WO2016197959A1 - 检测二氢乳清酸脱氢酶的化合物 - Google Patents

检测二氢乳清酸脱氢酶的化合物 Download PDF

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WO2016197959A1
WO2016197959A1 PCT/CN2016/085367 CN2016085367W WO2016197959A1 WO 2016197959 A1 WO2016197959 A1 WO 2016197959A1 CN 2016085367 W CN2016085367 W CN 2016085367W WO 2016197959 A1 WO2016197959 A1 WO 2016197959A1
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group
compound
dihydroorotate dehydrogenase
alkyl
formula
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徐玉芳
李洪林
赵振江
朱丽丽
许鸣豪
吴方舒
朱俊生
张晨
李诗良
陈卓
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East China University of Science and Technology
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4245—Oxadiazoles
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D413/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms
    • C07D413/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings
    • C07D413/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and oxygen atoms as the only ring hetero atoms containing two hetero rings linked by a chain containing hetero atoms as chain links
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D271/00—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms
    • C07D271/12—Heterocyclic compounds containing five-membered rings having two nitrogen atoms and one oxygen atom as the only ring hetero atoms condensed with carbocyclic rings or ring systems
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/531—Production of immunochemical test materials
    • G01N33/532—Production of labelled immunochemicals
    • G01N33/533—Production of labelled immunochemicals with fluorescent label
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the field of chemical synthesis; in particular, the invention relates to compounds for the detection of dihydroorotate dehydrogenase (DHODH).
  • DHODH dihydroorotate dehydrogenase
  • Malaria is a vector-borne infectious disease caused by parasitic Plasmodium that is a serious threat to human health and is still one of the most serious parasitic diseases in the world. Malaria continues to spread through infected female Anopheles mosquitoes and healthy people. There are four pathogenic Plasmodium species that can infect humans and cause malaria, Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, and Plasmodium vivax. ). Among them, the plague malaria caused by Plasmodium falciparum is rapid, the disease is strong and the mortality rate is extremely high.
  • Pyrimidine synthesis in organisms is closely related to many life activities. Pyrimidine synthesis provides the raw material for DNA and RNA production, and DNA and RNA are indispensable for cell growth and division.
  • DNA and RNA replication in vivo depends only on the pyrimidine source synthesis pathway, while Plasmodium falciparum dihydroorotate dehydrogenase (PfDHODH) is a key enzyme in the synthetic pathway of the Plasmodium pyrimidine source (Wu, T.;S.Nagle, A.; K. Chatterjee, A. Road Towards New Antimalarials-Overview of the Strategies and their Chemical Progress. Current Medicinal Chemistry.
  • the invention provides a compound of formula I:
  • A is a pigment group
  • L is a connecting arm
  • R is a dihydroorotate dehydrogenase-specific binding ligand.
  • A is a dye group having an emission wavelength between 400 nm and 800 nm, including various dyes, (fluorescent) dyes or stains.
  • L comprises a saturated or unsaturated alkyl chain of 2 to 10 carbon atoms, or a saturated or unsaturated alkyl chain containing oxygen, sulfur, nitrogen or a carbonyl, ester, or amide group.
  • R comprises an inhibitor, antagonist, activator, antibody, and the like of dihydroorotate dehydrogenase.
  • L is selected from the group consisting of an optionally substituted C2-C10 alkyl group, an optionally substituted C4-C10 olefin-containing alkyl group, an optionally substituted C4-C10 alkyne-containing alkyl group, and a C4-C10 oxygen-containing ether.
  • a chain such as -(CH 2 CH 2 (OCH 2 CH 2 ) n OCH 2 CH 2 )- or -(O(CH 2 ) n O(CH 2 ) n NH(CH 2 ) n )-, an ester-containing chain , such as -(CH 2 ) m COO(CH 2 ) p -, containing a carbonyl chain such as -(CH 2 ) m CO(CH 2 ) p -, O(CH 2 ) q O, O(CH 2 ) q NH , NH(CH 2 ) q O, NH(CH 2 ) q NH, OCO(CH 2 ) q COO, OCO(CH 2 ) q O, O(CH 2 ) q COO, NHCO(CH 2 ) q O, O(CH 2 ) q COO, NHCO(CH 2 ) q O, O (CH 2 ) q COO, NHCO(CH
  • A is selected from the group consisting of chromophores containing a suitable conjugated system, such as naphthalimide, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD) ), fluorescein, rhodamine, coumarin, fluoroboron fluorescent dye (BODIPY), Nile Red, Nile Blue, cyanine, phthalocyanine, phthalocyanine dye, multiple methyl dye, triaryl methine dye, Azo dyes and azo pigments, indigo dyes, aza [18] olefin dyes, nitro and nitroso dyes, anthraquinone carbonyl dyes.
  • a suitable conjugated system such as naphthalimide, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD) ), fluorescein, rhodamine, coumarin, fluoroboron fluorescent dye (BODIPY), Ni
  • the R moiety is a five-membered ring comprising a five-membered ring core structure DHODH inhibitor comprising O, S heteroatoms.
  • the compound is as shown in Formula II below:
  • R 1 is a fused ring group which is unsubstituted or substituted with a substituent selected from the group consisting of C1-C5 alkyl, halogen, carboxyl, ester, amino;
  • R 2 is selected from the group consisting of C1-C6 alkyl, C1-C6 unsaturated hydrocarbon, C1-C6 alkylcarbonyl, C1-C6 fluoroalkylcarbonyl, C1-C6 alkoxy, C1-C6 fluoroalkoxy, Optionally substituted benzoyl, aminocarbonyl, C1-C6 alkoxycarbonyl, C1-C6 aminocarbonyl, hydroxy, C1-C6 alkoxy, C1-C6 ester;
  • R 3 is selected from O, NH or S
  • R 4 is selected from H, C1-C6 alkyl, optionally substituted C2-C6 unsaturated hydrocarbon group, C1-C6 alkylcarbonyl group, optionally substituted benzoyl group, carboxyl group, aminocarbonyl group, C1-C6 alkoxycarbonyl group , C1-C6 aminocarbonyl, hydroxy, C1-C6 alkoxy;
  • R 5 is selected from the group consisting of O, S, NH and CH 2 .
  • R 1 is a fused ring group containing 2-3 benzene rings, preferably R 1 is a naphthyl group.
  • the pigment is selected from the group consisting of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein.
  • L is (HNCH 2 CH 2 OCH 2 CH 2 OOC) or (OCH 2 CH 2 OCH 2 CH 2 NHCH 2 ).
  • the compound is as follows:
  • the present invention provides a diagnostic kit for a dihydroorotate dehydrogenase-mediated disease, the kit comprising the compound of the first aspect of the invention.
  • the dihydroorotate dehydrogenase mediated diseases include falciparum malaria, vivax malaria, ovarian malaria, three-day malaria, monkey malaria, Trypanosoma cruzi, schistosomiasis, dengue fever Parasitic infections; and rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus, glomerular disease, melanoma, and host rejection caused by allogeneic or xenogeneic organ transplantation.
  • the disease is falciparum malaria.
  • the present invention provides the use of a compound according to the first aspect of the invention for the preparation of a diagnostic reagent for the dihydroorotate dehydrogenase detection or localization reagent dihydroorotate dehydrogenase mediated disease.
  • the dihydroorotate dehydrogenase mediated diseases include falciparum malaria, vivax malaria, Parasitic infections such as ovarian malaria, three-day malaria, monkey malaria, Trypanosoma cruzi, schistosomiasis, and dengue fever; and rheumatoid arthritis, colitis, psoriatic arthritis, lupus erythematosus, glomerular disease , melanoma and host rejection caused by allogeneic or xenogeneic organ transplantation.
  • Parasitic infections such as ovarian malaria, three-day malaria, monkey malaria, Trypanosoma cruzi, schistosomiasis, and dengue fever
  • rheumatoid arthritis colitis
  • psoriatic arthritis lupus erythematosus
  • glomerular disease melanoma
  • host rejection caused by allogeneic or xenogeneic organ transplantation.
  • the disease is falciparum malaria.
  • the present invention provides a pharmaceutical composition comprising:
  • a pharmaceutically acceptable carrier or excipient is provided.
  • Figure 1 shows the binding of the compound IIA (DH03A430) of the present invention to the PfDHODH protein
  • Figure 2 shows a fluorescent imaging image of the compound IIA (DH03A430) of the present invention and the erythrocytes infected with Plasmodium in vitro;
  • Figure 3 shows the detection and fluorescence imaging images of the blood of the compound IIA (DH03A430) of the present invention and mice infected with Plasmodium berghei.
  • Figure 4 shows the confocal experiment (in vitro culture) of the compound of the present invention and the 3D7 strain of Plasmodium falciparum (newly synthesized probe, fluorescence excited at 460 nm, emission at 520 nm; Mito Tracker: purchased mitochondrial dye, excited at 579 nm, 599 nm The fluorescence produced by the emission; Merge: the superposition of the fluorescence produced by the probe and the Mito Tracker dye; Bright field: bright field).
  • Figure 5 shows in vitro incubation of a compound of the invention with Plasmodium berghei in a confocal experiment (mouse) (fluorescence produced at 460 nm excitation, 520 nm emission; Mito Tracker: purchased mitochondrial dye, excited at 579 nm, fluorescence generated by 599 nm emission; Merge: superposition of the fluorescence produced by the probe and the Mito Tracker dye; Bright field: bright field).
  • alkyl refers to a saturated branched or straight-chain alkyl group having a carbon chain length of from 1 to 10 carbon atoms, and preferred alkyl groups include from 2 to 8 carbon atoms, from 1 to 6, and from 1 to 4 carbon atoms.
  • alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, heptyl, and the like.
  • the alkyl group may be substituted by one or more substituents, for example by halogen or haloalkyl.
  • the alkyl group may be an alkyl group substituted with 1 to 4 fluorine atoms, or the alkyl group may be an alkyl group substituted with a fluoroalkyl group.
  • ester group means a group represented by -COOR x wherein R x is an alkyl group as defined above.
  • aryl refers to a monocyclic, bicyclic or tricyclic aromatic group containing from 6 to 14 carbon atoms, including phenyl, naphthyl, phenanthryl, anthryl, fluorenyl, fluorenyl, tetrahydronaphthalene. Base, indanyl group, and the like.
  • the aryl group may be optionally substituted with from 1 to 5 (e.g., 1, 2, 3, 4 or 5) substituents selected from the group consisting of halogen, C1-C4 aldehyde group, C1-C6 linear or branched chain.
  • halogen substituted alkyl eg trifluoromethyl
  • halogen substituted alkoxy eg trifluoromethoxy
  • carboxyl C1-C4 Alkoxy
  • C1-C4 substituted indenyl morpholinyl
  • optionally substituted aryl eg, optionally substituted phenyl
  • optionally substituted aryloxy eg, optionally substituted phenoxy
  • an aryl group may be substituted with from 1 to 3 groups selected from the group consisting of fluorine, chlorine, bromine, C1-C4 alkyl, trifluoromethyl, morpholinyl, methoxy, phenyl, methoxy substituted Phenyl, phenoxy, benzyloxy, benzyloxy substituted by halogen, ethoxy, nitro, and the like.
  • heterocyclyl refers to a single or fused ring structure, which may be aromatic or non-aromatic in nature, and which preferably contains from 3 to 20 ring atoms, more preferably from 5 to 14
  • One or more ring atoms, at least one and preferably up to 4, are heteroatoms selected from the group consisting of O, S and N.
  • examples of the heterocyclic group include furyl, thienyl, pyrrolyl, pyrrolidinyl, imidazolyl, triazolyl, thiazolyl, tetrazolyl, oxazolyl, isoxazolyl, pyrazolyl, pyridine , pyrimidinyl, pyrazinyl, pyridazinyl, triazinyl, quinolinyl, isoquinolinyl, quinoxalinyl, benzothiazolyl, benzoxazolyl, benzothienyl, benzofuran Base, morpholinyl, oxazolyl, dibenzothiophene, coumarinyl and 1,2-methylenedioxyphenyl.
  • a heterocyclic group may be optionally substituted with from 1 to 3 substituents as described herein.
  • heteroatom as used herein includes O, S and N.
  • the hetero atom is N, the N atom may be further substituted with a group such as hydrogen or a C1-C10 alkyl group.
  • the hetero atom is S, the S atom may be further substituted with a group such as a C1-C10 alkyl group.
  • heteroaryl or “aromatic heterocyclic” as used herein, refers to those heterocyclic groups having aromatic character as described above, including but not limited to furyl, thienyl, pyrrolyl, pyridyl, oxazolyl. , pyrazinyl, pyridazinyl, pyrimidinyl and the like.
  • halogen as used herein includes fluoro, chloro, bromo and iodo.
  • optionally substituted means that the group to which it is modified may be optionally substituted with from 1 to 5 (typically 1, 2 or 3) substituents selected from the group consisting of: C1-C4 alkyl, carboxyl, halogen, C1-C4 alkoxy, cyano, nitro, amino, hydroxy, aldehyde, C1-C6 acyl, hydroxymethyl, halogen-substituted C1-C4 alkyl (eg three Fluoromethyl), halogen-substituted C1-C4 alkoxy (e.g., trifluoromethoxy), fluorenyl, and C1-C4 acyl.
  • substituents selected from the group consisting of: C1-C4 alkyl, carboxyl, halogen, C1-C4 alkoxy, cyano, nitro, amino, hydroxy, aldehyde, C1-C6 acyl, hydroxymethyl, halogen-substituted C1-
  • the amide group (aminocarbonyl group) itself or as part of another group means a "C1-C6 alkyl-CO-NH-" group, "C3-C8 cycloalkyl-CO-NH-" or "C3" -C8 cycloalkyl-C1-C6 alkyl-CO-NH-”.
  • exemplary Amide groups include, but are not limited to, formamide groups, acetamido groups, propionamide groups, butanamide groups, cyclopropionamido groups, cyclopropionamide groups, and the like.
  • the acyl group itself may contain, as a part of other groups, 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms.
  • exemplary acyl groups include, but are not limited to, acetyl, fluoroacetyl, fluoropropionyl, fluorobutyryl, and the like.
  • the present invention contemplates a class of fluorescent compounds that can be used to specifically detect PfDHODH at the cellular and in vivo levels.
  • Pharmacological test results indicate that the compound of the present invention not only has a certain inhibitory activity against Plasmodium falciparum at the cellular level, but also has affinity for PfDHODH, and it also specifically recognizes the marker DHODH in erythrocytes infected with Plasmodium and selectively localizes to mitochondria. in.
  • Blood tests by mice infected with Plasmodium berghei demonstrated that the compounds of the present invention can accurately label and trace the malaria parasites in red blood cells, and accurately discriminate the growth morphology of the malaria parasites at different stages, and quantify and simplify the detection process of the malaria parasite.
  • the design and discovery of these novel DHODH-labeled compounds will provide detailed and powerful evidence for the early diagnosis of malaria and the life cycle and pathogenesis of the malaria parasite. It can also be used for the early diagnosis and prevention of malaria.
  • the invention provides a compound of formula I:
  • A is a dye group, preferably a dye group having an emission wavelength of from 400 nm to 800 nm, including various dyes, (fluorescent) dyes or dyes;
  • L is a linking arm comprising a saturated or unsaturated alkyl chain of 2 to 10 carbon atoms, or a saturated or unsaturated alkyl chain containing oxygen, sulfur, nitrogen or a carbonyl group, an ester group or an amide group;
  • R is a dihydroorotate dehydrogenase-specific binding ligand, including inhibitors, antagonists, activators, antibodies, and the like of dihydroorotate dehydrogenase.
  • one of skill in the art can employ any suitable dye group, tether, and dihydroorotate dehydrogenase to specifically bind the ligand.
  • L is selected from the group consisting of an optionally substituted C2-C10 alkyl group, an optionally substituted C4-C10 olefin-containing alkyl group, an optionally substituted C4-C10 alkyne-containing alkyl group, and a C4-C10 oxygen-containing ether.
  • a chain such as -(CH 2 CH 2 (OCH 2 CH 2 ) n OCH 2 CH 2 )- or -(OCH 2 CH 2 OCH 2 CH 2 NHCH 2 )-, an ester-containing chain such as -(CH 2 ) m COO(CH 2 ) p -, containing a carbonyl chain such as -(CH 2 ) m CO(CH 2 ) p -, O(CH 2 ) q O, O(CH 2 ) q NH, NH(CH 2 ) q O , NH(CH 2 ) q NH, OCO(CH 2 ) q COO, OCO(CH 2 ) q O, O(CH 2 ) q COO, NHCO(CH 2 ) q O, O(CH 2 ) q CONH, NHCO (CH 2 ) q CONH, S(CH 2 ) q O, S(CH 2 ) q S, NH(CH 2 )
  • A is selected from chromophores containing a suitable conjugated system, such as naphthalimide, 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD), fluorescein, rhodamine, coumarin, fluoroboron fluorescent dye (BODIPY), Nile Red, Nile Blue, cyanine, phthalocyanine, phthalocyanine dye, multiple methyl dye, triaryl methine Dyes, azo dyes and azo pigments, indigo dyes, aza [18] olefin dyes, nitro and nitroso dyes, anthraquinone carbonyl dyes.
  • A is selected from the group consisting of 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD) or fluorescein.
  • the R moiety is a five-membered ring comprising a five-membered ring core structure DHODH inhibitor containing O, S heteroatoms.
  • R 1 is a fused ring group which is unsubstituted or substituted with a substituent selected from the group consisting of a C1-C5 alkyl group, a halogen, a carboxyl group, an ester group, an amino group, and R 1 is preferably a thick 2-3 ring.
  • R 2 is selected from the group consisting of: C1-C6 alkyl, C1-C6 unsaturated hydrocarbon group, C1-C6 alkylcarbonyl group, C1-C6 fluoroalkylcarbonyl group, C1-C6 alkoxy group , C1-C6 fluoroalkoxy, optionally substituted benzoyl, aminocarbonyl, C1-C6 alkoxycarbonyl, C1-C6 aminocarbonyl, hydroxy, C1-C6 alkoxy, C1-C6 ester; R 3 is selected from O, NH or S; R 4 is selected from H, C1-C6 alkyl, optionally substituted C2-C6 unsaturated hydrocarbon, C1-C6 alkylcarbonyl, optionally substituted benzoyl, carboxy, aminocarbonyl, C1-C6 alkoxycarbonyl group, C1-C6 aminocarbonyl group,
  • attachment arms of various configurations, such as (HNCH 2 CH 2 OCH 2 CH 2 OOC) or (OCH 2 CH 2 OCH 2 CH 2 NHCH 2 ).
  • the invention provides the following compounds:
  • the absorption and emission wavelengths of the compounds of the present invention cover all wavelength ranges and have substantially no background fluorescence interference, and can be used as a fluorescent probe for detecting DHODH;
  • the compound of the present invention can specifically bind to dihydroorotate dehydrogenase and generate a fluorescent signal.
  • the compound ZC-Y-3 (327 mg, 1 mmol) was placed in a 25 ml round bottom flask, 5 ml of anhydrous dichloromethane was added, and 1 ml of thionyl chloride was added dropwise under an argon-protected ice bath, and the mixture was stirred for 5 min.
  • the solution has a colorless dark green color, and the TLC is traced to the reaction of the starting material.
  • the mixture is dried, and extracted with dichloromethane and saturated sodium hydrogen carbonate solution.
  • Example 4 Inhibitory activity of the compound of the present invention on a cell strain
  • Chloroquine was purchased from Sigma, SYBR Green I was purchased from Life technologies, chloroquine-sensitive 3D7 cell line and chloroquine-resistant Dd2 cell line were used to test antimalarial activity in vitro, by Trager and Jensen culture method (Human malaria parasites in continuous Culture. Science. 1976, 193 (4254): 673-675) was cultured in a medium containing 0.5% Albumi II non-human serum. Anti-Plasmodium cell line activity was determined by fluorescence titration using SYBR Green I as a probe.
  • the parasites in different periods were in triplicate, cultured in 2% red blood cells and in the presence or absence of 100 ⁇ L of 1% parasitemia and a series of drugs, each compound concentration 0.15625 was changed to 20 ⁇ M. 0.2% DMSO was used as a back control and different concentrations of chloroquine were used as positive controls.
  • the culture dish was incubated at 37 ° C for 3 days, then the supernatant was removed, and 100 ⁇ L of SYBR Green I cell buffer (8.26 g/L NH 4 Cl, 1 g/L KHCO 3 , 0.037 g/L EDTA and 5 ⁇ ) was added. SYBR Green I).
  • the culture dish was further cultured for 1 hour at room temperature in a dark room, and the experiment was repeated 2 times by Synergy MX, Biotek fluorescence detector at 485/520 nm. Data analysis was performed according to the method reported by Michael et al. Briefly, the fluorescence data obtained from the negative control treatment group minus the background fluorescence data generated by the uninfected red blood cell group represents the maximum amount of DNA in the normal cultured Plasmodium falciparum in this experiment, and is recorded as the fluorescence data of the negative control well. The fluorescence data of each test group and positive control group were corrected as such.
  • inhibition rate (%) [1 - (average fluorescence count in test well / average fluorescence count in negative control well)] x 100%.
  • the curves were fitted by the Growth/Sigmoidal program in the software Origin 8.0 and calculated to obtain the half-inhibition rate (IC 50 values), and the mean and standard deviation were calculated by Microsoft Excel.
  • Example 5 Binding of a compound of the invention to a target protein
  • the fragment containing the PfDHODH gene was cloned into the PET-19b vector to obtain a recombinant plasmid, which was transformed into E. coli BL21 for expression, and purified by immobilized metal ion affinity chromatography and gel filtration chromatography to obtain a relatively pure protein.
  • the purified PfDHODH protein was diluted to a final concentration of 2 mg/mL, divided into two tubes, 500 ⁇ L per tube, one tube plus 25 ⁇ L of the mother liquor was 50 mM of the compound of the invention (IIA), and the other tube was added with 25 ⁇ L of DMSO as a control, on ice.
  • the protein was dispensed by PCR tube, and the protein of the compound and DMSO was separately dispensed into 12 tubes, 30 ⁇ L per tube, and the PCR apparatus was used at different temperatures (37 ° C, 40 ° C, 43 ° C, 46 ° C, 49 ° C, 52 ° C, 55 ° C, 58 ° C, 61 ° C, 64 ° C, 67 ° C, 70 ° C) respectively heated for 3 min, the compound and the control were treated the same, after heating, 4 ° C high-speed centrifugation for half an hour, respectively, the supernatant was taken 2 ⁇ L, The Buffer was diluted to 100 ⁇ L to prepare a protein sample for western blot, and finally developed by a chemiluminescence imaging system, and the results are shown in Fig. 1.
  • the compound of the invention (IIA, IIC) was dissolved in DMSO to 50 mM.
  • a certain amount of Plasmodium falciparum was cultured in a 37 ° C incubator (Mr. Zhou Hongchang, Huzhou Teachers College), divided into two dishes, labeled A and B; the Plasmodium falciparum in A and B dishes was separated by 5% sorbitol for one day.
  • culture for two days add the compound of the present invention to the A and B culture dishes to a final concentration of 10 ⁇ g/mL, and incubate for 3.5 hours in a 37 ° C incubator, and then add fluorescent dye to the A and B culture dishes.
  • Red CMXROS (purchased from Life Technologies) to a final concentration of 500 nM, and cultured in a 37 ° C incubator for 30 min; transfer the red blood cells infected with Plasmodium falciparum in two culture dishes to a 15 mL centrifuge tube, and centrifuge at 1800 rpm for 3 min. The supernatant was removed and washed three times with PBS; blood smears were separately prepared and positioned using a confocal microscope.
  • Red CMXROS excitation wavelength is 579 nm, emission wavelength is 599 nm
  • IIA excitation wavelength is 480 nm
  • emission wavelength is 530 nm. The result is shown in Figure 2.
  • Fig. 2 in severely infected red blood cells, hemoglobin is denatured and degraded into a black precipitate, that is, malaria pigment, which is a rough method for distinguishing normal red blood cells and infected red blood cells by the naked eye.
  • A-F is the localization effect of IIA in different fields of view. In red blood cells containing malaria pigment, the cells show green fluorescence, and Tracer is superimposed. IIA acts only on the mitochondria of cells, and is bright as a small star. In the red blood cells not infected with Plasmodium, no fluorescent signal appeared, indicating that the compound of the present invention has good cell-level targeting.
  • Example 7 Detection of blood of a compound of the present invention against mice infected with Plasmodium berghei and fluorescence imaging
  • Kunming mice male, 20 ⁇ 2g, purchased from Shanghai Jiesijie Experimental Animal Co., Ltd.), inoculated with 200 ⁇ L of Plasmodium berghei (a gift from Teacher Pan Weiqing of the Second Military Medical University); observe the infection of mice under light microscope. When the infection rate reaches 20%, blood is taken by eyeballs.
  • fluorescent dye Red CMXROS was weighed to a final concentration of 500 nM; incubated at 37 ° C for 15 min; centrifuged at 1800 rpm for 3 min, the supernatant was removed, and washed three times with PBS; blood smears were prepared and localized using a confocal microscope.
  • Plasmodium with different growth cycles can be seen in different fields of view of A-D.
  • the small circle in the red blood cell is the parasite in the ring stage; as shown in Fig. 3B or 3C, the red blood cells in the bright field have large melanin circles, that is, late large trophozoites, among which small Point is a small trophozoite.
  • IIA can selectively label it, and the fluorescence intensity is positively correlated with the morphology and volume of Plasmodium. Fluorescence is large in large trophozoites, and fluorescence is small in small trophozoites. Not only that, normal red blood cells are completely non-fluorescent, with strong specificity and extremely low background signal noise.
  • IIC can also selectively label cells infected with Plasmodium berghei and Plasmodium falciparum.
  • the present inventors further synthesized a plurality of compounds including other tethers, and when these compounds 5-5 were repeated using these compounds, it was found that these compounds could not bind to PfDHODH, or could not be fluorescently imaged with Plasmodium-infected red blood cells cultured in vitro. Fluorescence imaging of blood from mice infected with Plasmodium.
  • the present invention combines a dye group with a dihydroorotate dehydrogenase-specific ligand via a tether, the resulting compound can specifically bind to dihydroorotate dehydrogenase.
  • the function of generating a fluorescent signal is not reasonably predictable; in other words, the pigment group, the tether and the dihydroorotate dehydrogenase-specific binding ligand in the compound of the present invention may interact with each other, thereby adversely affecting the final The binding specificity of the resulting compound to dihydroorotate dehydrogenase or the ability of the pigment group to produce fluorescence.

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Abstract

本发明涉及用于检测二氢乳清酸脱氢酶的式I所示化合物，式中，A为色素基团，L为连接臂，R为二氢乳清酸脱氢酶的特异性结合配体。本发明的化合物可用于检测和定位二氢乳清酸脱氢酶，从而可用于诊断二氢乳清酸脱氢酶介导的相关疾病。

Description

检测二氢乳清酸脱氢酶的化合物 技术领域
本发明涉及化学合成领域；具体地说，本发明涉及用于检测二氢乳清酸脱氢酶(DHODH)的化合物。
背景技术
疟疾(Malaria)是由寄生性疟原虫所引起的严重危害人类健康的虫媒介传染病，至今依然是世界上最严重的寄生虫病之一。疟疾通过雌性按蚊叮咬被感染者和健康人群持续传播。能感染人类并导致疟疾的致病疟原虫有四种，分别是恶性疟原虫(Plasmodium falciparum)、卵形疟原虫(Plasmodium ovale)、三日疟原虫(Plasmodium malariae)和间日疟原虫(Plasmodium vivax)。其中，恶性疟原虫所导致的凶险型疟疾发病迅速，病症强烈且致死率极高。
生物体内的嘧啶合成与许多生命活动息息相关，嘧啶合成为DNA和RNA的生成提供原料，而DNA和RNA对于细胞的生长分裂是不可或缺的。就恶性疟原虫而言，其体内DNA和RNA复制只依赖于嘧啶源合成途径，而恶性疟原虫二氢乳清酸脱氢酶(PfDHODH)是疟原虫嘧啶源合成途径中的关键酶(Wu,T.；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)，对PfDHODH进行荧光检测对疟原虫的定位示踪及疟疾的早期预警、诊断有着极其重要的科学价值和研究意义(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)。
长期以来，疟原虫的生长周期、耐药机理研究尚不透彻，同时现有的疟疾快速诊断方法如镜检染色法、抗体抗原检测(ELISA)和聚合酶链式反应(PCR)等，这些方法缺点较多，主观偏差大、耗时长、成本高、操作复杂严重依赖仪器(Wilson,M.L.Malaria Rapid Diagnostic Tests.Clinical Infectious Diseases,2012,54(11):1637-1641)。化学发光检测手段简便、灵敏、快捷但利用化学探针识别定位疟原虫及研究其生命史和机理目前尚未有报道。
因此，本领域急需能够简便、灵敏、快捷地检测或定位疟原虫，从而用于研究其生命史和机理的物质手段和方法。
发明内容
本发明的目的在于提供一种简便、灵敏、快捷地检测或定位疟原虫的探针化合物以及利用所述化合物研究疟原虫的生命史和机理的方法。
在第一方面，本发明提供式I所示化合物：
                              A-L-R
                                    I
式中，
A为色素基团；
L为连接臂；
R为二氢乳清酸脱氢酶特异性结合配体。
在优选的实施方式中，A是发射波长介于400nm-800nm的色素基团，包括各种染料、(荧光)染料或染色剂。
在优选的实施方式中，L包括2-10个碳原子的饱和或不饱和烷基链，或含氧、含硫、含氮或含羰基、酯基、酰胺基的饱和或不饱和烷基链。
在优选的实施方式中，R包括二氢乳清酸脱氢酶的抑制剂、拮抗剂、激活剂、抗体等。
在优选的实施方式中，L选自任选取代的C2-C10烷基、任选取代的C4-C10含烯烷基、任选取代的C4-C10含炔烷基、C4-C10含氧醚链，如-(CH2CH2(OCH2CH2)nOCH2CH2)-或-(O(CH2)nO(CH2)nNH(CH2)n)-，含酯基链，如-(CH2)mCOO(CH2)p-，含羰基链，如-(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为独立选自0-2的整数，m为独立选自1-5的整数，p为独立选自1-5的整数，q为独立选自1-10的整数；
在优选的实施方式中，A选自含合适共轭体系的发色团，如萘酰亚胺、4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD)、荧光素、罗丹明、香豆素、氟硼荧光染料(BODIPY)、尼罗红、尼罗蓝、花菁、酞菁、噁菁染料、多次甲基染料、三芳次甲基染料、偶氮染料及偶氮颜料、靛蓝类染料、氮杂[18]轮烯染料、硝基及亚硝基染料、醌类羰基染料。
在优选的实施方式中，R部分是五元环，包括含O、S杂原子的五元环母核结构DHODH抑制剂。
在具体的实施方式中，所述化合物如下式II所示：
Figure PCTCN2016085367-appb-000001
式中，
R1为未取代的或被选自下组的取代基取代的稠环基团：C1-C5烷基、卤素、羧基、酯基、氨基；
R2选自：C1-C6烷基，C1-C6不饱和烃基，C1-C6烷基羰基，C1-C6氟代烷基羰基， C1-C6烷氧基，C1-C6氟代烷氧基，任选取代的苯甲酰基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基、C1-C6酯基；
R3选自O、NH或S；
R4选自H，C1-C6烷基，任选取代的C2-C6不饱和烃基，C1-C6烷基羰基，任选取代的苯甲酰基，羧基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基；
R5选自O，S，NH和CH2。
在具体的实施方式中，R1为含2-3个苯环的稠环基团，优选地，R1为萘基。
在具体的实施方式中，所述色素选自4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD)或荧光素。
在具体的实施方式中，L为(HNCH2CH2OCH2CH2OOC)或(OCH2CH2OCH2CH2NHCH2)。
在具体的实施方式中，所述化合物如下式所示：
Figure PCTCN2016085367-appb-000002
在第二方面，本发明提供一种二氢乳清酸脱氢酶介导的疾病的诊断试剂盒，所述试剂盒装有本发明第一方面所述的化合物。
在优选的实施方式中，所述二氢乳清酸脱氢酶介导的疾病包括恶性疟疾、间日疟、卵形疟疾、三日疟、猴型疟、克氏锥虫、血吸虫病、登革热等寄生虫传染病；以及类风湿性关节炎、结肠炎、银屑病关节炎、红斑狼疮、肾小球疾病、黑色素瘤和同种或异种器官移植引起的宿主排斥反应。
在具体的实施方式中，所述疾病是恶性疟疾。
在第三方面，本发明提供本发明第一方面所述的化合物在制备二氢乳清酸脱氢酶检测或定位试剂二氢乳清酸脱氢酶介导的疾病的诊断试剂中的用途。
在优选的实施方式中，所述二氢乳清酸脱氢酶介导的疾病包括恶性疟疾、间日疟、 卵形疟疾、三日疟、猴型疟、克氏锥虫、血吸虫病、登革热等寄生虫传染病；以及类风湿性关节炎、结肠炎、银屑病关节炎、红斑狼疮、肾小球疾病、黑色素瘤和同种或异种器官移植引起的宿主排斥反应。
在具体的实施方式中，所述疾病是恶性疟疾。
在第四方面，本发明提供一种药物组合物，所述药物组合物包含：
本发明第一方面所述的化合物或其药学上可接受的盐，和
药学上可接受的载体或赋形剂。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1显示了本发明的化合物IIA(DH03A430)与PfDHODH蛋白的结合情况；
图2显示了本发明化合物IIA(DH03A430)与体外培养的受疟原虫感染红细胞的荧光成像图像；
图3显示了本发明化合物IIA(DH03A430)与伯氏疟原虫感染小鼠的血液的检测以及荧光成像图像。
图4显示了本发明化合物与3D7株恶性疟原虫共聚焦实验(体外培养)(新合成的探针，在460nm激发，520nm发射产生的荧光；Mito Tracker:购买的线粒体染料，在579nm激发，599nm发射产生的荧光；Merge:探针和Mito Tracker染料产生的荧光的叠合；Bright field:明场)。
图5显示了本发明化合物与伯氏疟原虫体外孵育共聚焦实验(小鼠)(在460nm激发，520nm发射产生的荧光；Mito Tracker:购买的线粒体染料，在579nm激发，599nm发射产生的荧光；Merge:探针和Mito Tracker染料产生的荧光的叠合；Bright field:明场)。
具体实施方式
发明人经过广泛而深入的研究，出乎意料地发现了一类可用于在细胞及活体水平中特异性检测PfDHODH的荧光化合物，这些化合物不仅在细胞水平对恶性疟原虫有一定抑制活性，它们还能特异性识别并标记感染疟原虫的红细胞中的DHODH。在此基础上完成了本发明。
术语定义
本文中涉及到的一些基团定义如下：
本文中，“烷基”指碳链长度为1-10个碳原子的饱和的支链或直链烷基，优选的烷基包括长2-8个碳原子、1-6个、1-4个碳原子、1-3个碳原子不等的烷基。烷基的例子包括但不限于甲基、乙基、正丙基、异丙基、正丁基、异丁基、庚基等。烷基可以被1个或多个取代基取代，例如被卤素或卤代烷基取代。例如，烷基可以是被1-4个氟原子取代的烷基，或者烷基可以是被氟代烷基取代的烷基。
本文中，“酯基”是指-COORx所示的基团，其中，Rx是如上定义的烷基。
本文中，“芳基”指含有6到14个碳原子的单环、双环或三环芳族基团，包括苯基、萘基、菲基、蒽基、茚基、茀基、四氢化萘基、二氢化茚基等。芳基可任选地被1-5个(例如，1、2、3、4或5个)选自以下的取代基取代：卤素、C1-C4醛基、C1-C6的直链或支链烷基、氰基、硝基、氨基、羟基、羟甲基、卤素取代的烷基(例如三氟甲基)、卤素取代的烷氧基(例如三氟甲氧基)、羧基、C1-C4的烷氧基、C1-C4取代巯基、吗啉基、任选取代的芳基(例如任选取代的苯基)、任选取代的芳氧基(例如任选取代的苯氧基)和任选取代的苄氧基。例如，芳基可以被1-3个选自以下的基团取代：氟、氯、溴、C1-C4烷基、三氟甲基、吗啉基、甲氧基、苯基、甲氧基取代的苯基、苯氧基、苄氧基、被卤素取代的苄氧基、乙氧基和硝基等。
本文所使用的术语“杂环基”指单一或稠合的环结构，在性质上可以是芳族或非芳族的，并且其优选含有3-20个成环原子，更优选含有5-14个环原子，其中至少1个并且优选最多可至4个是选自O、S和N的杂原子。本文中，杂环基的例子包括呋喃基、噻吩基、吡咯基、吡咯烷基、咪唑基、三唑基、噻唑基、四唑基、噁唑基、异噁唑基、吡唑基、吡啶基、嘧啶基、吡嗪基、哒嗪基、三嗪基、喹啉基、异喹啉基、喹喔啉基、苯并噻唑基、苯并噁唑基、苯并噻吩基、苯并呋喃基、吗啉基、咔唑基、二苯并噻吩、香豆素基和1,2-亚甲基二氧苯基。本文中，杂环基可任选地被1-3个本文所述的取代基取代。
本文所使用的术语“杂原子”包括O、S和N。当杂原子是N时，此N原子可以进一步由例如氢或C1-C10烷基的基团所取代。当杂原子是S时，此S原子可以进一步由例如C1-C10烷基的基团所取代。
本文所使用的术语“杂芳基”或“芳香杂环基”指如上所述具有芳族特性的那些杂环基，包括但不限于呋喃基、噻吩基、吡咯基、吡啶基、噁唑基、吡嗪基、哒嗪基、嘧啶基等。
本文所使用的术语“卤素”包括氟、氯、溴和碘。
除非另有说明，本文所使用的术语“任选取代的”指其所修饰的基团可任选地被1-5个(通常为1、2或3个)选自以下的取代基取代：C1-C4烷基、羧基、卤素、C1-C4烷氧基、氰基、硝基、氨基、羟基、醛基、C1-C6酰基、羟甲基、卤素取代的C1-C4烷基(例如三氟甲基)、卤素取代的C1-C4烷氧基(例如三氟甲氧基)、巯基和C1-C4酰基。
本文中，酰胺基(氨基羰基)自身或作为其它基团的一部分，指“C1-C6烷基-CO-NH-”基团、“C3-C8环烷基-CO-NH-”或“C3-C8环烷基-C1-C6烷基-CO-NH-”。示例性的 酰胺基包括但不限于甲酰胺基、乙酰胺基、丙酰胺基、丁酰胺基、环丙酰胺基、环丙甲酰胺基等。
本文中，酰基自身或作为其它基团的一部分，可含有1-6个碳原子，优选1-4个碳原子。示例性的酰基包括但不限于乙酰基、氟代乙酰基、氟代丙酰基、氟代丁酰基等。
本发明化合物
本发明设计了一类可用于在细胞及活体水平中特异性检测PfDHODH的荧光化合物。药理测试结果表明本发明的化合物不仅在细胞水平对恶性疟原虫有一定抑制活性，对PfDHODH具有亲和力，同时其还其能在感染疟原虫的红细胞中特异性识别标记DHODH，并选择性定位于线粒体中。通过伯氏疟原虫感染小鼠的血液检测证明，本发明的化合物可以准确地标记和示踪红细胞中的疟原虫，并且准确辨别不同时期疟原虫的生长形态，量化并简化疟原虫的检测过程。综上，这类新型DHODH标记型化合物的设计和发现将会对于疟疾的早期诊断和疟原虫生命周期、致病机理研究提供详实、有力的证据，也可用于疟疾的早期诊断和预防。
在具体的实施方式中，本发明提供式I所示化合物：
                             A-L-R
                                   I
式中，
A为色素基团，优选发射波长介于400nm-800nm的色素基团，包括各种染料、(荧光)染料或染色剂；
L为连接臂，包括2-10个碳原子的饱和或不饱和烷基链，或含氧、含硫、含氮或含羰基、酯基、酰胺基的饱和或不饱和烷基链；
R为二氢乳清酸脱氢酶特异性结合配体，包括二氢乳清酸脱氢酶的抑制剂、拮抗剂、激活剂、抗体等。
鉴于本发明的教导，本领域技术人员可以采用任何合适的色素基团、连接臂和二氢乳清酸脱氢酶特异性结合配体。
在优选的实施方式中，L选自任选取代的C2-C10烷基、任选取代的C4-C10含烯烷基、任选取代的C4-C10含炔烷基、C4-C10含氧醚链，如-(CH2CH2(OCH2CH2)nOCH2CH2)-或-(OCH2CH2OCH2CH2NHCH2)-，含酯基链，如-(CH2)mCOO(CH2)p-，含羰基链，如-(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为独立选自0-2的整数、m为独立选自1-5的整数、p为独立选自1-5的整数、q为独立选自1-10的整数；
A选自含合适共轭体系的发色团，如萘酰亚胺、4-氯-7-硝基苯并-2-氧杂-1,3-二唑 (NBD)、荧光素、罗丹明、香豆素、氟硼荧光染料(BODIPY)、尼罗红、尼罗蓝、花菁、酞菁、噁菁染料、多次甲基染料、三芳次甲基染料、偶氮染料及偶氮颜料、靛蓝类染料、氮杂[18]轮烯染料、硝基及亚硝基染料、醌类羰基染料。在具体的实施方式中，A选自4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD)或荧光素。
R部分是五元环，包括含O、S杂原子的五元环母核结构DHODH抑制剂。
在进一步的具体实施方式中本发明的化合物如下式II所示：
Figure PCTCN2016085367-appb-000003
式中，
R1为未取代的或被选自下组的取代基取代的稠环基团：C1-C5烷基、卤素、羧基、酯基、氨基，R1优选为含2-3个苯环的稠环基团，更优选为萘基；R2选自：C1-C6烷基，C1-C6不饱和烃基，C1-C6烷基羰基，C1-C6氟代烷基羰基，C1-C6烷氧基，C1-C6氟代烷氧基，任选取代的苯甲酰基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基、C1-C6酯基；R3选自O、NH或S；R4选自H，C1-C6烷基，任选取代的C2-C6不饱和烃基，C1-C6烷基羰基，任选取代的苯甲酰基，羧基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基；R5选自O，S，NH和CH2。
本领域技术人员可以采用各种结构的连接臂，例如(HNCH2CH2OCH2CH2OOC)或(OCH2CH2OCH2CH2NHCH2)所示的连接臂。
在具体的实施方式中，本发明提供以下化合物：
Figure PCTCN2016085367-appb-000004
本发明的优点：
1.本发明化合物的吸收和发射波长涵盖所有波长范围，且基本没有背景荧光干扰，可用作检测DHODH的荧光探针；
2.本发明化合物既可特异性结合二氢乳清酸脱氢酶，又能产生荧光信号。
以下结合具体实施案例对本发明的技术方案进一步描述，但以下实施案例不构成对本发明的限制，所有依据本发明的原理和技术手段采用的各种施用方法，均属于本发明范围。在下列合成实施例中，除非另有说明，否则所用试剂均是从市场上直接购买所得，所用到溶剂使用前均经无水处理，所述的常温是指25℃。下列实施例中未注明具体条件的实验方法，通常按照常规条件，或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数按重量计算。
实施例1.化合物IIA的合成
Figure PCTCN2016085367-appb-000005
中间体A的合成
Figure PCTCN2016085367-appb-000006
氩气保护下，将996mg，5mmol的4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD-Cl)加入到2.5mL的无水DMF中，缓慢滴加630mg，6mmol的2-氨氧基乙醇的2.5mL无水DMF溶液和0.9mL的三乙胺，常温搅拌过夜，减压蒸除DMF，二氯甲烷萃取，饱和食盐水洗涤，有机层浓缩，柱层析(石油醚：乙酸乙酯＝1：2，v/v)得红色固体，收率35％。
1H NMR(400MHz,DMSO-d6):δ9.46(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.
中间体6-氨基甲酸叔丁酯-2-萘甲酸的合成
Figure PCTCN2016085367-appb-000007
将20mmol的6-氨基-2-萘甲酸溶于20mL叔丁醇和20mL水的混合溶液中，依次加入24mmol的二碳酸二叔丁酯和24mmol的氢氧化钠，常温反应过夜。用5％盐酸溶液调节至pH 2-3，加入大量二氯甲烷萃取，有机层干燥，浓缩，柱层析(石油醚：乙酸乙酯＝1：1，v/v)得米色固体，收率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).
中间体B的合成
Figure PCTCN2016085367-appb-000008
冰浴下将5mmol的6-氨基甲酸叔丁酯-2-萘甲酸溶于20mL无水二氯甲烷中，滴加7.5mmol的二甲氨基吡啶(DMAP)和6mmol中间体A的20mL的无水二氯甲烷混合溶液，搅拌半小时后再加入10mmol的1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDC)，2小时后撤去冰浴，常温反应过夜。加入大量二氯甲烷萃取，饱和食盐水洗涤，有机层干燥，浓缩，柱层析(石油醚：乙酸乙酯＝1：2，v/v)得黄色固体，收率50％。
1H NMR(400MHz,DMSO-d6):δ9.77(s,1H),9.43(brs,1H),8.37(d,J＝8.8Hz,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.
中间体C的合成
Figure PCTCN2016085367-appb-000009
将0.5mmol的中间体B溶于4mL二氯甲烷中，冰浴下滴加1.5mL三氟乙酸，常温反应过夜。用10％碳酸氢钠溶液调节至pH 7-8，加入大量二氯甲烷萃取，饱和食盐水洗涤，有机层干燥，浓缩，柱层析(二氯甲烷：甲醇＝50：1，v/v)得橙红色固体，收率72％。
1H NMR(400MHz,DMSO-d6):δ9.49(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.
中间体D的合成
Figure PCTCN2016085367-appb-000010
在50mL烧瓶中加入3mmol的钠氢(60％)约240mg和1.8mL的无水THF，冰浴下滴加1.8mL含有6mmol的丙二酸二乙酯的无水THF溶液，10分钟后滴加3mL含有3mmol的氯乙酰氯溶液，维持冰浴一小时，40-45摄氏度反应1-2小时，，乙酸乙酯萃取，食盐水洗涤2次，有机层浓缩，干燥，柱层析(100％EA)，柱层析得白色粉末，GC-MS验证无误。
化合物IIA的合成
Figure PCTCN2016085367-appb-000011
将0.22mmol的中间体D溶于5mL无水THF中，加入0.2mmol的中间体C，40℃下反应96小时。加入大量二氯甲烷萃取，饱和食盐水洗涤，有机层干燥，浓缩，柱层析分离纯化(二氯甲烷：甲醇＝60：1，v/v)得橙黄色固体。
1H NMR(400MHz,DMSO-d6):δ10.50(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.
实施例2.化合物IIB的合成
Figure PCTCN2016085367-appb-000012
中间体A-C的合成如前所述
中间体E的合成
Figure PCTCN2016085367-appb-000013
常温下将溶有4mmol的2,2-二氟丙酸中的5mL无水二氯甲烷溶液滴加到溶有324mg的N,N-二羰基咪唑(2mmol)的5mL无水二氯甲烷溶液，搅拌30min后滴加溶有68mg咪唑(1mmol)和98mg的1,3-环戊二酮(1mmol)，加毕，反应6小时。5％盐酸溶液，饱和食盐水依次洗涤，有机层浓缩，干燥，浓缩得粗品，黄色固体，GC-MS验证无误。
中间体F的合成
Figure PCTCN2016085367-appb-000014
常温下将0.7mL草酰氯加入到0.3mmol的中间体E中，常温反应4小时，常压蒸除余下的草酰氯，二氯甲烷萃取，饱和食盐水洗涤，有机层浓缩，干燥，快速柱层析(二氯甲烷：甲醇＝100：1，v/v)得黄色油状液体，收率55％，-35℃冰箱保存。
化合物IIB的合成
Figure PCTCN2016085367-appb-000015
将0.2mmol的中间体C溶于5mL无水THF中，加入0.2mmol的中间体F，40℃下反应96小时。加入大量二氯甲烷萃取，饱和食盐水洗涤，有机层干燥，浓缩，柱层析分离纯化(二氯甲烷：甲醇＝60：1，v/v)得橙黄色固体。
1H NMR(400MHz,DMSO-d6):δ8.45(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.
实施例3.化合物IIC的合成
Figure PCTCN2016085367-appb-000016
称取荧光素(1.66g，5mmol)置于50mL的圆底烧瓶中加入20mL的无水乙醇，再加入3ml浓硫酸后于80℃下加热搅拌5h。TLC跟踪至原料反应完全，旋干，用DCM和饱和碳酸氢钠溶液萃取，取有机相旋干，干燥，(DCM/MeOH＝50:3v/v)柱层析分离,得橙红色固体1.58g，产率为87.78％。1H NMR(400MHz,CDCl3)δ8.26(d,J＝7.2Hz,1H),7.748-7.659(m,2H),7.44(s,1H),7.31(d,J＝7.2Hz,1H),6.98(d,J＝9.2Hz,2H),6.89(s,2H),6.82(d,J＝9.2Hz,2H),3.99(q,J＝7.2Hz,2H),0.91(t,J＝7.1Hz,3H).
连接臂ZC-Z-1的合成
Figure PCTCN2016085367-appb-000017
称取二甘醇胺(1.26g,12mmol)置于25mL的圆底烧瓶中，在冰浴下搅拌，滴加Boc2O的二氯甲烷溶液，滴毕常温下搅拌10h,点板有新点产生原料基本反应完，lc-ms为目标产物，将反应液用CH2Cl2/水萃取5遍，取有机相，干燥，旋干，不进一步纯化。得无色油状液体912.8mg，产率为37.1％。1H NMR(400MHz,CDCl3)δ5.12(s,1H),3.74(t,J＝4.4Hz,2H),3.59-3.54(m,4H),3.34(d,J＝4.8Hz,2H),2.68(s,1H),1.45(s,9H).
化合物ZC-B-3的合成
Figure PCTCN2016085367-appb-000018
称取化合物ZC-B-2(360mg,1mmol),DIAD(0.606mL,3mL)，三苯基膦(790mg，3mmol)溶于无水THF中置于25mL的圆底烧瓶中，在氩气保护和避光下搅拌1h,再缓 慢滴加化合物ZC-Z-1(400.5mg,2mmol)的THF溶液，滴毕，继续搅拌10h，点板原料反应了大半,直接旋干，柱层析(EA:PE＝1:1v/v)得黄色固体420.5mg，产率为76.8％。1H NMR(400MHz,CDCl3)δ8.26(d,J＝7.6Hz,1H),7.76-7.67(m,2H),7.31(d,J＝7.6Hz,1H),6.99(d,J＝2Hz,1H),6.930-6.873(m,2H),6.79(d,J1＝8.8Hz,J2＝2.2Hz,1H),6.56(dd,J1＝9.7Hz,J2＝1.6Hz 1H),6.48(s,1H),4.98(s,1H),4.23(t,J＝4.4Hz,2H),4.06-3.99(m,2H),3.87(t,J＝4.2Hz,2H),3.63(d,J＝5.2Hz,2H),3.36(d,J＝4.2Hz,2H),1.44(s,9H),0.96(t,J＝7.1Hz,3H).
化合物ZC-B-4的合成
Figure PCTCN2016085367-appb-000019
将化合物ZC-A-3(100mg，0.15mmol)溶于三氟乙酸的二氯甲烷溶液中(三氟乙酸：二氯甲烷＝3:7v/v),室温下搅拌反应5h，旋干溶剂，饱和碳酸氢钠和二氯甲烷萃取，取有机相干燥，旋干得到黄色固体67mg，不做进一步纯化用于下一步反应。LC-MS(ESI)calcd for C26H25NO6[M+H]+:448.17,found 448.21.
化合物ZC-Y-2的合成
Figure PCTCN2016085367-appb-000020
称硼氢化钠(112mg，3mmol)于三口圆底烧瓶中加入10ml THF，在冰浴下恒压滴加溶于5ml THF的化合物ZC-Z-1(187mg，1mmol)溶液，滴加完毕后至气泡不再产生后，缓慢滴加溶于5ml THF的碘(304mg，1.2mmol)溶液，滴加完毕后，搅拌30分钟，升温至60℃，反应24小时，TLC跟踪至原料转化完全。在冰水浴中冷却，滴加甲醇到溶液澄清，旋去溶剂，剩余物中加入10ml的20％的KOH溶液，在50℃下搅拌2小时，用乙酸乙酯萃取，取有机层，无水硫酸钠干燥，旋干，产物经硅胶柱层析(PE/EA＝7/1，v/v)分离，得158mg白色固体,产率为91.3％。1H NMR(400MHz,MeOD)δ7.61–7.59(m,2H),7.53(d,J＝8.8Hz,1H),7.32(dd,J1＝8.4Hz,J2＝1.6Hz,1H),7.01-6.99(m,2H),4.66(s,2H),3.31(s,1H).
化合物ZC-Y-3的合成
Figure PCTCN2016085367-appb-000021
称取化合物ZC-Y-2(207.6mg,1.2mmol)和化合物ZC-Z-2(200mg,1mmol)置于25ml的圆底烧瓶中加入10ml的无水THF，在氩气保护下于45℃下搅拌12h,TLC跟踪至原料反应完，直接旋干柱层析(DCM:MeOH＝50:1v/v),得178.5g灰白色固体产率为54.9％。 1H NMR(400MHz,CDCl3)δ10.34(s,1H),7.84-7.78(m,4H),7.53(d,J＝8.4Hz,1H),7.38(dd,J1＝8.8Hz,J2＝1.8Hz,1H),4.85(s,2H),4.60(s,2H),4.41(q,J＝7.2Hz,2H),1.44(t,J＝7.2Hz,3H),1.26(s,1H).
化合物ZC-Y-4的合成
Figure PCTCN2016085367-appb-000022
取化合物ZC-Y-3(327mg,1mmol)置于25ml圆底烧瓶中，加入5ml无水二氯甲烷，在氩气保护冰浴下滴加1ml二氯亚砜，在冰浴下搅拌5min后溶液有无色变墨绿，TLC跟踪至原料反应完，旋干，用二氯甲烷和饱和碳酸氢钠溶液萃取，取有机相干燥，旋干，柱层析(DCM:MeOH＝100:1v/v),得235.6mg灰色固体，产率为68.4％。1H NMR(400MHz,CDCl3)δ10.49(s,1H),7.88–7.80(m,4H),7.55(d,J＝8.4Hz,1H),7.47(dd,J1＝8.8Hz,J2＝1.8Hz,1H),4.75(s,4H),4.41(q,J＝7.2Hz,2H),1.42(t,J＝7.1Hz,3H).
化合物IIC的合成
Figure PCTCN2016085367-appb-000023
称取化合物ZC-B-4(894mg,2mmol),化合物ZC-Y-4(345mg,1mmol),碳酸钾(690mg,5mmol),碘化钾(166mg，1mmol)置于50ml的圆底烧瓶中加入20ml无水乙腈，再加入三乙胺(101mg,1mmol)在氩气保护下回流搅拌反应，TLC跟踪至原料反应完。旋干，用二氯甲烷和水萃取，取有机相干燥，旋干柱层析(DCM:MeOH＝50:1v/v)，得112.7mg黄色固体产率为14.9％。1H NMR(500MHz,CDCl3)δ8.32(d,J＝7.5Hz,1H),7.90–7.73(m,6H),7.61(d,J＝8.0Hz,1H),7.48(d,J＝8.5Hz,1H),7.33(t,J＝8.5Hz,1H),7.01(s,1H),6.92(t,J＝7.5Hz,2H),6.81(d,J＝8.5Hz,1H),6.61(d,J＝9.5Hz,1H),6.48(s,1H),5.37(s,1H),4.80(s,2H),4.47(q,J＝7.0Hz,2H),4.30(s,2H),4.08(d,J＝7.1Hz,4H),3.95(s,2H),3.83(s,2H),3.01(s,2H),1.48(t,J＝6.9Hz,3H),1.32(s,1H),1.03(t,J＝6.9Hz,3H).
13C NMR(126MHz,CDCl3)δ188.4,185.7,177.7,165.65,165.31,163.20,158.87,154.10,150.23,137.04,134.19,132.79,132.53,132.26,131.23,130.74,130.37,130.31,129.89,129.67,129.35,128.97,128.01,127.97,126.76,120.59,118.48,117.73,115.08,113.65,105.74,100.99,87.89,75.60,70.40,69.17,68.15,61.35,60.70,53.55,48.45,29.69,14.55,13.61.HRMS(ESI)calcd for C44H40N2O10[M+H]+＝757.2761,found 757.2759.
实施例4.本发明化合物对细胞株的抑制活性
本发明提供的化合物对两种恶性疟原虫细胞株(3D7和Dd2)的体外活性的抑制效果：
氯喹购于Sigma公司，SYBR Green I购于Life technologies公司，氯喹敏感的3D7细胞株和氯喹抗性的Dd2细胞株用于体外测试抗疟活性，通过Trager and Jensen的培养方法(Human malaria parasites in continuous culture.Science.1976,193(4254):673-675)在含有0.5％Albumax II非人血清的介质中培养。以SYBR Green I为探针，通过荧光滴定方法测定抗疟原虫细胞株活性。首先，不同时期的寄生虫一式三份，在2％的血红细胞和在100μL的1％的寄生虫血症与一系列的药物存在或不存在条件下培养，稀释时每个化合物浓度0.15625变化到20μM。0.2％DMSO作为反向对照，不同浓度的氯喹作为正向对照。培养皿在37℃下培养3天，然后取出上清液，并加入100μL of SYBR Green I的细胞缓冲液(8.26g/L NH4Cl,1g/L KHCO3，0.037g/L EDTA和5×SYBR Green I)。培养皿在暗室中常温继续培养1小时，通过Synergy MX,Biotek荧光检测器在485/520nm下读数，重复2次本实验。数据分析是根据Michael等报道的方法进行改进获得。简述如下，阴性对照处理组获得的荧光数据减去未感染红细胞组所产生的背景荧光数据，代表本次实验中正常培养的恶性疟原虫中的DNA最大量，记为阴性对照孔的荧光数据，各测试组及阳性对照组的荧光数据均如此校正。不同浓度下抑制率的计算由下式所得：抑制率(％)＝〔1-(测试孔中平均荧光计数/阴性对照孔中平均荧光计数)〕×100％。通过软件Origin 8.0中的Growth/Sigmoidal程序拟合曲线并计算获得半数抑制率(IC50values)，通过Microsoft Excel计算平均值和标准差。
实施例4的结果如下表：
Figure PCTCN2016085367-appb-000024
实施例5.本发明化合物与靶蛋白的结合
将含有PfDHODH基因的片段克隆到PET-19b载体中，得到重组质粒，转入大肠杆菌BL21中进行表达，分别经过固定金属离子亲和色谱和凝胶过滤色谱纯化后，得到较纯的蛋白，将纯化好的PfDHODH蛋白稀释到终浓度为2mg/mL，分装成两管，每管500μL，一管加25μL母液为50mM的本发明化合物(IIA)，另一管加25μL DMSO作为对照，冰上孵育30min，用PCR管分装蛋白，将加化合物和DMSO的蛋白分别分装12管，每管30μL,用PCR仪在不同温度下(37℃，40℃，43℃，46℃，49℃，52℃，55℃，58℃，61℃，64℃，67℃，70℃)分别加热3min，化合物和对照进行相同的处理，加热完后，4℃高速离心半小时，分别取上清2μL，用Buffer稀释到100μL，制备蛋白样品进行western blot，最后用化学发光成像系统进行显影，得到结果如图1所示。
由图1所知，与只加DMSO的对照相比，加入本发明化合物后，对pfDHODH蛋白 起一定的稳定作用。加DMSO的对照组，Tm＝53.77℃，而加入本发明化合物的实验组，Tm＝56.94℃，使Tm值增大了3.2℃，即，本发明化合物对PfDHODH蛋白有结合，并且在一定程度上能够对其起稳定作用。
鉴于PfDHODH蛋白蛋白质结构复杂，构象处于不停变化中，不同构象下的能垒不同，稳定性亦不同；本实施例证明，加入本发明物(IIA)后，其与蛋白质结合，与之间未结合时相比，蛋白质更稳定，因此说明本发明化合物与靶蛋白的结合效果较好。
实施例6.本发明化合物与体外培养的受疟原虫感染红细胞的荧光成像
用DMSO溶解本发明化合物(IIA、IIC)至50mM。在37℃培养箱中培养一定量恶性疟原虫(湖州师范学院周洪昌老师惠赠)，平均分两皿，标记为A和B；将A、B培养皿中的恶性疟原虫用5％山梨醇间隔一天同步，培养两天；向A和B培养皿中加入本发明化合物至终浓度为10μg/mL，37℃培养箱中培养3.5小时，之后向A、B培养皿中加入荧光染料
Figure PCTCN2016085367-appb-000025
Red CMXROS(购自Life Technologies)至终浓度为500nM，放在37℃培养箱中培养30min；将两个培养皿中感染恶性疟原虫的红细胞转移到15mL离心管中，在1800rpm，3min条件下离心，去掉上清，用PBS洗三遍；分别制作血涂片，用共聚焦显微镜定位。
Figure PCTCN2016085367-appb-000026
Red CMXROS：激发波长为579nm，发射波长为599nm；IIA：激发波长为480nm，发射波长为530nm。结果如图2所示。
由图2所见，在严重感染的红细胞中，血红蛋白变性，降解成黑色沉淀物，即疟色素，这是肉眼区分正常红细胞和受感染红细胞的粗略方法。A-F为不同视野内IIA在细胞内的定位效果，在明显含有疟色素的红细胞中，细胞呈现绿色荧光，同时Tracer进行叠合发现，IIA仅作用于细胞线粒体中，明亮如小星。而在未感染疟原虫的红细胞中，没有出现荧光信号，说明本发明化合物具有良好的细胞水平靶向性。
实施例7.本发明化合物对伯氏疟原虫感染小鼠的血液的检测及荧光成像
昆明种小鼠(雄性，20±2g，购自上海杰思捷实验动物有限公司)，接种伯氏疟原虫(第二军医大学潘卫庆老师惠赠)200μL；在光学显微镜下观察小鼠感染情况，当感染率达到20％时，采用眼球取血的方法取血。加入本发明化合物(IIA)至终浓度为10μg/mL，荧光染料
Figure PCTCN2016085367-appb-000027
Red CMXROS至终浓度为500nM；在37℃条件下孵育15min；在1800rpm，3min条件下离心，去掉上清，用PBS洗三遍；制作血涂片，用共聚焦显微镜定位。
由图3所示，A-D不同视野下可见不同生长周期的疟原虫。如图3A所示，明场下红细胞内小圈即处于环状体阶段的疟原虫；如图3B或3C所示，明场下红细胞内带有黑色素大圈即晚期的大滋养体，其中小点即小滋养体。不同生长周期的疟原虫虽然形态、体积不一，但IIA都可对其进行选择性标记，且荧光强度与疟原虫形态、体积成一定的正相关。大滋养体中荧光亮而大，而小滋养体中荧光如小星。不仅如此，正常红细胞完全不显荧光，特异性强、背景信噪极低。
由图4和图5所示，IIC也可对伯氏疟原虫和恶性疟原虫感染的细胞进行选择性标记。
对比例
本发明人进一步合成了包含其它连接臂的多种化合物，当利用这些化合物重复以上实施例5-7时，发现这些化合物或无法结合PfDHODH，或无法与体外培养的受疟原虫感染红细胞进行荧光成像，或无法与疟原虫感染的小鼠血液进行荧光成像。
发明人认为，由于本发明将色素基团经连接臂与二氢乳清酸脱氢酶特异性配体结合起来，最终获得的化合物是否能够兼具与二氢乳清酸脱氢酶特异性结合以及产生荧光信号的功能是无法合理预知的；换言之，本发明化合物中色素基团、连接臂与二氢乳清酸脱氢酶特异性结合配体之间可能会互相影响，从而不利地影响最终所得化合物与二氢乳清酸脱氢酶的结合特异性或者色素基团产生荧光的能力。
在本发明提及的所有文献都在本申请中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本申请所附权利要求书所限定的范围。

Claims (11)

  1. 式I所示化合物：
    A——L——R
    I
    式中，
    A为色素基团；
    L为连接臂；
    R为二氢乳清酸脱氢酶特异性结合配体。
  2. 如权利要求1所述的化合物，其特征在于，所述化合物如下式II所示：
    Figure PCTCN2016085367-appb-100001
    式中，
    R1为未取代的或被选自下组的取代基取代的稠环基团：C1-C5烷基、卤素、羧基、酯基、氨基；
    R2选自：C1-C6烷基，C1-C6不饱和烃基，C1-C6烷基羰基，C1-C6氟代烷基羰基，C1-C6烷氧基，C1-C6氟代烷氧基，任选取代的苯甲酰基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基、C1-C6酯基；
    R3选自O、NH或S；
    R4选自H，C1-C6烷基，任选取代的C2-C6不饱和烃基，C1-C6烷基羰基，任选取代的苯甲酰基，羧基，氨基羰基，C1-C6烷氧基羰基，C1-C6氨基羰基，羟基，C1-C6烷氧基；
    R5选自O，S，NH和CH2。
  3. 如权利要求2所述的化合物，其特征在于，R1为含2-3个苯环的稠环基团，优选地，R1为萘基。
  4. 如权利要求1所述的化合物，其特征在于，所述色素选自4-氯-7-硝基苯并-2-氧杂-1,3-二唑(NBD)或荧光素。
  5. 如权利要求1-4中任一项所述的化合物，其特征在于，L为(HNCH2CH2OCH2CH2OOC)或(OCH2CH2OCH2CH2NHCH2)。
  6. 如权利要求5所述的化合物，其特征在于，所述化合物如下式所示：
    Figure PCTCN2016085367-appb-100002
  7. 一种二氢乳清酸脱氢酶介导的疾病的诊断试剂盒，所述试剂盒装有权利要求1-6中任一项所述的化合物。
  8. 如权利要求7所述的诊断试剂盒，其特征在于，所述疾病是恶性疟疾。
  9. 权利要求1-6中任一项所述的化合物在制备二氢乳清酸脱氢酶检测或定位试剂二氢乳清酸脱氢酶介导的疾病的诊断试剂中的用途。
  10. 如权利要求9所述的用途，其特征在于，所述疾病是恶性疟疾。
  11. 一种药物组合物，其特征在于，所述药物组合物包含：
    权利要求1-6中任一项所述的化合物或其药学上可接受的盐，和
    药学上可接受的载体或赋形剂。
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CN103127095A (zh) * 2011-11-23 2013-06-05 华东理工大学 二氢噻吩酮衍生物作为pfDHODH抑制剂的合成及应用
WO2015031824A1 (en) * 2013-08-30 2015-03-05 Icahn School Of Medicine At Mount Sinai Cyclic vinylogous amides as bromodomain inhibitors

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CN103127095A (zh) * 2011-11-23 2013-06-05 华东理工大学 二氢噻吩酮衍生物作为pfDHODH抑制剂的合成及应用
WO2015031824A1 (en) * 2013-08-30 2015-03-05 Icahn School Of Medicine At Mount Sinai Cyclic vinylogous amides as bromodomain inhibitors

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