate the invention and not to limit the scope of the invention.

[0057] Synthesis method

[0058] Unless otherwise specified, the starting materials can be obtained from commercial sources, or prepared by methods known in the art, or prepared according to the methods described herein. The structures of the compounds were determined by nuclear magnetic resonance ( 1 H-NMR) and / or mass spectrometry (MS) were used for confirmation. NMR determination was performed using a Varian INOVA (300 MHz) or Bruker Advance (400 MHz) nuclear magnetic resonance instrument, with deuterated dimethyl sulfoxide (DMSO-d6) as the measuring solvent and TMS as the internal standard. MS determination was performed using a Waters-Q-Tof micro mass spectrometer. Silica gel column chromatography used 200-300 mesh silica gel from Qingdao Ocean Chemical Plant.

[0059] The preparation method of the compounds of this invention is described in more detail below, but these specific methods do not constitute any limitation to the present invention. The compounds of this invention can also be optionally combined with various synthetic methods described in this specification or known in the art and easily prepared, and such combination can be easily carried out by those skilled in the art to which the present invention belongs.

[0060] Example 1 Preparation of 2-imino-6-methoxy-2H-chromene-3-thioamide (Compound DDO3753)

[0061]

[0062] After dissolving 5-methoxysalicylaldehyde (1.52 g, 10 mmol) in isopropanol, add the isopropanol solution of 2-cyanothioacetamide (1 g, 10 mmol) to the isopropanol solution of 5-methoxysalicylaldehyde, add a catalytic amount of piperidine, and reflux the mixture with stirring at 45 °C for 1 h. After the reaction, solid precipitation is observed in the reaction mixture, which is filtered and the filter cake is rinsed with isopropanol 3 times to obtain 1.98 g of a yellow solid compound 2-imino-6-methoxy-2H-chromene-3-thioamide with a yield of 85%. 1 HNMR(300MHz,DMSO-d6)δ12.10(s,1H),9.13(s,1H),8.05(s,1H),7.65(s,1H),7.51(d,J=8 .6Hz,1H),6.81(d,J=10.5Hz,1H),6.67(s,1H),3.91(s,3H).HRMS(ESI):Found235.05379(C 11 H 11 N2O2S[M+H + ], requires 235.05357).

[0063] Example 2 Preparation of 2-imino-5-methoxy-2H-chromene-3-thioamide (Compound DDO3721)

[0064]

[0065] This product is synthesized from 6-methoxy salicylaldehyde and 2-cyanothioacetamide according to the method of Example 1, with a yield of 65%. 1 HNMR(300MHz,DMSO-d6)δ11.81(s,1H),10.34(s,1H),9.19(s,1H),9.15(s,1H),7.57(t,J=8.4H z,1H),6.91(d,J=8.4Hz,1H),6.82(d,J=8.3Hz,1H),3.98(s,3H).HRMS(ESI):Found235.05379(C 11 H 11 N2O2S[M+H + ], requires 235.05357).

[0066] Example 3 Preparation of 6-hydroxy-2-imino-2H-chromene-3-thioamide (Compound DD03724)

[0067]

[0068] This product is synthesized from 5-hydroxysalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 85%. 1 H NMR(300MHz,DMSO-d6)δ11.81(s,1H),10.34(d,2H),9.19(s,1H),9.15(s,1H),7.57(t,J =8.4Hz,1H),6.91(d,J=8.4Hz,1H),6.82(d,J=8.3Hz,1H).HRMS(ESI):Found221.03815(C 10 H8N2O2S[M+H + ], requires 221.03792).

[0069] Example 4 Preparation of 2-Imino-2H-chromene-3-thioamide (Compound DDO3715)

[0070]

[0071] This product is synthesized from salicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 32%. 1 H NMR (300MHz, DMSO-d6) δ10.38(s,1H),9.83(s,1H),8.98(s,1H),8.01(d,J=7.8Hz,1H),7.76(q,J=11.3,9.8Hz,2H),7.55–7.42(m,2H).HRMS(ESI): Found 205.04313(C 10 H8N2OS[M+H + ], requires 205.04301).

[0072] Example 5 Preparation of 2-imino-6-methyl-2H-chromene-3-thioamide (Compound DDO3722)

[0073]

[0074] This product is synthesized from 5-methyl salicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 65%. 1 H NMR(300MHz,DMSO-d6)δ11.82(s,1H),10.38(s,1H),9.13(s,1H),8.91(s,1H),7.63(s,1H) ,7.44(d,J=8.4Hz,1H),7.15(d,J=8.4Hz,1H),2.36(s,3H).HRMS(ESI):Found219.05893(C 11 H 10 N2OS[M+H + ], requires 219.05866).

[0075] Example 6 Preparation of 6,8-dibromo-2-imino-2H-chromene-3-thioamide (Compound DDO3754)

[0076]

[0077] This product is synthesized from 3,5-dibromosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 85%. 1 H NMR (300MHz, DMSO-d6) δ10.42 (s, 1H), 9.71 (s, 1H), 8.75 (s, 1H), 8.28 (d, J = 2.4Hz, 1H), 8.27 (d, J = 2.3Hz, 1H). HRMS (ESI): Found 358.85101 (C 10 H6Br2N2OS[MH + ], requires 358.84948).

[0078] Example 7 Preparation of 6-bromo-2-imino-8-methoxy-2H-chromene-3-thioamide (Compound DDO3718)

[0079]

[0080] This product is synthesized from 3-methoxy-5-bromosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 82%. 1 H NMR (300MHz, DMSO-d6) δ10.40(s,1H),9.78(s,1H),8.82(s,1H),7.81(d,J=2.1Hz,1H),7.59(d,J=2.1Hz,1H),3.99(s,3H).HRMS(ESI):Found 312.96624(C 11 H9BrN2O2S[M+H + ], requires 312.96409).

[0081] Example 8 Preparation of 6,8-dichloro-2-imino-2H-chromene-3-thioamide (Compound DDO3727)

[0082]

[0083] This product is synthesized from 3,5-dichlorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 83%. 1 H NMR (300MHz, DMSO-d6) δ10.44(s,1H),9.73(s,1H),8.79(s,1H),8.13(s,0H),8.09(s,1H).HRMS(ESI):Found 270.95129(C 10 H6Cl2N2OS[M+H + ], requires 270.95051).

[0084] Example 9 Preparation of Compound 6-Fluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3731)

[0085]

[0086] This product was synthesized from 5-fluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 84%. 1HNMR (300MHz, DMSO-d6) δ11.94 (s, 1H), 9.43 (s, 1H), 9.03 (d, J = 1.8Hz, 1H), 8.65 (s, 1H), 7.64 (dd, J = 8.3, 3.0Hz, 1H), 7.45-7.34 (m, 1H), 7.25 (dd, J = 9.0, 4.4Hz, 1H). HRMS (ESI): Found 223.03383 (C 10 H7FN2OS[M+H + ], requires 223.03359).

[0087] Example 10 Preparation of 6,8-difluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3728)

[0088]

[0089] This product is synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 83%. 1 HNMR(300MHz,DMSO-d6)δ10.45(s,1H),9.76(s,1H),8.81(s,1H),7.95–7.81(m,1H),7.78(d,J=6.6Hz,1H).HRMS(ESI):Found 241.02458(C 10 H6F2N2OS[M+H + ], requires 241.02417).

[0090] Example 11 Preparation of 2-imino-7-nitro-2H-chromene-3-thioamide (Compound DDO3755)

[0091] This product is synthesized from 4-nitrosalicylicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 85%. 1 H NMR (300MHz, DMSO-d6) δ10.25(s,1H),9.75(s,1H),8.28(s,1H),8.14(d,J=8.6Hz,1H ),8.10(s,1H),7.83(dd,J=8.7,2.3Hz,1H),7.77(d,J=2.2Hz,1H).HRMS(ESI):Found 250.02813(C 10 H7N3O3S[M+H +], requires 250.02809).

[0092] Example 12 Preparation of 5,7-difluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3733)

[0093]

[0094] This product is synthesized from 2,6-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 73%. 1 H NMR (300MHz, DMSO-d6) δ10.46 (s, 1H), 9.77 (s, 1H), 8.90 (s, 1H), 7.51 (d, J = 2.4Hz, 1H), 7.47 (d, J = 3.1Hz, 1H). HRMS (ESI): Found 241.02533 (C 10 H6F2N2OS[M+H + ], requires 241.02417).

[0095] Example 13 Preparation of 5-ethoxy-2-imino-2H-chromene-3-thioamide (Compound DDO3756)

[0096]

[0097] This product is synthesized from 6-ethoxy salicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 88%. 1 H NMR (300MHz, DMSO-d6) δ10.40(s,1H),9.25(s,1H),8.92(d,J=1.7Hz,1H),7.38(d,J=7.6Hz,1H),7.32(d,J=7.2H z,1H),7.22(d,J=7.8Hz,1H),7.17(d,J=5.2Hz,1H),4.24–4.17(m,2H),1.42(d,J=7.0Hz,3H).HRMS(ESI):Found 249.06955(C 12 H 12 N2O2S[M+H + ], requires 249.06922).

[0098] Example 14 Preparation of 5-bromo-2-imino-2H-chromene-3-thioamide (Compound DDO3725)

[0099]

[0100] This product was synthesized from 4-bromosalicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 73%. 1HNMR(300MHz, DMSO-d6)δ11.20(s,1H),10.12(s,1H),8.31(s,1H),7.93(d,J=8.3Hz,1H),7.63–7.35(m,1H),7.21(d,J=8.0Hz,2H).HRMS(ESI):Found 282.95148(C 10 H7BrN2OS[M+H + ], requires 282.95352).

[0101] Example 15 Preparation of 7-bromo-2-imino-2H-chromene-3-thioamide (Compound DDO3726)

[0102]

[0103] This product is synthesized from 6-bromosalicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 73%. 1 HNMR(300MHz,DMSO-d6)δ11.87(s,1H),9.47(s,1H),9.39(s,1H),8.78(s,1H),7 .58–7.55(m,1H),7.50(d,J=8.0Hz,1H),7.23(d,J=7.9Hz,1H).HRMS(ESI):Found 282.95627(C 10 H7BrN2OS[M+H + ], requires 282.95352).

[0104] Example 16 Preparation of 2-imino-7-methoxy-2H-chromene-3-thioamide (DD03720)

[0105]

[0106] This product is synthesized from 4-methoxy salicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 88%. 1 H NMR (300MHz, DMSO-d6) δ11.81(s,1H),10.34(s,1H),9.19(s,1H),9.15(s,1H),7.57(t,J=8.4Hz ,1H),6.91(d,J=8.4Hz,1H),6.82(d,J=8.3Hz,1H),3.98(s,3H).HRMS(ESI):Found235.05332(C 11 H 11 N2O2S[M+H + ], requires 235.053334).

[0107] Example 17 Preparation of 2-imino-8-methoxy-2H-chromene-3-thioamide (Compound DDO3719)

[0108]

[0109] This product is synthesized from 3-methoxy salicylaldehyde and 2-cyanothioacetamide according to Example 1 with a yield of 82%. 1 H NMR (300MHz, DMSO-d6) δ11.81(s,1H),10.34(s,1H),9.19(s,1H),9.15(s,1H),7.57(t,J=8.4Hz ,1H),6.91(d,J=8.4Hz,1H),6.82(d,J=8.3Hz,1H),3.98(s,3H).HRMS(ESI):Found235.05347(C 11 H 11 N2O2S[M+H + ], requires 235.05433).

[0110] Example 18 Preparation of 6,7-difluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3734)

[0111]

[0112] This product is synthesized from 5,6-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 78%. 1 H NMR (300MHz, DMSO-d6) δ11.99(s,1H),9.07(s,1H),8.25(s,1H),7.86(s,1H),7.44(t,J=8.8Hz,1H),7.04(dd,J=10.0,6.3Hz,1H).HRMS(ESI):Found 241.02411(C 10 H6F2N2OS[M+H + ], requires 241.02417).

[0113] Example 19 Preparation of 5-fluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3732)

[0114]

[0115] This product is synthesized from 6-fluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 77%. 1 HNMR(300MHz,DMSO-d6)δ12.03(s,1H),9.36(d,J=1.5Hz,1H),8.28(s,1H),7.87(s,1H),7.60–7.44(m,1H),6.98(t,J=8.1Hz,2H).HRMS(ESI):Found 223.03401(C 10 H7FN2OS[M+H+ ], requires 223.03359).

[0116] Example 20 Preparation of 2-imino-2H-pyrano[3,2-b]pyridine-3-thioamide (Compound DDO3716)

[0117]

[0118] This product is synthesized from 3-hydroxypyridinealdehyde and 2-cyanothioacetamide according to Example 1, with a yield of 32%. 1 H NMR(300MHz,DMSO-d6)δ10.04(s,1H),9.36(s,1H),8.28(s,1H),8.02(s,1H),7.87(s,1H),7.60–7.44(m,1H).HRMS(ESI):Found 206.03434(C9H7N3OS[M+H + ], requires 206.03475).

[0119] Example 21 Preparation of 7-hydroxy-2-imino-2H-chromene-3-thioamide (Compound DDO3723)

[0120]

[0121] This product is synthesized from 4-hydroxysalicylaldehyde and 2-cyanothioacetamide with a yield of 22%. 1 H NMR(300MHz,DMSO-d6)δ11.81(s,1H),10.34(d,2H),9.19(s,1H),9.15(s,1H),7.5 7(t,J=8.4Hz,1H),6.91(d,J=8.4Hz,1H),6.82(d,J=8.3Hz,1H).HRMS(ESI):Found 221.03815(C 10 H8N2O2S[M+H + ], requires 221.03792).

[0122] Example 22 Preparation of 5,8-difluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3735)

[0123]

[0124] This product is synthesized from 3,6-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 55%. 1 H NMR (300MHz, DMSO-d6) δ11.88(s,1H),9.32(s,0H),8.23(s,1H),8.11(s,1H),7.30–7.25(m,0H),7.10–6.63(m,1H).HRMS(ESI): Found 241.02402(C 10H6F2N2O2S[M+H + ], requires 241.02417).

[0125] Example 23 Preparation of 8-fluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3729)

[0126]

[0127] This product is synthesized from 3-fluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 55%. 1 HNMR(300MHz,DMSO-d6)δ12.05(s,1H),9.17(d,J=1.8Hz,1H),8.22(s,1H),8.02(s,1H),7.41( d,J=7.7Hz,1H),7.38–7.34(m,1H),7.21(td,J=8.0,4.6Hz,1H).HRMS(ESI):Found223.03311(C 10 H7FN2OS[M+H + ], requires 223.03562).

[0128] Example 24 Preparation of 7-fluoro-2-imino-2H-chromene-3-thioamide (Compound DDO3730)

[0129]

[0130] This product is prepared from 4-hydroxysalicylaldehyde and 2-cyanothioacetamide with a yield of 77%. 1 H NMR(300MHz,DMSO-d6)δ12.07(s,1H),9.15(s,1H),8.21(s,0H),7.83(s,1H),7.66–7. 59(m,1H),7.01(td,J=8.5,2.4Hz,1H),6.92(dd,J=9.0,2.2Hz,2H).HRMS(ESI):Found 223.03401(C 10 H7FN2OS[M+H + ], requires 223.03359).

[0131] Example 25 Preparation of 3-Imino-3H-benzo[f]chromene-2-thioamide (Compound DDO3717)

[0132]

[0133] This product is synthesized from naphthalene salicylaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 63%. 1H NMR (300MHz, DMSO-d6) δ11.88(s,1H),10.45(s,1H),9.70(s,1H),9.22(s,1H),8.32(d,J=8.5Hz,1H),8.21(d,J=9.0 Hz,1H),8.06(d,J=8.1Hz,1H),7.79(t,J=7.7Hz,1H),7.62(t,J=7.5Hz,1H),7.44(d,J=8.9Hz,1H).HRMS(ESI):Found 255.05833(C 14 H 10 N2OS[M+H + ], requires 225.05866).

[0134] Example 26 Preparation of 2-amino-6,8-difluoro-2H-chromene-3-thioamide (Compound DDO3736)

[0135]

[0136] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. DDO3728 (0.24 g, 1 mmol) was dissolved in 1,4-dioxane, and sodium triacetoxyborohydride was added. The mixture was stirred at room temperature for 8 h. After the reaction was completed, the solvent was distilled off under reduced pressure, and the target product was purified by silica gel column chromatography (PE:EA=20:1) to obtain 55.66 mg of a yellow solid with a yield of 23%. 1 H NMR (300MHz, DMSO-d6) δ10.00 (s, 2H), 9.66 (s, 2H), 7.16 (t, J = 9.3Hz, 2H), 6.92 (d, J = 8.1Hz, 1H), 4.39 (s, 1H). HRMS (ESI): Found 260.06681 (C 10 H8F2N2OS[M+NH4 + ], requires 260.06637).

[0137] Example 27 Preparation of 6,8-difluoro-2-imino-2H-chromene-3-carboxamide (Compound DDO3740)

[0138]

[0139] This product is synthesized from 3,5-difluorosalicylaldehyde and 2-cyanoamide according to Example 1, with a yield of 88%. 1H NMR (300MHz, DMSO-d6) δ9.41(d,J=33.6Hz,1H),8.38(s,1H),7.99(s,1H),7.87(d,J=16.4Hz,1H),7.71–7.59(m,1H),7.59–7.47(m,2H).HRMS(ESI):Found 225.04686(C 10 H6F2N2O2[M+H + ], requires 225.04701).

[0140] Example 28 Preparation of (E)-6,8-difluoro-2-(methylimino)-2H-chromene-3-thioamide (Compound DDO3737)

[0141]

[0142] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was prepared by dissolving DDO3728 (200 mg, 0.83 mmol) in acetic acid (1 ml), stirring at 45°C for 7 h, and extracting with ethyl acetate (20 ml) and water (30 ml) after the reaction. The organic layer was dried over anhydrous sodium sulfate and concentrated, and then purified by silica gel column chromatography (PE:EA=20:1) to obtain 176 mg of the target compound as a light yellow solid with a yield of 83.52%. 1 H NMR(300MHz,DMSO-d6)δ11.33(s,1H),10.47(s,1H),8.67(s,1H),7.73–7.60(m,2H),3.20(s,3H).HRMS(ESI):Found255.03975(C 11 H9F2N2O S [M+NH4 + ], requires 255.03982).

[0143] Example 29 Preparation of (E)-6,8-difluoro-2-(methoxyimino)-2H-chromene-3-thioamide (Compound DDO3738)

[0144]

[0145] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was prepared by dissolving DDO3728 (200 mg, 0.83 mmol) in acetic acid (1 ml), stirring at 45°C for 7 h, and extracting with ethyl acetate (20 ml) and water (30 ml) after the reaction. The organic layer was dried over anhydrous sodium sulfate and concentrated, and then purified by silica gel column chromatography (PE:EA=20:1) to obtain 176 mg of the target compound as a light yellow solid with a yield of 83.52%.1 H NMR (300MHz, DMSO-d6) δ10.17(s,1H),8.40(d,J=1.4Hz,1H),7.36(ddd,J=11.3,8 .6,3.1Hz,1H),7.20(ddd,J=9.2,3.1,1.9Hz,1H),3.94(s,3H).HRMS(ESI):Found 271.03081(C 11 H8F2N2OS[M+H + ], requires 271.03182).

[0146] Example 30 Preparation of 6,8-difluoro-N-formyl-2-imino-2H-chromene-3-thioamide (Compound DDO3739 )

[0147]

[0148] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. DDO3728 (240 mg, 1 mmol) was dissolved in formic acid and heated under reflux at 65°C for 5 hours to undergo an amide condensation reaction. After the reaction was completed, the solvent was removed by distillation under reduced pressure to obtain 243.88 mg of a yellow solid with a yield of 92%. 1 H NMR (300MHz, DMSO-d6) δ10.44 (s, 1H), 9.75 (s, 1H), 8.82 (d, J = 1.5Hz, 1H), 7.94–7.80 (m, 1H), 7.80–7.75 (m, 1H). HRMS (ESI): Found 269.01516 (C 11 H6F2N2O2S[M+H + ], requires 269.05542).

[0149] Example 31 Preparation of 7,9-difluoro-2-(4-methoxyphenyl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine-4- Thioketone (Compound DDO3757)

[0150]

[0151] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was prepared by dissolving compound DDO3728 (240 mg, 1 mmol) and commercially available compound p-methylbenzaldehyde (204 mg, 1.5 mmol) in an appropriate amount of tetrahydrofuran, adding piperidine (5.95 mg, 0.07 mmol), and refluxed for 8 hours. After the reaction was completed, the reaction solution was cooled to room temperature, and yellow solid precipitation was observed. The filter cake was filtered, rinsed twice with tetrahydrofuran, and dried under an infrared lamp to constant weight to obtain 42.96 mg of the target product with a yield of 12%. 1HNMR(300MHz,DMSO-d6)δ14.23(s,1H),8.50–7.88(m,3H),7.41(d,J=5.0Hz,1H), 7.34–7.26(m,1H),7.20–7.10(m,1H),3.90(s,2H),3.22(s,3H).HRMS(ESI):Found 359.06211(C 18 H 12 F2N2O2S[M+H + ], requires 359.06312).

[0152] Example 32 Preparation of 7,9-difluoro-2-(4-(hydroxymethyl)phenyl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine- 4-Thione (Compound DDO3745)

[0153]

[0154] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 4-(hydroxymethyl)benzaldehyde according to Example 31 with a yield of 14%. 1 H NMR (300MHz, DMSO-d6) δ14.16(s,1H),8.14(d,J=8.2Hz,2H),7.61(d,J=9.7Hz,1H),7.51( d,J=8.1Hz,2H),7.40–7.28(m,1H),7.19(d,J=9.1Hz,2H),4.63(s,2H).HRMS(ESI):Found 357.05123(C 18 H 12 F2N2O2S[MH + ], requires 357.05148).

[0155] Example 33 Preparation of 7,9-difluoro-2-(4-fluorophenyl)-3,5-dihydro-4H-chromo[2,3-d]pyrimidine-4-thione (Compound DDO3758)

[0156]

[0157] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and p-fluorobenzaldehyde according to Example 31 with a yield of 6%. 1H NMR (300MHz, DMSO-d6) δ14.23 (s, 1H), 8.50–7.88 (m, 3H), 7.41 (d, J = 5.0Hz, 1H), 7.34–7.26 (m, 1H), 7.20–7.10 (m, 1H), 3.90 (s, 2H). HRMS (ESI): Found 347.04212(C 17 H9F3N2OS[M+H + ], requires 347.05218).

[0158] Example 34 Preparation of 7,9-difluoro-2-(piperazin-1-yl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine-4-sulfur Ketone (Compound DDO3744)

[0159]

[0160] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and piperazine-1-carboxaldehyde according to Example 31 with a yield of 15%. 1 H NMR (300MHz, DMSO-d6) δ8.07(s,1H),7.63(ddd,J=9.9,3.1,1.8Hz,1H),7.15(d,J=8.9Hz,1 H),3.92(s,2H),3.75–3.47(m,4H),3.20–3.13(m,2H),3.12–3.06(m,2H).HRMS(ESI):Found 337.08759(C 15 H 14 F2N4OS[M+H + ], requires 337.08899).

[0161] Example 35 Preparation of 7,9-difluoro-2-(4-(trifluoromethyl)phenyl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine Pyridine-4-thione (Compound DDO3759)

[0162]

[0163] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and p-trifluoromethylbenzaldehyde according to Example 31 with a yield of 12%. 1 H NMR (300MHz, DMSO-d6) δ14.23 (s, 1H), 8.50–7.88 (m, 3H), 7.41 (d, J = 5.0Hz, 1H), 7.34–7.26 (m, 1H), 7.20–7.10 (m, 1H), 3.90 (s, 2H). HRMS (ESI): Found 397.03658(C 18H9F5N2OS[M+H + ], requires 397.03893).

[0164] Example 36 Preparation of 7,9-difluoro-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-thione (Compound DDO3741)

[0165]

[0166] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and formaldehyde according to Example 31 with a yield of 6%. 1 H NMR (300MHz, DMSO-d6) δ17.29(s,1H),8.28(s,1H),7.64(d,J=8.8Hz,1H),7.16(d,J=8.8Hz,1H),3.94(s,2H).HRMS(ESI): Found253.02054(C 11 H6F2N2OS[M+H + ], requires 253.02024).

[0167] Example 37 Preparation of 4-(7,9-difluoro-4-thioloxy-3,5-dihydro-4H-chrome[2,3-d]pyrimidin-2-yl)benzene Acetonitrile (Compound DDO3760)

[0168]

[0169] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and p-cyanoformaldehyde according to Example 31 with a yield of 16%. 1 H NMR (300MHz, DMSO-d6) δ14.23 (s, 1H), 8.50–7.88 (m, 3H), 7.41 (d, J = 5.0Hz, 1H), 7.34–7.26 (m, 1H), 7.20–7.10 (m, 1H), 3.90 (s, 2H). HRMS (ESI): Found 354.04577(C 18 H9F2N3OS[M+H + ], requires 354.04679).

[0170] Example 38 Preparation of 7,9-difluoro-2-phenyl-3,5-dihydro-4H-chromo[2,3-d]pyrimidine-4-thione (Compound DDO3742 )

[0171]

[0172] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and benzaldehyde according to Example 31 with a yield of 11%. 1 H NMR(300MHz,DMSO-d6)δ14.25(s,1H),8.17(s,1H),8.14(s,1H),7.75–7.54(m,2H),7. 36(ddd,J=11.4,8.8,3.0Hz,1H),7.23(d,J=9.0Hz,1H),3.90(s,2H).HRMS(ESI):Found 327.03990(C 17 H 10 F2N2OS[MH - ], requires327.03869).

[0173] Example 39 Preparation of 7,9-difluoro-2-(3-methoxyphenyl)-3,5-dihydro-4H-chroma[2,3-d]pyrimidine-4- Thioketone (Compound DDO3743)

[0174]

[0175] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and benzaldehyde according to Example 31 with a yield of 6.9%. 1 H NMR (300MHz, DMSO-d6) δ14.23(s,1H),7.76(s,1H),7.74(s,1H),7.49(t,J=7.9Hz,1H) ,7.40–7.31(m,1H),7.21(d,J=7.5Hz,2H),3.94(s,2H),3.89(s,3H).HRMS(ESI):Found 357.05210(C 18 H 12 F2N2OS[MH - ], requires357.05262).

[0176] Example 40 Preparation of 7,9-difluoro-2-(3,4,5-trimethoxyphenyl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine Pyridine-4-thione (Compound DDO3761)

[0177]

[0178] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 4,5,6-trimethoxybenzaldehyde according to Example 31 with a yield of 5.5%. 1H NMR (300MHz, DMSO-d6) δ14.12 (s, 1H), 7.56 (s, 2H), 7.32 (t, J = 10.1Hz, 1H), 7.18 (d, J = 8.9Hz, 1H), 3.93 (s, 6H), 3.79 (s, 3H). HRMS (ESI): Found 419.09638(C 20 H 16 F2N2O4S[M+H + ], requires 419.08324).

[0179] Example 41 Preparation of 7,9-difluoro-2-(pyridin-4-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-thione (Compound DDO3762)

[0180]

[0181] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and pyridinecarboxaldehyde according to Example 31 with a yield of 8%. 1 H NMR(300MHz,DMSO-d6)δ14.25(s,1H),8.17(s,1H),8.14(s,1H),7.75–7.54(m,2H),7. 36(ddd,J=11.4,8.8,3.0Hz,1H),7.23(d,J=9.0Hz,1H),3.90(s,2H).HRMS(ESI):Found 330.04679(C 16 H9F2N3OS[M+H + ], requires330.04539).

[0182] Example 42 Preparation of 2-(2,5-difluorophenyl)-7,9-difluoro-3,5-dihydro-4H-chroma[2,3-d]pyrimidine-4- Thioketone (Compound DDO3763)

[0183]

[0184] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 2,5-difluorobenzaldehyde according to Example 31 with a yield of 12%. 1 H NMR (300MHz, DMSO-d6) δ14.87–14.21(m,1H),7.67(dd,J=9.5,4.3Hz,1H),7.54(dq,J=13.7 ,4.6Hz,2H),7.37(t,J=11.3Hz,1H),7.23(d,J=8.7Hz,1H),3.96(s,2H).HRMS(ESI):Found 365.03270(C17 H8F4N2OS[M+H + ], requires 365.03554).

[0185] Example 43 Preparation of 7,9-difluoro-2-(2-methoxypyridin-4-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine Pyridine-4-thione (Compound DDO3764)

[0186]

[0187] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 2-methoxyisonicotinaldehyde according to Example 31 with a yield of 6.3%. 1 H NMR(300MHz,DMSO-d6)δ14.25(s,1H),8.17(s,1H),7.75–7.54(m,2H),7.36(ddd,J=11 .4,8.8,3.0Hz,1H),7.23(d,J=9.0Hz,1H),3.90(s,3H),3.22(s,2H).HRMS(ESI):Found 360.05736(C 17 H 11 F2N3O2S[M+H + ], requires 360.055274).

[0188] Example 44 Preparation of 7,9-difluoro-2-(2H-pyrrol-5-yl)-3,5-dihydro-4H-chromo[2,3-d]pyrimidine-4-sulfur Ketone (Compound DDO3765)

[0189]

[0190] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 2H-pyrrole-5-carboxaldehyde according to Example 31 with a yield of 13%. 1 H NMR (300MHz, DMSO-d6) δ8.26 (s, 0H), 7.76–7.53 (m, 15H), 7.31 (t, J = 10.3Hz, 0H), 7.18 (d, J = 9.4Hz, 0H), 3.95 (s, 1H), 3.13–2.80 (m, 2H). HRMS (ESI): Found 318.04679(C 15 H9F2N3OS[M+H + ], requires318.045279).

[0191] Example 45 Preparation of 7-bromo-9-methoxy-2-phenyl-3,5-dihydro-4H-chromo[2,3-d]pyrimidine-4-thione (Chem. Compound DDO3766)

[0192] The intermediate 6-bromo-2-imino-8-methoxy-2H-chromene-3-thioamide was synthesized from 5-bromo-2-hydroxy-3-methoxybenzaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from 6-bromo-2-imino-8-methoxy-2H-chromene-3-thioamide and benzaldehyde according to Example 31 with a yield of 21%. 1 H NMR(300MHz,DMSO-d6)δ14.13(s,1H),8.41–7.93(m,2H),7.83–7.38(m,3H),7.17(s,2H),3.89(s,5H).HRMS(ESI):Found401.98606(C 18 H 13 BrN2O2S[M+H + ], requires 401.985629).

[0193] Example 46 Preparation of 7,9-difluoro-2-(quinolin-6-yl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine-4-sulfur Ketone (Compound DDO3767)

[0194]

[0195] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and quinoline-6-carboxaldehyde according to Example 31 with a yield of 13%. 1 H NMR (300MHz, DMSO-d6) δ9.06(s,1H),8.93(s,2H),8.59(d,J=9.2Hz,0H),8.47(d,J=8.7Hz,2H),8.17(d,J=1 2.0Hz,1H),7.67(s,0H),7.45–7.35(m,0H),7.33–7.18(m,2H),4.00(s,4H).HRMS(ESI):Found380.06244(C 20 H 11 F2N3OS[M+H + ], requires 380.064442).

[0196] Example 47 Preparation of 7,9-difluoro-2-(1H-indol-3-yl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine- 4-Thione (Compound DDO3768)

[0197]

[0198] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 3H-indole-3-carboxaldehyde according to Example 31 with a yield of 3.5%. 1H NMR (300MHz, DMSO-d6) δ14.20(s,1H),12.01(s,1H),7.94(s,1H),7.68(d,J=8.0Hz,1H),7.5 4(d,J=8.3Hz,1H),7.41–7.16(m,1H),7.10(t,J=7.5Hz,1H),3.95(s,2H).HRMS(ESI):Found 368.06244(C 19 H 11 F2N3OS[M+H + ], requires 368.06434).

[0199] Example 48 Preparation of 7,9-difluoro-2-(2-fluoropyridin-4-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-yl Thioketone (Compound DDO3769)

[0200]

[0201] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 2-fluoroisonicotinaldehyde according to Example 31 with a yield of 5%. 1 H NMR(300MHz,DMSO-d6)δ14.60(s,1H),9.12(s,1H),8.87(d,J=2.6Hz,1H),8.54–8.33 (m,1H),7.38(t,J=9.8Hz,1H),7.24(d,J=8.6Hz,1H),3.97(s,2H).HRMS(ESI):Found 348.03737(C 16 H8F3N3OS[M+H + ], requires348.03784).

[0202] Example 49 Preparation of 7,9-difluoro-2-(1H-pyrrol-3-yl)-3,5-dihydro-4H-chromo[2,3-d]pyrimidine-4-sulfur Ketone (Compound DDO3770)

[0203]

[0204] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 1H-pyrrole-3-carboxaldehyde according to Example 31 with a yield of 15%. 1H NMR (300MHz, DMSO-d6) δ13.57(s,1H),11.65(s,1H),8.01(s,0H),7.33(ddd,J=11.4,8. 8,3.0Hz,1H),7.18(d,J=9.6Hz,1H),6.98–6.89(m,2H),3.90(s,2H).HRMS(ESI):Found 318.04679(C 15 H9F2N3OS[M+H + ], requires 318.04774).

[0205] Example 50 Preparation of 7,9-difluoro-2-(pyrazin-2-yl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine-4-sulfur Ketone (Compound DDO3771)

[0206]

[0207] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and pyrazine-2-carboxaldehyde according to Example 31 with a yield of 5%. 1 H NMR (300MHz, DMSO-d6) δ13.55(s,1H),9.45(s,1H),8.98(d,J=2.2Hz,1H),8.91(s, 1H),7.38(t,J=11.0Hz,1H),7.23(d,J=8.3Hz,1H),3.99(s,2H).HRMS(ESI):Found 331.04204(C 15 H8F2N4OS[M+H + ], requires331.04274).

[0208] Example 51 Preparation of 7,9-difluoro-2-(1H-pyrazol-4-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-yl Thioketone (Compound DDO3772)

[0209]

[0210] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 1H-pyrazole-4-carboxaldehyde according to Example 31 with a yield of 6.6%. 1 H NMR (300MHz, DMSO-d6) δ13.97(s,1H),13.56(s,1H),8.78(s,1H),8.37(s,1H),7.38–7.27(m,1H),7.19(d,J=8.8Hz,1H),3.90(s,2H).HRMS(ESI):Found 319.04204(C 15H8F2N4OS[M+H + ], requires 319.04598).

[0211] Example 52 Preparation of 7,9-difluoro-2-(4-morpholinophenyl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine-4- Thioketone (Compound DDO3773)

[0212]

[0213] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 4-morpholinebenzaldehyde according to Example 31 with a yield of 4.5%. 1 H NMR (300MHz, DMSO-d6) δ13.78(s,1H),8.16(d,J=9.0Hz,2H),7.36(t,J=11.3Hz,1H),7.22(d,J=8.9Hz,2H),7.09 (d,J=9.1Hz,3H),3.95(s,2H),3.78(q,J=3.9Hz,8H),3.65(t,J=6.6Hz,1H),3.50–3.37(m,2H).HRMS(ESI):Found 414.10431(C 21 H 17 F2N3O2S[M+H + ], requires 414.10425).

[0214] Example 53 Preparation of 7,9-difluoro-2-(1H-imidazol-2-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-yl Thioketone (Compound DDO3774)

[0215]

[0216] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 1H-imidazole-2-carboxaldehyde according to Example 31 with a yield of 6%. 1 H NMR (300MHz, DMSO-d6) δ7.46 (s, 1H), 7.33 (t, J = 10.3Hz, 0H), 7.20 (d, J = 8.8Hz, 1H), 3.94 (s, 1H). HRMS (ESI): Found319.04204 (C 14 H8F2N4OS[M+H + ], requires 319.04224).

[0217] Example 54 Preparation of 2-(3-aminopyridin-4-yl)-7,9-difluoro-3,5-dihydro-4H-chrome[2,3-d]pyrimidine- 4-Thione (Compound DDO3746)

[0218]

[0219] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 3-aminoisonicotinaldehyde according to Example 31 with a yield of 3.4%. 1 H NMR (300MHz, DMSO-d6) δ13.25(s,1H),11.02(s,1H),9.10(s,1H),8.11(d,J=7.0Hz,1H),7.27 (t,J=9.9Hz,1H),7.14(d,J=8.8Hz,1H),7.00(d,J=6.9Hz,1H),3.92(s,2H).HRMS(ESI):Found 345.06127(C 16 H 10 F2N4OS[M+H + ], requires 345.06161).

[0220] Example 55 Preparation of 7,9-difluoro-2-(1H-indazol-3-yl)-3,5-dihydro-4H-chromeno[2,3-d]pyrimidine- 4-Thione (Compound DDO3775)

[0221]

[0222] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 1H-indazole-3-carboxaldehyde according to Example 31 with a yield of 4%. 1 H NMR (300MHz, DMSO-d6) δ14.23(s,1H),13.52(s,1H),8.34(d,J=8.1Hz,1H),7.73(d,J=8.4Hz,1H) ,7.54(t,J=7.4Hz,1H),7.45–7.37(m,2H),7.23(d,J=8.9Hz,1H),3.98(s,2H).HRMS(ESI):Found 369.05769(C 18 H 10 F2N4OS[M+H + ], requires 369.05729).

[0223] Example 56 Preparation of 7,9-difluoro-2-(furan-3-yl)-3,5-dihydro-4H-chrome[2,3-d]pyrimidine-4-thione (Compound DDO3776)

[0224]

[0225] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and furan-3-carboxaldehyde according to Example 31 with a yield of 10.3%.1 H NMR (300MHz, DMSO-d6) δ14.14(s,1H),8.80(s,0H),7.91(t,J=1.7Hz,1H),7.33(ddd,J=11.4,8. 7,3.0Hz,1H),7.26(d,J=2.0Hz,1H),7.19(dt,J=9.0,2.4Hz,1H),3.91(s,2H).HRMS(ESI):Found 319.03081(C 15 H8F2N2O2S[M+H + ], requires 319.03382).

[0226] Example 57 Preparation of 3-(7,9-difluoro-4-thioloxy-3,5-dihydro-4H-chrome[2,3-d]pyrimidin-2-yl)pyrrolidone 2-(1H- ) Ketone (Compound DDO3777)

[0227]

[0228] Compound DDO3728 was synthesized from 3,5-difluorosalicylaldehyde and 2-cyanothioacetamide according to Example 1. This product was synthesized from DDO3728 and 2-oxo-1,2-dihydropyridine-3-carboxaldehyde according to Example 31 with a yield of 5%. 1 H NMR (300MHz, DMSO-d6) δ15.00(s,1H),13.26(s,1H),8.81(d,J=8.8Hz,1H),8.02(d,J=6.1Hz,1H), 7.37(t,J=11.7Hz,1H),7.22(d,J=11.0Hz,1H),6.78–6.69(m,1H),3.95(s,2H).HRMS(ESI):Found 346.04171(C 16 H9F2N3O2S[M+H + ], requires 346.04273).

[0229] Example 58 Preparation of 8-fluoro-6-((4-fluorophenyl)amino)-2-imino-2H-chromene-3-thioamino (compound DDO3747)

[0230]

[0231] 5-Bromo-3-fluoro-2-hydroxybenzaldehyde (2.10 g, 9.60 mol) and (4-fluorophenyl)formamide (100 mg, 8.00 mmol) were dissolved in an appropriate amount of DMF. Then cesium carbonate (2.18 g, 16.0 mmol), Xantphos (231 mg, 0.4 mmol) and palladium acetate (89.8 mg, 0.4 mmol) were added and stirred at 120 ° C for 10 h under nitrogen protection. After the reaction was completed, ethyl acetate and water were used for extraction, and the organic layer was dried with anhydrous sodium sulfate. After drying, the organic layer was concentrated and the mixture was purified by silica gel column chromatography (PE: EA = 20: 1) to obtain 2.38 g of the intermediate 3-fluoro-5-((4-fluorobenzyl)amino)-2-hydroxybenzaldehyde with a yield of 86.23%. This product is synthesized from 3-fluoro-5-((4-fluorobenzyl)amino)-2-hydroxybenzaldehyde and 2-cyanothioacetamide according to Example 1, with a yield of 31.56%. 1 H NMR (300MHz, DMSO-d6) δ13.73 (s, 1H), 8.35 (s, 1H), 7.34–7.23 (m, 5H), 7.21 (s, 1H), 7.08 (t, J = 8.6Hz, 3H), 4.82 (s, 2H). HRMS (ESI): Found 346.07474 (C 17 H 13 F2N3OS[M+H + ], requires 346.07274).

[0232] Example 59 Preparation of 8-fluoro-6-(4-fluorophenyl)-2-imino-2H-chromene-3-carbothioamide (Compound DDO3748)

[0233]

[0234] 5-Bromo-3-fluorosalicylaldehyde (2.17 g, 10 mmol) and p-fluorophenyl borate (2.67 g, 12 mmol) were dissolved in an appropriate amount of DMF, cesium carbonate (16.29 g, 50 mmol) and triphenylphosphine palladium dichloride (200 mg, 0.5 mmol) were added, and the mixture was refluxed and stirred at 120°C for 8 h. After the reaction, the reaction solution was extracted with water and ethyl acetate, and the organic layer was dried over anhydrous sodium sulfate and concentrated to obtain the intermediate 4',5-difluoro-4-hydroxy-[1,1'-biphenyl]-3-carboxaldehyde as a crude product. It was not purified and was directly reacted with 2-cyanothioacetamide according to Example 1 to obtain 1.35 g of the target product as a yellow solid with a yield of 31.56%. 1HNMR(300MHz,DMSO-d6)δ12.08(s,1H),9.25(s,1H),8.26(s,1H),8.09(s,1H),7.76–7.48(m,5H),7.34(s,1H).HRMS(ESI):Found 317.05574(C 16 H 10 F2N2OS[M+H + ], requires317.05547).

[0235] Example 60 Preparation of 8-fluoro-6-(4-aminophenyl)-2-imino-2H-chromene-3-carbothioamide (Compound DDO3778)

[0236]

[0237] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and p-aminophenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1 with a yield of 28.78%. 1 H NMR(300MHz,DMSO-d6)δ12.08(s,2H),9.25(s,2H),8.26(s,1H),8.09(s,1H),7.76–7.48(m,5H),7.34(s,1H).HRMS(ESI):Found 314.07187(C 16 H 12 FN3OS[M+H + ], requires 314.07147).

[0238] Example 61 Preparation of 8-fluoro-6-(4-hydroxyphenyl)-2-imino-2H-chromene-3-carbothioamide (Compound DDO3752)

[0239]

[0240] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and p-hydroxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 40.21%. 1 H NMR(300MHz,DMSO-d6)δ11.89(s,1H),9.45(s,1H),9.14(s,1H),8.93(s,1H),8.61(s,1H),7.87 (s,1H),7.70(d,J=11.9Hz,1H),7.63(d,J=8.5Hz,2H),6.97(d,J=8.5Hz,2H).HRMS(ESI):Found 315.05975(C 16 H 11 FN2O2S[M+H +], requires 315.0598).

[0241] Example 62 Preparation of 4-(3-carbamoyl-8-fluoro-2-imino-2H-chromium-6-yl)benzoic acid (Compound DDO3750)

[0242]

[0243] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and p-carboxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 43.25%. 1 H NMR(300MHz,DMSO-d6)δ13.01(s,1H),11.20(s,1H),10.17(s,1H),9.68(s,1H),8.39(s,1 H),8.18(s,1H),8.03(d,J=8.2Hz,1H),7.9(s,1H),7.8(d,J=8.2Hz,2H).HRMS(ESI):Found 343.05447(C 17 H 11 FN2O3S,[M+H + ], requires 343.05527).

[0244] Example 63 Preparation of 8-fluoro-2-imino-6-(4-(trifluoromethyl)phenyl)-2H-chromene-3-thioamide (Chem. Compound DDO3749)

[0245]

[0246] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and p-trifluoromethylphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1 with a yield of 32.26%. 1 H NMR(300MHz,DMSO-d6)δ11.85(s,1H),9.49(s,1H),9.16(s,1H),9.04(s,1H),8.09( s,1H),8.03(d,J=8.1Hz,2H),7.92(s,1H),7.86(d,J=8.1Hz,2H).HRMS(ESI):Found 367.05238(C 17 H 10 F4N2OS,[M+H + ], requires 367.05227).

[0247] Example 64 Preparation of 2-(4-(3-carbamoyl-8-fluoro-2-imino-2H-chromen-6-yl)phenyl)acetic acid (Compound DDO3751)

[0248]

[0249] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and p-ethylcarboxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 43.25%. 1 H NMR(300MHz,DMSO-d6)δ11.91(s,1H),9.50(s,1H),9.18(s,1H),9.02(s,1H),7.76(d ,J=8.3Hz,1H),7.70–7.58(m,2H),7.54–7.38(m,4H),3.72(s,2H).HRMS(ESI):Found 357.07034(C 18 H 13 FN2O3S,[M+H + ], requires 357.07037).

[0250] Example 65 Preparation of 8-fluoro-6-(4-hydroxy-3-methoxyphenyl)-2-imino-2H-chromene-3-thioamide (Compound DDO3779)

[0251]

[0252] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and 4-hydroxy-3-methoxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1 with a yield of 32.75%. 1 H NMR (300MHz, DMSO-d6) δ11.90(s,1H),9.48(s,1H),9.13(s,1H),8.94(s,1H),7.93(s,1H),7.76(d,J=12 .1Hz,1H),7.40(s,1H),7.25(d,J=8.2Hz,1H),6.95(s,1H),6.92(s,1H),3.96(s,3H).HRMS(ESI):Found 345.06645(C 17 H 13 FN2O3S,[M+H + ], requires 345.065545).

[0253] Example 66 Preparation of 6-(3,5-dimethoxyphenyl)-8-fluoro-2-imino-2H-chromene-3-thioamide (Chemical Compound DDO3780)

[0254]

[0255] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and 3,5-dimethoxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 32.75%. 1H NMR(300MHz,DMSO-d6)δ11.85(s,1H),9.46(s,1H),9.10(s,1H),8.95(s,1H),7. 95(s,1H),7.80(s,1H),6.90(s,2H),6.52(s,1H),3.86(s,6H).HRMS(ESI):Found 359.08210(C 18 H 15 FN2O3S,[M+H + ], requires 359.08232).

[0256] Example 67 Preparation of 6-(2,4-dimethoxyphenyl)-8-fluoro-2-imino-2H-chromene-3-thioamide (Chemical Compound DDO3781)

[0257]

[0258] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and 2,4-dimethoxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 44.25%. 1 H NMR(300MHz,DMSO-d6)δ11.85(s,1H),9.46(s,1H),9.10(s,1H),8.95(s,1H),7. 95(s,1H),7.80(s,1H),6.90(s,2H),6.52(s,1H),3.86(s,6H).HRMS(ESI):Found 359.08210(C 18 H 15 FN2O3S,[M+H + ], requires 359.08232).

[0259] Example 68 Preparation of 6-(2,5-dimethoxyphenyl)-8-fluoro-2-imino-2H-chromene-3-thioamide (Chemical Compound DDO3782)

[0260]

[0261] Intermediate 1 was synthesized from 5-bromo-3-fluorosalicylaldehyde and 2,5-dimethoxyphenyl borate according to Example 59. This product was synthesized from Intermediate 1 and 2-cyanothioacetamide according to Example 1, with a yield of 24.35%. 1H NMR (300MHz, DMSO-d6) δ10.95(s,1H),10.13(s,1H),9.58(d,J=25.2Hz,1H),8.38(s,1H),8.00(s,1H),7.60(d,J=1 2.1Hz,1H),7.04(d,J=7.7Hz,1H),6.97(d,J=5.3Hz,1H),6.92(s,1H),3.74(s,3H),3.74(s,3H).HRMS(ESI):Found 359.08210(C 18 H 15 FN2O3S,[M+H + ], requires 359.08232).

[0262] Biological activity test

[0263] (I) Phosphatase activity test (pNPP method)

[0264] Table 1 Activation activity of compounds on PP5 enzyme activity

[0265]

[0266]

[0267] Note: Due to the solubility of the compound, a The data are for the test at 50 μM.

[0268] As shown in Table 1, the example compounds all showed activation activity on PP5. The activation activity of the compound at a concentration of 200 μM on PP5 ranged from 108% to 420%, which was basically at the same level as the positive compounds arachidonic acid and P5SA-2. This shows that the example compounds of this patent have strong PP5 enzyme activity. Figure 1 The pNPP method is used to analyze the EC of compounds DDO3728, DDO3733, DD03750, and DDO3752. 50 and E max Test, where DDO3728 EC 50 =51.92μM, E max =4.09folds; EC of DDO3733 50 =52.80μM, E max =4.28folds; EC of DDO3750 50 =198μM, E max =8.1folds; EC of DDO3752 50 =130.60μM, E max =4.71folds.

[0269] Among them, the pNPP method was used to test the single-site enzyme activity and EC of PP5 50 The operation method is as follows:

[0270] First, prepare 50mL of buffer solution containing 100mM Tris, 50mM NaCl, 0.5Mm MnCl2 (PH = 8.0), and use this buffer solution to dissolve the test compound (DMSO content is less than 10%), PP5 protein and pNPP substrate. Use a 96-well transparent plate, set up three duplicate wells, add 50μLPP5 protein solution (15μM), 50μL compound solution (600μM) to each well, and finally add 50μL substrate solution (1mg / mL); add 50μLPP5 protein solution, 50μL inhibitor positive drug LB100 solution, and 50μL substrate solution to the negative control well; add 50μL PP5 protein solution, 50μL buffer solution, and 50μL substrate solution to the blank well. Incubate the 96-well plate at room temperature (25℃) for 30min, use an ELISA reader to scan and collect data at a wavelength of 405nm, and analyze the results using GraphaPad software.

[0271] (II) Isothermal titration calorimetry (ITC) experiment

[0272] Isothermal Titration Calorimetry (ITC) is a thermodynamic analysis technique that uses a target compound to titrate a target protein. The binding of a protein to a molecule will reduce the entropy of the entire system, so the binding process will release heat. By measuring the change in the heat release of the reaction system, the affinity constant K of the compound molecule interacting with the protein can be calculated. d . This experiment was measured using an ITC instrument (MicroCal Itc200). The temperature of the entire measurement process was set at 25°C. The protein (PP5 full-length or truncated form) was condensed and stored in 10mM PBS buffer. The compound preparation concentration was 0.5mM, the preparation concentration of the PP5 full-length protein was 13μM, and the preparation concentration of the PP5 truncation was 18μM. The specific experimental process is as follows: In the first step, 300μL of the configured protein was injected into the sample pool, 2μL per drop for a total of 19 drops (in order to ensure the accuracy of the experiment, the first drop of titration volume was 0.5μL to exclude interference from other factors outside the system). The Origin software package (version 7.0, MicroCal) was used for data analysis, including the binding constant Ka, enthalpy change (ΔH) and entropy change (ΔS). The final dissociation constant K d According to the formula K d =1 / K a Calculated.

[0273] like Figure 2 The ITC binding affinity activity evaluation of compound DDO3728 with PP5 truncated protein and PP5 full-length protein. As can be seen from the figure, its binding affinity constant K for PP5 truncated protein is d =7.41 μM, binding affinity constant K for full length PP5 d =1.78μM.

[0274] (III) Western blots to test the effect of PP5 activators on the dephosphorylation level of intracellular PP5 substrate proteins

[0275] The operation method of Western blots test refers to the standard experimental process. The specific experimental process includes cell administration, cell lysis, total protein collection, preparation of polyacrylamide gel, SDS-PAGE analysis, primary antibody incubation, secondary antibody incubation and result scanning. The expression levels of ASK1, p-ASK1, Cdc37 and p-Cdc37 were tested in the experiment.

[0276] The dephosphorylation effects of the compounds of the present invention on ASK1 and Cdc37 are as follows:

[0277] like Figure 3 , 4 As shown in Figures 5 and 6, the test results showed that DDO3728 and DDO3733 dephosphorylated ASK1 and Cdc37 in a concentration- and time-dependent manner. Figure 3 ASK1 and p-ASK1 in HCT116 cells after administration of 50 μM positive compound P5SA-2 for different time periods T838 , CDC37 and p-CDC37 S13 Content; Figure 4 ASK1 and p-ASK1 in HCT116 cells after administration of compound DDO3728 at different concentrations for 24 hours T838 , CDC37 and p-CDC37 S13 Content; Figure 5 ASK1 and p-ASK1 in HCT116 cells after administration of compound DDO3733 at different concentrations for 24 hours T838 , CDC37 and p-CDC37 S13 Content; Figure 6 ASK1 and p-ASK1 in HCT116 cells after administration of 50 μM compound DDO3733 for different time periods T838 , CDC37 and p-CDC37 S13 The content.

[0278] It can be seen from the above examples that some compounds of the present invention as PP5 agonists have strong PP5 agonistic effects, show good activity in various biological activity tests, and can be used to prepare drugs for treating related diseases caused by PP5 abnormalities.

[0279] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A compound as a protein phosphatase 5 agonist, characterized in that The compound structure is selected from one of the following:

2. A pharmaceutical composition, characterized in that The active ingredient of the pharmaceutical composition is selected from the compound according to claim 1, or a pharmaceutically acceptable salt thereof.

3. Use of a compound or pharmaceutical composition in the preparation of a drug for treating / preventing PP5-related diseases, characterized in that: The compound comprises the compound according to claim 1, The pharmaceutical composition is the pharmaceutical composition according to claim 2.

4. The use according to claim 3, characterized in that The compound or its pharmaceutical composition is used in the preparation of medicines for treating any one or more of Alzheimer's disease, obesity and cancer caused by dephosphorylation dysfunction of certain transcription factors or proteases.

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