阅读说明：本技术 Sox18蛋白活性的抑制剂用于治疗血管生成相关疾病和/或***生成相关疾病 (The inhibitor of SOX18 protein active is for treating angiogenesis-associated diseases and/or lymphatic vessel generation related disease ) 是由 M·弗兰克斯 J·朱格 J·奥沃曼 S·K·玛米德雅拉 A·A·萨利姆 F·R·方丹 M 于 2017-12-21 设计创作，主要内容包括：本发明公开了本文所提供的式的化合物,其显示出抑制SOX18蛋白活性的功效,特别是有关SOX18结合DNA和/或特定蛋白质配偶体的能力。另外,本文提供了治疗诸如癌症转移和血管癌的血管生成相关疾病、病症或病况和/或淋巴管生成相关疾病、病症或病况的方法。(The invention discloses the compound of formula provided in this article, the effect of inhibiting SOX18 protein active is shown, more particularly to the ability of SOX18 combination DNA and/or specific protein gametophyte.In addition, there is provided herein the methods for the treatment of such as cancer metastasis and the angiogenesis-associated diseases of blood vessel cancer, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition.)

1. the compound or its pharmaceutically acceptable salt, solvate or prodrug of formula (I):

Wherein,

R₁Selected from OH and OR₆, wherein R₆For C₁–C₄Alkyl；

R₂Selected from H, COOR₇With C (O) NR₈R₉, wherein R₇、R₈And R₉It is independently selected from H and C₁–C₄Alkyl；

R₃For L-A, wherein L is selected from C₂–C₈Alkyl, C₂–C₈Alkenyl and C₂–C₈The connector of alkoxyalkyl, and A is selected from optionally Substituted phenyl and the naphthalene optionally replaced；

R₄Selected from H, OR₁₀, halogenated and C₁–C₄Alkyl, wherein R₁₀Selected from H and C₁–C₄Alkyl；And

R₅Selected from H, OR₁₁, halogenated and C₁–C₄Alkyl, wherein R₁₁Selected from H and C₁–C₄Alkyl,

Wherein, the compound is for inhibiting SOX18 active.

2. compound according to claim 1, wherein R₁Selected from OH and OMe.

3. the compound according in claim 1 or 2, wherein R₂Selected from H, COOH, COOMe and

4. compound according to any one of the preceding claims, wherein R₄Selected from H, OH, OMe, Cl and Me.

5. compound according to any one of the preceding claims, wherein R₅Selected from H, OH and OMe.

6. compound according to any one of the preceding claims, wherein R₄And R₅For H.

7. compound according to any one of the preceding claims, wherein L is selected from C₂–C₆Alkyl, C₂–C₆Alkenyl and C₂– C₆The connector of alkoxyalkyl.

8. compound according to any one of the preceding claims, wherein R₃It is selected from:

Wherein, dotted line indicates the connection of the ring from adjacent atom to Formulas I, and shown structure includes its E/Z isomers.

9. compound according to any one of the preceding claims, wherein compound is selected from:

10. compound according to any one of the preceding claims, wherein the SOX18 activity include with DNA sequence dna and/ Or protein contacts and/or combination.

11. compound according to claim 10, wherein the protein be selected from SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

12. pharmaceutical composition, it includes described in any one of preceding claims compound or its pharmaceutically acceptable salt, Solvate or prodrug and pharmaceutically acceptable carrier, diluent and/or excipient.

13. angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation related disease for the treatment of or prevention subject, The method of illness or the patient's condition, the method includes a effective amount of according to claim 1 to any in 11 to subject application Compound or its pharmaceutically effective salt, solvate or prodrug or pharmaceutical composition according to claim 12 described in Object, to treat or prevent the angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation related disease, illness Or the step of patient's condition.

14. compound according to any one of claim 1 to 11 or its pharmaceutically effective salt, solvate or prodrug Purposes in medicine preparation, the drug is for treating or preventing angiogenesis-associated diseases, illness or the patient's condition and/or lymph Pipe generates related disease, illness or the patient's condition.

15. purposes described in the method according to claim 11 or claim 14, wherein the angiogenesis correlation disease Disease, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition are ophthalmology disease, illness or the patient's condition or including eye Section's disease, illness or the patient's condition.

16. the method according to claim 11 or purposes, wherein the ophthalmology disease, illness or the patient's condition are selected from age correlation Property macular degeneration, diabetic retinopathy, ischemic retinopathies, retinopathy of prematurity, neovascular glaucoma, rainbow Film inflammation, cornea rebirth blood vessel, cyclitis, sickle cell retinopathy, pteryium, during corneal injury vascular reaction and Any combination thereof.

17. the method according to claim 11 or purposes according to claim 14, wherein the angiogenesis phase Related disorders, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition are cancers or including cancer.

18. the method according to claim 11 or purposes, wherein the cancer is selected from prostate cancer, lung cancer, breast cancer, wing Guang cancer, kidney, colon cancer, gastric cancer, cancer of pancreas, oophoroma, melanoma, liver cancer, hepatocellular carcinoma, sarcoma, leukaemia, lymthoma, Vascular tumor and any combination thereof.

19. method described in 7 or 18 or purposes according to claim 1, wherein the compound or pharmaceutical composition prevent and/or Inhibit the cancer metastasis.

20. the method according to claim 11 or purposes according to claim 14, wherein the angiogenesis phase Related disorders, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition are kidney trouble, illness or the patient's condition or packet Include kidney trouble, illness or the patient's condition.

21. the method according to claim 11 or purposes, wherein the kidney trouble, illness or the patient's condition are moved selected from chronic renal Plant dysfunction, primary kidney fibrosis illness, albuminuria, nephrosis, ephritis and any combination thereof.

22. the method for the cancer metastasis of inhibition or prevention subject comprising Xiang Suoshu subject's application is a effective amount of according to power Benefit require any one of 1 to 11 described in compound or its pharmaceutically effective salt, solvate or prodrug or wanted according to right Pharmaceutical composition described in asking 12, thus the step of inhibiting or preventing the cancer metastasis.

23. inhibiting, preventing or reducing the active method of SOX18 in subject comprising described a effective amount of to subject's application Compound according to any one of claim 1 to 11 or its pharmaceutically effective salt, solvate or prodrug or according to Pharmaceutical composition described in claim 12, to inhibit, prevent or reduce the step of the SOX18 activity in the subject.

24. according to the method for claim 23, wherein the SOX18 activity includes and DNA sequence dna and/or protein contacts And/or it combines.

25. according to the method for claim 24, wherein the protein be selected from SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

Invention field

The present invention relates to therapeutic treatment fields.More particularly, the present invention relate to inhibit SOX18 transcription factor activity Compound.

Background of invention

Herein to background technique it is any refer to be not necessarily to be construed as recognizing such technology Australia or elsewhere Constitute common knowledge.

It is still a long-term exploration that transcription factor (TF) is directly adjusted by small molecule.Earlier results be only limitted to core by Body contains the ligand binding domains that can be targeted by small molecule.These discoveries have been converted into controlling for hormone dependent cancer Treat application (Perissi and Rosenfeld, 2005).Current challenge be nuclear receptor is expanded to widely transcription because Son, the transcription factor lack the binding pocket for being used for small-molecule drug.Since many TF lack determining three-dimensional structure, especially It is its protein-protein binding structural domain, recombinantly expresses the difficulty of TF, and lack the measurement skill for measuring its binding mode Art, so that this task is extremely difficult (Fontaine etc., 2015).The active adjusting of TF is usually by changing them in nucleus In gene expression dose or concentration, or realized by changing them to the binding ability of DNA or chaperone, the latter is real The more promising strategy of existing TF selectivity.It is some to demonstrate this about the publication for destroying the small molecule that chaperone TF is raised Kind of method potentiality (Miyoshi etc., 2011, Vassilev etc., 2004, Filippakopoulos etc., 2010, Vogler etc., 2009, Liu et al., 2014).

In TF in human genome, it is outstanding for developing TF as attractive molecular target, because of their table It up to usually lacking of proper care under the conditions of adult particular pathologies, however is silencing in physiological conditions (for example, for manhood phenotype Maintenance is unwanted) (Boyadjiev and Jabs, 2000, Darnell, 2002, Lopez-Bigas etc., 2006, Vaquerizas etc., 2009).One kind development factor, i.e. SOX (SRY correlation HMG- box) TF occur being stem cell programming recently In key regulator and cancer related conditions molecular switch (Sarkar and Hochedlinger, 2013, Niwa etc., 2009).Previously SOX2 (Narasimhan etc., 2011) was focused primarily upon (in a kind of various cancers in the trial of targeting SOX albumen Potential oncogene) (key molecule for vascular development is opened by (Bass etc., 2009) and SOX18 (Klaus etc., 2016) Close) (Cermenati etc., 2008, Francois etc., 2008, Pennisi etc., 2000).The gloomy polyoxometallate in road has been demonstrated It can inhibit SOX2DNA combination, however, only showing the low selectivity to various TF families, and never in any external or body Test (Narasimhan etc., 2014, Narasimhan etc., 2011) was carried out in interior functional examination.Recently, SOX DNA bait SOX18 DNA has been used as it in conjunction with the selective depressant with SOX18 dependence trans-activation.Although these baits show ratio The non-higher selectivity of SOX TF, but their own cannot diffuse through cell membrane, to limit their application range (Klaus etc., 2016).How one aspect that do not explore of the pharmacological modulation of SOX TF and these protein raise its spouse Body is simultaneously therefore related by a series of protein-protein interactions (PPI) adjusting transcription.Normally, synthetic library is certain Structure diversity needed for not targeting PPI (Hopkins and Groom, 2002, Feher and Schmidt, 2003).

The SOXF group (SOX7, SOX17 and SOX18) of transcription factor (TF) is the key that endothelial cell differentiation in growth course Regulatory factor (Francois etc., 2008, Corada etc., 2013, Hosking etc., 2009, Matsui etc., 2006, Cermenati Deng 2008, Herpers etc., 2008), it is therefore, most important for the formation of vascular system.The mutation or missing of SoxF gene Arteriovenous specification, vascular integrity and lymphatic vessel generation can be made impaired, and inhibit tumour growth in animal model for cancer and turn It moves (Duong etc., 2012, Yang etc., 2013, Zhang etc., 2009, Young etc., 2006).Recently, high-caliber SOX18 and people The cancer prognosis of patient is bad related (Eom etc., 2012, Pula etc., 2013, Jethon etc., 2015).Therefore, SOX18 albumen function The pharmacology of energy inhibits to provide the potential treatment in the potential approach and blood vessel cancer for the vascular reaction in treating cancer Target.

Therefore, there is still a need for inhibit SOX18 protein active (such as by directly it is in connection, or close to its DNA combine tie Structure domain) to upset the compound that for example SOX18- protein partner is raised and/or SOX18 DNA is combined.

Summary of the invention

The present invention is based at least partially on following discovery: certain conjunction objects of formula provided herein, which have, inhibits SOX18 albumen The effect of active (especially with regard to the ability of SOX18 combination DNA and/or specific protein gametophyte).By extension, further Show these compounds treatment angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or It is effective in terms of the patient's condition such as cancer metastasis and blood vessel cancer.

In the first aspect of the present invention, formula (I) compound or its pharmaceutically acceptable salt, solvate or preceding are provided Medicine:

Wherein,

R₁Selected from OH and OR₆, wherein R₆For C₁–C₄Alkyl；

R₂Selected from H, COOR₇With C (O) NR₈R₉, wherein R₇、R₈And R₉It is independently selected from H and C₁–C₄Alkyl；

R₃For L-A, wherein L is selected from C₂–C₈Alkyl, C₂–C₈Alkenyl and C₂–C₈The connector of alkoxyalkyl, A are selected from optional Substituted phenyl and the naphthalene optionally replaced；

R₄Selected from H, OR₁₀, halogenated and C₁–C₄Alkyl, wherein R₁₀Selected from H and C₁–C₄Alkyl；And

R₅Selected from H, OR₁₁, halogenated and C₁–C₄Alkyl, wherein R₁₁Selected from H and C₁–C₄Alkyl,

Wherein, the compound is for inhibiting SOX18 active.

In embodiments, R₁Selected from OH and OMe.

Suitably, R₂Selected from H, COOH, COOMe and

Preferably, R₂Selected from COOH and

In embodiments, R₄Selected from H, OH, OMe, Cl and Me.

Suitably, R₅Selected from H, OH and OMe.

In certain embodiments, R₄And R₅For H.

In embodiments, L is selected from C₂–C₆Alkyl, C₂–C₆Alkenyl and C₂–C₆The connector of alkoxyalkyl.

In any embodiment, R₃It is selected from:

Wherein, dotted line indicates the connection of the ring from adjacent atom to Formulas I, and shown structure includes its E/Z isomers.

In one embodiment, the compound of first aspect is selected from:

It is highly preferred that the compound of first aspect is selected from:

Suitably, for the compound of present aspect, SOX18 activity include with DNA sequence dna and/or protein contacts and/ Or it combines.Preferably, protein is selected from SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

In the second aspect of the present invention, pharmaceutical composition is provided, it includes the compound of first aspect or its pharmacy Upper acceptable salt, solvate or prodrug and pharmaceutically acceptable carrier, diluent and/or excipient.

In the third aspect of the present invention, angiogenesis-associated diseases, illness or the disease for treating or preventing subject are provided The method of condition and/or lymphatic vessel generation related disease, illness or the patient's condition, including applying a effective amount of first aspect to subject Compound or its pharmaceutically the pharmaceutical composition of effective salt, solvate or prodrug or second aspect to treating or preventing blood The step of pipe generation and/or the relevant disease of lymphatic vessel generation, illness or the patient's condition.

In the fourth aspect of the present invention, the compound or its pharmaceutically effective salt, solvate of first aspect are provided Or prodrug is relevant for treating or preventing the relevant disease of angiogenesis, illness or the patient's condition and/or lymphatic vessel generation in preparation Purposes in the drug of disease, illness or the patient's condition.

When referring to the third and fourth aspect, the relevant disease of angiogenesis, illness or the patient's condition and/or lymphatic vessel generation phase Disease, illness or the patient's condition of pass are suitably or including ophthalmology disease, illness or the patient's condition.Preferably, ophthalmology disease, illness or disease Condition is selected from age-related macular degeneration, diabetic retinopathy, ischemic retinopathies, retinopathy of prematurity, new life Neovascular glaucoma, iritis, cornea rebirth blood vessel, cyclitis, sickle cell retinopathy, pteryium, corneal injury The vascular reaction and any combination thereof of period.

In the alternate embodiment of the invention of the third and fourth aspect, the relevant disease of angiogenesis, illness or disease Condition and/or the relevant disease of lymphatic vessel generation, illness or the patient's condition are or including cancer.Preferably, cancer is selected from prostate cancer, lung It is cancer, breast cancer, bladder cancer, kidney, colon cancer, gastric cancer, cancer of pancreas, oophoroma, melanoma, liver cancer, hepatocellular carcinoma, sarcoma, white Blood disease, acute T cell lymthoma, vascular tumor and any combination thereof.In specific embodiments, the compound of first aspect or The cancer metastasis is prevented and/or inhibited to the pharmaceutical composition of second aspect.

In two foregoing aspects of other embodiments, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching Hand shaft generates relevant disease, illness or the patient's condition suitably or including kidney trouble, illness or the patient's condition.Preferably, kidney disease Disease, illness or the patient's condition are selected from chronic renal portability function obstacle, primary kidney fibrosis illness, albuminuria, nephrosis, kidney Scorching and any combination thereof.

In two another foregoing aspects of embodiments, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching Hand shaft generates relevant disease, illness or the patient's condition.

In two another foregoing aspects of embodiments, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching Hand shaft generates relevant disease, illness or the patient's condition.

In the fifth aspect of the invention, the method for providing inhibition or preventing the cancer metastasis of subject, including to tested Person applies the first a effective amount of compound or its pharmaceutically medicine group of effective salt, solvate or prodrug or second aspect The step of object is closed to inhibit or prevent cancer metastasis.

Suitably, cancer is selected from prostate cancer, lung cancer, breast cancer, bladder cancer, kidney, colon cancer, gastric cancer, cancer of pancreas, ovum Nest cancer, melanoma, hepatoma, sarcoma, leukaemia, lymthoma, vascular tumor (for example, blood vessel swollen, angiosarcoma, hemangioma) and its Any combination.

In the sixth aspect of the present invention, provides inhibition, prevention or reduce the active method of SOX18 in subject, including The first a effective amount of compound or its pharmaceutically effective salt, solvate or prodrug or second aspect are applied to subject Pharmaceutical composition is to inhibit, prevent or reduce the step of the SOX18 activity in subject.

Suitably, SOX18 activity includes and DNA sequence dna and/or protein contacts and/or combination.Preferably, protein selects From SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

Brief Description Of Drawings

In order to should be readily appreciated that the present invention and try out, description is preferably implemented by way of example with reference to the drawings Scheme, in which:

Fig. 1: natural products, the inhibitor that SOX18-DNA is combined.A. the marine extracts from 2688 parts of 0.25mg/mL High-throughput FP screening representative data collection.To the complete of the SOX response element (mouse Prox1 introne 1) marked with FAM Long mouse SOX18 operation screening.The competitive binding of ligand and SOX18 reduce FP index, and (arrow is directed toward red dot activity and extracts Object).The chemical structure of B.Sm1 and Sm2.C.Sm1 and Sm2 FP concentration-response curve (overall length mouse SOX18, average value ± S.D..N=3).

Fig. 2: the focused libraries of analogue and utilization computer aggregation fallout predictor (aggregation predictor) With the secondary screening of critical micelle concentration (CMC) measurement.A. it is based on apparent o- hydroxybenzoic acid in compound Sm1 and Sm2 for first group (salicylic acid) motif.Second group is based on similar resorcinol bracket.Third group is containing similar salicylic acid or adjacent aminobenzene The NSAID of the approval of formic acid bracket.B. the allusion quotation of neutral detergent Triton X100 control and two kinds of compounds Sm4 and Sm10 Type CMC data.C.Sm4, Meclofenamic Acid, Niflumic Acid and Flufenamic acid inhibit SOX18-DNA to combine.

Fig. 3: compound and SOX protein-interacting, but do not interact with DNA.A., the biotinylated double-stranded DNA of B. Probe (about 40 base-pairs are long, there is SOX18 to share element (A.) or random sequence (B.), and flank genomic DNA) is used It is combined in test small molecule DNA.Probe is fixed on SPR Streptavidine chip.Positive control DAPI, bromination second Ingot and actinomycin D are in a manner of consistent with document in conjunction with DNA.Micromolecular inhibitor (Sm4,5 and 14) not with consensus sequence Or random DNA is combined.C. as measured by the difference static light scattering for being heated to 80 DEG C of protein complex from 25 DEG C, In The thermal stability of SOX18 [109] HMG segment in the presence of Prox1-DNA, Sm4,5 or Sm14.The combination of small molecule promotes Protein stability (Δ Tagg > 3 DEG C are considered as significantly stabilizing).The standardized light scattering of Boltzmann curve matching Triple data (goodness of fit R2 > 0.97).D. as measured by the DNA combination competition assay based on FP, Sm4 inhibits The DNA of SOX2,6,9,11,15 and 18-HMG segment is combined.

Fig. 4: Sm4, the shadow of Niflumic Acid, Flufenamic acid and Meclofenamic Acid to SOX18 protein-protein interaction It rings.A. left figure: such as by XRCC6 (negative control) and known two kinds of protein RBPJ with SOX18 interaction and MEF2C carries out the thermal map for the pairs of protein-protein interaction of SOX18 that α screening is tested.Right figure: protein complex Co-immunoprecipitation.Under the conditions of cell-free, SOX18-mCherry-cMyc GFP (yin with GFP-RBPJ, GFP-MEF2C or only Property control) coexpression, and with GFP Nanotrap pearl immunoprecipitation.Band: 1.RBPJ-GFP, 2.MEF2C-GFP, 3.SOX18- mCherry,4.GFP.The interaction of B.Sm4, Niflumic Acid, Flufenamic acid and Meclofenamic Acid to SOX18 and MEF2C and RBPJ Influence.

Fig. 5: Sm4 chemistry motif combines inhibition, SOX18-RBPJ to combine inhibition, cytotoxicity and aggregation SOX18 DNA Contribution.The table of upper figure describes Sm14-44 compound, and mode is summarized for four kinds of activity marks color-coded The result that (that is, protein-DNA and protein-protein combine inhibition, cytotoxicity and aggregation risk) obtains.Second bottom The SOX18-RBPJ protein-protein combination suppression result under 50 μM and 5 μM (when available) is described in detail in portion's bar chart (N=4, average value ± SD).(DNA combines inhibition, cytotoxicity and cLogP initial data to be summarized in table 3).Bottom bar chart Illustrate the inhibition of the SOX18-RBPJ protein-protein interaction (PPI) as measured by α the screening test method.It shows Result at 50 μM (PBS control, DMSO are compareed and the left stick of Sm4) and 5 μM (the right stick of Sm4-Sm44).It is worth noting Be that result (N=4, average value ± SD) is shown when compound can get.SOX18 homodimer is used in α screening test It is destroyed with the formation of SOX18/RBPJ heterodimer as the PPI for comparing the Sm4 series tested under 5 μM is read out.Some chemical combination The formation of object preferential destruction SOX18 homodimer, and other compounds form more specificity to SOX18 heterodimer, and Other compounds are then the general destruction factors of homodimer and heterodimeric complex.

Fig. 6: the computer modeling that the interface between SOX18-HMG and RBPJ carries out Sm4.To SOX18 dependence The external inhibition of trans-activation.A., in B.SOX18/Prox1DNA x-ray crystal structure Sm4 stable bond gesture, will inhibit Agent is placed in the open to the outside world pocket between protein and DNA.C. SOX18/DNA structure is docked to Notch transcription complex In structure.D. with Sox18 and the carrier transient transfection containing the Vcam1 promoter merged with Fluc gene Luciferase report gene in COS7 cell measures (Hosking etc., 2004).In the culture for containing maximum 1%DMSO (v/v) In base, handled cell 24 hours with small molecule with the concentration lower than CC10 (10% cytotoxicity).It describes for Sm4 and Buddhist nun's fluorine The result of acid.Meclofenamic Acid and Flufenamic acid are inactive under the concentration lower than CC10.

The inhibition of Fig. 7: COX-1/2 enzyme.SOX18 inhibitor inhibits COX-1 and COX-2 enzyme, including Meclofenamic Acid conduct Positive control.The PGH generated by arachidonic acid conversion₂The amount of prostanoid measures the inhibition of COX enzyme.

Fig. 8: Sm14-44 H NMR spectroscopy.

The positioning of Fig. 9: SOX18 meridian genomics and the destruction of Sm4 Thermodynamic parameters.(A) chromatin imrnunoprecipitation-is combined Mass spectrum (ChIP-MS) and amplification shine close to homogeneous assay method (Amplified Luminescent Proximity Homogeneous Assay) (α screening) method reality for making SOX18 dependence protein matter-protein interaction (PPI) deconvolute Test the schematic diagram of strategy.(B) to the identification in Human umbilical vein endothelial cells (HUVEC) by SOX18-cMyc ChIP-MS The GO-term of the molecular function of 289 kinds of protein is analyzed.Deduction finds non-specific in the transfection cell for containing only Myc label Property interaction factor.It is combined with nucleic acid or the protein of protein binding capacity (purple) is considered for continuously directly mutually Effect study identifies the similitude of the direct interaction factor to enhance.(C) left column: such as by α screening test about ChIP- The thermal map figure of the pairs of PPI of SOX18 of the selection of MS SOX18 GAP-associated protein GAP, the endothelial transcriptional factor and male/female reference protein Show.Right column: the active thermal map of Sm4 to interact about SOX18 dependence protein matter-protein such as tested under 100 μM Diagram.Interaction and damage threshold are indicated in scale bar with black line.Higher than the interaction of threshold value and destroy horizontal by '+' It distinguishes, is distinguished lower than threshold value by '-'.Tagged protein is at lizard Leishmania (Leishmania tarentolae) It is expressed in cell-free protein expression system.(D) representative α screening concentration-response curve that Sm4 destroys SOX18 PPI.It is aobvious The data shown are average value ± s.e.m..

The effect of the QC and Sm4 of Figure 10: SOX18 PPI.(A) utilize anti-cMyc antibody from cMyc-SOX18 in HUVEC Immunoprecipitation identification the representative tape double charge with sequence KAPILIATDVASRG (Muscat ion scoring be 51.6) DDX17 peptide mass spectrum wave spectrum.(B) identification selected from the SOX18 peptide of ChIP-MS and the coverage rate of interaction protein.(C) The amino acid sequence of DDX17, wherein the ChIP-MS peptide identified is indicated with green.(D) the typical α screening of protein dilution optimization Curve shows SOX9-SOX9 and SOX18-SOX18.The presence at peak (hook effect) shows to interact and represent continuous The ideal protein concentration of binding.Protein is expressed in lizard Leishmania cell-free protein expression system.(E) The molecular structure of SOX18 inhibitor Sm4.(F) α screening concentration-response curve that Sm4 destroys SOX18 PPI.The data of display It is average value ± s.e.m..

Figure 11: Sm4 destroys the difference of SOXF PPI.Left figure show by α screening measurement SOXF, MEF2C and The matrix of protein-protein interaction between RBPJ and OCT4.Right figure shows influence (blue of 50 μM of Sm4 to PPI =destroyed without PPI/, green/yellow=low PPI/ is destroyed, and orange/red=strong PPI/ is destroyed completely, and grey=lower than threshold value PPI, Sm4 effect can not determine).

Figure 12: Sm4 selectively influences SOX18 transcription output in vitro.(A) full-length genome TF ChIP-seq data with The schematic diagram of correlation analysis between the gene influenced by Sm4 from transcription group data.Have studied the base influenced by Sm4 Because of the chromatinic transcription factor combination peak (grey) around the transcription initiation site (TSS) of (purple), to calculate given transcription " from the TSS to the distance of nearest binding site " of the factor.This representative for being used as transcriptional regulatory possibility at a distance from TSS, because This makes to exist between the gene influenced by Sm4 and transcription factor and be associated with (Cusanovich etc., PLoS Genetics, 2014； Verbist etc., Drug Discov Today, 2015).It is included in SOX18 and SOX7 and can be obtained from Encode alliance All transcription factors (GATA2, c-FOS, c-JUN, CTCF, EZH2, MAX and c-MYC) the peak ChIP-seq in HUVEC into In capable analysis.It is analyzed using one group of random gene as being compareed as serendipitous.(B) it is influenced by Sm4 Gene be grouped into downward (Sm4- downward), uninfluenced (Sm4- has not been changed) and up-regulation (Sm4- up-regulation).The figure show by The TSS (purple line, absolute multiple variation >=2) and SOX18 of the gene that Sm4 influences and compare transcription factor SOX7 and GATA2 Binding site the cumulative distribution closest to the distance between genomic locations.By the TSS of difference expression gene and given turn The intermediate value distance closest to binding events of the record factor is compared with from the accidentally expected intermediate value distance of random gene group (green line) Compared with.The gene and the peak SOX18 that Sm4 is lowered are significantly closer (runic), but not closer with the peak SOX7 or GATA2.

The in-vitro transcription group surface analysis of Figure 13: Sm4 selectivity.(A) SOX18ChIP-seq carried out from HUVEC Most common (top) motif of peak (MEME software) identification.(B) representativeness of SOX18 target gene VCAM and PROX1 known to The UCSC browser view at the peak ChIP-seq (arrow).(C) condition of the transcript profile surface analysis of Sm4.Using DEseq2 in mistake Express the difference calculated between medium DMSO (SOX18oe) and the cell (Sm4) for receiving 25 μM of Sm4 in the HUVEC of SOX18 It expresses (DE).(D) principal component analysis of quadruplicate RNA-seq sample.The same terms (control, SOX18oe, Sm4) will be come from Repeat samples cluster together.(E) figure of the display DESeq2 compared between edgeR method, mark SOX18oe with The conspicuousness of DE gene between Sm4 condition.The transcript for changing >=1 or≤- 1 (dotted line) with DESeq2Log2 multiple is considered For further analyzing.(F) by the binding site of the gene (purple) influenced by Sm4 and given transcription factor closest to gene The distance between group position is plotted as cumulative distribution.From the TSS of difference expression gene to the nearest knot of transcription factor binding events The intermediate value distance of conjunction event is expressed as the ratio (random gene, green) relative to accidental expected intermediate value distance.

Figure 14: c-JUN motif is enriched in SOX18 binding site.(A) to the HOMER motif point at the peak SOX18ChIP-seq Analysis discloses the enrichment of c-JUN motif 5'-TGAC/GTCA-3'.(B) SOX18-c-JUN and SOX18-SOX18 (positive control) α screen binding curve, it was demonstrated that c-JUN has the ability with SOX18 direct interaction in vitro.Protein is assorted in lizard benefit It is expressed in graceful protozoon cell-free protein expression system.

Figure 15: Sm4 does not interfere SOX9 or SOX17 activity in vitro.(A) the active report based on cell of SOX9 homodimer Accuse genetic testing.COS-7 cell is transfected with Sox9 and Col2a1:luc reporter gene construct.Sox9 overexpression leads to > 8 times Col2a1 activation induction.Variation is not observed at the Sm4 of high concentration.(B) the active reporter gene based on cell of SOX17 is surveyed Fixed (Robinson etc., 2014).Bovine aortic is transfected with pTK- β-gal (pTK) or ECE1-TK- β-gal (ECE1) reporter gene Endothelial cell (BAEC) measures the endogenous activity of SOX17 (only ECE1).Variation is not observed under any test concentrations.X-axis On number be μM to indicate [Sm4].

Figure 16: Sm4 blocks SoxF transcriptional activity in vivo.(A) tg (- 6.5kdrl:eGFP) SoxF reporter gene is carried The lateral bright-field (top) and fluorescence (bottom) image of 60hpf zebrafish larvae.Existed with DMSO (negative control) or 1 μM of Sm4 Advanced stage, (20hpf) was started to process, or injected morpholino (dMO sox7/18) to the young for sox7 and sox18.Fluorescence intensity As shown in thermal map.200 μm of scale bar.(B) to the tg (- 6.5kdrl:eGFP) young of processing and sox7/18 morpholine mutant (morphant) qRT-PCR of the gfp transcript level in is analyzed, and shows that the activity of the transgenosis reduces.(C) tg is carried (Dll4in3:eGFP) side view of the zebrafish larvae of SoxF/Notch reporter gene, the reporter gene have Rbpj and Multiple binding sites of SoxF transcription factor.It uses from 13hpf for the morpholino injection young of rbpj and/or at 2 μM of Sm4 Manage the young.(D) qRT-PCR of the gfp transcript in tg (Dll4in3:eGFP) young is analyzed, is shown in the children of Sm4 processing The active inhibition of the SoxF/Notch combined in body.(E) from early stage (16hpf) to 72hpf with the children of Sm4 or DMSO control treatment Embryonic death rate quantifies in body.(F) from the 16hpf vascular phenotype (arteriovenous point of 1.5 μM of Sm4 48hpf young handled Stream) genepenetrance.(G) from the 16hpf genepenetrance of the circulation defect of 1.5 μM of Sm4 48hpf young handled.(H) relative to DMSO compares (dotted line), the endogenous endothelial transcript level in the young that 1.5 μM of Sm4 of 16hpf are handled when 48hpf QRT-PCR analysis.The data of display are average value ± s.e.m..* p < 0.001 p < 0.05, * * p < 0.01, * * *.

Figure 17: Sox9 activity is not by internal processing interference.(A) timeline: the continuous processing in cartilage formation process is handled Zebrafish larvae 4 days.Update culture medium daily in the entire experiment process to maintain Sm4 horizontal.(B)tg(col2a1:YFP) The Sox9 reporter gene young marks cartilage (Mitchell etc., 2013).YFP level is unaffected in the presence of Sm4, and And chondrogenetic variation is not observed.Mc: Meckel's cartilage, ch: cartilago ceratohyoidea, hs: pharynx bow hyomandibular (hyosymplectic).(C) yfp in a series of entire chondrogenetic stages in the young of DMSO control and Sm4 processing The qRT-PCR of transcript level.

Figure 18: Sm4 interferes SoxF active in vivo.(A) timeline that Sm4 is handled in zebrafish larvae.SOXF reports base Because the processing of research starts in 20hpf, and for phenotype research, processing starts from arteriovenous specification (arteriovenous Specification start before correct development window), and work during it.(B) DMSO control and Sm4 are handled The side view in the trunk area of tg (fli1:eGFP, -6.5kdrl:mCherry) young and cross section.The control DMSO young forms bright Aobvious isolated aorta dorsalis (DA) and vena postcardinalis (PCV).In the young of Sm4 processing, DA is compacted and/or merges with PCV (arrow).For artery marker efnb2a whole mount in situ hybridization show 48hpf when Sm4 processing the young in reduction table The DA for reaching and being damaged forms (arrow).DAPI dyeing (blue) is carried out to slice.Scale bar bright-field: 0.5mm, fluorescence and original 25 μm of position.(C) the concentration dependent effect of Sm4 shows the dominant phenotype (predominant phenotype) when 72hpf: light Degree (tail portion bending), medium (expansion of PCV) or serious (arteriovenous defect and/or recycle defect) quantify.The time frame of instruction Refer to Sm4 processing window and terminal.(D) quantifying with the cardiac edema frequency of the young of (1.5 μM) of Sm4 processing.(E) relative to DMSO compares (dotted line), Sox18 dependence -6.5kdr1:mCherry and endogenous endothelial transcript water in the young of Sm4 processing Flat qRT-PCR analysis, effect when being shown in pf for 24 hours to artery and vein marker.All expressions are directed to endothelium The expression of marker cdh5 is standardized.The data of display are average value ± s.e.m..* p < 0.05, * * p < 0.01, * * * p < 0.001。

Figure 19: Sm4 processing inhibits transfer and Tumor angiogenesis.(A) timeline of the mouse model of Metastasis in Breast Cancer.In 0th day inoculation 4T1.2 tumour, and cut off at the 12nd day.From the 3rd day to the 12nd day, Sm4 (25mg/ is administered orally once a day Kg/ days), aspirin (25mg/kg/ days) or intermedium control (PBS).Independent experiment is carried out to assess survival rate and transfer Rate.(B) show the good systemic delivery of drug in the plasma concentration of processing scheme (the 7th day and the 12nd day) period Sm4.(C) Expression of the SOX18 such as shown by situ hybridization in tumor vascular system.100 μm of scale bar.(D) survival of mouse is monitored Rate (every group of n=6-12 mouse).The mouse of Sm4 processing is analyzed relative to intermedium control and A Si by Log-Rank Test The improved survival rate (p < 0.001) of woods.(E) significant difference of tumor size is not found in any stage.(F) in any matchmaker The animal of the control of Jie's object or Sm4 processing dies of before burden of cancer, the metastatic tumo(u)r tubercle on the 28th day quantitative lung surface. The data of display are the average value ± s.e.m. of every group of 12-14 mouse.(G) blood on 300 μm of slices of entire tumour is investigated Pipe density.Brightfield image shows that whole brightfield image shows the configuration (by dotted line Outline) and red blood cell of tumour Presence, mark Major Vessels and hemorrhagic areas (asterisk).Scale bar 1mm.(H) endothelial specificity marker ERG and endothelium are viscous The Double immune fluorescent dyeing of albumen (EMCN) discloses the vascular pattern of in tumour and peripheral region and penetrates (penetrance).Left: entire tumor biopsy (scale bar 1mm) intermediate and right side: adds the enlarged drawing (scale bar 200 of frame region μm).(I) the EMCN volume (vessel density) of n=6 tumour and ERG positive nucleus (endothelial cell number) under the conditions of every kind It is quantitative.Each data point represents the average value of the 3-4 representative area (in figure H plus frame region) of each tumour.It shows Average value ± the s.e.m. of two kinds of conditions.* p < 0.01 p < 0.05, * *.(J) similar with upper figure (D), in application medium or it is incremented by The Sm4 (i.e. 5mg/kg, 10mg/kg, 25mg/kg and 50mg/kg) of concentration monitors survival rate (every group n=6-12 of mouse afterwards Mouse).The survival rate that the experiment determines that Sm4 improves is dose-dependent, and should be the result shows that internal specific middle target It engages (on-target engagement).

Figure 20: Sm4 effect is not the result of inflammation caused by performing the operation.In the 0th day inoculation 4T1.2 tumour, and the 12nd It is performed the operation, not tumor resection (n=6).(A) mouse (25mg/kg/ days) handled in PBS intermedium control mouse and Sm4 Middle monitoring survival rate (n=6).Difference (Log-Rank Test) is not observed.(C) in any stage before or after sham-operation The significant difference of tumor size is not found.

Figure 21: Sm4 damage blood vessel penetrates into 4T1.2 tumour.For the mouse with PBS medium or Sm4 processing, come from The brightfield image of the continuous shaking slice (300 μm) of entire 4T1.2 tumor of breast.Major Vessels and hemorrhagic areas are with red color area Not.

The mouse of Figure 22: Sm4 processing has reduced microvessel density.ERG and endothelium mucoprotein on tumor biopsy (EMCN) immunofluorescence dyeing.Show two representative areas of medium PBS and Sm4.Detailed enlarged drawing shows ERG Different nuclear stainings and EMCN film in leather dyeing.Endothelial cell is carried out to the image with identical XYZ dimension in Imaris Quantity and blood vessel volume quantify.Selection threshold value is accurately to capture total EMCN+ vascular system and (the ERG counting of total ERG+ nucleus With the EMCN volume of yellow).

Figure 23: Sm4 processing destroys the lymphatic vessel generation of tumor inducing.For with PBS medium or Sm4 (25mg/kg days) The mouse of processing, the lymphatic vessel image of the continuous shaking slice (200 μm) from entire 4T1.2 tumor of breast.Lymphatic vessel is special The immunofluorescence of property marker PROX1 and flatfoot albumen (PDPN) and blood vessel EC marker endothelium mucoprotein (EMCN) discloses It the vascular pattern of in tumour and peripheral region and penetrates.The entire tumor biopsy of control group (above) and Sm4 processing group (following figure). The PDPN+ endolymphatic duct region (density, upper figure) and PROX1+ nucleus (lymphatic endothelial cells of n >=6 tumour under the conditions of every kind Number, the following figure) quantify.Scale bar is left: 0.5mm, right: 0.1mm.Show the average value ± s.e.m. of two kinds of conditions.**p< 0.01, * * * p < 0.001.

Figure 24: (A) unimolecule tracks the principle of (SMT) experiment.SMT allow in living cells to transcription factor such as The chromatin binding kinetics of SOX18 carry out real time imagery.SMT determines the search pattern of SOX18 albumen, while it scans gene Group is in conjunction with the specific reaction element of its target gene.(B) figure B describes experimental work process, is related to molecule track Two-dimensional tracking and the analysis for using MATLAB.SOX18 molecule and DNA (fixed) combination or do not combine and in nucleus freely Diffusion.In fixed fraction, can defining 2 groups based on the residence time on chromatin, (specific binding is non-specific Property combination).(C) upper figure；Sm4 with concentration dependant manner with sacrifice non-specific binding grade be divided into cost increase SOX18 it is special Property combine fraction.The following figure；Sm4 increases the residence time of the SOX18 fraction of specific binding, while reducing non-specific binding Fraction residence time.(D): upper figure；Sm4 is selectively engaged the SOX18 dominant negative mutation for facilitating the rescue of its part Body Ra^op" Ragged Opossum " mutant, on the contrary, it is benefited from non-specific binding fraction with concentration dependant manner and subtracts Few Ra^opSpecifically bind fraction.The following figure；Ra of the Sm4 to specificity and non-specific binding^opThe residence time of fraction with it is right SOX18 fraction has phase same-action, but effect becomes apparent from.

It is described in detail

Definition

In the specification, term " include (comprises) ", " including (comprising) ", " including (includes) ", " including (including) " or similar terms are intended to indicate that nonexcludability includes, so that including element list Method or composition not only include these elements, and may further include unlisted other elements.

Unless otherwise defined, otherwise all technical and scientific terms used herein have with it is of the art general The logical identical meaning of the normally understood meaning of technical staff.

As used herein, term " alkyl " refers to containing such as 1 to about 8 carbon atom, preferably 1 to about 7 carbon atom, More preferable 1 to about 6 carbon atom, the linear or branched alkyl group substituent group of even more preferably 1 to about 4 carbon atom.Such substitution The example of base can be selected from methyl, ethyl, propyl, isopropyl, normal-butyl, sec-butyl, isobutyl group, tert-butyl, amyl, isopentyl, 2- methyl butyl, 3- methyl butyl, hexyl, heptyl, 2- methyl amyl, 3- methyl amyl, 4- methyl amyl, 2- ethyl-butyl, 3- ethyl-butyl, octyl, nonyl, decyl, undecyl, dodecyl etc..The number of mentioned carbon is related to carbon backbone chain and carbon Branch, but do not include belonging to the carbon atom of any substituent group, such as from the alkyl of main carbochain branch or alkoxy substituent Carbon atom.

Term " alkenyl " refers to the unsaturated linear chain or branched chain alkyl optionally replaced, with 2-8 carbon atom, preferably 2- 7 carbon atoms, more preferable 2-6 carbon atom or 2-4 carbon atom and have at least one carbon-to-carbon double bond.Where appropriate, alkenyl There can be the carbon atom specified number, for example, C₂-C₆Alkenyl comprising have 2,3,4,5 or 6 linear or branched arrangement The alkenyl of carbon atom.Mentioned carbon number number is related to carbon backbone chain and carbon branch, but does not include the carbon for belonging to any substituent group Atom.The example of such substituent group can be selected from vinyl, acrylic, isopropenyl, cyclobutenyl, secondary cyclobutenyl and tertiary cyclobutenyl, Pentenyl, hexenyl, hept- 1,3- diene, hex- 1,3- diene, nonyl- 1,3,5- triolefin etc..

As used herein, term " alkoxyalkyl " means the linear or branched alkyl group connected by oxygen atom (that is, alkane Base-O- alkyl, further referred to as " ether " base), wherein alkyl is as described above.In specific embodiments, alkoxyalkyl Refer to the oxygen linking group (" C1-8 alkoxyalkyl ") comprising 1-8 carbon atom.In a further embodiment, alkoxy alkane Base refers to comprising 2 to 8 carbon atoms (" C2-8 alkoxyalkyl "), 2 to 6 carbon atoms (" C2-6 alkoxyalkyl "), 2 to 4 The group of the oxygen of a carbon atom (" C2-4 " alkoxyalkyl ") or 2 to 3 carbon atoms (" C2-3 alkoxyalkyl ") connection.Institute It states carbon atom number and those of refers in entire alkoxyalkyl/ether chain carbon atom.

As used herein, term " optionally replacing " refers to the substituent group that can be extended from related group, such as phenyl Or naphthalene, and may include such functional group of such as halo groups including F, Cl and Br；C₁-C₄Alkyl；OR₁₂, wherein R₁₂It is C_1-4Alkyl；And NR₁₃R₁₄, wherein R₁₃And R₁₄It is independently selected from H and C₁-C₄Alkyl.

According to the first aspect of the invention, the compound or its pharmaceutically acceptable salt, solvate of formula (I) are provided Or prodrug:

Wherein,

R₁Selected from OH and OR₆, wherein R₆For C₁–C₄Alkyl；

R₂Selected from H, COOR₇With C (O) NR₈R₉, wherein R₇、R₈And R₉It is independently selected from H and C₁–C₄Alkyl；

R₃For L-A, wherein L is selected from C₂–C₈Alkyl, C₂–C₈Alkenyl and C₂–C₈The connector of alkoxyalkyl, A are selected from optional Substituted phenyl and the naphthalene optionally replaced；

R₄Selected from H, OR₁₀, halogenated and C₁–C₄Alkyl, wherein R₁₀Selected from H and C₁–C₄Alkyl；And

R₅Selected from H, OR₁₁, halogenated and C₁–C₄Alkyl, wherein R₁₁Selected from H and C₁–C₄Alkyl,

Wherein, compound is for inhibiting SOX18 active.

In embodiments, R₁Selected from OH and OMe.

Suitably, R₂Selected from H, COOH, COOMe and

Preferably, R₂Selected from COOH and

In embodiments, R₄Selected from H, OH, OMe, Cl and Me.

Suitably, R₅Selected from H, OH and OMe.

In certain embodiments, R₄And R₅For H.

In embodiments, L is selected from C₂–C₆Alkyl, C₂–C₆Alkenyl and C₂–C₆The connector of alkoxyalkyl.

In any embodiment, R₃It is selected from:

Wherein, dotted line indicates the connection of the ring from adjacent atom to Formulas I, and shown structure includes its E/Z isomers.

In one embodiment, the compound of first aspect is selected from:

In a preferred embodiment, the compound of present aspect is selected from:

It will be understood by those skilled in the art that SOX18 is the member of SOX (SRY correlation HMG- box) transcription factor family.These Transcription factor usually participates in the regulation of embryonic development and the determination of cell fate.Particularly, SOX18 albumen with other protein Transcriptional regulatory agent can be played after formation protein complex to work.Have shown that SOX18 is developed in hair, blood vessel and lymphatic vessel In work.Other titles of SASH1 may include SRY- box 18, HLTS and HLTRS.Referring to the nucleotide sequence of SOX18 gene Or the non-limiting example of the accession number of the protein of its coding includes as known in the art NG_ in people 008095.1, NM_018419.2 and NP_060889.1.Unless otherwise stated, " SOX18 " can as usually used herein To refer to the protein of SOX18 nucleic acid or coding.

Suitably, the SOX18 activity being conditioned is lymphatic vessel generation (that is, new lymphatic vessel is from existing vasculolymphatic life It is long), angiogenesis (that is, from the beginning formation of embryo's circulatory system) and/or angiogenesis be (that is, blood vessel is from existing vascular system Growth) activity.For this purpose, the compound of one or more formulas (I) can be used for treating, subtract in the embodiment of first aspect Less or prevention lymphatic vessel generation, angiogenesis and/or angiogenesis.

Therefore, the compound of one or more formulas (I) suitably have prevention and/or mitigate angiogenesis-associated diseases, The effect of the symptom severity of illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition.

In one embodiment, SOX18 activity includes and DNA sequence dna and/or protein contacts and/or combination.At this The compound of aspect, first aspect can be to angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation correlation disease One or more potential cell signaling pathways of disease, illness or the patient's condition have an impact, including but not limited to inhibition SOX18 DNA is combined and/or protein-protein interaction.

Combined about DNA, it should be understood that SOX18 be can with transcription factor DNA ins conjunction with, such as by its HMG box and Consensus sequence 5'-AACAAAG-3' is combined, to be transcribed by this in conjunction with trans-activation.In addition, SOX18 albumen can with one kind Or transcriptional regulatory agent is played after multiple proteins formation protein complex and is worked.

As used herein, " gene " is the nucleic acid of the Structure Heredity unit as genome, may include one or more The nucleotide sequence of a coding amino acid and one or more non-coding nucleotide sequences (including promoter and other 5' untranslateds Sequence, introne, Polyadenylation sequences and other 3' non-translated sequences, but not limited to this).In most cells biology, Gene is the nucleic acid comprising double-stranded DNA.

As used herein, term " nucleic acid " indicates single-stranded or double-stranded DNA and RNA.DNA includes genomic DNA and cDNA. RNA includes mRNA, RNA, RNAi, siRNA, cRNA and self-catalysis RNA.Nucleic acid is also possible to DNA RNA hybrid.Nucleic acid includes Generally comprise the nucleotide sequence of the nucleotide containing A, G, C, T or U base.However, nucleotide sequence may include other bases, Such as inosine, methylcystein, methylinosine, methyladenosine and/or sulphur urine glycosides, but not limited to this.

" protein " refers to amino acid polymer.As understood by those skilled in the art, term " protein " is in its model It further include the phosphorylation form (i.e. phosphoprotein) of protein and/or the glycoforms (i.e. glycoprotein) of protein in enclosing." peptide " It is with the protein no more than a amino acid in 50 (50)." polypeptide " is with the albumen no more than a amino acid in 50 (50) Matter.

Additionally provide the protein " variant " of SOX18, such as naturally occurring (such as allelic variant) and its directly to Homologue.Preferably, the amino acid sequence of protein variant and SOX18 disclosed herein or known in the art are shared at least 70% or 75%, preferably at least 80% or 85% or more preferably at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

As used herein, term " protein-protein interaction " or " PPI " refer to two or more protein Between close and stable association.It is usually directed to form Noncovalent chemical bonds, such as hydrogen bond.PPI can be (the two of binary A protein binding partner；Dimer) or ternary (tertiary) (three or more protein binding partners, example Such as tripolymer).Protein (i.e. binding partners) in PPI can be identical protein (such as homodimer or same trimerization Body) or different protein (such as heterodimer or miscellaneous tripolymer).Preferably, protein interaction is reversible, so that The dissociation of SOX18 and protein or protein subunit can occur under suitable conditions.Preferably, such power is very weak, for example, K with μM range_d, the compound of the present invention is allowed to destroy the interaction between SOX18 and protein.

Preferably, protein is selected from SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

In view of the foregoing, one of the compound of first aspect advantageous aspect is: depending on the base around core phenyl ring The selection of group, the clinical effectiveness that they show can be customized for passing through to a certain extent according to the selection of group SOX18 inhibits DNA combination and/or specific protein-protein interaction.

In some embodiments, it is possible to provide there is the compound of one or more chiral centres.Although chemical combination of the present invention The racemic mixture of object can be active, selective and bioavailable but isolated isomers and be also possible to enable People is interested.

The compound of the present invention further includes the stereoisomer of compound as described herein, and under applicable circumstances, Composition comprising more than one the compound of the present invention may include that such solid for individually or in any proportion mixing is different Structure body, such as E/Z isomers.Stereoisomer may include but be not limited to enantiomter, diastereoisomer, racemic mixing Object and combinations thereof.Such stereoisomer can be used routine techniques (by make enantiomer starting material react, or by point Isomers from the compound of the present invention and prodrug) it prepares and separates.Isomers may include geometric isomer.Geometric isomer Example include but is not limited to cross over the transisomer or cis-isomer (E/Z) of double bond.It is set in the compound of the present invention Other isomers are thought.Isomers can be mixed using or with other isomers of compound as described herein in a pure form to be made With.

It is known in the art to be used to prepare optical active forms and determine active various methods.Such method includes this paper institute The standard testing and other similar test well known in the art stated.It can be used for obtaining the optical siomerism of compound according to the present invention The example of the method for body include the following:

I) physical separation of crystal, in this way, macroscopical crystal of each enantiomer of manual separation.It is independent when existing Enantiomer crystal (that is, the material is racemic heap collective) and visually different crystal when, which can be spy It does not use；

Ii) crystallize simultaneously, in this way, each enantiomer crystallized from racemic solution respectively, only when after Person is likely to when being solid racemic heap collective；

Iii) Its Enzymatic Resolution, in this way, can be partially or wholly separated by the differential responses rate of enantiomer and enzyme Racemate；

Iv) enzymatic asymmetric syntheses, a kind of wherein at least one synthesis step obtain required mapping using enzymatic reaction The technology of the enantiomer-pure of body or enrichment synthesis precursor；

V) chemical asymmetric syntheses, in this way, from non-under conditions of generating asymmetry (i.e. chiral) in the product Enantiomer needed for chiral precursor synthesis, chiral catalyst can be used in this or chiral auxiliary is realized；

Vi) diastereoisomer separates, in this way, (chirality helps by the reagent of racemic compound and enantiomer-pure Agent) it reacts, each enantiomter is converted diastereoisomer by the reagent.Then had now according to them and become apparent from Architectural difference by chromatography or the resulting diastereoisomer of Crystallization Separation, it is required to obtain then to remove chiral auxiliary Enantiomter；

Vii) firsts and seconds asymmetric transformation, in this way, the diastereoisomer from racemate is balanced To generate advantage from required enantiomter or diastereoisomer in diastereo-isomerism liquid solution from required mapping Preferential crystallization in isomers upsets balance, so that final, all in principle by material to convert crystallization from required enantiomer non- Enantiomter.Then required enantiomter is released from diastereoisomer；

Viii) Kinetic Resolution comprising by enantiomer under dynamic conditions with chiral, non-racemic reagent or urge The reaction rates such as or not agent, partially or completely resolution of racemic object (or compound for further splitting partial resolution)；

Ix it) is synthesized by the enantiospecific that non-racemic precursor carries out, in this way, being obtained from achiral starting material Required enantiomer, and wherein stereochemical integrity is without damage in the synthesis process is or only by the damage of minimum Evil；

X) chiral liquid chromatography, in this way, the enantiomer of racemate is due to their phases different from stationary phase Interaction and separated in liquid mobile phase.Stationary phase can be made of chiral material or mobile phase may include other hand Property material is to cause different interactions；

Xi) chiral gas chromatography, in this way, making racemate volatilize, and since they are in gaseous flow phase In from the column containing fixed non-racemic chiral sorbent phase different interact and enantiomer separation；

Xii it) is extracted with chiral solvent, in this way, can be by the way that a kind of enantiomer is preferentially dissolved into specific chirality Carry out enantiomer separation in solvent；And

Xiii) across chiral film transhipment, in this way, racemic modification is contacted with thin barrier film.Barrier is generally separated two kinds Miscible fluid, one kind containing racemate, and driving force such as concentration or pressure difference lead to the preferential biography across envelope barrier It is defeated.Separation only allows a kind of enantiomer of racemate by occurring because of the non-racemic Chirality of film.

The compound of first aspect can optionally be provided in the composition of enrichment enantiomer or diastereomer In, the mixture of such as enantiomer or diastereomer, one of enantiomer or diastereomer excess In the presence of, particularly reach 95% or more, 96% or more, 97% or more, 98% or more or 99% or more (including 100%) degree.

Optionally, the compound of first aspect itself can use or with pharmaceutically acceptable ester, amide, salt, solvation The form of object, prodrug or isomers uses.For example, compound can be used as pharmaceutically acceptable salt offer.If used, medicine The salt of compounds should be pharmacologically with it is pharmaceutically acceptable, but acceptable salt may be conveniently used in non-pharmaceutical Free reactive compound or its pharmaceutically acceptable salt are prepared, and is not precluded within except the scope of the present invention.Such pharmacology On and the standard method that is described in detail in document can be used in pharmaceutically acceptable salt, passes through the anti-of drug and organic acid or inorganic acid It should prepare.

The example of the pharmaceutically acceptable salt of useful compound according to the present invention includes acid-addition salts.However, non- The salt of pharmaceutically acceptable acid can be used for the preparation and purification of such as compound.Suitable acid-addition salts packet according to the present invention Include organic acid and inorganic acid.Preferred salt includes by hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, citric acid, tartaric acid, lactic acid, acetone Acid, acetic acid, succinic acid, fumaric acid, maleic acid, oxaloacetic acid, methanesulfonic acid, ethanesulfonic acid, p-methyl benzenesulfonic acid, benzene sulfonic acid and ethoxy Those of sulfonic acid formation salt.Other useful acid-addition salts include propionic acid, glycolic, oxalic acid, malic acid, malonic acid, benzoic acid, Cinnamic acid, mandelic acid, salicylic acid etc..The particular instance of pharmaceutically acceptable salt include but is not limited to sulfate, pyrosulfate, Disulfate, sulphite, bisulfites, phosphate, dibasic alkaliine, dihydric phosphate, metaphosphate, pyrophosphate, Chloride, bromide, iodide, acetate, propionate, caprate, caprylate, acrylates, formates, isobutyrate, oneself Hydrochlorate, enanthate, propiolate, oxalates, malonate, succinate, suberate, sebacate, fumarate, Malaysia Hydrochlorate, butine -1,4- diacid salt, hexin -1,6- diacid salt, benzoate, chloro benzoate, methyl benzoate, dinitrobenzene Formates, hydroxy benzoate, methoxy benzoic acid salt, phthalate, sulfonate, xylenesulfonate, phenylacetate, Phenpropionate, phenyl butyrate, citrate, lactate, gamma hydroxybutyrate, glycollate, tartrate, mesylate, third Sulfonate, naphthalene -1- sulfonate, naphthalene-2-sulfonic acid salt and mandelate.

By that acid-addition salts can be then converted into free alkali with suitable alkali process.It can reside according to the present invention Pharmaceutically acceptable alkali (such as hydroxide can be used in the preparation of the basic salt of acid moieties on useful compound or prodrug Sodium, potassium hydroxide, ammonium hydroxide, calcium hydroxide, triethylamine etc.) it prepares in a similar way.

The ester of compound according to the present invention can be by being functionalized the hydroxyl and/or carboxyl that are likely to be present in compound To prepare.Also technology well known by persons skilled in the art can be used to prepare for amide and prodrug.For example, amide can be used suitably Amine reactant is prepared by ester or they can be by reacting with ammonia or low-grade alkylamine by acid anhydrides or acyl chlorides preparation.In addition, this The ester and amide of the compound of invention can by 0 DEG C to 60 DEG C at a temperature of with carbonylation agent (such as Ethyl formate, acetic acid Acid anhydride, methoxyacetyl chloride, chlorobenzoyl chloride, methyl isocyanate, ethyl chloroformate, mesyl chloride) and suitable alkali (for example, 4- Dimethyl aminopyridine, pyridine, triethylamine, potassium carbonate) in suitable organic solvent (for example, tetrahydrofuran, acetone, methanol, pyrrole Pyridine, N,N-dimethylformamide) in reaction to generate.

The example of pharmaceutically acceptable solvate includes but is not limited to compound according to the present invention and water, isopropyl The combination of alcohol, ethyl alcohol, methanol, DMSO, ethyl acetate, acetic acid or ethanol amine.

According to the second aspect of the invention, pharmaceutical composition is provided, it includes the compound of first aspect or its pharmacy Upper acceptable salt, solvate or prodrug and pharmaceutically acceptable carrier, diluent and/or excipient.

Suitably, pharmaceutically acceptable carrier, diluent and/or excipient can be or including diluent, solvent, pH Buffer, adhesive, filler, emulsifier, disintegrating agent, polymer, lubricant, oil, fat, wax, coating, viscosity modifier, One of glidant etc. is a variety of.

Since solubility improves, the salt form of the compounds of this invention can be particularly useful.

Diluent may include microcrystalline cellulose, lactose, mannitol, calcium phosphate, calcium sulfate, kaolin, dried starch, Icing Sugar etc. One of or it is a variety of.Adhesive may include one of povidone, starch, stearic acid, natural gum, hydroxypropyl methyl cellulose etc. Or it is a variety of.Disintegrating agent may include one of starch, croscarmellose sodium, Crospovidone, sodium starch glycollate etc. Or it is a variety of.Solvent may include one of ethyl alcohol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, methylene chloride, water etc. Or it is a variety of.Lubricant may include magnesium stearate, zinc stearate, calcium stearate, stearic acid, sodium stearyl fumarate, hydrogenated vegetable One of oil, Compritol 888 ATO etc. are a variety of.Glidant can be in colloidal silicon dioxide, talcum or cornstarch etc. It is one or more.Buffer may include phosphate buffer, borate buffer and carbonate buffer agent, but not limited to this.It fills out Material may include one or more gels (including gelatin), starch and synthetic polymer gel, but not limited to this.Coating may include into One of film, solvent, plasticizer etc. are a variety of.Suitable film forming agent can be hydroxypropyl methyl cellulose, methyl hydroxyl second In base cellulose, ethyl cellulose, hydroxypropyl cellulose, povidone, sodium carboxymethylcellulose, polyethylene glycol, acrylate etc. It is one or more.Suitable solvent may include water, ethyl alcohol, methanol, isopropanol, chloroform, acetone, methyl ethyl ketone, dichloromethane One of alkane etc. is a variety of.Plasticizer can be one in propylene glycol, castor oil, glycerol, polyethylene glycol, polysorbate etc. Kind is a variety of.

With reference to The Handbook of Excipients the 6th edition, Rowe, Sheskey&Quinn are edited (Pharmaceutical Press), provides the non-limiting example that can be useful excipient according to the present invention.

It should be understood that the selection of pharmaceutically acceptable carrier, diluent and/or excipient will depend, at least partially, on making The method of application of agent.Only for example, composition can be in tablet, capsule, cachet, powder, injectable liquids, suppository, sustained release Preparation, osmotic pump preparation form are effective and safe any other forms for application.

Suitably, pharmaceutical composition is used to treat or prevent the disease, illness or the patient's condition of mammal as described below.It is excellent Selection of land, pharmaceutical composition are used to treat or prevent angiogenesis-associated diseases, illness or the patient's condition and/or the lymph in mammal Pipe generates related disease, illness or the patient's condition.

The third aspect of the present invention is present in treatment or prevention by angiogenesis-associated diseases, illness or the patient's condition and/or leaching The method that hand shaft generates related disease, illness or the patient's condition comprising apply the compound or its pharmacy of a effective amount of first aspect The pharmaceutical composition of upper effective salt, solvate or prodrug or second aspect, to treat or prevent angiogenesis and/or leaching Hand shaft generates the step of relevant disease, illness or patient's condition.

The fourth aspect of the present invention provides the compound or its pharmaceutically effective salt, solvate or preceding of first aspect Medicine preparation for treat or prevent disease, illness or the patient's condition (the relevant disease of such as angiogenesis, illness or the patient's condition and/or The relevant disease of lymphatic vessel generation, illness or the patient's condition) drug in purposes.

Such as usually used, the term administering (administering) of this paper " or " application (administration) " etc. It describes and compound or composition is such as introduced into mammal by particular approach or medium.Administration method may include office Portion, parenteral and enteral route, including oral, oral cavity, sublingual, nose, anus, stomach and intestine, subcutaneous, intramuscular and intradermal administration way Diameter, but not limited to this.

As used herein, " treatment (treating) " (or " treatment (treat) " or " treatment (treatment) ") refers to Improve after angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition start development The therapeutic intervention of the S or S of the disease, illness or the patient's condition.About angiogenesis-associated diseases, illness or the patient's condition And/or lymphatic vessel generation related disease, illness or the patient's condition, term " improvement " refer to the beneficial effect of any observable for the treatment of. Treatment need not be absolutely beneficial to subject.Any method or standard known to those of ordinary skill can be used to determine beneficial to effect Fruit.

As used herein, " prevent (preventing) " (or " prevention (prevent) " or " prevention (prevention) ") refer to angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation related disease, illness or What symptom, aspect or the feature breaking-out of the patient's condition caused before, to prevent or mitigate the effect of symptom, aspect or feature (such as Apply one or more compounds as described herein of therapeutically effective amount) process.It should be understood that this prevention need not be to subject It is absolutely beneficial." preventative " treatment is that angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation occurs for reduction Related disease, the symptom of illness or the patient's condition, the purpose of the risk of aspect or feature and to do not show angiogenesis-associated diseases, The sign of illness or the patient's condition and/or lymphatic vessel generation related disease, illness or the patient's condition or the subject for only showing early stage sign The treatment of application.

As used herein, " effective quantity " refers to that the symptom for being enough to prevent the treated patient's condition occurs or deteriorates symptom The application of the amount of the related compound or composition of the seriousness of stopping or treatment and alleviation or at least mitigation symptom.Effective quantity It will change in the way understood by a person skilled in the art with patient age, gender, weight etc..It can be determined and be closed by routine test Suitable dosage or dosage.

As used herein, term " subject " or " individual " or " patient " can refer to any subject, especially vertebra Animal subjects, even more in particular for the mammalian subject for the treatment of.Suitable vertebrate includes but is not limited to spirit Long class animal, birds, livestock animals (for example, sheep, ox, horse, donkey, pig), laboratory test animal are (for example, rabbit, mouse, big Mouse, cavy, hamster), companion animals (for example, cat, dog) and wild animal in cage (for example, fox, deer, dingo).Such as this Described in text, preferred subject is to need to treat angiogenesis-associated diseases, illness or the patient's condition and/or lymphatic vessel generation correlation The people of disease, illness or the patient's condition.It should be appreciated, however, that above-mentioned term is not meant to certainly exist symptom.

As used herein, term " angiogenesis-associated diseases, illness or the patient's condition " indicate with abnormal vessel growth (including Excessive blood vessel development) relevant any illness.It should be understood that the control of angiogenesis can be sent out in certain diseases, illness or the patient's condition It is raw to change.Many such diseases are related to pathologic vessels and generate (that is, unsuitable, excessive or undesirable vascularization), It supports morbid state, and in many cases, facilitates cell relevant to such disease and tissue damage.Angiogenesis Related disease, illness or the patient's condition those of (that is, be related to pathologic vessels generate) can be diversified, and may include Such as various types of cancers, chronic inflammatory disease and neovascularization disease.The example of chronic inflammatory disease, illness or the patient's condition Including but not limited to inflammatory bowel disease, such as Crohn disease and ulcerative colitis, lupus, psoriasis, move rheumatoid arthritis Pulse atherosclerosis and diabetes.

As used herein, term " lymph vessels generate related disease, illness or the patient's condition " refers to raw with abnormal lymphatic vessel Long (including excessive lymphatic growth) relevant any illness.Lymphatic vessel generation is finally by growth factor, cell factor and chemotactic The complex network of the factor controls, and can be in many pathology patient's condition (including but not limited to growth of cancers and transfer, inflammation and transplanting Repel) under occur (see, e.g., El-Chemaly, Ann N Y Acad Sci (2008)；Patel,Seminars Ophtalmol(2009)；El-Chemaly,Lymphatic Res Biol(2009)；Pepper,Clin Cancer Res (2001)).About transfer, cancer cell can be transferred to lymph node and remote organ by lymphatic vessel, this typically represents cancer cell expansion The first step being scattered to except primary carcinoma.

In an embodiment of the third and fourth aspect, angiogenesis-associated diseases, illness or the patient's condition and/or lymph It is ophthalmology disease, illness or the patient's condition that pipe, which generates related disease, illness or the patient's condition, more particularly to those of neovascularization, or Person includes the disease, illness or the patient's condition.For this purpose, angiogenesis and/or lymphatic vessel generation can be in ophthalmology disease, illness or the patient's condition Such as age-related macular degeneration, diabetic retinopathy, ischemic retinopathies, retinopathy of prematurity, new green blood Pipe glaucoma, iritis, keratonosus, corneal neovascularization, cyclitis, sickle cell retinopathy become, cornea damages It plays a crucial role in vascular reaction and pteryium development during wound.As these ophthalmology diseases, illness or the patient's condition are in progress, The blood vessel of eyes not only can hyper-proliferative, but also new blood vessel can also be died down, be leaked and be easy bleeding.For this purpose, new abnormal blood Pipe bleeding and may cause subject then to blind.

In an embodiment of the third and fourth aspect, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching It is cancer or including cancer that hand shaft, which generates relevant disease, illness or the patient's condition,.For this purpose, the formation and transfer of cancer are usually directed to disease Pathological vascular generates.Similar with health tissues, it is useless to provide nutrients and oxygen and remove cell that cancer needs neovascularization Object.Therefore, pathologic vessels generation is vital for the growth and amplification of cancer.

As conventionally used herein, term " cancer ", " tumour ", " pernicious " and " pernicious ", which refers to, is characterized in that usual companion With the disease or the patient's condition of the abnormal or abnormal cell Proliferation, differentiation and/or migration that have abnormal or abnormal molecular phenotype, Or referring to cell or tissue relevant to disease or the patient's condition, the molecular phenotype includes one or more genetic mutations or sends out with tumour Other raw relevant heredity variations, tumor suppressor gene expression or active forfeiture and/or abnormal or abnormal cell surface Marker expression.

Cancer may include any invasion or potential invasive cancer, tumour or other malignant tumours, such as exist It is listed in the NCI cancer index of http://www.cancer.gov/cancertopics/alphalist, including all masters Cancer forms, such as sarcoma, cancer, lymthoma, leukaemia and enblastoma are wanted, but not limited to this.These may include breast cancer, packet Include the lung cancer including adenocarcinoma of lung, reproductive system cancers, brain including oophoroma, cervical carcinoma, uterine cancer and prostate cancer and Nervous system cancer, head and neck cancer, the gastrointestinal cancer including colon cancer, colorectal cancer and gastric cancer, liver cancer, kidney, such as Skin cancer, the haemocyte cancer including lymph sample cancer and myelomonocyte cancer, such as pancreas of melanoma and cutaneum carcinoma etc. The endocrine system cancers of gland cancer and hypophysis cancer etc., the muscle skeleton cancer including bone and soft tissue cancer, such as hemangioma, blood The swollen blood vessel cancer or tumour with angiosarcoma etc. of pipe, but not limited to this.

In specific embodiments, cancer is selected from prostate cancer, lung cancer, breast cancer, bladder cancer, kidney, colon cancer, stomach Cancer, cancer of pancreas, oophoroma, melanoma, hepatoma, hepatocellular carcinoma, sarcoma, leukaemia, lymthoma, vascular tumor (such as, blood vessel Tumor, blood vessel be swollen, angiosarcoma) and any combination thereof.

In one particular embodiment, the pharmaceutical composition of the compound of first aspect or second aspect prevention and/or Inhibit the cancer metastasis.

As used herein, " transfer " or " metastatic " refers to malignant tumor cells or tumour via the circulatory system or lymph System passes through natural body cavity, distant sites of the original focus usually formed from tumour, cancer or tumor into body, and then Develop one or more secondary tumors or the migration or transfer of its colony in one or more new positions." transfer stove " refers to As transfer result and the secondary tumors or colony that are formed, and including micrometastasis stove and region and DISTANT METASTASES IN stove.

It should be understood that pathologic vessels generate and lymphatic vessel generation can play an important role in cancer metastasis.For this purpose, primary The formation of cancer medium vessels not only allows for cancer cell to enter blood flow and in entire body body-internal-circulation, but also supports by from primary Property position transfer cancer cell inoculation metastatic cancer formation and growth.

In two foregoing aspects of other embodiments, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching Hand shaft generate relevant disease, illness or the patient's condition suitably kidney trouble, illness or the patient's condition or including kidney trouble, illness or The patient's condition.Preferably, kidney trouble, illness or the patient's condition are selected from chronic renal portability function obstacle, primary kidney fibrosis illness, albumen Urine, nephrosis, ephritis and any combination thereof.

Third and fourth aspect a specific embodiment in, angiogenesis-associated diseases, illness or the patient's condition and/or Lymphatic vessel generation related disease, illness or the patient's condition are atherosclerosis or including atherosclerosis.For this purpose, art technology Personnel should be understood that the pathological change of atherosclerosis can be at least partly due to chronic inflammation and new vessels are formed.In addition, Contacting between NF- κ B dependence atharosclerosis inflammatory reaction and SOX18 regulation has been had proven to, has shown that SOX18 may Work (Garcia-Ramirez etc., 2005) in the development of atherosclerosis.Sox18 is also proved in Atherosclerosis Change and be overexpressed in patch, it is thus possible to be the chief component (Brown etc., 2014) of the disease cause of disease.

In an embodiment of the third and fourth aspect, the relevant disease of angiogenesis, illness or the patient's condition and/or leaching Hand shaft generates relevant disease, illness or the patient's condition for oligotrichosis disease-lymphedema-telangiectasia syndrome or including institute State disease.In this respect, it should be understood that oligotrichosis disease-lymphedema-telangiectasia syndrome and SOX18 gene Mutation is related.

The 5th aspect, the present invention provides prevent or inhibit subject cancer metastasis method, the method includes The compound or its pharmaceutically effective salt, solvate or prodrug or second party of a effective amount of first aspect are applied to subject The step of pharmaceutical composition (thus inhibiting or prevent cancer metastasis) in face.

Suitably, cancer is cancer described above.

The sixth aspect of the present invention is to inhibit, prevent or reduce the active method of SOX18 in subject, including application has The compound of the first aspect of effect amount or its pharmaceutically effective salt, solvate or prodrug or pharmaceutical composition of second aspect Object, to inhibit, prevent or reduce the step of the SOX18 activity in subject.

Suitably, SOX18 activity includes and DNA sequence dna and/or protein contacts and/or combination.Preferably, protein selects From SOX7, RBPJ, XRCC5, SOX18, ILF3, DDX17 and any combination thereof.

The various features and embodiment of the invention referred in above-mentioned various pieces are subject to necessity in the appropriate case Modification is used for other parts.Therefore, in the appropriate case, can by a part specify feature and other parts in specify Feature combination.

The characterization and its effect of certain compounds of the invention is described in further detail in following experimental part.Purpose is explanation The certain specific embodiments and its effect of the compounds of this invention, without limiting the invention in any way.

Embodiment 1

Material and method

Compound preparation

The n-butanol fraction library that marine extracts library will be generated from the ocean library that Australia and the Antarctic Continent collect For screening.Active fraction is separated into pure compound, is redeterminated in a manner of identical with original level point.

The 2688 parts of oceanic invertebrates collected in Australian south and the Antarctic Continent and algae sample library are handled, with Generate the extract library for being suitble to high-throughput bioassay.EtOH extract is decanted, is concentrated, and be assigned to n-BuOH and H₂O Xiang Zhong is then transferred into deep 96 orifice plates, obtains the small molecule of > 10 times of concentration, while removing and desalting.In 10 times and 100 times of dilution After (2.5mg/mL and 0.25mg/mL), using n-BuOH fraction (dried residue of 25mg/mL w/v) for screening.It will live Property fraction hexane, CH₂Cl₂It is ground with MeOH, and pure compound is separated by HPLC.In a manner of identical with fraction Measure all compounds.

From the original aqueous EtOH extract of brown alga sample Caulocystis cephalornithos (CMB-01671) Purify Sm1 (6- undecyl salicylic acid) and Sm2 (6- tridecyl salicylic acid).By brown alga sample Caulocystis The aqueous EtOH extract vacuum (4.8g) of cephalornithos (CMB-01671) is concentrated and distributes to n-BuOH (0.80g) And H₂In O (4.0g) solable matter.The aliquot (40mg) that n-BuOH solubility is distributed carries out HPLC classification separation (5 μm of C18 of Agilent Zorbax SB, 250x 9.4mm column, with 4mL/ minutes through 10 minutes from 60%MeOH/H₂O is extremely 100%MeOH carries out gradient elution, was then eluted through 10 minutes with 100%MeOH, is washed with the 0.01%TFA modifying agent of constant gradient It is de-) to obtain 6- undecyl salicylic acid (Sm1,5.9mg, RT 12.8min) and 6- tridecyl salicylic acid (Sm2,11mg, RT 13.7min)。

The analog of Sm4-Sm14- focused libraries synthesis is purchased from EndoTherm GmbH (Germany) and Princeton BioMolecular Research (USA), and pass through HP-LC/MS purity assay.

The library Sm15-Sm44 SAR.The library SAR is designed to study the possibility substituent group of lipophilic tail and salicylic acid bracket Effect.Six steps being summarized below are synthesized since substituted benzoic acid, introduce lipid tail using Wittig olefination.Portion Shielded intermediate is divided to be also included in the library SAR.All compound (purity > 90%, UV/ELSD/ are purified by HPLC MS)。

i)SOCl₂, reflux, 3h；Et₂NH, CH₂Cl₂, 0 DEG C of-RT, 12h；Ii) s-BuLi/TMEDA, -78 DEG C, DMF；iii) AcOH/HCl；Iv) NaH, THF, 50 DEG C, 2h；v)BBr₃, CH₂Cl₂；Or 48%aq HBr, flow back 5h:vi) Pd/C, H₂Atm., RT, lh

Versatile material and method use reagent and anhydrous solvent (THF, methylene chloride and acetonitrile) as it is.Using passing through The Merck silica gel 60F-254 of UV detection, monitors reaction process by TLC.(Merck40-63 μm) of silica gel 60 is used for column chromatography. Other than the UV light with fluorescence TLC plate, following staining solution: phosphomolybdic acid, anisaldehyde/EtOH is also used.Under a nitrogen into Row needs the reaction of anhydrous condition.Collect NMR data and in Varian Unity 400MHz or Bruker Avance 600MHz In d4-MeOH or CDCl at 298K on spectrometer₃Alignment.HPLC and conventional mass spectrum are in Agilent Technologies It is obtained on 1200 series instruments, which is furnished with G1316A UV-Vis detector, 1200 series ELSD and 6110 quadrupole rod ESI- MS.High resolution mass spec (HRMS) is carried out on Bruker MicroTOF mass spectrograph.

N, the N that the universal method (4a-d) 1. of N- diethyl-benzamide is replaced by each substituted benzoic acid preparation are prepared, N- diethyl-benzamide.By sour (5g, 32.9mmol) and excessive thionyl chloride (50mL) together reflux until stopping discharging Hydrogen chloride out.Be removed under reduced pressure excessive thionyl chloride, and with toluene (3 × 15mL) condistillation.Acyl chlorides is dissolved in anhydrous CH₂Cl₂ Diethylamine (13.6mL, 131.5mmol) is added dropwise in (100mL) and at 0 DEG C, and is stirred at room temperature overnight.Reaction is mixed Object CH₂Cl₂(100mL) dilution, with water (50mL), aqueous salt solu-tion, and through anhydrous MgSO₄It is dry.On a rotary evaporator Organic layer is removed, crude compound is obtained.By flash column chromatography crude compound, pure diethyl-benzamide is obtained.

The universal method (5a-d) 2. of ortho-metalated reaction is oriented at -78 DEG C to TMEDA (0.55mL, 3.7mmol) Anhydrous THF (10mL) solution in be added s-BuLi (cyclohexane solution of 2.6mL, 3.7mmol, 1.5M) and stir 15 minutes, Then THF (5mL) solution of 1- methoxyl group-N, N- diethyl-benzamide (0.35g, 1.7mmol) is added.At the same temperature After stirring 2 hours, it is slowly added to anhydrous DMF (0.52mL, 6.8mmol).Reaction mixture is gradually heated to room temperature and is stirred 30 minutes.By the way that HCL aqueous solution (10mL) quenching reaction of 6N is added, then extracted with ethyl acetate (3x 15mL).It will merge Organic layer washed with salt water (10mL), and it is dry through MgSO4.After solvent is removed in vacuum, by flash column chromatography (it is n- oneself Alkane/ethyl acetate, 1/2) purifying residue, obtain product.

Crack N, the universal method (10a-d) 3. of N- diethyl-benzamide is by N, N- diethyl-benzamide (1.3mmol) is dissolved in ice AcOH (3mL), is added 10%HCl aqueous solution (3mL), and mixture is flowed back 12 hours.It is cooled to room Acetic acid is removed under reduced pressure in Wen Hou, uses H₂O dilution, is extracted with EtOAc (30mL).Organic layer is washed with brine, is separated and through anhydrous MgSO4 is dry.Solvent is removed under reduced pressure, obtains product.

The universal method (12a-d) 4. of Wittig olefination is at 0 DEG C to the THF of Wittig salt (1.0mmol) T-BuOK (2.0mmol) or NaH (2.0mmol) are added in (10mL) suspension and stirs 30 minutes.It is slowly added to be dissolved in THF In aldehyde (0.8mmol) and be stirred at room temperature overnight (reacted at 50 DEG C using NaH, entry 2,4,5,6).It will be anti- Using H₂O is quenched and is extracted into EtOAc (30mL).Organic layer is washed with salt water (20mL), through MgSO₄It dries, filters, and Vacuum concentration, obtains crude compound.By flash column chromatography crude compound, pure product is obtained.

The universal method of demethylation: method A (uses BBr₃) 5. BBr is used at -78 DEG C₃(the CH of 1.0M₂Cl₂Solution, 0.576mL, 0.576mmol) processing compound (74mg, 0.192mmol) CH₂Cl₂(70mL) solution.By mixture at -78 DEG C Lower stirring 2 hours, then with saturation NH₄Cl aqueous solution (10mL) is quenched.Mixture is warmed to room temperature and uses CH₂Cl₂(30mL) Dilution.Organic layer is washed with salt water (10mL), through MgSO₄It dries, filters, and is concentrated in vacuo.

The universal method of demethylation: method B (using HBr) 6. is by compound (100mg, 0.192mmol) 48% Solution in HBr aqueous solution (3.0mL) is heated to reflux 3 hours.After the reaction was completed, it warms to room temperature and is evaporated under reduced pressure, obtain Thick residue.Crude compound is extracted with EtOAc (30mL) and uses H₂O (20mL) washing.Separate organic layer, dry (MgSO₄) simultaneously Concentration, obtains product.

10%Pd/C is added into the olefin solution (EtOAc:MeOH) of 1:1 by universal method (14a-d) of olefin reduction (10mol%) and in H₂It is stirred at room temperature under atmosphere 2 hours.After the reaction was completed, it is filtered by bed of diatomaceous earth.By filtrate decompression Solvent is removed, product is obtained.

Protein formulation

Mouse SOX HMG segment is by mouse SOX2 (group B), SOX11 (group C), SOX6 (group D), SOX9 (group E), SOX18 The HMG structural domain of (group F) and SOX15 (group G) clone (IMAGE cDNA clone ID:Sox18:3967084 from cDNA template； Sox9:5354229；Sox4:6822618) BP is cloned into pDONRTM221pENTRY carrier, is used LR Technology (Ng etc., 2012) is sequenced and recombinates into pETG20A or pHisMBP expression plasmid.Construct is transformed into Escherichia coli In (Escherichia coli) BL21 (DE3) cell (Luria-Bertani, 100 μ g/ml ampicillins).

Overall length mouse SOX18. recombinantly expresses the tagged mouse HIS-GST-SOX18 of N-terminal in Sf9 cell, in GST It is purified on resin (GE Healthcare Life Sciences, Sweden), and in Tris buffer (50mM Tris, 500mM NaCl, 10mM reduced glutathione, pH 8.0) in elution.The cDNA clone of PCR amplification mouse Sox18 is simultaneously cloned into In pOPIN-GST carrier, to generate the tagged HIS-GST-SOX18 of N-terminal.By through sequence verification construct with FlashBACULTRA (Oxford Expression Technologies, Oxford, United Kingdom) baculoviral The HIS-GST-SOX18 of recombinant expression is obtained on DNA cotransfection to Spodopterafrugiperda Sf9 cell.By Sf-900^TMII is without blood First five high cell (BTI-TN-5B1-4) in clear culture medium is with 1.5 × 10⁶The cell density of a cell/mL infects, and infection is multiple Number (MOI) is 5PFU/ cell, and is incubated for 144 hours at 21 DEG C, is then harvested.Cell from 100mL expression culture is sunk Shallow lake is resuspended in 30mL phosphate lysis buffer (50mM sodium phosphate, 500mM sodium chloride, 1%Triton X-100,2mM chlorination Magnesium, a piece of cOmplete Protease Inhibitor Cocktail, pH 7.5) in and on ice be ultrasonically treated 20 seconds.It will Cell lysate is at 4 DEG C with 17,000 × g centrifugation 40 minutes.By supernatant and all-round nuclease (Benzonase Nuclease) (Merck Millipore) is incubated at room temperature 1 hour to carry out DNA degradation, then with 500 μ L GST resin (GE Healthcare Life Sciences, Sweden) mixing, and be incubated for 1 hour in rotating wheel at room temperature.By sample with 500 × g is centrifuged 1 minute to remove the protein being not associated in supernatant.With 50 times of resin volume (RV) washing buffer (50mM Sodium phosphate, 500mM NaCl, pH7.5) resin is further washed, unbonded protein is removed by centrifugation as described above.With 3 × 1Rv elution buffer (50mM Tris, 500mM NaCl, 10mM reduced glutathione, pH 8.0) elutes knot from resin The protein of conjunction, as described above by the way that supernatant is collected by centrifugation.

Use the DNA combination competition assay of fluorescence polarization (FP)

DNA combination competition assay is carried out in 384 orifice plate of black using mouse overall length SOX18 or SOX-HMG segment.It uses The Prox1-DNA element of fluorescent marker carries out all experiments.Compare DNA (low FP milli polarization index, mP), the In by free label The DNA (negative control, high mP) marked in the presence of protein and the feelings existing for the excessive unlabelled DNA of competition The DNA and protein (positive control, low mP) of label under condition are formed.

Use 30mM HEPES (N-2- hydroxyethyl piperazine-N'-2- ethanesulfonic acid) (pH 7.5, KCl containing 100mM, 40mM NaCl, 10mM NH4OAc, 10mM guanidine, 2mM MgCl2,0.5mM EDTA, 0.01%Np-40) (being used for mouse overall length SOX18), Tris-NaCl (10mM Tris pH 8.0 and 100mM NaCl) (being used for SOX-HMG segment) is in 384 orifice plate of black with 25 μ L Carry out DNA combination competition assay.Using with 5' fluorescein amidite (FAM) label (Sigma Proligo or InVitrogen 40bp double-strand Prox1-DNA element) carries out all experiments.In the presence of the DNA of 5nM label, In It is horizontal that best combination is obtained under the SOX-HMG segment of mouse the overall length SOX18 and 60nM of 200nM.Compare the DNA by free label (low FP milli polarization index, mP), the DNA marked in the presence of protein (negative control, high mP) and competing at 400 times Strive the DNA and protein (positive control, low mP) composition of the label in the presence of excessive unlabelled DNA.It depends on Compound, final DMSO concentration range are 2%v/v to 3.33%v/v.It, will after mixed protein, DNA probe and compound Plate sealing is simultaneously quickly shaken 5 minutes in the dark at room temperature, is quickly shaken at 37 DEG C 10 minutes, is quickly shaken at room temperature 30 minutes, fluorescence polarization then was read on Tecan M1000Pro (λ exc=485nm, λ em=525nm).It is all experiment with It is triplicate to carry out.

Functional examination based on cell.

Monkey kidney fibroblast-like cell (COS-7) is being contained into FBS, Sodium Pyruvate, L-Glutamine, penicillin, chain In 37 DEG C, 5%CO in the DMEM (Life technologies, 11995) of mycin, nonessential amino acid and HEPES₂Lower culture. Cell is grown to 80% to converge in 96 orifice plates, and use X-tremeGENE 9DNA transfection reagent (Roche, 06365787001), the cell is transfected with 1 promoter construct (VC1889) of mouse plasmid pGL2Vcam and pSG5Sox18 (Hosking etc., 2004, Duong etc., 2014).After transfection 4-6 hours, by cell and compound in 0.5%FBS culture medium It is incubated for again 24 hours, then carries out cracking and luciferase assay (Perkin Elmer, 6016711).

Cell-free expression and α screening.

GFP (GFP), the mCherry and cMyc (myc) label of plasmid preparation and cell-free expression enhancing are to all eggs White matter carries out genetic coding, and is cloned into cell-free expression vector (Gagoski etc., 2015, Sierecki etc., 2013).Such as It is preceding it is described preparation have translation ability lizard Leishmania extract (LTE) with co-express protein to (Kovtun etc., 2011, Mureev etc., 2009).

Genetic coding is carried out to protein with GFP (GFP), the mCherry of enhancing and cMyc (myc) label, and is cloned into In cell-free expression Gateway destination carrier: N-terminal adds (pCellFree_G03), the N-terminal of GFP label to add (pCellFree_G07) and C-terminal of Cherry-cMyc label add (pCellFree_G08) of Cherry-cMyc label The carrier of (Gagoski etc., 2015).(Sierecki etc., 2013) as previously described, people RBPJ (BC020780) and MEF2C (BC026341) open reading frame (ORF) derive from people ORFeome collection, 1.1 editions and 5.1 editions and Human Orfeome collaboration OCAAcollection (Open Biosystems), and in ARVEC facility, UQ Diamantina Institute clones it.It will by LR recombination (Life Technologies, Australia) Entry clones the ccdB gene swapping in pDONOR223 or pENTR201 carrier and expression plasmid.Overall length people SOX18 is synthesized Gene, and realized using Gateway PCR clone to the transfer of carrier.Preparation has the lizard benefit of translation ability assorted as previously described Graceful protozoon extract (LTE) (Kovtun etc., 2011, Mureev etc., 2009).By the GFP template plasmid for adding 30nM to LTE 3 hours are incubated for the Cherry template plasmid of 60nM and at 27 DEG C to co-express protein pair.

(Sierecki etc., 2014, Sierecki etc., 2013) carries out α screening as previously described.By expressing egg in LTE White matter is to and with test compound (0.3 to 300 μM) or DMSO (0.7%DMSO final concentration) in the buffer solution B of dilution range 1 hour is incubated for carry out destruction protein-protein interaction (IC₅₀) measurement.The following percentage for calculating interaction: (I_cpd/I_DMSO) x 100 comes from 3 independent experiments.

Using cMyc detection kit and Proxiplate-384Plus plate (PerkinElmer), as previously described (Sierecki etc., 2014, Sierecki etc., 2013) carries out α screening.The LTE lysate of target protein will be co-expressed slow Dilution in fliud flushing A (25mM HEPES, 50mM NaCl).For measurement, by the buffer solution B (25mM of 12.5 μ L (0.4 μ g) HEPES, 50mM NaCl, 0.001%NP40,0.001% casein) in the coated acceptor bead of anti-cMyc be distributed to each hole In.Then the GFP-Nanotrap of the dilute sample of 2 μ L various concentrations and the biotin labeling in the buffer solution A of 2 μ L is added.It will Plate is incubated at room temperature 45 minutes, and the diluted streptavidin packet in buffer solution A of 2 μ L (0.4 μ g) is then added The donor bead of quilt dilutes, and is incubated for 45 minutes in the dark at room temperature.Setting (the λ exc=680/ recommended using manufacturer 30nm, continues 0.18s, the λ em=570/100nm after 37ms) α is measured on Envision plate reader (PerkinElmer) Screen signal.Gained bell curve indicates positive interaction, and flat wire indicates to lack interaction between protein.With one Formula repeats the measurement of each protein pair in part.

According to the formula calculations incorporated index:

I is that highest signal is horizontal (top of hook effect curve), and Ineg is minimum (background) signal level.By signal It is standardized for the Iref signal obtained for most strong interaction.By in LTE express protein to and with dilute Test compound (0.3 to 300 μM or 50 μM) or DMSO (0.7%DMSO in the buffer solution B of the concentration of range or single concentration Final concentration) 1 hour is incubated for carry out destruction protein-protein interaction (IC₅₀) measurement.Following calculating interaction Percentage:It is fitted in GraphPad Prism 6.0 editions using 3- parameter nonlinear regression from 3 The data of a independent experiment.

Co-immunoprecipitation (Co-IP).

(Sierecki etc., 2014) carries out co-immunoprecipitation as previously described.In short, by SOX18-Cherry-cMyc with GFP-RBPJ, GFP-MEF2C or GFP construct (as negative control bait) are expressed in lizard Leishmania acellular albumen It is co-expressed in system.NaCl is added in the protein (200mM) of expression, and by sample and 10 μ L GFP-nanotrap packets The bead (agarose with the NHS- activation of MBP-GFP-Nanotrap coupling) of quilt is incubated for 30 minutes at 4 DEG C, is passed through simultaneously Rotation is gently mixed.Then, it is washed with 200 μ L washing buffers (PBS containing 0.1%Triton X-100 and 200mM NaCl) It washs bead 6 times.Albumen is discharged from bead by heating 3 minutes in 15 μ L 2x NuPAGE LDS sample-loading buffers in 72 DEG C Matter, and separated on NuPage Novex 4-12% gel (Life Technologies, Australia).It uses GFP the and Cherry fluorescence of ChemiDoc MP system (Bio-Rad, Australia) scanning gel.

Critical micelle concentration (CMC).

Based on incorporation of the fluorescence 1,6- diphenyl -1,3,5- hexatriene (DPH) into micella is dense to measure critical micell It spends (Chattopadhyay and London, 1984).By small molecule and positive control (neutral detergent Triton X100 and yin from Sub- detergent SDS) in 96 orifice plate of low combination from 1000 μM to 0.1 μM cascade dilution.In 200mM NaCl or FP buffer It is diluted.DPH is added in 384 orifice plate of black with 5 μM.Fluorescence intensity (λ exc is measured after being incubated at room temperature 30 minutes =360nm, λ em=430nm), CMC transformation is estimated to graphically.

Cytotoxicity assay.

(McMillian etc., 2002) as previously described measures cytotoxicity using Alamar indigo plant.By COS-7 cell with every hole 7000 cells are inoculated into black wall in the DMEM culture medium (Life Technologies Australia) with 10%FBS In 96 orifice plate of clear bottom.By HepG2 the and HEK293 cell line from ATCC with 5000, every hole cell in black wall clear bottom 384 It is seeded in porocyte culture plates in the DMEM containing 1%FBS.By cell in 37 DEG C, 5%CO₂Lower culture 24 hours.Additionization The serial dilution thing for closing object, is finally adjusted to 0.5%v/v for DMSO concentration.Cell is incubated for again 24 hours.By 1%Triton X-100 is used as negative control, and 0.5%DMSO is used as positive control.5 μM of Alamar indigo plants are added into each hole, and at 37 DEG C Fluorescence (λ exc=560nm, λ em=590nm) is measured after being incubated for 2 hours.Use Prism software analysis data.

Direct DNA binding assay is carried out using surface plasma resonance (SPR).

HBS-EP buffer (10mM HEPES, 150mM NaCl, 3mM EDTA, 0.005%v/v polysorbate20, PH 7.4) in 1%v/v DMSO under test compound.The same buffer that the DMSO of 1%v/v will be supplemented with is used as flowing Phase.DNA minor groove binding DAPI (4', 6- diamidino -2-phenylindone), DNA intercalator and minor groove binding actinomycin D and DNA intercalator ethidium bromide is used as positive control.It is moved back using the standard of the single-stranded antiparallel DNA probe purchased from Geneworks Fiery program (5 minutes at 100 DEG C, ambient temperature overnight is simultaneously stored at -20 DEG C) prepares biotinylated (one label of each probe) Double chain DNA probe, and be fixed on CM5-SA Streptavidine chip according to the recommendation of manufacturer.For chemical combination Running buffer flowing is set as L/ minutes 25 μ by object and actinomycin D, and circulation was associated by 4 minutes, was dissociated within 4 minutes, then It is regenerated with the pulse of 10 seconds 10mM Gly-HCl pH 2.5 and 1 minute stabilisation forms.DAPI does not need any Regeneration, and for ethidium bromide, need the pulse of the SDS of 40 seconds 25 L/ minutes 0.5%v/v of μ to carry out before stabilisation Regeneration.All samples and control circulation to carry out in triplicate.DMSO calibration is carried out according to the recommendation of manufacturer.Per injection Afterwards, additional running system is carried out with the DMSO of 50%v/v to wash to avoid residual.Experiment is in Biacore T200 (GE Healthcare, USA) on carry out, a flow cell is retained as referring to.

Direct protein binding assay (heat accumulation).

In presence and there is no in the case where Prox1-DNA or the smaller ligand of presumption, mouse SOX18-HMG is used (109aa) carries out the research of difference static light scattering on Harbinger Biotech StarGazer.Initial experiment is carried out to comment The variation for estimating aggregation temperature is no longer dependent on the compound concentration of concentration.The final concentration range used is 500 μM (Sm4 and Sm14) To 1.5mM (Sm5), this depends on the necessity that DMSO concentration is limited to 3%.17bp is used with 10 μM of final concentration Prox1-DNA oligonucleotides.To be combined in triplicate there are ligand, and the Tagg for passing through > 2 DEG C (assembles Temperature) increase detect combine.Tagg is measured with identical protein batch in a single operation.Containing 3%v/v's It is carried out in the Tris-NaCl buffer (10mM Tris-HCL, 150mM NaCl, pH 8) of DMSO, end reaction volume is 45 μ L, mouse SOX18-HMG concentration are 154 μM.These reagents are incubated at room temperature 1 hour before measuring.By protein with every The rate of 1 DEG C of minute is heated to 80 DEG C from 25 DEG C.The overall strength of each sample is mapped relative to temperature, and is passed through non-linear time Return and Boltzmann equation model.

Computer simulation docking and molecular dynamics

Ligand-protein docks and carries out Sm4 to SOX18/ using LeadIT/FlexX (BioSolveIT, Germany) The computer simulation of DNA compound is docked.It is all by being removed from the structure of SOX18-HMG/Prox1-DNA (pdb:4Y60) Entire protein/compound is simultaneously defined as possible binding site to dock by hydrone.Analyze best 20 of Sm4 Docking posture is simultaneously grouped as 4 clusters.Since SOX18/DNA does not include classical binding pocket, so passing through 200ns's long Each posture cluster is further verified in MD simulation.MD simulation is carried out using complete SOX18/DNA structure, and wherein Sm4 has not Same combination posture.To MD simulation analysis shows that three kinds in the posture be it is unstable, wherein Sm4 is 3 before simulation Its interaction with protein is destroyed in 14ns, is retained in aqueous solvent for remaining simulation.Only for one of them In posture, MD simulation generates stable combination posture during entire 200ns simulation for Sm4.Similarly, with the Sm4 for being free of DNA Docking and MD simulation are carried out with SOX18.However, not generating stable combination orientation in 4- posture cluster during MD simulation.For Compare, in the case where Sm4 is not present, to SOX18/DNA and the SOX18 without DNA carries out MD simulation, generates and has The similar conformation of the structure of Sm4 ligand and dynamic behaviour.

Protein-protein docks and uses ClusPro line server version (cluspro.bu.edu), for It is compound that the structure of Notch1 transcription complex and SOX18-HMG use pdb:3V79 and pdb:4Y60 to carry out Notch1 transcription respectively Protein-protein between object and SOX18 docks.DNA molecular is removed before docking, because ClusPro can not handle it , and restore after docking.Reject has the docking solution of conflict DNA molecular.Docking posture is 50ns's long at the top of gained It is used as starting conformation in MD simulation to optimize docking posture, and verifies the stability of multiprotein complex.

MD simulates and uses AMBER MD software " pmemd ", (is used for using the field of force ff99SB (protein and DNA), TIP3 Implicit water) and application periodic boundary condition (NTP), particle mesh screen Ewald (particle-mesh Ewald) (PME) side Method (being used for remote electrostatic interaction), isotropism coupling pressure and Langevin thermostat (gamma_ln=1/ps) (coupling for temperature) carries out any MD simulation.Simulation is related to the key of hydrogen with the operation of 2fs step-length, using the constraint of SHAKE algorithm.It is right It is simulated in using the MD of Sm4, the cup from AMBER packet is used to calculate the force field parameter of ligand.By minimizing structure simultaneously Them are balanced by slowly reducing position constraint in 5ns and carries out all MD simulations.It is right to carry out each simulation in triplicate Different random numbers is used in the distribution of initial velocity.

The measurement of COX1/COX2 enzymatic

Use COX1 the and COX2 inhibitor screening assay kit (Ref# from Cayman Chemical Company 701090 and 701080) measure COX inhibitory activity.It is tested in quadruplicate in 2%v/v end DMSO with single 200 μM of concentration All compounds.By compound and sheep COX1 or people COX2 37 DEG C preincubate 10 minutes, then with COX substrate arachidonic Acid is incubated for 3 minutes again.Concentrated hydrochloric acid is added and terminates reaction.Prostaglandin standard curve is prepared, and is carried out according to the description of manufacturer Enzyme immunoassay (EIA).Including DMSO control, the Meclofenamic Acid for inhibiting control and 200 μM using the 100% of heat-inactivated COX enzyme Positive control (potent non-selective COX1/2 inhibitor) is for reference.Prostanoid standard value is drawn relative to log concentration It is made as logit (B/B0), and uses 6.0 editions benefits of GraphPad Prism and linear regression fit.Use standard curve Linear Quasi It closes to calculate each sample concentration.

The tracking of SOX18 unimolecule

Figure 24 B depicts experimental work process, is related to the two-dimensional tracking of molecule track and the analysis using MATLAB, such as (the Cell 156,1274-1285 such as Chen；2014) it is summarized in.It was noticed that fixed DNA combination fraction (%, with Pie chart is shown) there are two types of type (specific and nonspecific), and this depends on the residence time in the same position DNA； If the time is short (averaged less than 1 second), this is considered as non-specific binding (that is, instantaneous combination with random dna site), If for a long time (averagely be more than 5-6 second), this be considered as specific binding (that is, have transcribe effect with target DNA site Longer combination).The residence time (being shown with bar chart) of fixed dna combination fraction, also there are two types of types, specific and non- Specificity: SOX18 molecule and DNA (are averagely more than 5-6 seconds) combination non-specifically (averaged less than 1 second) or specifically Time span (in seconds).Unimolecule is carried out after with SOX18-Halotag reporter gene construct transfection HeLa cell Tracking.The expression vector enables us to detect monomolecular SOX18 albumen after adding ligand, and the ligand is passing through Become to fluoresce after the processing of Halotag system enzyme.Using ZEISS ELYRA super-resolution microscope, the TIRF of modified version is utilized Microscopy (HiLo) carries out real time imagery.

As a result

The screening for the natural products for inhibiting SOX18-DNA to combine.

Using based on fluorescence polarization measuring method (FP) screening marine extracts library SOX18 albumen (overall length mouse) with The inhibitor that DNA is combined.We have selected the oligonucleotides of the fluorescent marker with shared SOX motif, and the motif is present in It is the direct target (Francois etc., 2008) (Figure 1A) of SOX18 in the First Intron of Prox1 gene.Library packet Include 2,000 kind of purifying metabolin and 2,688 parts of marine extracts, containing having more than 50,000 kind of structure.Preliminary screening identification Never 16 kinds of activity extracts that fellow disciple collects, i.e. sponge (10 kinds), algae (5 kinds) and tunicate (a kind) (hit rate The factor=0.62 0.6%, preliminary screening Z'-) (Zhang etc., 1999).Effect then based on one or more bioactive molecules Power and abundance, which deconvolute to extract, carries out order of priority differentiation, wherein most active extract comes from brown alga Caulocystis cephalornithos generates two kinds of reactive compounds: 6- undecyl salicylic acid (Sm1) and 6- tridecane Base salicylic acid (Sm2) (Figure 1B).The dose response screening of Sm1 and Sm2 leads to the inhibiting effect (IC in high micro-molar range₅₀) (Fig. 1 C).Two kinds of reactive compounds contain the salicylic acid bracket with big aliphatic group.

The design and preliminary screening of focused libraries.

In next step, we devise a small-sized analog library to verify salicylic acid (hydroxybenzoic acid) activity branch Frame, and study its structure-activity relation spectrum.Selection (as shown in Figure 2 A) further includes having similar resorcinol bracket (in addition Hydroxyl substituted carboxylic acid) compound and many approvals (taken with amine containing similar salicylic acid or ortho-aminobenzoic acid bracket For hydroxyl) NSAID.Buy library, and the inhibition combined using FP measurement screening SOX18-DNA.In the measurement, Gao Qinhe The destruction of power protein-DNA interaction needs the inhibitor concentration in high micro-molar range, can gather in the range Collection, especially in the case where amphipathic and high logP molecule (Irwin etc., 2015).Therefore, the critical micell of secondary screening compound Concentration (CMC), using eliminate as false positive formation aggregation compound or formed micella compound (table the 1, the 2nd column with 5th column and Fig. 2 B).

CMC measuring method eliminates five kinds of compounds: Sm3, Sm6, Sm7, Sm9 and Sm10, it is shown that under 20 μM and 30 μM CMC (column of table the 1, the 2nd).Rest part does not show up to 1000 μM of micelle forma-tion, and is included in SOX18-DNA binding assay In.Due to compound insufficient supply, the CMC of Sm1 and Sm2 can not be measured.SOX18-DNA binding assay identifies 7 kinds of IC₅₀It is worth low In 1000 μM of compound, wherein 2 kinds of Sm4 and Sm14 and original hit Sm1 and Sm2 (IC₅₀About 350 μM) it compares, display is about 100 μM of improved IC₅₀(table the 1, the 1st arranges value；Fig. 2 C).It is interesting that all three anthranilic acid analogs, first chlorine are fragrant That acid, Niflumic Acid and Flufenamic acid also show IC₅₀It is worth the activity (column of table the 1, the 1st within the scope of 100-400 μM；Fig. 2 C).

In order to accurately find the possibility binding site of small molecule, with it is shorter by DNA combine centered on protein piece Duan Chongfu FP measurement.The DNA binding structural domain of SOX TF is made of high speed swimming race (HMG) box of 79 amino acid longs.109 The SOX18 segment (SOX18 [109]) of amino acid long corresponds to residue 69-177 (numbering by mouse SOX18) comprising HMG The N-terminal flanking sequence and C-terminal flanking sequence of box (residue 78-149) and respectively 9 and 28 amino acid.Use SOX18 [109] the FP measurement of segment shows the IC almost the same with overall length SOX18₅₀Value (data are not shown) shows that small molecule interference should The part of the 109aa of protein.

In conjunction with selectivity.

From the perspective of molecule, SOX18-DNA combine inhibitor can by directly with DNA or protein phase interaction With or between protein and DNA interface interaction work.It is reported even for other TF, small molecule (Leung etc., 2013) also there is non-specificity DNA to combine directly in conjunction with DNA, lead to possible genetoxic, mutagenicity Or the potential of carcinogenicity.For this reason, we have developed determine reactive compound whether with DNA or the direct phase interaction of protein Bind directly measurement.Method based on surface plasma body resonant vibration has been developed to analysis small molecule and is fixed on strepto- antibiosis The combination of biotinylated double-stranded DNA on object fibroin chip surface.This method measures association rate (kon) constant, dissociation (koff) constant and binding constant (KD), and for measuring and the combination of two kinds of different DNA sequence dnas: SOX binding site shares DNA With random DNA.Intercalator ethidium bromide and actinomycin D and minor groove binding 4', 6- diamino -2-phenylindone (DAPI) The positive control combined as non-selective DNA.All positive controls show that KD is unrelated with DNA sequence dna, consistent with document.It should Analysis shows that SOX18 inhibitor does not show any combination to consensus sequence or random DNA (Fig. 3 A and 3B).

In order to study inhibitor whether with protein direct interaction, we by assessment to protein unfolding balance Higher temperature offset come measure realized SOX18 thermal stability increase (Shrake and Ross, 1992, Shrake and Ross, 1990, Brandts and Lin, 1990, Fukada etc., 1983).For this purpose, we use static light scattering as protein- Inhibitor complexes aggregation reading (Mittal etc., 2014, Senisterra etc., 2006, Senisterra etc., 2008, Senisterra and Finerty, 2009, Senisterra etc., 2012).Use the only SOX18 of HMG [109] segment and use The DNA probe of SOX motif modification measures thermal stability as positive control.It is being saturated the dense of all potential binding sites of SOX18 Sm4, Sm5 and Sm14 are tested under degree (being respectively 500 μM for Sm4 and Sm14, be 1.5mM for Sm5).In these maximum ligands Under conjugation condition, temperature dependency protein aggregation, which is no longer occupied by binding site, to be limited, and it is measured until Reach platform.Every kind of inhibitor shows the increase of the Tagg more than 3 DEG C, consistent with the direct interaction of protein with inhibitor (Fig. 3 C).Two kinds of bindings are combined together, the results showed that micromolecular inhibitor and SOX18 protein-interacting, without With DNA direct interaction.In addition, the data splitting from FP measurement and thermal stability determination shows inhibitor-protein phase Interaction site is located in SOX18-HMG box or closest to SOX18-HMG box.

SOX DNA, which is combined, inhibits selectivity.

The DNA binding structural domain of all SOX albumen is highly conserved, with mammal testicular determinant SRY HMG The sequence identity (Bowles etc., 2000, Gubbay etc., 1990) of structural domain shared 46%, and in HMG structural domain two sides Remaining structural domain of SOX TF shows low-level similitude.In order to study the selectivity of SOX18 inhibitor, using containing The oligonucleotides of the fluorescent marker of the SOX motif 5'AACAAT3' known, using from difference in the measurement based on DNA combination FP The only protein fragments of HMG of SOX albumen.Different SOX TF include: SOX2 (group B), SOX11 (group C), SOX6 (group D), SOX9 (group E), SOX18 (group F) and SOX15 (group G).Most active inhibitor Sm4 is assessed for all different SOX TF, It shows that DNA is combined in all cases and inhibits (IC₅₀About 200-300 μM), slightly prefer to SOX18 and SOX15 (about 200 to 220 μM of IC₅₀Compare 270 to 310 μM for other inhibitor), show that, for DNA decohesion, inhibitor exists It is non-selective (Fig. 3 D) in SOX TF.

It misses the target analysis.

It is a kind of important NSAID that COX, which inhibits salicylate, by directly or indirectly inhibiting proinflammatory prostaglandins Cyclo-oxygenase (COX) dependence generation work (Pillinger etc., 1998).In our current research, we identify and COX Inhibitor has the SOX18 inhibitor of structural similarity, and in order to study appointing between NSAID and novel SOX18 inhibitor What function overlapping, we include the similar NSAID of structure in SOX18 research.Similarly, we have studied novel SOX18 suppressions Whether preparation can inhibit COX1/2 enzymatic activity.Use business COX-1/2ELISA measuring method and uses Meclofenamic Acid as sun Property control assess COX-1 the and COX-2 inhibiting effect of Sm4, Sm5, Sm8, Sm11, Sm12, Sm13 and Sm14.Until 200 μM Concentration, SOX18 inhibitor do not show any COX-1 or COX-2 inhibitory activity (figure S1A, S1B).

Missing the target, we are extensive potential for participating in using Eurofins-CEREP/Panlabs group by characteristic spectrum analysis The various receptors of undershooting-effect, enzyme and transport protein (including g protein coupled receptor (GPCR), ion channel, membrane receptor, kinases With nonkinase and nuclear receptor) radioligand binding assay further probed into our the potential of guide Sm4 and do not depended on In the effect (table 4) of SOX18.Also many epigenetic modification objects are tested using a series of recombinase fluorimetries.In 10 μ It is not observed when M and significantly inhibits (> 50%), show Sm4 selectivity and the potential for further drug development.

SOX18 protein-protein interaction

It is reported that the highly conserved HMG structural domain of SOX transcription factor participates in protein-DNA and protein-protein It interacts (Agresti and Bianchi, 2003, Prokop etc., 2012, Huang etc., 2015).Therefore, directly with HMG structure Domain combines or adjusts closest to the compound of HMG structural domain with the allosteric change directly or by SOX18 protein conformation The potentiality of SOX18-DNA and SOX18- protein-interacting.In order to which the small molecule adjusting SOX18- protein for studying us is mutual The potentiality of effect, we selected known to two participate in endothelial cell transcriptional control SOX18 PPI:MEF2C (it is reported that GST- drop-down test in conjunction with SOX18 (Hosking etc., 2001)) and RBPJ (it is reported that genetically mutual with SOX18 Effect, and not yet identify and bind directly (Sacilotto etc., 2013)).In addition, being selected as the XRCC6 of non-binding gametophyte (ATP dependent DNA helicase II) is used as negative control.

We are inhibited using the protein of Cell free expression system expression label to analyze PPI, and by itself and α screening technique (amplification shine close to homogeneous assay method) combination, enable us to the protein of measurement markers the degree of approach (Sierecki etc., 2013).This method confirms direct at Thermodynamic parameters between SOX18 and its known gametophyte MEF2C, and discloses Direct PPI (Fig. 4 A, left figure) between SOX18 and RBPJ.Direct physics is further demonstrated using standard immunoassay co-precipitation It interacts (Fig. 4 A, right figure).Next, we have studied our most efficient small compound Sm4 and Meclofenamic Acids, Buddhist nun Fluoric acid and Flufenamic acid destroy the ability of SOX18-MEF2C or SOX18-RBPJ interaction.Sm4 is with 42.3 μM of IC₅₀It destroys SOX18-RBPJ interaction, but interact SOX18-MEF2C without influencing (Fig. 4 B, the picture left above and top right plot).On the contrary, Flufenamic acid destroys SOX18-MEF2C interaction (IC₅₀It is 29.1 μM), and only weakly destroy SOX18-RBPJ interaction (IC₅₀It is about 444 μM) (Fig. 4 B, the picture left above and bottom-right graph).Another NSAID has little effect any PPI or without shadow It rings.

The library SAR

With individual analog library have studied in more detail SOX18-DNA combination salicylic acid type inhibitor with The structure-activity relation (SAR) of SOX18/RBPJ PPI.The library is designed to probe into some Sm4 and other salicylates are only The importance of feature, the specific characteristic include: the electron density of salicylic acid aromatic ring, two acidity of β hydroxycarboxylic acid motif The importance of hydrogen is saturated that bonded or ethylene is bonded and the structure and lipophilicity of lipophilic tail with lipophilic tail.We 30 kinds of analog Sm15-Sm44 (Fig. 5) have been synthesized, the suppression of its combination to SOX18 [109] segment of DNA and only HMG is screened System and the destruction (upper figure and the following figure) to interact to SOX18-RBPJ.In addition, also being tested perhaps using above-mentioned α the screening test method (i.e. Sm4, Sm17 to Sm24, Sm26, Sm31, Sm34, Sm37 and Sm40 are same to SOX18-SOX18 to Sm42) for these more analogs The destruction of dimerization interaction.As shown in figure 5, Sm4, Sm17 are to Sm23, Sm26, Sm31, Sm34, Sm37, Sm40 and Sm41 Certain inhibitory activity is shown to SOX18-SOX18 homodimerization when concentration is 5 μM.That also screens the library is directed to two kinds The general cytotoxicity of cell line HEK293 and HepG2, to assess their potentiality to be exploited (table 3).

Use computer simulation fallout predictor " FAF-Drugs3 " (http://fafdrugs3.mti.univ-paris- Diderot.fr/) (Baell and Holloway, 2010, Lagorce etc., 2008) analyzes potential PAINS (general measurement interference Compound) chemical part, the chemical part is common in the frequent hit person mixed, in many biochemical high flux screenings As false positive.Only have 3 kinds of compounds by PAINS part label (Sm14, Sm40 and Sm44) in total.Sm14 contains methylene- Thiazolone motif or reactivity α, beta-unsaturated carbonyl, and Sm40 and Sm44 contain the unstable catechol group (table of oxidation 2).Although Sm40 and Sm44, without activity, is not measured Sm14 other than binding assay in FP measurement.Separately Outside, use the new library of " Aggregator Advisor " database analysis potential aggregation, however none display with Know the similitude of aggregation, and all show medium lipophilicity, logP value is lower than 5.8 (tables 3).

Combined for SOX18-DNA the SAR. activity data inhibited show SOX18-DNA combine inhibit centainly clearly SAR, wherein reducing or eliminating activity by both β hydroxyl or carboxylic acid or any one any etherificate, esterification or amidation.It is interesting Even if which reduce the inhibitory activity of compound, also tolerable hydroxyl substituted carboxylic acid.Similarly, by small electron The salicylic acid that group contraposition the replaces activity similar to display has tolerance；However, reduced acidity seems to increase cell toxicant Property.For aromatics tail portion, it is active that institute is completely eliminated with phenyl substituted naphthyl (Sm22).In the library small from this, only Sm20 (a kind of unsaturated analog of Sm4) shows the SOX18-DNA binding inhibition activity and low cytotoxicity similar with Sm4 (Fig. 5).

The SAR. inhibited is combined to test analog under 50 μM first for SOX18-RBPJ protein-protein.Then compared with The compound that display inhibits more than 50% is retested under low 5 μM.By activity in the medium with or without lead compound Sm4 The control level of existing SOX18-RBPJ is compared in the presence of object solvent, is also tested under two kinds of concentration.Such as from The IC described in Fig. 4 B (top right plot)₅₀Figure is desired, inhibition of the Sm4 under 50 μM be nearly completed (11.9 ± 5.6%, Fig. 5, The small figure in bottom) and under 5 μM be in edge.Under 50 μM of high concentration, the half of all test compounds shows strong work Property, however, only Sm18, Sm19, Sm26, Sm34 and Sm40 keep high activity under 5 μM, wherein Sm26 keeps appropriateness activity.Have It is interesting, also protein-DNA is inhibited to combine without a kind of in this five kinds efficient PPi inhibitor.

The mode of SOX18-RBPJ protein-protein interaction is inhibited to be not so good as the suppression to interact in SOX18-DNA It is clear like that in system.Several compounds containing vinyl naphthalene have PPI inhibitory activity；However, it is not clear that activity whether be by Still caused due to vinyl-aromatic group as the reactivity of michael acceptor caused by the rigidity for increasing connector 's.One specific characteristic in the library is that PPI activity does not need free carboxy acid's salt, because diethylamide or containing poly- hydroxyl/first The compound of oxygroup shows that PPI inhibits under 50 μM, but does not inhibit DNA to combine.Finally, PPI inhibit in conjunction with DNA between almost It is not overlapped, except Sm4 and Sm20, they inhibit both DNA and PPI.

Energy structure survey

The mouse in conjunction with the DNA containing SOX motif has been parsed more recently by X-ray crystallography (Klaus etc., 2016) The three-dimensional structure of SOX18-HMG structural domain shows the high similarity with other HMG structural domains.However, existing for the Sm4 In the case of SOX18HMG structural domain of the cocrystallization in conjunction with DNA trial fail identify inhibitor binding pocket because not having Detect the electron density of inhibitor.This may be attributed to protein/DNA destruction characteristic of Sm4.In order to assess the possibility of Sm4 Binding site, we carry out computer simulation docking and Molecular Dynamics Calculation using SOX18/DNA crystal structure.In SOX18- In the case where the binding pocket not determined in HMG structural domain, computer simulation docking produces several possible combination postures, The combination posture is further verified by molecular dynamics (MD) simulation.These simulation determined one in the entire of 200ns Stable combination posture is kept in simulated time.In contrast, in every other posture, inhibitor destroys after 3 to 14ns The interaction of its protein, is retained in surrounding solvent without protein contacts.Similarly, using the SOX18- for being free of DNA HMG structure does not find stable Sm4 combination posture.

In SOX18/DNA structure the stable bond posture of Sm4 inhibitor is placed in the solvent between protein and DNA can In close pocket, the pocket described in x-ray crystal structure is occupied by hydrone originally.Sm4 dominant polarity interacts Using Arg136 and Lys147, its interaction is exchanged with dG15, and there is the conformation of certain induction to become His143 Change, its side chain is made to rotate (Fig. 6 A, B) towards Sm4.However, other main conformation changes, the conformation change is not observed It will directly explain the DNA combination suppression mode of Sm4.

In order to study the possible protein-protein interaction site of SOX18, we use computer mould albuminoid Matter-protein docking, simulates in conjunction with MD, constructs the complex model of SOX18/DNA and its protein partner RBPJ.For RBPJ, the X-ray that we are used in a part for people's Notch transcription complex that 2012 (Choi et al., 2012) illustrate are brilliant Body structure.The part includes ankyrin (ANK) repetitive structure domain, the RBPJ-J relevant molecule (RAM) of Notch intracellular domain Structural domain (together activity factor MAML1 combine), and with its share DNA in conjunction with transcription factor RBPJ.By SOX18/DNA Structure is docked in the structure of the Notch transcription complex, is carried out MD simulation then to optimize, is obtained as shown in Figure 6 C Complex model.The model shows that RBPJ/SOX18 compound can be mediated by HMG structural domain, and it is capable of forming not by two kinds The protein complex of DNA molecular interference.In fact, two DNA chain from RBPJ and SOX18 are nearly parallel to each other orientation, Similar to the DNA orientation on nucleosome.In addition, the C of HMG structural domain and N-terminal tail portion are orientated both facing to solvent, allow to add lacking The SOX18 structural domain of mistake is without direct interference RBPJ compound.

Interaction between SOX18 and RBPJ is by the C-terminal part of spiral 3 and the C-terminal tail from HMG structural domain Residue (residue Gln138, Arg141, Asp142 and His143) provide.Therefore, the protein-protein interface and the region DNA combination interface it is opposite.The hypothesis binding site of Sm4 is navigated on RBPJ/SOX18 compound, inhibitor is positioned at spiral shell The combined area SOX18 DNA of rotation -3 and C-terminal tail, it is opposite with its main protein-protein interface, show the combination of Sm4 Protein-protein and protein-DNA interaction may be upset.

The adjusting of SOX18 transcriptional activity

The functional effect of SOX18 inhibitor for further evaluation, we are made using the reporting system based on cell in vitro For the reading of SOX18 transcriptional activity.Started with containing the Vcam-1 merged with luciferase report gene and SOX18 expression vector The construct of sub-piece transfects COS-7 cell.By Sm4 (we PPI destroy specificity in terms of lead compound) together with And first chlorine it is fragrant that, Buddhist nun's fluorine amine and Flufenamic acid test in the measurement based on cell.In these four small molecules, Sm4 Show the most effective inhibition to SOX18 transcriptional activity, wherein IC₅₀Value is 5.2 μM (Fig. 6 D).Can be observed any concentration according to Before relying property SOX18 to inhibit, the cytotoxicity of Meclofenamic Acid and Flufenamic acid is reached.Every other test compound is shown (20 μM < IC of lower effect out₅₀< 50 μM, table the 1, the 3rd arranges).

Sm4 with inhibit its transcriptional activity in vitro needed for selectively upset in the similar concentration range of concentration range it is specific The observation of PPI shows that the mode of action of the small compound may be by the recruitment of interferencing protein gametophyte.

SOX18 target engagement .SMT technology using unimolecule tracking (SMT) assessment SM4 enables us in the thin of living cells Show SOX18 albumen in karyon in real time under single-molecule resolution to the search pattern of the target gene on its chromatin.It can make The fact that SM 4 combines dynamic (dynamical) disturbance visual SOX18 chromatin clearly demonstrates that the middle target of SM4 engages.In This effect is observed under the concentration for not having any cytotoxicity.The effect of SM4 causes SOX18 on chromatin in specific site Place stops the longer time, and this variation of protein dynamics may be mutual by the SOX18 protein-protein of SM4 damage The result of the variation (as shown in previously passed α screening test) of effect.Transcription factor binding mode is by protein-protein phase The code of interaction drives, the code indicator target selectivity.If this code is changed, transcription factor activity without Effect.

It discusses

Transcription factor is (even endogenous in some cases to match with DNA binding structural domain, multiple protein partners Body) protein.The activity of TF is to find that the molecule for adjusting its function is opened up dependent on the concept of such interaction Different approach, such as screening protein-DNA or protein-protein interaction inhibitor.Although participating in it about TF In genetic approach information it is very much, but about their molecular action mode, especially with regard to multiple proteins gametophyte It raises, seldom has been reported that.Therefore, up to date, the screening of DNA binding inhibitors provides the main choosing for finding TF regulator It selects, even if DNA binding structural domain is highly conserved in TF family and constitutes the region with low selectivity potentiality.

During this investigation it turned out, we screen chemically diversified natural product libraries using high throughput DNA binding assay, Identification is able to suppress the compound that the DNA of transcription factor SOX18 is combined.Screening and identification goes out two kinds of chemical combination with similar structure Object both has salicylic acid core and lipophilic tail.Focused libraries around the design of two kinds of reactive compounds identify more It is extensive that there is different degrees of active similar compound, it was demonstrated that these compounds form SOX18DNA binding inhibitors cluster. By focused libraries, we further demonstrate that inhibitory activity be the combination by small compound and protein rather than with DNA sheet Caused by the combination of body.Bioactive molecule and SOX18 protein-interacting increase its thermal stability when combining.In addition, using The albumen of SOX18 overall length or only HMG box, we demonstrate compound directly with DNA binding domain or its immediate area phase interaction With.

We think that the destruction for using DNA to combine can generate the high-level sequence preservative because of HMG box as filter before Property and between SOX transcription factor with low selectivity compound.It is consistent with this, when we use high concentration (200-300 μ When M), our lead compound Sm4 can destroy the DNA binding activity of the various HMG boxes of various SOX albumen.SOX albumen HMG box is made of three alpha-helixes, and two of them provide the DNA major interfaces combined.However it is reported that SOX9HMG box is also joined With protein-protein interaction (Huang etc., 2015, Agresti and Bianchi, 2003, Prokop etc., 2012), Middle third alpha-helix is proposed as the major interfaces for partner protein matter.During this investigation it turned out, we apply albumen The in-vitro method of matter-protein interaction (PPI) detection studies the destruction of TF protein partner recruitment.Transcription factor The direct protein-protein interaction of SOX18 only has been reported that (Hosking etc., 2001) in MEF2C, and with RBPJ's Genetic interaction is only illustrated in the trans-activation of Dll4 gene (Sacilotto etc., 2013).Two kinds of protein are in cell-free/α It shows in screening and co-immunoprecipitation measurement and is bound directly with SOX18.Importantly, some small molecule othernesses destroy spy Fixed PPI.Therefore, the Sm4 with salicylic acid bracket has higher selectivity in terms of destroying SOX18/RBPJ compound, and Flufenamic acid with ortho-aminobenzoic acid bracket has higher selectivity to SOX18/MEF2C compound.In addition, computer Simulation provides the binding pocket of presumption to being connected in Sm4, close to the C-terminal tail of HMG box, wedges the of DNA and protein Between three spirals.The position shows that the inhibitor as Sm4 can change the conformation of SOX18 albumen, not only influences DNA knot It closes, has an effect on the interactive surfaces with protein partner such as RBPJ.

Influence of the small compound to SOX18 transcriptional activity is further studied, discloses to work as and melt with luciferase report gene When conjunction, Sm4 can block SOX18 dependence Vcam-1 promoter activity.Flufenamic acid blocks SOX18 transcription and only shows very little Effect.This report genetic testing is carried out in Sox18 and the COS-7 cell of luciferase expression vector transfection, therefore is limited The explanation for the Latent destruction raised to SOX18 endogenous gametophyte is made.However, Sm4 is to the inhibition of the SOX18 transcription regulated and controled Small molecule may interfere with the clear index of the transcription factor activity in the environment based on cell.

The synthesis and screening for extending library further demonstrate that some clear structures-for inhibiting SOX18/DNA to combine Activity relationship.Although can be resistant to a degree of variation in lipophilic tail and its connector, hydroxyl or carboxylic acid group must Its hydrogen bond supply capacity must be kept, because any ester, ether or amide all can elimination activities.Changed with the electron donating group that contraposition replaces Become carboxylic acid almost without effect, replaces acid to be also with hydroxyl (resorcinol bracket) in this way, the two, which remains them, inhibits SOX18 The ability that DNA is combined.Select change of the compound with ortho-aminobenzoic acid bracket as salicylic acid bracket and NSAID compound Learn the extension of similitude.Although should be studies have shown that SOX18 inhibitor have inhibitory activity, some NSAID to COX1 or COX2 Compound shows SOX18 DNA combination inhibiting effect.However, SOX18- protein binding inhibit (SOX18-MEF2C inhibit without SOX18-RBPJ) difference show different combination or binding mode.

Compound inhibits concentration of the effect of TF transcriptional activity depending on both TF in nucleus and compound.The concentration of TF It can reach almost mM level (Chen etc., 2014) in nucleus, and compound concentration depends on its distribution and passes through cell The ability of film and nucleus.Initial drug is found, IC is measured in homogeneous determination₅₀More information is provided (that is, building SAR Model), there is defined compound concentrations.However, for the optimization of further drug, it is also necessary to consider compound to nucleus In infiltration measure effective inhibition concentration (IC of compound using prediction model or based on the measurement of cell₅₀)。

Which kind of PPI is another to be considered factor be mainly by compounds affect when developing TF inhibitor.Protein-albumen Matter interaction is relatively weak (including the interaction between TF).For example, interaction between p32 and HDM2 have down to The KD (Dawson etc., 2003, Chen etc., 2013) of middle micro-molar range.In contrast, antibody-antigene interaction or endogenous Interaction between property peptide ligand and receptor (such as EGF-EGFR) wants much better than, and wherein KD is in low nanomole to high picomole In range (Mian etc., 1991, Lax etc., 1988).Similarly, the interaction between protein and DNA is in low nanomole model In enclosing, mainly due to the strong electrostatic interaction between negatively charged DNA and usually positively charged DNA binding structural domain And cause.Sm4 is able to suppress SOX18/DNA and SOX18/RBPJ interaction, however, more to the inhibiting effect of weaker PPI Greatly.

One significant consideration of exploitation TF inhibitor is the ability of selective depression PPI, especially because TF can Raise six kinds of different protein partners (Gamper and Roeder, 2008).Although TF be substantially it is unordered (Wright and Dyson, 2015), but these protein show the domain module (Reichmann of different protein-protein interfaces Deng 2005).This means that some interaction shared structure similitudes, while activating different downstream pathways.With other adjustings Albumen is similar with enzyme (such as kinases), needs to consider the selectional feature spectrum that TF protein-protein inhibits.Although may not want that resistance Break all PPI, but single PPI inhibition may cannot achieve maximum effect, but passes through while inhibiting selected protein partner Subgroup recruitment can be achieved maximum effect.

In short, salicyclic acid derivatives are accredited as the main pharmacodynamics group of SOX18 inhibition by this research, and Sm4 is identified For lead compound.Following drug optimization should follow protein-DNA in conjunction with carrying out with PPI setting-out line rope, with into one Step improves selectivity and effect.

Table 1: focused libraries compound inhibits SOX18-DNA to combine the ability with SOX18 dependence trans-activation in vitro.

*: similar with known aggregation

^#: without known aggregation, "high" LogP；Possible aggregation

About upper table 1, in first row, it is fitted using variable Hill slope curve to estimate compound FP IC₅₀.In base Different concentration ranges (0.2-200 μM, 10-500 μM, 10 μM of -3mM) is tested in the DNA combination competition assay of FP, wherein DMSO concentration range is 0 to 3.33%v/v.Experiment independent is repeated with three.Secondary series summarises compound Sm1-14 In salt water (200mM NaCl) or fluorescence polarization buffer (30mM HEPES pH7.5,100mM KCl, 40mM NaCl, 10mM NH₄OAc, 10mM guanidine salt, 2mM MgCl₂, 0.5mM EDTA, 0.01%NP-40)) in threshold concentration when initially forming micella. Third and fourth column summarise 50% inhibition concentration-IC of the luciferase SOX18 dependence trans-activation based on cell₅₀With And in COS7 fibroblast all reactive compounds cytotoxicity-CC50-.In last column, we are used " Aggregator Advisor " (Irwin etc., 2015) carrys out physical property (P > 3 CLog) based on known aggregation and with 12, 600 kinds of compound-Qiang Wenku similitude prediction aggregation (*: the compound similar with known aggregation, #: with it is any known Aggregation is dissimilar, but since there may be risks by "high" LogP.: the forecasting risk without aggregation.).

Table 2 --- PAINS analysis

The just assessment containing PAINS substructure to compound Sm1-Sm44, that lists discovery mixes combination with possible Three kinds of compounds of structure.

Table 3 --- DNA is combined and cytotoxicity

Sm15-Sm44 inhibits the experiment of SOX18/DNA combination (FP) and the cytotoxicity to two kinds of mammal cell lines Data.Also display is with the lipophilicity of the cLogP prediction indicated.

The activity profile that misses the target of table 4 --- Sm4

Use the Hit for coming from Eurofins CEREP/Panlabs (France, USA, Taiwan)Software package characterizes miss the target activity profile of the Sm4 in 10uM.

The chemical characterization of the compound Sm15-Sm44 of table 5 --- synthesis

The chemical analysis of compound Sm15-Sm44 measures compound purity by HPLC (ESI-MS/UV/ELSD), and leads to It crosses HighRes MS (ESI microTOF-LC) and measures its molecular formula.¹H and¹³C NMR is tested for confirming structure (referring to annex A)。

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