CN106279141A - The compound of detection dihydroorate dehydrogenase - Google Patents
The compound of detection dihydroorate dehydrogenase Download PDFInfo
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- CN106279141A CN106279141A CN201510319723.2A CN201510319723A CN106279141A CN 106279141 A CN106279141 A CN 106279141A CN 201510319723 A CN201510319723 A CN 201510319723A CN 106279141 A CN106279141 A CN 106279141A
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Abstract
The present invention relates to compound shown in the Formulas I for detecting dihydroorate dehydrogenase, in formula, A is pigment group, and L is linking arm, and R is the specific binding ligand of dihydroorate dehydrogenase.The compound of the present invention can be used for detecting and positioning dihydroorate dehydrogenase, thus can be used for diagnosing the relevant disease of dihydroorate dehydrogenase mediation.
Description
Technical field
The present invention relates to the field of chemical synthesis；Specifically, the present invention relates to for detect dihydrooratic acid take off
The compound of hydrogen enzyme (DHODH).
Background technology
Malaria (Malaria) is to be situated between by the entomophila of the serious harm human health caused by parasitics plasmodium to pass
Catch an illness, be still that one of the most serious parasitic disease so far.Malaria is bitten by female Anopheles mosquitoes and is felt
Dye person and healthy population steady spread.The mankind can be infected and cause the pathogenic plasmodium of malaria to have four kinds, respectively
Be Plasmodium falciparum (Plasmodium falciparum), Plasmodium ovale (Plasmodium ovale), three
Day plasmodium (Plasmodium malariae) and Plasmodium vivax (Plasmodium vivax).Wherein, dislike
Property the pernicious malaria morbidity that caused of plasmodium rapidly, disease is strong and fatality rate is high.
Pyrimidine synthesis in organism is closely bound up with many vital movements, and pyrimidine synthesizes DNA's and RNA
Generate provide raw material, and DNA and RNA for cell grow divide be indispensable.Former with regard to subtertian malaria
For worm, its internal DNA and rna replicon only depend on pyrimidine source route of synthesis, and Plasmodium falciparum dihydro
Orotic acid dehydrogenase (PfDHODH) is key enzyme (Wu, the T. in the route of synthesis of plasmodium pyrimidine source；S.
Nagle,A.；K.Chatterjee,A.Road Towards New Antimalarials-Overview of the
Strategies and their Chemical Progress.Current Medicinal Chemistry.2011,18(6),
853-871), PfDHODH is carried out fluoroscopic examination to the spike of plasmodial location and the early warning of malaria,
Diagnosis has extremely important scientific value and Research Significance (Malmquist, N.A.；Gujjar,R.；Rathod,
P.K.；Phillips,M.A.Analysis of flavin oxidation and electron-transfer inhibition
in Plasmodium falciparum dihydroorotate dehydrogenase.Biochemistry.2008,47
(8),2466-2475)。
For a long time, the research of plasmodial growth cycle, resistance mechanism is not the most thorough, the most existing malaria
Fast diagnosis method such as microscopy staining, antibody antigen detection (ELISA) and polymerase chain reaction (PCR)
Deng, these method shortcomings are more, and subjective bias length big, time-consuming, cost are high, operation complicated and severe relies on instrument
Device (Wilson, M.L.Malaria Rapid Diagnostic Tests.Clinical Infectious Diseases,
2012,54(11):1637-1641).Chemiluminescence detection means are easy, sensitive, quick but utilize chemistry to visit
Pin identification location plasmodium and study its life cycle and mechanism the most not yet has been reported that.
Therefore, this area is badly in need of detecting or to position plasmodium easy, sensitive, quickly, thus is used for
Study its life cycle and the physical means of mechanism and method.
Summary of the invention
It is an object of the invention to provide a kind of easy, sensitive, detect or position plasmodial probe quickly
Compound and utilize described compound to study plasmodial life cycle and the method for mechanism.
In first aspect, the present invention provides compound shown in Formulas I:
A——L——R
I
In formula,
A is pigment group；
L is linking arm；
R is dihydroorate dehydrogenase specific binding ligand.
In a preferred embodiment, A is the pigment group launching wavelength between 400nm-800nm, including
Various dyestuffs, (fluorescence) dyestuff or stain.
In a preferred embodiment, L includes the saturated of 2-10 carbon atom or unsaturated alkyl chain, or contains
Oxygen, sulfur-bearing, nitrogenous or containing carbonyl, ester group, the saturated or unsaturated alkyl chain of amide groups.
In a preferred embodiment, R includes the inhibitor of dihydroorate dehydrogenase, antagonist, activation
Agent, antibody etc..
In a preferred embodiment, L is selected from optionally substituted C2-C10 alkyl, optionally substituted C4-C10
Alkynes alkyl, C4-C10 oxygen-containing ether chain is contained containing allylic alkylation, optionally substituted C4-C10, as
-(CH2CH2(OCH2CH2)nOCH2CH2)-, is containing ester group chain, such as-(CH2)mCOO(CH2)p-, containing carbonyl chain,
Such as-(CH2)mCO(CH2)p-、O(CH2)qO、O(CH2)qNH、NH(CH2)qO、NH(CH2)qNH、
OCO(CH2)qCOO、OCO(CH2)qO、O(CH2)qCOO、NHCO(CH2)qO、O(CH2)qCONH、
NHCO(CH2)qCONH、S(CH2)qO、S(CH2)qS、NH(CH2)mO(CH2)mOOC(CH2)n；N=0-2,
M=1-5, p=1-5, q=1-10；
In a preferred embodiment, A is selected from the chromophore containing proper conjugation system, such as naphthalimide, 4-
Chloro-7-nitro benzo-2-oxa--1,3-diazole (NBD), fluorescein, rhodamine, coumarin, fluorine boron fluorescence
Dyestuff (BODIPY), Nile red, Nile blue, Hua Jing, phthalocyanine, cyanine dyes, polymethin dye, three
Virtue methine dyes, azo dye and AZOpigments, indigo, azepine [18] annulene dyes, nitro
And nitroso-dyes, quinones carbonyl dyes.
In a preferred embodiment, R-portion is five-membered ring, including containing O, S heteroatomic five-membered ring parent nucleus
Structure DHODH inhibitor.
In a particular embodiment, described compound is as shown in Formula Il:
In formula,
R1For unsubstituted or be selected from the substituent group substituted condensed ring group of lower group: C1-C5 alkyl, halogen,
Carboxyl, ester group, amino；
R2It is selected from: C1-C6 alkyl, C1-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl, C1-C6 fluoroalkane
Base carbonyl, C1-C6 alkoxyl, C1-C6 fluoroalkyl, optionally substituted benzoyl, amino carbonyl,
C1-C6 alkoxy carbonyl, C1-C6 amino carbonyl, hydroxyl, C1-C6 alkoxyl, C1-C6 ester group；
R3Selected from O, NH or S；
R4Selected from H, C1-C6 alkyl, optionally substituted C2-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl,
Optionally substituted benzoyl, carboxyl, amino carbonyl, C1-C6 alkoxy carbonyl, C1-C6 amino carbonyl,
Hydroxyl, C1-C6 alkoxyl；
R5Selected from O, S, NH and CH2。
In a particular embodiment, R1For the condensed ring group containing 2-3 phenyl ring, it is preferable that R1For naphthyl.
In a particular embodiment, L is (HNCH2CH2OCH2CH2OOC)。
In a particular embodiment, described compound is shown below:
In second aspect, the present invention provides the diagnostic reagent of the disease that a kind of dihydroorate dehydrogenase mediates
Box, described test kit is equipped with the compound described in first aspect present invention.
In a preferred embodiment, described dihydroorate dehydrogenase mediation disease include pernicious malaria,
The parasites such as tertian malaria, avette malaria, quartan malaria, monkey type malaria, schizotrypanum cruzi, schistosomicide, dengue fever
Infectious disease；And rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus, glomerule disease
The host rejection reaction that sick, melanoma and of the same race or xenotransplant cause.
In a particular embodiment, described disease is pernicious malaria.
In the third aspect, the present invention provides the compound described in first aspect present invention preparing dihydrooratic acid
Purposes in the diagnostic reagent of the disease of dehydrogenase detection or the mediation of location reagent dihydroorate dehydrogenase.
In a preferred embodiment, described dihydroorate dehydrogenase mediation disease include pernicious malaria,
The parasites such as tertian malaria, avette malaria, quartan malaria, monkey type malaria, schizotrypanum cruzi, schistosomicide, dengue fever
Infectious disease；And rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus, glomerule disease
The host rejection reaction that sick, melanoma and of the same race or xenotransplant cause.
In a particular embodiment, described disease is pernicious malaria.
In fourth aspect, the present invention provides a kind of pharmaceutical composition, and described pharmaceutical composition comprises:
Compound described in first aspect present invention or its pharmaceutically acceptable salt, and
Pharmaceutically acceptable carrier or excipient.
In should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and below (such as embodiment)
Can be combined with each other between each technical characteristic of middle specific descriptions, thus constitute new or preferred technical side
Case.As space is limited, the most tired at this state.
Accompanying drawing explanation
Fig. 1 shows the compound of the present invention and the combination situation of PfDHODH albumen；
Fig. 2 show the compounds of this invention and In vitro culture by Infected With Plasmodium erythrocytic fluorescence imaging figure
Picture；
Fig. 3 shows that the compounds of this invention becomes with detection and the fluorescence of the blood of P. berghei infecting mouse
As image.
Detailed description of the invention
Inventor is through extensively in-depth study, it has surprisingly been found that a class can be used in cell and work
The fluorescent chemicals of specific detection PfDHODH in body level, these compounds not only at cellular level to evil
Property plasmodium have certain inhibitory activity, they can also specific recognition labelling infect in plasmodial erythrocyte
DHODH.Complete the present invention on this basis.
Term defines
Some group definition arrived referred to herein are as follows:
Herein, " alkyl " refers to the saturated branched-chain or straight-chain alkyl that carbon chain lengths is 1-10 carbon atom,
Preferably alkyl include that long 2-8 carbon atom, 1-6 be individual, 1-4 carbon atom, 1-3 carbon atom alkyl not etc..
The example of alkyl includes but not limited to methyl, ethyl, n-pro-pyl, isopropyl, normal-butyl, isobutyl group, heptan
Base etc..Alkyl can be replaced by one or more substituent groups, such as, replaced by halogen or haloalkyl.Such as,
The alkyl that alkyl can be replaced by 1-4 fluorine atom, or the alkane that alkyl can be replaced by fluoro-alkyl
Base.
Herein, " ester group " refers to-COORxShown group, wherein, RxIt it is alkyl as defined above.
Herein, " aryl " refers to the monocycle containing 6 to 14 carbon atoms, dicyclo or tricyclic aromatic group,
Including phenyl, naphthyl, phenanthryl, anthryl, indenyl, base, tetrahydro naphthyl, indanyl etc..Virtue
Base is optionally replaced selected from following substituent group by 1-5 (such as, 1,2,3,4 or 5): halogen,
C1-C4 aldehyde radical, the straight or branched alkyl of C1-C6, cyano group, nitro, amino, hydroxyl, methylol, halogen
Element substituted alkyl (such as trifluoromethyl), the alkoxyl (such as trifluoromethoxy) of halogen substiuted, carboxyl,
The alkoxyl of C1-C4, C1-C4 substituted sulfhydryl, morpholinyl, optionally substituted aryl are (the most optionally substituted
Phenyl), optionally substituted aryloxy group (the most optionally substituted phenoxy group) and optionally substituted benzyloxy.Example
As, aryl can be replaced selected from following group by 1-3: fluorine, chlorine, bromine, C1-C4 alkyl, fluoroform
Base, morpholinyl, methoxyl group, phenyl, the phenyl of methoxy substitution, phenoxy group, benzyloxy, taken by halogen
The benzyloxy in generation, ethyoxyl and nitro etc..
Term as used herein " heterocyclic radical " refers to ring structure that is single or that condense, can be virtue in nature
Race or non-aromatic, and it preferably comprises 3-20 ring member nitrogen atoms, more preferably contains 5-14 annular atoms,
At least a part of which 1 and preferably up to can to 4 be the hetero atom selected from O, S and N.Herein, heterocyclic radical
Example include furyl, thienyl, pyrrole radicals, pyrrolidinyl, imidazole radicals, triazolyl, thiazolyl,
Tetrazole radical, oxazolyl, isoxazolyl, pyrazolyl, pyridine radicals, pyrimidine radicals, pyrazinyl, pyridazinyl, three
Piperazine base, quinolyl, isoquinolyl, quinoxalinyl, benzothiazolyl, benzoxazolyl group, benzothienyl,
Benzofuranyl, morpholinyl, carbazyl, dibenzothiophenes, coumarin base and 1,2-methylenedioxyphenyl.
Herein, heterocyclic radical is optionally replaced by 1-3 substituent group as herein described.
Term as used herein " hetero atom " includes O, S and N.When hetero atom is N, this atom N
Can be replaced by the group of such as hydrogen or C1-C10 alkyl further.When hetero atom is S, this S atom can
To be replaced by the group of such as C1-C10 alkyl further.
Term as used herein " heteroaryl " or " fragrant heterocyclic radical " refer to have as mentioned above aromatic character
Those heterocyclic radicals, include but not limited to furyl, thienyl, pyrrole radicals, pyridine radicals, oxazolyl, pyrrole
Piperazine base, pyridazinyl, pyrimidine radicals etc..
Term as used herein " halogen " includes fluorine, chlorine, bromine and iodine.
Except as otherwise noted, term as used herein " optionally substituted " refers to that its group modified can be appointed
Selection of land is replaced selected from following substituent group by 1-5 (usually 1,2 or 3): C1-C4 alkyl, carboxyl,
Halogen, C1-C4 alkoxyl, cyano group, nitro, amino, hydroxyl, aldehyde radical, C1-C6 acyl group, methylol,
The C1-C4 alkyl (such as trifluoromethyl) of halogen substiuted, C1-C4 alkoxyl (the such as fluoroform of halogen substiuted
Epoxide), sulfydryl and C1-C4 acyl group.
Herein, amide groups (amino carbonyl) self or the part as other group, refer to " C1-C6 alkane
Base-CO-NH-" group, " C3-C8 cycloalkyl-CO-NH-" or " C3-C8 cycloalkyl-C1-C6 alkyl
-CO-NH-”.Exemplary amide groups include but not limited to formamido, acetamido, propionamido-,
Amide-based small, ring propionamido-, ring propyl formamide base etc..
Herein, acyl group self or the part as other group, can contain 1-6 carbon atom, preferably 1-4
Individual carbon atom.Exemplary acyl group includes but not limited to acetyl group, fluoro acetyl group, fluoro propiono, fluorine
For bytyry etc..
The compounds of this invention
The present invention devises a class and can be used for the glimmering of in cell and live body level specific detection PfDHODH
Optical compounds.Pharmacology test result shows that Plasmodium falciparum is not only had by the compound of the present invention at cellular level
Certain inhibitory activity, has affinity to PfDHODH, and it is and it can infect plasmodial erythrocyte simultaneously
Middle specific recognition labelling DHODH, and selectivity is positioned in mitochondrion.Infected by P. berghei
The blood testing of mice proves, the compound of the present invention can exactly the malaria in labelling and spike erythrocyte former
Worm, and the plasmodial growthform of accurate discrimination different times, quantify and simplify plasmodial detection process.
To sum up, design and the discovery of this kind of novel DHODH marking type compound will be for the early diagnosis of malaria
There is provided full and accurate, strong evidence it can also be used to the morning of malaria with plasmodium life cycle, pathogenesis
Phase diagnosis and prevention.
In a particular embodiment, the present invention provides compound shown in Formulas I:
A——L——R
I
In formula,
A is pigment group, and preferred emission wavelength is between the pigment group of 400nm-800nm, including various dyes
Material, (fluorescence) dyestuff or stain；
L is linking arm, including the saturated of 2-10 carbon atom or unsaturated alkyl chain or oxygen-containing, sulfur-bearing, contains
Nitrogen or containing carbonyl, ester group, the saturated or unsaturated alkyl chain of amide groups；
R is dihydroorate dehydrogenase specific binding ligand, including dihydroorate dehydrogenase inhibitor,
Antagonist, activator, antibody etc..
In view of the teachings of the present invention, those skilled in the art can use any suitable pigment group, connection
Arm and dihydroorate dehydrogenase specific binding ligand.
In a preferred embodiment, L is selected from optionally substituted C2-C10 alkyl, optionally substituted C4-C10
Alkynes alkyl, C4-C10 oxygen-containing ether chain is contained containing allylic alkylation, optionally substituted C4-C10, as
-(CH2CH2(OCH2CH2)nOCH2CH2)-, is containing ester group chain, such as-(CH2)mCOO(CH2)p-, containing carbonyl chain,
Such as-(CH2)mCO(CH2)p-、O(CH2)qO、O(CH2)qNH、NH(CH2)qO、NH(CH2)qNH、
OCO(CH2)qCOO、OCO(CH2)qO、O(CH2)qCOO、NHCO(CH2)qO、O(CH2)qCONH、
NHCO(CH2)qCONH、S(CH2)qO、S(CH2)qS、NH(CH2)mO(CH2)mOOC(CH2)n；N=0-2,
M=1-5, p=1-5, q=1-10；
A is selected from the chromophore containing proper conjugation system, such as naphthalimide, 4-chloro-7-nitro benzo-2-oxa--1,3-
Diazole (NBD), fluorescein, rhodamine, coumarin, fluorine boron fluorescent dye (BODIPY), Nile red,
Nile blue, Hua Jing, phthalocyanine, cyanine dyes, polymethin dye, three virtue methine dyes, azo dyes
And AZOpigments, indigo, azepine [18] annulene dyes, nitro and nitroso-dyes, quinones carbonyl
Dyestuff；
R-portion is five-membered ring, including containing O, S heteroatomic five-membered ring mother nucleus structure DHODH inhibitor.
In further embodiment, the compound of the present invention is as shown in Formula Il:
In formula,
R1For unsubstituted or be selected from the substituent group substituted condensed ring group of lower group: C1-C5 alkyl, halogen,
Carboxyl, ester group, amino, R1It is preferably the condensed ring group containing 2-3 phenyl ring, more preferably naphthyl；R2It is selected from:
C1-C6 alkyl, C1-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl, C1-C6 fluoro-alkyl carbonyl, C1-C6
Alkoxyl, C1-C6 fluoroalkyl, optionally substituted benzoyl, amino carbonyl, C1-C6 alkoxyl carbonyl
Base, C1-C6 amino carbonyl, hydroxyl, C1-C6 alkoxyl, C1-C6 ester group；R3Selected from O, NH or S；
R4Selected from H, C1-C6 alkyl, optionally substituted C2-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl, optionally
Substituted benzoyl, carboxyl, amino carbonyl, C1-C6 alkoxy carbonyl, C1-C6 amino carbonyl, hydroxyl,
C1-C6 alkoxyl；R5Selected from O, S, NH and CH2。
Those skilled in the art can use the linking arm of various structure, such as
(HNCH2CH2OCH2CH2OOC) linking arm shown in.
In a particular embodiment, the present invention provides following compound:
Advantages of the present invention:
1. absorption and the transmitting wavelength of the compounds of this invention contains all wavelengths scope, and does not substantially have background glimmering
Light disturbs, and can be used as detecting the fluorescent probe of DHODH；
2. the compounds of this invention both can specific binding dihydroorate dehydrogenase, fluorescence signal can be produced again.
Below in conjunction with being embodied as case, technical scheme is further described, but following case study on implementation
It is not construed as limiting the invention, the various sides of using that all principles according to the present invention and technological means use
Method, belongs to the scope of the invention.In following synthetic example, unless otherwise stated, agents useful for same
Being all the most directly to buy gained, all through Non-aqueous processing before the use of used solvent, described room temperature is
Refer to 25 DEG C.The experimental technique of unreceipted actual conditions in the following example, generally according to normal condition, or presses
According to the condition proposed by manufacturer.Unless otherwise indicated, otherwise percentage ratio and number are calculated by weight.
The synthesis of embodiment 1. compound IIA
The synthesis of intermediate A
Under argon shield, by 4-chloro-7-nitro benzo-2-oxa--1 of 996mg, 5mmol, 3-diazole
(NBD-Cl) join in the dry DMF of 2.5mL, be slowly added dropwise the 2-aminooxy group second of 630mg, 6mmol
The 2.5mL anhydrous DMF solution of alcohol and the triethylamine of 0.9mL, stirring at normal temperature overnight, removes DMF under reduced pressure,
Dichloromethane extracts, and saturated aqueous common salt washs, and organic layer concentrates, column chromatography (petroleum ether: ethyl acetate=1:
2, v/v) red solid, yield 35% are obtained.
1H NMR(400MHz,DMSO-d6): δ 9.46 (s, 1H), 8.50 (d, J=8.0Hz, 1H), 6.47
(d, J=8.8Hz, 1H), 4.61 (s, 1H), 3.72-3.66 (m, 4H), 3.49 (brs, 4H).
HRMS(ESI)calcd for C10H12N4O5[M-H]-267.0729,found 267.0506.
The synthesis of intermediate 6-t-butyl carbamate-2-naphthoic acid
6-amino-2-the naphthoic acid of 20mmol is dissolved in the mixed solution of the 20mL tert-butyl alcohol and 20mL water,
Being sequentially added into the Bis(tert-butoxycarbonyl)oxide of 24mmol and the sodium hydroxide of 24mmol, normal-temperature reaction is overnight.With
5% hydrochloric acid solution regulates to pH 2-3, adds the extraction of a large amount of dichloromethane, and organic layer is dried, and concentrates, post layer
Analysis (petroleum ether: ethyl acetate=1:1, v/v) obtains beige solid, yield 75%.
1H NMR(400MHz,DMSO-d6):δ12.90(s,1H),9.75(s,1H),8.48(s,1H),
8.18 (s, 1H), 7.99 (d, J=8.8Hz, 1H), 7.90 (dd, J1=1.6Hz, J2=8.4Hz, 1H), 7.84
(d, J=8.4Hz, 1H), 6.47 (dd, J1=2.0Hz, J2=9.2Hz, 1H), 1.52 (s, 9H).
The synthesis of intermediate B
Under ice bath, the 6-t-butyl carbamate-2-naphthoic acid of 5mmol is dissolved in 20mL anhydrous methylene chloride
In, the dimethylamino naphthyridine (DMAP) of dropping 7.5mmol and anhydrous the two of the 20mL of 6mmol intermediate A
Chloromethanes mixed solution, stirring adds 1-(3-the dimethylamino-propyl)-3-ethyl carbon of 10mmol after half an hour
Diimmonium salt hydrochlorate (EDC), recession in 2 hours removes ice bath, and normal-temperature reaction is overnight.Add a large amount of dichloromethane extraction
Taking, saturated aqueous common salt washs, and organic layer is dried, and concentrates, column chromatography (petroleum ether: ethyl acetate=1:2,
V/v) yellow solid, yield 50% are obtained.
1H NMR(400MHz,DMSO-d6): δ 9.77 (s, 1H), 9.43 (brs, 1H), 8.37 (d, J=8.8
Hz, 1H), 8.34 (s, 1H), 8.15 (s, 1H), 7.91 (d, J=8.8Hz, 1H), 7.74 (s, 2H), 7.57 (dd,
J1=2.0Hz, J2=8.8Hz, 1H), 6.44 (d, J=8.8Hz, 1H), 4.44-4.42 (m, 2H),
3.84-3.81(m,4H),3.68(brs,2H),1.53(s,9H).
13C NMR(100MHz,DMSO-d6):δ165.7,152.7,148.8,139.7,135.9,130.0,
129.8,127.8,127.2,124.9,120.1,112.7,106.6,79.6,68.3,63.7,55.9,28.1,18.5.
HRMS(ESI)calcd for C26H27N5O8[M+Na]+560.1757,found 560.1757.
The synthesis of intermediate C
The intermediate B of 0.5mmol is dissolved in 4mL dichloromethane, under ice bath, drips 1.5mL trifluoroacetic acid,
Normal-temperature reaction is overnight.Regulate to pH 7-8 with 10% sodium bicarbonate solution, add the extraction of a large amount of dichloromethane,
Saturated aqueous common salt washs, and organic layer is dried, and concentrates, column chromatography (dichloromethane: methanol=50:1, v/v)
Obtain Orange red solid, yield 72%.
1H NMR(400MHz,DMSO-d6): δ 9.49 (s, 1H), 8.41 (d, J=8.8Hz, 1H), 8.20
(s, 1H), 7.66 (d, J=8.8Hz, 1H), 7.59 (d, J=8.4Hz, 1H), 7.41 (d, J=8.4Hz, 1H),
6.98(dd,J1=2.0Hz, J2=8.8Hz, 1H), 6.80 (d, J=1.6Hz, 1H), 6.46 (d, J=8.8Hz,
1H),5.86(s,2H),4.40-4.38(m,2H),3.82-3.80(m,4H),3.68(brs,2H).
13C NMR(100MHz,DMSO-d6):δ166.5,149.9,145.8,144.8,144.4,138.0,
130.9,130.8,125.3,125.1,121.6,119.5,105.5,99.9,68.9,63.8,56.5,43.8,19.0.
HRMS(ESI)calcd for C21H19N5O6[M-H]-436.1257,found 436.1263.
The synthesis of intermediate D
The anhydrous THF of sodium hydrogen (60%) about 240mg and 1.8mL of 3mmol, ice is added in 50mL flask
The lower dropping 1.8mL of bath contains the anhydrous THF solution of the diethyl malonate of 6mmol, drips 3 after 10 minutes
ML contains the chloracetyl chloride solution of 3mmol, maintains ice bath one hour, and 40-45 degree Celsius is reacted 1-2 hour,
Ethyl acetate extracts, brine It 2 times, and organic layer concentrates, and is dried, column chromatography (100%EA), post layer
Analysing to obtain white powder, GC-MS checking is errorless.
The synthesis of compound IIA
The intermediate D of 0.22mmol is dissolved in the anhydrous THF of 5mL, adds the intermediate C of 0.2mmol,
React 96 hours at 40 DEG C.Adding the extraction of a large amount of dichloromethane, saturated aqueous common salt washs, and organic layer is dried,
Concentrating, column chromatographic isolation and purification (dichloromethane: methanol=60:1, v/v) obtains orange/yellow solid.
1H NMR(400MHz,DMSO-d6): δ 10.50 (s, 1H), 9.41 (d, J=2.4Hz, 1H),
8.42 (s, 1H), 8.29 (d, J=8.8Hz, 1H), 8.05 (d, J=8.8Hz, 1H), 7.91-7.80 (m, 3H),
7.58(dd,J1=2.0Hz, J2=8.8Hz, 1H), 6.41 (d, J=9.2Hz, 1H), 5.30 (s, 2H),
4.48-4.45 (m, 2H), 4.29 (q, J=7.2Hz, 2H), 3.86-3.82 (m, 4H), 3.67 (brs, 2H), 1.30
(t, J=7.2Hz, 3H).
13C NMR(100MHz,DMSO-d6):δ188.7,177.3,165.5,163.9,135.1,130.3,
130.1,128.0,125.4,123.0,119.5,99.4,87.4,75.4,68.3,68.2,65.5,63.9,59.6,56.0,
43.3,18.4,14.4,14.3.
HRMS(ESI)calcd for C28H25N5O10[M-H]-590.1523,found 590.1526.
The synthesis of embodiment 2. compound IIB
The synthesis of intermediate A-C is as previously mentioned
The synthesis of intermediate E
To be added drop-wise to molten dissolved with the 5mL anhydrous methylene chloride solution in the 2,2-difluoro propanoic acid of 4mmol under room temperature
There is the 5mL anhydrous methylene chloride solution of the N of 324mg, N-dicarbapentaborane imidazoles (2mmol), after stirring 30min
Dropping, dissolved with 68mg imidazoles (1mmol) and the 1 of 98mg, 3-cyclopentanedione (1mmol), finishes, and reaction 6 is little
Time.5% hydrochloric acid solution, saturated aqueous common salt washs successively, and organic layer concentrates, and is dried, is concentrated to give crude product, yellow
Color solid, GC-MS checking is errorless.
The synthesis of intermediate F
Under room temperature, 0.7mL oxalyl chloride is joined in the intermediate E of 0.3mmol, normal-temperature reaction 4 hours, often
Pressure is evaporated off remaining oxalyl chloride, and dichloromethane extracts, and saturated aqueous common salt washs, and organic layer concentrates, and is dried,
Rapid column chromatography (dichloromethane: methanol=100:1, v/v) yellow oily liquid, yield 55% ,-35 DEG C
Refrigerator store.
The synthesis of compound IIB
The intermediate C of 0.2mmol is dissolved in the anhydrous THF of 5mL, the intermediate F of addition 0.2mmol, 40
React 96 hours at DEG C.Adding the extraction of a large amount of dichloromethane, saturated aqueous common salt washs, and organic layer is dried, dense
Contracting, column chromatographic isolation and purification (dichloromethane: methanol=60:1, v/v) obtains orange/yellow solid.
1H NMR(400MHz,DMSO-d6): δ 8.45 (s, 1H), 8.29 (d, J=9.2Hz, 1H), 8.10
(s, 1H), 8.07 (s, 1H), 7.86 (q, J=8.0Hz, 2H), 7.66 (d, J=8.4Hz, 1H), 6.40 (d, J=
9.2Hz, 1H), 4.48-4.46 (m, 2H), 4.39 (d, J=4.0Hz, 2H), 3.86-3.81 (m, 4H), 3.67
(brs, 2H), 2.42-2.39 (m, 2H), 1.95 (t, J=18.8Hz, 3H).
13C NMR(100MHz,DMSO-d6):δ197.2,165.4,137.5,134.9,130.5,130.4,
130.0,128.1,127.1,125.4,124.3,122.3,117.9,107.7,68.2,63.8,55.9,54.8,47.7,
47.5,47.3,33.6,25.9,19.9,18.5.
HRMS(ESI)calcd for C29H25N5O8F2[M-H]-608.1590,found 608.1593.
Embodiment 3. the compounds of this invention inhibitory activity to cell strain
The compound that the present invention provides is to the external activity of two kinds of Plasmodium falciparum cell strains (3D7 and Dd2)
Inhibition:
Chloroquine is purchased from Sigma company, and SYBR Green I is purchased from Life technologies company, chloroquine-sensitive
3D7 cell strain and the Dd2 cell strain of chloroquine resistance for testing in vitro antimalarial active, by Trager and
The cultural method of Jensen (Human malaria parasites in continuous culture.Science.1976,
193 (4254): 673-675) cultivate in the medium containing 0.5%Albumax II non-human serum.With SYBR
Green I is probe, measures antiplasmodial cell strain activity by fluorescence titration method.First, different times
Parasite triplicate, erythrocyte 2% and 100 μ L 1% parasitemia with a series of
Medicine presence or absence under the conditions of cultivate, during dilution, each compound concentration 0.15625 changes to 20 μMs.
0.2%DMSO compares as forward as reversely comparison, the chloroquine of variable concentrations.Culture dish is at 37 DEG C
Cultivate 3 days, then take out supernatant, and add the Cell Buffer (8.26 of 100 μ L of SYBR Green I
g/L NH4Cl,1g/L KHCO3, 0.037g/L EDTA and 5 × SYBR Green I).Culture dish is in darkroom
Middle room temperature continues to cultivate 1 hour, is read under 485/520nm by Synergy MX, Biotek fluorescence detector
Number, is repeated 2 times this experiment.Data analysis is that the method reported according to Michael etc. improves acquisition.Letter
Stating as follows, the fluorescence data that negative control process group obtains deducts that to be uninfected by background produced by erythrocyte group glimmering
Light data, represent the DNA maximum in the normal Plasmodium falciparum cultivated in this experiment, and it is negative right to be designated as
According to the fluorescence data in hole, the fluorescence data of each test group and positive controls the most so corrects.Under variable concentrations
The calculating of suppression ratio is by following formula gained: suppression ratio (%)=(1-(mean fluorecence counting/negative control in instrument connection
Mean fluorecence counting in hole)) × 100%.By the Growth/Sigmoidal program in software Origin 8.0
Matched curve also calculates acquisition half suppression ratio (IC50Values), meansigma methods is calculated by Microsoft Excel
And standard deviation.
The result of embodiment 3 such as following table:
Embodiment 4. the compounds of this invention and the combination of target protein
Fragment containing PfDHODH gene is cloned in PET-19b carrier, obtains recombiant plasmid, proceed to
E. coli bl21 is expressed, pure respectively through fixing metal ions affinity chromatography and gel filtration chromatography
After change, obtain purer albumen, PfDHODH albumen good for purification be diluted to final concentration of 2mg/mL,
Being distributed into two pipes, often pipe 500 μ L, a pipe adds the compounds of this invention (IIA) that 25 μ L mother solutions are 50mM, separately
One pipe adds 25 μ L DMSO as comparison, hatches 30min on ice, with PCR pipe subpackage albumen, will add chemical combination
The albumen of thing and DMSO subpackage 12 respectively is managed, often pipe 30 μ L, with PCR instrument at different temperatures (37 DEG C,
40 DEG C, 43 DEG C, 46 DEG C, 49 DEG C, 52 DEG C, 55 DEG C, 58 DEG C, 61 DEG C, 64 DEG C, 67 DEG C, 70 DEG C) respectively
Heating 3min, compound and comparison carry out identical process, after having heated, and 4 DEG C of high speed centrifugation half an hour,
Take supernatant 2 μ L respectively, be diluted to 100 μ L with Buffer, prepare protein sample and carry out western blot, finally
Develop by chemiluminescence imaging system, obtain result as shown in Figure 1.
Known to Fig. 1, compared with the comparison only adding DMSO, after adding the compounds of this invention, to pfDHODH
Albumen plays certain Stabilization.Add the matched group of DMSO, Tm=53.77 DEG C, and add chemical combination of the present invention
The experimental group of thing, Tm=56.94 DEG C, make TmValue increases 3.2 DEG C, i.e. the compounds of this invention is to PfDHODH
Albumen has combination, and it can be played Stabilization to a certain extent.
In view of PfDHODH protein matter structure is complicated, conformation is in does not stops in change, tripe systems as under
Energy barrier different, stability is the most different；This example demonstrates that, after adding thing of the present invention (IIA), itself and albumen
Matter combines, with between uncombined time compared with, protein is more stable, therefore explanation the compounds of this invention and target egg
White combination effect is preferable.
Embodiment 5. the compounds of this invention and In vitro culture by the erythrocytic fluorescence imaging of Infected With Plasmodium
The compounds of this invention (IIA) is dissolved to 50mM with DMSO.A certain amount of evil is cultivated in 37 DEG C of incubators
Property plasmodium (Huzhou Teachers College teacher Zhou Hongchang give), average mark two ware, it is labeled as A and B；By A, B
Plasmodium falciparum in culture dish synchronizes with between 5% sorbitol every two days, cultivates two days；To A and B culture dish
Middle addition the compounds of this invention to final concentration of 10 μ g/mL, is cultivated 3.5 hours in 37 DEG C of incubators, backward
A, B culture dish adds fluorescent dyeRed CMXROS (purchased from Life Technologies)
To final concentration of 500nM, it is placed in 37 DEG C of incubators cultivation 30min；It is pernicious by two culture dishs infect
Plasmodial erythrocyte is transferred in 15mL centrifuge tube, centrifugal under the conditions of 1800rpm, 3min, removes
Supernatant, washes three times with PBS；Make blood smear respectively, position with Laser Scanning Confocal Microscope.Red
CMXROS: excitation wavelength is 579nm, a length of 599nm of transmitted wave；IIA: excitation wavelength is 480nm,
The a length of 530nm of transmitted wave.Result is as shown in Figure 2.
By Fig. 2 finding, in the erythrocyte of severe infections, hemoglobin degeneration, it is degraded into black precipitate,
I.e. malarial pigment, this is that naked eyes distinguish normocyte and infected erythrocytic rough method.A-F is different
In the visual field, IIA is at intracellular locating effect, and in the erythrocyte substantially containing malarial pigment, cell presents green
Fluorescence, Tracer carries out overlapping discovery simultaneously, and IIA acts only in cell mitochondrial, bright such as starlet.And
In being uninfected by plasmodial erythrocyte, fluorescence signal does not occur, illustrate that the compounds of this invention has well
Cellular level targeting.
Embodiment 6. the compounds of this invention is to the detection of the blood of P. berghei infecting mouse and fluorescence imaging
Kunming mouse (male, 20 ± 2g, purchased from Shanghai Jie Sijie laboratory animal company limited), inoculates Bai Shi
Plasmodium (The 2nd Army Medical College teacher Pan Weiqing give) 200 μ L；Observe mouse infection under an optical microscope
Situation, when infection rate reaches 20%, the method using eyeball to take blood takes blood.Add the compounds of this invention (IIA)
To final concentration of 10 μ g/mL, fluorescent dyeRed CMXROS to final concentration of 500
nM；15min is hatched under the conditions of 37 DEG C；It is centrifuged under the conditions of 1800rpm, 3min, removes supernatant,
Three times are washed with PBS；Make blood smear, position with Laser Scanning Confocal Microscope.
As shown in Figure 3, the plasmodium of visible different growth periods under the A-D difference visual field.As shown in Figure 3A,
Under light field, in erythrocyte, ringlet is i.e. in the plasmodium in ring bodies stage；As shown in Fig. 3 B or 3C, red under light field
The intracellular big trophozoite with melanin great circle i.e. late period, the wherein the least trophozoite of point.Different growth weeks
Although the plasmodium form of phase, volume differ, but IIA can carry out selected marker, and fluorescence intensity to it
Certain positive correlation is become with plasmodium form, volume.In big trophozoite, fluorescence is bright and big, and in little trophozoite
Fluorescence such as starlet.Moreover, normocyte does not the most show fluorescence, and high specificity, background noise are extremely low.
Comparative example
The present inventor has synthesized the multiple compounds comprising other linking arm further, when utilizing these compounds
When repeating above example 4-6, transmitting these compounds maybe cannot be in conjunction with PfDHODH, or cannot be with external
That cultivates is carried out fluorescence imaging by Infected With Plasmodium erythrocyte, or cannot enter with the mouse blood of Infected With Plasmodium
Row fluorescence imaging.
It has been recognised by the inventors that due to the present invention by pigment group through linking arm and dihydroorate dehydrogenase specificity
Part combines, and whether the final compound obtained can have concurrently is tied with dihydroorate dehydrogenase specificity
The function closing and producing fluorescence signal cannot rationally be predicted；In other words, pigment in the compounds of this invention
May interact between group, linking arm and dihydroorate dehydrogenase specific binding ligand, thus
Negatively affect final gained compound and the binding specificity of dihydroorate dehydrogenase or pigment group
Produce the ability of fluorescence.
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each document
It is individually recited as with reference to like that.In addition, it is to be understood that after the above-mentioned teachings having read the present invention,
The present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values fall within this Shen equally
Please appended claims limited range.
Claims (10)
1. compound shown in Formulas I:
A-L-R
I
In formula,
A is pigment group；
L is linking arm；
R is dihydroorate dehydrogenase specific binding ligand.
2. compound as claimed in claim 1, it is characterised in that described compound is as shown in Formula Il:
In formula,
R1For unsubstituted or be selected from the substituent group substituted condensed ring group of lower group: C1-C5 alkyl, halogen,
Carboxyl, ester group, amino；
R2It is selected from: C1-C6 alkyl, C1-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl, C1-C6 fluoroalkane
Base carbonyl, C1-C6 alkoxyl, C1-C6 fluoroalkyl, optionally substituted benzoyl, amino carbonyl,
C1-C6 alkoxy carbonyl, C1-C6 amino carbonyl, hydroxyl, C1-C6 alkoxyl, C1-C6 ester group；
R3Selected from O, NH or S；
R4Selected from H, C1-C6 alkyl, optionally substituted C2-C6 unsaturated alkyl, C1-C6 alkyl-carbonyl,
Optionally substituted benzoyl, carboxyl, amino carbonyl, C1-C6 alkoxy carbonyl, C1-C6 amino carbonyl,
Hydroxyl, C1-C6 alkoxyl；
R5Selected from O, S, NH and CH2。
3. compound as claimed in claim 2, it is characterised in that R1For the condensed ring group containing 2-3 phenyl ring,
Preferably, R1For naphthyl.
4. compound as claimed in claim 2 or claim 3, it is characterised in that L is
(HNCH2CH2OCH2CH2OOC)。
5. compound as claimed in claim 4, it is characterised in that described compound is shown below:
6. a diagnostic kit for the disease of dihydroorate dehydrogenase mediation, described test kit is equipped with right
Require the compound according to any one of 1-5.
7. diagnostic kit as claimed in claim 6, it is characterised in that described disease is pernicious malaria.
8. the compound according to any one of claim 1-5 is preparing dihydroorate dehydrogenase detection or fixed
Purposes in the diagnostic reagent of the disease of position reagent dihydroorate dehydrogenase mediation.
9. purposes as claimed in claim 8, it is characterised in that described disease is pernicious malaria.
10. a pharmaceutical composition, it is characterised in that described pharmaceutical composition comprises:
Compound according to any one of claim 1-5 or its pharmaceutically acceptable salt, and
Pharmaceutically acceptable carrier or excipient.
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