WO2019015475A1 - 化合物及其在结核分枝杆菌检测中的用途 - Google Patents

化合物及其在结核分枝杆菌检测中的用途 Download PDF

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WO2019015475A1
WO2019015475A1 PCT/CN2018/094422 CN2018094422W WO2019015475A1 WO 2019015475 A1 WO2019015475 A1 WO 2019015475A1 CN 2018094422 W CN2018094422 W CN 2018094422W WO 2019015475 A1 WO2019015475 A1 WO 2019015475A1
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alkyl
group
compound
phenyl
tuberculosis
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刘刚
穆然
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清华大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
    • C07D493/04—Ortho-condensed systems
    • C—CHEMISTRY; METALLURGY
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    • C09K11/00—Luminescent, e.g. electroluminescent, chemiluminescent materials
    • C09K11/06—Luminescent, e.g. electroluminescent, chemiluminescent materials containing organic luminescent materials
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
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    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1029—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom
    • C09K2211/1033—Heterocyclic compounds characterised by ligands containing one nitrogen atom as the heteroatom with oxygen
    • C—CHEMISTRY; METALLURGY
    • C09—DYES; PAINTS; POLISHES; NATURAL RESINS; ADHESIVES; COMPOSITIONS NOT OTHERWISE PROVIDED FOR; APPLICATIONS OF MATERIALS NOT OTHERWISE PROVIDED FOR
    • C09K—MATERIALS FOR MISCELLANEOUS APPLICATIONS, NOT PROVIDED FOR ELSEWHERE
    • C09K2211/00—Chemical nature of organic luminescent or tenebrescent compounds
    • C09K2211/10—Non-macromolecular compounds
    • C09K2211/1018—Heterocyclic compounds
    • C09K2211/1025—Heterocyclic compounds characterised by ligands
    • C09K2211/1088—Heterocyclic compounds characterised by ligands containing oxygen as the only heteroatom
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6432—Quenching

Definitions

  • the invention relates to the technical field of detection of Mycobacterium tuberculosis, in particular to a compound and its use in the detection of Mycobacterium tuberculosis.
  • Mycobacterium tuberculosis is the causative agent of tuberculosis.
  • Mycobacterium tuberculosis is abbreviated as Mycobacterium tuberculosis, and human lung infection is the most common. Common symptoms of lung infection include chest pain and long-term multi-cough cough.
  • WHO World Health Organization
  • MDR-TB multidrug-resistant Mycobacterium tuberculosis
  • the present invention aims to solve at least one of the technical problems existing in the prior art. To this end, it is an object of the present invention to provide a means and method for detecting M. tuberculosis quickly and efficiently.
  • the main methods of detecting and confirming tuberculosis patients are the techniques of sputum smear and chest X-ray examination.
  • the use of these technologies has been nearly a hundred years old, and there are obvious limitations: 1) the positive rate is low. At present, the positive accuracy rate of sputum smear is only 30%-40%; the average missed diagnosis rate of X-ray chest radiograph is 21.5%, and the average over-diagnosis rate is 19.5%; 2) the result is susceptible to the examiner. The quality of the smear and the quality of the examiner and reader will affect the report of the test results. Studies have shown that for X-ray chest X-ray, the diagnostic error of experts before and after the individual and each expert can be as high as 20%-30%.
  • the compound of formula I (sometimes referred to herein simply as “compound I”) is not only for replicating Mycobacterium tuberculosis (R-Mtb) and non-replicating Mycobacterium tuberculosis (NR-Mtb). It has high inhibitory activity and also has high inhibitory activity against many clinically resistant bacteria. In addition, compound I exhibits Mtb (R-Mtb and NR-Mtb) compared to other common Gram-positive and negative bacteria. a high degree of selective inhibition. The latest study by the inventors also found that this highly selective inhibition of Mtb by Compound I is associated with a thioredox-reduced oxidoreductase (Rv2466c) of M. tuberculosis, and this inhibition is also dependent on Actinomycete-specific amino acid thiol Mycothiol (MSH) (M. tuberculosis belongs to Actinobacteria).
  • Rv2466c thioredox-reduced
  • Rv2466c (the amino acid sequence thereof, or the preparation method, can be found in the following literature: Marcelo E. Guerin et al., ACS Chem. Biol. 2014, 9, 1567-1575) is a nitroreductase inherent in Mycobacterium tuberculosis, and is related to the redox pressure in the bacteria. Its expression level in the bacteria is directly regulated by the Sig H factor, and can be in an exogenous oxidant such as thiol. The oxidant diamide (or diammened Diamide) is overexpressed under pressure ( Figure 1).
  • Rv2466c is in a state of equilibrium between the oxidized state and the reduced state in M. tuberculosis, that is, the oxidized Rv2466c and the reduced Rv2466c are in a state of dynamic equilibrium, and in the presence of MSH (M. tuberculosis self-secrets MSH)
  • MSH M. tuberculosis self-secrets MSH
  • the nitro group in the compound I can be reduced by the Rv2466c in a reduced state, and the active oxygen or reactive nitrogen intermediate is released to efficiently kill the Mtb; while the reduced state Rv2466c is oxidized to the oxidized state, thereby continuing the next round. Catalytic cycling process.
  • amino acid sequence of Rv2466c is as follows:
  • the inventors further studied the above reduction process and found that Compound I exhibited a detectable difference in fluorescence spectral properties before and after reduction by the thioredox-like oxidoreductase Rv2466c inherent in the M. tuberculosis:
  • the compound shown by I is in a fluorescence quenching state before being reduced by Rv2466c, and is capable of emitting fluorescence under specific excitation light (320-400 nm) and emission light (420-510 nm) after being reduced by Rv2466c. This is because (see Fig.
  • I-6a, I-6b, I-6c, I-6d (or the thioether product obtained by the addition of an oxime group, such as I-22 and I-, after addition of a double bond of a furan ring by an electron-rich affinity reagent 69 can also emit fluorescence of another specific wavelength under a specific excitation light wavelength), and the electron cloud inside the entire molecule of the compound can be rearranged, so that the coumarin ring (mother core) becomes an electron-rich part, and The compound resumes fluorescence (fluorescence on state). That is, the compound of Formula I exhibits a significant difference in detectable fluorescence spectral properties before and after reduction by the thioredox-like oxidoreductase Rv2466c inherent in M.
  • the "off and on” effect of the above-mentioned “fluorescent switch” (the difference in the spectral properties of the detectable fluorescence) can also be achieved in the presence of Mycobacterium tuberculosis (because the M. tuberculosis contains both The reagents necessary for the reduction of the nitro group in the nitrofuran structure: Rv2466c and MSH), thus, based on the significant difference in the detectable fluorescence spectral properties, the compound of formula I can be detected quickly and efficiently by fluorescence detection.
  • the M. tuberculosis in the sample to be tested, and the detection information can be effectively used to diagnose whether the suspected patient from the sample source is suspected of having M.
  • tuberculosis-related diseases thereby realizing a tuberculosis-positive patient (active tuberculosis patient) And the detection of tuberculosis carriers.
  • derivatives of the compounds of formula I such as nitro reduction products and corresponding hydrolysates, also have detectable changes in fluorescence (before and after contact with M. tuberculosis) and can therefore also be utilized for detection. Diagnosis of M. tuberculosis and further M. tuberculosis-related diseases in the sample to be tested.
  • the invention provides a compound.
  • the compound is a compound of formula I or a derivative thereof:
  • R 1 is hydrogen, halogen, C 1-6 alkyl, substituted or unsubstituted phenyl, benzyl or substituted or unsubstituted heterocyclic group, each of which is optionally independently selected from a substituent substituted with a C 1-6 alkyl group, a C 1-6 alkoxy group, a nitro group, an amino group, a nitrile group or a halogen;
  • R 2 is hydrogen, halogen, C 1-6 alkyl, substituted or unsubstituted phenyl, benzyl or substituted or unsubstituted heterocyclic group, each of which is optionally independently selected from a substituent substituted with a C 1-6 alkyl group, a C 1-6 alkoxy group, a nitro group, an amino group, a nitrile group or a halogen;
  • R 3 is hydrogen, hydroxy, C 1-6 alkoxy, C 1-6 acyloxy, substituted or unsubstituted phenyl, benzyl, substituted or unsubstituted heterocyclic, alkynyl or alkenyl,
  • the phenyl and heterocyclic groups are each independently optionally substituted with a substituent selected from a C 1-6 alkyl group, a C 1-6 alkoxy group, a nitro group, an amino group, a nitrile group or a halogen;
  • R 4 is hydrogen, halogen or C 1-6 alkyl
  • R 5 and R 6 are each independently hydroxy, alkyl, hydrogen, substituted or unsubstituted alkyl, alkoxy, ester or substituted or unsubstituted fluorenyl, each independently and optionally Substituted with a substituent selected from a C 1-6 alkyl group, a C 1-6 alkyl alcohol, a C 1-6 alkyl thiol, a hydroxyl group, an acyloxy group, an amino group, a nitrile group or a halogen.
  • the inventors have found that the compound of the formula I and its derivatives, such as the nitro reduction product and the corresponding hydrolyzate, exhibit before and after reduction by the thioredox-reduced oxidoreductase Rv2466c inherent in M. tuberculosis.
  • the detectable fluorescence change and thus the compound of the formula I or a derivative thereof is used as a detection reagent, and the sample to be tested is contacted with the detection reagent, and based on whether the sample to be tested has a fluorescence change before and after the contact, the determination can be effectively determined.
  • the presence or absence of M. tuberculosis in the sample was measured and the results were accurate and reliable.
  • the detection information can be effectively used for diagnosing whether a suspected patient whose source of the sample is tested has a Mycobacterium tuberculosis-related disease, thereby achieving detection of a tuberculosis-positive patient (active tuberculosis patient) and a tuberculosis carrier.
  • the fluorescence change described above is at least one of the following: the sample to be tested is in a fluorescence quenching state before the contact, and can be excited by the excitation light of 320-400 nm after the contact and emits 420- The fluorescence of 510 nm; the sample to be tested is in a fluorescence quenching state before the contact, and can be excited by the excitation light of 320-400 nm after the contact and emit fluorescence of 420-510 nm, and the emitted fluorescence exhibits a strong and weak change.
  • the compound of the formula I or a derivative thereof is contacted with the sample to be tested, and fluorescence detection is performed, and based on the fluorescence detection results before and after the contact, it is possible to effectively determine whether M. tuberculosis is present in the sample to be tested.
  • R 1 and R 2 are joined as a 4-8 membered ring.
  • R 4 is hydrogen and R 3 is R 1 O-, R 1 OCH 2 O-, R 1 COO-, HO-, HOCH 2 CH 2 O-,
  • the invention provides a compound which is a compound of formula I or a derivative thereof or a compound acceptable salt of formula I:
  • R 1 is hydrogen, halogen, C 1-6 alkyl, substituted or unsubstituted phenyl, benzyl or substituted or unsubstituted heterocyclic group, each of which is optionally independently selected from a substituent substituted with a C 1-6 alkyl group, a C 1-6 alkoxy group, a nitro group, an amino group, a nitrile group or a halogen;
  • R 2 is hydrogen, halogen, C 1-6 alkyl, substituted or unsubstituted phenyl, benzyl or substituted or unsubstituted heterocyclic group, each of which is optionally independently selected from a substituent substituted with a C 1-6 alkyl group, a C 1-6 alkoxy group, a nitro group, an amino group, a nitrile group or a halogen;
  • R 1 and R 2 are bonded to a C 4-8 saturated carbocyclic ring;
  • R 4 is hydrogen, halogen or C 1-6 alkyl
  • R 3 and R 4 are bonded to a C 4-8 saturated or unsaturated carbocyclic ring or a C 4-8 saturated or unsaturated heterocyclic ring wherein the C 4-8 saturated or unsaturated carbocyclic ring or C 4-8 is saturated or
  • the unsaturated heterocyclic ring is unsubstituted or optionally substituted with 1 to 3 substituents selected from a C 1-6 alkyl group or a phenyl group
  • the above compound may further comprise at least one of the following additional technical features:
  • each R 1 , R 2 is independently H, halo, C 1-3 alkyl, phenyl, benzyl, C 5-6 heterocyclyl, wherein said C 1-3 alkyl , phenyl, benzyl, C 5-6 heterocyclyl are each independently unsubstituted or optionally 1 or 2 C 1-3 alkyl, C 1-3 alkoxy, nitro, amino, CN , halogen substituted; or R 1 and R 2 are attached to a C 5-6 carbocyclic ring.
  • each of R 1 and R 2 is independently H, methyl, ethyl, n-propyl, isopropyl, phenyl, benzyl, halogen, wherein the methyl, ethyl, and The propyl, isopropyl, phenyl, benzyl groups are each independently unsubstituted or optionally substituted with 1 C 1-3 alkyl group, C 1-3 alkoxy group, nitro group, amino group, CN, halogen.
  • R 4 is hydrogen and R 3 is R a O-, R a OCH 2 O-, R a COO-, HO-, HOCH 2 CH 2 O-, or -N(C 1-3 alkyl) 2 ,
  • Ra is a C 1-6 alkyl group
  • n1, n2, n3, and n4 is independently an integer between 0 and 3;
  • Each Rc, Rd is independently a C 1-6 alkyl group, or Rc is bonded to Rd to form a C 5-6 saturated carbocyclic ring,
  • Re is H, halogen
  • R 3 and R 4 are linked to a structure selected from the group consisting of:
  • Rf is C 1-3 alkyl or phenyl, and n5 is 0, 1 or 2.
  • the derivative of the compound of formula (I) has the structure of one of the following:
  • each of R x and R y is independently H, a hydroxyl group, a halogen, a CN, a C 1-6 alkyl group, a C 1-6 alkenyl group, or a C 1-6 alkynyl group. According to an embodiment of the present invention, each of R x and R y is independently H and a hydroxyl group.
  • the compound of formula I has the structure of one of the following:
  • R 4 and R 3 are joined as a 4-8 membered ring.
  • the compound of formula I has the structure of one of the following:
  • derivatives of the compounds of formula I include, but are not limited to, stereoisomers, geometric isomers, tautomers, racemates, oxynitrides, of the compounds of formula I, Hydrates, solvates, metabolites, pharmaceutically acceptable salts or prodrugs, nitro reduction products and their corresponding hydrolysates.
  • the derivative of the compound of formula I has the structure of one of the following:
  • the compound of the above Ia structure is a derivative of the corresponding compound I, a nitro reduction product, which, as described above, is also detectable before and after reduction by the Rv2466c enzyme inherent in M. tuberculosis. Fluorescence change, thus can be used as a detection reagent, after the sample to be tested is in contact with it, based on whether there is fluorescence change before and after the test sample, it is possible to effectively determine whether there is live M. tuberculosis in the sample to be tested, and The result is accurate and reliable.
  • the derivative of the compound of formula I has the structure of one of the following:
  • the compound of the above Ib structure is also a derivative of the corresponding compound I, a nitro reduction product, which, as described above, also has detectable fluorescence, and thus can be used for detection of Mycobacterium tuberculosis .
  • the derivative of the compound of formula I has the structure of one of the following:
  • the compound of the above Ic structure is also a derivative of the corresponding compound I, a hydrolyzate of a nitro reduction product thereof, as described above, which also has detectable fluorescence and thus can be used for tuberculosis Detection of Mycobacterium.
  • the derivative of the compound of formula I has the structure of one of the following:
  • the compound of the above Id structure is also a derivative of the corresponding compound I, a hydrolyzate of a nitro reduction product thereof, as described above, which also has detectable fluorescence and thus can be used for tuberculosis Detection of Mycobacterium.
  • the derivative of the compound of formula I has the structure of one of the following:
  • the compound of the above Ie structure is also a derivative of the corresponding compound I, a nitro reduction product, and a tautomer of the compound of the Ib structure (imine-enamine isomerization). As previously mentioned, it also has detectable fluorescence and can therefore be used for the detection of M. tuberculosis.
  • the invention also provides a kit for detecting M. tuberculosis.
  • the kit comprises the compound described above as a detection reagent.
  • the compound of Formula I exhibits a detectable difference in fluorescence spectral properties before and after reduction by the thioredox-like oxidoreductase Rv2466c inherent in M. tuberculosis, using the kit of the present invention,
  • the compound or a derivative thereof is used as a detection reagent, and the sample to be tested is contacted with the detection reagent, and based on whether the sample to be tested has a fluorescence change before and after the contact, the presence or absence of M.
  • tuberculosis in the sample to be tested can be effectively determined, and the result Accurate and reliable. Furthermore, the detection information can be effectively used for diagnosing whether a suspected patient whose source of the sample is tested has a Mycobacterium tuberculosis-related disease, thereby achieving detection of a tuberculosis-positive patient (active tuberculosis patient) and a tuberculosis carrier.
  • the invention also provides a method of detecting M. tuberculosis.
  • the method comprises: contacting a sample to be tested with the compound described above; and determining whether there is a tuberculosis branch in the sample to be tested based on whether the sample to be tested has a fluorescence change before and after the contact Bacillus.
  • the compound of the formula I and its derivatives are all in a fluorescence quenching state before being reduced by Rv2466c, and are restored after being reduced by Rv2466c, i.e., capable of emitting fluorescence under excitation light of a specific wavelength.
  • fluorescence detection of the excitation light of a specific wavelength is performed, based on whether or not the emission of the corresponding wavelength is detected. Light or even specific changes in intensity and fluorescence can effectively determine the presence of M. tuberculosis in the sample to be tested, and the results are accurate, reliable, and reproducible.
  • the detection information can be effectively used for diagnosing whether a suspected patient whose source of the sample is tested has a Mycobacterium tuberculosis-related disease, thereby achieving detection of a tuberculosis-positive patient (active tuberculosis patient) and a tuberculosis carrier.
  • the fluorescence change is at least one of the following: the sample to be tested is in a fluorescence quenching state before contact, and can be excited by excitation light of 320-400 nm and emits 420-510 nm after contact. Fluorescence; the sample to be tested is in a fluorescence quenching state before contact, and can be excited by excitation light of 320-400 nm and emit fluorescence of 420-510 nm after contact, and the emitted fluorescence exhibits a strong and weak change.
  • the compound of the formula I or a derivative thereof is contacted with the sample to be tested, and fluorescence detection is performed, and based on the fluorescence detection results before and after the contact, it is possible to effectively determine whether M. tuberculosis is present in the sample to be tested.
  • the sample to be tested is a sample suspected to contain Mycobacterium tuberculosis, including but not limited to a sample of sputum.
  • the source of the sample is also not particularly limited and may be derived from humans or from other animals, such as livestock.
  • the invention also provides the use of a compound as described above for screening a drug, detecting a drug sensitivity or detecting a dead/live bacteria for treating or preventing M. tuberculosis-associated Human and animal diseases.
  • screening a drug using the compound of the present invention can be carried out by contacting the drug to be tested with Mycobacterium tuberculosis (for example, for a certain period of time), then adding the compound of the present invention, and the control group ( Fluorescence intensity after addition of the compound of the present invention was compared without adding a drug to be tested.
  • the fluorescence of the compound of the present invention was increased after the addition of the compound of the present invention relative to the control group (as described above, the drug was not contacted but was in contact with the drug.
  • the significant decrease in fluorescence of the inoculant of the inventive compound is an indication that the test drug has an effect of preventing or treating a disease associated with M. tuberculosis infection.
  • the use of the compound of the present invention for detecting drug sensitivity can also be carried out according to the above method, except that a series of concentration gradients are set for the drug to be tested, and a series of concentrations of the drug to be tested are separately associated with the tuberculosis under the same conditions of the same tuberculosis amount.
  • Contact with bacteria for example, for a certain period of time, then add the compound of the present invention, and compare the fluorescence intensity of the compound of the present invention and the control group (without the drug to be tested) after the addition of the compound of the present invention.
  • the treatment group of the present invention has no change in fluorescence compared with the control group after the addition of the compound of the present invention, it can be judged that the amount of Mycobacterium tuberculosis in the system is lower than the minimum detection limit of the compound, and accordingly, the concentration of the drug to be tested corresponding to the amount of the bacteria is MIC (minimum inhibitory concentration of the drug to be tested).
  • the Mycobacterium tuberculosis infection-related diseases are pediatric tuberculosis, tuberculosis, intestinal tuberculosis, lymphatic tuberculosis, bone tuberculosis, renal tuberculosis, tuberculous peritonitis, tuberculous meningitis, drug-susceptible tuberculosis, and more Drug-resistant tuberculosis, extensively drug-resistant tuberculosis, latent tuberculosis, or HIV-infected tuberculosis.
  • the Mung tuberculosis infection-related disease is an animal tuberculosis-related disease.
  • the medicament is an anti-tuberculosis lead compound or an anti-tuberculosis drug.
  • the method for detecting M. tuberculosis of the present invention has at least one of the following advantages with respect to the prior art:
  • the traditional acid-fast staining method and the gold amine "O" staining method require 300 fields of view to be collected under a microscope.
  • the diagnostic technique involved in the fluorescence method of the present invention is simple, quick, and safe to operate. High and capable of high throughput diagnostics.
  • the growth rate of Mycobacterium tuberculosis is slow, so the clinical time for pregnancy is generally one month, and the time for reporting yin is two months.
  • the diagnostic technique involved in the present invention has the characteristics that the result report is significantly accelerated, and it is expected to achieve efficient and rapid detection of outpatients.
  • the detection principle of the method of the present invention is based on the "off and on” of the fluorescent "switch" before and after the reduction of Rv2466c, and has a high signal-to-noise ratio and high specificity. specialty.
  • the method of the present invention is expected to achieve full detection of activity and latent Mtb, all resistant Mtb, and greatly improve the sensitivity and accuracy of detection.
  • the organic small molecule for detection (the compound of the formula I) according to the invention is chemically stable, can be repeatedly freeze-thawed, resistant to strong acid, strong alkali, protease, and nuclease resistant, and can be processed in clinical samples. Use under any conditions.
  • the organic small molecule for detection (the compound of the formula I) according to the present invention has an advantage of extremely high synthesis efficiency and extremely low synthesis cost, and has conditions for mass production.
  • the small organic molecule for detection (the compound of the formula I) according to the invention has remarkable bacteriostatic activity and has significant safety against other detection methods.
  • the detection method involved in the invention does not require special large-scale instruments and equipment, and has obvious low cost and is not easily contaminated compared with other molecular biological methods (technologies).
  • the detection method of the present invention has a high specificity compared to other immunological methods.
  • Stereoisomer refers to a compound that has the same chemical structure but differs in the way the atoms or groups are spatially aligned. Stereoisomers include enantiomers, diastereomers, conformational isomers (rotomers), geometric isomers (cis/trans) isomers, atropisomers, etc. .
  • Enantiomer refers to two isomers of a compound that are not superimposable but are mirror images of each other.
  • Diastereomer refers to a stereoisomer that has two or more centers of chirality and whose molecules are not mirror images of each other. Diastereomers have different physical properties such as melting point, boiling point, spectral properties and reactivity. The mixture of diastereomers can be separated by high resolution analytical procedures such as electrophoresis and chromatography, such as HPLC.
  • “Chirality” is a molecule that has properties that cannot overlap with its mirror image; “non-chiral” refers to a molecule that can overlap with its mirror image.
  • optically active compounds Many organic compounds exist in optically active forms, i.e., they have the ability to rotate a plane of plane polarized light.
  • the prefixes D and L or R and S are used to indicate the absolute configuration of the molecule with respect to one or more of its chiral centers.
  • the prefixes d and l or (+) and (-) are symbols for specifying the rotation of plane polarized light caused by the compound, wherein (-) or l indicates that the compound is left-handed.
  • Compounds prefixed with (+) or d are dextrorotatory.
  • a particular stereoisomer is an enantiomer and a mixture of such isomers is referred to as a mixture of enantiomers.
  • a 50:50 mixture of enantiomers is referred to as a racemic mixture or a racemate, which can occur when there is no stereoselectivity or stereospecificity in a chemical reaction or process.
  • any asymmetric atom (e.g., carbon, etc.) of the compounds disclosed herein may exist in racemic or enantiomerically enriched form, such as the (R)-, (S)- or (R, S)-configuration presence.
  • each asymmetric atom has at least 50% enantiomeric excess in the (R)- or (S)-configuration, at least 60% enantiomeric excess, at least 70% enantiomeric excess, at least 80% enantiomeric excess, at least 90% enantiomeric excess, at least 95% enantiomeric excess, or at least 99% enantiomeric excess.
  • the compounds of the invention may be one of the possible isomers or mixtures thereof, such as racemates and mixtures of diastereomers (depending on the number of asymmetric carbon atoms) The form exists.
  • Optically active (R)- or (S)-isomers can be prepared using chiral synthons or chiral reagents, or resolved using conventional techniques. If the compound contains a double bond, the substituent may be in the E or Z configuration; if the compound contains a disubstituted cycloalkyl group, the substituent of the cycloalkyl group may have a cis or trans configuration.
  • the resulting mixture of any stereoisomers can be separated into pure or substantially pure geometric isomers, enantiomers, diastereomers, for example, by chromatography, depending on the difference in physicochemical properties of the components. Method and / or step crystallization.
  • racemate of any of the resulting end products or intermediates can be resolved into the optical antipodes by methods known to those skilled in the art by known methods, for example, by obtaining the diastereomeric salts thereof. Separation. Racemic products can also be separated by chiral chromatography, such as high performance liquid chromatography (HPLC) using a chiral adsorbent.
  • HPLC high performance liquid chromatography
  • enantiomers can be prepared by asymmetric synthesis, for example, see Jacques, et al., Enantiomers, Racemates and Resolutions (Wiley Interscience, New York, 1981); Principles of Asymmetric Synthesis (2 nd Ed. Robert) E.
  • tautomer or "tautomeric form” refers to structural isomers having different energies that are interconvertible by a low energy barrier. If tautomerism is possible (as in solution), the chemical equilibrium of the tautomers can be achieved.
  • proton tautomers also known as prototropic tautomers
  • Valence tautomers include interconversions by recombination of some bonding electrons.
  • keto-enol tautomerization is the interconversion of a pentane-2,4-dione and a 4-hydroxypent-3-en-2-one tautomer.
  • Another example of tautomerization is phenol-keto tautomerization.
  • a specific example of phenol-keto tautomerization is the interconversion of pyridin-4-ol and pyridine-4(1H)-one tautomers. All tautomeric forms of the compounds of the invention are within the scope of the invention unless otherwise indicated.
  • the compounds of the present invention may be optionally substituted with one or more substituents, such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • substituents such as the compounds of the above formula, or specific examples, subclasses, and inclusions of the present invention.
  • a class of compounds A class of compounds.
  • C 1-6 alkyl refers particularly to the disclosure independently methyl, ethyl, C 3 alkyl, C 4 alkyl, C 5 alkyl, and C 6 alkyl.
  • linking substituents are described.
  • the Markush variable recited for that group is understood to be a linking group.
  • the Markush group definition for the variable recites “alkyl”, it should be understood that the "alkyl” represents a linked alkylene group.
  • an alkyl group means a linear and branched saturated aliphatic group having a specific number of carbon atoms.
  • Alkoxy means an alkyl-O- group having the specified number of carbon atoms.
  • Halogen or halo refers to fluorine, chlorine, bromine or iodine as a substituent. When a halogen atom is used as a substituent, the number of substitutions may be one, two or three.
  • the aryl group includes a phenyl group, a substituted phenyl group (here substituted phenyl group includes one, two or three groups as follows: C 1-6 alkyl group, C 1-6 alkoxy group, nitrile group, nitro group, amino group Or halogen).
  • heterocycle denotes a stable 5- to 7-membered monocyclic ring which may be saturated or unsaturated and which consists of a carbon atom and any one selected from N, O and S. 4 hetero atom compositions in which nitrogen and sulfur heteroatoms can be selectively oxidized, and nitrogen heteroatoms can be selectively quaternized, preferably 6-membered heterocycles such as pyridine, piperidine, pyrazine, piperazine , morpholine or thiomorpholine.
  • the solvate refers to a form of a solvent formed by a compound of the formula and a reaction organic solvent or water, or a molecule containing water or a solvent contained in a molecular formula, which is obtained by means of instrumental analysis.
  • the pharmaceutically acceptable solvents employed in the present invention include those which do not interfere with the biological activity of the compounds of the present invention (e.g., water, ethanol, acetic acid, N,N-dimethylformamide, dimethyl sulfoxide, and those skilled in the art) Known or easily determined solvent).
  • the compounds of the invention may form hydrates or other solvates.
  • Metal product refers to a product obtained by metabolism of a specific compound or a salt thereof in vivo. Metabolites of a compound can be identified by techniques well known in the art, and the activity can be characterized by experimental methods as described herein. Such a product may be obtained by administering a compound by oxidation, reduction, hydrolysis, amidation, deamidation, esterification, defatting, enzymatic cleavage and the like. Accordingly, the invention includes metabolites of a compound, including metabolites produced by intimate contact of a compound of the invention with a mammal for a period of time.
  • the "pharmaceutically acceptable salt” as used in the present invention means an organic salt and an inorganic salt of the compound of the present invention.
  • Pharmaceutically acceptable salts are well known in the art, as described in the literature: SMBerge et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 1977, 66: 1-19.
  • Salts formed by pharmaceutically acceptable non-toxic acids include, but are not limited to, mineral acid salts formed by reaction with amino groups, hydrochloride, hydrobromide, phosphate, sulfate, perchlorate, And organic acid salts such as acetates, oxalates, maleates, tartrates, citrates, succinates, malonates, or by other methods described in the literature, such as ion exchange These salts.
  • salts include adipate, alginate, ascorbate, aspartate, besylate, benzoate, disulfate, borate, butyrate, camphoric acid Salt, camphor sulfonate, cyclopentylpropionate, digluconate, lauryl sulfate, ethanesulfonate, formate, fumarate, glucoheptonate, glycerophosphate Salt, gluconate, hemisulfate, heptanoate, hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate, lactate, laurate, lauryl sulfate, Malate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate, palmitate, pamoate, pectate, persulphate, 3 -Phenylpropionate
  • Salts obtained by appropriate bases include the alkali metal, alkaline earth metal, ammonium and N + (C 1-4 alkyl) 4 salts.
  • the present invention also contemplates quaternary ammonium salts formed from any of the compounds comprising a group of N. Water soluble or oil soluble or dispersed products can be obtained by quaternization.
  • the alkali metal or alkaline earth metal salts include sodium, lithium, potassium, calcium, magnesium, and the like.
  • Pharmaceutically acceptable salts further comprise suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion, such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -8 sulfonate and aromatic sulfonate.
  • suitable amine cation nontoxic ammonium, quaternary ammonium, and the counterion such as halide, hydroxide, carboxylate, sulfated, phosphorylated compounds, nitrate compounds, C 1 -8 sulfonate and aromatic sulfonate.
  • treating any disease or condition, in some embodiments, “treating” refers to ameliorating a disease or condition (ie, slowing or preventing or alleviating the progression of a disease or at least one of its clinical symptoms). In other embodiments, “treating” refers to alleviating or ameliorating at least one physical parameter, including physical parameters that may not be perceived by the patient. In other embodiments, “treating” refers to modulating a disease or condition from the body (eg, stabilizing a detectable symptom) or physiologically (eg, stabilizing the body's parameters) or both. In other embodiments, “treating” refers to preventing or delaying the onset, onset, or exacerbation of a disease or condition.
  • Figure 1 is a schematic diagram showing the principle of luminescence reduction of a small molecule (compound I) by a thioredox-like oxidoreductase-Rv2466c;
  • Figure 2 is a graph showing the results of fluorescence enhancement of compounds I-1 to I-28 under the action of Rv2466c;
  • Figure 3 is a fluorescence emission spectrum of Compound I-6 and its nitro reduction product I-6b and corresponding hydrolysis products I-6c and I-6d at 390 nm;
  • Figure 5 is a graph showing the results of fluorescence luminescence ratio measurement of Compound I-6 at different concentrations for 45 hours after treatment with different concentrations of M. bovis BCG in the presence or absence of BCG;
  • Figure 6 is a fluorescent photograph of Compound I-6 (8048 in the figure) at different concentrations for 72 hours after treatment with different concentrations of M. bovis BCG, where the light source is a 385-390 nm LED lamp, which is photographed by Huawei mobile phone. ,
  • Picture A is the result of placing the sample to be tested in a 96-well microplate.
  • Figure B shows the results of the sample after the sample to be tested was placed in an eppendorf tube and filtered using a 540 nm bandpass filter (wave width of 80 nm).
  • Reagents and materials compounds (I-1 to I-28, I-69), Rv2466c, MSH, Tris-Cl (pH 7.5), DMSO, 96-well microtiter plate (Corning, 3603)
  • the other two wells were working groups containing enzymes, and each group was tested for two replicate wells. 4) 180 ⁇ L of the enzyme-containing working solution and 180 ⁇ L of the enzyme-free control solution were respectively added to the above wells. 5) After 3 hours, the fluorescence spectrum was studied on a microplate reader.
  • compounds I-1 to I-28 and I-69 exhibit a detectable difference in fluorescence spectral properties before and after reduction by Rv2466c: a compound of formula I It is in a fluorescence quenching state before being reduced by Rv2466c, and returns to fluorescence after being reduced by Rv2466c, that is, it is capable of emitting fluorescence under excitation light of a specific wavelength.
  • 2 is the result of fluorescence enhancement of compounds I-1 to I-28 under the action of Rv2466c, and Table 1 shows the maximum excitation light and emission of compounds I-1 to I-28 and I-69 after reduction by Rv2466c.
  • Example 1 On the basis of Example 1, the inventors studied the fluorescent properties of the compound I of the present invention and its nitro reduction product by taking the compound I-6 as an example according to the following procedure:
  • Reagents and materials compounds (I-6, I-6a, I-6b, I-6c, I-6d), DMSO, 7H9 medium, 96-well microtiter plate (Corning, 3603)
  • Figure 3 shows the fluorescence emission spectra of compound I-6 and its nitro reduction products I-6a, I-6b, I-6c and I-6d at 390 nm
  • Compounds I-6, I-6a, I-6b, I-6c, and I-6d correspond to different maximum excitation light and maximum emission light, respectively, wherein compound I-6 is in fluorescence quenching.
  • the maximum excitation and emission of the compound I-6a are 340 ( ⁇ 30) nm and 457 ( ⁇ 30) nm, respectively; the maximum excitation and emission of the compound I-6b are 390 ( ⁇ 30) nm and 470, respectively. ( ⁇ 30) nm; compounds I-6d are 354 ( ⁇ 30) nm and 470 ( ⁇ 30) nm, respectively; 2) the fluorescence intensity of the emission is also shown when excited with the respective maximum excitation light.
  • Example 1-2 the inventors studied the fluorescence response of the compound I of the present invention to the Rv2466c enzyme by taking the compound I-6 as an example according to the following procedure:
  • Reagents and materials Compound I-6, Rv2466c, DTT (dithiothreitol), MSH, Tris-Cl (pH 7.5), DMSO, 96-well microtiter plate (Corning, 3603)
  • Step 1)-4) In the same manner as in Example 2, in step 5), the fluorescence value was traced at 390 nm excitation and 470 nm emission.
  • the reaction conditions were: 0.5 mM MSH, 1 mM DTT, 200 ⁇ g/mL Rv 2466c, and 2.5 ⁇ g/mL of Example Compound I-6.
  • MSH is unstable and easily oxidized.
  • a disulfide bond reducing agent DTT or NADPH, ie, nicotinamide adenine dinucleotide phosphate
  • DTT or NADPH, ie, nicotinamide adenine dinucleotide phosphate
  • Reagents and materials Compound I-6, BCG, 7H9 medium, DMSO, 96-well microtiter plate (Corning, 3603)
  • Figure 5 shows the results of fluorescence luminescence ratio detection of compound I-6 at different concentrations for 45 hours after treatment with different concentrations of M. bovis BCG in the presence or absence of BCG.
  • Figure 6 shows a fluorescence image taken by a cell phone after excitation of a 385-390 nm LED lamp, showing compound I-6 (labeled 8048 in the figure) at different concentrations for 72 hours after treatment with different concentrations of M. bovis BCG. Fluorescence detection results.
  • Reagents and materials Compound I-6, sputum sample, 7H9 medium, DMSO, 96-well microtiter plate (Corning, 3603)
  • the fluorescence characteristics of the compound I and its derivatives of the present invention, the Rv2466c enzyme and the tuberculosis were studied by the methods of Examples 2-5, taking the compounds I-1 to I-28 and their reduction products and the corresponding hydrolysates as examples.
  • the inventors have found that the compounds I-1 to I-28 and their nitro reduction products I-1a to I-28a exhibit detectable fluorescence changes before and after reduction by the Rv2466c enzyme inherent in M. tuberculosis, and thus It can be used as a detection reagent. After the sample to be tested is in contact with it, whether there is fluorescence change before and after the test based on the sample to be tested can effectively determine whether M.
  • tuberculosis is present in the sample to be tested, and the result is accurate and reliable; compound I-
  • the nitro reduction products I-1b to I-28b of 1 to I-28, and their corresponding hydrolysates I-1c to I-28c, I-1d to I-28d also have detectable fluorescence and can be used for nodules Detection of mycobacteria.

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Abstract

公开了化合物及其在结核分枝杆菌检测中的用途，其中，该化合物为式I所示化合物或其衍生物。利用式I所示化合物或其衍生物作为检测试剂，将待测样本与该检测试剂接触，基于该待测样本在接触前后是否存在荧光变化，即可有效确定待测样本中是否存在结核分枝杆菌，且结果准确可靠。进而该检测信息能够有效用于诊断待测样本来源的疑似患者是否患有结核分枝杆菌相关的疾病，从而实现对结核阳性患者（活动性结核患者）以及结核杆菌携带者的检出，式I化合物如下所示：

Description

化合物及其在结核分枝杆菌检测中的用途
优先权信息
本申请请求2017年07月17日向中国国家知识产权局提交的、专利申请号为201710582928.9的专利申请的优先权和权益，并且通过参照将其全文并入此处。
技术领域
本发明涉及结核分枝杆菌检测技术领域，具体涉及化合物及其在结核分枝杆菌检测中的用途。
背景技术
1882年，德国科学家Robert Koch首次证明结核分枝杆菌(Mycobacterium tuberculosis,Mtb)是结核病的病原菌。结核分枝杆菌简称为结核杆菌，人类以肺部感染最常见。肺部感染的常见症状包括胸痛和长期的多痰性咳嗽。据世界卫生组织(World health organization,WHO)估计，2015年全球新发结核病案例约1040万，其中120万病人合并感染艾滋病，140万人死于结核病。在新发现案例中约有4.6％为多药耐药型结核杆菌(MDR-TB)感染，累积有105个国家确证有广泛耐药型结核杆菌(XDR-TB)感染的案例，世界范围内约有1/3的人是潜伏性结核杆菌的携带者。
我国结核病疫情严重，在全球居第二位。全国第五次结核病流行病学抽样调查报告研究发现：2010年15岁及以上人群中活动性肺结核的患病率为459/10万，涂片阳性肺结核患病率为66/10万；耐多药率为6.8％(19/280)。调查提示我国结核病特别是耐药结核病负担非常严重。另据最新世界卫生组织估计，2015年实际报告的新发病例仅为估计值的2/3，这意味着全世界约有37％的病例未得到诊断或者没有报告；而实际发现的耐药结核病人和报告的病例数也仅约为估计值的1/4。由此可见，优良的早期发现和早期诊断是结核病防控的重点内容。
然而，目前的结核病诊断方法甚至是结核分枝杆菌检测技术仍有待发展。
发明内容
本发明旨在至少解决现有技术中存在的技术问题之一。为此，本发明的一个目的在于提出一种能够快捷有效地检测结核分枝杆菌的手段和方法。
首先，需要说明的是，本发明是基于发明人的下列发现和工作而完成的：
目前发现及确诊肺结核患者的主要方式是痰涂片法以及胸部X-线检查等技术。这些技术的使用已有近百年的历史，存在明显的局限性：1)阳性率低。目前痰涂片阳性准确率只有30％-40％；而X-线胸片平均漏诊率21.5％，平均过诊率19.5％；2)结果易受检查者的 影响。涂片质量及镜检者、阅片者的素质均会影响检查结果的报告。有研究表明，对于X-线胸片，专家个人前后以及各专家相互间的诊断误差可高达20％-30％。3)检测速度慢。使用经典的痰培养(罗氏培养法)和药物敏感试验确定耐药结核病患者需要近3个月时间，往往不仅增加了患者传播的危险，也增加了患者死亡的概率。可见，传统的结核病诊断方法存在着诊断时间长、阳性率低、干扰因素多等缺点。
除了传统的细菌学方法，人们也发展了其他多种诊断方法，包括免疫学方法和分子生物学诊断方法；其中，基于结核分枝杆菌(Mycobacterium tuberculosis,Mtb)核酸扩增为基础的全自动分子诊断方法—Xpert Mtb/RIF技术目前已经获得WHO认可和重点推荐。该方法主要原理是通过PCR技术对Mtb的DNA进行扩增，因而灵敏度高，但样品在采集和制备过程中容易污染，从而易导致假阳性结果，且其昂贵的仪器、对技术人员的高要求、检测试剂的短缺以及检测机器需要严格地校对和定期维护等缺点致使其目前无法在结核发病率较高的发展中国家得到广泛应用。
在实际的临床诊断过程中，Mtb的诊断通常仍然需要多种方法联合应用，以互相弥补各自的不足，提高检出率以及检测的准确度。因此，研发新型的结核诊断试剂正在朝向简单、准确、快速、低成本、高通量、无(低)污染、对技术人员无伤害的方向发展。
发明人在针对本实验室于2014年首次报道的式I所示化合物的抗结核活性研究实验中时发现，
Figure PCTCN2018094422-appb-000001
式I所示化合物(在本文中有时也简称为“化合物I”)不仅对复制性结核杆菌(replicating Mycobacterium tuberculosis,R-Mtb)和非复制性结核杆菌(non-replicating Mycobacterium tuberculosis,NR-Mtb)具有高抑制活性，而且对多种临床耐药菌也同样具有高抑制活性，此外，相对于其他常见的革兰氏阳性菌和阴性菌，化合物I展示出对Mtb(R-Mtb和NR-Mtb)的高度的选择性抑制作用。而发明人的最新研究还发现：化合物I对Mtb的这种高选择性的抑制作用与结核分枝杆菌的一种硫氧还原样氧化还原酶(Rv2466c)相关，而这种抑制作用还依赖于放线菌特异性分泌的氨基酸硫醇Mycothiol(MSH)(结核分枝杆菌属于放线菌纲)。
发明人进一步对上述发现进行了机理研究和实验探索，结果发现：Rv2466c(其氨基酸序列，或制备方法，可参见下列文献获知：Marcelo E.Guerin et al.,ACS Chem.Biol.2014,9,1567-1575)为结核分枝杆菌中固有的硝基还原酶，并与菌内的氧化还原压力有关，其在菌内的表达水平受Sig H因子的直接调节，可在外源性氧化剂如硫醇类氧化剂二酰氨(或称联胺Diamide)的压力下过表达(图1)。Rv2466c在结核分枝杆菌内处于氧化态和还原态平衡 的状态，也即氧化态的Rv2466c和还原态的Rv2466c处于动态平衡的状态，而在MSH存在的条件下(结核分枝杆菌自行分泌MSH)，化合物I中的硝基可被处于还原态的Rv2466c还原，并释放出活性氧或活性氮中间体从而高效杀死Mtb；而处于还原态的Rv2466c则被氧化为氧化态，从而继续下一轮的催化循环过程。
其中，Rv2466c的氨基酸序列如下：
Figure PCTCN2018094422-appb-000002
而发明人在对上述还原过程进行进一步的研究时发现：化合物I在被该结核分枝杆菌中固有的硫氧还原样氧化还原酶Rv2466c还原前后表现出可检测到的荧光光谱性质的差异：式I所示化合物在被Rv2466c还原前处于荧光淬灭状态，在被Rv2466c还原后能够在特定激发光(320-400nm)和发射光(420-510nm)条件下发射荧光。这是因为(参见图1)：化合物I的分子结构中存在强吸电子的硝基呋喃结构，其强吸电子作用使化合物I的香豆素母核内的共轭电子云分布产生偏移，因而，该类化合物在硝基被还原前时荧光处于淬灭的状态(也即荧光关闭状态)；而当硝基呋喃结构中的硝基被Rv2466c还原成为其他形式后，如实施例2中的I-6a、I-6b、I-6c、I-6d(或者呋喃环的双键被富电子的亲和试剂加成后，比如巯基加成得到的硫醚产物，如I-22及I-69也可以在特定的激发光波长条件下发射另一特定波长的荧光)，其化合物整体分子内部的电子云得以重新排布，从而使香豆素环(母核)成为富电子部分，而使得化合物恢复荧光(荧光开启状态)。即式I所示化合物在被结核分枝杆菌中固有的硫氧还原样氧化还原酶Rv2466c还原前后表现出可检测的荧光光谱性质的显著差异。而上述的这种“荧光开关”的“关和开”(off and on)效应(即可检测的荧光光谱性质的差异)也可以在结核杆菌存在的条件下实现(因为结核杆菌内同时包含了硝基呋喃结构中的硝基被还原必需的试剂：Rv2466c和MSH)，因而，基于该可检测的荧光光谱性质的显著差异，利用式I所示化合物，通过荧光检测法即可快捷有效地检测待测样本中的结核分枝杆菌，进而该检测信息能够有效地运用于诊断待测样本来源的疑似患者是否患有结核分枝杆菌相关的疾病，从而实现对结核阳性患者(活动性结核患者)以及结核携带者的检出。此外，发明人还发现，式I所示化合物的衍生物例如硝基还原产物及相应的水解产物，同样具有可检测到的荧光变化(与结核杆菌接触前后)，因而同样也能够被利用于检测待测样本中的结核分枝杆菌以及进一步的结核分枝杆菌相关疾病诊断。
由此，在本发明的第一方面，本发明提供了一种化合物。根据本发明的实施例，该化合物为式I所示化合物或其衍生物：
Figure PCTCN2018094422-appb-000003
其中：
R 1为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
R 2为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
R 3为氢、羟基、C 1-6烷氧基、C 1-6酰氧基、取代或未取代的苯基、苄基、取代或未取代的杂环基、炔基或烯基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
R 4为氢、卤素或C 1-6烷基；
R 5和R 6各自独立地为羟基、烷基、氢、取代或未取代的烷基、烷氧基、酯基或者取代或未取代的巯基，所述烷基和巯基各自独立任选地被选自C 1-6烷基、C 1-6烷基醇、C 1-6烷基硫醇、羟基、酰氧基、氨基、腈基或卤素的取代基取代。
如前所述，发明人发现，式I所示化合物及其衍生物例如硝基还原产物及相应的水解产物，在被结核分枝杆菌中固有的硫氧还原样氧化还原酶Rv2466c还原前后表现出可检测到的荧光变化，因而利用式I所示化合物或其衍生物作为检测试剂，将待测样本与该检测试剂接触，基于该待测样本在接触前后是否存在荧光变化，即可有效确定待测样本中是否存在结核分枝杆菌，且结果准确可靠。进而该检测信息能够有效用于诊断待测样本来源的疑似患者是否患有结核分枝杆菌相关的疾病，从而实现对结核阳性患者(活动性结核患者)以及结核携带者的检出。
其中，需要说明的是，前面所述的荧光变化为下列的至少之一：所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光；所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光，且发射的荧光呈现出强弱变化。由此，利用式I所示化合物或其衍生物与待测样本接触，并进行荧光检测，基于接触前后的荧光检测结果，即可有效确定待测样本中是否存在结核分枝杆菌。
根据本发明的实施例，R 1与R 2连接为4-8元环。
根据本发明的实施例，R 4为氢，R 3为
Figure PCTCN2018094422-appb-000004
R 1O-、R 1OCH 2O-、R 1COO-、HO-、HOCH 2CH 2O-、
Figure PCTCN2018094422-appb-000005
在本发明的另一方面，本发明提出了一种化合物，其为式I所示化合物或其衍生物或式I所示的化合物上可接受的盐：
Figure PCTCN2018094422-appb-000006
其中：
R 1为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
R 2为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
或R 1与R 2连接为C 4-8饱和碳环；
R 3为氢、羟基、C 1-6烷氧基、C 1-6酰氧基、苯基、苄基、C 4-8杂环基、C 2-6炔基、C 2-6烯基、-O-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基-C 1-6烷基-苯基、-O-C 1-6烷基-C 4-8杂环基-(C＝O)-苯基、-O-C 1-6烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-6烷氧基、-O-C(＝O)C 1-6烷基、-O-C 1-6烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基，所述苯基、苄基、C 4-8杂环基、C 2-6炔基、C 2-6烯基、-O-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基-C 1-6烷基-苯基、-O-C 1-6烷基-C 4-8杂环基-(C＝O)-苯基、-O-C 1-6烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-6烷氧基、-O-C(＝O)C 1-6烷基、-O-C 1-6烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基各自独立未被取代或任选地被1～3个选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
R 4为氢、卤素或C 1-6烷基；
或R 3与R 4连接为C 4-8饱和或不饱和碳环或C 4-8饱和或不饱和杂环，其中所述C 4-8饱和或不饱和碳环或C 4-8饱和或不饱和杂环未被取代或任选地被1～3个选自C 1-6烷基或苯基的取代基取代；R 5和R 6各自独立地为羟基、C 1-6烷基、氢、C 1-6烷氧基、-C 1-6烷基酯基、-S-C 1-6 烷基羟基、-C(＝O)-O-C 1-6烷基、-S-C 1-6烷基巯基或者巯基，所述C 1-6烷基、C 1-6烷氧基、-C 1-6烷基酯基、-S-C 1-6烷基羟基、-C(＝O)-O-C 1-6烷基、-S-C 1-6烷基巯基各自独立未被取代或任选地被选自1～3个C 1-6烷基、C 1-6烷基醇、C 1-6烷基硫醇、羟基、酰氧基、氨基、腈基或卤素的取代基取代。
根据发明的实施例，上述化合物还可以包括如下附加技术特征至少之一：
根据本发明的实施例，各R 1、R 2独立地为H、卤素、C 1-3烷基、苯基、苄基、C 5-6杂环基，其中所述C 1-3烷基、苯基、苄基、C 5-6杂环基各自独立地未被取代或任选地被1或2个C 1-3烷基、C 1-3烷氧基、硝基、氨基、CN、卤素取代；或R 1与R 2连接为C 5-6碳环。
根据本发明的实施例，各R 1、R 2独立地为H、甲基、乙基、正丙基、异丙基、苯基、苄基、卤素，其中所述甲基、乙基、正丙基、异丙基、苯基、苄基各自独立地未被取代或任选地被1个C 1-3烷基、C 1-3烷氧基、硝基、氨基、CN、卤素取代。
根据本发明的实施例，R 3为氢、羟基、C 1-3烷氧基、C 1-3酰氧基、苯基、苄基、C 5-6杂环基、C 2-4炔基、C 2-4烯基、-O-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基-C 1-3烷基-苯基、-O-C 1-3烷基-C 5-6杂环基-(C＝O)-苯基、-O-C 1-3烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-3烷氧基、-O-C(＝O)C 1-3烷基、-O-C 1-3烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基，所述苯基、苄基、C 5-6杂环基、C 2-4炔基、C 2-4烯基、-O-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基-C 1-3烷基-苯基、-O-C 1-3烷基-C 5-6杂环基-(C＝O)-苯基、-O-C 1-3烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-3烷氧基、-O-C(＝O)C 1-3烷基、-O-C 1-3烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基各自独立未被取代或任选地被1或2个选自C 1-3烷基、C 1-3烷氧基、硝基、氨基、腈基或卤素的取代基取代；R 4为氢、卤素或C 1-3烷基；或R 3与R 4连接为C 5-6饱和或不饱和碳环或C 5-6饱和或不饱和杂环，其中所述C 5-6饱和或不饱和碳环或C 5-6饱和或不饱和杂环未被取代或任选地被1或2个选自C 1-3烷基或苯基的取代基取代。
根据本发明的实施例，R 4为氢，R 3为
Figure PCTCN2018094422-appb-000007
R aO-、R aOCH 2O-、R aCOO-、HO-、HOCH 2CH 2O-、或
Figure PCTCN2018094422-appb-000008
-N(C 1-3烷基) 2、
Figure PCTCN2018094422-appb-000009
其中，Ra为C 1-6烷基，
各n1、n2、n3、n4独立地为0～3之间的整数；
R b为苯基，-C(＝O)-苯基；其中各苯基未被取代或任选地被1或2个卤素、CN、C 1-3烷基取代；
各Rc、Rd各自独立地为C 1-6烷基，或Rc与Rd连接成C 5-6饱和碳环，
Re为H、卤素；
或R 3与R 4连接为选自如下所示的结构：
Figure PCTCN2018094422-appb-000010
其中，Rf为C 1-3烷基或苯基，n5为0、1或2。
根据本发明的实施例，式(I)所示化合物的衍生物具有如下之一的结构：
Figure PCTCN2018094422-appb-000011
其中，各R x、R y各自独立地为H、羟基、卤素、CN、C 1-6烷基、C 1-6烯基、C 1-6炔基。根据本发明的实施例，各R x、R y各自独立地为H、羟基。
根据本发明的一些具体示例，式I所示化合物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000012
根据本发明的实施例，R 4与R 3连接为4-8元环。根据本发明的一些具体示例，式I所示化合物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000013
根据本发明的实施例，式I所示化合物的衍生物包括但不限于：式I所示化合物的立体异构体、几何异构体、互变异构体、消旋体、氮氧化物、水合物、溶剂合物、代谢产物、药学上可接受的盐或前药、硝基还原产物及其相应的水解产物。
根据本发明的一些实施例，式I所示化合物的衍生物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000014
根据本发明的实施例，上述I-a结构的化合物为相应化合物I的衍生物——硝基还原产物，如前所述，其同样会在被结核分枝杆菌中固有的Rv2466c酶还原前后呈现可检测的荧光变化，因而可以作为检测试剂，在将待测样本与其接触后，基于待测样本在接触前后是否存在荧光变化，即可有效确定该待测样本中是否存在活的结核分枝杆菌，且结果准确可靠。
根据本发明的一些实施例，式I所示化合物的衍生物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000015
根据本发明的实施例，上述I-b结构的化合物同样为相应化合物I的衍生物——硝基还原产物，如前所述，其同样具有可检测的荧光，因而可以用于结核分枝杆菌的检测。
根据本发明的一些实施例，式I所示化合物的衍生物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000016
根据本发明的实施例，上述I-c结构的化合物同样为相应化合物I的衍生物——其硝基还原产物的水解产物，如前所述，其同样具有可检测的荧光，因而可以用于结核分枝杆菌的检测。
根据本发明的一些实施例，式I所示化合物的衍生物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000017
根据本发明的实施例，上述I-d结构的化合物同样为相应化合物I的衍生物——其硝基还原产物的水解产物，如前所述，其同样具有可检测的荧光，因而可以用于结核分枝杆菌的检测。
根据本发明的一些实施例，式I所示化合物的衍生物具有以下其中之一的结构：
Figure PCTCN2018094422-appb-000018
根据本发明的实施例，上述I-e结构的化合物同样为相应化合物I的衍生物——硝基还原产物，同时也为I-b结构的化合物的互变异构体(亚胺-烯胺异构化)，如前所述，其同样具有可检测的荧光，因而可以用于结核分枝杆菌的检测。
在本发明的第二方面，本发明还提供了一种用于检测结核分枝杆菌的试剂盒。根据本发明的实施例，该试剂盒包括前面所述的化合物作为检测试剂。由此，基于式I所示化合物在被结核分枝杆菌中固有的硫氧还原样氧化还原酶Rv2466c还原前后表现出可检测的荧光光谱性质的差异，利用本发明的试剂盒，以式I所示化合物或其衍生物作为检测试剂，将待测样本与该检测试剂接触，基于该待测样本在接触前后是否存在荧光变化，即可有效确定待测样本中是否存在结核分枝杆菌，且结果准确可靠。进而该检测信息能够有效用于诊断待测样本来源的疑似患者是否患有结核分枝杆菌相关的疾病，从而实现对结核阳性患者(活动性结核患者)以及结核携带者的检出。
其中，式I所示化合物及其衍生物的结构和活性前面已详细描述，后面均不再赘述。
在本发明的第三方面，本发明还提供了一种检测结核分枝杆菌的方法。根据本发明的实施例，该方法包括：将待测样本与前面所述的化合物接触；以及基于所述待测样本在接触前后是否存在荧光变化，确定所述待测样本中是否存在结核分枝杆菌。
如前所述，式I所示化合物及其衍生物均是在被Rv2466c还原前处于荧光淬灭状态，在被Rv2466c还原后恢复荧光，即能够在特定波长的激发光下发射荧光。因而，根据本发明的实施例，利用本发明的方法，利用式I所示化合物或其衍生物与待测样本接触后，进行特定波长的激发光的荧光检测，基于是否检测到相应波长的发射光甚至特定的强弱荧光变化，即可有效确定待测样本中是否存在结核分枝杆菌，且结果准确可靠，可重复性好。进而该检测信息能够有效用于诊断待测样本来源的疑似患者是否患有结核分枝杆菌相关的疾病，从而实现对结核阳性患者(活动性结核患者)以及结核携带者的检出。
根据本发明的实施例，所述荧光变化为下列的至少之一：所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光；所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光，且发射的荧光呈现出强弱变化。由此，利用式I所示化合物或其衍生物与待测样本接触，并进行荧光检测，基于接触前后的荧光检测结果，即可有效确定待测样本中是否存在结核分枝杆菌。
其中，需要说明的是，所述待测样本为疑似包含结核分枝杆菌的样本，包括但不限于痰样本。且其样本来源也不受特别限制，可以来源于人，也可以来源于其他动物，例如家畜。
在本发明的第四方面，本发明还提供了前面所述的化合物在筛选药物、检测药物敏感性或死/活菌检测中的用途，所述药物用于治疗或者预防结核分枝杆菌相关的人畜疾病。
根据本发明的一些具体示例，利用本发明的化合物筛选药物，可以按照如下方法进行：将待测药物与结核杆菌接触(例如共孵育一定时间)，然后加入本发明的化合物，并与对照组(不加待测药物，其他条件一致)比较本发明的化合物加入后的荧光强度，其中，本发明化合物加入后产生的荧光相对于对照组(如前所述，未经待测药物接触但接触本发明化合物的结核杆菌)的荧光显著减弱是该待测药物具有预防或治疗结核分枝杆菌感染相关疾 病的作用的指示。
而利用本发明的化合物检测药物敏感性，同样可以按照上述方法进行，只是将待测药物设置系列浓度梯度，实验时，在相同结核菌量的条件下，将系列浓度的待测药物分别与结核杆菌接触(例如共孵育一定时间)，然后加入本发明的化合物，并比较各处理的待测药物组以及对照组(不加待测药物)之间在本发明的化合物加入后的荧光强度，当一处理组在本发明化合物加入后与对照组相比荧光恰好无变化时，可判断体系内结核杆菌量已低于化合物的最低检测限，相应地，该菌量对应的待测药物浓度即为MIC(待测药物的最低抑菌浓度)。
根据本发明的实施例，所述结核分枝杆菌感染相关的疾病为小儿结核、肺结核、肠结核、淋巴结核、骨结核、肾结核、结核性腹膜炎、结核性脑膜炎、药物敏感性结核病、多药耐药性结核病、广泛耐药性结核病、潜伏性结核病或HIV合并感染的结核病。
根据本发明的另一些实施例，所述结核分枝杆菌感染相关的疾病为动物结核相关的疾病。
根据本发明的实施例，所述药物为抗结核先导化合物或抗结核药物。
此外，需要说明的是，本发明的结核分枝杆菌检测方法，相对于现有技术具有下列优点的至少之一：
(1)传统的抗酸染色法和金胺“O”染色法需要在显微镜下收集300个视野，相比较而言，本发明的荧光方法所涉及到的诊断技术具有操作简便，快捷，安全性高、且能够实现高通量诊断的优点。
(2)相对于临床检测的金标准—培养法，因结核分枝杆菌的生长速度缓慢，所以其临床报阳时间一般为一个月，而报阴则需要两个月。而本发明所涉及到的诊断技术具有结果报告明显加快的特点，有望实现对门诊病人高效快速地检测。
(3)相对于已报道的基于有机小分子的荧光探针法，本发明的方法的检测原理是基于Rv2466c还原前后荧光“开关”的“关和开”，具有信噪比高，特异性高的特点。
(4)相对于已报道的基于有机小分子的荧光探针法，本发明的方法有望实现对活动及潜伏Mtb、所有耐药Mtb全部检出，并大大提高检出的灵敏性以及准确率。
(5)发明所涉及到的检测用有机小分子(式I所示化合物)的化学性质稳定，可重复冻融，耐强酸、强碱，耐蛋白酶，耐核酸酶，可在临床样本处理时的任何条件下使用。
(6)此发明所涉及到的检测用有机小分子(式I所示化合物)具有合成效率极高且合成成本极低的优点，具备大规模生产的条件。
(7)此发明所涉及到的检测用有机小分子(式I所示化合物)具有显著的抑菌活性，相对于其他检测方法具有明显的安全性。
(8)此发明所涉及到的检测方法，无需特殊的大型仪器设备，相较于其他分子生物学方法(技术)具有明显的低成本及不易被污染的特点。
(9)此发明所涉及到的检测方法，相较于其他免疫学方法具有特异性高的特点。
另外，为方便理解，现对本文涉及的术语进行说明：
术语“包含”为开放式表达，即包括本发明所指明的内容，但并不排除其他方面的内容。
“立体异构体”是指具有相同化学构造，但原子或基团在空间上排列方式不同的化合物。立体异构体包括对映异构体、非对映异构体、构象异构体(旋转异构体)、几何异构体(顺/反)异构体、阻转异构体，等等。
“对映异构体”是指一个化合物的两个不能重叠但互成镜像关系的异构体。
“非对映异构体”是指有两个或多个手性中心并且其分子不互为镜像的立体异构体。非对映异构体具有不同的物理性质，如熔点、沸点、光谱性质和反应性。非对映异构体混合物可通过高分辨分析操作如电泳和色谱，例如HPLC来分离。
“手性”是具有与其镜像不能重叠性质的分子；而“非手性”是指与其镜像可以重叠的分子。
本发明所使用的立体化学定义和规则一般遵循S.P.Parker,Ed.,McGraw-Hill Dictionary of Chemical Terms(1984)McGraw-Hill Book Company,New York；and Eliel,E.and Wilen,S.,"Stereochemistry of Organic Compounds",John Wiley&Sons,Inc.,New York,1994。
许多有机化合物以光学活性形式存在，即它们具有使平面偏振光的平面发生旋转的能力。在描述光学活性化合物时，使用前缀D和L或R和S来表示分子关于其一个或多个手性中心的绝对构型。前缀d和l或(+)和(-)是用于指定化合物所致平面偏振光旋转的符号，其中(-)或l表示化合物是左旋的。前缀为(+)或d的化合物是右旋的。一种具体的立体异构体是对映异构体，这种异构体的混合物称作对映异构体混合物。对映异构体的50:50混合物称为外消旋混合物或外消旋体，当在化学反应或过程中没有立体选择性或立体特异性时，可出现这种情况。
本发明公开化合物的任何不对称原子(例如，碳等)都可以以外消旋或对映体富集的形式存在，例如(R)-、(S)-或(R,S)-构型形式存在。在某些实施方案中，各不对称原子在(R)-或(S)-构型方面具有至少50％对映体过量，至少60％对映体过量，至少70％对映体过量，至少80％对映体过量，至少90％对映体过量，至少95％对映体过量，或至少99％对映体过量。
依据起始物料和方法的选择，本发明化合物可以以可能的异构体中的一个或它们的混合物，例如外消旋体和非对应异构体混合物(这取决于不对称碳原子的数量)的形式存在。光学活性的(R)-或(S)-异构体可使用手性合成子或手性试剂制备，或使用常规技术拆分。如果化合物含有一个双键，取代基可能为E或Z构型；如果化合物中含有二取代的环烷基，环烷基的取代基可能有顺式或反式构型。
所得的任何立体异构体的混合物可以依据组分物理化学性质上的差异被分离成纯的或基本纯的几何异构体，对映异构体，非对映异构体，例如，通过色谱法和/或分步结晶法。
可以用已知的方法将任何所得终产物或中间体的外消旋体通过本领域技术人员熟悉的方法拆分成光学对映体，如，通过对获得的其非对映异构的盐进行分离。外消旋的产物也可以通过手性色谱来分离，如，使用手性吸附剂的高效液相色谱(HPLC)。特别地，对映异 构体可以通过不对称合成制备，例如，可参考Jacques,et al.,Enantiomers,Racemates and Resolutions(Wiley Interscience,New York,1981)；Principles of Asymmetric Synthesis(2 nd Ed.Robert E.Gawley,Jeffrey Aubé,Elsevier,Oxford,UK,2012)；Eliel,E.L.Stereochemistry of Carbon Compounds(McGraw-Hill,NY,1962)；Wilen,S.H.Tables of Resolving Agents and Optical Resolutions p.268(E.L.Eliel,Ed.,Univ.of Notre Dame Press,Notre Dame,IN 1972)；Chiral Separation Techniques:A Practical Approach(Subramanian,G.Ed.,Wiley-VCH Verlag GmbH&Co.KGaA,Weinheim,Germany,2007)。
术语“互变异构体”或“互变异构形式”是指具有不同能量的可通过低能垒(low energy barrier)互相转化的结构异构体。若互变异构是可能的(如在溶液中)，则可以达到互变异构体的化学平衡。例如，质子互变异构体(protontautomer)(也称为质子转移互变异构体(prototropic tautomer))包括通过质子迁移来进行的互相转化，如酮-烯醇异构化和亚胺-烯胺异构化。价键互变异构体(valence tautomer)包括通过一些成键电子的重组来进行的互相转化。酮-烯醇互变异构的具体实例是戊烷-2,4-二酮和4-羟基戊-3-烯-2-酮互变异构体的互变。互变异构的另一个实例是酚-酮互变异构。酚-酮互变异构的一个具体实例是吡啶-4-醇和吡啶-4(1H)-酮互变异构体的互变。除非另外指出，本发明化合物的所有互变异构体形式都在本发明的范围之内。
像本发明所描述的，本发明的化合物可以任选地被一个或多个取代基所取代，如上面的通式化合物，或者像实施例里面特殊的例子，子类，和本发明所包含的一类化合物。应了解“任选取代的”这个术语与“取代或非取代的”这个术语可以交换使用。一般而言，术语“取代的”表示所给结构中的一个或多个氢原子被具体取代基所取代。除非其他方面表明，一个任选的取代基团可以在基团各个可取代的位置进行取代。当所给出的结构式中不只一个位置能被选自具体基团的一个或多个取代基所取代，那么取代基可以相同或不同地在各个位置取代。
在本说明书的各部分，本发明公开化合物的取代基按照基团种类或范围公开。特别指出，本发明包括这些基团种类和范围的各个成员的每一个独立的次级组合。例如，术语“C 1-6烷基”特别指独立公开的甲基、乙基、C 3烷基、C 4烷基、C 5烷基和C 6烷基。
在本发明的各部分，描述了连接取代基。当该结构清楚地需要连接基团时，针对该基团所列举的马库什变量应理解为连接基团。例如，如果该结构需要连接基团并且针对该变量的马库什基团定义列举了“烷基”，则应该理解，该“烷基”代表连接的亚烷基基团。
在本发明中，烷基是指具有特定碳原子数的直链和支链饱和脂肪基。
烷氧基是指具有指定碳原子数的烷基-O-基团。
“卤素”或“卤代”是指作为取代基的氟、氯、溴或碘。当卤原子作为取代基的时候，其取代的数目可以是一个、两个或三个。
芳基包括苯基、取代苯基(此处取代苯基包括下述一个、两个或三个基团：C 1-6烷基，C 1-6烷氧基，腈基，硝基，氨基或卤素)。
本发明使用的术语了“杂环”表示稳定的5-至7-元单环，这些杂环可以是饱和的或不 饱和的，并由碳原子和任选自N、O和S的1至4个杂原子组成，其中的氮和硫杂原子可被选择性地氧化，且氮杂原子可被选择性地季铵化，优选6元杂环，例如吡啶、哌啶、吡嗪、哌嗪、吗啉或硫吗啉等。
本发明中，当结构式中带有
Figure PCTCN2018094422-appb-000019
或
Figure PCTCN2018094422-appb-000020
或类似符号时，指的是该共价键处于纸面上方或者下方的位置，但是需要注意的是，若非特别说明，这种构型仅具有相对的含义。同样，当提及“顺”或者“反”时，若非特别说明，也仅仅是说基团的相对位置。
溶剂合物是指通式化合物与反应有机溶剂或者水等形成的包结溶剂或者在晶格中含有水，或者通过仪器分析手段获取的在分子式中包含的水或者溶剂的分子的形式。本发明采用的药学上可接受的溶剂包括不干扰本发明化合物的生物活性的那些溶剂(例如水、乙醇、乙酸、N,N-二甲基甲酰胺、二甲亚砜以及该领域技术人员所知的或容易确定的溶剂)。本发明的化合物可形成水合物或其他溶剂合物。本领域技术人员已知将化合物与水一起冻干时形成水合物或在溶液中与合适的有机溶剂浓缩时形成溶剂合物的方法。因此，在本发明也包括本发明化合物的水合物和溶剂合物。
“代谢产物”是指具体的化合物或其盐在体内通过代谢作用所得到的产物。一个化合物的代谢产物可以通过所属领域公知的技术来进行鉴定，其活性可以通过如本发明所描述的那样采用试验的方法进行表征。这样的产物可以是通过给药化合物经过氧化，还原，水解，酰氨化，脱酰氨作用，酯化，脱脂作用，酶裂解等等方法得到。相应地，本发明包括化合物的代谢产物，包括将本发明的化合物与哺乳动物充分接触一段时间所产生的代谢产物。
本发明所使用的“药学上可接受的盐”是指本发明的化合物的有机盐和无机盐。药学上可接受的盐在所属领域是为我们所熟知的，如文献：S.M.Berge et al.,describe pharmaceutically acceptable salts in detail in J.Pharmaceutical Sciences,1977,66:1-19.所记载的。药学上可接受的无毒的酸形成的盐包括，但并不限于，与氨基基团反应形成的无机酸盐有盐酸盐，氢溴酸盐，磷酸盐，硫酸盐，高氯酸盐，和有机酸盐如乙酸盐，草酸盐，马来酸盐，酒石酸盐，柠檬酸盐，琥珀酸盐，丙二酸盐，或通过书籍文献上所记载的其他方法如离子交换法来得到这些盐。其他药学上可接受的盐包括己二酸盐，藻酸盐，抗坏血酸盐，天冬氨酸盐，苯磺酸盐，苯甲酸盐，重硫酸盐，硼酸盐，丁酸盐，樟脑酸盐，樟脑磺酸盐，环戊基丙酸盐，二葡萄糖酸盐，十二烷基硫酸盐，乙磺酸盐，甲酸盐，反丁烯二酸盐，葡庚糖酸盐，甘油磷酸盐，葡萄糖酸盐，半硫酸盐，庚酸盐，己酸盐，氢碘酸盐，2-羟基-乙磺酸盐，乳糖醛酸盐，乳酸盐，月桂酸盐，月桂基硫酸盐，苹果酸盐，丙二酸盐，甲磺酸盐，2-萘磺酸盐，烟酸盐，硝酸盐，油酸盐，棕榈酸盐，扑酸盐，果胶酸盐，过硫酸盐，3-苯基丙酸盐，苦味酸盐，特戊酸盐，丙酸盐，硬脂酸盐，硫氰酸盐，对甲苯磺酸盐，十一酸盐，戊酸盐，等等。通过适当的碱得到的盐包括碱金属，碱土金属，铵和N +(C 1-4烷基) 4的盐。本发明也拟构思了任何所包含N的基团的化合物所形成的季铵盐。水溶性或油溶性或分散产物可以通过季铵化作用得到。碱金属或碱土金属盐包括钠，锂，钾，钙，镁， 等等。药学上可接受的盐进一步包括适当的、无毒的铵，季铵盐和抗平衡离子形成的胺阳离子，如卤化物，氢氧化物，羧化物，硫酸化物，磷酸化物，硝酸化物，C 1-8磺酸化物和芳香磺酸化物。
如本发明所使用的术语“治疗”任何疾病或病症，在其中一些实施方案中，“治疗”指改善疾病或病症(即减缓或阻止或减轻疾病或其至少一种临床症状的发展)。在另一些实施方案中，“治疗”指缓和或改善至少一种身体参数，包括可能不为患者所察觉的身体参数。在另一些实施方案中，“治疗”指从身体上(例如稳定可察觉的症状)或生理学上(例如稳定身体的参数)或上述两方面调节疾病或病症。在另一些实施方案中，“治疗”指预防或延迟疾病或病症的发作、发生或恶化。
本发明的附加方面和优点将在下面的描述中部分给出，部分将从下面的描述中变得明显，或通过本发明的实践了解到。
附图说明
本发明的上述和/或附加的方面和优点从结合下面附图对实施例的描述中将变得明显和容易理解，其中：
图1是硫氧还原样氧化还原酶—Rv2466c还原小分子(化合物I)而发光的原理示意图；
图2是化合物I-1至I-28在Rv2466c作用下荧光增强的结果；
图3是化合物I-6及其硝基还原产物I-6b及相应水解产物I-6c及I-6d在390nm下的荧光发射光谱；
图4在DTT及MSH存在的条件下，Rv2466c催化还原化合物I-6后，在激发光及发射光(390/470nm)条件下跟踪检测的结果；
图5是在有无BCG存在的条件下，不同浓度下化合物I-6对不同浓度梯度的牛分枝杆菌BCG处理48小时后的荧光发光比值检测结果；
图6是不同浓度下化合物I-6(图中标注的8048)对不同浓度梯度的牛分枝杆菌BCG处理72小时后的荧光拍摄照片，其中，光源为385-390nm LED灯，采用华为手机拍摄，
其中，
A图为：将待测样本置于96孔酶标板中的拍摄结果，
B图为：将待测样本置于eppendorf管中并使用540nm带通滤光片(波宽为80nm)过滤后的拍摄结果。
具体实施方式
下面将结合实施例对本发明的方案进行解释。本领域技术人员将会理解，下面的实施例仅用于说明本发明，而不应视为限定本发明的范围。实施例中未注明具体技术或条件的，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。所用试剂或仪器未注明生产厂商者，均为可以通过市购获得的常规产品。
实施例1 式I所示化合物对Rv2466c酶的荧光响应
仪器：多功能酶标仪(
Figure PCTCN2018094422-appb-000021
Flash，Thermo Scientific)
试剂与材料：化合物(I-1至I-28、I-69)，Rv2466c，MSH，Tris-Cl(pH 7.5)，DMSO，96孔酶标板(Corning，3603)
操作：1)配制含有0.5mM MSH的50mM Tris-Cl(pH 7.5)缓冲液，各取20mL缓冲液于两个无菌管中。2)向其中一管中加入一定量的Rv2466c，使其终浓度为200μg/mL，此为含酶工作液，向另一管中加入等量的Tris-Cl缓冲液作为不含酶对照液。3)将实施例化合物分别溶于DMSO，配制为25μg/mL的溶液，每个化合物均各取20μL加于96孔板中的四个孔中，其中两个孔作不含酶的对照组，另两个孔为含酶的工作组，每组实验两个复孔。4)分别取180μL含酶工作液和180μL不含酶对照液加于上述孔中。5)3小时后于酶标仪上进行荧光光谱的研究。
结果，发明人发现：化合物I-1至I-28以及I-69(即式I所示的部分化合物)在被Rv2466c还原前后均表现出可检测的荧光光谱性质的差异：式I所示化合物在被Rv2466c还原前处于荧光淬灭状态，在被Rv2466c还原后恢复荧光，即能够在特定波长的激发光下发射荧光。其中，图2为化合物I-1至I-28在Rv2466c作用下荧光增强的结果，表1示出了化合物I-1至I-28及I-69在被Rv2466c还原后的最大激发光及发射光的波长。由此，表明式I所示化合物在结核分枝杆菌中固有的硫氧还原样氧化还原酶Rv2466c还原前后表现出可检测的荧光光谱性质的差异。
表1.部分化合物I在Rv2466c作用下的最大激发光及发射光的波长
Figure PCTCN2018094422-appb-000022
Figure PCTCN2018094422-appb-000023
实施例2
在实施例1的基础上，发明人按照以下步骤，以化合物I-6为例，研究本发明的化合物I及其硝基还原产物的荧光特性：
根据质谱分析结果可知，化合物I-6在Mtb存在的条件下被还原为I-6a、I-6b以及I-6d，而根据I-6d的结构推测存在中间体I-6c，因此，推测I-6存在如下还原及水解过程：
Figure PCTCN2018094422-appb-000024
然后进行如下：
仪器：多功能酶标仪(
Figure PCTCN2018094422-appb-000025
Flash，Thermo Scientific)
试剂与材料：化合物(I-6、I-6a、I-6b、I-6c、I-6d)，DMSO，7H9培养基，96孔酶标板(Corning，3603)
操作：1)取适量化合物I-6、I-6a、I-6b、I-6c、I-6d粉末，将其配制成10mg/mL的DMSO溶液。2)将此DMSO溶液使用7H9培养基进行1000倍稀释，即得10μg/mL的测试液，分别取200μL置于96孔酶标板，于全波长酶标仪上进行三个化合物荧光光谱性质的研究。结果见图3。
图3显示了化合物I-6及其硝基还原产物I-6a、I-6b、I-6c及I-6d在390nm下的荧光发射光谱
由上述结果可知：1)化合物I-6、I-6a、I-6b、I-6c、I-6d分别对应不同的最大激发光和最大发射光，其中，化合物I-6处于荧光淬灭的状态；化合物I-6a的最大激发光和发射光分别为340(±30)nm和457(±30)nm；化合物I-6b的最大激发光和发射光分别为390(±30)nm和470(±30)nm；化合物I-6d则分别为354(±30)nm和470(±30)nm；2)在使用各自的最大激发光对其进行激发时，其发射的荧光强度也显示出显著性的差异，其中，以390nm激发，收集470nm的荧光信号，我们发现化合物I-6b的荧光强度最强，约为其母体分子I-6的300倍。
实施例3
在实施例1-2的基础上，发明人按照以下步骤，以化合物I-6为例，研究本发明的化合物I对Rv2466c酶的荧光响应：
仪器：多功能酶标仪(
Figure PCTCN2018094422-appb-000026
Flash，Thermo Scientific)
试剂与材料：化合物I-6，Rv2466c，DTT(二硫苏糖醇)，MSH，Tris-Cl(pH 7.5)，DMSO，96孔酶标板(Corning，3603)
操作：步骤1)-4)同实施例2，步骤5)中需在390nm激发和470nm发射下跟踪检测荧光值。其中，反应条件为：0.5mM MSH,1mM DTT，200μg/mL Rv2466c以及2.5μg/mL实施例化合物I-6。
结果见图4。
图4在DTT及MSH存在的条件下，Rv2466c催化还原化合物I-6后，在激发光及发射光(390/470nm)条件下跟踪检测的结果。由此，表明在DTT及MSH存在的条件下，Rv2466c催化还原实施例化合物I-6并发射荧光。
其中，MSH不稳定，易被氧化，为保持其活性，反应中添加二硫键还原剂DTT(或NADPH，即烟酰胺腺嘌呤二核苷酸磷酸)，以便将已氧化的MSH还原。
实施例4
在实施例1-3的基础上，发明人按照以下步骤，以化合物I-6为例，研究本发明的化合物I对结核分枝杆菌的荧光响应：
仪器：多功能酶标仪(
Figure PCTCN2018094422-appb-000027
Flash，Thermo Scientific)，LED灯(385-390nm)
试剂与材料：化合物I-6，BCG，7H9培养基，DMSO，96孔酶标板(Corning，3603)
操作：1)取处于对数生长期的牛分枝杆菌BCG测量OD 580，并将菌液密度从4.93×10 7以2倍梯度稀释至1.92×10 5(4.93×10 7,2.46×10 7,1.23×10 7,6.16×10 6,3.08×10 6,1.54×10 6,7.70×10 5,3.85×10 5,1.92×10 5cell/mL)。2)移取200μL各浓度梯度的菌液至96孔酶标板中，加入2.0μL不同浓度梯度的实施例化合物I-6(200,100,50,25,12.5 6.25μg/mL)，混匀，使其终浓度分别为2.0，1.0，0.5，0.25，0.125，0.0625μg/mL，每组实验两个复孔。3)设定激发光为390nm，发射光为470nm，立刻于酶标仪上读取其荧光值，37℃下孵育48小时后再次读取荧光值。结果见图5-图6。
图5显示的是在有无BCG存在的条件下，不同浓度下化合物I-6对不同浓度梯度的牛分枝杆菌BCG处理48小时后的荧光发光比值检测结果。图6显示的是385-390nm的LED灯激发后由手机拍摄的荧光图，显示了不同浓度下化合物I-6(图中标注为8048)对不同浓度梯度的牛分枝杆菌BCG处理72小时后的荧光检测结果。
结果表明：在BCG存在的条件下，无论是采用荧光酶标的方法还是使用相应波长的激发光照射后直接肉眼观看的方法，均可以在有菌存在的条件下检测到明显的荧光。
实施例5本发明涉及的化合物对临床痰样本的检测
仪器：多功能酶标仪(
Figure PCTCN2018094422-appb-000028
Flash，Thermo Scientific)，LED灯(385-390nm)
试剂与材料：化合物I-6，痰样本，7H9培养基，DMSO，96孔酶标板(Corning，3603)
操作:1)取5mL痰将其按照临床痰样本处理规程处理。2)将处理后的痰浓缩至500μL，后加入I-6，使其终浓度为1μg/mL。3)于37℃下共孵育72h，使用LED灯照射并观察结果。结果表2。
表2.部分临床痰样本检测结果
Figure PCTCN2018094422-appb-000029
Figure PCTCN2018094422-appb-000030
结果表明：在上述12份痰样本中，涂片法检出的阳性样本为2份，而使用本发明的方法进行检测可检测到的阳性样本为5份。与临床最常见的涂片法相比，本发明所提到的方法大大提高了检出率。
实施例6
按照实施例2-5的方法，以化合物I-1至I-28及其还原产物及相应的水解产物为例，研究本发明的化合物I及其衍生物的荧光特性、对Rv2466c酶和结核分枝杆菌的荧光响应，以及临床痰样本的检测。
结果，发明人发现，化合物I-1至I-28及其硝基还原产物I-1a至I-28a均会在被结核分枝杆菌中固有的Rv2466c酶还原前后呈现可检测的荧光变化，因而可以作为检测试剂，在将待测样本与其接触后，基于待测样本在接触前后是否存在荧光变化，即可有效确定该待测样本中是否存在结核分枝杆菌，且结果准确可靠；化合物I-1至I-28的硝基还原产物I-1b至I-28b，及其相应的水解产物I-1c至I-28c、I-1d至I-28d同样具有可检测的荧光，可以用于结核分枝杆菌的检测。
其中，还原产物I-1a至I-28a的结构如下：
Figure PCTCN2018094422-appb-000031
还原产物I-1b至I-28b的结构如下：
Figure PCTCN2018094422-appb-000032
相应的水解产物I-1c至I-28c的结构如下：
Figure PCTCN2018094422-appb-000033
相应的水解产物I-1d至I-28d的结构如下：
Figure PCTCN2018094422-appb-000034
实施例7 I-a，I-b，I-c以及I-d的制备
40mg I溶解在5mL乙酸乙酯中，向溶液中加入5％Pd/C(40mg)，反应混合液置于氢气环境中于50℃反应1至3小时，TLC监测反应的进行，原料转化完后，反应液过滤后，浓缩，之后柱层析纯化，即可得到I-a和I-b。
将0.061mmol I-b溶解于1mL冰醋酸中，向体系中加入0.61mmol H 2O，反应于80℃下搅拌3.5小时，TLC监测反应的进行，反应结束后，加入2mL二氯甲烷，之后分别水洗，饱和氯化钠洗，收集有机相，无水硫酸钠干燥，随后过滤，滤液浓缩，甲基叔丁基醚/乙醚重结晶即可得到I-c。
将0.061mmol I-b加入4mL冰醋酸/水(体积比为1:1)的溶液中，80℃下反应2小时，TLC监测反应的进行，反应结束后，加入2mL水，过滤，收集滤液，真空减压浓缩，之后再加入4mL水，可看到灰色固体析出，过滤，水洗，真空干燥过夜，即可得到I-d。
在本说明书的描述中，参考术语“一个实施例”、“一些实施例”、“示例”、“具体示例”、或“一些示例”等的描述意指结合该实施例或示例描述的具体特征、结构、材料或者 特点包含于本发明的至少一个实施例或示例中。在本说明书中，对上述术语的示意性表述不一定指的是相同的实施例或示例。而且，描述的具体特征、结构、材料或者特点可以在任何的一个或多个实施例或示例中以合适的方式结合。
尽管已经示出和描述了本发明的实施例，本领域的普通技术人员可以理解：在不脱离本发明的原理和宗旨的情况下可以对这些实施例进行多种变化、修改、替换和变型，本发明的范围由权利要求及其等同物限定。

Claims (25)

  1. 一种化合物，其为式I所示化合物或其衍生物：
    Figure PCTCN2018094422-appb-100001
    其中：
    R 1为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 2为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 3为氢、羟基、C 1-6烷氧基、C 1-6酰氧基、取代或未取代的苯基、苄基、取代或未取代的杂环基、炔基或烯基、，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 4为氢、卤素或C 1-6烷基；
    R 5和R 6各自独立地为羟基、烷基、氢、取代或未取代的烷基、烷氧基、酯基或者取代或未取代的巯基，所述烷基和巯基各自独立任选地被选自C 1-6烷基、C 1-6烷基醇、C 1-6烷基硫醇、羟基、酰氧基、氨基、腈基或卤素的取代基取代。
  2. 根据权利要求1所述的化合物，其特征在于，R 1与R 2连接为4-8元环。
  3. 根据权利要求1所述的化合物，其特征在于，R 4为氢，R 3为
    Figure PCTCN2018094422-appb-100002
    R 1O-、R 1OCH 2O-、R 1COO-、HO-、HOCH 2CH 2O-、
    Figure PCTCN2018094422-appb-100003
  4. 一种化合物，其为式I所示化合物或其衍生物或式I所示的化合物上可接受的盐：
    Figure PCTCN2018094422-appb-100004
    其中：
    R 1为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 2为氢、卤素、C 1-6烷基、取代或未取代的苯基、苄基或者取代或未取代的杂环基，所述苯基和杂环基各自独立任选地被选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    或R 1与R 2连接为C 4-8饱和碳环；
    R 3为氢、羟基、C 1-6烷氧基、C 1-6酰氧基、苯基、苄基、C 4-8杂环基、C 2-6炔基、C 2-6烯基、-O-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基-C 1-6烷基-苯基、-O-C 1-6烷基-C 4-8杂环基-(C＝O)-苯基、-O-C 1-6烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-6烷氧基、-O-C(＝O)C 1-6烷基、-O-C 1-6烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基，所述苯基、苄基、C 4-8杂环基、C 2-6炔基、C 2-6烯基、-O-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基、-O-C 1-6烷基-C 4-8杂环基-C 1-6烷基-苯基、-O-C 1-6烷基-C 4-8杂环基-(C＝O)-苯基、-O-C 1-6烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-6烷氧基、-O-C(＝O)C 1-6烷基、-O-C 1-6烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基各自独立未被取代或任选地被1～3个选自C 1-6烷基、C 1-6烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 4为氢、卤素或C 1-6烷基；
    或R 3与R 4连接为C 4-8饱和或不饱和碳环或C 4-8饱和或不饱和杂环，其中所述C 4-8饱和或不饱和碳环或C 4-8饱和或不饱和杂环未被取代或任选地被1～3个选自C 1-6烷基或苯基的取代基取代；R 5和R 6各自独立地为羟基、C 1-6烷基、氢、C 1-6烷氧基、-C 1-6烷基酯基、-S-C 1-6烷基羟基、-C(＝O)-O-C 1-6烷基、-S-C 1-6烷基巯基或者巯基，所述C 1-6烷基、C 1-6烷氧基、-C 1-6烷基酯基、-S-C 1-6烷基羟基、-C(＝O)-O-C 1-6烷基、-S-C 1-6烷基巯基各自独立未被取代或任选地被选自1～3个C 1-6烷基、C 1-6烷基醇、C 1-6烷基硫醇、羟基、酰氧基、氨基、腈基或卤素的取代基取代。
  5. 根据权利要求4所述的化合物，其特征在于，各R 1、R 2独立地为H、卤素、C 1-3烷基、苯基、苄基、C 5-6杂环基，其中所述C 1-3烷基、苯基、苄基、C 5-6杂环基各自独立地未被取代或任选地被1或2个C 1-3烷基、C 1-3烷氧基、硝基、氨基、CN、卤素取代；
    或R 1与R 2连接为C 5-6碳环。
  6. 根据权利要求4所述的化合物，其特征在于，各R 1、R 2独立地为H、甲基、乙基、正丙基、异丙基、苯基、苄基、卤素，其中所述甲基、乙基、正丙基、异丙基、苯基、苄基各自独立地未被取代或任选地被1个C 1-3烷基、C 1-3烷氧基、硝基、氨基、CN、卤素取代。
  7. 根据权利要求4所述的化合物，其特征在于，R 3为氢、羟基、C 1-3烷氧基、C 1-3酰氧基、苯基、苄基、C 5-6杂环基、C 2-4炔基、C 2-4烯基、-O-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基-C 1-3烷基-苯基、-O-C 1-3烷基-C 5-6杂环基-(C＝O)-苯基、-O-C 1-3 烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-3烷氧基、-O-C(＝O)C 1-3烷基、-O-C 1-3烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基，所述苯基、苄基、C 5-6杂环基、C 2-4炔基、C 2-4烯基、-O-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基、-O-C 1-3烷基-C 5-6杂环基-C 1-3烷基-苯基、-O-C 1-3烷基-C 5-6杂环基-(C＝O)-苯基、-O-C 1-3烷基-O(C＝O)-C 5-6杂芳基、羟基-C 1-3烷氧基、-O-C(＝O)C 1-3烷基、-O-C 1-3烷基氨基、-O-C 5-6杂环基-C(＝O)-苯基、氨基各自独立未被取代或任选地被1或2个选自C 1-3烷基、C 1-3烷氧基、硝基、氨基、腈基或卤素的取代基取代；
    R 4为氢、卤素或C 1-3烷基；
    或R 3与R 4连接为C 5-6饱和或不饱和碳环或C 5-6饱和或不饱和杂环，其中所述C 5-6饱和或不饱和碳环或C 5-6饱和或不饱和杂环未被取代或任选地被1或2个选自C 1-3烷基或苯基的取代基取代。
  8. 根据权利要求4所述的化合物，其特征在于，R 4为氢，R 3为
    Figure PCTCN2018094422-appb-100005
    R aO-、R aOCH 2O-、R aCOO-、HO-、HOCH 2CH 2O-、或
    Figure PCTCN2018094422-appb-100006
    Figure PCTCN2018094422-appb-100007
    -N(C 1-3烷基) 2、
    Figure PCTCN2018094422-appb-100008
    其中，Ra为C 1-6烷基，
    各n1、n2、n3、n4独立地为0～3之间的整数；
    R b为苯基，-C(＝O)-苯基；其中各苯基未被取代或任选地被1或2个卤素、CN、C 1-3烷基取代；
    各Rc、Rd各自独立地为C 1-6烷基，或Rc与Rd连接成C 5-6饱和碳环，
    Re为H、卤素；
    或R 3与R 4连接为选自如下所示的结构：
    Figure PCTCN2018094422-appb-100009
    其中，Rf为C 1-3烷基或苯基，n5为0、1或2。
  9. 根据权利要求1-8任一项所述的化合物，其特征在于，式(I)所示化合物的衍生物具有如下之一的结构：
    Figure PCTCN2018094422-appb-100010
    其中，各R x、R y各自独立地为H、羟基、卤素、CN、C 1-6烷基、C 1-6烯基、C 1-6炔基。
  10. 根据权利要求9所述的化合物，其特征在于，各R x、R y各自独立地为H、羟基。
  11. 根据权利要求3-10任一项所述的化合物，其特征在于，式I所示化合物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100011
  12. 根据权利要求1所述的化合物，其特征在于，R 4与R 3连接为4-8元环。
  13. 根据权利要求12或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100012
  14. 根据权利要求1或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物的衍生物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100013
  15. 根据权利要求1或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物的衍生物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100014
  16. 根据权利要求1或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物的衍生物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100015
  17. 根据权利要求1或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物的衍生物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100016
  18. 根据权利要求1或权利要求4-10任一项所述的化合物，其特征在于，式I所示化合物的衍生物具有以下其中之一的结构：
    Figure PCTCN2018094422-appb-100017
  19. 一种用于检测结核分枝杆菌的试剂盒，其特征在于，包括权利要求1-18任一项所述的化合物作为检测试剂。
  20. 一种检测结核分枝杆菌的方法，其特征在于，包括：
    将待测样本与权利要求1-18任一项所述的化合物接触；以及
    基于所述待测样本在接触前后是否存在荧光变化，确定所述待测样本中是否存在结核分枝杆菌。
  21. 根据权利要求20所述的方法，其特征在于，所述荧光变化为下列的至少之一：
    所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光；
    所述待测样本在接触前处于荧光淬灭状态，在接触后能够被320-400nm的激发光激发并发射420-510nm的荧光，且发射的荧光呈现出强弱变化。
  22. 权利要求1-18任一项所述的化合物在筛选药物、检测药物敏感性或死/活菌检测中的用途，所述药物用于治疗或者预防结核分枝杆菌相关的人畜疾病。
  23. 根据权利要求22所述的用途，其特征在于，所述结核分枝杆菌感染相关的疾病为小儿结核、肺结核、肠结核、淋巴结核、骨结核、肾结核、结核性腹膜炎、结核性脑膜炎、药物敏感性结核病、多药耐药性结核病、广泛耐药性结核病、潜伏性结核病或HIV合并感染的结核病。
  24. 根据权利要求22所述的用途，其特征在于，所述结核分枝杆菌感染相关的疾病为动物结核相关的疾病。
  25. 根据权利要求22所述的用途，其特征在于，所述药物为抗结核先导化合物或抗结核药物。
PCT/CN2018/094422 2017-07-17 2018-07-04 化合物及其在结核分枝杆菌检测中的用途 WO2019015475A1 (zh)

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