CN104031055B - A kind of compound that can be used as Wnt signal pathway activator and preparation and application thereof - Google Patents
A kind of compound that can be used as Wnt signal pathway activator and preparation and application thereof Download PDFInfo
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Abstract
The present invention provides a kind of compound as Wnt signal pathway activator and preparation and application thereof.The compound of the present invention is the compound of formula (I) or formula (II).The compound of the present invention can be used for the medicine of preparation regulation canonical Wnt signal pathway and can be used for studying canonical Wnt signal pathway.The present invention also provides new thinking for the medicine screening the medicine regulating Wnt signal pathway, preventing and treat canonical Wnt signal pathway relevant disease, research Wnt signal pathway application in stem cell.
Description
Technical field
The present invention relates to can be used as compound and the preparation and application thereof of Wnt signal pathway activator.
Background technology
Wnt signal transduction pathway is the signal transduction pathway that a class is guarded at organism evolutionary process camber, regulation control
Make numerous vital movement process.In animal body early development, the formation of Wnt signal deciding dorsoventral axis, germinal layer are set up, body segment
Differentiation, tissue or a series of critical events such as orga-nogenesis, and directly control propagation, break up, polarize, apoptosis and anti-apoptotic etc.
The destiny of cell [Clevers, H.and R.Nusse, Wnt/beta-Catenin Signaling and Disease.Cell,
2012.149(6):p.1192-205].Ten several members of Wnt albumen be take part in by the receptor acting different from cell membrane
Different signal transduction pathways.These approach are broadly divided into the classical Wnt signal depending on β-catenin/TCF transcription complex
Transduction pathway (Wnt/ β-catenin approach) and do not rely on the non-classical Wnt signal of β-catenin/TCF transcription complex and turn
Lead approach (Wnt/Ca2+Approach and Wnt/JNK approach) [Barker, N.and H.Clevers, Mining the Wnt
pathway for cancer therapeutics.Nature reviews.Drug discovery,2006.5(12):
p.997-1014]。
For the process of Wnt/ β-catenin signal transduction pathway, we can understand from two states.When this way
When footpath is not activated, intracytoplasmic transcription activator β-catenin albumen can be degraded by " degraded complex ".At this
In " degraded complex ", Axin and APC(adenomatous polyposis coli) as skelemin, they combine energy
Enough kinase c K1 α (casein-kinase1 α) and GSK3(glycogen that β-catenin is carried out phosphorylation modification
Synthase kinase3) [Liu, C., et al., Control of beta-catenin phosphorylation/
degradation by a dual-kinase mechanism.Cell,2002.108(6):p.837-47].Both kinases
Successively the specific site of the N end of β-catenin albumen is carried out phosphorylation modification so that the β-catenin of phosphorylation can be by
Ubiquitination E3 ligase β-Trcp identifies, and it is proceeded ubiquitination and then by proteasomal degradation.So Cytoplasm and
β-catenin in core maintains a relatively low level all the time, so that transcription inhibitory factor combines transcription factor Tcf/
Lef(T-cell factor/lymphoid enhancer factor), hinder under Wnt/ β-catenin signal transduction pathway
The expression of the genes of interest of trip.When Wnt/ β-catenin signal transduction pathway is activated, i.e. Wnt protein binding seven times across
Membrane receptor Frizzled(Fz) and its co-receptor LRP5/6(low-density lipoprotein receptor-related
Protein5/6).On film, the formation of Wnt-Fz-LRP5/6 complex result in skelemin Dishevelled(Dvl) and seven times
Region in the cell membrane of transmembrane receptor Fz combines and then has promoted the phosphorylation of LRP5/6.The phosphorylation of receptor LRP5/6 enters one
Step recruited region combination in the complex (GSK3-Axin-CK1) and the cell membrane of its phosphorylation that Axin combined [Zeng,
X.,et al.,A dual-kinase mechanism for Wnt co-receptor phosphorylation and
activation.Nature,2005.438(7069):p.873-7.Mao,J.,et al.,Low-density
lipoprotein receptor-related protein-5binds to Axin and regulates the
Canonical Wnt signaling pathway.Molecular cell, 2001.7 (4): p.801-9.], result in
LRP5/6 is by multiple phosphorylation thus can recruit more intracytoplasmic GSK3-Axin-CK1 and be attached to receptor LRP5/6 cell
Region in film.The key members combining the degraded complex of β-catenin in Cytoplasm is thus made constantly to reduce, and then
β-catenin is not phosphorylated namely is not degraded by ubiquitination.Intracytoplasmic β-catenin will constantly accumulate and go forward side by side one
Step transport is combined with transcription factor Tcf/Lef in nucleus, activates the purpose in Wnt/ β-catenin signal transduction pathway downstream
The expression of gene.
Owing to Wnt/ β-catenin signal pathway has relation, so regulating Wnt/ out of control with numerous cancers and disease
β-catenin signal pathway can as treatment and fine means of Wnt/ β-catenin signal pathway relevant disease, but
It is that the research of present stage does not also have anything to be significantly in progress.Such as, the pathological process of Alzheimer along with Wnt/ β-
The exception inactivation of catenin signal transduction pathway.The Presenilins that Ahl tribulus sea silent sickness is closely related can with-
Catenin and GSK3 forms complex [Kang, D.E., et al., Presenilin couples the paired
phosphorylation of beta-catenin independent of axin:implications for beta-
catenin activation in tumorigenesis.Cell,2002.110(6):p.751-62].So for this signal
The molecular targeted targeted therapy of approach is expected to provide new effective way for disease associated therewith.It addition, Wnt believes in stem cell
Number transduction pathway also plays a part to maintain stem cell versatility, is expected to carry for the application of stem cell so regulating this signal pathway
For new effective ways.
Summary of the invention
It is an object of the invention to provide new Wnt signal pathway activator and preparation and application thereof.
Present invention firstly provides a kind of compound, its structural formula is:
Or
Hereinafter the compound that structural formula is I being referred to as compound I, structural formula is that the compound of II is referred to as compound II, knot
Structure formula is that the intermediate of III is referred to as intermediate III, by that analogy.
Invention further provides the preparation method of compound I, its syntheti c route is:
Comprise the following steps:
1) it is initiation material through being methylated by N-and intermediate III is prepared in Hofmann degradation with lycorine (Lycorine)
And separate product；
2) intermediate III hydro-reduction is made to obtain product Compound I.
Invention further provides the preparation method of compound II, its syntheti c route is:
Comprise the following steps:
1) with lycorine (Lycorine) be initiation material through N-methylate with Hofmann degradation prepare intermediate III and point
From product；
2) intermediate III hydro-reduction is made to obtain compound I and separate product；
3) compound I is prepared intermediate compound IV by degraded dioxymethylene and is separated product；
4) make intermediate compound IV be alkylated reaction with propargyl bromide prepare intermediate V and separate product；
5) intermediate V and compound VI is reacted acquisition product Compound II by Click.
Wherein, the syntheti c route of compound VI is:
Comprise the following steps:
1) it is that initiation material is prepared intermediate VII through condensation reaction and separates product with biotin (Biotin)；
2) intermediate VII is made to obtain compound VI with compound VIII class ester exchange reaction and separate product.
Wherein, the syntheti c route of compound VIII is:
For with 2-bromine ethylamine hydrobromide (2-Bromoethylamine hydrobromide) as initiation material and nitrine
Sodium (sodium azide) nucleophilic displacement of fluorine is prepared compound VIII and separates product.
Present invention also offers described compound I and compound II use in preparing canonical Wnt signal pathway agonist
On the way.
Further, described canonical Wnt signal pathway agonist can activate reporter gene or the mesh of canonical Wnt signal pathway
The expression activity of gene.
Present invention also offers described compound I and compound II application in preparing medicine, described medicine selected from
Under arbitrary:
1) prevent or treat by the disease caused by the abnormal inactivation of canonical Wnt signal pathway or the medicine of imbalance；
2) disease of specific activation canonical Wnt signal pathway or the medicine of imbalance are needed；
3) stem cell population amplification medicine.
" disease or imbalance " herein includes any disease that can benefit from the process of the present invention.Its including, but not limited to
Various chronic and acute imbalance or disease.In one preferably embodiment, described disease or imbalance are senile dementia, wind
Wet arthritis or osteoporosis.In another preferably embodiment, described disease or imbalance include hematopoietic stem cell
Transplant.
Further, described medicine is selected from following arbitrary: senile dementia, rheumatoid arthritis agents, sclerotin are dredged
Pine disease drug, hematopoietic stem cell transplantation medicine, stem cell versatility maintain medicine or Brachydanio rerio developmental regulation medicine.
Invention further provides a kind of that cause by the abnormal inactivation of canonical Wnt signal pathway for prevention or treatment or
The pharmaceutical composition of disease or imbalance for needing specific activation canonical Wnt signal pathway: containing the change of therapeutically effective amount
Compound I or compound II and the most acceptable excipient.
Term " comprises " and refers to " containing " and " by constituting ", and the compositions such as " comprising " X can be made up of X completely, or
Person can be containing the material outside X, such as X-Y.Term used herein " therapeutically effective amount " refers to therapeutic agent treats, alleviation or pre-
Anti-target disease or the amount of situation, or show the amount of detectable treatment or preventive effect.This effect such as can be by changing
Learn labelling or antigen levels detects.Therapeutic effect also includes alleviating of physical symptoms.For a certain object accurately effectively
Dosage depends on build and the health status of this object, the character of disease or degree and select the therapeutic agent given and/or control
Treat the combination of agent.Therefore, it is useless for preassigning effective dose accurately.But, for the symptom that certain is given, Ke Yiyong
Normal experiment determines this effective dose, and clinicist can interpolate that out.
Described pharmaceutically acceptable excipient (or carrier) refers to the excipient for Therapeutic Administration, itself and
Consumption should not induce the antibody that the individual generation accepting said composition is harmful, and does not has undue toxicity after administration.Pharmaceutically may be used
The excipient accepted generally includes nontoxic solid, semisolid or liquid filler, diluent, lapping or any conventional class
The formulation auxiliary of type.Suitably excipient includes but are not limited to water, glucose, glycerol, saline, ethanol or a combination thereof.Institute
State excipient also include other reagent such as wetting agent or emulsifying agent, pH buffer agent or strengthen the adjuvant of preparation effect.Other materials
Expect can be added as needed on such as antioxidant, wetting agent, viscosity stabiliser and similar reagents.Liposome is also included within pharmaceutically may be used
In the definition of the excipient accepted.
Generally, pharmaceutical composition can be made injectable agent, such as liquid solution or suspension；May also be fabricated which before the injection
Be suitable for allocating in solution or suspension, the solid form of liquid-carrier.
The compound of the present invention can be used for the medicine of preparation regulation canonical Wnt signal pathway and can be used for studying classical Wnt
Signal pathway.The present invention also regulates the medicine of Wnt signal pathway for screening, prevents and the treatment relevant disease of canonical Wnt signal pathway
Sick medicine, research Wnt signal pathway application in stem cell provide new thinking.
Accompanying drawing explanation
Fig. 1: compound I affects result to the activity of canonical Wnt signal pathway reporter gene TOPFlash:
HLY78: compound I
DMSO: dimethyl sulfoxide
In HEK293T cell, transfect TOPFlash reporter gene after 18 hours, add HLY78 and the solvent of certain concentration
The Wnt conditioned medium of comparison DMSO or control medium.Cell detection reporter gene activity is collected after 6 hours.
Result shows: by being expressed as in mammalian cell laboratory report genescreen system discovery compound I(figure
HLY78) can be with the activity of a kind of form deexcitation canonical Wnt signal pathway reporter gene TOPFlash depending on receptor.
Fig. 2: the compound I result that affects on canonical Wnt signal pathway target gene mRNA level in-site:
HLY78: compound I
DMSO: dimethyl sulfoxide
The mRNA level in-site of mRNA level in-site expression standardized A XIN2 of AXIN2/GAPDH: house-keeping gene GAPDH
The mRNA level in-site of the mRNA level in-site expression standardization DKK1 of DKK1/GAPDH: house-keeping gene GAPDH
HEK293T cell adds HLY78 and the Wnt conditioned medium of solvent control DMSO of certain concentration or compares cultivation
Base.Collect cell after 6 hours and make RT-PCR detection target gene mRNA level in-site.
Result illustrates: compound I can be with a kind of form deexcitation canonical Wnt signal pathway target gene depending on receptor
MRNA level in-site.
Fig. 3: compound I affects result to the protein level of β-catenin
HLY78: compound I
DMSO: dimethyl sulfoxide
Ctr: comparison
Alpha-beta-catenin: with the detection of specific antibody albumen of β-catenin；
Alpha-beta-Actin: with the detection of specific antibody albumen of β-Actin；
α-SP1: with the detection of specific antibody albumen of SP1；
HEK293T cell adds HLY78 and solvent control DMSO of certain concentration.Add containing same medicine after 1 hour
The Wnt conditioned medium of concentration or collating condition culture fluid.After 6 hours collect cell separation Cytoplasm and nucleus detection β-
The protein level of catenin.Wherein β-actin is as Cytoplasm label, and SP1 is as nuclear marker thing.
Result shows: compound I can remove to affect the protein level of β-catenin with a kind of form depending on receptor.
Fig. 4: medicine on canonical Wnt signal pathway reporter gene TOPFlash activity affect result
HLY78: compound I
HLY179: compound II
DMSO: dimethyl sulfoxide
In HEK293T cell, transfect TOPFlash reporter gene after 17 hours, add the HLY179 of 10 μMs, biotin,
The Wnt conditioned medium of HLY78 and solvent control DMSO or control medium.Collect cell detection reporter gene after 6 hours to live
Property.Result shows: compound I and II can be with a kind of form deexcitation canonical Wnt signal pathway report base depending on receptor
Activity because of TOPFlash.
Fig. 5: after compound I effect Brachydanio rerio hematopoietic stem cell, the label runx1/cmyb to performance hematopoietic stem cell
MRNA level in-site express native staining result of the test figure.
40/45: 45 fishes of sum have 40 as shown；
23/32: 32 fishes of sum have 23 as shown；
HLY78 and solvent control DMSO of 80 μMs is added when Brachydanio rerio three body segment.Zebrafish embryo is collected after 24 hours
Doing native staining experiment, the mRNA level in-site of the label runx1/cmyb of detection hematopoietic stem cell is expressed.
Result shows: compound I strengthens the growth of Brachydanio rerio hematopoietic stem cell.
Detailed description of the invention
Inventor, through further investigation, finds that compound I and compound II can be with a kind of shape depending on receptor first
Formula deexcitation canonical Wnt signal pathway.Due to canonical Wnt signal pathway and multiple disease, such as senile dementia, rheumatic is closed
The growth of joint inflammation, osteoporosis and regulation and control hematopoietic stem cell is relevant, consequently found that compound I and compound II can be with one
It is to develop new medicine to provide new to treat these diseases that kind depends on the form deexcitation canonical Wnt signal pathway of receptor
Thinking.
Further illustrate the essentiality content of the present invention below in conjunction with the embodiment of the present invention, but do not limit this with this
Bright.Should be understood that these embodiments are only for the scope illustrated the present invention without limiting the present invention.It is concrete real that embodiment is used
Proved recipe method and experiment condition are this area conventional method and condition, or the method recommended of manufacturer and condition.Unless otherwise
Definition, specialty used in literary composition and scientific words are identical with known to those skilled in the art.
Following compound and intermediate are characterized by liquid chromatography-mass spectrography (LC-MS) and nuclear magnetic resonance, NMR (NMR), in preparation
The initial substance that uses in described compound and reagent can be buied from supplier or by well known by persons skilled in the art
Prepared by method.The most general synthetic route merely illustrates the method that can synthesize the compounds of this invention by it, and
And for the those skilled in the art by reference to the disclosure of invention, the multiple amendment of described synthetic route is to make
Come and take a hint.
Unless otherwise indicated, the experimental technique that disclosed in this invention, detection method, preparation method all use this technology to lead
Conventional molecular biology, biochemistry, chromatin Structure and the analysis in territory, analytical chemistry, cell are cultivated, recombinant DNA technology and
The routine techniques of association area.These technology have improved explanation in existing document, specifically can be found in Sambrook etc.
MOLECULAR CLONING:A LABORATORY MANUAL, Second edition, Cold Spring
Harbor Laboratory Press, 1989and Third edition, 2001；Ausubel etc., CURRENT
PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley&Sons, New York, 1987and periodic
updates；The series METHODS IN ENZYMOLOGY, Academic Press, San Diego；Wolffe,
CHROMATIN STRUCTURE AND FUNCTION, Third edition, Academic Press, San Diego, 1998；
METHODS IN ENZYMOLOGY, Vol.304, Chromatin (P.M.Wassarman and A.P.Wolffe, eds.),
Academic Press, San Diego, 1999；With METHODS IN MOLECULAR BIOLOGY, Vol.119,
Chromatin Protocols (P.B.Becker, ed.) Humana Press, Totowa, 1999 etc..
Embodiment 1
1. experiment material
1) plasmid
TOPFlash plasmid is bought from the company of Millipore.
GFP plasmid is bought from the company of Millipore.
2) antibody
β-actin(sc-47778) antibody purchased from Santa Cruz company；SP1(S9809) antibody is public purchased from Sigma
Department；β-catenin(610154) antibody purchased from BD Biosci.Pharmingen company.
3) reagent
Protease inhibitor (adequate proteins enzyme inhibitor mixing tablet) and NP-40 are purchased from Roche company；
Lipofectamine and PLUS transfection reagent is purchased from Invitrogen company；
Wnt3a conditioned medium is prepared from Wnt3a cell strain (CRL-2647, purchased from U.S.'s ATCC cell bank): uses and adds
The DMEM cell culture medium of 10% calf serum cultivates Wnt3a cell 4 days, is then centrifuged for collecting supernatant culture medium.
Collating condition culture medium is prepared from L cell strain (CRL-2648, purchased from U.S.'s ATCC cell bank): uses and has added 10%
The DMEM cell culture medium of calf serum cultivates L cell 4 days, is then centrifuged for collecting supernatant culture medium.
4) preparation of compound:
Synthetic route:
A. intermediate III: 5-methyl-4-vinyl-5,6-dihydro-[1,3] is prepared with Lycorine for raw material
dioxolo[4,5-j]phenanthridine
Lycorine (Lycorine) (28.7mg, 0.1mmol) dissolves solution in DMF (DMF) (5mL),
Add iodomethane (MeI) (17.0mg, 0.12mmol), stirring reaction 12h at 50 DEG C, it is directly used in after decompression distilling off solvent
Next step reaction；Under nitrogen protective condition, compound (30.2mg, the 0.1mmol) solvent upper step prepared is in the tert-butyl alcohol (t-
BuOH) in (5mL), adding potassium tert-butoxide (t-BuOK) (30mg) back flow reaction 4h afterwards, reaction adds ammonium chloride solution after terminating,
It is subsequently adding ether 2 times (2 × 10ml) of extraction, then washs 2 times (2 × 10ml) with ammonium chloride solution, wash 2 with saturated aqueous common salt
Secondary (2 × 10ml), is dried with anhydrous magnesium sulfate after the washing of organic layer saturated common salt, and decompression uses silicon after removing volatile solvent
Glue chromatography (ethyl acetate/petroleum ether 200:1, Rf=0.23), intermediate III, 21.2mg, yield 80% are obtained.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,CDCl3)δ:7.58(d,J=7.7Hz,1H),7.47(d,J=7.7Hz,1H),7.26(dt,J=
10.7,7.1Hz,2H),7.16(t,J=7.7Hz,1H),6.72(s,1H),5.99(s,2H),5.75(d,J=17.8Hz,1H),
5.32(d,J=11.1Hz,1H),4.03(s,2H),2.51(s,3H);13C NMR(100MHz,CDCl3)δ:147.4(C),
145.1(C),133.4(CH),133.2(C),129.2(C),126.4(C),125.8(C),124.9(CH),124.3(CH),
122.7(CH),114.3(CH2),107.1(CH),103.6(CH),100.9(CH2),54.8(CH2),41.5(CH),EIMS m/
z:266[M+H]+.
B. preparing compound I:N-methyl-4-ethyl-5,6-dihydro-8 from intermediate III further, 9-dioxy is sub-
Methyl-phenanthridines (4-ethyl-5-methyl-5,6-dihydro-[1,3] dioxolo [4,5-j] phenanthridine)
Under nitrogen protective condition, intermediate III (26.5mg, 0.1mmol) is dissolved in oxolane (THF)/H2O
In (5mL, 4:1), it is subsequently adding unifor (TsNHNH2) (37.2mg, 0.2mmol) back flow reaction 4h, it is cooled to room
Temperature is subsequently adding after sodium acetate (NaOAc) (73.8mg, 0.3mmol) reaction terminates and adds ammonium chloride solution, is subsequently adding ether
Extract 2 times (2 × 10ml), then wash 2 times (2 × 10ml) with ammonium chloride solution, with being full of brine It 2 times (2 × 10ml),
Being dried with anhydrous magnesium sulfate after the washing of organic layer saturated common salt, decompression uses silica gel chromatography column purification after removing volatile solvent
(ethyl acetate/petroleum ether 200:1, Rf=0.21) compound I, is obtained.Yellow solid 26.8mg, yield 95%.Product m.p.(melts
Point) 179-180 ° of C.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,MeOD)δ:7.51(t,J=8.4Hz,1H),7.28(d,J=6.4Hz,2H),7.20(t,J=
7.9Hz,2H),6.75(s,1H),6.01(s,1H),4.01(s,2H),2.83(q,J=7.5Hz,2H),2.50(s,3H),1.32
(dd,J=15.7,8.2Hz,3H)；13C NMR(100MHz,CDCl3)δ:147.3(C),147.1(C),145.4(C),139.4
(C),129.3(C),127.7(CH),126.6(C),126.3(C),124.6(CH),121.0(CH),107.2(CH),103.7
(CH),100.9(CH2),55.2(CH2),41.02(CH),23.1(CH2),14.8(CH3),HREIMS m/z217.1261[M]+
(calcd for C17H17NO2,267.1259).
C. from compound I preparation intermediate compound IV: 4-ethyl-5-methyl-5,6-
dihydrophenanthridine-8,9-diol
Under the conditions of-78 DEG C, it is dissolved into dichloromethane (CH at compound I (26.7mg, 0.1mmol)2Cl2) in (2mL), add
Enter Boron tribromide (BBr3) (49.4mg, 0.2mmol) react 2h, reaction adds sodium bicarbonate solution after terminating, and is subsequently adding second
Ether extraction 2 times (2 × 10ml), then with sodium bicarbonate solution wash 2 times (2 × 10ml), with saturated aqueous common salt wash 2 times (2 ×
10ml), being dried with anhydrous magnesium sulfate after the washing of organic layer saturated common salt, decompression uses silica gel chromatography after removing volatile solvent
Column purification (ethyl acetate/petroleum ether 1:1, Rf=0.22), intermediate compound IV, yellow solid 17.9mg, yield 70% are obtained.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,CDCl3)δ:7.46(d,J=7.1Hz,1H),7.27(d,J=2.7Hz,1H),7.17-7.11
(m,2H),6.75(s,1H),3.96(s,2H),2.79(q,J=7.5Hz,2H),2.47(s,3H),1.28(dd,J=14.7,
7.1Hz,3H)；13C NMR(100MHz,CDCl3)δ:143.8(C),143.0(C),139.4(C),129.0(C),127.6
(CH),125.7(C),125.5(C),124.7(CH),120.9(CH),113.8(CH),113.4(C),110.4(CH),54.6
(CH2),41.2(CH3),23.1(CH2),14.8(CH3);EIMS m/z:256[M+H]+.
D. from intermediate compound IV prepare intermediate V:4-ethyl-5-methyl-8,9-bis (prop-2-ynyloxy)-
5,6-dihydrophenanthridine
Under nitrogen protective condition, intermediate compound IV (25.5mg, 0.1mmol) is dissolved in THF(5mL) in, it is subsequently adding hydrogen
Change sodium (NaH) (180mg, 0.3mmol, 40%) and propine bromine (14mg, 0.3mmol, 80%), react 8 hours under room temperature, reaction knot
After bundle add ammonium chloride solution, be subsequently adding ether extraction 2 times (2 × 10ml), then with ammonium chloride solution wash 2 times (2 ×
10ml), wash 2 times (2 × 10ml) with saturated aqueous common salt, be dried with anhydrous magnesium sulfate after the washing of organic layer saturated common salt, subtract
Pressure uses silica gel chromatography column purification (ethyl acetate/petroleum ether 200:1, R after removing volatile solventf=0.21), intermediate is obtained
V, yellow solid 26.4mg, yield 80%.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,CDCl3)δ1H NMR(400MHz,CDCl3)δ7.54(d,J=6.9Hz,1H),7.46(s,
1H),7.23–7.12(m,2H),6.91(s,1H),4.83(s,2H),4.80(s,2H),4.02(s,2H),3.09–2.99(m,
2H),2.86–2.74(m,2H),2.47(s,3H),1.29–1.17(m,3H);；13C NMR(100MHz,CDCl3)δ:147.4
(C),146.8(C),145.6(C),139.5(C),128.9(C),127.9(CH),126.8(C),126.3(C),124.6
(CH),121.0(CH),113.1(CH),110.5(CH),78.6(C),78.4(C),75.9(CH),75.8(CH),57.2
(CH2),56.9(CH2),54.8(CH2),41.31(CH3),23.1(CH2),14.8(CH3);ESIMS m/z:330[M-H]-.
E. compound VIII:2-Azidoethylamine is prepared
2-bromine ethylamine hydrobromide (2-Bromoethylamine hydrobromide) (500mg, 2.44mmol) and folded
Nitrogen sodium (sodium azide) (475.9mg, 7.32mmol) is dissolved into H2In O (2mL), under 75 ° of C, stirring reaction 21h is the coldest
But to 0 ° of C, potassium hydroxide (KOH) (800mg) and Et are added2O (2mL), water layer ether extracts 2 times (2 × 10ml), organic layer
Be dried with anhydrous magnesium sulfate after washing with saturated common salt, decompression remove after volatile solvent with silica gel chromatography column purification (chloroform/
Methanol 20:1, Rf=0.21), compound VIII, yellow liquid 171mg, 1.99mmol, yield 82% are obtained.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,CDCl3)δ:1.27(s,2H,NH2),2.80–2.84(m,2H,CH2N3),3.30(t,J=
5.7Hz,2H,CH2NH2)；13C NMR(100MHz,CDCl3)δ:41.2(CH2NH2),54.6(CH2N3)。
F. intermediate VII:2,5-dioxopyrrolidin-1-yl5-((3aS, 4S, 6aR)-2-is prepared
oxohexahydro-1H-thieno[3,4-d]imidazol-4-yl)pentanoate
Biotin (24.4mg, 0.1mmol) is dissolved in DMF (DMF) (10mL), adds pyridine
(Py) (2mL) and N, N'-Dicyclohexylcarbodiimide (DCC) (41.2mg, 0.2mmol), reaction adds N-hydroxyl after half an hour
Butanimide (N-Hydroxysuccinimide) (13.8mg, 0.12mmol), stopped reaction after reaction 24h, filter after filtration
Liquid decompression distillation removes DMF, then with the dissolving of hot isopropanol solvent, then filters, and filtrate precipitates 2 days in 4 DEG C of refrigerators, finally
It is filtrated to get white solid precipitation 10.2mg, yield 30%.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,[D6]DMSO)δ:6.42(s,1H,3-NH),6.36(s,1H,1-NH),4.27–4.32(m,
1H,6a-H),4.11–4.16(m,1H,3a-H),3.06–3.12(m,1H,SCH),2.78–2.85(m,5H,CH2CH2
(succinyl),SCH2),2.66(t,J=7.3Hz,2H,2'-H),2.57(d,J=11.4Hz,1H,SCH2),1.58–1.68(m,
3H,3'-H,5'H),1.35–1.54(m,3H,4'-H,5'-H);13C NMR(100MHz,[D6]DMSO)δ:170.3(N(CO)
2),168.9(CO2),162.7((HN)2CO),61.0(C-3a),59.2(C-6a),55.2(SCH),39.9(SCH2),30.0
(C-2'),27.8(C-4'),27.6(C-5'),25.4(CH2CH2(succinyl)),24.3(C-3');EIMS m/z:342[M+
H]+。
G. compound VI:N-(2-azidoethyl)-5-((3aS, 4S, 6aR)-2-oxohexahydro-1H-is prepared
thieno[3,4-d]imidazol-4-yl)pentanamide
Intermediate VII(34.1mg, 0.1mmol) it is dissolved in DMF (2mL), add Et3N(12.0mg, 1.2mmol), so
Rear addition compound VIII(17.2mg, 0.2mmol) at room temperature react 24h, decompression distillation uses silica gel chromatographic column after removing DMF
Purification (chloroform/methanol 20:1, Rf=0.23), compound VI, yellow solid 21.8mg, yield 70% are obtained.
NMR, MS are analyzed to identify product:
1H NMR(400MHz,[D6]DMSO)δ:8.03(t,J=5.3Hz,1H,CONH),6.42(s,1H,3-NH),6.35
(s,1H,1-NH),4.26–4.32(m,1H,6a-H),4.08–4.14(m,1H,3a-H),3.31(d,J=7.6Hz,2H,
CH2N3),3.19–3.24(m,2H,CH2CH2N3),3.05–3.11(m,1H,SCH),2.80(dd,J=12.4,5.1Hz,1H,
SCH2),2.56(d,J=12.9Hz,1H,SCH2),2.06(t,J=7.3Hz,2H,2'-H),1.55–1.65(m,1H,5'-H),
1.39–1.55(m,3H,3'-H,5'-H),1.20–1.38(m,2H,4'-H);13C NMR(100MHz,[D6]DMSO)δ:172.4
(CONH),162.7((HN)2CO),61.0(C-3a),59.2(C-6a),55.4(SCH),50.0(CH2N3),39.9(SCH2),
38.1(CH2CH2N3),35.1(C-2'),28.2(C-4'),28.0(C-5'),25.2(C-3');HRESI+MS:m/
z313.14419[M+H]+(calcd for C12H20N6O2S+H,313.14412).
H. compound II:(R, S, S are prepared)-N, N'-(2,2'-(4,4'-(4-ethyl-5-methyl-5,6-
dihydrophenanthridine-8,9-diyl)bis(oxy)bis(methylene)bis(1H-1,2,3-triazole-4,
1-diyl))bis(ethane-2,1-diyl))bis(5-((3aS,4S,6aR)-2-oxohexahydro-1H-thieno[3,
4-d]imidazol-4-yl)pentanamide)
Intermediate V(33.1mg, 0.1mmol) and compound VI(68.6,0.22mmol) be dissolved to tBuOH(5mL) in, anti-
Bad hematic acid sodium (sodium ascorbate) (19.8mg, 0.1mmol) and copper sulfate (1.8mg, 0.01) add water (2mL), so
Afterwards aqueous solution being added in t-BuOH solution, stirring reaction 12 hours at 50 DEG C, decompression distillation uses silica gel chromatography after removing solvent
Column purification (chloroform/methanol 25:1, Rf=0.23), compound 12, yellow solid 57.3mg, yield 60% are obtained.m.p.196-
197°C。
NMR, MS are analyzed to identify product:
1H NMR(400MHz,MeOD)δ7.96(s,1H),7.95(s,1H),7.58–7.51(m,1H),7.46(s,1H),
7.19–7.11(m,2H),6.97(s,1H),5.24(s,2H),5.21(s,2H),4.55–4.48(m,6H),4.46–4.39(m,
2H),4.31–4.22(m,2H),4.02–3.94(m,2H),3.71–3.62(m,4H),3.35–3.26(m,4H),2.80–2.72
(m,2H),2.43(s,3H),2.20–2.04(m,4H),1.75–1.46(m,8H),1.39–1.32(m,4H),1.28(t,J=
7.1Hz,3H)；13C NMR(101MHz,CDCl3)δ176.42,170.23,165.98,163.95,153.09,152.51,
152.04,149.87,149.69,147.94,147.35,147.29,144.84,143.41,143.20,142.15,138.90,
128.20,127.50,126.39,126.01,125.20,124.26,123.96,120.57,119.87,110.56,108.66,
92.87,89.43,62.94,62.41,61.37,59.65,55.06,54.05,41.63,40.49,39.58,38.68,
34.89,27.76,27.51,24.85,22.55,14.04,10.24;HREIMS m/z955.4321[M]+(calcd for
C46H61N13O6S2,955.4307).
2. experimental technique
1) cell is cultivated
HEK293T, cell is at the DMEM(Invitrogen containing 10% hyclone) culture fluid is cultivated, 37 DEG C, CO2Dense
Degree 5%.The L cell strain of stably excreting mice Wnt3a and comparison strain are purchased from U.S.'s ATCC cell bank, and this cell is growing to about
During 70% density, it is changed liquid (containing the DMEM of 10% hyclone), after cultivating four days continuously, collects culture fluid and be centrifuged,
-80 DEG C long-term preservations after liquid nitrogen flash freezer.
2) cell transfecting
HEK293T cell is 24 little time-division dish before transfection, transfects after cultivating one day.Transfection plasmid transfection reagent used
Calculate with the consumption in the 24 every holes of porose disc: plasmid total amount is 250ng/ hole, as transfected plasmids amount deficiency lacZ plasmid is supplied；Matter
Grain is firstly added mixing in the culture medium (25 μ L/ hole) of serum-free, then according to 1 μ L/ hole adds PLUS reagent
(Invitrogen) mixing, stands 15 minutes；According to the amount in 1 μ L/ hole by Lipofectamine(Invitrogen) liposome adds
Enter mixing in plasma-free DMEM medium (25 μ L/ hole), then mix with the mixed solution of above-mentioned plasmid and PLUS, stand 15 points
Clock；Cell is changed to plasma-free DMEM medium (200 μ L/ hole), be added dropwise in cell final plasmid, PLUS and
The package combination of Lipofectamine, hatches 3 hours and transfects, later by spline in the culture medium containing 10% hyclone
Dye reaction.
3) reporter gene is surveyed and is lived
Within first 24 hours, it is divided into being used for the HEK293T cell measuring reporter gene activity in 24 porose discs in transfection, and with 2)
Method transfect.Each plasmid amount of transfection is that 2.5ng/ hole TOPFlash and 2.5ng/ hole are as interior target GFP plasmid.
Remaining with lacZ polishing 250ng/ hole.Within 18 hours, add the Wnt3a added with the small-molecule drug less than or equal to 20uM after transfection
Conditioned medium stimulates 6-8 hour, with Boehringer Mannheim Luci-ferase Assay Kit cell lysis (200
μ L/ hole), with exometer FL600(BIO-TEK Inc.Winooski, VT) survey the intensity of GFP albumen in cell pyrolysis liquid, as
The internal standard of cell expression, then with the substrate of every hole 20 μ L luciferase, with Micro Lumate Plus(Perkin
Elmer Inc.Wellesley, MA) luminometer measures uciferase activity, activity the highest expression Wnt reporter gene table
The amount of reaching is the highest.Finally with the activity data that the fluorescence volume of GFP is internal standard correction luciferase.Simultaneously right with containing primary dcreening operation medicine
Carry out parallel test as comparison according to culture medium, and the Wnt3a conditioned medium parallel test of only solubilizer comparison DMSO is made
For comparison.
4) endocellular liberation β-catenin detection
After the false suitably stimulation of HEK293T cell (6 porose disc), discard culture medium, be placed on ice.Receive with the PBS of pre-cooling
In EP pipe, after 4 DEG C of 3000rpm × 5 minute are centrifugal, remove PBS, with hypotonic buffer liquid (10mMHEPES-KOH pH7.9,1.5mM
MgCl2, 1mM EDTA, 10mM KCl, 1mM pyrophosphoric acid, with front adding protease inhibitor, NaF and Na3VO4) 120 μ L suspensions,
Lash 6-8 time with insulin needle after placing 10 minutes on ice.4 DEG C of 3000rpm × 5 minute are centrifuged, and this step is centrifuged obtain upper
Being used for clearly preparing cytoplasm component, precipitation is used for preparing nuclear components.Cytoplasm component exceeds the speed limit under the conditions of 100,000g4 DEG C
Centrifugal 1 hour, the supernatant that last ultracentrifugation obtains was cytoplasm sample.Nuclear components is washed with 300 μ L hypotonic buffer liquid,
4 DEG C of 3000rpm × 5 minute are centrifuged, add in precipitation 50 μ L high osmotic buffers (20mM HEPES pH7.9,420mM NaCl,
0.2mM EDTA, 25% glycerol, 1mM pyrophosphoric acid, with front adding protease inhibitor, NaF and Na3VO4), place 30 minutes on ice
After, ultracentrifugation 1 hour under the conditions of 100,000g4 DEG C, the supernatant that last ultracentrifugation obtains is core component.Through SDS-PAGE electricity
β-catenin specific antibody is utilized to carry out Western Blot after swimming, the free β in detection cytoplasm component and core component-
catenin。
5)Western Blot
The SDS-PAGE glue of preparation suitable concn, adds protein sample and is separated by electrophoresis.Turn through electricity and protein is turned
On nitrocellulose membrane, nitrocellulose membrane confining liquid (0.5% skim milk) is closed 1 hour, with TTBS wash 3 times, each 5
Minute, then add anti-(specific antibody of β-catenin, β-Actin or SP1) incubated at room 1 hour of correspondence (if one resists
Need 4 DEG C of overnight incubation for the antibody for intrinsic protein).With TTBS washing 3 times after one process resistant, each 5 minutes,
Rear addition resists for the HRP bis-of an anti-species, incubated at room 1 hour, after washing three times, and the nitre of the anti-immunoblotting of HRP coupling two
Acid cellulose film uses FujiFilm Las4000 exposure scan after adding reaction substrate.
6) reverse transcriptional PCR and real-time quantitative PCR detection
Utilize TRIzol reagent (Invitrogen) to be extracted from cell by total serum IgE to obtain, recycle superscriptTM
III first strand sythesis system(Invitrogen) its reverse transcription is cDNA by test kit.CDNA is the dilutest
After releasing, use the preparation of Quantitative SYBR green PCR kit (Takara SYBR premix Ex Taq) test kit
Real-time quantitative PCR reaction system.The instrument that real-time quantitative PCR uses is Rotor-gene RG-3000A apparatus
(Corbett Research)。
For Quantitative Real-Time PCR(qPCR) primer of corresponding gene tested respectively:
human GAPDH:5’-GCACCACCAACTGCTTA-3’(SEQ ID NO:1)
5’-AGTAGAGGCAGGGATGA-3’(SEQ ID NO:2)；
human AXIN2:5’-AGTGTGAGGTCCACGGAAAC-3’(SEQ ID NO:3)
5’-CTTCACACTGCGATGCATTT-3’(SEQ ID NO:4)
human DKK1:5’-CTGCAAAAATGGAATATGTGT-3’(SEQ ID NO:5)
5’-CTTCTTGTCCTTTGGTGTGA-3’；(SEQ ID NO:6)
7) strain of Brachydanio rerio and condition of culture
The Brachydanio rerio of T ü ebingen strain is cultivated in the water of 28.5 ° of C.
8) hybridization in situ experiment of Brachydanio rerio
In situ hybridization is a kind of method that can quantitatively position specific nucleic acid in tissue.Zebrafish embryo leads to
Chang Suoyong's is all rna probe, is used for detecting the expression of specific mRNA.The most frequently used outer rim label is digoxin
(Digoxigenin, Roche), concrete hybridization in situ technique the most ripe (Thisse, C.and B.Thisse, High-
resolution in situ hybridization to whole-mount zebrafish embryos.Nat Protoc,
2008.3(1):p.59-69.).Probe used in this experiment is runx1 and c-myb, and they are all labelling Brachydanio rerio Hematopoietic Stems
Gene (North, T.E., et al., the Prostaglandin E2regulates vertebrate of cell
haematopoietic stem cell homeostasis.Nature,2007.447(7147):p.1007-11.;Burns,
C.E.,et al.,A genetic screen in zebrafish defines a hierarchical network of
pathways required for hematopoietic stem cell emergence.Blood,2009.113(23):
p.5776-82.).Plasmid PBSK-runx1 and PBSK-cmyb by Hind III and Bamh I linearisation, uses T7 respectively after purified templates
In vitro transcription enzyme (Ambion) synthesizes corresponding probe.After in situ hybridization, all of photo is all at room temperature to pass through
The DP71 camera of Olympus shoot under the microscopic system of Olympus (SZX16,10 × or 40 ×;Olympus).
3. experimental result
1) compound I can activate the Reporter System of canonical Wnt signal pathway to depend on the form of Wnt3a receptor
Inventor utilizes the Reporter System SCREENED COMPOUND of canonical Wnt signal pathway.Find that compound I can work in coordination with
The Reporter System (Fig. 1) activating canonical Wnt signal pathway of Wnt3a dose dependent；And there is no Wnt3a receptor
In the case of do not affect the Reporter System (Fig. 1) of canonical Wnt signal pathway.
2) compound I can be to depend on the endogenous target gene of the form activation canonical Wnt signal pathway of Wnt3a receptor
Express
In order to further confirm that compound I can activate canonical Wnt signal pathway, inventor is examined by real-time quantitative PCR
Survey the expression of canonical Wnt signal pathway, find that compound I can work in coordination with the activation classical Wnt letter of Wnt3a dose dependent
The expression (Fig. 2) of the endogenous target gene (AXIN2 and DKK1) of number approach；And in the presence of there is no Wnt3a receptor not shadow
Ring the expression (Fig. 2) of the endogenous target gene of canonical Wnt signal pathway.
3) compound I can improve Cytoplasm to depend on the form of Wnt3a receptor and endonuclear β-catenin amasss
Tired
Inventor is found that by nucleus and cytoplasmic separating experiment, and compound I can actually improve classical Wnt letter
The transmission molecule β-catenin of number approach is in Cytoplasm and endonuclear accumulation, and this depend on Wnt3a when improving and be subject to
Body (Fig. 3).
4) compound I, compound II can activate the report of canonical Wnt signal pathway to depend on the form of Wnt3a receptor
Accuse genic system
Inventor utilizes the Reporter System of canonical Wnt signal pathway.Find that compound II can work in coordination with Wnt3a dosage
The Reporter System (Fig. 4) of dependent activation canonical Wnt signal pathway；And in the presence of there is no Wnt3a receptor
Do not affect the Reporter System (Fig. 4) of canonical Wnt signal pathway.
5) compound I can improve the formation of hematopoietic stem cell in Brachydanio rerio
In order to verify that the compound that we screen can work really in vivo, we have chosen Brachydanio rerio
Hematopoietic stem cell system.Existing document report improves classical Wnt signal and can promote the formation of hematopoietic stem cell.Grinding at us
In studying carefully, we really see compound I can significance improve hematopoietic stem cell label runx1/cmyb
[Goessling,W.,et al.,Genetic interaction of PGE2and Wnt signaling regulates
developmental specification of stem cells and regeneration.Cell,2009.136(6):
P.1136-47], namely compound I can significance improve hematopoietic stem cell quantity (Fig. 5).
Above embodiment illustrates that embodiment disclosed by the invention, can not be interpreted as the limit to the present invention
System.Additionally, various amendments listed herein and invention in method, the change of compositions, without departing from the scope of the present invention
It is apparent from for those skilled in the art with on the premise of spirit.Although combined the present invention multiple specifically
Preferred embodiment has carried out concrete description to the present invention, it is to be understood that, the present invention should not be limited only to these specific embodiments.
It is true that various as above for those skilled in the art obvious amendment obtain invention and all should include
Within the scope of the invention.
Claims (9)
1. a compound, its structural formula is:
2. the preparation method of compound II, its syntheti c route is:
Specifically include the following step:
1) methylate through N-with lycorine for initiation material and prepare intermediate III with Hofmann degradation and separate product；
2) intermediate III hydro-reduction is made to obtain compound I and separate product；
3) compound I by dioxymethylene degraded preparation intermediate compound IV and separates product；
4) intermediate compound IV and propargyl bromide is made to prepare intermediate V through alkylated reaction and separate product；
5) intermediate V and compound VI is prepared product Compound II by Click reaction.
3. the preparation method of compound II as claimed in claim 2, it is characterised in that the syntheti c route of described compound VI is:
4. the preparation method of compound II as claimed in claim 3, it is characterised in that wherein, the syntheti c route of compound VIII
For:
5. compound purposes in preparing canonical Wnt signal pathway agonist as claimed in claim 1.
6. purposes as claimed in claim 5, it is characterised in that described canonical Wnt signal pathway agonist can activate classical Wnt
The reporter gene of signal pathway or the expression activity of genes of interest.
7. compound purposes in preparing medicine as claimed in claim 1, described medicine is selected from following arbitrary:
1) prevent or treat by the disease caused by the abnormal inactivation of canonical Wnt signal pathway or the medicine of imbalance；
2) disease of specific activation canonical Wnt signal pathway or the medicine of imbalance are needed；
3) stem cell population amplification medicine.
8. the purposes of compound as claimed in claim 7, it is characterised in that described medicine is selected from: senile dementia, rheumatism
Property arthritis drug, osteoporosis agents, hematopoietic stem cell transplantation medicine, stem cell population amplification medicine or Brachydanio rerio grow
Regulating medicine.
9. one kind that cause by the abnormal inactivation of canonical Wnt signal pathway for prevention or treatment or for needs specific activation warp
The disease of allusion quotation Wnt signal pathway or the pharmaceutical composition of imbalance, compound described in the claim 1 containing therapeutically effective amount and
The most acceptable excipient.
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| EP1267616B1 (en) * | 2000-03-31 | 2007-08-08 | The General Hospital Corporation | Methods of increasing hair growth by wnt polypeptide |
| CN103145617A (en) * | 2013-03-01 | 2013-06-12 | 中国科学院昆明植物研究所 | Phenanthridine derivative as well as medicinal composition, preparation method and application thereof |
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